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Sample records for acids including serine

  1. The bioactive acidic serine- and aspartate-rich motif peptide.

    PubMed

    Minamizaki, Tomoko; Yoshiko, Yuji

    2015-01-01

    The organic component of the bone matrix comprises 40% dry weight of bone. The organic component is mostly composed of type I collagen and small amounts of non-collagenous proteins (NCPs) (10-15% of the total bone protein content). The small integrin-binding ligand N-linked glycoprotein (SIBLING) family, a NCP, is considered to play a key role in bone mineralization. SIBLING family of proteins share common structural features and includes the arginine-glycine-aspartic acid (RGD) motif and acidic serine- and aspartic acid-rich motif (ASARM). Clinical manifestations of gene mutations and/or genetically modified mice indicate that SIBLINGs play diverse roles in bone and extraskeletal tissues. ASARM peptides might not be primary responsible for the functional diversity of SIBLINGs, but this motif is suggested to be a key domain of SIBLINGs. However, the exact function of ASARM peptides is poorly understood. In this article, we discuss the considerable progress made in understanding the role of ASARM as a bioactive peptide.

  2. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor.

    PubMed

    Dounay, A B; Forsyth, C J

    2002-11-01

    As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

  3. D-amino acids in the brain: the biochemistry of brain serine racemase.

    PubMed

    Baumgart, Florian; Rodríguez-Crespo, Ignacio

    2008-07-01

    It has been recently established that in various brain regions D-serine, the product of serine racemase, occupies the so-called 'glycine site' within N-methyl D-aspartate receptors. Mammalian brain serine racemase is a pyridoxal-5' phosphate-containing enzyme that catalyzes the racemization of L-serine to D-serine. It has also been shown to catalyze the alpha,beta-elimination of water from L-serine or D-serine to form pyruvate and ammonia. Serine racemase is included within the group of type II-fold pyridoxal-5' phosphate enzymes, together with many other racemases and dehydratases. Serine racemase was first purified from rat brain homogenates and later recombinantly expressed in mammalian and insect cells as well as in Escherichia coli. It has been shown that serine racemase is activated by divalent cations like calcium, magnesium and manganese, as well as by nucleotides like ATP, ADP or GTP. In turn, serine racemase is also strongly inhibited by reagents that react with free sulfhydryl groups such as glutathione. Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein interacting with C kinase 1 and Golga3 to bind to serine racemase, having different effects on its catalytic activity or stability. In addition, it has also been proposed that serine racemase is regulated by phosphorylation. Thus, d-serine production in the brain is tightly regulated by various factors pointing at its physiologic importance. In this minireview, we will focus on the regulation of brain serine racemase and d-serine synthesis by the factors mentioned above.

  4. Detection systems for carbapenemase gene identification should include the SME serine carbapenemase.

    PubMed

    Bush, Karen; Pannell, Megan; Lock, John L; Queenan, Anne Marie; Jorgensen, James H; Lee, Ryan M; Lewis, James S; Jarrett, Deidre

    2013-01-01

    Carbapenemase detection has become a major problem in hospitals that encounter outbreaks of infections caused by carbapenem-resistant Gram-negative bacteria. Rapid detection systems have been reported using multiplex PCR analyses and DNA microarray assays. Major carbapenemases that are detected by these systems include the KPC and OXA serine carbapenemases, and the IMP, VIM and NDM families of metallo-β-lactamases. However, increasing numbers of the SME serine carbapenemase are being reported from Serratia marcescens, especially from North and South America. These organisms differ from many of the other carbapenemase-producing pathogens in that they are generally susceptible to the expanded-spectrum cephalosporins ceftazidime and cefepime while retaining resistance to almost all other β-lactam antibiotics. Thus, multiplex PCR assays or DNA microarray testing of carbapenem-resistant S. marcescens isolates should include analyses for production of the SME carbapenemase. Confirmation of the presence of this enzyme may provide reassurance that oxyimino-cephalosporins can be considered for treatment of infections caused by these carbapenem-resistant pathogens.

  5. Efficient asymmetric synthesis of N-protected-β-aryloxyamino acids via regioselective ring opening of serine sulfamidate carboxylic acid.

    PubMed

    Malhotra, Rajesh; Dey, Tushar K; Dutta, Swarup; Basu, Sourav; Hajra, Saumen

    2014-09-07

    First regioselective ring opening of serine derived cyclic sulfamidate by hard nucleophiles like ArONa is developed, where β-elimination of serine sulfamidate ester by stronger nucleophiles is overcome by reversal of the electronic effect of the carboxylate anion. This method provides easy and direct access to a variety of N-Boc- and N-PMB protected β-aryloxy-α-amino acids with complete retention of enantiopurity in moderate to high yields.

  6. The effects of a low protein diet on amino acids and enzymes in the serine synthesis pathway in mice.

    PubMed

    Antflick, Jordan E; Baker, Glen B; Hampson, David Richard

    2010-06-01

    L-serine is required for cellular and tissue growth and is particularly important in the immature brain where it acts as a crucial neurotrophic factor. In this study, the levels of amino acids and enzymes in the L-serine biosynthetic pathway were examined in the forebrain, cerebellum, liver, and kidney after the exposure of mice to protein-restricted diets. The levels of L-serine, D-serine, and L-serine-O-phosphate were quantified by HPLC and quantitative Western blotting was used to measure changes in protein levels of five enzymes in the pathway. The L-serine biosynthetic enzyme phosphoserine phosphatase was strongly upregulated, while the serine degradative enzymes serine racemase and serine dehydratase were downregulated in the livers and kidneys of mice fed low (6%) or very low (2%) protein diets for 2 weeks compared with mice fed a normal diet (18% protein). No changes in these enzymes were seen in the brain. The levels of L-serine increased in the livers of mice fed 2% protein; in contrast, D-serine levels were reduced below the limit of detection in the livers of mice given either the 6 or 2% diets. D-Serine is a co-agonist at the NMDA class of glutamate receptors; no alterations in NMDA-R1 subunit expression were observed in liver or brain after protein restriction. These findings demonstrate that the expression of L-serine synthetic and degradative enzymes display reciprocal changes in the liver and kidney to increase L-serine and decrease D-serine levels under conditions of protein restriction, and that the brain is insulated from such changes.

  7. 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

  8. Synthesis of unnatural amino acids from serine derivatives by beta-fragmentation of primary alkoxyl radicals.

    PubMed

    Boto, Alicia; Gallardo, Juan A; Hernández, Dacil; Hernández, Rosendo

    2007-09-14

    The fragmentation of primary alkoxyl radicals has been scarcely used in synthesis since other competing processes (such as oxidation or hydrogen abstraction) usually predominate. However, when serine derivatives were used as substrates, the scission took place in excellent yields. Tandem scission-allylation, -alkylation, or -arylation reactions were subsequently developed. This one-pot methodology was applied to the synthesis of unnatural amino acids, which are useful synthetic blocks or amino acid surrogates in peptidomimetics.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. The epidermis of scales in gecko lizards contains multiple forms of beta-keratins including basic glycine-proline-serine-rich proteins.

    PubMed

    Toni, M; Dalla Valle, L; Alibardi, L

    2007-05-01

    The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.

  15. Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

    PubMed Central

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

  16. Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A.

    PubMed

    Saino, Tomoyuki; Watson, Eileen L

    2009-01-01

    Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.

  17. Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases.

    PubMed

    Yin, DeLu Tyler; Kazlauskas, Romas J

    2012-06-25

    Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (β(0)=170  M(-1)) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (β(0)=330  M(-1)), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by (18)O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T(d)1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes.

  18. An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations

    NASA Astrophysics Data System (ADS)

    Pusterla, Ivano; Bode, Jeffrey W.

    2015-08-01

    Amide-forming ligation reactions allow the chemical synthesis of proteins by the union of unprotected peptide segments, and enable the preparation of protein derivatives not accessible by expression or bioengineering approaches. The native chemical ligation (NCL) of thioesters and N-terminal cysteines is unquestionably the most successful approach, but is not ideal for all synthetic targets. Here we describe the synthesis of an Fmoc-protected oxazetidine amino acid for use in the α-ketoacid-hydroxylamine (KAHA) amide ligation. When incorporated at the N-terminus of a peptide segment, this four-membered cyclic hydroxylamine can be used for rapid serine-forming ligations with peptide α-ketoacids. This ligation operates at low concentration (100 μM-5 mM) and mild temperatures (20-25 °C). The utility of the reaction was demonstrated by the synthesis of S100A4, a 12 kDa calcium-binding protein not easily accessible by NCL or other amide-forming reactions due to its primary sequence and properties.

  19. 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.

  20. Chemoattraction of Vibrio fischeri to Serine, Nucleosides, and N-Acetylneuraminic Acid, a Component of Squid Light-Organ Mucus

    PubMed Central

    DeLoney-Marino, Cindy R.; Wolfe, Alan J.; Visick, Karen L.

    2003-01-01

    Newlyhatched juveniles of the Hawaiian squid Euprymna scolopes rapidly become colonized by the bioluminescent marine bacterium Vibrio fischeri. Motility is required to establish the symbiotic colonization, but the role of chemotaxis is unknown. In this study we analyzed chemotaxis of V. fischeri to a number of potential attractants. The bacterium migrated toward serine and most sugars tested. V. fischeri also exhibited the unusual ability to migrate to nucleosides and nucleotides as well as to N-acetylneuraminic acid, a component of squid mucus. PMID:14660408

  1. Targeting Class A and C Serine β-Lactamases with a Broad-Spectrum Boronic Acid Derivative

    PubMed Central

    2015-01-01

    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

  2. N-terminal amino acid sequences of D-serine deaminases of wild-type and operator-constitutive strains of Escherichia coli K-12.

    PubMed Central

    Heincz, M C; McFall, E

    1975-01-01

    The N-terminal amino acid sequences of the D-serine deaminases from strains of Escherichia coli K-12 that harbor wild-type and high-level constitutive catabolite-insensitive operator-initiator regions are identical: Met-Ser-GluNH2-Ser-Gly-Arg-His-Cys. This result indicates that the operator-initiator region is probably distinct from the D-serine deaminase structural gene. Images PMID:1099073

  3. Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine.

    PubMed

    Ovchinnikova, Olga G; Kocharova, Nina A; Torzewska, Agnieszka; Blaszczyk, Aleksandra; Shashkov, Alexander S; Knirel, Yuriy A; Rozalski, Antoni

    2005-05-23

    The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.

  4. An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor.

    PubMed

    Hedley, M L; Amrein, H; Maniatis, T

    1995-12-05

    We have identified an amino acid sequence in the Drosophila Transformer (Tra) protein that is capable of directing a heterologous protein to nuclear speckles, regions of the nucleus previously shown to contain high concentrations of spliceosomal small nuclear RNAs and splicing factors. This sequence contains a nucleoplasmin-like bipartite nuclear localization signal (NLS) and a repeating arginine/serine (RS) dipeptide sequence adjacent to a short stretch of basic amino acids. Sequence comparisons from a number of other splicing factors that colocalize to nuclear speckles reveal the presence of one or more copies of this motif. We propose a two-step subnuclear localization mechanism for splicing factors. The first step is transport across the nuclear envelope via the nucleoplasmin-like NLS, while the second step is association with components in the speckled domain via the RS dipeptide sequence.

  5. β-Amino acid catalyzed asymmetric Michael additions: design of organocatalysts with catalytic acid/base dyad inspired by serine proteases.

    PubMed

    Yang, Hui; Wong, Ming Wah

    2011-09-16

    A new type of chiral β-amino acid catalyst has been computationally designed, mimicking the enzyme catalysis of serine proteases. Our catalyst approach is based on the bioinspired catalytic acid/base dyad, namely, a carboxyl and imidazole pair. DFT calculations predict that this designed organocatalyst catalyzes Michael additions of aldehydes to nitroalkenes with excellent enantioselectivities and remarkably high anti diastereoselectivities. The unusual stacked geometry of the enamine intermediate, hydrogen bonding network, and the adoption of an exo transition state are the keys to understand the stereoselectivity.

  6. Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of Type 2 diabetes: 2. Optimization of serine and threonine ether amino acid residues.

    PubMed

    Sparks, Steven M; Banker, Pierette; Bickett, David M; Clancy, Daphne C; Dickerson, Scott H; Garrido, Dulce M; Golden, Pamela L; Peat, Andrew J; Sheckler, Lauren R; Tavares, Francis X; Thomson, Stephen A; Weiel, James E

    2009-02-01

    Optimization of the amino acid residue of a series of anthranilimide-based glycogen phosphorylase inhibitors is described leading to the identification of serine and threonine ether analogs. t-Butylthreonine analog 20 displayed potent in vitro inhibition of GPa, low potential for P450 inhibition, and excellent pharmacokinetic properties.

  7. Homology modeling and identification of amino acids involved in the catalytic process of Mycobacterium tuberculosis serine acetyltransferase.

    PubMed

    Qiu, Juanjuan; Zang, Shizhu; Ma, Yufang; Owusu, Lawrence; Zhou, Lei; Jiang, Tao; Xin, Yi

    2017-03-01

    Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of L‑cysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for anti‑tuberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, site‑directed mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished ~75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective anti‑TB drugs.

  8. 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.

  9. Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

    PubMed Central

    Schaap, D; van der Wal, J; van Blitterswijk, W J; van der Bend, R L; Ploegh, H L

    1993-01-01

    In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor. Images Figure 2 Figure 3 Figure 4 PMID:7679574

  10. 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

  11. Application of serine- and threonine-derived cyclic sulfamidates for the preparation of S-linked glycosyl amino acids in solution- and solid-phase peptide synthesis.

    PubMed

    Cohen, Scott B; Halcomb, Randall L

    2002-03-20

    Cyclic sulfamidates were synthesized in 60% yield from L-serine and allo-L-threonine, respectively. These sulfamidates reacted with a variety of unprotected 1-thio sugars in aqueous bicarbonate buffer (pH 8) to afford the corresponding S-linked serine- and threonine-glycosyl amino acids with good diastereoselectivity (> or =97%) after hydrolysis of the N-sulfates. The serine-derived sulfamidate was incorporated into a simple dipeptide to generate a reactive dipeptide substrate that underwent chemoselective ligation with a 1-thio sugar to afford an S-linked glycopeptide. This sulfamidate was also incorporated into a peptide on a solid support in conjunction with solid-phase peptide synthesis. Chemoselective ligation of a 1-thio sugar with the cyclic sulfamidate was achieved on the solid support, followed by removal of the N-sulfate. Finally, the peptide chain of the resulting support-bound S-linked glycopeptide was extended using standard peptide synthesis procedures.

  12. Identification of Genes, including the Gene Encoding p27Kip1, Regulated by Serine 276 Phosphorylation of the p65 Subunit of NF-κB

    PubMed Central

    Prasad, Ratna Chakraborty; Wang, Xiaohui L.; Law, Brian K.; Davis, Bradley; Green, Gail; Boone, Braden; Sims, Lauren; Law, Mary

    2009-01-01

    Phosphorylation of the p65 subunit of NF-κB is required for its transcriptional activity. Recent reports show that phosphorylation of p65 at serine 276 regulates only a subset of genes, such as those encoding IL-6, IL-8, Gro-β, and ICAM-1. In order to identify additional genes regulated by serine 276 phosphorylation, HepG2 hepatoma cells were infected with adenoviruses encoding either wild-type p65 or the S276A mutant of p65, followed by DNA microarray analysis. The results show that mutation of serine 276 affected the expression of several genes that encode proteins involved in cell cycle regulation, signal transduction, transcription, and metabolism. Notably, expression of S276A increased the mRNA and protein level of p27, a cell cycle inhibitory protein, which led to an increased association of p27 with cdk2, and inhibition of cdk2 activity. Furthermore, while wild-type NF-κB is known to increase cell proliferation in a number of different cancer cell lines, our data show that S276A inhibits cell proliferation. Evidence is mounting that NF-κB plays a pivotal role in oncogenesis. Therapeutic agents that regulate the phosphorylation of serine 276 and p27 gene expression, therefore, may be useful as anti-cancer agents in the future. PMID:19038492

  13. Structure-based approach for identification of novel phenylboronic acids as serine-β-lactamase inhibitors

    NASA Astrophysics Data System (ADS)

    Sgrignani, Jacopo; De Luca, Filomena; Torosyan, Hayarpi; Docquier, Jean-Denis; Duan, Da; Novati, Beatrice; Prati, Fabio; Colombo, Giorgio; Grazioso, Giovanni

    2016-10-01

    β-Lactamases are bacterial enzymes conferring resistance to β-lactam antibiotics in clinically-relevant pathogens, and represent relevant drug targets. Recently, the identification of new boronic acids (i.e. RPX7009) paved the way to the clinical application of these molecules as potential drugs. Here, we screened in silico a library of 1400 boronic acids as potential AmpC β-lactamase inhibitors. Six of the most promising candidates were evaluated in biochemical assays leading to the identification of potent inhibitors of clinically-relevant β-lactamases like AmpC, KPC-2 and CTX-M-15. One of the selected compounds showed nanomolar K i value with the clinically-relevant KPC-2 carbapenemase, while another one exhibited broad spectrum inhibition, being also active on Enterobacter AmpC and the OXA-48 class D carbapenemase.

  14. 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).

  15. D-serine is distributed in neurons in the brain of the sea lamprey.

    PubMed

    Villar-Cerviño, Verona; Barreiro-Iglesias, Antón; Rodicio, María Celina; Anadón, Ramón

    2010-05-15

    The amino acid D-serine is an endogenous coagonist of N-methyl-D-aspartate (NMDA) receptors in mammals that has been shown to play an important role in synaptic function, behavior, learning, and memory. The distribution and cellular location of D-serine in the brain of the sea lamprey was investigated by using immunofluorescence methods. One major finding of our study, unlike early studies of mammals, was the localization of D-serine immunoreactivity in perikarya and dendrites of neurons, whereas D-serine immunoreactivity was not generally observed in the lamprey glia. D-serine-immunoreactive neurons were observed in different brain regions, including the olfactory bulb, medial pallium, thalamus, torus semicircularis, isthmus, and reticular formation. The colocalization of D-serine with gamma-aminobutyric acid (GABA) was also studied with a double-immunofluorescence technique. The relationship between D-serine and glycine immunoreactivities was studied in alternate parallel series of sections stained for either D-serine/GABA or glycine/GABA. Colocalization with GABA was observed in various D-serine-immunoreactive populations, and codistribution and possible colocalization with glycine was also observed in some populations, mainly in the dorsal isthmic gray, medial octavolateral nucleus, dorsal column nucleus, interpeduncular nucleus, and reticular formation. Although numerous fibers were strongly GABA- and glycine-immunoreactive, D-serine immunoreactivity was observed mostly in cell perikarya and dendrites. The present results indicate that the D-serine immunoreactive cells are small to medium-sized neurons, some exhibiting classical inhibitory neurotransmitters, in which D-serine might be acting as a modulator. The neuronal distribution of D-serine and its frequent colocalization and/or codistribution with the two main inhibitory neurotransmitters appeared early in vertebrates.

  16. Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase.

    PubMed Central

    Mikkelsen, J; Højrup, P; Rasmussen, M M; Roepstorff, P; Knudsen, J

    1985-01-01

    Goat mammary fatty acid synthetase was labelled in the acyltransferase domain by formation of O-ester intermediates by incubation with [1-14C]acetyl-CoA and [2-14C]malonyl-CoA. Tryptic-digest and CNBr-cleavage peptides were isolated and purified by high-performance reverse-phase and ion-exchange liquid chromatography. The sequences of the malonyl- and acetyl-labelled peptides were shown to be identical. The results confirm the hypothesis that both acetyl and malonyl groups are transferred to the mammalian fatty acid synthetase complex by the same transferase. The sequence is compared with those of other fatty acid synthetase transferases. PMID:3922356

  17. Cobalamin deficiency results in severe metabolic disorder of serine and threonine in rats.

    PubMed

    Ebara, S; Toyoshima, S; Matsumura, T; Adachi, S; Takenaka, S; Yamaji, R; Watanabe, F; Miyatake, K; Inui, H; Nakano, Y

    2001-12-05

    Dietary cobalamin (vitamin B12; Cbl) deficiency caused significant increases in plasma serine, threonine, glycine, alanine, tyrosine, lysine and histidine levels in rats. In particular, the serine and threonine levels were over five and eight times, respectively, higher in the Cbl-deficient rats than those in the sufficient controls. In addition, some amino acids, including serine and threonine, were excreted into urine at significantly higher levels in the deficient rats. When Cbl was supplemented into the deficient rats for 2 weeks, in coincidence with the disappearance of the urinary excretion of methylmalonic acid (an index of Cbl deficiency), the plasma serine and threonine levels were normalized. These results indicate that Cbl deficiency results in metabolic disorder of certain amino acids, including serine and threonine. The expression level of hepatic serine dehydratase (SDH), which catalyzes the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, was significantly lowered by Cbl deficiency, even though Cbl does not participate directly in the enzyme reaction. The SDH activity in the deficient rats was less than 20% of that in the sufficient controls, and was normalized 2 weeks after the Cbl supplementation. It is thus suggested that the decrease of the SDH expression relates closely with the abnormalities in the plasma and urinary levels of serine and threonine in the Cbl-deficient rats.

  18. Modification of histones by sugar β-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.

    PubMed

    Zhang, Suisheng; Roche, Kevin; Nasheuer, Heinz-Peter; Lowndes, Noel Francis

    2011-10-28

    The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

  19. l-Serine Deficiency Elicits Intracellular Accumulation of Cytotoxic Deoxysphingolipids and Lipid Body Formation*

    PubMed Central

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

    2015-01-01

    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

  20. 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

  1. In vitro evidence that D-serine disturbs the citric acid cycle through inhibition of citrate synthase activity in rat cerebral cortex.

    PubMed

    Zanatta, Angela; Schuck, Patrícia Fernanda; Viegas, Carolina Maso; Knebel, Lisiane Aurélio; Busanello, Estela Natacha Brandt; Moura, Alana Pimentel; Wajner, Moacir

    2009-11-17

    The present work investigated the in vitro effects of D-serine (D-Ser) on important parameters of energy metabolism in cerebral cortex of young rats. The parameters analyzed were CO(2) generation from glucose and acetate, glucose uptake and the activities of the respiratory chain complexes I-IV, of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase and of creatine kinase and Na(+),K(+)-ATPase. Our results show that D-Ser significantly reduced CO(2) production from acetate, but not from glucose, reflecting an impairment of the citric acid cycle function. Furthermore, D-Ser did not affect glucose uptake. We also observed that the activity of the mitochondrial enzyme citrate synthase from mitochondrial preparations and purified citrate synthase was significantly inhibited by D-Ser, whereas the other activities of the citric acid cycle as well as the activities of complexes I-III, II-III, II and IV of the respiratory chain, creatine kinase and Na(+),K(+)-ATPase were not affected by this D-amino acid. We also found that L-serine did not affect citrate synthase activity from mitochondrial preparations and purified enzyme. The data indicate that D-Ser impairs the citric acid cycle activity via citrate synthase inhibition, therefore compromising energy metabolism production in cerebral cortex of young rats. Therefore, it is presumed that this mechanism may be involved at least in part in the neurological damage found in patients affected by disorders in which D-Ser metabolism is impaired, with altered cerebral concentrations of this D-amino acid.

  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. 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

  4. The cooperative effect between active site ionized groups and water desolvation controls the alteration of acid/base catalysis in serine proteases.

    PubMed

    Shokhen, Michael; Khazanov, Netaly; Albeck, Amnon

    2007-08-13

    What is the driving force that alters the catalytic function of His57 in serine proteases between general base and general acid in each step along the enzymatic reaction? The stable tetrahedral complexes (TC) of chymotrypsin with trifluoromethyl ketone transition state analogue inhibitors are topologically similar to the catalytic transition state. Therefore, they can serve as a good model to study the enzyme catalytic reaction. We used DFT quantum mechanical calculations to analyze the effect of solvation and of polar factors in the active site of chymotrypsin on the pKa of the catalytic histidine in FE (the free enzyme), EI (the noncovalent enzyme inhibitor complex), and TC. We demonstrated that the acid/base alteration is controlled by the charged groups in the active site--the catalytic Asp102 carboxylate and the oxyanion. The effect of these groups on the catalytic His is modulated by water solvation of the active site.

  5. 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.

  6. Nucleotide and deduced amino acid sequences of a subtilisin-like serine protease from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T).

    PubMed

    Kurata, Atsushi; Uchimura, Kohsuke; Shimamura, Shigeru; Kobayashi, Tohru; Horikoshi, Koki

    2007-11-01

    The acpI gene encoding an alkaline protease (AcpI) from a deep-sea bacterium, Alkalimonas collagenimarina AC40(T), was shotgun-cloned and sequenced. It had a 1,617-bp open reading frame encoding a protein of 538 amino acids. Based on analysis of the deduced amino acid sequence, AcpI is a subtilisin-like serine protease belonging to subtilase family A. It consists of a prepropeptide, a catalytic domain, and a prepeptidase C-terminal domain like other serine proteases from the genera Pseudomonas, Shewanella, Alteromonas, and Xanthomonas. Heterologous expression of the acpI gene in Escherichia coli cells yielded a 28-kDa recombinant AcpI (rAcpI), suggesting that both the prepropeptide and prepeptidase C-terminal domains were cleaved off to give the mature form. Analysis of N-terminal and C-terminal amino acid sequences of purified rAcpI showed that the mature enzyme would be composed of 273 amino acids. The optimal pH and temperature for the caseinolytic activity of the purified rAcpI were 9.0-9.5 and 45 degrees C in 100 mM glycine-NaOH buffer. Calcium ions slightly enhanced the enzyme activity and stability. The enzyme favorably hydrolyzed gelatin, collagen, and casein. AcpI from A. collagenimarina AC40(T) was also purified from culture broth, and its molecular mass was around 28 kDa, indicating that the cleavage manner of the enzyme is similar to that in E. coli cells.

  7. 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).

  8. Serine 157, a retinoic acid receptor alpha residue phosphorylated by protein kinase C in vitro, is involved in RXR.RARalpha heterodimerization and transcriptional activity.

    PubMed

    Delmotte, M H; Tahayato, A; Formstecher, P; Lefebvre, P

    1999-12-31

    Retinoic acid (RA) regulation of cellular proliferation and differentiation is mediated, at least in part, through two related nuclear receptors, RAR and RXR. RA-induced modulation of gene expression leads generally to cellular differentiation, whereas stimulation of the protein kinase C (PKC) signaling pathway is associated with cellular proliferation. Pursuant to our discovery that prolonged activation of PKCs induced a strong decrease in RA responsiveness of a retinoid-inducible reporter gene, we have further investigated the connections between these two signaling pathways. We demonstrate that PKC isoforms alpha and gamma are able to phosphorylate human RARalpha (hRARalpha) in vitro on a single serine residue located in the extended DNA binding domain (T box). The introduction of a negative charge at this position (serine 157) strongly decreased hRARalpha transcriptional activity, whereas a similar mutation at other PKC consensus phosphorylation sites had no effect. The effect on transcriptional activation was correlated with a decrease in the capacity of hRARalpha to heterodimerize with hRXRalpha. Thus hRARalpha is a direct target for PKCalpha and gamma, which may control retinoid receptor transcriptional activities during cellular proliferation and differentiation.

  9. 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.

  10. Conformers of Gaseous Serine.

    PubMed

    He, Kedan; Allen, Wesley D

    2016-08-09

    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.

  11. 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.

  12. 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

  13. 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.

  14. Serine recombinases as tools for genome engineering.

    PubMed

    Brown, William R A; Lee, Nicholas C O; Xu, Zhengyao; Smith, Margaret C M

    2011-04-01

    The serine recombinases differ mechanistically from the tyrosine recombinases and include proteins such as ϕC31 integrase which, unlike Cre and Flp, promote unidirectional reactions. The serine recombinase family is large and includes many other proteins besides ϕC31 integrase with the potential to be widely used in genome engineering. Here we review the details of the mechanism of the reactions promoted by the serine recombinases and discuss how these not only limit the utility of this class of recombinase but also creates opportunities for the engineering of new enzymes. We discuss the unanswered questions posed by genome engineering experiments in a variety of systems in which the serine recombinases have been used and finally describe more recently discovered serine recombinases that have the potential to be used in genome engineering.

  15. Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues.

    PubMed Central

    Bigler, T. L.; Lu, W.; Park, S. J.; Tashiro, M.; Wieczorek, M.; Wynn, R.; Laskowski, M.

    1993-01-01

    In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. beta-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of gamma-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6-18 peptide GlyNH2 were

  16. Serine proteases inhibiting cyanopeptides.

    PubMed

    Radau, G

    2000-08-01

    There are many compounds inhibiting serine proteases which play an important role in the human organism. This article reviews publications on the low-molecular weight, serine protease inhibitory cyanopeptides and reports on new developments in establishing structure-activity relationships.

  17. Arjunolic acid ameliorates reactive oxygen species via inhibition of p47phox-serine phosphorylation and mitochondrial dysfunction

    PubMed Central

    Miriyala, Sumitra; Chandra, Mini; Maxey, Benjamin; Day, Alicia; St. Clair, Daret K.; Panchatcharam, Manikandan

    2015-01-01

    Impaired cardiovascular function during acute myocardial infarction (MI) is partly associated with recruitment of activated polymorphonuclear neutrophils. The protective role of arjunolic acid (AA; 2:3:23-Trihydroxy olean-12-en-28-oic acid) is studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation. Neutrophils were isolated from normal and acute MI mice to find out the efficacy of AA in reducing oxidative stress. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) resulted in an oxidative burst of superoxide anion (O2•−) and enhanced release of lysosomal enzymes. The treatment of neutrophils with PMA induced phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase. Furthermore, we observed activated ERK induced phosphorylation of Ser345 in MI neutrophils. Treatment with AA significantly inhibited the phosphorylation of P47phox and ERK in the stimulated controls and MI neutrophils. Oxidative phosphorylation activities in MI cells were lower than in control, while the glycolysis rates were elevated in MI cells compared to the control. In addition, we observed AA decreased intracellular oxidative stress and reduced the levels of O2•− in neutrophils. This study therefore identifies targets for AA in activated neutrophils mediated by the MAPK pathway on p47phox involved in ROS generation. PMID:26319153

  18. L-Serine deaminase activity is induced by exposure of Escherichia coli K-12 to DNA-damaging agents.

    PubMed Central

    Newman, E B; Ahmad, D; Walker, C

    1982-01-01

    The synthesis of L-serine deaminase in Escherichia coli K-12 was induced after exposure of cells to a variety of DNA-damaging agents, including UV irradiation, nalidixic acid, and mitomycin C. Synthesis was also induced during growth at high temperature. A mutant constitutive for SOS functions showed an elevated level of L-serine deaminase activity. The response to DNA-damaging agents thus may be mediated via the SOS system. PMID:6813312

  19. 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)

  20. 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.

  1. Gas chromatographic analysis of infant formulas for total fatty acids, including trans fatty acids.

    PubMed

    Satchithanandam, Subramaniam; Fritsche, Jan; Rader, Jeanne I

    2002-01-01

    Twelve powdered and 13 liquid infant formulas were analyzed by using an extension of AOAC Official Method 996.01 for fat analysis in cereal products. Samples were hydrolyzed with 8 N HCl and extracted with ethyl and petroleum ethers. Fatty acid methyl esters were prepared by refluxing the mixed ether extracts with methanolic sodium hydroxide in the presence of 14% boron trifluoride in methanol. The extracts were analyzed by gas chromatography. In powdered formulas, saturated fatty acid (SFA) content (mean +/- SD; n = 12) was 41.05 +/- 3.94%, monounsaturated fatty acid (MUFA) content was 36.97 +/- 3.38%, polyunsaturated fatty acid (PUFA) content was 20.07 +/- 3.08%, and total trans fatty acid content was 1.30 +/- 1.27%. In liquid formulas, SFA content (mean +/- SD; n = 13) was 42.29 +/- 2.98%, MUFA content was 36.05 +/- 2.47%, PUFA content was 20.65 +/- 2.40%, and total trans fatty acid content was 0.88 +/- 0.54%. Total fat content in powdered formulas ranged from 4.4 to 5.5 g/100 kcal and linoleic acid content ranged from 868 to 1166 mg/100 kcal. In liquid formulas, total fat content ranged from 4.1 to 5.1 g/100 kcal and linoleic acid content ranged from 820 to 1100 mg/100 kcal. There were no significant differences between powdered and liquid infant formulas in concentrations of total fat, SFA, MUFA, PUFA, or trans fatty acids.

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

    DOEpatents

    Nasarabadi, Shanavaz [Livermore, CA

    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.

  3. Simultaneous quantification of D- vs. L-serine, taurine, kynurenate, phosphoethanolamine and diverse amino acids in frontocortical dialysates of freely-moving rats: differential modulation by N-methyl-D-aspartate (NMDA) and other pharmacological agents.

    PubMed

    Gobert, Alain; Rivet, Jean-Michel; Billiras, Rodolphe; Parsons, Francesca; Millan, Mark J

    2011-11-15

    This study describes a novel analytical method permitting simultaneous HPLC-fluorimetric quantification of multiple (15) D- and L-amino acids, kynurenate, taurine and phosphoethanolamine (a marker of membrane integrity) in microdialysates of prefrontal cortex of freely-moving rats. Levels of GABA were elevated by the transporter inhibitor, nipecotic acid, and by the transaminase inhibitor, vigabatrine.Supporting a neuronal origin, they were decreased by the GABAB autoreceptor agonist, baclofen,yet unaffected by fluoroacetate which disrupts glial metabolism. Glutamate levels were elevated by the transporter inhibitor, L-trans-PDC, and mainly neuronal since they were not decreased by fluoroacetate,yet reduced by baclofen (which recruits GABAB receptors on glutamatergic terminals) and elevated by the NMDA receptor antagonist, dizocilpine. By contrast, levels of glutamine were reduced by L-trans-PDC.Consistent with glial origin, they were unaffected by baclofen, yet reduced by fluoroacetate. Administration of D-serine selectively increased its levels over L-serine, and vice versa. D-serine modestly decreased levels of glycine, which were enhanced by administration of glycine itself and of the glycine transporter-1 inhibitor, sarcosine. Kynurenate levels were increased by its precursor, kynurenine, an effect abolished by the amino-transferase inhibitor, amino-oxyacetate. Taurine and the energy drink, Red Bull®, selectively elevated levels of taurine, which were only slightly reduced by fluoroacetate. Finally, administration of NMDA increased levels of taurine, kynenurate and phosphoethanolamine, while reducing D-serine. These actions were abolished by the competitive NMDA receptor antagonist, CPP, which was inactive alone. This broad-based dialysis system should prove instructive for exploring actions of psychotropic drugs, and for characterising animal models of CNS disorders.

  4. Supplemental safflower oil affects the fatty acid profile, including conjugated linoleic acid, of lamb.

    PubMed

    Boles, J A; Kott, R W; Hatfield, P G; Bergman, J W; Flynn, C R

    2005-09-01

    The objective of this study was to determine whether increasing levels of dietary safflower oil would alter unsaturated fat (especially CLA) and tocopherol content of lamb, animal performance, carcass characteristics, or color stability of lamb muscle tissue. Targhee x Rambouillet wethers (n = 60) were assigned to one of three diets (four pens per treatment with five lambs per pen) in a completely random design. Diets were formulated with supplemental safflower oil at 0 (control), 3, or 6% (as-fed basis) of the diet. Diets containing approximately 80% concentrate and 20% roughage were formulated, on a DM basis, to be isocaloric and isonitrogenous and to meet or exceed NRC requirements for Ca, P, and other nutrients. A subsample of 12 wethers per treatment was selected based on average BW (54 kg) and slaughtered. Carcass data (LM area, fat thickness, and internal fat content) and wholesale cut weight (leg, loin, rack, shoulder, breast, and foreshank), along with fatty acid, tocopherol, and color analysis, were determined on each carcass. The LM and infraspinatus were sampled for fatty acid profile. Increasing safflower oil supplementation from 0 to 3 or 6% increased the proportion of linoleic acid in the diet from 49.93 to 55.32 to 62.38%, respectively, whereas the percentage of oleic acid decreased from 27.94 to 23.80 to 20.73%, respectively. The percentage of oil in the diet did not (P > or = 0.11) alter the growth and carcass characteristics of lambs, nor did it alter the tocopherol content or color stability of meat. Increasing levels of safflower oil in lamb diets decreased (P < 0.01) the weight percentage of oleic acid in the infraspinatus and LM, and increased linoleic acid (P < 0.01). Oil supplementation increased (P < 0.01) the weight percentage of various isomers of CLA in muscle, with the greatest change in the cis-9,trans-11 isomer. Supplementation of sheep diets with safflower oil, up to 6% of the diet, resulted in increasing levels of unsaturated fatty

  5. 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.

  6. 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.

  7. 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

  8. Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

    PubMed

    Karakawa, Sachise; Shimbo, Kazutaka; Yamada, Naoyuki; Mizukoshi, Toshimi; Miyano, Hiroshi; Mita, Masashi; Lindner, Wolfgang; Hamase, Kenji

    2015-11-10

    A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples.

  9. Serine synthesis by an isolated perfused rat kidney preparation.

    PubMed Central

    Scaduto, R C; Davis, E J

    1985-01-01

    The isolated perfused rat kidney was shown to synthesize serine from aspartate or glutamate, both of which are also precursors of glucose. The major products of aspartate metabolism were ammonia, serine, glutamate, glucose, glutamine and CO2. Perfusion of kidneys with aspartate in the presence of amino-oxyacetate resulted in a near-complete inhibition of aspartate metabolism, illustrating the essential role of aspartate aminotransferase in the metabolism of this substrate. Radioactivity from 14C-labelled aspartate and from 14C-labelled glycerol was incorporated into serine and glucose. Production of both glucose and serine from aspartate was suppressed in the presence of 3-mercaptopicolinic acid. These data provide evidence for the operation of the phosphorylated and/or non-phosphorylated pathway for serine production to the presence of 3-mercaptopicolinic acid. This is explained by simultaneous glycolysis. The rate of glucose production, but not that of serine, was greater in kidneys perfused with glutamate or with aspartate plus glycerol than the rates obtained by perfusion with aspartate alone. These data are taken to suggest that serine synthesis occurred at a near-maximal rate, and that the capacity of the kidney for serine synthesis from glucose precursors is lower than that for glucose synthesis. PMID:2864920

  10. Serine ADP-Ribosylation Depends on HPF1.

    PubMed

    Bonfiglio, Juan José; Fontana, Pietro; Zhang, Qi; Colby, Thomas; Gibbs-Seymour, Ian; Atanassov, Ilian; Bartlett, Edward; Zaja, Roko; Ahel, Ivan; Matic, Ivan

    2017-03-02

    ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.

  11. Revealing the multiple structures of serine

    PubMed Central

    Blanco, Susana; Sanz, M. Eugenia; López, Juan C.; Alonso, José L.

    2007-01-01

    We explored the conformational landscape of the proteinogenic amino acid serine [CH2OHCH(NH2)COOH] in the gas phase. Solid serine was vaporized by laser ablation, expanded in a supersonic jet, and characterized by Fourier transform microwave spectroscopy. In the isolation conditions of the jet there have been discovered up to seven different neutral (non-zwitterionic) structures of serine, which are conclusively identified by the comparison between the experimental values of the rotational and quadrupole coupling constants with those predicted by ab initio calculations. These seven forms can serve as a basis to represent the shape of serine in the gas phase. From the postexpansion abundances we derived the conformational stability trend, which is controlled by the subtle network of intramolecular hydrogen bonds formed between the polar groups in the amino acid backbone and the hydroxy side chain. It is proposed that conformational cooling perturbs the equilibrium conformational distribution; thus, some of the lower-energy forms are “missing” in the supersonic expansion. PMID:18077350

  12. Molecular markers of serine protease evolution

    PubMed Central

    Krem, Maxwell M.; Di Cera, Enrico

    2001-01-01

    The evolutionary history of serine proteases can be accounted for by highly conserved amino acids that form crucial structural and chemical elements of the catalytic apparatus. These residues display non- random dichotomies in either amino acid choice or serine codon usage and serve as discrete markers for tracking changes in the active site environment and supporting structures. These markers categorize serine proteases of the chymotrypsin-like, subtilisin-like and α/β-hydrolase fold clans according to phylogenetic lineages, and indicate the relative ages and order of appearance of those lineages. A common theme among these three unrelated clans of serine proteases is the development or maintenance of a catalytic tetrad, the fourth member of which is a Ser or Cys whose side chain helps stabilize other residues of the standard catalytic triad. A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains. PMID:11406580

  13. Effect of omega-3 fatty acids on the modification of erythrocyte membrane fatty acid content including oleic acid in peritoneal dialysis patients.

    PubMed

    An, W S; Lee, S M; Son, Y K; Kim, S E; Kim, K H; Han, J Y; Bae, H R; Park, Y

    2012-01-01

    Erythrocyte membrane fatty acids (FA), such as oleic acid, are related to acute coronary syndrome. There is no report about the effect of omega-3 FA on oleic acid in peritoneal dialysis (PD) patients. We hypothesized that omega-3 FA can modify erythrocyte membrane FA, including oleic acid, in PD patients. In a double-blind, randomized, placebo-controlled study, 18 patients who were treated with PD for at least 6 months were randomized to treatment for 12 weeks with omega-3 FA or placebo. Erythrocyte membrane FA content was measured by gas chromatography at baseline and after 12 weeks. The erythrocyte membrane content of eicosapentaenoic acid and docosahexaenoic acid was significantly increased and saturated FA and oleic acid were significantly decreased in the omega-3 FA supplementation group after 12 weeks compared to baseline. In conclusion, erythrocyte membrane FA content, including oleic acid, was significantly modified by omega-3 FA supplementation for 12 weeks in PD patients.

  14. 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.

  15. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.

    PubMed

    Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N

    2012-09-01

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.

  16. Assignment of the Perfluoropropionic Acid-Formic Acid Complex and the Difficulties of Including High K_a Transitions.

    NASA Astrophysics Data System (ADS)

    Obenchain, Daniel A.; Lin, Wei; Novick, Stewart E.; Cooke, S. A.

    2016-06-01

    We recently began an investigation into the perfluoropropionic acid\\cdotsformic acid complex using broadband microwave spectroscopy. This study aims to examine the possible double proton transfer between the two interacting carboxcyclic acid groups. The spectrum presented as a doubled set of lines, with spacing between transitions of < 1 MHz. Transitions appeared to be a-type, R branch transitions for an asymmetric top. Assignment of all K_a=1,0 transitions yields decent fits to a standard rotational Hamiltonian. Treatment of the doubling as either a two state system (presumably with a double proton transfer) or as two distinct, but nearly identical conformations of the complex produce fits of similar quality. Including higher K_a transitions for the a-type, R-branch lines greatly increases the error of these fits. A previous study involving the trifluoroacetic acid\\cdotsformic acid complex published observed similar high K_a transitions, but did not include them in the published fit. We hope to shed more light on this conundrum. Similarities to other double-well potential minimum systems will be discussed. Martinache, L.; Kresa, W.; Wegener, M.;, Vonmont, U.; and Bauder, A. Chem. Phys. 148 (1990) 129-140.

  17. 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.

  18. Changes in plasma D-serine, L-serine, and glycine levels in treatment-resistant schizophrenia before and after clozapine treatment.

    PubMed

    Yamamori, Hidenaga; Hashimoto, Ryota; Fujita, Yuko; Numata, Shusuke; Yasuda, Yuka; Fujimoto, Michiko; Ohi, Kazutaka; Umeda-Yano, Satomi; Ito, Akira; Ohmori, Tetsuro; Hashimoto, Kenji; Takeda, Masatoshi

    2014-10-17

    Hypofunction of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors may be involved in the pathophysiology of schizophrenia. Many studies have investigated peripheral NMDA receptor-related glutamatergic amino acid levels because of their potential as biological markers. Peripheral d-serine levels and the ratio of d-serine to total serine have been reported to be significantly lower in patients with schizophrenia than in controls. Peripheral d-serine levels and the d-/l-serine ratio have also been reported to significantly increase in patients with schizophrenia as their clinical symptoms improve from the time of admission to the time of discharge. In this study, we examined whether peripheral NMDA receptor-related glutamatergic amino acids levels were altered in patients with treatment-resistant schizophrenia compared to controls and whether these peripheral amino acids levels were altered by clozapine treatment. Twenty-two patients with treatment-resistant schizophrenia and 22 age- and gender-matched healthy controls were enrolled. The plasma levels of d-serine, l-serine, glycine, glutamate, and glutamine were measured before and after clozapine treatment. We found that the plasma levels of d-serine and the d-/l-serine ratio were significantly lower in the patients before clozapine treatment than in the controls. The d-/l-serine ratio was significantly increased by clozapine treatment in patients, and no significant difference was observed in the plasma levels of d-serine and the d-/l-serine ratio between the patients after clozapine treatment and the controls. We also found that plasma glycine levels and the glycine/l-serine ratio were significantly increased following clozapine treatment in the patients, and the glycine/l-serine ratio was significantly higher in the patients after clozapine treatment than in the controls. There was no significant difference in the plasma levels of glutamate and glutamine both between the controls and patients and

  19. CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

    PubMed

    Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

    2011-04-25

    We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

  20. Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

    PubMed Central

    Bavan, Tharmatha; Oberli, Andrea; Roetschi, Alexandra; Badertscher, René; Guggenbühl, Barbara; Berthoud, Hélène

    2013-01-01

    The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from l- and d-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate. PMID:23241976

  1. Down Regulation of Asparagine Synthetase and 3-Phosphoglycerate Dehydrogenase, and the Up-Regulation of Serine Dehydratase in Rat Liver from Intake of Excess Amount of Leucine Are Not Related to Leucine-Caused Amino Acid Imbalance.

    PubMed

    Yoshimura, Ryoji; Takai, Marie; Namaki, Hiroya; Minami, Kimiko; Imamura, Wataru; Kato, Hisanori; Kamei, Yasutomi; Kanamoto, Ryuhei

    2015-01-01

    Asparagine synthetase (ASNS), 3-phosphoglycerate dehydrogenase (PHGDH) and serine dehydratase (SDS) in rat liver are expressed in response to protein and amino acid intake. In the present study, we examined the expression of these enzymes in relation to amino acid imbalance caused by leucine. Rats were subjected to leucine administration in the diet or orally between meals. Consumption of more than 2% leucine in a 6% casein diet suppressed food intake and caused growth retardation in a dose-dependent manner, but this was not seen in a 12% or 40% casein diet. ASNS and PHGDH expression in the liver was significantly induced by the 6% casein diet and was suppressed by leucine in a dose-dependent manner, whereas the SDS expression was induced. These effects were leucine specific and not seen with ingestion of isoleucine or valine. However, leucine orally administered between meals did not change the food intake or growth of rats fed a 6% casein die, though it similarly affected the expression of ASNS, PHGDH and SDS in the liver. These results suggest that the growth retardation caused by leucine imbalance was mainly because of the suppression of food intake, and demonstrated that there are no causal relationships between ASNS, PHGDH and SDS expression and amino acid imbalance caused by leucine.

  2. Adsorption of O-Phospho-L-Serine and L-Serine onto Poorly Crystalline Apatite.

    PubMed

    Benaziz, Lhaj; Barroug, Allal; Legrouri, Ahmed; Rey, Christian; Lebugle, Albert

    2001-06-01

    The adsorption of phosphoserine and serine was studied to determine the effect of amino acid functional groups on the surface reactivity of synthetic poorly crystalline apatite similar to bone mineral. The experimental results for phosphoserine and serine uptake agree respectively with the Langmuir and Freundlich models. Phosphoserine exhibits stronger adsorption capacity and a higher affinity constant for the surface crystals compared to serine molecules. The enhanced adsorption capacity noted for phosphoserine might be related to the presence of phosphate groups in the molecule, which are specific attachment sites. This observation suggests that the strength of phosphate bonds to the solid surface, especially to calcium ions, is higher than that of carboxyl and hydroxyl ones. Spectroscopic observations provide evidence of an adsorption mechanism involving the anionic species of the amino acids and the surface of the crystals. Thus, a change in the position of the band of carboxyl groups occurred for the adsorbed molecules compared to the native amino acids. This revealed that the molecular residues do interact with apatite surface calcium. The shift noted in the frequencies of the bands associated with carboxylate vibrations is more pronounced for phosphoserine, confirming the stronger interaction noted for this molecule. Based on these results, one can conclude that the sorbent and sorbate charged species play an important role in the mechanism of uptake of the amino acids onto crystal surfaces. This may contribute to a better understanding of the mechanism by which phosphoproteins could influence mineralization processes and caries. Copyright 2001 Academic Press.

  3. 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...

  4. The intrinsic disorder alphabet. III. Dual personality of serine

    PubMed Central

    Uversky, Vladimir N

    2015-01-01

    Proteins are natural polypeptides consisting of 20 major amino acid residues, content and order of which in a given amino acid sequence defines the ability of a related protein to fold into unique functional state or to stay intrinsically disordered. Amino acid sequences code for both foldable (ordered) proteins/domains and for intrinsically disordered proteins (IDPs) and IDP regions (IDPRs), but these sequence codes are dramatically different. This difference starts with a very general property of the corresponding amino acid sequences, namely, their compositions. IDPs/IDPRs are enriched in specific disorder-promoting residues, whereas amino acid sequences of ordered proteins/domains typically contain more order-promoting residues. Therefore, the relative abundances of various amino acids in ordered and disordered proteins can be used to scale amino acids according to their disorder promoting potentials. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and represents serine, which is the third most disorder-promoting residue. Similar to previous publications, this review represents some physico-chemical properties of serine and the roles of this residue in structures and functions of ordered proteins, describes major posttranslational modifications tailored to serine, and finally gives an overview of roles of serine in structure and functions of intrinsically disordered proteins. PMID:28232888

  5. Phytosphingosine degradation pathway includes fatty acid α-oxidation reactions in the endoplasmic reticulum.

    PubMed

    Kitamura, Takuya; Seki, Naoya; Kihara, Akio

    2017-03-28

    Although normal fatty acids (FAs) are degraded via β-oxidation, unusual FAs such as 2-hydroxy (2-OH) FAs and 3-methyl-branched FAs are degraded via α-oxidation. Phytosphingosine (PHS) is one of the long-chain bases (the sphingolipid components) and exists in specific tissues, including the epidermis and small intestine in mammals. In the degradation pathway, PHS is converted to 2-OH palmitic acid and then to pentadecanoic acid (C15:0-COOH) via FA α-oxidation. However, the detailed reactions and genes involved in the α-oxidation reactions of the PHS degradation pathway have yet to be determined. In the present study, we reveal the entire PHS degradation pathway: PHS is converted to C15:0-COOH via six reactions [phosphorylation, cleavage, oxidation, CoA addition, cleavage (C1 removal), and oxidation], in which the last three reactions correspond to the α-oxidation. The aldehyde dehydrogenase ALDH3A2 catalyzes both the first and second oxidation reactions (fatty aldehydes to FAs). In Aldh3a2-deficient cells, the unmetabolized fatty aldehydes are reduced to fatty alcohols and are incorporated into ether-linked glycerolipids. We also identify HACL2 (2-hydroxyacyl-CoA lyase 2) [previous name, ILVBL; ilvB (bacterial acetolactate synthase)-like] as the major 2-OH acyl-CoA lyase involved in the cleavage (C1 removal) reaction in the FA α-oxidation of the PHS degradation pathway. HACL2 is localized in the endoplasmic reticulum. Thus, in addition to the already-known FA α-oxidation in the peroxisomes, we have revealed the existence of FA α-oxidation in the endoplasmic reticulum in mammals.

  6. A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, characterization and l-serine production.

    PubMed

    Jiang, Wei; Xia, Bingzhao; Liu, Ziduo

    2013-10-01

    Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50°C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40°C for 3h. The enzyme showed better stability under alkaline environment (pH 6.5-9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0-7.5). The SaSHMT can produce 77.76mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.

  7. A novel serine protease inhibitor from Bungarus fasciatus venom.

    PubMed

    Lu, Jia; Yang, Hailong; Yu, Haining; Gao, Weikai; Lai, Ren; Liu, Jingze; Liang, Xingcai

    2008-03-01

    By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

  8. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

    PubMed

    Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela; Gráf, László; Naude, Ryno

    2014-10-01

    One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

  9. 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...

  10. A snake-like serine proteinase (PmSnake) activates prophenoloxidase-activating system in black tiger shrimp Penaeus monodon.

    PubMed

    Monwan, Warunthorn; Amparyup, Piti; Tassanakajon, Anchalee

    2017-02-01

    Clip domain serine proteinases (ClipSPs) play critical roles in the activation of proteolytic cascade in invertebrate immune systems including the prophenoloxidase (proPO) activating system. In this study, we characterized a snake-like serine protease, namely PmSnake, from the shrimp Penaeus monodon which has previously been identified based on the subtractive cDNA library of proPO double-stranded RNA (dsRNA)-treated hemocytes. An open reading frame of PmSnake contains 1068 bp encoding a predicted protein of 355 amino acid residues with a putative signal peptide of 22 amino acids and two conserved domains (N-terminal clip domain and C-terminal trypsin-like serine proteinase domain). Sequence analysis revealed that PmSnake was closest to the AeSnake from ant Acromyrmex echinatior (53% similarity), but was quite relatively distant from other shrimp PmclipSPs. PmSnake transcript was mainly expressed in shrimp hemocytes and up-regulated after systemic Vibrio harveyi infection indicating that it is an immune-responsive gene. Suppression of PmSnake expression by dsRNA interference reduced both transcript and protein levels leading to a reduction of the hemolymph phenoloxidase (PO) activity (36%), compared to the control, suggesting that the PmSnake functions as a clip-SP in shrimp proPO system. Western blot analysis using anti-PmSnake showed that the PmSnake was detected in hemocytes but not in cell-free plasma. In vitro PO activity and serine proteinase activity assays showed that adding rPmSnake into the shrimp hemolymph could increase PO activity as well as serine proteinase activity suggesting that the rPmSnake activates the proPO system via serine proteinase cascade.

  11. [Antiinflammatory therapy in ostheoarthritis including omega 3 and omega 6 fatty acids].

    PubMed

    Dzielska-Olczak, Małgorzata; Nowak, Jerzy Z

    2012-05-01

    Osteoarthritis (ostheoarthrosis, OA) is characterized by progressive destruction of articular cartilage, remodeling of the periarticular bone and inflammation of the synovial membrane. In patients occur joints pain, impaired joints motion and disability. The results of many studies indicate an inflammation as foundation of this disease. The management of OA include a combination of pharmacological treatments and nonpharmacological interventions. Pharmacological treatments include used paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs) and chondroprotectives (glucosamine, chondroitin sulfate and so on). NSAIDs long-term use associated with serious adverse effects. OA symptoms are effectively reduced by nutrients such omega 3 and omega 6 fatty acids (PUFAs as EPA, DHA), which decrease the need for non-steroidal drugs and may less adverse events. They exerts, particularly EPA, anti-inflammatory effect, inhibit catabolic processes, stimulate the anabolic process in the cartilage in the joint. Many different evidence validate that omega 3 alleviate the progression of osteoarthritis and have exciting therapeutic potential for preventing cartilage degradation associated with chronic inflammatory in joints.

  12. 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

    2016-09-01

    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.

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

    PubMed Central

    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

  14. Acidity constant determination of novel drug precursor benzothiazolon derivatives including acyl and piperazine moieties

    NASA Astrophysics Data System (ADS)

    Sıdır, İsa; Gülseven Sıdır, Yadigar; Berber, Halil

    2013-07-01

    In this study, protonation and deprotonation behaviors of eight new drug precursor benzothiazolon derivatives in all of acidic and basic scale (super acidic, pH, super basic regions) are analyzed by using UV-visible spectrophotometric technique. Acidity constants (pKa), elucidation of the structure and protonation mechanisms of the studied molecules are obtained. Substituent effect on acidity constant values is discussed. These molecules are protonated from oxygen atom of acetamide group in the keto form. The protonation is found to be considerably contributed by the keto form.

  15. Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model

    PubMed Central

    Laurido, Claudio; Hernández, Alejandro; Pelissier, Teresa; Constandil, Luis

    2012-01-01

    N-methyl-D-aspartic acid receptor (NMDAr) activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase (SR). D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS) and L-erythro-3-hydroxyaspartate (LEHA), among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 μg/10 μL) and LEHA (100 μg/10 μL) in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 μg/10 μL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain. PMID:22536130

  16. D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

    PubMed Central

    Schell, M J; Molliver, M E; Snyder, S H

    1995-01-01

    Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7732010

  17. 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

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

    PubMed Central

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

    2016-01-01

    Summary 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

  19. 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.

  20. 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.

  1. Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease.

    PubMed

    de Almeida Nogueira, Natália Pereira; Morgado-Díaz, José Andrés; Menna-Barreto, Rubem Figueiredo Sadok; Paes, Marcia Cristina; da Silva-López, Raquel Elisa

    2013-10-01

    It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced parasite viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.

  2. 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…

  3. 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.

  4. 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

  5. Export of aquatic productivity, including highly unsaturated fatty acids, to terrestrial ecosystems via Odonata.

    PubMed

    Popova, Olga N; Haritonov, Anatoly Y; Sushchik, Nadezhda N; Makhutova, Olesia N; Kalachova, Galina S; Kolmakova, Anzhelika A; Gladyshev, Michail I

    2017-03-01

    Based on 31-year field study of the abundance and biomass of 18 species of odonates in the Barabinsk Forest-Steppe (Western Siberia, Russia), we quantified the contribution of odonates to the export of aquatic productivity to surrounding terrestrial landscape. Emergence varied from 0.8 to 4.9g of wet biomass per m(2) of land area per year. Average export of organic carbon was estimated to be 0.30g·m(-2)·year(-1), which is comparable with the average production of herbivorous terrestrial insects in temperate grasslands. Moreover, in contrast to terrestrial insects, emerging odonates contained high quantities of highly unsaturated fatty acids (HUFA), namely eicosapentaenoic acid (20:5n-3, EPA), and docosahexaenoic acid (22:6n-3, DHA), which are known to be essential for many terrestrial animals, especially for birds. The export of EPA+DHA by odonates was found to be 1.92-11.76mg·m(-2)·year(-1), which is equal to an average general estimation of the export of HUFA by emerging aquatic insects. Therefore, odonates appeared to be a quantitatively and qualitatively important conduit of aquatic productivity to forest-steppe ecosystem.

  6. Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

    PubMed Central

    Mamidi, Ranganath; Gollapudi, Sampath K.; Mallampalli, Sri Lakshmi; Chandra, Murali

    2012-01-01

    Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~36% decrease in Ca2+-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics. PMID:22684024

  7. 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

  8. Protection of folic acid through encapsulation in mesoporous silica particles included in fruit juices.

    PubMed

    Ruiz-Rico, María; Pérez-Esteve, Édgar; Lerma-García, María J; Marcos, María D; Martínez-Máñez, Ramón; Barat, José M

    2017-03-01

    Folic acid (FA) is a synthetic vitamin commonly used for food fortification. However, its vulnerability to processing and storage implies loss of efficiency, which would induce over-fortification by processors to obtain a minimum dose upon consumption. Recent studies have indicated potential adverse effects of FA overdoses, and FA protection during processing and storage could lead to more accurate fortification. In addition, sustained vitamin release after consumption would help improve its metabolism. The objective of this work was to study controlled FA delivery and stability in fruit juices to reduce potential over-fortification risks by using gated mesoporous silica particles (MSPs). The obtained results indicated that FA encapsulation in MSPs significantly improved its stability and contributed to controlled release after consumption by modifying vitamin bioaccessibility. These results confirmed the suitability of MSPs as support for controlled release and protection of bioactive molecules in food matrices in different food production and storage stages.

  9. 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.

  10. Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family.

    PubMed Central

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

    1990-01-01

    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 approximately 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' 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. Images PMID:2187193

  11. Improvement in Regional CBF by L-Serine Contributes to Its Neuroprotective Effect in Rats after Focal Cerebral Ischemia

    PubMed Central

    Jiang, Zheng-Lin; Wang, Guo-Hua; Sun, Li; Jiang, Rui; Zhao, Guang-Wei; Han, Le-Yang

    2013-01-01

    To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF). Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1) reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2) improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3) increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca2+-activated K+ channels on the cerebral blood vessel endothelium. PMID:23825613

  12. Fate of N-nitrosodimethylamine, trihalomethane and haloacetic acid precursors in tertiary treatment including biofiltration.

    PubMed

    Farré, Maria José; Reungoat, Julien; Argaud, Francois Xavier; Rattier, Maxime; Keller, Jürg; Gernjak, Wolfgang

    2011-11-01

    The presence of disinfection by-products (DBPs) such as trihalomethanes (THMs), haloacetic acids (HAAs) and N-nitrosamines in water is of great concern due to their adverse effects on human health. In this work, the removal of N-nitrosodimethylamine (NDMA), total THM and five HAA precursors from secondary effluent by biological activated carbon (BAC) is investigated at full and pilot scale. In the pilot plant two filter media, sand and granular activated carbon, are tested. In addition, we evaluate the influence of ozonation prior to BAC filtration on its performance. Among the bulk of NDMA precursors, the fate of four pharmaceuticals containing a dimethylamino moiety in the chemical structure are individually investigated. Both NDMA formation potential and each of the studied pharmaceuticals are dramatically reduced by the BAC even in the absence of main ozonation prior to the filtration. The low removal of NDMA precursors at the sand filtration in comparison to the removal of NDMA precursors at the BAC suggests that adsorption may play an important role on the removal of NDMA precursors by BAC. Contrary, the precursors for THM and HAA formation are reduced in both sand filtration and BAC indicating that the precursors for the formation of these DBPs are to some extent biodegradable.

  13. Occurrence of phosphatidyl-D-serine in the rat cerebrum.

    PubMed

    Omori, Taketo; Mihara, Hisaaki; Kurihara, Tatsuo; Esaki, Nobuyoshi

    2009-05-01

    Phosphatidylserine (PS), a relatively abundant component of mammalian cell membranes, plays important roles in biological processes including apoptosis and cell signaling. It is believed that phosphatidyl-L-serine is the only naturally occurring PS. Here, we describe for the first time the occurrence of phosphatidyl-D-serine (D-PS) in rat cerebrum. Quantitative HPLC analysis of the derivatives of serine liberated from PS by hydrolysis revealed that the amount of D-PS was approximately 1% of the total PS in the cerebrum. Enzymatic cleavage of cerebrum PS with phospholipase D and phospholipase C resulted in the release of both isomers of serine and phosphoserine, respectively, providing additional evidence for the existence of D-PS. Free D-serine was incorporated into PS in an in vitro system using a cerebrum extract, and this activity was inhibited by EDTA, suggesting the occurrence of a divalent cation-dependent enzyme that synthesizes D-PS by a base-exchange reaction.

  14. 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

  15. 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.

  16. Liver protective effect of ursodeoxycholic acid includes regulation of ADAM17 activity

    PubMed Central

    2013-01-01

    Background Ursodeoxycholic acid (UDCA) is used to treat primary biliary cirrhosis, intrahepatic cholestasis, and other cholestatic conditions. Although much has been learned about the molecular basis of the disease pathophysiology, our understanding of the effects of UDCA remains unclear. Possibly underlying its cytoprotective, anti-apoptotic, anti-oxidative effects, UDCA was reported to regulate the expression of TNFα and other inflammatory cytokines. However, it is not known if this effect involves also modulation of ADAM family of metalloproteinases, which are responsible for release of ectodomains of inflammatory cytokines from the cell surface. We hypothesized that UDCA modulates ADAM17 activity, resulting in amelioration of cholestasis in a murine model of bile duct ligation (BDL). Methods The effect of UDCA on ADAM17 activity was studied using the human liver hepatocellular carcinoma cell line HepG2. Untransfected cells or cells ectopically expressing human ADAM17 were cultured with or without UDCA and further activated using phorbol-12-myristate-13-acetate (PMA). The expression and release of ADAM17 substrates, TNFα, TGFα, and c-Met receptor (or its soluble form, sMet) were evaluated using ELISA and quantitative real-time (qRT) PCR. Immunoblotting analyses were conducted to evaluate expression and activation of ADAM17 as well as the level of ERK1/2 phosphorylation after UDCA treatment. The regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by UDCA was studied using zymography and qRT-PCR. A mouse model of acute cholestasis was induced by common BDL technique, during which mice received daily orogastric gavage with either UDCA or vehicle only. Liver injury was quantified using alkaline phosphatase (ALP), relative liver weight, and confirmed by histological analysis. ADAM17 substrates in sera were assessed using a bead multiplex assay. Results UDCA decreases amount of shed TNFα, TGFα, and sMet in cell culture media and the phosphorylation of

  17. Reduced Folate Supply as a Key to Enhanced l-Serine Production by Corynebacterium glutamicum▿

    PubMed Central

    Stolz, Michael; Peters-Wendisch, Petra; Etterich, Helga; Gerharz, Tanja; Faurie, Robert; Sahm, Hermann; Fersterra, Holger; Eggeling, Lothar

    2007-01-01

    The amino acid l-serine is required for pharmaceutical purposes, and the availability of a sugar-based microbial process for its production is desirable. However, a number of intracellular utilization routes prevent overproduction of l-serine, with the essential serine hydroxymethyltransferase (SHMT) (glyA) probably occupying a key position. We found that constructs of Corynebacterium glutamicum strains where chromosomal glyA expression is dependent on Ptac and lacIQ are unstable, acquiring mutations in lacIQ, for instance. To overcome the inconvenient glyA expression control, we instead considered controlling SHMT activity by the availability of 5,6,7,8-tetrahydrofolate (THF). The pabAB and pabC genes of THF synthesis were identified and deleted in C. glutamicum, and the resulting strains were shown to require folate or 4-aminobenzoate for growth. Whereas the C. glutamicum ΔsdaA strain (pserACB) accumulates only traces of l-serine, with the C. glutamicum ΔpabABCΔsdaA strain (pserACB), l-serine accumulation and growth responded in a dose-dependent manner to an external folate supply. At 0.1 mM folate, 81 mM l-serine accumulated. In a 20-liter controlled fed-batch culture, a 345 mM l-serine accumulation was achieved. Thus, an efficient and highly competitive process for microbial l-serine production is available. PMID:17142381

  18. 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.

  19. Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis.

    PubMed

    Miyoshi, Takeharu; Tsuji, Naotoshi; Islam, M Khyrul; Kamio, Tsugihiko; Fujisaki, Kozo

    2004-08-01

    Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.

  20. Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase.

    PubMed

    Xue, H H; Fujie, M; Sakaguchi, T; Oda, T; Ogawa, H; Kneer, N M; Lardy, H A; Ichiyama, A

    1999-06-04

    L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver.

  1. Evolutionary switches between two serine codon sets are driven by selection

    PubMed Central

    Rogozin, Igor B.; Belinky, Frida; Pavlenko, Vladimir; Shabalina, Svetlana A.; Kristensen, David M.; Koonin, Eugene V.

    2016-01-01

    Serine is the only amino acid that is encoded by two disjoint codon sets so that a tandem substitution of two nucleotides is required to switch between the two sets. Previously published evidence suggests that, for the most evolutionarily conserved serines, the codon set switch occurs by simultaneous substitution of two nucleotides. Here we report a genome-wide reconstruction of the evolution of serine codons in triplets of closely related species from diverse prokaryotes and eukaryotes. The results indicate that the great majority of codon set switches proceed by two consecutive nucleotide substitutions, via a threonine or cysteine intermediate, and are driven by selection. These findings imply a strong pressure of purifying selection in protein evolution, which in the case of serine codon set switches occurs via an initial deleterious substitution quickly followed by a second, compensatory substitution. The result is frequent reversal of amino acid replacements and, at short evolutionary distances, pervasive homoplasy. PMID:27799560

  2. Selected nutrient contents, fatty acid composition, including conjugated linoleic acid, and retention values in separable lean from lamb rib loins as affected by external fat and cooking method.

    PubMed

    Badiani, Anna; Montellato, Lara; Bochicchio, Davide; Anfossi, Paola; Zanardi, Emanuela; Maranesi, Magda

    2004-08-11

    Proximate composition and fatty acid profile, conjugated linoleic acid (CLA) isomers included, were determined in separable lean of raw and cooked lamb rib loins. The cooking methods compared, which were also investigated for cooking yields and true nutrient retention values, were dry heating of fat-on cuts and moist heating of fat-off cuts; the latter method was tested as a sort of dietetic approach against the more traditional former type. With significantly (P < 0.05) lower cooking losses, dry heating of fat-on rib-loins produced slightly (although only rarely significantly) higher retention values for all of the nutrients considered, including CLA isomers. On the basis of the retention values obtained, both techniques led to a minimum migration of lipids into the separable lean, which was higher (P < 0.05) in dry heating than in moist heating, and was characterized by the prevalence of saturated and monounsaturated fatty acids. On the whole, the response to cooking of the class of CLA isomers (including that of the nutritionally most important isomer cis-9,trans-11) was more similar to that of the monounsaturated than the polyunsaturated fatty acids.

  3. Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

    PubMed

    Okano, K; Aoki, Y; Sakurai, T; Kajitani, M; Kanai, S; Shimazu, T; Shimizu, H; Naruto, M

    1987-07-01

    Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

  4. Glycine transport accounts for the differential role of glycine vs. D-serine at NMDA receptor coagonist sites in the salamander retina

    PubMed Central

    Stevens, Eric R.; Gustafson, Eric C.; Miller, Robert F.

    2010-01-01

    In this study, we demonstrate that D-serine interacts with N-methyl-D-aspartate receptor (NMDAR) coagonist sites of retinal ganglion cells of the tiger salamander retina by showing that exogenous D-serine overcomes the competitive antagonism of 7-chlorokynurenic acid for this site. Additionally, we show that exogenous D-serine was more than 30 times as effective at potentiating NMDAR currents compared with glycine. We thus examined the importance of glycine transport through the application of selective antagonists of the GlyT1 (NFPS) and GlyT2 (ALX-5670) transport systems, while simultaneously evaluating the degree of occupancy of the NMDAR coagonist binding sites. Analysis was carried out with electrophysiological recordings from the inner retina, including whole-cell recordings from retinal ganglion cells and extracellular recordings of the proximal negative field potential. Blocking the GlyT2 transport system had no effect on the light-evoked NMDAR currents or on the sensitivity of these currents to exogenous D-serine. In contrast, when the GlyT1 system was blocked, the coagonist sites of NMDARs showed full occupancy. These findings clearly establish the importance of the GlyT1 transporter as an essential component for maintaining the coagonist sites of NMDARs in a non-saturated state. The normal, unsaturated state of the NMDAR coagonist binding sites allows modulation of the NMDAR currents, by release of either D-serine or glycine. These results are discussed in light of contemporary findings which favor D-serine over glycine as the major coagonist of the NMDARs found in ganglion cells of the tiger salamander retina. PMID:20374282

  5. Plasma ω-3 fatty acid levels negatively and ω-6 fatty acid levels positively associated with other cardiovascular risk factors including homocysteine in severe obese subjects.

    PubMed

    Mehmetoglu, Idris; Yerlikaya, F Hümeyra; Kurban, Sevil; Polat, Hakkı

    2012-01-01

    Obesity and homocysteine (tHcy) are important risk factors for cardiovascular diseases (CVD). Plasma omega-3 fatty acids (ω-3 FAs) and omega-6 fatty acids (ω-6 FAs) are essential fatty acids with diverse biological effects in human health and disease. We have investigated the relation of plasma ω-3 FAs and ω-6 FAs levels with other cardiovascular risk factors including tHcy in severe obese subjects. This study was performed on 96 severe obese and 65 normal weight subjects. Plasma fatty acid composition was measured by GC/MS and serum tHcy level was measured by HPLC methods. There were no differences between groups in terms of concentrations of serum tHcy, plasma ω-3 FAs, ω-6 FAs and ω-3/ω-6 ratio, whereas serum vitamin B-12 (p<0.01) and folic acid (p<0.05) levels were lower than those of the normal weight subjects. Homocysteine positively correlated with ω-6 FAs and negatively correlated with ω-3 FAs in severe obese and normal weight subjects. Serum vitamin B-12 positively correlated with ω-3 FAs (p<0.01) and ω-3/ω-6 ratio (p<0.01) and negatively correlated with ω-6 FAs (p<0.05) in severe obese subjects. Serum folic acid positively correlated with ω-3 FAs (p<0.01) in severe obese subjects. Our results suggest an association between the plasma ω-3 FAs and ω-6 FAs and serum tHcy concentrations in severe obese and normal weight subjects. Low levels vitamin B-12 and folic acid may have been responsible for the elevated tHcy levels in severe obese subjects, increasing the risk for future development of cardiovascular diseases.

  6. An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism

    PubMed Central

    Makino, Yuki; Sato, Takaaki; Kawamura, Hiroki; Hachisuka, Shin-ichi; Takeno, Ryo; Imanaka, Tadayuki; Atomi, Haruyuki

    2016-01-01

    Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization. PMID:27857065

  7. First-principles study of interaction of serine with nucleobases of DNA and RNA.

    PubMed

    Abbas, Haider

    2017-03-01

    The nature of interaction between serine-a vital molecule for cancer cell proliferation and nucleic acid bases-adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) is investigated within the framework of Møller-Plesset perturbation theory (MP2) and density functional theory (DFT). To quantify the interaction strength between serine and nucleobases, the corresponding binding energies were computed, showing energetic ordering such that G > C > T > A > U. This shows that the interaction energy of serine with guanine is the highest, while with uracil it is the lowest. The amount of charge transferred is the lowest in case of the serine-guanine complex and highest in case of the serine-uracil complex. The results show the serine-guanine complex to be more stable and to have a salt bridge structure involving the -COOH group. Theoretical analysis based on MP2 and DFT shows that the interaction between the serine and nucleobases is mainly determined by hydrogen bonding.

  8. FT-midIR determination of fatty acid profiles, including trans fatty acids, in bakery products after focused microwave-assisted Soxhlet extraction.

    PubMed

    Ruiz-Jiménez, J; Priego-Capote, F; Luque de Castro, M D

    2006-08-01

    A study of the feasibility of Fourier transform medium infrared spectroscopy (FT-midIR) for analytical determination of fatty acid profiles, including trans fatty acids, is presented. The training and validation sets-75% (102 samples) and 25% (36 samples) of the samples once the spectral outliers have been removed-to develop FT-midIR general equations, were built with samples from 140 commercial and home-made bakery products. The concentration of the analytes in the samples used for this study is within the typical range found in these kinds of products. Both sets were independent; thus, the validation set was only used for testing the equations. The criterion used for the selection of the validation set was samples with the highest number of neighbours and the most separation between them (H<0.6). Partial least squares regression and cross validation were used for multivariate calibration. The FT-midIR method does not require post-extraction manipulation and gives information about the fatty acid profile in two min. The 14:0, 16:0, 18:0, 18:1 and 18:2 fatty acids can be determined with excellent precision and other fatty acids with good precision according to the Shenk criteria, R (2)>/=0.90, SEP=1-1.5 SEL and R (2)=0.70-0.89, SEP=2-3 SEL, respectively. The results obtained with the proposed method were compared with those provided by the conventional method based on GC-MS. At 95% significance level, the differences between the values obtained for the different fatty acids were within the experimental error.

  9. Multifunctional role of His159in the catalytic reaction of serine palmitoyltransferase.

    PubMed

    Shiraiwa, Yuka; Ikushiro, Hiroko; Hayashi, Hideyuki

    2009-06-05

    Serine palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5'-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-serine and palmitoyl-CoA. Like other alpha-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His(159)) instead of an aromatic amino acid residue. His(159) was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-serine. These results indicate that His(159) is not only the anchoring site for l-serine but regulates the alpha-deprotonation of l-serine by fixing the conformation of the PLP-l-serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His(159) promotes both the Claisen-type condensation as an acid catalyst and the protonation at Calpha of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542-7553), lead us to propose a novel mechanism, in which His(159) plays multiple roles by exploiting the stereochemistry of Dunathan's conjecture.

  10. Multifunctional Role of His159in the Catalytic Reaction of Serine Palmitoyltransferase*

    PubMed Central

    Shiraiwa, Yuka; Ikushiro, Hiroko; Hayashi, Hideyuki

    2009-01-01

    Serine palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5′-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-serine and palmitoyl-CoA. Like other α-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His159) instead of an aromatic amino acid residue. His159 was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-serine. These results indicate that His159 is not only the anchoring site for l-serine but regulates the α-deprotonation of l-serine by fixing the conformation of the PLP-l-serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His159 promotes both the Claisen-type condensation as an acid catalyst and the protonation at Cα of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542–7553), lead us to propose a novel mechanism, in which His159 plays multiple roles by exploiting the stereochemistry of Dunathan's conjecture. PMID:19346561

  11. The Saccharomyces cerevisiae poly(A)-binding protein is subject to multiple post-translational modifications, including the methylation of glutamic acid.

    PubMed

    Low, Jason K K; Hart-Smith, Gene; Erce, Melissa A; Wilkins, Marc R

    2014-01-10

    Poly(A)-binding protein in mouse and man was recently found to be highly post-translationally modified. Here we analysed an ortholog of this protein, Pab1 from Saccharomyces cerevisiae, to assess the conservation and thus likely importance of these modifications. Pab1 showed the presence of six sites of methylated glutamate, five sites of lysine acetylation, and one phosphorylation of serine. Many modifications on Pab1 showed either complete conservation with those on human or mouse PABPC1, were present on nearby residues and/or were present in the same domain(s). The conservation of methylated glutamate, an unusual modification, was of particular note and suggests a conserved function. Comparison of methylated glutamate sites in human, mouse and yeast poly(A)-binding protein, along with methylation sites catalysed by CheR L-glutamyl protein methyltransferase from Salmonella typhimurium, revealed that the methylation of glutamate preferentially occurs in EE and DE motifs or other small regions of acidic amino acids. The conservation of methylated glutamate in the same protein between mouse, man and yeast suggests the presence of a eukaryotic l-glutamyl protein methyltransferase and that the modification is of functional significance.

  12. Evaluation of the number of ionogenic groups of inulinase by acid-base titration.

    PubMed

    Kovaleva, T A; Holyavka, M G; Rezvan, S G; Kozhedub, S V

    2008-06-01

    Acid base titration showed that Aspergillus awamori inulinase includes 178 asparaginic and glutamic acid residues, 20 histidine, 10 serine, and 34 lysine and tyrosine residues. Denaturation temperature for this enzyme was calculated using analysis of the proportion of stabilizing and destabilizing amino acids in the molecule.

  13. 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.

  14. 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

  15. Serine protease activity in developmental stages of Eimeria tenella.

    PubMed

    Fetterer, R H; Miska, K B; Lillehoj, H; Barfield, R C

    2007-04-01

    A number of complex processes are involved in Eimeria spp. survival, including control of sporulation, intracellular invasion, evasion of host immune responses, successful reproduction, and nutrition. Proteases have been implicated in many of these processes, but the occurrence and functions of serine proteases have not been characterized. Bioinformatic analysis suggests that the Eimeria tenella genome contains several serine proteases that lack homology to trypsin. Using RT-PCR, a gene encoding a subtilisin-like and a rhomboid protease-like serine protease was shown to be developmentally regulated, both being poorly expressed in sporozoites (SZ) and merozoites (MZ). Casein substrate gel electrophoresis of oocyst extracts during sporulation demonstrated bands of proteolytic activity with relative molecular weights (Mr) of 18, 25, and 45 kDa that were eliminated by coincubation with serine protease inhibitors. A protease with Mr of 25 kDa was purified from extracts of unsporulated oocysts by a combination of affinity and anion exchange chromatography. Extracts of SZ contained only a single band of inhibitor-sensitive proteolytic activity at 25 kDa, while the pattern of proteases from extracts of MZ was similar to that of oocysts except for the occurrence of a 90 kDa protease, resistant to protease inhibitors. Excretory-secretory products (ESP) from MZ contained AEBSF (4-[2-Aminoethyl] benzenesulphonyl fluoride)-sensitive protease activity with a specific activity about 10 times greater than that observed in MZ extracts. No protease activity was observed in the ESP from SZ. Pretreatment of SZ with AEBSF significantly reduced SZ invasion and the release of the microneme protein, MIC2. The current results suggest that serine proteases are present in all the developmental stages examined.

  16. A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes.

    PubMed

    Iketani, Aya; Nakamura, Mayumi; Suzuki, Yuya; Awai, Koichiro; Shioi, Yuzo

    2013-03-01

    A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.

  17. 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.

  18. Identification of codon-specific serine to asparagine mistranslation in recombinant monoclonal antibodies by high-resolution mass spectrometry.

    PubMed

    Yu, X Christopher; Borisov, Oleg V; Alvarez, Melissa; Michels, David A; Wang, Yajun Jennifer; Ling, Victor

    2009-11-15

    Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.

  19. Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

    PubMed

    Pariani, Sebastián; Contreras, Marisol; Rossi, Franco R; Sander, Valeria; Corigliano, Mariana G; Simón, Francisco; Busi, María V; Gomez-Casati, Diego F; Pieckenstain, Fernando L; Duschak, Vilma G; Clemente, Marina

    2016-04-01

    Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.

  20. 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.

  1. Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma.

    PubMed

    Guo, Min; Chong, Yeeting E; Shapiro, Ryan; Beebe, Kirk; Yang, Xiang-Lei; Schimmel, Paul

    2009-12-10

    Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps-free-standing, genome-encoded editing proteins that clear Ser-tRNA(Ala). The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the alpha-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.

  2. 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.

  3. Interspecific variation in mitochondrial serine transfer RNA (UCN) in Euptychiina butterflies (Lepidoptera: Satyrinae): structure and alignment.

    PubMed

    Marín, Mario Alejandro; López, Andrés; Uribe, Sandra Inés

    2012-06-01

    The nucleotide variation and structural patterns of mitochondrial RNA molecule have been proposed as useful tools in molecular systematics; however, their usefulness is always subject to a proper assessment of homology in the sequence alignment. The present study describes the secondary structure of mitochondrial tRNA for the amino acid serine (UCN) on 13 Euptychiina species and the evaluation of its potential use for evolutionary studies in this group of butterflies. The secondary structure of tRNAs showed variation among the included species except between Hermeuptychia sp1 and sp2. Variation was concentrated in the ribotimidina-pseudouridine-cystosine (TψC), dihydrouridine (DHU) and variable loops and in the DHU and TψC arms. These results suggest this region as a potential marker useful for taxonomic differentiation of species in this group and also confirm the importance of including information from the secondary structure of tRNA to optimize the alignments.

  4. Microstructure and nanomechanical properties of enamel remineralized with asparagine-serine-serine peptide.

    PubMed

    Chung, Hsiu-Ying; Li, Cheng Che

    2013-03-01

    A highly biocompatible peptide, triplet repeats of asparagine-serine-serine (3 NSS) was designed to regulate mineral deposition from aqueous ions in saliva for the reconstruction of enamel lesions. Healthy human enamel was sectioned and acid demineralized to create lesions, then exposed to the 3 NSS peptide solution, and finally immersed in artificial saliva for 24h. The surface morphology and roughness were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. X-ray diffraction (XRD) was used to identify the phases and crystallinity of the deposited minerals observed on the enamel surface. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively analyze the mineral variation by calculating the relative integrated-area of characteristic bands. Nanohardness and elastic modulus measured by nanoindentation at various treatment stages were utilized to evaluate the degree of recovery. Biomimetic effects were accessed according to the degree of nanohardness recovery and the amount of hydroxyapatite deposition. The charged segments in the 3 NSS peptide greatly attracted aqueous ions from artificial saliva to form hydroxyapatite crystals to fill enamel caries, in particular the interrod areas, resulting in a slight reduction in overall surface roughness. Additionally, the deposited hydroxyapatites were of a small crystalline size in the presence of the 3 NSS peptide, which effectively restrained the plastic deformations and thus resulted in greater improvements in nanohardness and elastic modulus. The degree of nanohardness recovery was 5 times greater for remineralized enamel samples treated with the 3 NSS peptide compared to samples without peptide treatment.

  5. 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

  6. 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

  7. 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

  8. AMPA receptor mediated D-serine release from retinal glial cells

    PubMed Central

    Sullivan, Steve J.; Miller, Robert F.

    2010-01-01

    The NMDA receptor coagonist D-serine is important in a number of different processes in the central nervous system, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major coagonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated D-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an AMPA receptor dependent mechanism. AMPA-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration of a glial toxin. These findings provide the first evidence that extracellular D-serine levels in the retina can be modulated, and that such modulation is contingent upon glial cell activity. PMID:20969576

  9. 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

  10. 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

  11. 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

  12. Inhibition of serine and proline racemases by substrate-product analogues.

    PubMed

    Harty, Matthew; Nagar, Mitesh; Atkinson, Logan; Legay, Christina M; Derksen, Darren J; Bearne, Stephen L

    2014-01-01

    d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (Ki=167±21mM, Ki'=661±81mM, cf. Km=19±2mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (Ki=111±15mM, cf. Km=5.7±0.5mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible.

  13. Discovery libraries targeting the major enzyme classes: the serine hydrolases.

    PubMed

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L

    2014-08-15

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent structural diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki=100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas.

  14. Discovery libraries targeting the major enzyme classes: the serine hydrolases

    PubMed Central

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L.

    2014-01-01

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible structural inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki = 100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas. PMID:25037918

  15. Overexpression of halophilic serine hydroxymethyltransferase in fresh water cyanobacterium Synechococcus elongatus PCC7942 results in increased enzyme activities of serine biosynthetic pathways and enhanced salinity tolerance.

    PubMed

    Waditee-Sirisattha, Rungaroon; Kageyama, Hakuto; Tanaka, Yoshito; Fukaya, Minoru; Takabe, Teruhiro

    2017-01-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the conversion of serine to glycine and provides activated one-carbon units required for synthesis of nucleic acids, proteins and numerous biological compounds. SHMT is involved in photorespiratory pathway of oxygenic photosynthetic organisms. Accumulating evidence revealed that SHMT plays vital role for abiotic stresses such as low CO2 and high salinity in plants, but its role in cyanobacteria remains to be clarified. In this study, we examined to overexpress the SHMT from halotolerant cyanobacterium Aphanothece halophytica in freshwater cyanobacterium, Synechococcus elongatus PCC7942. The transformed cells did not show an obvious phenotype under non-stress condition, but exhibited more tolerance to salinity than the control cells harboring vector only under high salinity. Elevated levels of enzymes in phosphorylated serine biosynthetic pathway and photorespiration pathway were observed in the transformed cells. Glycine level was also increased in the transformed cells. Physiological roles of SHMT for salt tolerance were discussed.

  16. Bioaccumulation of perfluoroalkyl acids including the isomers of perfluorooctane sulfonate in carp (Cyprinus carpio) in a sediment/water microcosm.

    PubMed

    Fang, Shuhong; Zhang, Yifeng; Zhao, Shuyan; Qiang, Liwen; Chen, Meng; Zhu, Lingyan

    2016-12-01

    Carp (Cyprinus carpio) were exposed to perfluoroalkyl acids (PFAAs) including perfluorooctane sulfonate (PFOS) isomers in an artificially contaminated sediment/water microcosm. The uptake constant of PFAAs increased with increasing carbon chain length, whereas the elimination coefficient displayed the opposite trend, suggesting that carbon chain length plays an important role in the bioaccumulation of PFAAs. When the contribution of suspended particulate matter was taken into account, the bioaccumulation factors (BAFs) became lower (3.61-600 L/kg) compared with BAFs derived from only considering the absorption from free PFAAs in water (3.85-97000 L/kg). The results indicate that suspended particulate matter in water constitutes an important source of exposure for aquatic organisms to long-chain PFAAs. Linear (n-)PFOS was preferentially accumulated compared with branched isomers in carp. Among the branched isomers, 1m-PFOS displayed the greatest bioaccumulation, whereas m2 -PFOS had the lowest. Linear PFOS displayed greater partitioning ability from blood to other tissues over branched PFOS (br-PFOS) isomers, leading to a relatively lower n-PFOS proportion in blood. In summary, suspended particulate matter made a contribution to the accumulation of long-chain PFAAs in aquatic organisms, and n-PFOS was preferentially accumulated compared with br-PFOS isomers. Environ Toxicol Chem 2016;35:3005-3013. © 2016 SETAC.

  17. Chemical Cues which Include Amino Acids Mediate Species-Specific Feeding Behavior in Invasive Filter-Feeding Bigheaded Carps.

    PubMed

    Claus, Aaron W; Sorensen, Peter W

    2017-03-15

    This study tested whether and how dissolved chemicals might assist food recognition in two filter-feeding fishes, the silver (Hypophthalmichthys molitrix) and the bighead carp (H. nobilis). These species evolved in Asia, are now invasive in the Mississippi River, and feed voraciously on microparticles including plankton. The food habits and biology of these carps are broadly similar to many filter-feeding fish, none of whose chemical ecology has been examined. We conducted five experiments. First, we demonstrated that buccal-pharngeal pumping (BPP), a behavior in which fish pump water into their buccal cavities, is responsible for sampling food: BPP activity in both silver and bighead carps was low and increased nearly 25-fold after exposure to a filtrate of a planktonic food mixture (P < 0.01) and over 35-fold when planktonic food was added (P < 0.001). Next, we showed that of nine food filtrates, the one containing chemicals released by spirulina, a type of cyanobacterium, was the most potent planktonic component for both species. The potency of filtrates varied between species in ways that reflected their different chemical compositions. While L-amino acids could explain about half of the activity of food filtrate, other unknown chemical stimuli were also implicated. Finally, occlusion experiments showed the olfactory sense has a very important, but not exclusive, role in bigheaded carp feeding behaviors and this might be exploited in both their control and culture.

  18. Cellular mechanisms of insulin resistance: role of stress-regulated serine kinases and insulin receptor substrates (IRS) serine phosphorylation.

    PubMed

    Tanti, Jean-François; Jager, Jennifer

    2009-12-01

    Insulin receptor substrates (IRS) serine phosphorylation is a time-controlled physiological feedback mechanism in insulin signaling that is hijacked by metabolic and inflammatory stresses to promote insulin resistance. Kinases, including IKKbeta, JNK, ERK, mTOR, and S6K, activated by the inducers of insulin resistance induce uncontrolled IRS serine phosphorylation. Studies with genetically modified mice reveal that these kinases integrate signals from metabolic and inflammatory stresses in adipose tissue, liver, and hypothalamus leading to peripheral and central insulin resistance. Moreover, IKKbeta/NF-kappaB and JNK1 pathways in myeloid cells represent a core mechanism involved in inflammation linked to obesity. These kinases are thus potential drug targets against insulin resistance and the targeting of the IKKbeta/NF-kappaB or the JNK pathway may evolve into future diabetes medication.

  19. 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

  20. 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

  1. 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

  2. A serine proteinase inhibitor from frog eggs with bacteriostatic activity.

    PubMed

    Han, Yaoping; Yu, Haining; Yang, Xinbo; Rees, Huw H; Liu, Jingze; Lai, Ren

    2008-01-01

    By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.

  3. Isolation and identification of microorganisms including lactic acid bacteria and their use in microbial deacidification of wines from domestic vineyards.

    PubMed

    Drozdz, Iwona; Makarewicz, Malgorzata; Tuszyński, Tadeusz

    2013-01-01

    The aim of this study was to identify various bacteria isolated from grapes and their wines. Additionally we investigated the capacity of lactic acid bacteria for microbiological deacidification of wines produced in Poland. We have identified Oenococcus oeni, Lactobacillus acidophilus and Lactobacillus delbrueckii. During the microbial deacidification process, we observed decreases of total acidity and increases of volatile acidity, with statistically significant changes noted for O. oeni in Marechal Foch and Seyval Blanc, and for Lb. acidophilus in Frontenac. On the other hand, a statistically significant increase in pH was observed in Marechal Foch and Seyval Blanc following deacidification by O. oeni.

  4. Hyaluronic Acid--an "Old" Molecule with "New" Functions: Biosynthesis and Depolymerization of Hyaluronic Acid in Bacteria and Vertebrate Tissues Including during Carcinogenesis.

    PubMed

    Tsepilov, R N; Beloded, A V

    2015-09-01

    Hyaluronic acid is an evolutionarily ancient molecule commonly found in vertebrate tissues and capsules of some bacteria. Here we review modern data regarding structure, properties, and biological functions of hyaluronic acid in mammals and Streptococcus spp. bacteria. Various aspects of biogenesis and degradation of hyaluronic acid are discussed, biosynthesis and degradation metabolic pathways for glycosaminoglycan together with involved enzymes are described, and vertebrate and bacterial hyaluronan synthase genes are characterized. Special attention is given to the mechanisms underlying the biological action of hyaluronic acid as well as the interaction between polysaccharide and various proteins. In addition, all known signaling pathways involving hyaluronic acid are outlined. Impaired hyaluronic acid metabolism, changes in biopolymer molecular weight, hyaluronidase activity, and enzyme isoforms often accompany carcinogenesis. The interaction between cells and hyaluronic acid from extracellular matrix that may be important during malignant change is discussed. An expected role for high molecular weight hyaluronic acid in resistance of naked mole rat to oncologic diseases and the protective role of hyaluronic acid in bacteria are discussed.

  5. 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.

  6. A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

    PubMed

    Burlini, N; Magnani, P; Villa, A; Macchi, F; Tortora, P; Guerritore, A

    1992-08-21

    A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

  7. 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

  8. Isolation and characterization of the hyperthermostable serine protease, pyrolysin, and its gene from the hyperthermophilic archaeon Pyrococcus furiosus.

    PubMed

    Voorhorst, W G; Eggen, R I; Geerling, A C; Platteeuw, C; Siezen, R J; Vos, W M

    1996-08-23

    The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.

  9. 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

  10. Pleiotropic aspartate taxis and serine taxis mutants of Escherichia coli.

    PubMed

    Reader, R W; Tso, W W; Springer, M S; Goy, M F; Adler, J

    1979-04-01

    Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.

  11. Inhibition of serine proteases by peptidyl fluoromethyl ketones

    SciTech Connect

    Imperiali, B.; Abeles, R.H.

    1986-07-01

    Peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases ..cap alpha..-chymotrypsin and porcine pancreatic elastase were synthesized. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the ..gamma..-OH group of the active site serine to form a stable hemiacetal. /sup 19/F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF/sub 3//sup 1/ and Ac-ambo-Phe-CF/sub 3/ clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrat or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of K/sub i/ concomitant with the change from a dipeptide analogue to a tetrapeptide analogue correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues. In addition to chain length, the number of fluorine substituents also affects the K/sub i/. In the case of chymotrypsin, the K/sub i/ for Ac-Leu-ambo-Phe-CF/sub 3/ is 30-fold lower than that for Ac-Leu-ambo-Phe-CF/sub 2/H. With elastase this trend is not as profound. In all cases, however, the difluoro- and trifluoromethyl ketones are better inhibitors than the monofluoromethyl and nonfluorinated analogues. This improvement must be associated with both the degree of hydration of the fluoromethyl ketones and the significant effect that fluorine substitution has on lowering the first pK/sub a/ of the hemiacetal hydroxyl. The monofluoromethyl ketone inhibitor of chymotrypsin, Ac-Leu-ambo-Phe-CFH/sub 2/, is a weak competitive inhibitor.

  12. Novel compounds reducing IRS-1 serine phosphorylation for treatment of diabetes.

    PubMed

    Simon-Szabó, Laura; Kokas, Márton; Greff, Zoltán; Boros, Sándor; Bánhegyi, Péter; Zsákai, Lilián; Szántai-Kis, Csaba; Vantus, Tibor; Mandl, József; Bánhegyi, Gábor; Vályi-Nagy, István; Őrfi, László; Ullrich, Axel; Csala, Miklós; Kéri, György

    2016-01-15

    Activation of various interacting stress kinases, particularly the c-Jun N-terminal kinases (JNK), and a concomitant phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307 play a central role both in insulin resistance and in β-cell dysfunction. IRS-1 phosphorylation is stimulated by elevated free fatty acid levels through different pathways in obesity. A series of novel pyrido[2,3-d]pyrimidin-7-one derivatives were synthesized as potential antidiabetic agents, preventing IRS-1 phosphorylation at serine 307 in a cellular model of lipotoxicity and type 2 diabetes.

  13. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    PubMed

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  14. Health information impact on the relative importance of beef attributes including its enrichment with polyunsaturated fatty acids (omega-3 and conjugated linoleic acid).

    PubMed

    Kallas, Zein; Realini, Carolina E; Gil, José Maria

    2014-08-01

    This paper uses Choice Experiments (CE) to investigate Spanish consumers' preferences towards beef meat enriched with polyunsaturated fatty acids (omega-3 and conjugated linoleic acid). Data were gathered from self-completed questionnaires in a controlled environment with two different samples (320 and 322 consumers) differentiated by the information received. The surveys were carried out in three main Spanish cities (Barcelona, Zaragoza and Pamplona), representing the average consumer. A variation of the "Dual Response Choice Experiments" (DRCE) design was used due to its ability to emphasize the purchase context. Results showed that consumers who received information attach higher preference for enriched meat with polyunsaturated fatty acids. The utility associated with the higher content of fat increase for informed consumers, showing a substitute effect. Informed consumers are willing to accept meat with a higher amount of visible fat if it is enriched with beneficial fatty acids.

  15. Serine biosynthesis by photorespiratory and non-photorespiratory pathways: an interesting interplay with unknown regulatory networks.

    PubMed

    Ros, R; Cascales-Miñana, B; Segura, J; Anoman, A D; Toujani, W; Flores-Tornero, M; Rosa-Tellez, S; Muñoz-Bertomeu, J

    2013-07-01

    Photorespiration is a primary metabolic pathway, which, given its energy costs, has often been viewed as a wasteful process. Despite having reached the consensus that one important function of photorespiration is the removal of toxic metabolite intermediates, other possible functions have emerged, and others could well emerge in the future. As a primary metabolic pathway, photorespiration interacts with other routes; however the nature of these interactions is not well known. One of these interacting pathways could be the biosynthesis of serine, since this amino acid is synthesised through photorespiratory and non-photorespiratory routes. At present, the exact contribution of each route to serine supply in different tissues and organs, their biological significance and how pathways are integrated and/or regulated remain unknown. Here, we review the non-photorespiratory serine biosynthetic pathways, their interactions with the photorespiratory pathway, their putative role in plants and their biotechnological interest.

  16. 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

  17. Regulatory properties of O-acetyl-L-serine sulfhydrylase of Cephalosporium acremonium: evidence of an isoenzyme and its importance in cephalosporin C biosynthesis.

    PubMed

    Döbeli, H; Nüesch, J

    1980-07-01

    O-Acetyl-L-serine sulfhydrylase catalyzes the final step in the biosynthesis of cysteine from H2S and O-acetyl-L-serine in the fungus Cephalosporsium acremonium, a cephalosporin C-producing organism. We separated this enzyme from the closely related but less specific O-acetyl-L-homoserine sulfhydrylase and showed that O-acetyl-L-homoserine sulfhydrylase also catalyzes the formation of cysteine from O-acetyl-L-serine and H2S. The expression of O-acetyl-L-serine sulfhydrylase was regulated by exogenous methionine. In addition, this enzyme was inhibited by S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid. The inhibition of both S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid was noncompetitive. Results obtained with gel filtraton experiments in various buffer systems indicate an association-dissociation behavior of O-acetyl-L-serine sulfhydrylase.

  18. Regulatory properties of O-acetyl-L-serine sulfhydrylase of Cephalosporium acremonium: evidence of an isoenzyme and its importance in cephalosporin C biosynthesis.

    PubMed Central

    Döbeli, H; Nüesch, J

    1980-01-01

    O-Acetyl-L-serine sulfhydrylase catalyzes the final step in the biosynthesis of cysteine from H2S and O-acetyl-L-serine in the fungus Cephalosporsium acremonium, a cephalosporin C-producing organism. We separated this enzyme from the closely related but less specific O-acetyl-L-homoserine sulfhydrylase and showed that O-acetyl-L-homoserine sulfhydrylase also catalyzes the formation of cysteine from O-acetyl-L-serine and H2S. The expression of O-acetyl-L-serine sulfhydrylase was regulated by exogenous methionine. In addition, this enzyme was inhibited by S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid. The inhibition of both S-adenosyl-L-methionine and 5-formylpteroyl monoglutamic acid was noncompetitive. Results obtained with gel filtraton experiments in various buffer systems indicate an association-dissociation behavior of O-acetyl-L-serine sulfhydrylase. PMID:7191238

  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. Meat texture and antioxidant status are improved when carnosic acid is included in the diet of fattening lambs.

    PubMed

    Morán, Lara; Andrés, Sonia; Bodas, Raúl; Prieto, Nuria; Giráldez, F Javier

    2012-08-01

    Thirty-two Merino lambs fed barley straw and a concentrate alone (CONTROL group) or enriched with carnosic acid [0.6 g kg(-1) dry matter (DM), CARN006 group; 1.2 g kg(-1) DM, CARN012 group] or vitamin E (0.6 g kg(-1) DM, VITE006 group) were used to assess the effect of these antioxidant compounds on meat quality. After being fed the experimental diets for at least 5 weeks, the animals were slaughtered with the 25 kg intended body weight and the different muscles (longissimus lumborum; LL, gluteus medius; GM) were sliced and kept refrigerated under modified atmosphere packaging during 0, 7 and 14 days. The results indicate that carnosic acid seemed to be useful to delay lipid peroxidation in a medium colour-stable muscle such as GM, but this effect was lower than that observed when vitamin E was supplemented to fattening lambs. On the contrary, meat texture and protection against cholesterol oxidation were equally improved with both compounds.

  1. Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii.

    PubMed

    Yu, Gang; Liu, Jin-Liang; Xie, Li-Qin; Wang, Xue-Liang; Zhang, Shi-Hong; Pan, Hong-Yu

    2012-12-01

    The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

  2. Induction of CYP1A and cyp2-mediated arachidonic acid epoxygenation and suppression of 20-hydroxyeicosatetraenoic acid by imidazole derivatives including the aromatase inhibitor vorozole.

    PubMed

    Diani-Moore, Silvia; Papachristou, Fotini; Labitzke, Erin; Rifkind, Arleen B

    2006-08-01

    Cytochrome P450 (P450) enzymes metabolize the membrane lipid arachidonic acid to stable biologically active epoxides [eicosatrienoic acids (EETs)] and 20-hydroxyeicosatetraenoic acid (20-HETE). These products have cardiovascular activity, primarily acting as vasodilators and vasoconstrictors, respectively. EET formation can be increased by the prototype CYP1A or CYP2 inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or phenobarbital (PB), respectively. We report here that imidazole derivative drugs: the anthelminthics, albendazole and thiabendazole; the proton pump inhibitor, omeprazole; the thromboxane synthase inhibitor, benzylimidazole; and the aromatase (CYP19) inhibitor vorozole (R76713, racemate; and R83842, (+) enantiomer) increased hepatic microsomal EET formation in a chick embryo model. Albendazole increased EETs by transcriptional induction of CYP1A5 and the others by combined induction of CYP1A5 and CYP2H, the avian orthologs of mammalian CYP1A2 and CYP2B, respectively. All inducers increased formation of the four EET regioisomers, but TCDD and albendazole had preference for 5,6-EET and PB and omeprazole for 14,15-EET. Vorozole, benzylimidazole, and TCDD also suppressed 20-HETE formation. Vorozole was a remarkably effective and potent inducer of multiple hepatic P450s at a dose range which overlapped its inhibition of ovarian aromatase. Increased CYP1A activity in mouse Hepa 1-6 and human HepG2 cells by vorozole and other imidazole derivatives demonstrated applicability of the findings to mammalian cells. The findings suggest that changes in P450-dependent arachidonic acid metabolism may be a new source of side effects for drugs that induce CYP1A or CYP2. They demonstrate further that in vivo induction of multiple hepatic P450s produces additive increases in arachidonic acid epoxygenase activity and can occur concurrently with inhibition of ovarian aromatase activity.

  3. Growth and development of the arborescent cactus Stenocereus queretaroensis in a subtropical semiarid environment, including effects of gibberellic acid.

    PubMed

    Pimienta, Eulogio; Hernandez, Gerardo; Domingues, Alejandro; Nobel, Park S.

    1998-01-01

    In Stenocereus queretaroensis (Weber) Buxbaum, an arborescent cactus cultivated in Jalisco, Mexico, for its fruits but studied here in wild populations, stem extension occurred in the autumn at the beginning of the dry season, flowering and fruiting occurred in the spring at the end of the dry season, and new roots grew in the summer during the wet season. The asynchrony of vegetative and reproductive growth reduces competitive sink effects, which may be advantageous for wild populations growing in infertile rocky soils. Seasonal patterns of sugars in the roots and especially the stems of S. queretaroensis were closely related to the main phenological stages, becoming lower in concentration during periods of major stem extension. Cessation of stem extension occurred in 100-year-old plants for which injection of GA(3) reinitiated such growth. Isolated chlorenchyma cylinders had maximum extension in a bathing solution containing 0.1 &mgr;M gibberellic acid.

  4. Far infrared spectra of solid state L-serine, L-threonine, L-cysteine, and L-methionine in different protonation states.

    PubMed

    Gaillard, Thomas; Trivella, Aurélien; Stote, Roland H; Hellwig, Petra

    2015-01-01

    In this study, experimental far infrared measurements of L-serine, L-threonine, L-cysteine, and L-methionine are presented showing the spectra for the 1.0-13.0 pH range. In parallel, solid state DFT calculations were performed on the amino acid zwitterions in the crystalline form. We focused on the lowest frequency far infrared normal modes, which required the most precision and convergence of the calculations. Analysis of the computational results, which included the potential energy distribution of the vibrational modes, permitted a detailed and almost complete assignment of the experimental spectrum. In addition to characteristic signals of the two main acid-base couples, CO2H/CO2(-) and NH3(+)/NH2, specific side chain contributions for these amino acids, including CCO and CCS vibrational modes were analyzed. This study is in line with the growing application of FIR measurements to biomolecules.

  5. Multi-species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation.

    PubMed

    Park, Seongjun; Bae, Wookeun; Rittmann, Bruce E

    2010-04-15

    A multi-species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite-oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia-oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients.

  6. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE1[OPEN

    PubMed Central

    Van de Wouwer, Dorien; Decou, Raphaël; Audenaert, Dominique; Nguyen, Long

    2016-01-01

    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

  7. Ribosome bypassing at serine codons as a test of the model of selective transfer RNA charging

    PubMed Central

    Lindsley, Dale; Bonthuis, Paul; Gallant, Jonathan; Tofoleanu, Teodora; Elf, Johan; Ehrenberg, Måns

    2005-01-01

    Recently, a model of the flux of amino acids through transfer RNAs (tRNAs) and into protein has been developed. The model predicts that the charging level of different isoacceptors carrying the same amino acid respond very differently to variation in supply of the amino acid or of the rate of charging. It has also been shown that ribosome bypassing is specifically stimulated at ‘hungry' codons calling for an aminoacyl-tRNA in short supply. We have constructed two reporters of bypassing, which differ only in the identity of the serine codon subjected to starvation. The stimulation of bypassing as a function of starvation differed greatly between the two serine codons, in good agreement with the quantitative predictions of the model. PMID:15678161

  8. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    PubMed

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.

  9. The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of D-serine.

    PubMed

    Sakinç, Türkân; Michalski, Nadine; Kleine, Britta; Gatermann, Sören G

    2009-10-01

    Human urine contains a relatively high concentration of d-serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d-serine deaminase gene (dsdA) of UPEC. We found the gene in several clinical isolates of S. saprophyticus; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii, phylogenetically close relatives of S. saprophyticus, and could also not be detected in isolates of Staphylococcus aureus, Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d-serine, whereas S. aureus, S. epidermidis and other staphylococcal species could not. The association of the dsdA gene with growth in media including d-serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d-serine utilization and detoxification may be a general property of uropathogenic bacteria.

  10. Simultaneous optimization of monolayer formation factors, including temperature, to significantly improve nucleic acid hybridization efficiency on gold substrates.

    PubMed

    Pris, Andrew D; Ostrowski, Sara G; Garaas, Sarah D

    2010-04-20

    Past literature investigations have optimized various single factors used in the formation of thiolated, single stranded DNA (ss-DNA) monolayers on gold. In this study a more comprehensive approach is taken, where a design of experiment (DOE) is employed to simultaneously optimize all of the factors involved in construction of the capture monolayer used in a fluorescence-based hybridization assay. Statistical analysis of the fluorescent intensities resulting from the DOE provides empirical evidence for the importance and the optimal levels of traditional and novel factors included in this investigation. We report on the statistical importance of a novel factor, temperature of the system during monolayer formation of the capture molecule and lateral spacer molecule, and how proper usage of this temperature factor increased the hybridization signal 50%. An initial theory of how the physical factor of heat is mechanistically supplementing the function of the lateral spacer molecule is provided.

  11. The N terminus of PA polymerase of swine-origin influenza virus H1N1 determines its compatibility with PB2 and PB1 subunits through a strain-specific amino acid serine 186.

    PubMed

    Wanitchang, Asawin; Jengarn, Juggragarn; Jongkaewwattana, Anan

    2011-01-01

    Despite several lines of evidence suggesting possible mechanisms by which the influenza virus polymerase complex, comprising PB2, PB1 and PA, work in concert during virus replication, exactly how they function is not entirely understood. The N terminal region of the PA subunit has been shown to play a key role in various functions through a number of conserved amino acid residues. However, little is known about the role of amino acids reported to be unique for a virus strain. Here, we investigated the functional implication of an amino acid (S186) present uniquely in the N terminus of the PA subunit of the pandemic H1N1 influenza virus and determined the effect of its mutation in terms of polymerase activity as well as virus growth. Using chimeric constructs of PA derived from A/PR/8/34 (H1N1) (PR8) and the swine-origin influenza virus (S-OIV) H1N1, we found that, when complexed with PB2 and PB1 of PR8, the chimeric PA protein containing the N terminus of S-OIV (1-213) with the remaining region from PR8 showed significantly reduced polymerase activity. Recombinant viruses harboring the chimeric PA also grew poorly in MDCK cells and embryonated eggs. Likewise, the chimeric PA in which the N terminus of PA of PR8 (1-213) was assembled with the remaining region of PA of S-OIV showed a similar phenotype when complexed with PB2 and PB1 of S-OIV. Interestingly, when S186 in the N terminus was altered to the residue common in most strains of influenza virus (G186), the chimeric as well as wild-type PA of S-OIV showed severely impaired polymerase activity when assayed with PB2 and PB1 of S-OIV. Collectively, this finding suggests that S186 at the N terminal region of PA of S-OIV is necessary for the protein to function optimally.

  12. 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.

  13. Hepatic serine and alanine metabolism during endotoxin-induced fever in sheep.

    PubMed Central

    Southorn, B G; Thompson, J R

    1987-01-01

    Time course changes in plasma amino acid concentrations and the hepatic metabolism of serine and alanine were measured in six mature wethers during endotoxin-induced fever. In separate trials, the animals' responses to injections of saline and endotoxin were measured. The endotoxin was from Escherichia coli serotype 055:B5 and was injected intravenously (4 micrograms/kg body weight). Liver biopsies were obtained from the sheep at 6 h postinjection during both endotoxin and saline injection trials. Rectal temperature in the endotoxin treated animals was increased (P less than 0.05, above that in control animals from 4.25 h to 9 h postinjection, with a maximum rise of 2.43 degrees C at 5.5 h postinjection. Glucose concentration in jugular plasma decreased (P less than 0.05) by 3 h postinjection and remained depressed throughout the 24 h postinjection sampling period. Plasma serine concentration was decreased (P less than 0.05) by 3 h postinjection. Plasma alanine concentration was decreased significantly (P less than 0.05) only at 24 h postinjection. Endotoxin injection increased (P less than 0.05) hepatic oxidation of 14C-serine (162%) and the net incorporation of 14C-serine carbon into hepatic protein (173%) and glycogen (275%). The net incorporation of 14C-alanine carbon into hepatic protein (172%) and glycogen (323%) were increased (P less than 0.05) by endotoxin injection, while alanine oxidation was not affected by endotoxin treatment (P greater than 0.05). The increased hepatic use of serine may explain, in part, the dramatic decrease in plasma concentrations of this amino acid following endotoxin injection into sheep. PMID:3115552

  14. 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

  15. The CUG codon is decoded in vivo as serine and not leucine in Candida albicans.

    PubMed Central

    Santos, M A; Tuite, M F

    1995-01-01

    Previous studies have shown that the yeast Candida albicans encodes a unique seryl-tRNA(CAG) that should decode the leucine codon CUG as serine. However, in vitro translation of several different CUG-containing mRNAs in the presence of this unusual seryl-tRNA(CAG) result in an apparent increase in the molecular weight of the encoded polypeptides as judged by SDS-PAGE even though the molecular weight of serine is lower than that of leucine. A possible explanation for this altered electrophoretic mobility is that the CUG codon is decoded as modified serine in vitro. To elucidate the nature of CUG decoding in vivo, a reporter system based on the C. albicans gene (RBP1) encoding rapamycin-binding protein (RBP), coupled to the promoter of the C. albicans TEF3 gene, was utilized. Sequencing and mass-spectrometry analysis of the recombinant RBP expressed in C. albicans demonstrated that the CUG codon was decoded exclusively as serine while the related CUU codon was translated as leucine. A database search revealed that 32 out of the 65 C. albicans gene sequences available have CUG codons in their open reading frames. The CUG-containing genes do not belong to any particular gene family. Thus the amino acid specified by the CUG codon has been reassigned within the mRNAs of C. albicans. We argue here that this unique genetic code change in cellular mRNAs cannot be explained by the 'Codon Reassignment Theory'. Images PMID:7784200

  16. Regulation of human serine racemase activity and dynamics by halides, ATP and malonate.

    PubMed

    Marchetti, Marialaura; Bruno, Stefano; Campanini, Barbara; Bettati, Stefano; Peracchi, Alessio; Mozzarelli, Andrea

    2015-01-01

    D-Serine is a non-proteinogenic amino acid that acts as a co-agonist of the NMDA receptors in the central nervous system. D-Serine is produced by human serine racemase (hSR), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme that also catalyzes the physiologically relevant β-elimination of both L- and D-serine to pyruvate and ammonia. After improving the protein purification yield and stability, which had so far limited the biochemical characterization of hSR, we found that the catalytic activity is affected by halides, in the order fluoride > chloride > bromide. On the contrary, iodide elicited a complete inhibition, accompanied by a modulation of the tautomeric equilibrium of the internal aldimine. We also investigated the reciprocal effects of ATP and malonate, an inhibitor that reversibly binds at the active site, 20 Å away from the ATP-binding site. ATP increased ninefold the affinity of hSR for malonate and malonate increased 100-fold that of ATP, confirming an allosteric interaction between the two binding sites. To further investigate this allosteric communication, we probed the active site accessibility by quenching of the coenzyme fluorescence in the absence and presence of ATP. We found that ATP stabilizes a closed conformation of the external aldimine Schiff base, suggesting a possible mechanism for ATP-induced hSR activation.

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

    PubMed

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

    2016-08-04

    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.

  18. Structural basis of serine/threonine phosphatase inhibition by the archetypal small molecules cantharidin and norcantharidin.

    PubMed

    Bertini, I; Calderone, V; Fragai, M; Luchinat, C; Talluri, E

    2009-08-13

    The inhibition of a subgroup of human serine/threonine protein phosphatases is responsible for the cytotoxicity of cantharidin and norcantharidin against tumor cells. It is shown that the anhydride rings of cantharidin and norcantharidin are hydrolyzed when bound to the catalytic domain of the human serine/threonine protein phosphatases 5 (PP5c), and the high-resolution crystal structures of PP5c complexed with the corresponding dicarboxylic acid derivatives of the two molecules are reported. Norcantharidin shows a unique binding conformation with the catalytically active Mn2PP5c, while cantharidin is characterized by a double conformation in its binding mode to the protein. Different binding modes of norcantharidin are observed depending of whether the starting ligand is in the anhydride or in the dicarboxylic acid form. All these structures will provide the basis for the rational design of new cantharidin-based drugs.

  19. Conserved water molecules in bacterial serine hydroxymethyltransferases.

    PubMed

    Milano, Teresa; Di Salvo, Martino Luigi; Angelaccio, Sebastiana; Pascarella, Stefano

    2015-10-01

    Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments.

  20. [Isolation of a specific inhibitor of microbial serine proteinase from kidney bean seeds].

    PubMed

    Mosolov, V V; Malova, E L; Cheban, A N

    1983-10-01

    A protein acting as a specific inhibitor of microbial serine proteinases was isolated from kidney bean seeds. The purification procedure included complex formation between the inhibitor and Aspergillus oryzae proteinase. The protein with a Mr approximately 10 000 inhibits subtilisin and Asp. oryzae proteinase but does not affect trypsin and chymotrypsin. The inhibitor molecule contains no half-cystine residues.

  1. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

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

    2014-06-01

    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. In this study, 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 targets, whereas adding RNA to recombinant RNA binding protein substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Preincubation 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. During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps, which bind NE and cathepsin G. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and neutrophil extracellular traps 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.

  2. An isozyme of earthworm serine proteases acts on hydrolysis of triacylglycerol.

    PubMed

    Nakajima, Nobuyoshi; Sugimoto, Manabu; Tsuboi, Sadao; Tsuji, Hideaki; Ishihara, Kohji

    2005-10-01

    An enzyme catalyzing the hydrolysis of triacylglycerol was purified from an earthworm. The N-terminal amino acid sequence and the catalytic function of the purified enzyme were identical to those of Isozyme C, an isozyme of the earthworm-serine proteases. No other lipase proteins were found in the earthworm cells. The isozyme might act on the hydrolysis of triacylglycerol as well as the protein decomposition.

  3. Serine is a new target residue for endogenous ADP-ribosylation on histones

    PubMed Central

    Colby, Thomas; Zhang, Qi; Atanassov, Ilian; Zaja, Roko; Palazzo, Luca; Stockum, Anna; Ahel, Ivan; Matic, Ivan

    2016-01-01

    ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones. We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage. PMID:27723750

  4. Serine is a new target residue for endogenous ADP-ribosylation on histones.

    PubMed

    Leidecker, Orsolya; Bonfiglio, Juan José; Colby, Thomas; Zhang, Qi; Atanassov, Ilian; Zaja, Roko; Palazzo, Luca; Stockum, Anna; Ahel, Ivan; Matic, Ivan

    2016-12-01

    ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones. We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage.

  5. 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.

  6. Serine 71 of the glycoprotein HEF is located at the active site of the acetylesterase of influenza C virus.

    PubMed

    Herrler, G; Multhaup, G; Beyreuther, K; Klenk, H D

    1988-01-01

    The acetylesterase of influenza C virus has been reported recently to be inhibited by diisopropylfluorophosphate (DFP) [Muchmore EA, Varki A (1987) Science 236: 1293-1295]. As this inhibitor is known to bind covalently to the serine in the active site of serine esterases, we attempted to determine the serine in the active site of the influenza C acetylesterase. Incubation of purified influenza C virus with 3H-DFP resulted in the selective labelling of the influenza C glycoprotein HEF. The labelled glycoprotein was isolated from a SDS-polyacrylamide gel. Following reduction and carboxymethylation, tryptic peptides of HEF were prepared and analyzed by reversed phase HPLC. The peptide containing the 3H-DFP was subjected to sequence analysis. The amino acids determined from the NH2-terminus were used to locate the peptide on the HEF polypeptide. Radiosequencing revealed that 3H-DFP is attached to amino acid 17 of the tryptic peptide. These results indicate that serine 71 is the active-site serine of the acetylesterase of influenza C virus.

  7. Enteropeptidase, a type II transmembrane serine protease.

    PubMed

    Zheng, X Long; Kitamoto, Yasunori; Sadler, J Evan

    2009-06-01

    Enteropeptidase, a type II transmembrane serine protease, is localized to the brush border of the duodenal and jejunal mucosa. It is synthesized as a zymogen (proenteropeptidase) that requires activation by another protease, either trypsin or possibly duodenase. Active enteropeptidase then converts the pancreatic precursor, trypsinogen, to trypsin by cleavage of the specific trypsinogen activation peptide, Asp-Asp-Asp-Asp-Lys- Ile that is highly conserved in vertebrates. Trypsin, in turn, activates other digestive zymogens such as chymotrypsinogen, proelastase, procarboxypeptidase and prolipase in the lumen of the gut. The important biological function of enteropeptidase is highlighted by the manifestation of severe diarrhea, failure to thrive, hypoproteinemia and edema as a result of congenital deficiency of enteropeptidase activity in the gut. Conversely, duodenopancreatic reflux of proteolytically active enteropeptidase may cause acute and chronic pancreatitis.

  8. Highly potent fibrinolytic serine protease from Streptomyces.

    PubMed

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

    2011-01-05

    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.

  9. 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.

  10. Hepatitis C virus NS3-4A serine protease inhibitors: SAR of P'2 moiety with improved potency.

    PubMed

    Arasappan, A; Njoroge, F G; Chan, T-Y; Bennett, F; Bogen, S L; Chen, K; Gu, H; Hong, L; Jao, E; Liu, Y-T; Lovey, R G; Parekh, T; Pike, R E; Pinto, P; Santhanam, B; Venkatraman, S; Vaccaro, H; Wang, H; Yang, X; Zhu, Z; Mckittrick, B; Saksena, A K; Girijavallabhan, V; Pichardo, J; Butkiewicz, N; Ingram, R; Malcolm, B; Prongay, A; Yao, N; Marten, B; Madison, V; Kemp, S; Levy, O; Lim-Wilby, M; Tamura, S; Ganguly, A K

    2005-10-01

    We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.

  11. Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

    PubMed

    Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

    2015-08-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Purification and characterization of serine proteinase 2 from Bacillus intermedius 3-19.

    PubMed

    Balaban, N P; Mardanova, A M; Sharipova, M R; Gabdrakhmanova, L A; Sokolova, E A; Rudenskaya, G N; Leshchinskaya, I B

    2004-04-01

    A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.

  17. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    PubMed

    Ogawara, Hiroshi

    2016-09-01

    PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

  18. 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

  19. 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

  20. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication.

    PubMed Central

    Koonin, E V

    1993-01-01

    A new superfamily of (putative) DNA-dependent ATPases is described that includes the ATPase domains of prokaryotic NtrC-related transcription regulators, MCM proteins involved in the initiation of eukaryotic DNA replication, and a group of uncharacterized bacterial and chloroplast proteins. MCM proteins are shown to contain a modified form of the ATP-binding motif and are predicted to mediate ATP-dependent opening of double-stranded DNA in the replication origins. In a second line of investigation, it is demonstrated that the products of unidentified open reading frames from Marchantia mitochondria and from yeast, and a domain of a baculovirus protein involved in viral DNA replication are related to the superfamily III of DNA and RNA helicases that previously has been known to include only proteins of small viruses. Comparison of the multiple alignments showed that the proteins of the NtrC superfamily and the helicases of superfamily III share three related sequence motifs tightly packed in the ATPase domain that consists of 100-150 amino acid residues. A similar array of conserved motifs is found in the family of DnaA-related ATPases. It is hypothesized that the three large groups of nucleic acid-dependent ATPases have similar structure of the core ATPase domain and have evolved from a common ancestor. PMID:8332451

  1. Synthesis, characterization, quantum chemical calculations and evaluation of antioxidant properties of 1,3,4-thiadiazole derivatives including 2- and 3-methoxy cinnamic acids

    NASA Astrophysics Data System (ADS)

    Gür, Mahmut; Muğlu, Halit; Çavuş, M. Serdar; Güder, Aytaç; Sayıner, Hakan S.; Kandemirli, Fatma

    2017-04-01

    A series of 1,3,4-thiadiazole derivatives including 2- and 3-methoxy cinnamic acids were synthesized, and their structures were elucidated by the UV, IR, 1H NMR, 13C NMR spectroscopies and elemental analysis. The UV and IR calculations of the molecules were performed by using B3LYP, HF and MP2 methods with selected 6-311++G(2d,2p), 6-311++G(3df,3pd) and cc-pvtz basis sets. Dipole moment, polarizability, chemical hardness/softness and electronegativity were also calculated and analyzed. Experimental FT-IR spectra and UV-Vis spectrum of the compounds were compared with theoretical data. Furthermore, antioxidant activities of the compounds were practised via different test methods such as 2,2-diphenyl-1-picryl-hydrazyl (DPPHrad), N,N-dimethyl-p-phenylenediamine (DMPDrad +), and 2,2‧-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTSrad +) scavenging activity assays. When compared with standards (BHA-Butylated hydroxyanisole, RUT-Rutin, and TRO-Trolox), it was observed that especially XIII and XIV which include methoxy groups at the o- and m-positions, respectively, had effective activities.

  2. Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29.

    PubMed Central

    Hermoso, J M; Méndez, E; Soriano, F; Salas, M

    1985-01-01

    B. subtilis phage phi 29 has a terminal protein, p3, covalently linked to the 5' ends of the DNA through a phosphodiester bond between a serine residue and 5'-dAMP. This protein acts as a primer in DNA replication by forming an initiation complex with the 5'-terminal nucleotide dAMP. The amino acid sequence of the terminal protein, deduced from the nucleotide sequence of gene 3, showed the presence of 18 serine residues in a total of 266 amino acids. In this paper we have identified the serine involved in the linkage with the DNA as the residue 232, located close to the C-terminus of the molecule. This result was obtained by amino acid analysis of the peptide that remains linked to the DNA after proteinase K digestion of the terminal protein-phi 29 DNA complex and automated Edman degradation of the corresponding [125I]-labeled tryptic peptide. Prediction of the secondary structure of the terminal protein suggested that the serine residue involved in the linkage with the DNA is placed in a beta-turn, probably located on the external part of the molecule, as indicated by hydropathic values. Images PMID:3934646

  3. Characteristics of the transport of alanine, serine and glutamine across the plasma membrane of isolated rat liver cells.

    PubMed Central

    Joseph, S K; Bradford, N M; McGivan, J D

    1978-01-01

    1. Alanine, glutamine and serine were actively accumulated in liver cells isolated from starved rats. 2. This accumulation was inhibited when either Na+ or HCO3- ions were omitted from the incubation medium. In general the degree of dependence on Na+ was quantitatively similar to that on HCO3-. 3. The apparent Km values for the transport of all three amino acids were in the range 3--5mM with Vmax. values in the range 15--25nmol/min per mg of cell protein at 37 degrees C. 4. Alanine and serine transport were mutually competitive; glutamine inhibited the transport of alanine and serine non-competitively. 5. The initial rate of transport of these amino acids was inhibited when the intracellular content of ATP was decreased. 6. Ouabain inhibited the rate of alanine transport without inhibiting the rate of alanine metabolism. 7. It is concluded that a minimum of three transport systems must be postulated to exist in the liver cell plasma membrane to account for the transport of alanine, serine and glutamine. The rate of transport of these amino acids in isolated hepatocytes is unlikely to limit the rate at which they are metabolized. PMID:747655

  4. Purification, characterization and gene cloning of Da-36, a novel serine protease from Deinagkistrodon acutus venom.

    PubMed

    Zheng, Ying; Ye, Feng-Ping; Wang, Jie; Liao, Guo-Yang; Zhang, Yun; Fan, Quan-Shui; Lee, Wen-Hui

    2013-06-01

    A serine protease termed Da-36 was isolated from crude venom of Deinagkistrodon acutus. The enzyme was a single chain protein with an apparent molecular weight of 36,000 on SDS-PAGE with an isoelectric point of 6.59. Da-36 could clot human plasma by cleaving the Aα, Bβ and γ chains of fibrinogen and also exhibited arginine esterase activity. The proteolytic activity of Da-36 toward TAME was strongly inhibited by PMSF and moderately affected by benzamidine and aprotinin, indicating that it was a serine protease. Meanwhile, Da-36 showed stability with wide temperature (20-50 °C) and pH value ranges (pH 6-10). Divalent metal ions of Ca(2+), Mg(2+), and Mn(2+) had no effects but Zn(2+) and Cu(2+) inhibited the arginine esterase activity of Da-36. Total DNA was extracted directly from the lyophilized crude venom and the gene (5.5 kbp) coding for Da-36 had been successfully cloned. Sequence analysis revealed that the Da-36 gene contained five exons and four introns. The mature Da-36 was encoded by four separate exons. The deduced mature amino acid sequence of Da-36 was in good agreement with the determined N-terminal sequence of the purified protein and shared high homology with other serine proteases isolated from different snake venoms. Blast search using amino acid sequence of Da-36 against public database revealed that Da-36 showed a maximal identity of 90% with both Dav-X (Swiss-Prot: Q9I8W9.1) and thrombin-like protein 1 (GenBank: AAW56608.1) from the same snake species, indicating that Da-36 is a novel serine protease.

  5. 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.

  6. Selective Michael-aldol reaction by use of sterically hindered aluminum aryloxides as Lewis acids: an easy approach to cyclobutane amino acids.

    PubMed

    Avenoza, Alberto; Busto, Jesús H; Canal, Noelia; Peregrina, Jesús M; Pérez-Fernández, Marta

    2005-08-04

    A formal [2 + 2] cycloaddition of 2-amidoacrylates with monosubstituted donor olefins, including its asymmetric version, is described. The stereoselectivity of this reaction can be modulated by the use of sterically hindered aluminum aryloxides or methylaluminoxane as Lewis acids. The reaction was applied to the synthesis of both stereoisomers of 2-benzyloxycyclobutane-alpha-amino acid, which are protected serine analogues c(4)Ser(OBn).

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

    PubMed

    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, and leptin/adiponectin).

  8. 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.

  9. Serine 421 regulates mutant huntingtin toxicity and clearance in mice

    PubMed Central

    Kratter, Ian H.; Zahed, Hengameh; Lau, Alice; Daub, Aaron C.; Weiberth, Kurt F.; Gu, Xiaofeng; Humbert, Sandrine; Yang, X. William; Osmand, Alex; Steffan, Joan S.; Masliah, Eliezer

    2016-01-01

    Huntington’s disease (HD) is a progressive, adult-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (HTT). There are no cures or disease-modifying therapies for HD. HTT has a highly conserved Akt phosphorylation site at serine 421, and prior work in HD models found that phosphorylation at S421 (S421-P) diminishes the toxicity of mutant HTT (mHTT) fragments in neuronal cultures. However, whether S421-P affects the toxicity of mHTT in vivo remains unknown. In this work, we used murine models to investigate the role of S421-P in HTT-induced neurodegeneration. Specifically, we mutated the human mHTT gene within a BAC to express either an aspartic acid or an alanine at position 421, mimicking tonic phosphorylation (mHTT-S421D mice) or preventing phosphorylation (mHTT-S421A mice), respectively. Mimicking HTT phosphorylation strongly ameliorated mHTT-induced behavioral dysfunction and striatal neurodegeneration, whereas neuronal dysfunction persisted when S421 phosphorylation was blocked. We found that S421 phosphorylation mitigates neurodegeneration by increasing proteasome-dependent turnover of mHTT and reducing the presence of a toxic mHTT conformer. These data indicate that S421 is a potent modifier of mHTT toxicity and offer in vivo validation for S421 as a therapeutic target in HD. PMID:27525439

  10. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion

    PubMed Central

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-01-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed). PMID:22638583

  11. Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion.

    PubMed

    Thomsen, Martin Christen Frølund; Nielsen, Morten

    2012-07-01

    Seq2Logo is a web-based sequence logo generator. Sequence logos are a graphical representation of the information content stored in a multiple sequence alignment (MSA) and provide a compact and highly intuitive representation of the position-specific amino acid composition of binding motifs, active sites, etc. in biological sequences. Accurate generation of sequence logos is often compromised by sequence redundancy and low number of observations. Moreover, most methods available for sequence logo generation focus on displaying the position-specific enrichment of amino acids, discarding the equally valuable information related to amino acid depletion. Seq2logo aims at resolving these issues allowing the user to include sequence weighting to correct for data redundancy, pseudo counts to correct for low number of observations and different logotype representations each capturing different aspects related to amino acid enrichment and depletion. Besides allowing input in the format of peptides and MSA, Seq2Logo accepts input as Blast sequence profiles, providing easy access for non-expert end-users to characterize and identify functionally conserved/variable amino acids in any given protein of interest. The output from the server is a sequence logo and a PSSM. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo (14 May 2012, date last accessed).

  12. 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

  13. Serine one-carbon catabolism with formate overflow

    PubMed Central

    Meiser, Johannes; Tumanov, Sergey; Maddocks, Oliver; Labuschagne, Christiaan Fred; Athineos, Dimitris; Van Den Broek, Niels; Mackay, Gillian M.; Gottlieb, Eyal; Blyth, Karen; Vousden, Karen; Kamphorst, Jurre J.; Vazquez, Alexei

    2016-01-01

    Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors. PMID:27819051

  14. Catalysis of poliovirus VP0 maturation cleavage is not mediated by serine 10 of VP2.

    PubMed Central

    Harber, J J; Bradley, J; Anderson, C W; Wimmer, E

    1991-01-01

    The maturation of the poliovirus capsid occurs as the result of a single unexplained proteolytic event during which 58 to 59 copies of the 60 VP0 capsid protein precursors are cleaved. An autocatalytic mechanism for cleavage of VP0 to VP4 and VP2 was proposed by Arnold et al. (E. Arnold, M. Luo, G. Vriend, M. G. Rossman, A. C. Palmenberg, G. D. Parks, M. J. Nicklin, and E. Wimmer, Proc. Natl. Acad. Sci. USA 84:21-25, 1987) in which serine 10 of VP2 is activated by virion RNA to catalyze VP4-VP2 processing. The hypothesis rests on the observation that a hydrogen bond was observed between serine 10 of VP2 (S2010) and the carboxyl terminus of VP4 in three mature picornaviral atomic structures: rhinovirus 14, mengovirus, and poliovirus type 1 (Mahoney). We constructed mutant viruses with cysteine (S2010C) or alanine (S2010A) replacing serine 10 of VP2; these exhibited normal proteolytic processing of VP0. While our results do not exclude an autocatalytic mechanism for the maturation cleavage, they do eliminate the conserved S2010 residue as the catalytic amino acid. Images PMID:1845893

  15. Contributions of spinal D-amino acid oxidase to chronic morphine-induced hyperalgesia.

    PubMed

    Ma, Shuai; Li, Xin-Yan; Gong, Nian; Wang, Yong-Xiang

    2015-12-10

    Spinal D-amino acid oxidase (DAAO) is an FAD-dependent peroxisomal flavoenzyme which mediates the conversion of neutral and polar D-amino acids (including D-serine) to the corresponding α-keto acids, and simultaneously produces hydrogen peroxide and ammonia. This study has aimed to explore the potential contributions of spinal DAAO and its mediated hydrogen peroxide/D-serine metabolism to the development of morphine-induced hyperalgesia. Bi-daily subcutaneous injections of morphine to mice over 7 days induced thermal hyperalgesia as measured by both the hot-plate and tail-immersion tests, and spinal astroglial activation with increased spinal gene expression of DAAO, glial fibrillary acidic protein (GFAP) and pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). Subcutaneous injections of the potent DAAO inhibitor CBIO (5-chloro-benzo[D]isoxazol-3-ol) prevented and reversed the chronic morphine-induced hyperalgesia. CBIO also inhibited both astrocyte activation and the expression of pro-inflammatory cytokines. Intrathecal injection of the hydrogen peroxide scavenger PBN (phenyl-N-tert-butylnitrone) and of catalase completely reversed established morphine hyperalgesia, whereas subcutaneous injections of exogenous D-serine failed to alter chronic morphine-induced hyperalgesia. These results provided evidence that spinal DAAO and its subsequent production of hydrogen peroxide rather than the D-serine metabolism contributed to the development of morphine-induced hyperalgesia.

  16. An update on potato crisps contents of moisture, fat, salt and fatty acids (including trans-fatty acids) with special emphasis on new oils/fats used for frying.

    PubMed

    Gonçalves Albuquerque, Tânia; Sanches-Silva, Ana; Santos, Lèlita; Costa, Helena S

    2012-09-01

    Eighteen brands of potato crisps, frequently consumed, were analyzed to establish their nutritional value in relation to salt, fat and fatty acid (FA) composition. The purpose of the present study was to determine moisture, total fat, salt contents and FA profiles (including trans-FAs), and to identify the oil/fat used for frying of the 18 brands of potato crisps. Our results show that salt content ranged from 0.127 to 2.77 g/100 g and total fat content of potato crisps varied between 20.0 and 42.8 g/100 g. With respect to FAs analysis, palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2) were the major FAs found in the analyzed potato crisps. It is clear from our work that nowadays most potato crisps are currently produced using oils with high contents in unsaturated FAs, which can be considered as healthier from a nutritional point of view. Nevertheless, some brands of potato crisps still use palm oil or a blend of palm oil and other fats/oils, which are very rich in saturated FAs.

  17. Serine protease activities in Leishmania (Leishmania) chagasi promastigotes.

    PubMed

    da Silva-López, Raquel Elisa; dos Santos, Tatiana Resende; Morgado-Díaz, José Andrés; Tanaka, Marcelo Neves; de Simone, Salvatore Giovanni

    2010-10-01

    The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-L: -arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.

  18. The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase.

    PubMed Central

    Goring, D R; Rothstein, S J

    1992-01-01

    An S-receptor kinase (SRK) cDNA, SRK-910, from the active S-locus in a self-incompatible Brassica napus W1 line has been isolated and characterized. The SRK-910 gene is predominantly expressed in pistils and segregates with the W1 self-incompatibility phenotype in an F2 population derived from a cross between the self-incompatible W1 line and a self-compatible Westar line. Analysis of the predicted amino acid sequence demonstrated that the extracellular receptor domain is highly homologous to S-locus glycoproteins, whereas the cytoplasmic kinase domain contains conserved amino acids present in serine/threonine kinases. An SRK-910 kinase protein fusion was produced in Escherichia coli and found to contain kinase activity. Phosphoamino acid analysis confirmed that only serine and threonine residues were phosphorylated. Thus, the SRK-910 gene encodes a functional serine/threonine receptor kinase. PMID:1332796

  19. Plasma glycine and serine levels in schizophrenia compared to normal controls and major depression: relation to negative symptoms.

    PubMed

    Sumiyoshi, Tomiki; Anil, A Elif; Jin, Dai; Jayathilake, Karu; Lee, Myung; Meltzer, Herbert Y

    2004-03-01

    Previous studies have suggested decreased N-methyl-D-aspartate (NMDA)-type glutamate receptor function may contribute to increased negative symptoms in patients with schizophrenia. Consistent with this hypothesis, glycine, a co-agonist at NMDA receptors, has been reported to improve negative symptoms associated with the illness. This study was performed to determine if plasma levels of glycine or its ratio to serine, a precursor of glycine, are decreased in patients with schizophrenia compared to normal control subjects or patients with major depression. We also tested the hypothesis that these amino acids were correlated with negative symptoms in subjects with schizophrenia. Plasma levels of glycine, serine, and their ratio, were compared in 144 patients with schizophrenia, 44 patients with major depression, and 49 normal control subjects. All subjects were medication-free. Psychopathology was evaluated using the Brief Psychiatric Rating Scale (BPRS). Plasma glycine levels and glycine/serine ratios were decreased in patients with schizophrenia relative to control subjects and patients with major depression. By contrast, serine levels were increased in patients with schizophrenia compared to normal subjects but not compared to major depression. Patients with major depression also had increased plasma serine levels and decreased glycine/serine ratios compared to normal controls, but glycine levels were not different from those of normal controls. In subjects with schizophrenia, glycine levels predicted the Withdrawal-Retardation score (BPRS), whereas no such correlation was found in subjects with major depression. These results provide additional evidence that decreased availability of glycine may be related to the pathophysiology of negative symptoms. The decreases in plasma glycine levels support the evidence for an abnormality in the glutamatergic system in schizophrenia, and provide additional support for efforts to improve negative symptoms by augmentation of

  20. Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine.

    PubMed

    Gilbert, G E; Drinkwater, D

    1993-09-21

    Phosphatidylserine, a negatively charged lipid, is exposed on the platelet membrane following cell stimulation, correlating with the expression of factor VIII receptors. We have explored the importance of the negative electrostatic potential of phosphatidylserine vs chemical moieties of phosphatidylserine for specific membrane binding of factor VIII. Fluorescein-labeled factor VIII bound to membranes containing 15% phosphatidic acid, a negatively charged phospholipid, with low affinity compared to phosphatidylserine-containing membranes. Binding was not specific as it was inhibited by other proteins in plasma. Factor VIII bound to membranes containing 10% phosphatidylserine in spite of a varying net charge provided by 0-15% stearylamine, a positively charged lipid. The soluble phosphatidylserine moiety, O-phospho-L-serine, inhibited factor VIII binding to phosphatidylserine-containing membranes with a Ki of 20 mM, but the stereoisomer, O-phospho-D-serine, was 5-fold less effective. Furthermore, binding of factor VIII to membranes containing synthetic phosphatidyl-D-serine was 5-fold less than binding to membranes containing phosphatidyl-L-serine. Membranes containing synthetic phosphatidyl-L-homoserine, differing from phosphatidylserine by a single methylene, supported high-affinity binding, but it was not specific as factor VIII was displaced by other plasma proteins. O-Phospho-L-serine also inhibited the binding of factor VIII to platelet-derived microparticles with a Ki of 20 mM, and the stereoisomer was 4-fold less effective. These results indicate that membrane binding of factor VIII is mediated by a stereoselective recognition O-phospho-L-serine of phosphatidylserine and that negative electrostatic potential is of lesser importance.

  1. 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.

  2. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

    PubMed Central

    2010-01-01

    Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

  3. 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...

  4. Pathogenic disruption of DISC1-serine racemase binding elicits schizophrenia-like behavior via D-serine depletion

    PubMed Central

    Ma, T.M.; Abazyan, S.; Abazyan, B.; Nomura, J.; Yang, C.; Seshadri, S.; Sawa, A.; Snyder, S.H.; Pletnikov, M.V.

    2012-01-01

    Perturbation of Disrupted-In-Schizophrenia-1 (DISC1) and D-serine/NMDA receptor hypofunction have both been implicated in the pathophysiology of schizophrenia and other psychiatric disorders. In the present study, we demonstrate that these two pathways intersect with behavioral consequences. DISC1 binds to and stabilizes serine racemase (SR), the enzyme that generates D-serine, an endogenous co-agonist of the NMDA receptor. Mutant DISC1 fails to bind to SR, facilitating ubiquitination and degradation of SR and a decrease in D-serine production. To elucidate DISC1-SR interactions in vivo, we generated a mouse model of selective and inducible expression of mutant DISC1 in astrocytes, the main source of D-serine in the brain. Expression of mutant DISC1 down-regulates endogenous DISC1 and decreases protein but not mRNA levels of SR, resulting in diminished production of D-serine. In contrast, mutant DISC1 does not alter levels of ALDH1L1, connexins, GLT-1 or binding partners of DISC1 and SR, LIS1 or PICK1. Adult male and female mice with life-long expression of mutant DISC1 exhibit behavioral abnormalities consistent with hypofunction of NMDA neurotransmission. Specifically, mutant mice display greater responses to an NMDA antagonist, MK-801, in open field and pre-pulse inhibition of the acoustic startle tests and are significantly more sensitive to the ameliorative effects of D-serine. These findings support a model wherein mutant DISC1 leads to SR degradation via dominant-negative effects, resulting in D-serine deficiency that diminishes NMDA neurotransmission thus linking DISC1 and NMDA pathophysiologic mechanisms in mental illness. PMID:22801410

  5. Pathogenic disruption of DISC1-serine racemase binding elicits schizophrenia-like behavior via D-serine depletion.

    PubMed

    Ma, T M; Abazyan, S; Abazyan, B; Nomura, J; Yang, C; Seshadri, S; Sawa, A; Snyder, S H; Pletnikov, M V

    2013-05-01

    Perturbation of Disrupted-In-Schizophrenia-1 (DISC1) and D-serine/NMDA receptor hypofunction have both been implicated in the pathophysiology of schizophrenia and other psychiatric disorders. In the present study, we demonstrate that these two pathways intersect with behavioral consequences. DISC1 binds to and stabilizes serine racemase (SR), the enzyme that generates D-serine, an endogenous co-agonist of the NMDA receptor. Mutant DISC1 fails to bind to SR, facilitating ubiquitination and degradation of SR and a decrease in D-serine production. To elucidate DISC1-SR interactions in vivo, we generated a mouse model of selective and inducible expression of mutant DISC1 in astrocytes, the main source of D-serine in the brain. Expression of mutant DISC1 downregulates endogenous DISC1 and decreases protein but not mRNA levels of SR, resulting in diminished production of D-serine. In contrast, mutant DISC1 does not alter levels of ALDH1L1, connexins, GLT-1 or binding partners of DISC1 and SR, LIS1 or PICK1. Adult male and female mice with lifelong expression of mutant DISC1 exhibit behavioral abnormalities consistent with hypofunction of NMDA neurotransmission. Specifically, mutant mice display greater responses to an NMDA antagonist, MK-801, in open field and pre-pulse inhibition of the acoustic startle tests and are significantly more sensitive to the ameliorative effects of D-serine. These findings support a model wherein mutant DISC1 leads to SR degradation via dominant negative effects, resulting in D-serine deficiency that diminishes NMDA neurotransmission thus linking DISC1 and NMDA pathophysiological mechanisms in mental illness.

  6. 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.

  7. Distribution and enantiomeric composition of amino acids in the Murchison meteorite

    NASA Technical Reports Server (NTRS)

    Engel, M. H.; Nagy, B.

    1982-01-01

    Studies of the amino acid contents and enantiomeric compositions of a single stone from the Murchison meteorite are reported. Water-extracted and 6M HCl-extracted samples from the meteorite interior of meteorite fragments were analyzed by gas chromatography and combined gas chromatography-chemical ionization mass spectrometry. Examination of the D/L ratios of glutamic acid, aspartic acid, proline, leucine and alanine reveals those amino acids extractable by water to be partially racemized, whereas the acid-extracted amino acids were less racemized. The amino acid composition of the stone is similar to those previously reported, including the absence of serine, threonine, tyrosine phenylalanine and methionine and the presence of unusual amino acids including such as isovaline, alpha-aminoisobutyric acid and pseudoleucine. It is concluded that the most likely mechanism accounting for the occurrence of nonracemic amino acid mixtures in the Murchison meteorite is by extraterrestrial stereoselective synthesis or decomposition reactions.

  8. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation.

    PubMed

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-18

    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.

  9. On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

    PubMed Central

    Henriques, Elsa S.; Fonseca, Nelson; Ramos, Maria João

    2004-01-01

    Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors α-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors. PMID:15322279

  10. Controlled synthesis of phosphorylcholine derivatives of poly(serine) and poly(homoserine).

    PubMed

    Yakovlev, Ilya; Deming, Timothy J

    2015-04-01

    We report methods for the synthesis of polypeptides that are fully functionalized with desirable phosphorylcholine, PC, groups. Because of the inherent challenges in the direct incorporation of the PC group into α-amino acid N-carboxyanhydride (NCA) monomers, we developed a synthetic approach that combined functional NCA polymerization with efficient postpolymerization modification. While poly(L-phosphorylcholine serine) was found to be unstable upon synthesis, we successfully prepared poly(L-phosphorylcholine homoserine) with controlled chain lengths and found these to be water-soluble with disordered chain conformations.

  11. 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

  12. Changes in free amino acid content and activities of amination and transamination enzymes in yeasts grown on different inorganic nitrogen sources, including hydroxylamine.

    PubMed

    Norkrans, B; Tunblad-Johansson, I

    1981-01-01

    This study concerns inter- and intraspecific differences between yeasts at assimilation of different nitrogen sources. Alterations in the content of free amino acids in cells and media as well as in the related enzyme activities during growth were studied. The hydroxylamine (HA)-tolerant Endomycopsis lipolytica was examined and compared with the nitrate-reducing Cryptococcus albidus, and Saccharomyces cerevisiae, requiring fully reduced nitrogen for growth. Special attention was paid to alanine, aspartic acid, and glutamic acid, the amino acids closely related to the Krebs cycle keto acids. The amino acids were analyzed as their n-propyl N-acetyl esters by gas-liquid chromatography (GLC). The composition of the amino acid pool was similar for the three yeasts. Glutamic acid was predominant; in early log-phase cells of E. lipolytica contents of 200-234 micromol . g(-1) dry weight were found. A positive correlation between the specific growth rate and the size of the amino acid pool was observed. The assimilation of ammonia was mediated by glutamate dehydrogenase (GDH). The NADP-GDH was the dominating enzyme in all three yeasts showing the highest specific activity in Cr. albidus grown on nitrate (6980 nmol . (min(-1)).(mg protein(-1)). Glutamine synthetase (GS) displayed a high specific activity in S. cerevisiae, which also had a high amount of glutamine. The assimilation of HA did not differ greatly from the assimilation of ammonium in E. lipolytica. The existing differences could rather be explained as provoked by the concentration of available nitrogen.

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

    PubMed

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

    2015-10-08

    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.

  14. 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

  15. [Properties of glycyrrhizin in Kampo extracts including licorice root and changes in the blood concentration of glycyrrhetic acid after oral administration of Kampo extracts].

    PubMed

    Miyamura, M; Ono, M; Kyotani, S; Nishioka, Y

    1996-03-01

    We investigated in vitro the properties of glycyrrhizin (GL), such as dissolution, absorption and resolution, using a Sho-Seiryu-To extract, a Sho-Saiko-To extract, both including a licorice root, and licorice extract. The dissolution of GL differed with the pH of the solvent. The absorption (partition coefficient) of GL decreased with an increase in pH, and increased in the presence of other active constituents, such as baicalin, baicalein, and ephedrine. In the case of the Sho-Saiko-To extract, the conversion from GL to glycyrrhetic acid (GA) by beta-glucuronidase originated from E. coli occurred slowly. It was also suppressed by adding baicalin. We determined in vivo the pharmacokinetics of GA after oral administration of Kampo extracts in healthy volunteers. In each Kampo extract, the time of administration had no influence on the mean maximum blood concentration (Cmax) and the area under the blood concentration-time curve (AUC). Tmax was delayed in the case of the administration after meal (p < 0.05).

  16. 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

    2016-01-01

    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.

  17. Transport systems of serine at the brain barriers and in brain parenchymal cells.

    PubMed

    Kasai, Yasuyuki; Tachikawa, Masanori; Hirose, Shirou; Akanuma, Shin-ichi; Hosoya, Ken-ichi

    2011-07-01

    D-Serine is a co-agonist for NMDA-type glutamate receptors. Although D-serine levels in CSF and interstitial fluid (ISF) affect CNS function, the regulatory system remains to be fully understood. Therefore, the purpose of this study was to investigate d-serine transport across the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) and in brain parenchymal cells. D-Serine microinjected into the cerebrum was not eliminated, suggesting a negligible contribution of D-serine efflux transport at the BBB. In contrast, D-serine was taken up from the circulating blood across the BBB via a carrier-mediated process. D-Serine elimination clearance from CSF was fourfold greater than that of d-mannitol, which is considered to reflect CSF bulk flow. The characteristics of D-serine uptake by isolated choroid plexus were consistent with those of Na(+)-independent alanine-serine-cysteine transporter 1 (asc-1). Uptake of D-serine by brain slices appeared to occur predominantly via asc-1 and Na(+)-dependent alanine-serine-cysteine transporter 2. These findings suggest that the regulatory system of D-serine levels in ISF and CSF involves (i) asc-1 at the BCSFB, acting as a major pathway of D-serine elimination from the CSF, (ii) blood-to-brain and blood-to-CSF influx transport of D-serine across the BBB and BCSFB, and (iii) concentrative uptake of D-serine by brain parenchymal cells.

  18. 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 ...

  19. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  20. Bovine papillomavirus with a mutation in the E2 serine 301 phosphorylation site replicates at a high copy number.

    PubMed Central

    McBride, A A; Howley, P M

    1991-01-01

    The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes at least three proteins with transcriptional regulatory properties. The full-length E2 open reading frame encodes a transcriptional transactivator, and the 3' region encodes two smaller polypeptides that repress E2-mediated transactivation. The full-length gene product is also required for viral DNA replication. We have demonstrated that the BPV-1 E2 polypeptides are phosphorylated primarily on two serine residues at a site adjacent to the carboxy-terminal DNA binding domain, which is common to all three E2 proteins (A. A. McBride, J. B. Bolen, and P. M. Howley, J. Virol. 63:5076-5085, 1989). These serine residues, at amino acid positions 298 and 301, were substituted with alanine residues in the context of the entire BPV-1 genome. The mutated BPV-1 genomes were introduced into rodent cell lines and assayed for focus formation, viral gene expression, and extrachromosomal viral DNA replication. Viral DNAs containing the E2 serine-to-alanine substitution mutants transformed C127 cells with efficiencies comparable to that of wild-type BPV-1. However, the viral genome containing the serine-to-alanine substitution at position 301 of the E2 polypeptide replicated to a copy number 20-fold higher than that of wild-type DNA. Images PMID:1658358

  1. [Development of highly stereoselective reactions utilizing heteroatoms--asymmetric synthesis of alpha-substituted serines].

    PubMed

    Sano, S

    2000-01-01

    This article reviews the efficient methods for diastereoselective and enantioselective syntheses of alpha-substituted serines. A newly designed bislactim ether, ethyl (5S)- or (5R)-3,6-diethoxy-2,5-dihydro-5-isopropyl-2-pyrazinecarboxylate, was treated with base or Lewis acid-tertiary amine to generate an enolate or enaminate (imine anion, metalloenamine). Alkylation or aldol-type reaction with the resultant enolate or enaminate proceeded in a highly diastereoselective manner to give the corresponding alkylated or aldol products, respectively. Reduction of these products with diisobutylaluminum hydride (DIBAL) followed by hydrolysis with hydrochloric acid afforded the desirable alpha-substituted serines. The enantioselective aldol-type reaction of an achiral bislactim ether, ethyl 3,6-diethoxy-2,5-dihydro-2-pyrazinecarboxylate, was also investigated by employing Sn(OSO2CF3)2-triethylamine in the presence of an external chiral ligand, (--)-sparteine. Not only a stoichiometric amount, but also a catalytic amount of (--)-sparteine promoted the highly enantioselective aldol-type reactions. Interestingly, the stereoselective outcome of the Sn(II)-mediated reaction differed from that of the Mg(II)-mediated one in the aldol-type reaction of the bislactim ethers with aliphatic aldehydes. On the other hand, chemoenzymatic synthesis of enantiomerically pure alpha-substituted serines must also be a convenient and useful procedure. Porcine liver esterase (PLE) or rabbit liver esterase (RLE) catalyzed hydrolysis of the pro-S ester group of diethyl alpha-alkyl-alpha-(benzyloxycarbonylamino)malonates to afford (R)-ethyl alpha-alkyl-alpha-(benzyloxycarbonylamino)malonates each in excellent enantiomeric excess. Enantiodivergent reductions of these acid esters readily proceeded to furnish both the corresponding enantiomeric alpha-substituted serines. Finally, the application of these methodologies mentioned above to a total synthesis of ISP-I (a potent immunosuppressive principle in

  2. 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

  3. 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.

  4. Functional suppression of HAMP domain signaling defects in the E. coli serine chemoreceptor.

    PubMed

    Lai, Run-Zhi; Parkinson, John S

    2014-10-23

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia 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, and 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 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a 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 and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and 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.

  5. 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

  6. Structural insights into the polypharmacological activity of quercetin on serine/threonine kinases

    PubMed Central

    Baby, Bincy; Antony, Priya; Al Halabi, Walaa; Al Homedi, Zahrah; Vijayan, Ranjit

    2016-01-01

    Polypharmacology, the discovery or design of drug molecules that can simultaneously interact with multiple targets, is gaining interest in contemporary drug discovery. Serine/threonine kinases are attractive targets for therapeutic intervention in oncology due to their role in cellular phosphorylation and altered expression in cancer. Quercetin, a naturally occurring flavonoid, inhibits multiple cancer cell lines and is used as an anticancer drug in Phase I clinical trial. Quercetin glycosides have also received some attention due to their high bioavailability and activity against various diseases including cancer. However, these have been studied to a lesser extent. In this study, the structural basis of the multitarget inhibitory activity of quercetin and isoquercitrin, a glycoside derivative, on serine/threonine kinases using molecular modeling was explored. Structural analysis showed that both quercetin and isoquercitrin exhibited good binding energies and interacted with aspartate in the highly conserved Asp–Phe–Gly motif. The results indicate that isoquercitrin could be a more potent inhibitor of several members of the serine/threonine kinase family. In summary, the current structural evaluation highlights the multitarget inhibitory property of quercetin and its potential to be a chemical platform for oncological polypharmacology. PMID:27729770

  7. 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

    2015-01-07

    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.

  8. Serine racemase deletion disrupts memory for order and alters cortical dendritic morphology

    PubMed Central

    DeVito, Loren M.; Balu, Darrick T.; Kanter, Benjamin R.; Lykken, Christine; Basu, Alo C.; Coyle, Joseph T.; Eichenbaum, Howard

    2012-01-01

    There is substantial evidence implicating N-methyl-d-aspartate receptors (NMDARs) in memory and cognition. It has also been suggested that NMDAR hypofunction might underlie the cognitive deficits observed in schizophrenia since morphological changes, including alterations in the dendritic architecture of pyramidal neurons in the prefrontal cortex (PFC), have been reported in the schizophrenic brain post mortem. Here, we used a genetic model of NMDAR hypofunction, a serine racemase knockout (SR−/−) mouse in which the first coding exon of the mouse serine racemase gene has been deleted, to explore the role of d-serine in regulating cognitive functions as well as dendritic architecture. SR −/− mice exhibited a significantly disrupted representation of the order of events in distinct experiences as revealed by object recognition and odor sequence tests; however, SR −/− animals were unimpaired in the detection of novel objects and in spatial displacement, and showed intact relational memory in a test of transitive inference. In addition, SR −/− mice exhibited normal sociability and preference for social novelty. Neurons in the medial PFC of SR−/− mice displayed reductions in the complexity, total length, and spine density of apical dendrites. These findings demonstrate that d-serine is important for specific aspects of cognition, as well as in regulating dendritic morphology of pyramidal neurons in the mPFC. Moreover, they suggest that NMDAR hypofunction might, in part, be responsible for the cognitive deficits and synaptic changes associated with schizophrenia, and highlight this signaling pathway as a potential target for therapeutic intervention. PMID:21029376

  9. Regulation of the actin-activated MgATPase activity of Acanthamoeba myosin II by phosphorylation of serine 639 in motor domain loop 2.

    PubMed

    Liu, Xiong; Lee, Duck-Yeon; Cai, Shutao; Yu, Shuhua; Shu, Shi; Levine, Rodney L; Korn, Edward D

    2013-01-02

    It had been proposed previously that only filamentous forms of Acanthamoeba myosin II have actin-activated MgATPase activity and that this activity is inhibited by phosphorylation of up to four serine residues in a repeating sequence in the C-terminal nonhelical tailpiece of the two heavy chains. We have reinvestigated these issues using recombinant WT and mutant myosins. Contrary to the earlier proposal, we show that two nonfilamentous forms of Acanthamoeba myosin II, heavy meromyosin and myosin subfragment 1, have actin-activated MgATPase that is down-regulated by phosphorylation. By mass spectroscopy, we identified five serines in the heavy chains that can be phosphorylated by a partially purified kinase preparation in vitro and also are phosphorylated in endogenous myosin isolated from the amoebae: four serines in the nonhelical tailpiece and Ser639 in loop 2 of the motor domain. S639A mutants of both subfragment 1 and full-length myosin had actin-activated MgATPase that was not inhibited by phosphorylation of the serines in the nonhelical tailpiece or their mutation to glutamic acid or aspartic acid. Conversely, S639D mutants of both subfragment 1 and full-length myosin were inactive, irrespective of the phosphorylation state of the serines in the nonhelical tailpiece. To our knowledge, this is the first example of regulation of the actin-activated MgATPase activity of any myosin by modification of surface loop 2.

  10. Meiofaunal Richness in Highly Acidic Hot Springs in Unzen-Amakusa National Park, Japan, Including the First Rediscovery Attempt for Mesotardigrada.

    PubMed

    Suzuki, Atsushi C; Kagoshima, Hiroshi; Chilton, Glen; Grothman, Gary T; Johansson, Carl; Tsujimoto, Megumu

    2017-02-01

    Extreme environments sometimes support surprisingly high meiofaunal diversity. We sampled runoff from the acidic hot springs of Unzen, Japan. This is the type locality of Thermozodium esakii Rahm, 1937, the only tardigrade in the class Mesotardigrada, which remains contentious in the absence of corroboration or supporting specimens. Our sampling revealed at least three species of arthropods, four rotifers, and five nematodes living in the hot (ca. 40°C) and acidic (ca. pH 2.5) water, but no tardigrades.

  11. Azurocidin and a homologous serine protease from neutrophils. Differential antimicrobial and proteolytic properties.

    PubMed Central

    Campanelli, D; Detmers, P A; Nathan, C F; Gabay, J E

    1990-01-01

    Two 29-kD polypeptides, azurocidin and p29b, were purified to homogeneity from human neutrophils by acid extraction of azurophil granule membrane-associated material followed by gel filtration and reverse-phase chromatography. Azurocidin and p29b share NH2-terminal sequence homology with each other as well as with elastase, cathepsin G, and other serine proteases. p29b bound [3H]diisopropyl fluorophosphate and hydrolyzed elastin, casein, and hemoglobin. A peptide substrate for p29b could not be identified. Azurocidin neither bound [3H]diisopropyl fluorophosphate nor hydrolyzed any of the proteins, peptides, or esters tested. In microbicidal assays, purified azurocidin was comparable to p29b in activity against Escherichia coli, Streptococcus faecalis, and Candida albicans. The antimicrobial activity of azurocidin was enhanced under mildly acidic conditions, but was inhibited in a dose-dependent manner by NaCl, CaCl2, or serum. Immunoblot analysis with monospecific antibodies localized greater than 90% of the azurocidin and greater than 75% of the p29b to azurophil granule-rich fractions of PMN lysates. Immunoelectron microscopy confirmed the localization of azurocidin to the azurophil granules. Azurocidin associated with the azurophil granule membrane, but did not appear to be an integral membrane protein. Thus, azurocidin and p29b are members of a family of serine protease homologs stored in azurophil granules and may play a role in inflammatory and antimicrobial processes involving PMN. Images PMID:2312733

  12. Characterization of a novel serine protease inhibitor gene from a marine metagenome.

    PubMed

    Jiang, Cheng-Jian; Hao, Zhen-Yu; Zeng, Rong; Shen, Pei-Hong; Li, Jun-Fang; Wu, Bo

    2011-01-01

    A novel serine protease inhibitor (serpin) gene designated as Spi1C was cloned via the sequenced-based screening of a metagenomic library from uncultured marine microorganisms. The gene had an open reading frame of 642 base pairs, and encoded a 214-amino acid polypeptide with a predicted molecular mass of about 28.7 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Spi1C and some partial proteinase inhibitor I4 serpins were closely related. Functional characterization demonstrated that the recombinant Spi1C protein could inhibit a series of serine proteases. The Spi1C protein exhibited inhibitory activity against α-chymotrypsin and trypsin with K(i) values of around 1.79 × 10(-8) and 1.52 × 10(-8) M, respectively. No inhibition activity was exhibited against elastase. Using H-d-Phe-Pip-Arg-pNA as the chromogenic substrate, the optimum pH and temperature of the inhibition activity against trypsin were 7.0-8.0 and 25 °C, respectively. The identification of a novel serpin gene underscores the potential of marine metagenome screening for novel biomolecules.

  13. Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila.

    PubMed Central

    Martín, J; Slade, A; Aitken, A; Arche, R; Virden, R

    1991-01-01

    The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2'-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis. PMID:1764029

  14. Convergent Synthesis of a Deuterium Labeled Serine Dipeptide Lipid for Analysis of Biological Samples.

    PubMed

    Dietz, Christopher; Clark, Robert B; Nichols, Frank C; Smith, Michael B

    2017-03-08

    Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-L-serine, (3S)-L-serine] isolated from Porphyromonas gingivalis,(1) in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria.

  15. 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.

  16. 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

  17. Serine acts as a metabolic signal for the transcriptional control of photorespiration-related genes in Arabidopsis.

    PubMed

    Timm, Stefan; Florian, Alexandra; Wittmiß, Maria; Jahnke, Kathrin; Hagemann, Martin; Fernie, Alisdair R; Bauwe, Hermann

    2013-05-01

    Photosynthetic carbon assimilation including photorespiration is dynamically regulated during the day/night cycle. This includes transcriptional regulation, such as the light induction of corresponding genes, but little is known about the contribution of photorespiratory metabolites to the regulation of gene expression. Here, we examined diurnal changes in the levels of photorespiratory metabolites, of enzymes of the photorespiratory carbon cycle, and of corresponding transcripts in wild-type plants of Arabidopsis (Arabidopsis thaliana) and in a mutant with altered photorespiratory flux due to the absence of the peroxisomal enzyme Hydroxypyruvate Reductase1 (HPR1). Metabolomics of the wild type showed that the relative amounts of most metabolites involved in photorespiration increased after the onset of light, exhibited maxima at the end of the day, and decreased during the night. In accordance with those findings, both the amounts of messenger RNAs encoding photorespiratory enzymes and the respective protein contents showed a comparable accumulation pattern. Deletion of HPR1 did not significantly alter most of the metabolite patterns relative to wild-type plants; only serine accumulated to a constitutively elevated amount in this mutant. In contrast, the hpr1 mutation resulted in considerable deregulation of the transcription of photorespiration-related genes. This transcriptional deregulation could also be induced by the external application of l-serine but not glycine to the Arabidopsis wild type, suggesting that serine acts as a metabolic signal for the transcriptional regulation of photorespiration, particularly in the glycine-to-serine interconversion reactions.

  18. Serine Acts as a Metabolic Signal for the Transcriptional Control of Photorespiration-Related Genes in Arabidopsis1[W

    PubMed Central

    Timm, Stefan; Florian, Alexandra; Wittmiß, Maria; Jahnke, Kathrin; Hagemann, Martin; Fernie, Alisdair R.; Bauwe, Hermann

    2013-01-01

    Photosynthetic carbon assimilation including photorespiration is dynamically regulated during the day/night cycle. This includes transcriptional regulation, such as the light induction of corresponding genes, but little is known about the contribution of photorespiratory metabolites to the regulation of gene expression. Here, we examined diurnal changes in the levels of photorespiratory metabolites, of enzymes of the photorespiratory carbon cycle, and of corresponding transcripts in wild-type plants of Arabidopsis (Arabidopsis thaliana) and in a mutant with altered photorespiratory flux due to the absence of the peroxisomal enzyme Hydroxypyruvate Reductase1 (HPR1). Metabolomics of the wild type showed that the relative amounts of most metabolites involved in photorespiration increased after the onset of light, exhibited maxima at the end of the day, and decreased during the night. In accordance with those findings, both the amounts of messenger RNAs encoding photorespiratory enzymes and the respective protein contents showed a comparable accumulation pattern. Deletion of HPR1 did not significantly alter most of the metabolite patterns relative to wild-type plants; only serine accumulated to a constitutively elevated amount in this mutant. In contrast, the hpr1 mutation resulted in considerable deregulation of the transcription of photorespiration-related genes. This transcriptional deregulation could also be induced by the external application of l-serine but not glycine to the Arabidopsis wild type, suggesting that serine acts as a metabolic signal for the transcriptional regulation of photorespiration, particularly in the glycine-to-serine interconversion reactions. PMID:23471132

  19. An Investigation of the Complexity of Maillard Reaction Product Profiles from the Thermal Reaction of Amino Acids with Sucrose Using High Resolution Mass Spectrometry

    PubMed Central

    Golon, Agnieszka; Kropf, Christian; Vockenroth, Inga; Kuhnert, Nikolai

    2014-01-01

    Thermal treatment of food changes its chemical composition drastically with the formation of “so-called” Maillard reaction products, being responsible for the sensory properties of food, along with detrimental and beneficial health effects. In this contribution, we will describe the reactivity of several amino acids, including arginine, lysine, aspartic acid, tyrosine, serine and cysteine, with carbohydrates. The analytical strategy employed involves high and ultra-high resolution mass spectrometry followed by chemometric-type data analysis. The different reactivity of amino acids towards carbohydrates has been observed with cysteine and serine, resulting in complex MS spectra with thousands of detectable reaction products. Several compounds have been tentatively identified, including caramelization reaction products, adducts of amino acids with carbohydrates, their dehydration and hydration products, disproportionation products and aromatic compounds based on molecular formula considerations. PMID:28234331

  20. Spinal D-Serine Increases PKC-Dependent GluN1 Phosphorylation Contributing to the Sigma-1 Receptor-Induced Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain.

    PubMed

    Choi, Sheu-Ran; Moon, Ji-Young; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kwon, Soon-Gu; Choi, Hoon-Seong; Kang, Suk-Yun; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

    2017-04-01

    We have recently shown that spinal sigma-1 receptor (Sig-1R) activation facilitates nociception via an increase in phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). The present study was designed to examine whether the Sig-1R-induced facilitative effect on NMDA-induced nociception is mediated by D-serine, and whether D-serine modulates spinal pGluN1 expression and the development of neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the D-serine degrading enzyme, D-amino acid oxidase attenuated the facilitation of NMDA-induced nociception induced by the Sig-1R agonist, 2-(4-morpholinethyl)1-phenylcyclohexane carboxylate. Exogenous D-serine increased protein kinase C (PKC)-dependent (Ser896) pGluN1 expression and facilitated NMDA-induced nociception, which was attenuated by preteatment with the PKC inhibitor, chelerythrine. In CCI mice, administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt or D-amino acid oxidase on postoperative days 0 to 3 suppressed CCI-induced mechanical allodynia (MA) and pGluN1 expression on day 3 after CCI surgery. Intrathecal administration of D-serine restored MA as well as the GluN1 phosphorylation on day 3 after surgery that was suppressed by the Sig-1R antagonist, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide or the astrocyte inhibitor, fluorocitrate. In contrast, D-serine had no effect on CCI-induced thermal hyperalgesia or GluN1 expression. These results indicate that spinal D-serine: 1) mediates the facilitative effect of Sig-1R on NMDA-induced nociception, 2) modulates PKC-dependent pGluN1 expression, and 3) ultimately contributes to the induction of MA after peripheral nerve injury.

  1. 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...

  2. Temporal trends of perfluoroalkane sulfonic acids and their sulfonamide-based precursors in herring from the Swedish west coast 1991-2011 including isomer-specific considerations.

    PubMed

    Ullah, Shahid; Huber, Sandra; Bignert, Anders; Berger, Urs

    2014-04-01

    A method was developed for simultaneous analysis of perfluoroalkane sulfonic acids (PFSAs) and their sulfonamide-based precursors (perfluoroalkane sulfonamidoacetic acids (FASAAs), sulfonamides (FASAs), and sulfonamidoethanols (FASEs)) in fish muscle. Extraction was performed with acetonitrile followed by a clean-up and fractionation step and instrumental analysis by UPLC/MS/MS and GC/MS. Time trends of PFSAs and their precursors in herring muscle samples originating from the Kattegat at the west coast of Sweden were investigated covering the years 1991-2011. The following analytes were detected, all with decreasing or unchanged trends between 1991 and 2011: Perfluorobutane sulfonic acid (PFBS, below the method detection limit (acid (PFHxS, 9-38pg/g), perfluorooctane sulfonic acid (PFOS, 240-930pg/g), perfluorodecane sulfonic acid (PFDS, acid (MeFOSAA and EtFOSAA, 2-39 and 2-31pg/g, respectively) and perfluorooctane sulfonamide (FOSA, 78-920pg/g). The highest concentrations were found for PFOS and FOSA around the turn of the century. Shorter disappearance half-lives were observed for precursors compared to PFSAs. Assuming that these trend differences are representative for fish consumed by the general Swedish population, this observation suggests that the relative contribution of precursors to total human exposure to PFOS via fish intake has decreased in Sweden over the study period. PFOS precursors in fish may have constituted a significant indirect exposure pathway for PFOS in the 1990s. Isomer-specific analysis of PFOS and FOSA revealed <10% relative contributions of branched isomers to total PFOS and total FOSA. Furthermore, the percentage branched isomers decreased over time for both compounds. These findings are contrary to patterns and temporal trends of PFOS isomers commonly found in human serum. In combination with literature data on isomer patterns

  3. 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

    2016-07-05

    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.

  4. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

    PubMed

    Takayama, S; White, M F; Kahn, C R

    1988-03-05

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

  5. Cardioprotection by a novel recombinant serine protease inhibitor in myocardial ischemia and reperfusion injury.

    PubMed

    Murohara, T; Guo, J P; Lefer, A M

    1995-09-01

    Polymorphonuclear neutrophils (PMN) play an important role in myocardial ischemia/reperfusion (MI/R) injury; however, the role of neutrophilic proteases is less understood. The effects of a novel serine protease inhibitor (serpin), LEX032, were investigated in a murine model of MI (20 min) and R (24 hr) injury in vivo. LEX032 is a recombinant human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of alpha-1 protease inhibitor. LEX032 has the ability to inhibit both neutrophil elastase and cathepsin G, two major neutral serine proteases in neutrophils, as well as superoxide generation. LEX032 (25 or 50 mg/kg) administered i.v. 1 min before reperfusion significantly attenuated myocardial necrotic injury evaluated by cardiac creatine kinase loss compared to MI/R rats receiving only vehicle (P < .001). Moreover, cardiac myeloperoxidase activity, an index of PMN accumulation, in the ischemic myocardium was significantly attenuated by LEX032 as compared with rats receiving vehicle (P < .001). LEX032 also moderately attenuated leukotriene B4-stimulated PMN adherence to rat superior mesenteric artery endothelium and markedly diminished superoxide radical release from LTB4-stimulated PMN in vitro. In a glycogen-induced rat peritonitis model, LEX032 (50 mg/kg) significantly attenuated PMN transmigration into the peritoneal cavity in vivo. In conclusion, the recombinant serine protease inhibitor, LEX032, appears to be an effective agent for attenuating MI/R injury by inhibiting neutrophil-accumulation into the ischemic-reperfused myocardium and by inactivating cytotoxic metabolites (proteases and superoxide radical) released from neutrophils.

  6. Regulation of divalent metal transporter-1 by serine phosphorylation

    PubMed Central

    Seo, Young Ah; Kumara, Ruvin; Wetli, Herbert; Wessling-Resnick, Marianne

    2016-01-01

    Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake. PMID:27681840

  7. 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

  8. Vaccinia virus F16 protein, a predicted catalytically inactive member of the prokaryotic serine recombinase superfamily, is targeted to nucleoli

    PubMed Central

    Senkevich, Tatiana G.; Koonin, Eugene V.; Moss, Bernard

    2011-01-01

    The F16L gene of vaccinia virus (VACV) is conserved in all chordopoxviruses except avipoxviruses. The crocodile poxvirus F16 protein ortholog has highly significant similarity to prokaryotic serine recombinases and contains all amino acids that comprise the catalytic site. In contrast, F16 orthologs encoded by other poxviruses show only marginally significant similarity to serine recombinases, lack essential amino acids of the active site and are most likely inactive derivatives of serine recombinases. Nevertheless, the conservation of F16L in non-avian poxviruses suggested an important function. However, a VACV mutant with the F16L gene knocked out replicated normally in dividing and quiescent cells. The F16 protein was synthesized early after infection and detected in virus cores. When expressed in infected or uninfected cells, F16 accumulated in nucleoli depending on the level of expression and confluency of cells. Evidence was obtained that F16 forms multimers, which might regulate concentration-dependent intracellular localization. PMID:21752417

  9. Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

    PubMed

    Major, G N; Gardner, E J; Carne, A F; Lawley, P D

    1990-03-25

    DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).

  10. 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.

  11. A comparison of an optimised sequential extraction procedure and dilute acid leaching of elements in anoxic sediments, including the effects of oxidation on sediment metal partitioning.

    PubMed

    Larner, Bronwyn L; Palmer, Anne S; Seen, Andrew J; Townsend, Ashley T

    2008-02-11

    The effect of oxidation of anoxic sediment upon the extraction of 13 elements (Cd, Sn, Sb, Pb, Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As) using the optimised Community Bureau of Reference of the European Commission (BCR) sequential extraction procedure and a dilute acid partial extraction procedure (4h, 1 molL(-1) HCl) was investigated. Elements commonly associated with the sulfidic phase, Cd, Cu, Pb, Zn and Fe exhibited the most significant changes under the BCR sequential extraction procedure. Cd, Cu, Zn, and to a lesser extent Pb, were redistributed into the weak acid extractable fraction upon oxidation of the anoxic sediment and Fe was redistributed into the reducible fraction as expected, but an increase was also observed in the residual Fe. For the HCl partial extraction, sediments with moderate acid volatile sulfide (AVS) levels (1-100 micromolg(-1)) showed no significant difference in element partitioning following oxidation, whilst sediments containing high AVS levels (>100 micromolg(-1)) were significantly different with elevated concentrations of Cu and Sn noted in the partial extract following oxidation of the sediment. Comparison of the labile metals released using the BCR sequential extraction procedure (SigmaSteps 1-3) to labile metals extracted using the dilute HCl partial extraction showed that no method was consistently more aggressive than the other, with the HCl partial extraction extracting more Sn and Sb from the anoxic sediment than the BCR procedure, whilst the BCR procedure extracted more Cr, Co, Cu and As than the HCl extraction.

  12. The Clinical Spectrum of Missense Mutations of the First Aspartic Acid of cbEGF-like Domains in Fibrillin-1 Including a Recessive Family

    PubMed Central

    Hilhorst-Hofstee, Yvonne; Rijlaarsdam, Marry EB; Scholte, Arthur JHA; Swart-van den Berg, Marietta; Versteegh, Michel IM; van der Schoot-van Velzen, Iris; Schäbitz, Hans-Joachim; Bijlsma, Emilia K; Baars, Marieke J; Kerstjens-Frederikse, Wilhelmina S; Giltay, Jacques C; Hamel, Ben C; Breuning, Martijn H; Pals, Gerard

    2010-01-01

    Marfan syndrome (MFS) is a dominant disorder with a recognizable phenotype. In most patients with the classical phenotype mutations are found in the fibrillin-1 gene (FBN1) on chromosome 15q21. It is thought that most mutations act in a dominant negative way or through haploinsufficiency. In 9 index cases referred for MFS we detected heterozygous missense mutations in FBN1 predicted to substitute the first aspartic acid of different calcium-binding Epidermal Growth Factor-like (cbEGF) fibrillin-1 domains. A similar mutation was found in homozygous state in 3 cases in a large consanguineous family. Heterozygous carriers of this mutation had no major skeletal, cardiovascular or ophthalmological features of MFS. In the literature 14 other heterozygous missense mutations are described leading to the substitution of the first aspartic acid of a cbEGF domain and resulting in a Marfan phenotype. Our data show that the phenotypic effect of aspartic acid substitutions in the first position of a cbEGF domain can range from asymptomatic to a severe neonatal phenotype. The recessive nature with reduced expression of FBN1 in one of the families suggests a threshold model combined with a mild functional defect of this specific mutation. © 2010 Wiley-Liss, Inc. PMID:20886638

  13. Development of activity-based probes for trypsin-family serine proteases.

    PubMed

    Pan, Zhengying; Jeffery, Douglas A; Chehade, Kareem; Beltman, Jerlyn; Clark, James M; Grothaus, Paul; Bogyo, Matthew; Baruch, Amos

    2006-06-01

    A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

  14. Conserved serines in simian immunodeficiency virus capsid are required for virus budding.

    PubMed

    Rue, Sarah M; Roos, Jason W; Clements, Janice E; Barber, Sheila A

    2005-05-25

    The simian immunodeficiency virus (SIV) capsid protein (CA), a constituent of the Pr55Gag polyprotein, is phosphorylated in virions but not in virus-producing cells (Rue, S.M., Roos, J.W., Tarwater, P.M., Clements, J.E., Barber, S.A., 2005. Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus. J. Virol. 79 (4), 2484-2492.). Using phosphoamino acid analysis of CA, we show that serine is the primary phosphate acceptor. A series of substitution mutants of serines in the CA domain of Pr55Gag were constructed in the infectious viral clone SIVmac239. These virus mutants were examined for defects in virus replication and virion infectivity, release, and morphology, as well as alterations in phosphorylation of CA-containing proteins. Although the virus mutants exhibited a number of replication defects, none of these defects could be directly attributed to aberrant CA phosphorylation. A novel defect was a block in early budding, which was common among several virus mutants with substitutions in the CA N terminus. Together, these results indicate that certain residues in the CA N terminus are crucial for early budding events.

  15. Cloning and molecular characterization of a human intracellular serine proteinase inhibitor.

    PubMed Central

    Coughlin, P; Sun, J; Cerruti, L; Salem, H H; Bird, P

    1993-01-01

    We describe a cDNA encoding a serine proteinase inhibitor present in placental tissue and the cytosolic fraction of K562 cells. On the basis of its interaction with thrombin, through which it was discovered, the inhibitor has been operationally named the placental thrombin inhibitor (PTI). Amino acid sequence comparisons suggest that its reactive center is located at Arg-341 and Cys-342, that it lacks a classical N-terminal signal sequence, and that it has the highest degree of similarity to intracellular serine proteinase inhibitors (serpins), such as the human monocyte/neutrophil elastase inhibitor and the equine leukocyte elastase inhibitor. PTI also resembles these inhibitors in that it contains oxidation-sensitive residues adjacent to the reactive site. The PTI cDNA was expressed in rabbit reticulocyte lysate and in COS-7 cells and a 42-kDa protein was produced. Recombinant PTI formed a 67-kDa complex when incubated with thrombin. The ability of native PTI to bind thrombin was destroyed by incubation with iodoacetamide. Analysis of human tissue mRNA indicated that PTI is expressed widely with the highest levels in cardiac and skeletal muscle and placenta. We conclude that PTI is a member of an emerging class of intracellular serpins. Images Fig. 2 Fig. 3 Fig. 4 PMID:8415716

  16. 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.

  17. SufA--a novel subtilisin-like serine proteinase of Finegoldia magna.

    PubMed

    Karlsson, Christofer; Andersson, Marie-Louise; Collin, Mattias; Schmidtchen, Artur; Björck, Lars; Frick, Inga-Maria

    2007-12-01

    Finegoldia magna is an anaerobic Gram-positive bacterium and commensal, which is also associated with clinically important conditions such as skin and soft tissue infections. This study describes a novel subtilisin-like extracellular serine proteinase of F. magna, denoted SufA (subtilase of Finegoldia magna), which is believed to be the first subtilase described among Gram-positive anaerobic cocci. SufA is associated with the bacterial cell surface, but is also released in substantial amounts during bacterial growth. Papain was used to release SufA from the surface of F. magna and the enzyme was purified by ion-exchange chromatography and gel filtration. A protein band on SDS-PAGE corresponding to the dominating proteolytic activity on gelatin zymography was analysed by MS/MS. Based on the peptide sequences obtained, the sufA gene was sequenced. The gene comprises 3466 bp corresponding to a preprotein of 127 kDa. Like other members of the subtilase family, SufA contains the catalytic triad of aspartic acid, histidine and serine with surrounding conserved residues. A SufA homologue was identified in 33 of 34 investigated isolates of F. magna, as revealed by PCR and immunoprinting. The enzyme forms dimers, which are more proteolytically active than the monomeric protein. SufA was found to efficiently cleave and inactivate the antibacterial peptide LL-37 and the CXC chemokine MIG/CXCL9, indicating that the enzyme promotes F. magna survival and colonization.

  18. Leishmania (Leishmania) amazonensis: purification and enzymatic characterization of a soluble serine oligopeptidase from promastigotes.

    PubMed

    de Andrade, A S; Santoro, M M; de Melo, M N; Mares-Guia, M

    1998-06-01

    A soluble proteinase was purified 90-fold from extracts of promastigotes of Leishmania (Leishmania) amazonensis using a combination of ion-exchange chromatography in Q-Sepharose Fast Flow, gel filtration chromatography in Sephacryl HR S-200, and chromatofocusing. The enzyme appeared as a single band with an apparent molecular weight of 101 kDa by silver staining following SDS-PAGE, under both reducing and nonreducing conditions. The proteinase has a pH optimum between 8.0 and 8.5 and an isoelectric point between 5.12 and 5.23, belongs to the serine proteinase class, and is inhibited by Mg2+, Ca2+, and K+. The primary specificity determined using synthetic substrates is for basic amino acids. The kinetic parameters for the Bz-L-Arg-Nam substrate are Km = 26 microM, kcat = 32 min(-1), and Ksi = 1270 microM (the proteinase showed inhibition by excess substrate). The enzyme does not hydrolyze casein, albumin, and gelatin or large peptides like the oxidized insulin B chain, but hydrolyzes small peptides like bradykinin and fragment 4-10 of the adrenocorticotropic hormone, at the carboxyl side of basic residues and aromatic residues preceding basic residues. The enzyme appears, thus, to be restricted in its action, cleaving only small peptide substrates, which characterizes the proteinase as an oligopeptidase. This is the first report of purification of a serine peptidase from Leishmania species and it increases the short list of known oligopeptidases.

  19. The Role of the Microbial Metabolites Including Tryptophan Catabolites and Short Chain Fatty Acids in the Pathophysiology of Immune-Inflammatory and Neuroimmune Disease.

    PubMed

    Morris, Gerwyn; Berk, Michael; Carvalho, Andre; Caso, Javier R; Sanz, Yolanda; Walder, Ken; Maes, Michael

    2016-06-27

    There is a growing awareness that gut commensal metabolites play a major role in host physiology and indeed the pathophysiology of several illnesses. The composition of the microbiota largely determines the levels of tryptophan in the systemic circulation and hence, indirectly, the levels of serotonin in the brain. Some microbiota synthesize neurotransmitters directly, e.g., gamma-amino butyric acid, while modulating the synthesis of neurotransmitters, such as dopamine and norepinephrine, and brain-derived neurotropic factor (BDNF). The composition of the microbiota determines the levels and nature of tryptophan catabolites (TRYCATs) which in turn has profound effects on aryl hydrocarbon receptors, thereby influencing epithelial barrier integrity and the presence of an inflammatory or tolerogenic environment in the intestine and beyond. The composition of the microbiota also determines the levels and ratios of short chain fatty acids (SCFAs) such as butyrate and propionate. Butyrate is a key energy source for colonocytes. Dysbiosis leading to reduced levels of SCFAs, notably butyrate, therefore may have adverse effects on epithelial barrier integrity, energy homeostasis, and the T helper 17/regulatory/T cell balance. Moreover, dysbiosis leading to reduced butyrate levels may increase bacterial translocation into the systemic circulation. As examples, we describe the role of microbial metabolites in the pathophysiology of diabetes type 2 and autism.

  20. Serine proteinases of mast cell and leukocyte granules. A league of their own.

    PubMed

    Caughey, G H

    1994-12-01

    Serine proteinases are hydrolases that use serine's side chain hydroxyl group to attack and cleave internal peptide bonds in peptides and proteins. They reside in all mammalian tissues, including the lung and airway. As a group, they vary tremendously in form and target specificity and have a vast repertoire of functions, many of which are critical for life. A subset of these proteinases is expressed primarily in the cytosolic granules of leukocytes from bone marrow, including mast cells. Examples are elastase-related proteinases and cathepsin G of monocytes and neutrophils, the many "granzymes" of cytotoxic T lymphocytes and natural killer (NK) cells, and the tryptases and chymases of mast cells. The pace of discovery and characterization of these granule-associated serine proteinases, fueled by technical advances in molecular biology, has accelerated rapidly in the past few years. Progress has been made in assigning possible functions to individual proteinases. However, the burgeoning numbers of these enzymes; their cell, tissue and species-dependent differences in expression; and their variety of action in vitro (despite, in many cases, shared modes of activation and recent divergence in protein evolution) have vexed and challenged those of us who are anxious to establish their roles in mammalian biology. Certainly, much remains to be discovered and clarified. The purpose of this overview is to capture the state of the art in this field, stressing the similarities as well as the differences among individual granule-associated proteinases and focusing particularly on those enzymes likely to be important in the human lung and airways.

  1. Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase

    PubMed Central

    Lassy, Rachel A. L.; Miller, Charles G.

    2000-01-01

    Salmonella enterica serovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) in Escherichia coli and have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in the Archaea or in any of the other eukaryotes for which genome sequences are available. PMID:10762256

  2. Membrane-anchored serine proteases in health and disease

    PubMed Central

    Bugge, Thomas; Wu, Qingyu

    2013-01-01

    Serine proteases of the trypsin-like family have long been recognized to be critical effectors of biological processes as diverse as digestion, blood coagulation, fibrinolysis, and immunity. In recent years, a subgroup of these enzymes has been identified that are anchored directly to plasma membranes, either by a carboxy-terminal transmembrane domain (Type I), an amino-terminal transmembrane domain with a cytoplasmic extension (Type II or TTSP), or through a glycosyl-phosphatidylinositol (GPI) linkage. Recent biochemical, cellular, and in vivo analyses have now established that membrane-anchored serine proteases are key pericellular contributors to processes vital for development and the maintenance of homeostasis. This chapter will review our current knowledge of the biological and physiological functions of these proteases, their molecular substrates, and their contributions to disease. PMID:21238933

  3. Site-specific DNA Inversion by Serine Recombinases

    PubMed Central

    2015-01-01

    Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized. PMID:25844275

  4. 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.

  5. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    PubMed Central

    Zhang, Huan; Fei, Rui; Xue, Baigong; Yu, Shanshan; Zhang, Zuoming; Zhong, Sheng; Gao, Yuanqi; Zhou, Xiaoli

    2017-01-01

    Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles. PMID:28067849

  6. 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.

  7. cis-acting DNA regulatory elements, including the retinoic acid response element, are required for tissue specific laminin B1 promoter/lacZ expression in transgenic mice.

    PubMed

    Sharif, K A; Li, C; Gudas, L J

    2001-05-01

    The LAMB1 gene encodes the laminin beta1 subunit of laminin, an extracellular matrix protein. Using several transgenic mouse lines containing various lengths of the LAMB1 promoter driving lacZ reporter gene expression, regions of LAMB1 promoter that contain cis-acting DNA regulatory element(s) have been identified. The 3.9LAMB1betagal transgene is expressed in various tissues during development. LAMB1 transgene expression is observed in a selective set of nephrons of the neonatal and adult kidneys. The cis-acting DNA regulatory elements responsible for LAMB1 transgene expression in ovaries and in juvenile kidneys are present between -'1.4 and -0.7 kb relative to the transcription start site, while those of adult kidneys are located between -2.5 and -1.4 kb. The LAMB1 transgene is also expressed in the epididymis of 1 week old transgenic mice. Mutation of the retinoic acid response element (RARE) in the context of the 3.9LAMB1betagal transgene results in loss of LAMB1 transgene expression in all tissues. Thus, sequences between -2.5 and -0.7 kb plus the RARE are required for appropriate expression of the LAMB1 transgene in mice.

  8. Rubidium penta-aqua-(l-serine)cobalt(II) hexa-hydrogenhexa-molybdocobaltate(III) l-serine monosolvate deca-hydrate.

    PubMed

    Iijima, Jun; Naruke, Haruo; Takiyama, Hiroshi

    2013-10-19

    The Co(2+) ion in the title compound, Rb[Co(C3H7NO3)(H2O)5][H6CoMo6O24]·C3H7NO3·10H2O, is coordinated by five water mol-ecules and one O-monodentate l-serine ligand in a slightly distorted octahedral geometry. The Rb(+) ion is irregularly coordinated by nine O atoms. In the crystal, the [H6Co(III)Mo6O24](3-) polyanions are stacked along the b-axis direction, mediated by bridging Rb-O bonds. N-H⋯O and O-H⋯O hydrogen bonds are observed involving the l-serine mol-ecules.

  9. 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.

  10. Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi.

    PubMed

    Vieira Bard, Gabriela C; Nascimento, Viviane V; Ribeiro, Suzanna F F; Rodrigues, Rosana; Perales, Jonas; Teixeira-Ferreira, André; Carvalho, André O; Fernandes, Katia Valevski S; Gomes, Valdirene M

    2015-04-01

    Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.

  11. The use of serine protease from Yarrowia lipolytica yeast in the production of biopeptides from denatured egg white proteins.

    PubMed

    Pokora, Marta; Zambrowicz, Aleksandra; Zabłocka, Agnieszka; Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Eckert, Ewelina; Trziszka, Tadeusz; Chrzanowska, Józefa

    2017-04-07

    Deriving non-conventional enzymes from cheaper sources than those used for commercially available enzymes may result in the production of hydrolysates with beneficial features, while drastically reducing the cost of hydrolysis. This is especially significant for enzymatic hydrolysis as a method of protein waste utilization. We have previously described the ability of non-commercial serine protease from Yarrowia lipolytica yeast to produce/release bioactive peptides from egg white protein by-products (EP). The enzymatic hydrolysis of EP was carried out for 24 h using the serine protease at an enzyme: substrate ratio of 1:30 (w/w). The obtained hydrolysate was characterized by protein degradation of 38% and also exhibited an antioxidant and cytokine-inducing activity. The isolation procedure (ultrafiltration and RP-HPLC) of bioactive peptides from the EP hydrolysate provided peptide fractions with significant antioxidant and ACE inhibitory activities. Three homogeneous and three heterogeneous peptide fractions were identified using MALDI-TOF/MS and the Mascot Search Results database. The peptides, mainly derived from ovalbumin, were composed of 2-19 amino-acid residues. We have thus demonstrated a novel ability of serine protease from Y. lipolytica to release biopeptides from an EP by-product.

  12. 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.

  13. Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis.

    PubMed

    Calik, P; Calik, G; Ozdamar, T H

    2000-08-05

    The effects of oxygen transfer on the production and product distribution in serine alkaline protease (SAP) fermentation by Bacillus licheniformis and oxygen-transfer strategy in relation to the physiology of the bacilli were investigated on a defined medium with citric acid as sole carbon source in 3.5-dm(3) batch bioreactor systems. By forming a 3 x 3 matrix with the parameters air-inlet rates of Q(O)/V(R) = 0.2, 0.5, 1.0 vvm, and agitation rates of N = 150, 500, 750 min(-1), the effects of oxygen transfer were investigated at nine different conditions. The concentrations of the product SAP and by-products, i.e., neutral protease, alpha-amylase, amino acids, and organic acids, and SAP activities were determined throughout the bioprocess. Among the constant air-flow and agitation-rate fermentations, Q(O)/V(R) = 0.5 vvm, N = 750 min(-1) oxygen-transfer conditions produced maximum SAP activity that was 500 U cm(-3), at t = 37 h. With the increase in Q(O)/V(R) and/or N, Damköhler number that is the oxygen-transfer limitation decreases; and the process passes from oxygen-transfer limited conditions to biochemical-reaction limited conditions. Further increase in SAP activity, A = 680 U cm(-3) was achieved by applying an oxygen-transfer strategy based on the analysis of the data obtained with the constant oxygen-transfer condition experiments, with a step increase in air-inlet rate, from Q(O)/V(R) = 0.2 to Q(O)/V(R) = 0.5 vvm at N = 750 min(-1) constant agitation rate at t = 24 h. Organic acids and amino acids that were excreted to the fermentation medium varied depending on the oxygen-transfer conditions. With the increase in oxygen-transfer rate acetic acid concentration increased; contrarily, with the decrease in the oxygen-transfer rate the TCA-cycle organic acids alpha-ketoglutaric and succinic acids, and gluconic acid were excreted to the fermentation broth; nevertheless, the application of the oxygen-transfer strategy prevented the increase in acetic acid

  14. D-Amino Acids in the Nervous and Endocrine Systems

    PubMed Central

    Kiriyama, Yoshimitsu

    2016-01-01

    Amino acids are important components for peptides and proteins and act as signal transmitters. Only L-amino acids have been considered necessary in mammals, including humans. However, diverse D-amino acids, such as D-serine, D-aspartate, D-alanine, and D-cysteine, are found in mammals. Physiological roles of these D-amino acids not only in the nervous system but also in the endocrine system are being gradually revealed. N-Methyl-D-aspartate (NMDA) receptors are associated with learning and memory. D-Serine, D-aspartate, and D-alanine can all bind to NMDA receptors. H2S generated from D-cysteine reduces disulfide bonds in receptors and potentiates their activity. Aberrant receptor activity is related to diseases of the central nervous system (CNS), such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Furthermore, D-amino acids are detected in parts of the endocrine system, such as the pineal gland, hypothalamus, pituitary gland, pancreas, adrenal gland, and testis. D-Aspartate is being investigated for the regulation of hormone release from various endocrine organs. Here we focused on recent findings regarding the synthesis and physiological functions of D-amino acids in the nervous and endocrine systems. PMID:28053803

  15. A Pulmonary Perspective on GASPIDs: Granule-Associated Serine Peptidases of Immune Defense

    PubMed Central

    Caughey, George H.

    2008-01-01

    Airways are protected from pathogens by forces allied with innate and adaptive immunity. Recent investigations establish critical defensive roles for leukocyte and mast cell serine-class peptidases garrisoned in membrane-bound organelles-here termed Granule-Associated Serine Peptidases of Immune Defense, or GASPIDs. Some better characterized GASPIDs include neutrophil elastase and cathepsin G (which defend against bacteria), proteinase-3 (targeted by antineutrophil antibodies in Wegener’s vasculitis), mast cell β-tryptase and chymase (which promote allergic inflammation), granzymes A and B (which launch apoptosis pathways in infected host cells), and factor D (which activates complement’s alternative pathway). GASPIDs can defend against pathogens but can harm host cells in the process, and therefore become targets for pharmaceutical inhibition. They vary widely in specificity, yet are phylogenetically similar. Mammalian speciation supported a remarkable flowering of these enzymes as they co-evolved with specialized immune cells, including mast cells, basophils, eosinophils, cytolytic T-cells, natural killer cells, neutrophils, macrophages and dendritic cells. Many GASPIDs continue to evolve rapidly, providing some of the most conspicuous examples of divergent protein evolution. Consequently, students of GASPIDs are rewarded not only with insights into their roles in lung immune defense but also with clues to the origins of cellular specialization in vertebrate immunity. PMID:18516248

  16. [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.

  17. 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.

  18. Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function.

    PubMed

    Du, Ping; Loulakis, Pat; Luo, Chun; Mistry, Anil; Simons, Samuel P; LeMotte, Peter K; Rajamohan, Francis; Rafidi, Kristina; Coleman, Kevin G; Geoghegan, Kieran F; Xie, Zhi

    2005-12-01

    High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.

  19. Antidepressant-like effect of bright light is potentiated by L-serine administration in a mouse model of seasonal affective disorder.

    PubMed

    Kawai, Misato; Goda, Ryosei; Otsuka, Tsuyoshi; Iwamoto, Ayaka; Uotsu, Nobuo; Furuse, Mitsuhiro; Yasuo, Shinobu

    2015-09-01

    Bright light therapy is used as the primary treatment for seasonal affective disorder; however, the mechanisms underlying its antidepressant effect are not fully understood. Previously, we found that C57BL/6J mice exhibit increased depression-like behavior during a short-day condition (SD) and have lowered brain serotonin (5-HT) content. This study analyzed the effect of bright light on depression-like behaviors and the brain serotonergic system using the C57BL/6J mice. In the mice maintained under SD, bright light treatment (1000 lx, daily 1 h exposure) for 1 week reduced immobility time in the forced swimming test and increased intake of saccharin solution in a saccharin intake test. However, the light treatment did not modify 5-HT content and selective 5-HT uptake in the amygdala, or temporal patterns of core body temperature and wheel-running activity throughout a day. In the next experiment, we attempted to enhance the effect of bright light by using L-serine, a precursor of D-serine that acts as an N-methyl-D-aspartic acid receptor coagonist. Daily subcutaneous injection of L-serine for 2 weeks prior to the bright light strongly reduced the immobility time in the forced swimming test, suggesting a synergistic effect of light and L-serine. Furthermore, bright light increased the total number of 5-HT-immunoreactive cells and cells that had colocalized 5-HT and c-Fos immunosignals in several subregions of the raphe nuclei. These effects were potentiated by prior injection of L-serine. These data suggest that the bright light may elicit an antidepressant-like effect via enhanced 5-HT signals in the brain and L-serine can enhance these effects.

  20. Inframolecular acid base studies of the tris and tetrakis myo-inositol phosphates including the 1,2,3-trisphosphate motif

    NASA Astrophysics Data System (ADS)

    Dozol, Hélène; Blum-Held, Corinne; Guédat, Philippe; Maechling, Clarisse; Lanners, Steve; Schlewer, Gilbert; Spiess, Bernard

    2002-12-01

    The intrinsic acid-base properties of the phosphate groups of three myo-inositol derivatives which display the 1,2,3-trisphosphate motif, i.e. (±)- myo-inositol 1,2,3-trisphosphate (Ins(1,2,3)P 3), (±)- myo-inositol 1,2,3,6-tetrakisphosphate (Ins(1,2,3,6)P 4), and (±)- myo-inositol 1,2,3,5-tetrakisphosphate (Ins(1,2,3,5)P 4) are reported. The studies were performed in 0.2 M KCl solution at 37 °C, near physiological ionic strength and temperature. In addition, in order to shed light on the transition metal complexation properties of Ins(1,2,3)P 3, the influence of the Zn 2+ cations on its 31P NMR titration curves was investigated. From the titration curves as well as from the determined protonation microconstants, it appears that for Ins(1,2,3)P 3, the two lateral P1 and P3 phosphates strongly contribute to stabilise a proton on the central P2 phosphate. However, in the fully deprotonated form of Ins(1,2,3)P 3, P1 and P3 repulse each other so that they establish hydrogen bonds with, respectively, their neighbouring OH6 and OH4 hydroxyls. The 1,2,3-trisphosphate motif of Ins(1,2,3,5)P 4 behaves very similarly to that of Ins(1,2,3)P 3 indicating a poor interaction with the distant P5 phosphate. By contrast, moving a phosphate group from position 5 to position 6 on the myo-inositol ring as in Ins(1,2,3,6)P 4, leads to major changes in the basicity and cooperativity of the phosphate groups. Finally, the presence of Zn 2+ cations has a marked influence on the 31P NMR titration curves of Ins(1,2,3)P 3, leading to the conclusion that two equatorial phosphates, assisted by a middle axial one, afford an optimal chelating moiety that is able to occupy all sites of the metal coordination polyhedron which could be the reason for its antioxidant properties.

  1. [Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

    PubMed

    Kirillova, Iu M; Mikhaĭlova, E O; Balaban, N P; Mardanova, A M; Kaiumov, A R; Rudenskaia, G N; Kostrov, S V; Sharipova, M R

    2006-01-01

    The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

  2. The alteration of serine transporter activity in a cell line model of amyotrophic lateral sclerosis (ALS).

    PubMed

    Lee, Na-Young; Kim, Yunha; Ryu, Hoon; Kang, Young-Sook

    2017-01-29

    The alteration of d-serine levels is associated with the pathogenesis of sporadic ALS and mutant SOD1 (G93A) animal model of ALS. However, the exact mechanism of d-serine transport is not known in ALS. To better understand the distribution of d-serine in ALS, we determined the activity and the expression of serine transporter in a motor neuronal cell line model of ALS (NSC-34/hSOD1(G93A) cells). The uptake of [(3)H]d-serine was significantly lower in NSC-34/hSOD1(G93A) cells than in control NSC-34 and NSC-34/hSOD1(wt) cells. In contrast, the uptake of [(3)H]l-serine, precursor of d-serine, was markedly increased in NSC-34/hSOD1(G93A) cells compared to control NSC-34 and NSC-34/hSOD1(wt) cells. Both [(3)H]d-serine and [(3)H]l-serine uptake were saturable in these cells. The estimated Michaelis-Menten constant, Km, for d-serine uptakes was higher in NSC-34/hSOD1(G93A) cells than in NSC-34/hSOD1(wt) cells while the Km for l-serine uptake was 2 fold lower in NSC-34/hSOD1(G93A) cells than in control cells. [(3)H]d-serine and [(3)H]l-serine uptakes took place in a Na(+)-dependent manner, and both uptakes were significantly inhibited by system ASC (alanine-serine-cysteine) substrates. As a result of small interfering RNA experiments, we found that ASCT2 (SLC1A5) and ASCT1 (SLC1A4) are involved in [(3)H]d-serine and [(3)H]l-serine uptake in NSC-34/hSOD1(G93A) cells, respectively. The level of SLC1A4 mRNA was significantly increased in NSC-34/hSOD1(G93A) compared to NSC-34 and NSC-34/hSOD1(wt) cells. In contrast, the level of SLC7A10 mRNA was relatively lower in NSC-34/hSOD1(G93A) cells than the control cells. Together, these data suggest that the pathological alteration of d- and l-serine uptakes in ALS is driven by the affinity change of d-and l-serine uptake system.

  3. 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.

  4. 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.

  5. 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

  6. The effects of ursodeoxycholic acid treatment for intrahepatic cholestasis of pregnancy on maternal and fetal outcomes: a meta-analysis including non-randomized studies.

    PubMed

    Grand'Maison, Sophie; Durand, Madeleine; Mahone, Michèle

    2014-07-01

    Objectif : Les avantages de l’utilisation d’acide ursodésoxycholique (AUDC) pour la prise en charge de la cholestase intrahépatique de la grossesse (CIG) demeurent incertains. Une analyse Cochrane de 2010 ayant porté sur des essais comparatifs randomisés n’a pas été en mesure de se prononcer pour ou contre l’utilisation d’AUDC pour la prise en charge de la CIG. Nous avons mené une méta-analyse de la littérature, en englobant tant les études non randomisées (ENR) que les ECR. Nous avions pour objectif de déterminer si les patientes ayant participé aux ENR étaient comparables à celles qui avaient participé aux ECR; nous avions également pour objectif de déterminer si l’inclusion des ENR pouvait renforcer les données probantes disponibles et orienter la pratique clinique quant à l’utilisation d’AUDC chez les femmes qui présentent une CIG. Sources de données : Nous avons mené des recherches dans Medline (Ovid), Embase (Ovid), EMB Reviews, Cinahl (Ebsco) et Web of Knowledge (Thomson Reuters) en vue d’en tirer les articles publiés entre 1966 et juin 2012. Sélection des études : Nous avons inclus tous les ECR admissibles ayant comparé l’AUDC à un placebo ou à d’autres traitements et toutes les ENR ayant comparé l’AUDC à tout autre traitement chez des femmes présentant une CIG. Synthèse des données : Nous avons inclus 11 ECR (n = 625 grossesses) et six ENR (n = 211 grossesses). Bien que les femmes ayant participé aux ECR et aux ENR aient été comparables, la qualité des études était plus faible dans le cas des ENR. De façon générale, les femmes traitées à l’AUDC ont connu une atténuation du prurit dans 73 % des ECR et dans 100 % des ENR disposant de données disponibles. Les épreuves de fonction hépatique ont présenté une amélioration dans 82 % des ECR et dans 100 % des ENR disposant de données disponibles. Bien que l’utilisation d’AUDC n’ait pas affecté le taux de c

  7. 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

  8. [Proprotein convertases - family of serine proteases with a broad spectrum of physiological functions].

    PubMed

    Małuch, Izabela; Walewska, Aleksandra; Sikorska, Emilia; Prahl, Adam

    2016-01-01

    A large group of secretory proteins involved in proper functioning of living organisms, is synthesized as inactive precursor molecules. Their biologically active forms are obtained as a result of numerous post-translational modifications. Some of these processes occur irreversibly, permanently changing the initial compound structure. An example of such modifications is catalytic treatment of proteins performed by proteolytic enzymes. Among five separate classes of these enzymes, the most numerous are serine proteases. Mammalian proprotein convertases (PCs), which include: furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7, PCSK9, SKI-1, represent serine endoproteases family. PCs play a key role in the activation of a number of precursor proteins causing formation of biologically active forms of enzymes, hormones, signaling molecules, transcription and growth factors. This article summarizes current state of knowledge on biosynthesis, structure and substrate specificity of PCs, identifies the relationship between location and intracellular activity of these enzymes, and their physiological functions in mammals.

  9. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    SciTech Connect

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  10. 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

  11. The serine/threonine phosphatase PPM1B (PP2Cβ) selectively modulates PPARγ activity.

    PubMed

    Tasdelen, Ismayil; van Beekum, Olivier; Gorbenko, Olena; Fleskens, Veerle; van den Broek, Niels J F; Koppen, Arjen; Hamers, Nicole; Berger, Ruud; Coffer, Paul J; Brenkman, Arjan B; Kalkhoven, Eric

    2013-04-01

    Reversible phosphorylation is a widespread molecular mechanism to regulate the function of cellular proteins, including transcription factors. Phosphorylation of the nuclear receptor PPARγ (peroxisome-proliferator-activated receptor γ) at two conserved serine residue (Ser(112) and Ser(273)) results in an altered transcriptional activity of this transcription factor. So far, only a very limited number of cellular enzymatic activities has been described which can dephosphorylate nuclear receptors. In the present study we used immunoprecipitation assays coupled to tandem MS analysis to identify novel PPARγ-regulating proteins. We identified the serine/threonine phosphatase PPM1B [PP (protein phosphatase), Mg(2+)/Mn(2+) dependent, 1B; also known as PP2Cβ] as a novel PPARγ-interacting protein. Endogenous PPM1B protein is localized in the nucleus of mature 3T3-L1 adipocytes where it can bind to PPARγ. Furthermore we show that PPM1B can directly dephosphorylate PPARγ, both in intact cells and in vitro. In addition PPM1B increases PPARγ-mediated transcription via dephosphorylation of Ser(112). Finally, we show that knockdown of PPM1B in 3T3-L1 adipocytes blunts the expression of some PPARγ target genes while leaving others unaltered. These findings qualify the phosphatase PPM1B as a novel selective modulator of PPARγ activity.

  12. Photoreceptor Neuroprotection: Regulation of Akt Activation Through Serine/Threonine Phosphatases, PHLPP and PHLPPL.

    PubMed

    Rajala, Raju V S; Kanan, Yogita; Anderson, Robert E

    2016-01-01

    Serine/threonine kinase Akt is a downstream effector of insulin receptor/PI3K pathway that is involved in many processes, including providing neuroprotection to stressed rod photoreceptor cells. Akt signaling is known to be regulated by the serine/threonine phosphatases, PHLPP (PH domain and leucine rich repeat protein phosphatase) and PHLPPL (PH domain and leucine rich repeat protein phosphatase-like). We previously reported that both phosphatases are expressed in the retina, as well as in photoreceptor cells. In this study, we examined the PHLPP and PHLPPL phosphatase activities towards non-physiological and physiological substrates. Our results suggest that PHLPP was more active than PHLPPL towards non-physiological substrates, whereas both PHLPP and PHLPP dephosphorylated the physiological substrates of Akt1 and Akt3 with similar efficiencies. Our results also suggest that knockdown of PHLPPL alone does not increase Akt phosphorylation, due to a compensatory increase of PHLPP, which results in the dephosphorylation of Akt. Therefore, PHLPP and PHLPPL regulate Akt activation together when both phosphatases are expressed.

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

    PubMed

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

    1995-08-15

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

  14. Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.

    PubMed

    Peters, Haley L; Tripathi, Satyendra C; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R; St John, Lisa S; Federico, Lorenzo; Meraz, Ismail M; Roth, Jack A; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L; Heymach, John V; Swisher, Stephen G; Bernatchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J

    2017-03-02

    Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 1-11. ©2017 AACR.

  15. Characterization of a chemostable serine alkaline protease from Periplaneta americana

    PubMed Central

    2013-01-01

    Background Proteases are important enzymes involved in numerous essential physiological processes and hold a strong potential for industrial applications. The proteolytic activity of insects’ gut is endowed by many isoforms with diverse properties and specificities. Thus, insect proteases can act as a tool in industrial processes. Results In the present study, purification and properties of a serine alkaline protease from Periplaneta americana and its potential application as an additive in various bio-formulations are reported. The enzyme was purified near to homogeneity by using acetone precipitation and Sephadex G-100 gel filtration chromatography. Enzyme activity was increased up to 4.2 fold after gel filtration chromatography. The purified enzyme appeared as single protein-band with a molecular mass of ~ 27.8 kDa in SDS-PAGE. The optimum pH and temperature for the proteolytic activity for purified protein were found around pH 8.0 and 60°C respectively. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and compatibility towards detergents, oxidizing, reducing, and bleaching agents. In addition, enzyme also showed stability towards organic solvents and commercial detergents. Conclusion Several important properties of a serine protease from P. Americana were revealed. Moreover, insects can serve as excellent and alternative source of industrially important proteases with unique properties, which can be utilized as additives in detergents, stain removers and other bio-formulations. Properties of the P. americana protease accounted in the present investigation can be exploited further in various industrial processes. As an industrial prospective, identification of enzymes with varying essential properties from different insect species might be good approach and bioresource. PMID:24229392

  16. 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.

  17. 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.

  18. A New Class of Serine and Cysteine Protease Inhibitor with Chemotherapeutic Potential

    DTIC Science & Technology

    1999-06-01

    also be used to produce a serine protease inhibitor. Similar to the cysteine inhibitors, a dipeptide side chain is attached to the ring which is...which relieves the 7 strain (Figure 3). Serine and cysteine proteases use a mechanism to cleave peptide bonds which involves addition of a catalytic...serine and cysteine proteases share a similar mechanism for hydrolyzing amide bonds , we expect that 4-heterocyclohexanones should be good inhibitors

  19. Molecular orbital studies of enzyme activity: catalytic mechanism of serine proteinases.

    PubMed Central

    Scheiner, S; Lipscomb, W N

    1976-01-01

    The catalytic activity of the serine proteinases is studied using molecular orbital methods on a model of the enzyme-substrate complex. A mechanism is employed in which Ser-195, upon donating a proton to the His-57-Asp-102 dyad, attacks the substrate to form the tetrahedral intermediate. As His-57 then donates a proton to the leaving group, the intermediate decomposes to the acyl enzyme. An analogous process takes place during deacylation, as a water molecule takes the place of Ser-195 as the nucleophile. The motility of the histidine is found to be an important factor in both steps. An attempt is made to include the effects of those atoms not explicitly included in the calculations and to compare the reaction rate of the proposed mechanism with that of the uncatalyzed hydrolysis. This mechanism is found to be in good agreement with structural and kinetic data. PMID:1061145

  20. 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.

  1. Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani.

    PubMed

    Chandrasekaran, Murugesan; Chandrasekar, Raman; Sa, Tongmin; Sathiyabama, Muthukrishnan

    2014-07-01

    Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 β-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing.

  2. Antibacterial activity of silver nanoparticles synthesized from serine.

    PubMed

    Jayaprakash, N; Judith Vijaya, J; John Kennedy, L; Priadharsini, K; Palani, P

    2015-04-01

    Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV-Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443nm. The emission spectrum of Ag NPs showed an emission band at 484nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO3 against Gram-positive and Gram-negative bacteria.

  3. Molecular Characterization of Membrane-Associated Soluble Serine Palmitoyltransferases from Sphingobacterium multivorum and Bdellovibrio stolpii▿

    PubMed Central

    Ikushiro, Hiroko; Islam, Mohammad Mainul; Tojo, Hiromasa; Hayashi, Hideyuki

    2007-01-01

    Serine palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl coenzyme A (CoA) to form 3-ketodihydrosphingosine (KDS). Eukaryotic SPTs comprise tightly membrane-associated heterodimers belonging to the pyridoxal 5′-phosphate (PLP)-dependent α-oxamine synthase family. Sphingomonas paucimobilis, a sphingolipid-containing bacterium, contains an abundant water-soluble homodimeric SPT of the same family (H. Ikushiro et al., J. Biol. Chem. 276:18249-18256, 2001). This enzyme is suitable for the detailed mechanistic studies of SPT, although single crystals appropriate for high-resolution crystallography have not yet been obtained. We have now isolated three novel SPT genes from Sphingobacterium multivorum, Sphingobacterium spiritivorum, and Bdellovibrio stolpii, respectively. Each gene product exhibits an ∼30% sequence identity to both eukaryotic subunits, and the putative catalytic amino acid residues are conserved. All bacterial SPTs were successfully overproduced in Escherichia coli and purified as water-soluble active homodimers. The spectroscopic properties of the purified SPTs are characteristic of PLP-dependent enzymes. The KDS formation by the bacterial SPTs was confirmed by high-performance liquid chromatography/mass spectrometry. The Sphingobacterium SPTs obeyed normal steady-state ordered Bi-Bi kinetics, while the Bdellovibrio SPT underwent a remarkable substrate inhibition at palmitoyl CoA concentrations higher than 100 μM, as does the eukaryotic enzyme. Immunoelectron microscopy showed that unlike the cytosolic Sphingomonas SPT, S. multivorum and Bdellovibrio SPTs were bound to the inner membrane of cells as peripheral membrane proteins, indicating that these enzymes can be a prokaryotic model mimicking the membrane-associated eukaryotic SPT. PMID:17557831

  4. 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.

  5. Structural and functional adaptation of vancomycin resistance VanT serine racemases

    DOE PAGES

    Meziane-Cherif, Djalal; Stogios, Peter J.; Evdokimova, Elena; ...

    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

  6. 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

  7. Kinetic Behavior of Leucine and Other Amino Acids Modulating Cognitive Performance via mTOR Pathway

    DTIC Science & Technology

    2011-12-02

    Acid BBB Transporter Type Structure Leucine L1 Essential Neutral Non-polar (hydrophobic) Branched chain Aspartic Acid Acidic X- Acidic Polar Glutamine...compared with other tissues. • Effects of leucine on other amino acids were analyzed. Those measured were aspartic acid , glutamic acid, serine, histidine

  8. A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides.

    PubMed

    Thekkiniath, Jose C; Zabet-Moghaddam, Masoud; San Francisco, Susan K; San Francisco, Michael J

    2013-06-01

    Batrachochytrium dendrobatidis (B. dendrobatidis), a chytrid fungus, is one of the major contributors to the global amphibian decline. The fungus infects both tadpoles and adult amphibians. Tadpoles are infected in their keratinized mouthparts, and infected adults exhibit hyperkeratosis and loss of righting reflex. Infections of adults may result in death from cardiac arrest in susceptible species. Thyroid hormone plays a key role in amphibian metamorphosis. The occurrence of B. dendrobatidis in tadpoles during metamorphosis may result in exposure of the fungus to host morphogens including TH. This exposure may induce gene expression in the fungus contributing to invasion and colonization of the host. Here, we demonstrate movement of fungal zoospores toward TH. Additionally, expression of a subtilisin-like serine protease is up-regulated in B. dendrobatidis cells exposed to TH. A gene encoding this protease was cloned from B. dendrobatidis and expressed in Escherichia coli. The protein was partially purified and characterized. The similarity between subtilases of human dermatophytes and the B. dendrobatidis subtilisin-like serine protease suggests the importance of this enzyme in B. dendrobatidis pathogenicity. Cleavage of frog skin antimicrobial peptides (AMPs) by this B. dendrobatidis subtilisin-like serine protease suggests a role for this enzyme in fungal survival and colonization.

  9. 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.

  10. 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

  11. Preliminary Study on Cost-Effective L-Tryptophan Production from Indole and L-Serine by E. coli Cells

    PubMed Central

    Sadeghiyan-Rizi, Tahereh; Fooladi, Jamshid; Sadrai, Sima

    2016-01-01

    Background: L-tryptophan is used widespread in the pharmaceutical industry. The majority of L-Trp production depends on microbial processes that produce L-tryptophan from indole and L-serine. These processes are very costly due to the costs of precursors, especially L-serine. Use of inexpensive substitutions as the L-serine source of L-tryptophan production enables us to reach a cost-effective process. In this paper, effect of Triton X-100 on L-Trp production and the ability to use Iranian cane molasses as inexpensive L-serine source was investigated. Methods: Escherichia coli (E. coli) ATCC 11303 cells were grown in 10-L fermenter containing minimal medium supplemented with beet molasses as an inexpensive carbon source and indole as tryptophan synthase inducer. Whole cells of stationary phase were used as biocatalyst for L-Trp production. Triton X-100 addition to the production medium as indole reservoir was investigated. Then, cane molasses was used as L-Ser source in L-Trp production medium. Amount of L-Tryptophan and theoretical yield of L-Trp production was determined by HPLC and by a colorimetrically method on the basis of remaining indole assay, respectively. Results: As a result, triton X-100 increased L-Trp production three times. Also, the result showed that 0.68 mM L-Tryptophan was produced in the presence of cane molasses at 37°C for 8 hr. Conclusion: This result showed that cane molasses of Qazvin sugar factory includes significant amounts of L-Ser that makes it a suitable substitution for L-Ser in L-Trp production. Therefore, it has the potential to be used for cost-effective L-Trp production in industrial scale. PMID:27920887

  12. The borohydride-reducible compounds of human aortic elastin. Demonstration of a new cyclic amino acid in alkali hydrolysate, and changes with age and in patients with annulo-aortic ectasia including one with Marfan syndrome.

    PubMed Central

    Halme, T; Jutila, M; Vihersaari, T; Oksman, P; Light, N D; Penttinen, R

    1985-01-01

    Human aortic elastin reduced with [3H]borohydride was analysed by ion-exchange chromatography after alkali or acid hydrolysis. Alkali hydrolysates of elastins contained a radioactive peak that was eluted between proline and leucine. This peak was not present in foetal elastin, but its proportion increased steadily during aging. Aortic samples from patients with annulo-aortic ectasia (aneurysm of the ascending aorta), including one with classical Marfan syndrome, contained less elastin (CNBr-insoluble material) than did the age-matched controls. The proportion of radioactivity in the new peak of all these aortas was low when compared with age-matched controls. Gas-chromatographic/mass-spectrometric analysis suggested that it contained a cyclic derivative of a hydrated aldol-condensation product. The concentration of the cross-link precursors, lysine aldehyde and aldol-condensation product (estimated from the acid-hydrolysis product 6-chloronorleucine and the acid-degradation product of reduced aldol-condensation product) was high in very young aortas but remained quite stable after childhood. No differences were observed in cross-link profiles of acid hydrolysates between pathological and control aortas. A low proportion of radioactivity in the new peak may indicate the presence of young or immature elastin in the pathological aortas. PMID:4084226

  13. 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.

  14. 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

  15. Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production.

    PubMed

    Lai, Shujuan; Zhang, Yun; Liu, Shuwen; Liang, Yong; Shang, Xiuling; Chai, Xin; Wen, Tingyi

    2012-04-01

    L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH(r)). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22 ± 1.41) μmol g(CDM) (-1), which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH(r) improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C(1) units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.

  16. Influence of dietary phytic acid and source of microbial phytase on ileal endogenous amino acid flows in broiler chickens.

    PubMed

    Cowieson, A J; Ravindran, V; Selle, P H

    2008-11-01

    The effects of phytic acid and 2 sources of exogenous phytase (bacterial vs. fungal) on the flow of endogenous amino acids at the terminal ileum of broilers were assessed using the enzyme-hydrolyzed casein method. Phytic acid (as the sodium salt) was included in a purified diet at 8.5 and 14.5 g/kg, and each diet was fed without or with a fungal (Aspergillus niger-derived) or a bacterial (Escherichia coli-derived) microbial phytase at 500 phytase units/kg of diet. Increasing the concentration of phytic acid in the diet from 8.5 to 14.5 g/kg increased (P < 0.001) the flow of all measured amino acids by an average of 68%, with a range from 17% for proline to 145% for phenylalanine. The flow of endogenous aspartic acid, serine, glutamic acid, glycine, leucine, tyrosine, phenylalanine, and histidine were increased by more than the mean, indicating changes in the composition of endogenous protein in response to the presence of higher concentrations of phytic acid. Supplementation of both phytases reduced (P < 0.001) the flow of endogenous amino acids, but the reduction (P = 0.06) was greater for the bacterial phytase compared with the fungal phytase. These data suggest that a substantial part of the amino acid and energy responses observed following phytase supplementation in broiler chickens stems from reduced endogenous amino acid flows and that the capacity of different phytases to counteract the antinutritive properties of phytic acid vary.

  17. The role of imidazole in peptide cyclization by transesterification: parallels to the catalytic triads of serine proteases.

    PubMed

    Byler, Kendall G; Li, Yangmei; Houghten, Richard A; Martinez-Mayorga, Karina

    2013-05-14

    The improved bioavailability, stability and selectivity of cyclic peptides over their linear counterparts make them attractive structures in the design and discovery of novel therapeutics. In our previous work, we developed an imidazole-promoted preparation of cyclic depsipeptides in which we observed that increasing the concentration of imidazole resulted in the concomitant increase in the yield of cyclic product and reduction in dimerization, but also resulted in the generation of an acyl-substituted side product. In this work, we used transition state analysis to explore the mechanism of the imidazole-catalyzed esterification of one such peptide, Ac-SAFYG-SCH2φ, and determined the acyl substitution product to be an intermediate in a competing reaction pathway involving acyl substitution of the thioester by imidazole. Our findings indicate that imidazole plays an essential role in this side-chain to C-terminal coupling, and by extension, in transesterifications in general, through a concerted mechanism wherein imidazole deprotonates the nucleophile as the nucleophile attacks the carbonyl. The system under study is identical to the histidine-serine portion of the catalytic triads in serine proteases and it is likely that these enzymes employ the same concerted mechanism in the first step of peptide cleavage. Additionally, relatively high concentrations of imidazole must be used to effectively catalyze reactions in aprotic solvents since the overall reaction involves imidazole acting both as an acid and as a base, existing in solution as an equilibrium distribution between the neutral form and its conjugate acid.

  18. Evidence that the catalytic activity of prokaryote leader peptidase depends upon the operation of a serine-lysine catalytic dyad.

    PubMed Central

    Black, M T

    1993-01-01

    Leader peptidase (LP) is the enzyme responsible for proteolytic cleavage of the amino acid leader sequence from bacterial preproteins. Recent data indicate that LP may be an unusual serine proteinase which operates without involvement of a histidine residue (M. T. Black, J. G. R. Munn, and A. E. Allsop, Biochem. J. 282:539-543, 1992; M. Sung and R. E. Dalbey, J. Biol. Chem. 267:13154-13159, 1992) and that, therefore, one or more alternative residues must perform the function of a catalytic base. With the aid of sequence alignments, site-specific mutagenesis of the gene encoding LP (lepB) from Escherichia coli has been employed to investigate the mechanism of action of the enzyme. Various mutant forms of plasmid-borne LP were tested for their abilities to complement the temperature-sensitive activity of LP in E. coli IT41. Data are presented which indicate that the only conserved amino acid residue possessing a side chain with the potential to ionize, and therefore with the potential to transfer protons, which cannot be substituted with a neutral side chain is lysine at position 145. The data suggest that the catalytic activity of LP is dependent on the operation of a serine-lysine catalytic dyad. Images PMID:8394311

  19. Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines

    PubMed Central

    Lee, Joon No; Gong, Yi; Zhang, Xiangyu; Ye, Jin

    2006-01-01

    Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum that play crucial roles in cholesterol homeostasis by inhibiting excessive cholesterol synthesis and uptake. In sterol-depleted cells Insig-1 is degraded at least 15 times more rapidly than Insig-2, owing to ubiquitination of Lys-156 and Lys-158 in Insig-1. In this study, we use domain-swapping methods to localize amino acid residues responsible for this differential degradation. In the case of Insig-2, Glu-214 stabilizes the protein by preventing ubiquitination. When Glu-214 is changed to alanine, Insig-2 becomes ubiquitinated, but it is still not degraded as rapidly as ubiquitinated Insig-1. The difference in the degradation rates is traced to two amino acids: Ser-149 in Insig-1 and Ser-106 in Insig-2. Ser-149, which lies NH2-terminal to the ubiquitination sites, accelerates the degradation of ubiquitinated Insig-1. Ser-106, which is COOH-terminal to the ubiquitination sites, retards the degradation of ubiquitinated Insig-2. The current studies indicate that the degradation of ubiquitinated Insigs is controlled by serine residues flanking the sites of ubiquitination. PMID:16549805

  20. Regulated proteolysis by cortical granule serine protease 1 at fertilization.

    PubMed

    Haley, Sheila A; Wessel, Gary M

    2004-05-01

    Cortical granules are specialized organelles whose contents interact with the extracellular matrix of the fertilized egg to form the block to polyspermy. In sea urchins, the granule contents form a fertilization envelope (FE), and this construction is critically dependent upon protease activity. An autocatalytic serine protease, cortical granule serine protease 1 (CGSP1), has been identified in the cortical granules of Strongylocentrotus purpuratus eggs, and here we examined the regulation of the protease activity and tested potential target substrates of CGSP1. We found that CGSP1 is stored in its full-length, enzymatically quiescent form in the granule, and is inactive at pH 6.5 or below. We determined the pH of the cortical granule by fluorescent indicators and micro-pH probe measurements and found the granules to be pH 5.5, a condition inhibitory to CGSP1 activity. Exposure of the protease to the pH of seawater (pH 8.0) at exocytosis immediately activates the protease. Activation of eggs at pH 6.5 or lower blocks activation of the protease and the resultant FE phenotypes are indistinguishable from a protease-null phenotype. We find that native cortical granule targets of the protease are beta-1,3 glucanase, ovoperoxidase, and the protease itself, but the structural proteins of the granule are not proteolyzed by CGSP1. Whole mount immunolocalization experiments demonstrate that inhibition of CGSP1 activity affects the localization of ovoperoxidase but does not alter targeting of structural proteins to the FE. The mistargeting of ovoperoxidase may lead to spurious peroxidative cross-linking activity and contribute to the lethality observed in protease-null cells. Thus, CGSP1 is proteolytically active only when secreted, due to the low pH of the cortical granules, and it has a small population of targets for cleavage within the cortical granules.

  1. The Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and serine phosphorylation.

    PubMed

    Meijer, Inez M J; Kerperien, JoAnn; Sotoca, Ana M; van Zoelen, Everardus J J; van Leeuwen, Jeroen E M

    2013-04-01

    The ERBB1-ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1-504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14-3-3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14-3-3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14-3-3 to Usp8. Our findings are most consistent with the model that MIT

  2. l-Serine and glycine serve as major astroglia-derived trophic factors for cerebellar Purkinje neurons

    PubMed Central

    Furuya, Shigeki; Tabata, Toshihide; Mitoma, Junya; Yamada, Keiko; Yamasaki, Miwako; Makino, Asami; Yamamoto, Toshifumi; Watanabe, Masahiko; Kano, Masanobu; Hirabayashi, Yoshio

    2000-01-01

    Glial cells support the survival and development of central neurons through the supply of trophic factors. Here we demonstrate that l-serine (l-Ser) and glycine (Gly) also are glia-derived trophic factors. These amino acids are released by astroglial cells and promote the survival, dendritogenesis, and electrophysiological development of cultured cerebellar Purkinje neurons. Although l-Ser and Gly are generally classified as nonessential amino acids, 3-phosphoglycerate dehydrogenase (3PGDH), a key enzyme for their biosynthesis, is not expressed in Purkinje neurons. By contrast, the Bergman glia, a native astroglia in the cerebellar cortex, highly expresses 3PGDH. These data suggest that l-Ser and Gly mediate the trophic actions of glial cells on Purkinje neurons. PMID:11016963

  3. Serine-selective aerobic cleavage of peptides and a protein using a water-soluble copper-organoradical conjugate.

    PubMed

    Seki, Yohei; Tanabe, Kana; Sasaki, Daisuke; Sohma, Youhei; Oisaki, Kounosuke; Kanai, Motomu

    2014-06-16

    The site-specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine-selective peptide-cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water-soluble copper-organoradical conjugate. The method is applicable to the site-selective cleavage of polypeptides that possess various functional groups. Peptides comprising D-amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site-selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.

  4. 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.

  5. 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.

  6. Concordance between isolated cleft palate in mice and alterations within a region including the gene encoding the beta 3 subunit of the type A gamma-aminobutyric acid receptor.

    PubMed

    Culiat, C T; Stubbs, L; Nicholls, R D; Montgomery, C S; Russell, L B; Johnson, D K; Rinchik, E M

    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 5 and beta 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 p83FBFo 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 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.

  7. Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution: a small serine α/β-hydrolase

    SciTech Connect

    Yang, Chao-Yu; Chin, Ko-Hsin; Chou, Chia-Cheng; Wang, Andrew H.-J.; Chou, Shan-Ho

    2006-06-01

    The crystal structure of a conserved hypothetical protein from X. campestris has been determined to a resolution of 1.6 Å. The determined X. campestris structure shows that it belongs to the superfamily of serine α/β hydrolase, with an extra strand preceding the first β-strand to lead to extensive subunit interactions in the crystal. XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. The protein consists of 220 amino acids and its structure has been determined to 1.6 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Although it has very low sequence identity to protein sequences in the PDB (less than 20%), the determined structure nevertheless shows that it belongs to the superfamily of serine α/β-hydrolases, with an active site that is fully accessible to solvent owing to the absence of a lid domain. Modelling studies with the serine esterase inhibitor E600 indicate that XC6422 adopts a conserved Ser-His-Asp catalytic triad common to this superfamily and has a preformed oxyanion hole for catalytic activation. These structural features suggest that XC6422 is most likely to be a hydrolase active on a soluble ester or a small lipid. An extra strand preceding the first β-strand in the canonical α/β-hydrolase fold leads to extensive subunit interactions between XC6422 monomers, which may explain why XC6422 crystals of good diffraction quality can grow to dimensions of up to 1.5 mm in a few days.

  8. Reduction of phosphorylated histone H3 serine 10 and serine 28 cell cycle marker intensities after DNA damage.

    PubMed

    Ozawa, Kazuo

    2008-06-01

    Phosphorylated histone H3 at serine 10 and serine 28 (H3Ser10 and H3Ser28) have been recognized as cell cycle markers to evaluate the late-G(2)/M status of cell and tissue samples. I report about the reduction phenomena of H3Ser10 and Ser28 phosphorylation (H3Ser10P and 28P) at the late-G(2) through M cell cycle phases in association with DNA damage caused by hydrogen peroxide (H(2)O(2)). The levels of H3Ser10P and Ser28P decreased between 15 and 60 min after H(2)O(2) addition in an inverse correlation manner with H2AX Ser139 phosphorylation (gammaH2AX). Experiments using wortmannin suggested that the reduction events of H3Ser10P/28P are under the control of phosphatidylinositol 3-kinase-like kinases. Fluorescence microscopic observation showed that H3Ser10 and Ser28 on telomeric portions of condensed M-phase chromosomes retained their strongly phosphorylated status even after 60 min of H(2)O(2) treatment. In addition, these chromosome parts were poorly gammaH2AX positive showing mutually exclusive distribution patterns between H3Ser10P/28P and gammaH2AX. Considering these data, I hypothesize that the reduction of the H3Ser10P/28P is a part of the DNA damage response processes. It is advisable to pay careful attention to these phenomena at the time of designing cell cycle assay protocols with H3Ser10P or Ser28P mitosis markers when DNA damaging process is expected to occur.

  9. Target validation of highly conserved Amblyomma americanum tick saliva serine protease inhibitor 19.

    PubMed

    Kim, Tae K; Radulovic, Zeljko; Mulenga, Albert

    2016-04-01

    Amblyomma americanum tick serine protease inhibitor (serpin, AAS) 19, is a highly conserved protein that is characterized by its functional domain being 100% conserved across tick species. We also reported that AAS19 was an immunogenic tick saliva protein with anti-haemostatic functions and an inhibitor of trypsin-like proteases including five of the eight serine protease factors in the blood clotting cascade. In this study the goal was to validate the importance of AAS19 in A. americanum tick physiology, assess immunogenicity and investigate tick vaccine efficacy of yeast-expressed recombinant (r) AAS19. We confirm that AAS19 is important to A. americanum fitness and blood meal feeding. AAS19 mRNA disruption by RNAi silencing caused ticks to obtain blood meals that were 50% smaller than controls, and treated ticks being morphologically deformed with 100% of the deformed ticks dying in incubation. We show that rAAS19 is highly immunogenic in that two 500 μg inoculations mixed with TiterMax Gold adjuvant provoked antibody titers of more than 1:320,000 that specifically reacted with native AAS19 in unfed and partially fed tick tissue. Since AAS19 is injected into animals during tick feeding, we challenge infested immunized rabbits twice to test if tick infestations of immunized rabbits could act as booster. While in the first infestation significantly smaller tick blood meals were observed on one of the two immunized rabbits, smaller blood meals were observed on both rabbits, but 60% of ticks that engorged on immunized rabbits in the second infestation failed to lay eggs. It is notable that ticks fed faster on immunized animals despite obtaining smaller blood meals. We conclude that rAAS19 is a potential component of cocktail tick vaccine.

  10. The outwardly rectifying chloride channel in rat peritoneal mast cells is regulated by serine/threonine kinases and phosphatases.

    PubMed

    Seebeck, Jörg; Tritschler, Stefan; Roloff, Tim; Kruse, Marie-Luise; Schmidt, Wolfgang E; Ziegler, Albrecht

    2002-02-01

    A slowly activating, outwardly rectifying Cl- channel (ORCC) has been described in rat peritoneal mast cells (RPMCs). This channel is activated by intracellular application of cAMP, an effect that might be mediated by a PKA-type serine/threonine protein kinase. To test this hypothesis, whole-cell patch-clamp experiments (nystatin-perforated patch) were performed and 8-bromoadenosine 3',5'-cyclic monophosphothioate, Sp-enantiomer (Sp-8Br-cAMPS), a cell membrane-permeable activator of PKA, and three inhibitors of different serine/threonine protein phosphatases (okadaic acid, cantharidin, calyculin A), were tested. In RPMCs application of repetitive series of step hyper- and depolarizations (holding potential 0 mV, test potentials -80 to +80 mV, step size +20 mV) induced a slowly increasing, [half-maximal activation time ( t0.5) 11.0+/-1.1 min, Imax (at +80 mV) 18.7+/-3.1 pA pF-1], DIDS-sensitive, outwardly rectifying Cl- current I(Cl,OR). The activation of this current could be accelerated by Sp-8Br-cAMPS, okadaic acid or cantharidin in the extracellular solution. Co-application of Sp-8Br-cAMPS and okadaic acid increased Imax supra-additively. Calyculin A and higher concentrations of cantharidin inhibited the Cl- current via unknown mechanisms. Our findings suggest that I(Cl,OR) in RPMCs is activated by a PKA-type protein kinase, a process which is antagonized functionally by okadaic acid- and cantharidin-sensitive protein phosphatases.

  11. Enantiomer-specific selection of amino acids

    PubMed Central

    Ren, Xueying; Tellez, Luis A; de Araujo, Ivan E

    2013-01-01

    Dietary intake of L-amino acids impacts on several physiological functions, including the control of gastrointestinal motility, pancreatic secretion, and appetite. However, the biological mechanisms regulating behavioral predilections for certain amino acid types remain poorly understood. We tested the hypothesis that, in mice, the potency with which a given glucogenic amino acid increases glucose utilization reflects its rewarding properties. We have found that: 1. During long-, but not short-, term preference tests, L-alanine and L-serine were preferred over their D-enantiomer counterparts, while no such effect was observed for L-threonine vs. D-threonine; 2. These behavioral patterns were closely associated with the ability of L-amino acids to promote increases in respiratory exchange ratios such that those, and only those, L-amino acids able to promote increases in respiratory exchange ratios were preferred over their D-isomers; 3. These behavioral preferences were independent of gustatory influences, since taste-deficient Trpm5 knockout mice displayed ingestive responses very similar to those of their wild-type counterparts. We conclude that the ability to promote increases in respiratory exchange ratios enhances the reward value of nutritionally relevant amino acids, and suggest a mechanistic link between substrate utilization and amino acid preferences. PMID:24072505

  12. The serine proteinase chain of human complement component C1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments.

    PubMed Central

    Carter, P E; Dunbar, B; Fothergill, J E

    1983-01-01

    Human complement component C1s was purified from fresh blood by conventional methods of precipitation and chromatography. The single-chain zymogen form was activated by treatment with C1r. Reduction and carboxymethylation then allowed the light chain and heavy chain to be separated on DEAE-Sepharose CL-6B in 8 M-urea. Liquid-phase sequencing of the light chain determined 50 residues from the N-terminus. CNBr-cleavage fragments of the light chain were separated by high-pressure liquid chromatography on gel-permeation and reverse-phase columns. N-Terminal sequencing of these fragments determined the order of a further 138 residues, giving a total of 188 residues or about 75% of the light chain. Seven of these eight sequences could be readily aligned with the amino acid sequences of other serine proteinases. The typical serine proteinase active-site residues are clearly conserved in C1s, and the specificity-related side chain of the substrate-binding pocket is aspartic acid, as in trypsin, consistent with the proteolytic action of C1s on C4 at an arginine residue. Somewhat surprisingly, when the C1s sequence is compared with that of complement subcomponent C1r, the percentage difference (59%) is approximately the same as that found between the other mammalian serine proteinases (56-71%). PMID:6362661

  13. Physiological relevance of endogenous free D-serine in the mammalian brain: are scientists on a royal road for the treatment of glutamatergic-related brain disorders?

    PubMed

    Mothet, J P

    2001-10-01

    Over the last century, it has been considered that amino acids in mammalian tissues and body fluids occur solely in the L-configuration whether free or as components of peptides and proteins. However, the recent discovery that high levels of D-serine and D-aspartate are present in Mammals overturns this long-cherished theory. In this review, we focus on recent findings regarding the physiological relevance of D-serine, a new neurotransmitter formed in glial cells, that serves as the endogenous ligand for the accessory strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. This unusual molecule not only questions our basic ideas about how nerve cells converse but also offers a novel way to treat some brain disorders as both over-stimulation and down regulation of NMDA receptors has been implicated in a large number of acute and chronic degenerative conditions.

  14. Critical role of serine 465 in isoflurane-induced increase of cell-surface redistribution and activity of glutamate transporter type 3.

    PubMed

    Huang, Yueming; Feng, Xiaorong; Sando, Julianne J; Zuo, Zhiyi

    2006-12-15

    Glutamate transporters (also called excitatory amino acid transporters, EAATs) bind extracellular glutamate and transport it to intracellular space to regulate glutamate neurotransmission and to maintain extracellular glutamate concentrations below neurotoxic levels. We previously showed that isoflurane, a commonly used anesthetic, enhanced the activity of EAAT3, a major neuronal EAAT. This effect required a protein kinase C (PKC) alpha-dependent EAAT3 redistribution to the plasma membrane. In this study, we prepared COS7 cells stably expressing EAAT3 with or without mutations of potential PKC phosphorylation sites in the putative intracellular domains. Here we report that mutation of threonine 5 or threonine 498 to alanine did not affect the isoflurane effects on EAAT3. However, the mutation of serine 465 to alanine abolished isoflurane-induced increase of EAAT3 activity and redistribution to the plasma membrane. The mutation of serine 465 to aspartic acid increased the expression of EAAT3 in the plasma membrane and also abolished the isoflurane effects on EAAT3. These results suggest an essential role of serine 465 in the isoflurane-increased EAAT3 activity and redistribution and a direct effect of PKC on EAAT3. Consistent with these results, isoflurane induced an increase in phosphorylation of wild type, T5A, and T498A EAAT3, and this increase was absent in S465A and S465D. Our current results, together with our previous data that showed the involvement of PKCalpha in the isoflurane effects on EAAT3, suggest that the phosphorylation of serine 465 in EAAT3 by PKCalpha mediates the increased EAAT3 activity and redistribution to plasma membrane after isoflurane exposure.

  15. Analysis of free and total myo-inositol in foods, feeds, and infant formula by high-performance anion exchange chromatography with pulsed amperometric detection, including a novel total extraction using microwave-assisted acid hydrolysis and enzymatic treatment.

    PubMed

    Ellingson, David; Pritchard, Ted; Foy, Pamela; King, Kathryn; Mitchell, Barbara; Austad, John; Winters, Doug; Sullivan, Darryl

    2012-01-01

    A method for the analysis of free and total myo-inositol in foods, feeds, and infant formulas has been developed and validated using high-performance anion exchange chromatography with pulsed amperometric detection. The option of a free myo-inositol determination or a complete total myo-inositol determination from main bound sources can be achieved. These sources include phytates, lower'phosphorylated forms, and phosphatidylinositol. This approach gives the option for subtraction of myo-inositol from nonbioavailable sources when it is quantified using other methods if a total bioavailable myo-inositol result is desired for nutritional labeling of a product. The free analysis was validated in a milk-based infant formula, giving RSD(R) of 2.29% and RSD, of 2.06%. A mean recovery of 97.9% was achieved from various spike levels of myo-inositol. Certified National Institute of Standards and Technology reference material verified the method's compatibility and specificity. Two different total analyses were validated in a soy-based infant formula and compared. One technique involved using a conventional acid hydrolysis with autoclave incubation for 6 h, while the other used a novel technique of microwave-assisted acid hydrolysis with enzymatic treatment that can minimize extraction to 1 day. The autoclave analysis had RSD(R) of 2.08% and RSDr of 1.55%, along with a mean spike recovery of 102.1% at various myo-inositol spike levels. The microwave/enzyme total analysis had RSD(R) of 4.34% and RSD, of 4.70%, along with a mean spike recovery of 104.2% at various spike levels of myo-inositol. Main sources of myo-inositol including phytic acid and phosphatidylinositol were tested with both total analyses. Mean recoveries of phytic acid and phosphatidylinositol through the autoclave total analysis were 90.4 and 98.3%, respectively. Mean spike recoveries for these same sources in soy- based infant formula through the microwave/enzyme total analysis were 97.2 and 96

  16. Serine protease SP105 activates prophenoloxidase in Asian corn borer melanization, and is regulated by serpin-3

    PubMed Central

    Chu, Yuan; Hong, Fang; Liu, Qizhi; An, Chunju

    2017-01-01

    Melanization reaction, resulting from the activation of prophenoloxidase, is a vital immune response in insects for encapsulating and killing the invasive organisms. Prophenoloxidase needs to be proteolytically activated by its upstream prophenoloxidase-activating protease (PAP) in melanization. Identification and characterization of PAPs facilitates the understanding of the molecular mechanisms involved in insect immunity. We here cloned a full-length cDNA for a serine protease, named as SP105, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of SP105 encodes 424-amino acid residue protein with a 19-residue signal peptide. Sequence comparison indicates that SP105 is most similar to Manduca sexta PAP3, a defined prophenoloxidase-activating protease. qRT-PCR analysis showed that SP105 mRNA levels increased significantly after a bacterial injection. Recombinant SP105 directly cleaved and activated Asian corn borer prophenoloxidase and therefore acted as the prophenoloxidase-activating protease. Additionally, SP105 formed SDS-stable complexes with a serine protease inhibitor, serpin-3, and its activity in activating prophenoloxidase was efficiently inhibited by serpin-3. Our work thus illustrated a prophenoloxidase-activating protease and revealed its regulation by serpin-3. The results would allow further advances in the understanding of the melanization in Asian corn borer and other insects. PMID:28358031

  17. PLD1 regulates adipogenic differentiation through mTOR - IRS-1 phosphorylation at serine 636/639

    PubMed Central

    Song, Hae-In; Yoon, Mee-Sup

    2016-01-01

    Phospholipase D1 (PLD1) plays a known role in several differentiation processes, but its role in adipogenic differentiation remains unknown. In the present study, we identified PLD1 as a negative regulator of adipogenic differentiation. We showed that PLD activity was downregulated by both 3-Isobutyl-1-methylxanthine (IBMX) and insulin upon induction of differentiation in 3T3-L1 adipogenic cells. In line with this observation, PLD activity decreased in both high fat diet (HFD)-fed mice and ob/ob mice. We also found that differentiation of 3T3-L1 preadipocytes was enhanced by the depletion of PLD1 levels or inhibition of PLD1 activity by VU0155069, a PLD1-specific inhibitor. Conversely, treatment with phosphatidic acid (PA), a PLD product, and overexpression of PLD1 both caused a decrease in adipogenic differentiation. Moreover, the elevated differentiation in PLD1-knockdown 3T3-L1 cells was reduced by either PA treatment or PLD1 expression, confirming negative roles of PLD1 and PA in adipogenic differentiation. Further investigation revealed that PA displaces DEP domain-containing mTOR-interacting protein (DEPTOR) from mTORC1, which subsequently phosphorylates insulin receptor substrate-1 (IRS-1) at serine 636/639 in 3T3-L1 cells. Taken together, our findings provide convincing evidence for a direct role of PLD1 in adipogenic differentiation by regulating IRS-1 phosphorylation at serine 636/639 through DEPTOR displacement and mTOR activation. PMID:27872488

  18. Serine hydroxymethyltransferase from the cold adapted microorganism Psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity.

    PubMed

    Angelaccio, Sebastiana; Florio, Rita; Consalvi, Valerio; Festa, Guido; Pascarella, Stefano

    2012-01-01

    Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  19. A Kazal-type serine proteinase inhibitor from Cyclina sinensis is involved in immune response and signal pathway initiation.

    PubMed

    Ren, Yipeng; Zhang, Hao; Pan, Baoping; Yan, Chuncai

    2015-11-01

    Serine protease inhibitors (SPIs) are an important group of protease inhibitors involved in a variety of biological processes. In the present study, a Kazal-type serine protease inhibitor homolog gene (designated as CsKPI) was identified from a Cyclina sinensis cDNA library. The open reading frame consists of 456 bp and encodes a protein of 151 amino acid residues with a theoretical molecular mass of 16.85 kDa and an isoelectric point of 5.74. Furthermore, using quantitative real-time PCR, we focused on the expression patterns of CsKPI found in tissues and on the stimulation of this gene's expression by bacteria. The results show that a higher-level mRNA expression of CsKPI was detected in hemocytes (P < 0.05) and was significantly upregulated at 3 h (P < 0.01) upon receiving bacterial challenges with Vibrio anguillarum. In addition, after the CsKPI gene was silenced by RNA interference, the expression of the CsTLR2 and CsMyD88 genes was extremely significantly decreased (P < 0.01) in C. sinensis. Finally, the recombinant CsKPI (rCsKPI) protein was purified and shown to exhibit less inhibitory activity than C-lyz against V. anguillarum in vitro. Hence, we propose that CsKPI plays an important role in the innate immunity and mediates TLR2 and MyD88-dependent pathway initiation in C. sinensis.

  20. Thermostability and reactivity in organic solvent of O-phospho-L-serine sulfhydrylase from hyperthermophilic archaeon Aeropyrum pernix K1.

    PubMed

    Nakamura, Takashi; Asai, Shinji; Nakata, Kaori; Kunimoto, Kohei; Oguri, Masateru; Ishikawa, Kazuhiko

    2015-01-01

    O-phospho-l-serine sulfhydrylase (OPSS) from archaeon Aeropyrum pernix K1 is able to synthesize l-cysteine even at 80 °C. In this article, we compared thermal stability and reactivity in organic solvent of OPSS with those of O-acetyl-l-serine sulfhydrylase B (OASS-B) from Escherichia coli. As a result, the thermostability of OPSS was much higher than that of OASS-B. Moreover, the activity of OPSS increased in the reaction mixture containing the organic solvent, such as N, N'-dimethyl formamide and 1,4-dioxane, whereas that of OASS-B gradually decreased as the content of organic solvent increased. From the crystal structural analysis, the intramolecular electrostatic interactions of N-terminal domain in OPSS seemed to be correlated with the tolerance of OPSS to high temperature and organic solvent. These results indicate that OPSS is more superior to OASS-B for the industrial production of l-cysteine and unnatural amino acids that are useful pharmaceuticals in the presence of organic solvent.

  1. 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.

  2. 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

  3. Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux.

    PubMed

    Ost, Mario; Keipert, Susanne; van Schothorst, Evert M; Donner, Verena; van der Stelt, Inge; Kipp, Anna P; Petzke, Klaus-Jürgen; Jove, Mariona; Pamplona, Reinald; Portero-Otin, Manuel; Keijer, Jaap; Klaus, Susanne

    2015-04-01

    Recent studies on mouse and human skeletal muscle (SM) demonstrated the important link between mitochondrial function and the cellular metabolic adaptation. To identify key compensatory molecular mechanisms in response to chronic mitochondrial distress, we analyzed mice with ectopic SM respiratory uncoupling in uncoupling protein 1 transgenic (UCP1-TG) mice as model of muscle-specific compromised mitochondrial function. Here we describe a detailed metabolic reprogramming profile associated with mitochondrial perturbations in SM, triggering an increased protein turnover and amino acid metabolism with induced biosynthetic serine/1-carbon/glycine pathway and the longevity-promoting polyamine spermidine as well as the trans-sulfuration pathway. This is related to an induction of NADPH-generating pathways and glutathione metabolism as an adaptive mitohormetic response and defense against increased oxidative stress. Strikingly, consistent muscle retrograde signaling profiles were observed in acute stress states such as muscle cell starvation and lipid overload, muscle regeneration, and heart muscle inflammation, but not in response to exercise. We provide conclusive evidence for a key compensatory stress-signaling network that preserves cellular function, oxidative stress tolerance, and survival during conditions of increased SM mitochondrial distress, a metabolic reprogramming profile so far only demonstrated for cancer cells and heart muscle.

  4. Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation.

    PubMed

    Petsalaki, Eleni; Dandoulaki, Maria; Morrice, Nick; Zachos, George

    2014-09-15

    Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM.

  5. The circadian Clock gene regulates acrosin activity of sperm through serine protease inhibitor A3K

    PubMed Central

    Cheng, Shuting; Liang, Xin; Wang, Yuhui; Jiang, Zhou; Liu, Yanyou; Hou, Wang; Li, Shiping; Zhang, Jing

    2015-01-01

    Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements. PMID:26264441

  6. Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance

    PubMed Central

    Hou, Ming-Zhe; Shen, Guang-Mao; Wei, Dong; Li, Ya-Li; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 μg/g β-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with β-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis. PMID:24566149

  7. Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.

    PubMed

    Ahn, Mi Young; Hahn, Bum-Soo; Ryu, Kang Sun; Hwang, Jae Sam; Kim, Yeong Shik

    2005-07-01

    Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

  8. 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

  9. 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

  10. Serine-Rich Repeat Adhesins Contribute to Streptococcus gordonii-Induced Maturation of Human Dendritic Cells

    PubMed Central

    Ko, Eun Byeol; Kim, Sun Kyung; Seo, Ho Seong; Yun, Cheol-Heui; Han, Seung Hyun

    2017-01-01

    Dendritic cells (DCs) play a pivotal role in the induction of immunity by recognition, capture, process, and presentation of antigens from infectious microbes. Streptococcus gordonii is able to cause life-threatening systemic diseases such as infective endocarditis. Serine-rich repeat (SRR) glycoproteins of S. gordonii are sialic acid-binding adhesins mediating the bacterial adherence to the host and the development of infective endocarditis. Thus, the SRR adhesins are potentially involved in the bacterial adherence to DCs and the maturation and activation of DCs required for the induction of immunity to S. gordonii. Here, we investigated the phenotypic and functional changes of human monocyte-derived DCs treated with wild-type S. gordonii or the SRR adhesin-deficient mutant. The mutant poorly bound to DCs and only weakly increased the expression of CD83, CD86, MHC class II, and PD-L1 on DCs compared with the wild-type. In addition, the mutant induced lower levels of TNF-α, IL-6, and IL-12 than the wild-type in DCs. When DCs sensitized with the mutant were co-cultured with autologous T cells, they induced weaker proliferation and activation of T cells than DCs stimulated with the wild-type. Blockade of SRR adhesin with 3′-sialyllactose markedly reduced S. gordonii binding and internalization, causing attenuation of the bacterial immunostimulatory potency in DC maturation. Collectively, our results suggest that SRR adhesins of S. gordonii are important for maturation and activation of DCs.

  11. Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168.

    PubMed Central

    Roitsch, C A; Hageman, J H

    1983-01-01

    Bacillopeptidase F is a serine endopeptidase excreted by Bacillus subtilis 168 after the end of exponential growth. As a step toward discovering a physiological function for this protease, an enzymological and immunological study was undertaken. When bacillopeptidase F was purified at pH 10, a number of enzymically active, rapidly moving electrophoretic forms were observed, as had been previously reported. Rabbit antiserum was prepared against one form. When the enzyme was purified at pH 6.0 in the presence of the covalent inhibitor phenylmethylsulfonyl fluoride, using the rabbit antiserum to detect the bacillopeptidase F protein, no fast-moving electrophoretic forms were observed. Instead, only two forms of the enzyme were isolated. One form had a molecular weight of 33,000, and the other had a molecular weight of 50,000, as determined by equilibrium sedimentation methods. Both forms appeared to be glycoproteins, both contained compounds, released on acid hydrolysis, which cochromatographed with phosphoserine and galactosamine, and the two gave identical immunoprecipitin lines in Ouchterlony double-diffusion tests. The smaller form had a pI of 4.4, whereas the larger had a pI of 5.4. The data suggest that bacillopeptidase F is distinct from all other proteases of B. subtilis. Images PMID:6408058

  12. Mechanism-Based Inhibitors of Serine Proteases with High Selectivity Through Optimization of S’ Subsite Binding

    PubMed Central

    Li, Yi; Dou, Dengfeng; He, Guijia; Lushington, Gerald H.; Groutas, William C.

    2009-01-01

    A series of mechanism-based inhibitors designed to interact with the S’ subsites of serine proteases was synthesized and their inhibitory activity toward the closely-related serine proteases human neutrophil elastase (HNE) and proteinase 3 (PR 3) was investigated. The compounds were found to be time-dependent inhibitors of HNE and were devoid of any inhibitory activity toward PR 3. The results suggest that highly selective inhibitors of serine proteases whose primary substrate specificity and active sites are similar can be identified by exploiting differences in their S’ subsites. The best inhibitor (compound 16) had a kinact/KI value of 4580 M−1 s−1. PMID:19394830

  13. Solubility, thermal, photoconductivity and laser damage threshold studies on L-serine acetate (LSA) single crystal

    NASA Astrophysics Data System (ADS)

    Rajesh, K.; Thayanithi, V.; Mani, A.; Amudha, M.; Kumar, P. Praveen

    2015-06-01

    L-serine acetate crystal was grown by slow evaporation technique. Solubility of L-Serine Acetate was determined at different temperatures. L-Serine Acetate was characterized by SEM is to identify the morphology of the crystal. TG and DTA study reveals the thermal stability of the grown crystal. Dielectric measurement was carried out for different temperature ranges. Photo conductivity study revealed the nature of conductivity of the crystal under halogen light. Laser damage threshold of the crystal was measured using Nd:YAG laser source. NLO property of the crystal is confirmed by Kurtz-Perry powder technique.

  14. Metabolic flux analyses for serine alkaline protease production.

    PubMed

    Çalik; Çalik; Takaç; Özdamar

    2000-12-01

    The intracellular metabolic fluxes through the central carbon pathways in Bacillus licheniformis in serine alkaline protease (SAP) production were calculated to predict the potential strategies for increasing the performance of the bacilli, by using two optimization approaches, i.e. the theoretical data-based (TDA) and the theoretical data-based capacity (TDC) analyses based on respectively minimum in-vivo SAP accumulation rate and maximum SAP synthesis rate assumptions, at low-, medium-, and high-oxygen transfer conditions. At all periods of low-oxygen transfer condition, in lag and early exponential periods of medium-oxygen transfer (MOT) condition, and SAP synthesis period of high-oxygen transfer (HOT) condition, the TDA and TDC analyses revealed that SAP overproduction capacity is almost equal to the observed SAP production due to the regulation effect of the oxygen transfer. In the growth and early SAP synthesis period and in SAP synthesis period at MOT condition the calculated results of the two analyses reveal that SAP synthesis rate of the microorganism can be increased 7.2 and 16.7 folds, respectively; whereas, in the growth and early SAP synthesis period at HOT condition it can be increased 12.6 folds. The diversions in the biochemical reaction network and the influence of the oxygen transfer on the performance of the bacilli were also presented. The results encourage the application of metabolic engineering for lifting the rate limitations and for improving the genetic regulations in order to increase the SAP production.

  15. Serine phosphorylation and arginine methylation at the crossroads to neurodegeneration.

    PubMed

    Basso, Manuela; Pennuto, Maria

    2015-09-01

    Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, polyglutamine diseases and motor neuron diseases, are late-onset and progressive disorders characterized by the accumulation of misfolded proteins inside and outside neurons. No effective therapies exist to delay the onset or arrest the progression of these diseases. One novel and promising therapeutic approach consists of targeting disease-causing proteins at the post-translational level. Here we illustrate this concept using the example of spinal and bulbar muscular atrophy, a neurodegenerative disease caused by polyglutamine expansion in the androgen receptor. Emerging evidence suggests that two key post-translational modifications of polyglutamine-expanded androgen receptor, namely serine phosphorylation by protein kinase B/Akt and arginine methylation by protein arginine methyltransferases, occur at the same consensus site, are mutually exclusive, and have opposing effects on neurotoxicity. Because several proteins linked to neurodegenerative diseases have canonical Akt consensus site motifs, these findings may have a broad impact in the field of neurological diseases caused by misfolded proteins.

  16. 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).

  17. 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.

  18. Phosphorylation at serine 331 is required for Aurora B activation

    PubMed Central

    Petsalaki, Eleni; Akoumianaki, Tonia; Black, Elizabeth J.; Gillespie, David A.F.

    2011-01-01

    Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora BS331A, in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation. PMID:22024163

  19. 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

  20. Loop B serine of a plasma membrane aquaporin type PIP2 but not PIP1 plays a key role in pH sensing.

    PubMed

    Yaneff, Agustín; Sigaut, Lorena; Gómez, Natalia; Aliaga Fandiño, Cecilia; Alleva, Karina; Pietrasanta, Lía Isabel; Amodeo, Gabriela

    2016-11-01

    In the plant kingdom, the plasma membrane intrinsic aquaporins (PIPs) constitute a highly conserved group of water channels with the capacity of rapidly adjusting the water permeability (Pf) of a cell by a gating response. Most evidence regarding this mechanism was obtained by different biophysical approaches including the crystallization of a Spinaca olaracea PIP2 aquaporin (SoPIP2;1) in an open and close conformation. A close state seems to prevail under certain stimuli such as cytosolic pH decrease, intracellular Ca(2+) concentration increase and dephosphorylation of specific serines. In this work we decided to address whether the state of phosphorylation of a loop B serine - highly conserved in all PIPs - combined with cytosolic acidification can jointly affect the gating response. To achieve this goal we generated loop B serine mutants of two PIP types of Fragaria×ananassa (FaPIP2;1S121A and FaPIP1;1S131A) in order to simulate a dephosphorylated state and characterize their behavior in terms of Pf and pH sensitivities. The response was tested for different co-expressions of PIPs (homo and heterotetramers combining wild-type and mutant PIPs) in Xenopus oocytes. Our results show that loop B serine phosphorylation status affects pH gating of FaPIP2;1 but not of FaPIP1;1 by changing its sensitivity to more alkaline pHs. Therefore, we propose that a counterpoint of different regulatory mechanisms - heterotetramerization, serine phosphorylation status and pH sensitivity - affect aquaporin gating thus ruling the Pf of a membrane that expresses PIPs when fast responses are mandatory.

  1. Phosphoacceptors Threonine 162 and Serines 170 and 178 within the Carboxyl-Terminal RRRS/T Motif of the Hepatitis B Virus Core Protein Make Multiple Contributions to Hepatitis B Virus Replication

    PubMed Central

    Jung, Jaesung; Hwang, Seong Gyu; Chwae, Yong-Joon; Park, Sun; Shin, Ho-Joon

    2014-01-01

    and in the overlapping consensus RxxS/T, RRxS/T, and TP motifs. Here we show, for the first time, that in addition to the well-known phosphoacceptor serines 157, 164, and 172 in SPRRR motifs, threonine 162 and serines 170 and 178 in the RRRS/T motif are phosphorylated in cells. We also show that, like serines 157, 164, and 172, phosphorylated and dephosphorylated threonine 162 and serines 170 and 178 contribute to multiple steps of HBV replication, including pgRNA encapsidation, minus-strand and plus-strand DNA synthesis, and relaxed-circular DNA synthesis. Of these residues, threonine 162 is the most important. Furthermore, we show that phosphorylation of C protein is required for efficient completion of HBV replication. PMID:24850741

  2. 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

  3. 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.

  4. Genome-wide survey of putative Serine/Threonine protein kinases in cyanobacteria

    PubMed Central

    Zhang, Xiaowen; Zhao, Fangqing; Guan, Xiangyu; Yang, Yu; Liang, Chengwei; Qin, Song

    2007-01-01

    Background Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence

  5. Serine-based gemini surfactants with different spacer linkages: from self-assembly to DNA compaction.

    PubMed

    Silva, Sandra G; Oliveira, Isabel S; do Vale, M Luísa C; Marques, Eduardo F

    2014-12-14

    Cationic gemini surfactants have strong potential as compaction agents of nucleic acids for efficient non-viral gene delivery. In this work, we present the aggregation behavior of three novel cationic serine-based gemini surfactants as well as their ability to compact DNA per se and mixed with a helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). All the surfactants have a 12-12-12 configuration, i.e. two main 12-carbon alkyl chains linked to the nitrogen atom of the amino acid residue and a 12 methylene spacer, but they differ in the nature of the spacer linkage: for (12Ser)2N12, an amine bond; for (12Ser)2CON12, an amide bond; and for (12Ser)2COO12, an ester bond. Interestingly, while the amine-based gemini aggregates into micelles, the amide and ester ones spontaneously form vesicles, which denotes a strong influence of the type of linkage on the surfactant packing parameter. The size, ζ-potential and stability of the vesicles have been characterized by light microscopy, cryogenic scanning electron microscopy (cryo-SEM) and dynamic light scattering (DLS). The interaction of the gemini aggregates with DNA at different charge ratios and in the absence and presence of DOPE has been studied by DLS, fluorescence spectroscopy and cryo-SEM. All the compounds are found to efficiently compact DNA (complexation > 90%), but relevant differences are obtained in terms of the size, ζ-potential and stability of the lipoplexes formed. Results are rationalized in terms of headgroup differences and the type of aggregates present prior to DNA condensation.

  6. Domain Requirements of the JIL-1 Tandem Kinase for Histone H3 Serine 10 Phosphorylation and Chromatin Remodeling in Vivo*

    PubMed Central

    Li, Yeran; Cai, Weili; Wang, Chao; Yao, Changfu; Bao, Xiaomin; Deng, Huai; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2013-01-01

    The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH2-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo. We show that a JIL-1 construct without the NH2-terminal domain is without H3S10 phosphorylation activity despite the fact that it localizes properly to polytene interband regions and that it contains both kinase domains. JIL-1 is a double kinase, and we demonstrate that both kinase domains of JIL-1 are required to be catalytically active for H3S10 phosphorylation to occur. Furthermore, we provide evidence that JIL-1 is phosphorylated at serine 424 and that this phosphorylation is necessary for JIL-1 H3S10 phosphorylation activity. Thus, these data are compatible with a model where the NH2-terminal domain of JIL-1 is required for chromatin complex interactions that position the kinase domain(s) for catalytic activity in the context of the state of higher order nucleosome packaging and chromatin structure and where catalytic H3S10 phosphorylation activity mediated by the first kinase domain is dependent on autophosphorylation of serine 424 by the second kinase domain. Furthermore, using a lacO repeat tethering system to target mutated JIL-1 constructs with or without catalytic activity, we show that the epigenetic H3S10 phosphorylation mark itself functions as a causative regulator of chromatin structure independently of any structural contributions from the JIL-1 protein. PMID:23723094

  7. Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

    PubMed Central

    Burnside, Kellie; Lembo, Annalisa; de los Reyes, Melissa; Iliuk, Anton; BinhTran, Nguyen-Thao; Connelly, James E.; Lin, Wan-Jung; Schmidt, Byron Z.; Richardson, Anthony R.; Fang, Ferric C.; Tao, Weiguo Andy; Rajagopal, Lakshmi

    2010-01-01

    Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. PMID:20552019

  8. Confluence induced threonine41/serine45 phospho-β-catenin dephosphorylation via ceramide-mediated activation of PP1cγ

    PubMed Central

    Marchesini, Norma; Jones, Jeffrey A.; Hannun, Yusuf A.

    2008-01-01

    It was previously observed that cell confluence induced up-regulation of neutral sphingomyelinase 2 (nSMase2) and increased ceramide levels (Marchesini N, Osta W, Bielawski J, Luberto C, Obeid LM and Hannun YA. (2004) J Biol Chem, 279, 25101−11). In this study, we show that, in MCF7 cells, confluence induces the dephosphorylation of phosphorylated-β-catenin at threonine41/serine45. The effect of confluence on β-catenin dephosphorylation was prevented by down regulation of nSMase2 using siRNA; reciprocally, exogenous addition of short or very long chain ceramides induced dephosphorylation of β-catenin. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid prevented β-catenin dephosphorylation during confluence. The specific phosphatase involved was determined by studies using siRNA against the major serine/threonine phosphatases, and the results showed that a specific siRNA against PP1cγ prevented dephosphorylation of β-catenin. Moreover, exogenous ceramides and confluence were found to induce the translocation of PP1cγ to the plasma membrane. All together these results establish: A) a specific intracellular pathway involving the activation of PP1 to mediate the effects of confluence-induced β-catenin dephosphorylation and B) PP1 as a lipid-regulated protein phosphatase downstream of nSMase2/ceramide. Finally, evidence is provided for a role for this pathway in regulating cell motility during confluence. PMID:17996206

  9. O-Phospho-L-serine and the Thiocarboxylated Sulfur Carrier Protein CysO-COSH are Substrates for CysM, a Cysteine Synthase from Mycobacterium tuberculosis†

    PubMed Central

    O’Leary, Seán E.; Jurgenson, Christopher T.; Ealick, Steven E.; Begley, Tadhg P.

    2009-01-01

    The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, the expression of which is upregulated under conditions of oxidative stress, was studied by transient-state kinetic techniques. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine sulfhydrylases and employs a similar chemical mechanism involving a stable α-aminoacrylate intermediate. However, we show that specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine, suggesting that O-phospho-L-serine is the likely substrate in vivo. We also investigated the kinetics of the carbon-sulfur bond-forming reaction between the CysM-bound α-aminoacrylate intermediate and the thiocarboxylated sulfur-carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than three orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, whilst the reaction with bisulfide is consistent with a rapid equilibrium binding model. We interpret this finding to suggest that the CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer. This study represents the first detailed kinetic characterization of sulfide transfer from a sulfide carrier protein. PMID:18842002

  10. O-phospho-L-serine and the thiocarboxylated sulfur carrier protein CysO-COSH are substrates for CysM, a cysteine synthase from Mycobacterium tuberculosis.

    PubMed

    O'Leary, Seán E; Jurgenson, Christopher T; Ealick, Steven E; Begley, Tadhg P

    2008-11-04

    The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, was studied by transient-state kinetic techniques. The expression of which is upregulated under conditions of oxidative stress. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine sulfhydrylase family and employs a similar chemical mechanism involving a stable alpha-aminoacrylate intermediate. However, we show that specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine, suggesting that O-phospho-L-serine is the likely substrate in vivo. We also investigated the kinetics of the carbon-sulfur bond-forming reaction between the CysM-bound alpha-aminoacrylate intermediate and the thiocarboxylated sulfur carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than 3 orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, while the reaction with bisulfide is consistent with a rapid equilibrium binding model. We interpret this finding to suggest that the CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer. This study represents the first detailed kinetic characterization of sulfide transfer from a sulfide carrier protein.

  11. 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.

  12. Change in haloacetic acid formation potential during UV and UV/H2O2 treatment of model organic compounds.

    PubMed

    Sakai, Hiroshi; Autin, Olivier; Parsons, Simon

    2013-07-01

    Haloacetic acids (HAAs) are disinfection by-products produced by the chlorination of organic matter, including amino acids. Advanced oxidation processes are expected to be effective for the destruction of HAA precursors; however, recent studies have reported the possible failure of these processes to reduce HAA formation potential. This study examined HAA formation potential during the course of UV or UV/H2O2 treatment of three organic compounds: leucine, serine, and resorcinol. HAA formation potential decreased in the treatment of resorcinol, while the potential increased slightly in the treatment of serine and greatly increased for leucine. The chemical structure required for HAA formation was assumed to be produced during the course of UV/H2O2 treatment of leucine and serine. Also, H abstraction from the δ carbon was assumed to result from the initial degradation of leucine by the hydroxyl radical during the UV/H2O2 treatment. The hydroxyl radical may have further reacted with leucine moiety to shorten its carbon chain. This would have produced a chemical structure capable of forming HAA, thus increasing HAA formation potential.

  13. Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction.

    PubMed

    Martínez-Florensa, Mario; García-Blesa, Antonio; Yélamos, José; Muñoz-Suano, Alba; Domínguez-Villar, Margarita; Valdor, Rut; Alonso, Antonio; García-Cózar, Francisco; Aparicio, Pedro; Malissen, Bernard; Aguado, Enrique

    2011-01-01

    The adaptor protein LAT has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine-phosphorylated and thereby, recruits to the membrane several proteins implicated in the activation of downstream signaling pathways. However, little is known about the role of other conserved motifs present in the LAT sequence. Here, we report that the adaptor LAT contains several conserved serine-based motifs, which are essential for proper signal transduction through the TCR. Mutation of these serine motifs in the human T cell line Jurkat prevents proper calcium influx, MAPK activation, and IL-2 production in response to TCR/CD3 stimulation. Moreover, this mutant form of LAT has a reduced ability to bind to PLC-γ1 and SLP-76, although phosphorylation of tyrosine residues 132, 171, and 191 is not decreased, raising a possible role for the serine-based motifs of LAT for the binding of important partners. The functional role of LAT serine-based motifs in signal transduction could be mediated by an effect on tyrosine phosphorylation, as their mutation significantly diminishes the phosphorylation of tyrosine residue 226. In addition, these serine motifs seem to have a regulatory role, given that upon their mutation, ZAP-70 shows enhanced phosphorylation. Therefore, the LAT serine-based motifs likely regulate signaling pathways that are essential for T cell physiology.

  14. Fluoxetine and citalopram decrease microglial release of glutamate and D-serine to promote cortical neuronal viability following ischemic insult.

    PubMed

    Dhami, K S; Churchward, M A; Baker, G B; Todd, K G

    2013-09-01

    Depression is one of the most common disorders appearing following a stroke, and is also a major factor limiting recovery and rehabilitation in stroke patients. Antidepressants are the most common prescribed treatment for depression and have shown to have anti-inflammatory properties within the central nervous system (CNS). The major source of pro-inflammatory factors within the CNS is from activated microglia, the innate immune cells of the CNS. Antidepressants have been shown to promote midbrain and hippocampal neuronal survival following an ischemic insult and this survival is mediated through the anti-inflammatory effects on microglia, but the effects on cortical neuronal survival after this insult have yet to be investigated. The present study aimed to test and compare antidepressants from three distinct classes (tricyclics, monoamine oxidase inhibitors, and selective serotonin-reuptake inhibitors [SSRIs]) on the release of inflammatory factors and amino acids from activated microglia and whether altering this release could affect cortical neuronal viability after an ischemic insult. Primary microglia were treated with 1 μg/ml LPS and/or 10 μM antidepressants, and the various factors released into medium were assayed. Co-cultures consisting of microglia and primary cortical neurons were used to assess the effects of antidepressant-treated activated microglia on the viability of ischemic injured neurons. Of the antidepressants tested, most decreased the release of the proinflammatory factors nitric oxide, tumor necrosis factor-alpha, and interleukin 1-beta from activated microglia. Fluoxetine and citalopram, the SSRIs, decreased the release of the amino acids glutamate and d-serine from LPS-activated microglia. oxygen-glucose deprived (OGD) cortical neurons cocultured with LPS-activated microglia pre-treated with fluoxetine and citalopram showed greater survival compared to injured neurons co-cultured with untreated activated microglia. Microglial release of

  15. Single crystals of L-O-serine phosphate X-irradiated at low temperatures: EPR, ENDOR, EIE, and DFT studies.

    PubMed

    Øhman, Kjell Tage; Sanderud, Audun; Hole, Eli Olaug; Sagstuen, Einar

    2006-08-10

    Single crystals of the phosphorylated amino acid L-O-serine phosphate were X-irradiated and studied at 10 K and at 77 K using EPR, ENDOR, and EIE techniques. Two radicals, R1(10 K) and R1(77 K), were detected and characterized as two different geometrical conformations of the protonated reduction product >CH-C(OH)(2). R1(10 K) is only observed after irradiation at 10 K, and upon heating to 40 K, R1(10 K) transforms rapidly and irreversibly into R1(77 K). The transition from R1(10 K) to R1(77 K) strongly increases the isotropic hyperfine coupling of the C-CH(beta) coupling (Delta = 32 MHz) and the major C-OH(beta) coupling (Delta = 47 MHz), in sharp contrast to the their much reduced anisotropic hyperfine couplings after the transition. An umbrella-like inversion of the carboxylic acid center, accompanied by minor geometrical adjustments, explains the changes of these observed isotropic and anisotropic couplings. DFT calculations were done on the reduced and protonated L-O-serine phosphate radical at the B3LYP/6-311+G(2df,p)//B3LYP/6-31+G(d) level of theory in order to support the experimental observations. Two different conformations of the anion radical, related by an inversion at the carboxylic center, could be found within the single molecule partial energy-optimization scheme. These two conformations reproduce the experimental hyperfine couplings from radicals R1(10 K) and R1(77 K). A third radical, radical R2, was observed experimentally at both 10 and 77 K and was shown to be due to the decarboxylated L-O-serine phosphate oxidation product, a conclusion fully supported from the DFT calculations. Upon thermal annealing from 77 to 295 K, radicals R1(77 K) and R2 disappeared and all three previously observed room-temperature radicals could be observed. No phosphate-centered radicals could be observed at any temperatures, indicating that the phosphate-ester bond break for one of the room-temperature radicals does not occur by dissociative electron capture at the

  16. Neurotransmitter transporter family including SLC6A6 and SLC6A13 contributes to the 5-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX and photodamage, through uptake of ALA by cancerous cells.

    PubMed

    Tran, Tai Tien; Mu, Anfeng; Adachi, Yuka; Adachi, Yasushi; Taketani, Shigeru

    2014-01-01

    δ-Aminolevulinic acid (ALA)-induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA-induced accumulation of protoporphyrin by the treatment of colon cancer DLD-1 and epithelial cancer HeLa cells with γ-aminobutyric acid (GABA)-related compounds. When the cells were treated with GABA, taurine and β-alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA- and ALA methylester-dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA-treated cells were exposed to white light, the extent of photodamage increased in SLC6A6- and SLC6A13-expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD-1 and HeLa cells decreased the ALA-induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA-induced accumulation of protoporphyrin in cancerous cells.

  17. Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils

    PubMed Central

    1990-01-01

    Closely similar but nonidentical NH2-terminal amino acid sequences have been reported for a protein or proteins in human neutrophils whose bioactivities is/are diverse (as a serine protease, antibiotic, and Wegener's granulomatosis autoantigen) but that share(s) several features: localization in the azurophil granules, a molecular mass of approximately 29 kD, reactivity with diisopropylfluorophosphate, and the ability to degrade elastin. We previously purified one such entity, termed p29b. Using a monospecific antibody, we have cloned from human bone marrow a cDNA encoding the complete p29b protein in its mature form, along with pre- and pro-sequences. The predicted amino acid sequence agrees closely with the NH2-terminal sequence obtained previously from purified p29b, as well as with sequences newly obtained from CNBr fragments. The primary structure is highly homologous to elastase, cathepsin G, T cell granzymes, and other serine proteases, and shares both the catalytic triad and substrate binding pocket of elastase. Hybridization of the full-length cDNA with restriction enzyme digests of human genomic DNA revealed only one fragment. This suggests that the closely related species described previously are the same, and can be subsumed by the term used for the first-described activity, proteinase 3. Proteinase 3 is more abundant in neutrophils than elastase and has a similar proteolytic profile and specific activity. Thus, proteinase 3 may share the role previously attributed to neutrophil elastase in tissue damage, and has the potential to function as an antimicrobial agent. PMID:2258701

  18. 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 ...

  19. Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

    PubMed

    Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

    2014-09-15

    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.

  20. 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.

  1. The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish

    PubMed Central

    van der Velden, Yme U.; Wang, Liqin; Zevenhoven, John; van Rooijen, Ellen; van Lohuizen, Maarten; Giles, Rachel H.; Clevers, Hans; Haramis, Anna-Pavlina G.

    2011-01-01

    Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz–Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions. AMPK phosphorylation in turn, affects several processes, including inhibition of the target of rapamycin (TOR) pathway, and leads to proliferation inhibition. To gain insight into how LKB1 mediates its effects during development, we generated zebrafish mutants in the single LKB1 ortholog. We show that in zebrafish lkb1 is dispensable for embryonic survival but becomes essential under conditions of energetic stress. After yolk absorption, lkb1 mutants rapidly exhaust their energy resources and die prematurely from starvation. Notably, intestinal epithelial cells were polarized properly in the lkb1 mutants. We show that attenuation of metabolic rate in lkb1 mutants, either by application of the TOR inhibitor rapamycin or by crossing with von Hippel–Lindau (vhl) mutant fish (in which constitutive hypoxia signaling results in reduced metabolic rate), suppresses key aspects of the lkb1 phenotype. Thus, we demonstrate a critical role for LKB1 in regulating energy homeostasis at the whole-organism level in a vertebrate. Zebrafish models of Lkb1 inactivation could provide a platform for chemical genetic screens to identify compounds that target accelerated metabolism, a key feature of tumor cells. PMID:21368212

  2. 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

  3. Identification and characterization of a novel serine-threonine kinase gene from the Xp22 region.

    PubMed

    Montini, E; Andolfi, G; Caruso, A; Buchner, G; Walpole, S M; Mariani, M; Consalez, G; Trump, D; Ballabio, A; Franco, B

    1998-08-01

    Eukaryotic protein kinases are part of a large and expanding family of proteins. Through our transcriptional mapping effort in the Xp22 region, we have isolated and sequenced the full-length transcript of STK9, a novel cDNA highly homologous to serine-threonine kinases. A number of human genetic disorders have been mapped to the region where STK9 has been localized including Nance-Horan (NH) syndrome, oral-facial-digital syndrome type 1 (OFD1), and a novel locus for nonsyndromic sensorineural deafness (DFN6). To evaluate the possible involvement of STK9 in any of the above-mentioned disorders, a 2416-bp full-length cDNA was assembled. The entire genomic structure of the gene, which is composed of 20 coding exons, was determined. Northern analysis revealed a transcript larger than 9.5 kb in several tissues including brain, lung, and kidney. The mouse homologue (Stk9) was identified and mapped in the mouse in the region syntenic to human Xp. This location is compatible with the location of the Xcat mutant, which shows congenital cataracts very similar to those observed in NH patients. Sequence homologies, expression pattern, and mapping information in both human and mouse make STK9 a candidate gene for the above-mentioned disorders.

  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. 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 Central

    Mégroz, Marianne; Kleifeld, Oded; Wright, Amy; Powell, David; Harrison, Paul; Adler, Ben; Harper, Marina

    2016-01-01

    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

  6. A novel O-phospho-L-serine sulfhydrylation reaction catalyzed by O-acetylserine sulfhydrylase from Aeropyrum pernix K1.

    PubMed

    Mino, Koshiki; Ishikawa, Kazuhiko

    2003-09-11

    O-Acetylserine sulfhydrylase (OASS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. O-Acetyl-L-serine is labile at high temperatures at which hyperthermophilic archaea live. Herein, a study of the substrate specificity of OASS from Aeropyrum pernix K1 with respect to O-acetyl-L-serine in L-cysteine synthesis is described. L-Azaserine, 3-chloro-L-alanine, and O-phospho-L-serine reacted with A. pernix OASS in a PLP-dependent manner. Sulfhydrylation reactions using these substrates reached a maximum in the pH range between 7.3 and 8.1. L-Azaserine and O-phospho-L-serine were found to be heat-stable substrates. The presence of FeCl3 or NiCl2 strongly inhibited the O-acetyl-L-serine sulfhydrylation reaction, whereas the O-phospho-L-serine sulfhydrylation reaction was only slightly inhibited. Kinetic analyses revealed that the O-phospho-L-serine sulfhydrylation reaction as well as the O-acetyl-L-serine sulfhydrylation reaction for A. pernix OASS followed a ping-pong bi-bi mechanism. In the case of the O-phospho-L-serine sulfhydrylation reaction at 85 degrees C, the K(m) values for O-phospho-L-serine and sulfide, and the rate constant were 250 mM, 12.5 mM, and 14000 s(-1), respectively. The reactivity of O-phospho-L-serine in the L-cysteine synthetic reaction provides a key for understanding the biosynthesis of L-cysteine by hyperthermophilic archaea. This is the first report of an enzyme that catalyzes the O-phospho-L-serine sulfhydrylation reaction.

  7. Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver.

    PubMed

    Wolfe, L; Bradford, A P; Klarlund, J K; Czech, M P

    1992-05-15

    A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to

  8. 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

  9. A Serine Protease Homolog Negatively Regulates TEP1 Consumption in Systemic Infections of the Malaria Vector Anopheles gambiae

    PubMed Central

    Yassine, Hassan; Kamareddine, Layla; Chamat, Soulaima; Christophides, George K.; Osta, Mike A.

    2016-01-01

    Clip domain serine protease homologs are widely distributed in insect genomes and play important roles in regulating insect immune responses, yet their exact functions remain poorly understood. Here, we show that CLIPA2, a clip domain serine protease homolog of Anopheles gambiae, regulates the consumption of the mosquito complement-like protein TEP1 during systemic bacterial infections. We provide evidence that CLIPA2 localizes to microbial surfaces in a TEP1-dependent manner whereby it negatively regulates the activity of a putative TEP1 convertase, which converts the full-length TEP1-F form into active TEP1cut. CLIPA2 silencing triggers an exacerbated TEP1-mediated response that significantly enhances mosquito resistance to infections with a broad class of microorganisms including Plasmodium berghei, Escherichia coli and the entomopathogenic fungus Beauveria bassiana. We also provide further evidence for the existence of a functional link between TEP1 and activation of hemolymph prophenoloxidase during systemic infections. Interestingly, the enhanced TEP1-mediated immune response in CLIPA2 knockdown mosquitoes correlated with a significant reduction in fecundity, corroborating the existence of a trade-off between immunity and reproduction. In sum, CLIPA2 is an integral regulatory component of the mosquito complement-like pathway which functions to prevent an overwhelming response by the host in response to systemic infections. PMID:25012124

  10. Use of tissue-engineered nerve grafts consisting of a chitosan/poly(lactic-co-glycolic acid)-based scaffold included with bone marrow mesenchymal cells for bridging 50-mm dog sciatic nerve gaps.

    PubMed

    Ding, Fei; Wu, Jian; Yang, Yumin; Hu, Wen; Zhu, Qi; Tang, Xin; Liu, Jie; Gu, Xiaosong

    2010-12-01

    Bone marrow mesenchymal cells (MSCs) have attracted increasing research interest due to their possible use as support cells for nerve tissue-engineering approaches. We developed a novel design of tissue-engineered nerve grafts consisting of a chitosan/poly(lactic-co-glycolic acid) (PLGA)-based neural scaffold included with autologous MSCs. The graft was used as an alternative to nerve autografts for bridging 50-mm-long gaps in dog sciatic nerve, and the repair outcome at 6 months after nerve grafting was evaluated by a combination of electrophysiological assessment, FluoroGold retrograde tracing, and histological investigation to regenerated nerve tissue and reinnervated target muscle. The experimental results indicated that introduction of autologous MSCs to the chitosan/PLGA-based neural scaffold promoted sciatic nerve regeneration and functional recovery, demonstrating significant efficacy that was, to a certain degree, close to that by nerve autografting, a gold standard for treating large peripheral nerve gaps, and better than that by grafting with the chitosan/PLGA-based scaffold alone.

  11. Beyond the Dopamine Receptor: Regulation and Roles of Serine/Threonine Protein Phosphatases

    PubMed Central

    Walaas, Sven Ivar; Hemmings, Hugh Caroll; Greengard, Paul; Nairn, Angus Clark

    2011-01-01

    Dopamine plays an important modulatory role in the central nervous system, helping to control critical aspects of motor function and reward learning. Alteration in normal dopaminergic neurotransmission underlies multiple neurological diseases including schizophrenia, Huntington’s disease, and Parkinson’s disease. Modulation of dopamine-regulated signaling pathways is also important in the addictive actions of most drugs of abuse. Our studies over the last 30 years have focused on the molecular actions of dopamine acting on medium spiny neurons, the predominant neurons of the neostriatum. Striatum-enriched phosphoproteins, particularly dopamine and adenosine 3′:5′-monophosphate-regulated phosphoprotein of 32 kDa (DARPP-32), regulator of calmodulin signaling (RCS), and ARPP-16, mediate pleiotropic actions of dopamine. Notably, each of these proteins, either directly or indirectly, regulates the activity of one of the three major subclasses of serine/threonine protein phosphatases, PP1, PP2B, and PP2A, respectively. For example, phosphorylation of DARPP-32 at Thr34 by protein kinase A results in potent inhibition of PP1, leading to potentiation of dopaminergic signaling at multiple steps from the dopamine receptor to the nucleus. The discovery of DARPP-32 and its emergence as a critical molecular integrator of striatal signaling will be discussed, as will more recent studies that highlight novel roles for RCS and ARPP-16 in dopamine-regulated striatal signaling pathways. PMID:21904525

  12. Global Analysis of Serine-Threonine Protein Kinase Genes in Neurospora crassa ▿ †

    PubMed Central

    Park, Gyungsoon; Servin, Jacqueline A.; Turner, Gloria E.; Altamirano, Lorena; Colot, Hildur V.; Collopy, Patrick; Litvinkova, Liubov; Li, Liande; Jones, Carol A.; Diala, Fitz-Gerald; Dunlap, Jay C.; Borkovich, Katherine A.

    2011-01-01

    Serine/threonine (S/T) protein kinases are crucial components of diverse signaling pathways in eukaryotes, including the model filamentous fungus Neurospora crassa. In order to assess the importance of S/T kinases to Neurospora biology, we embarked on a global analysis of 86 S/T kinase genes in Neurospora. We were able to isolate viable mutants for 77 of the 86 kinase genes. Of these, 57% exhibited at least one growth or developmental phenotype, with a relatively large fraction (40%) possessing a defect in more than one trait. S/T kinase knockouts were subjected to chemical screening using a panel of eight chemical treatments, with 25 mutants exhibiting sensitivity or resistance to at least one chemical. This brought the total percentage of S/T mutants with phenotypes in our study to 71%. Mutants lacking apg-1, an S/T kinase required for autophagy in other organisms, possessed the greatest number of phenotypes, with defects in asexual and sexual growth and development and in altered sensitivity to five chemical treatments. We showed that NCU02245/stk-19 is required for chemotropic interactions between female and male cells during mating. Finally, we demonstrated allelism between the S/T kinase gene NCU00406 and velvet (vel), encoding a p21-activated protein kinase (PAK) gene important for asexual and sexual growth and development in Neurospora. PMID:21965514

  13. UBE4B targets phosphorylated p53 at serines 15 and 392 for degradation

    PubMed Central

    Du, Cheng; Wu, Hong; Leng, Roger P.

    2016-01-01

    Phosphorylation of p53 is a key mechanism responsible for the activation of its tumor suppressor functions in response to various stresses. In unstressed cells, p53 is rapidly turned over and is maintained at a low basal level. After DNA damage or other forms of cellular stress, the p53 level increases, and the protein becomes metabolically stable. However, the mechanism of phosphorylated p53 regulation is unclear. In this study, we studied the kinetics of UBE4B, Hdm2, Pirh2, Cop1 and CHIP induction in response to p53 activation. We show that UBE4B coimmunoprecipitates with phosphorylated p53 at serines 15 and 392. Notably, the affinity between UBE4B and Hdm2 is greatly decreased after DNA damage. Furthermore, we observe that UBE4B promotes endogenous phospho-p53(S15) and phospho-p53(S392) degradation in response to IR. We demonstrate that UBE4B and Hdm2 repress p53S15A, p53S392A, and p53-2A(S15A, S392A) functions, including p53-dependent transactivation and growth inhibition. Overall, our results reveal that UBE4B plays an important role in regulating phosphorylated p53 following DNA damage. PMID:26673821

  14. Protein serine/threonine phosphotase-1 is essential in governing normal development of vertebrate eye.

    PubMed

    Liu, W-B; Yan, Q; Liu, F-Y; Tang, X-C; Chen, H-G; Liu, J; Nie, L; Zhang, X-W; Ji, W-K; Hu, X-H; Hu, W-F; Woodward, Z; Wu, K-L; Wu, M-X; Liu, X-L; Luo, L-X; Yu, M-B; Liu, Y-Z; Liu, S-J; Li, D W-C

    2012-12-01

    Protein serine/threonine phosphatase-1 (PP-1) is one of the key enzymes responsible for dephosphorylation in vertebrates. Protein dephosphorylation via PP-1 is implicated in many different biological processes including gene expression, cell cycle control, transformation, neuronal transmission, apoptosis, autophage and senescence. However, whether PP-1 directly controls animal development remains to be investigated. Here, we present direct evidence to show that PP-1 plays an essential role in regulating eye development of vertebrates. Using goldfish as a model system, we have shown the following novel results. First, inhibition of PP-1 activity leads to death of a majority of the treated embryos, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of each catalytic subunit of PP-1 with morpholino oligomers leads to partial (PP-lα knockdown) or complete (PP-lβ or PP-lγ knockdown) death of the injected embryos. The survived embryos from PP-1α knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each subunit of PP-1 also causes death of majority of the injected embryos and leads to abnormal development of goldfish eye. Mechanistically, Pax-6 is one of the major downstream targets mediating the effects of PP-1 function since the eye phenotype in Pax-6 knockdown fish is similar to that derived from overexpression of PP-1. Together, our results for the first time provide direct evidence that protein phosphatase-1 plays a key role in governing normal eye formation during goldfish development.

  15. Protein serine/threonine Phosphotase-2A is differentially expressed and regulates eye development in vertebrates.

    PubMed

    Liu, W-B; Hu, X-H; Zhang, X-W; Deng, M-X; Nie, L; Hui, S-S; Duan, W; Tao, M; Zhang, C; Liu, J; Hu, W-F; Huang, Z-X; Li, L; Yi, M; Li, T-T; Wang, L; Liu, Y; Liu, S-J; Li, D W-C

    2013-09-01

    Protein serine/threonine phosphatase-2A (PP-2A) is one of the key enzymes responsible for dephosphorylation in vertebrates. PP-2A-mediated dephosphorylation participates in many different biological processes including cell proliferation, differentiation, transformation, apoptosis, autophage and senescence. However, whether PP-2A directly controls animal development remains to be explored. Here, we present direct evidence to show that PP-2A displays important functions in regulating eye development of vertebrates. Using goldfish as a model system, we have demonstrated the following novel information. First, inhibition of PP-2A activity leads to significant death of the treated embryos, which is derived from blastomere apoptosis associated with enhanced phosphorylation of Bcl-XL at Ser-62, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of PP-2A with morpholino oligomers leads to significant death of the injected embryos. The survived embryos from PP-2A knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each catalytic subunit of PP-2A also causes death of majority of the injected embryos and leads to absence of goldfish eye lens or severely disturbed differentiation. Together, our results provide direct evidence that protein phosphatase-2A is important for normal eye development in goldfish.

  16. Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

    PubMed Central

    Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

    2016-01-01

    ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

  17. Serine/threonine-kinase 33 (Stk33) - Component of the neuroendocrine network?

    PubMed

    Reuss, Stefan; Brauksiepe, Bastienne; Disque-Kaiser, Ursula; Olivier, Tim

    2017-01-15

    The present study was conducted to investigate the expression of serine/threonine-kinase 33 (Stk33) in neuronal structures of the central nervous system in rat and hamster as well as the presence of the protein in the brain of higher mammals, using a polyclonal antibody on cryosections of fixed brains. We found a distinct immunostaining pattern that included intense fluorescence of the ependymal lining of cerebral ventricles, and of hypothalamic tanycytes and their processes. We further observed intense staining of magnocellular neurons in the hypothalamic paraventricular, supraoptic and accessory neurosecretory nuclei, in particular the circular nuclei, and less intense stained neurons in other diencephalic regions. Double-immunostaining experiments showed a partial colocalization of Stk33 with arginine-vasopressin, oxytocin or neuronal nitric oxide-synthase in a large number of neurons of the hypothalamic nuclear regions. Colocalization of Stk33 with substance P or the catecholamine-synthesizing enzyme tyrosine-hydroxylase was not observed. Immunofluorescence was not found in autonomic regions of the lateral horn, suggesting that Stk33 does not contribute to hypothalamo-spinal connections. However, large Stk33-immunoreactive axonal projections from magnocellular hypothalamus to the neurohypophysis were evident. These functionally important connections provide the bridge from neuronal to humoral regulation of the endocrine system. Additionally, Western blots from mouse brain showed two distinct bands representing two Stk33 isoforms. We also present first evidence for the presence of Stk33/STK33 in neuronal structures, ependymal cells and tanycytes in tree shrew, baboon, and human brain.

  18. The serine/threonine kinase Ndr2 controls integrin trafficking and integrin-dependent neurite growth.

    PubMed

    Rehberg, Kati; Kliche, Stefanie; Madencioglu, Deniz A; Thiere, Marlen; Müller, Bettina; Meineke, Bernhard Manuel; Freund, Christian; Budinger, Eike; Stork, Oliver

    2014-04-09

    Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of β1-integrin to stimulate the PKC- and CaMKII-dependent activation of β1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated β1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.

  19. Mammary serine protease inhibitor and CD138 immunohistochemical expression in ovarian serous and clear cell carcinomas.

    PubMed

    Hasby, Eiman Adel

    2016-04-01

    This study aims to investigate the immunohistochemical expression of mammary serine protease inhibitor (maspin) and CD138 in primary ovarian high-grade serous carcinomas (HGSC) as compared to low-grade serous carcinomas (LGSC) and clear cell carcinomas and investigate if the studied markers have a correlation to International Federation of Gynaecology and Obstetrics (FIGO) stage, Ki67 proliferation index, and to each other. Maspin cellular location varied significantly between studied groups with only nuclear expression seen in 46.7 % of LGSC group, mixed nuclear and cytoplasmic in 13.3, 28.6, and 20 % of LGSC, HGSC, and clear cell carcinoma, respectively, and was only cytoplasmic in 26.7, 71.4, and 80 % of LGSC, HGSC, and clear cell carcinoma, respectively. Mean maspin and CD138 counts were significantly higher in HGSC and clear cell carcinoma compared to LGSC. Both maspin and CD138 scores varied significantly between studied groups and were positively correlated with adverse prognostic factors in studied carcinomas including FIGO stage and Ki67 proliferation index. Besides, both maspin and CD138 had significant correlation to each other. These findings suggest that epithelial cytoplasmic expression of maspin and CD138 may have a significant role in tumorigenesis in ovarian high-grade serous carcinomas and clear cell carcinomas; these markers may regulate tumor cell proliferation, and their significant correlation to each other may suggest that CD138 probably induces maspin expression to protect tumor growth factors from being lysed by proteolytic enzymes.

  20. Ku70 Serine 155 mediates Aurora B inhibition and activation of the DNA damage response

    PubMed Central

    Fell, Victoria L.; Walden, Elizabeth A.; Hoffer, Sarah M.; Rogers, Stephanie R.; Aitken, Amelia S.; Salemi, Louisa M.; Schild-Poulter, Caroline

    2016-01-01

    The Ku heterodimer (Ku70/Ku80) is the central DNA binding component of the classical non-homologous end joining (NHEJ) pathway that repairs DNA double-stranded breaks (DSBs), serving as the scaffold for the formation of the NHEJ complex. Here we show that Ku70 is phosphorylated on Serine 155 in response to DNA damage. Expression of Ku70 bearing a S155 phosphomimetic substitution (Ku70 S155D) in Ku70-deficient mouse embryonic fibroblasts (MEFs) triggered cell cycle arrest at multiple checkpoints and altered expression of several cell cycle regulators in absence of DNA damage. Cells expressing Ku70 S155D exhibited a constitutive DNA damage response, including ATM activation, H2AX phosphorylation and 53BP1 foci formation. Ku70 S155D was found to interact with Aurora B and to have an inhibitory effect on Aurora B kinase activity. Lastly, we demonstrate that Ku and Aurora B interact following ionizing radiation treatment and that Aurora B inhibition in response to DNA damage is dependent upon Ku70 S155 phosphorylation. This uncovers a new pathway where Ku may relay signaling to Aurora B to enforce cell cycle arrest in response to DNA damage. PMID:27849008

  1. 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

    2010-12-07

    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.

  2. Two distinct transmembrane serine/threonine kinases from Drosophila melanogaster form an activin receptor complex.

    PubMed Central

    Wrana, J L; Tran, H; Attisano, L; Arora, K; Childs, S R; Massagué, J; O'Connor, M B

    1994-01-01

    A transmembrane protein serine/threonine kinase, Atr-I, that is structurally related to receptors for members of the transforming growth factor-beta (TGF-beta) family has been cloned from Drosophila melanogaster. The spacing of extracellular cysteines and the cytoplasmic domain of Atr-I resemble most closely those of the recently described mammalian type I receptors for TGF-beta and activin. When expressed alone in test cells, Atr-I is unable to bind TGF-beta, activin, or bone morphogenetic protein 2. However, Atr-I binds activin efficiently when coexpressed with the distantly related Drosophila activin receptor Atr-II, with which it forms a heteromeric complex. Atr-I can also bind activin in concert with mammalian activin type II receptors. Two alternative forms of Atr-I have been identified that differ in an ectodomain region encompassing the cysteine box motif characteristic of receptors in this family. Comparison of Atr-I with other type I receptors reveals the presence of a characteristic 30-amino-acid domain immediately upstream of the kinase region in all these receptors. This domain, of unknown function, contains a repeated Gly-Ser sequence and is therefore referred to as the GS domain. Maternal Atr-I transcripts are abundant in the oocyte and widespread during embryo development and in the imaginal discs of the larva. The structural properties, binding specificity, and dependence on type II receptors define Atr-I as an activin type I receptor from D. melanogaster. These results indicate that the heteromeric kinase structure is a general feature of this receptor family. Images PMID:8289834

  3. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    PubMed

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.

  4. A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins.

    PubMed Central

    Stepanov, V M; Rudenskaya, G N; Revina, L P; Gryaznova, Y B; Lysogorskaya, E N; Filippova IYu; Ivanova, I I

    1992-01-01

    A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins). Images Fig. 3. PMID:1637313

  5. D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period

    PubMed Central

    Meunier, Claire N. J.; Dallérac, Glenn; Le Roux, Nicolas; Sacchi, Silvia; Levasseur, Grégoire; Amar, Muriel; Pollegioni, Loredano; Mothet, Jean-Pierre; Fossier, Philippe

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors. PMID:27003418

  6. D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

    PubMed

    Meunier, Claire N J; Dallérac, Glenn; Le Roux, Nicolas; Sacchi, Silvia; Levasseur, Grégoire; Amar, Muriel; Pollegioni, Loredano; Mothet, Jean-Pierre; Fossier, Philippe

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

  7. Functional roles of endogenous D-serine in pain-induced ultrasonic vocalization.

    PubMed

    Tsuzuki, Hitomi; Maekawa, Masao; Konno, Ryuichi; Hori, Yuuichi

    2012-11-14

    The N-methyl-D-aspartate receptor (NMDAR) is crucial for pain-related behaviors. D-Serine is synthesized from L-serine by serine racemase (SR) and modulates NMDAR functions by acting as an agonist at the glycine-binding site. We analyzed noxious stimulus-induced ultrasonic vocalization and locomotor activity in the open-field test using SR knockout (SR-KO) mice to examine the role of endogenous D-serine in mammalian behaviors. SR-KO mice emitted less ultrasonic vocalization after noxious stimulation (VAS) than wild-type (WT) mice. The locomotor activity of WT mice decreased with repeated daily exposures to the open field, whereas that of SR-KO mice remained unchanged. VAS was significantly enhanced during arthritis in WT mice, whereas it was not enhanced during arthritis in SR-KO mice. These results indicate that mice lacking the ability to produce D-serine endogenously in the brain differ from normal mice with respect to the chronic pain-induced behavioral changes.

  8. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.

    PubMed

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  9. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei.

    PubMed

    Qin, Zhen; Yan, Qiaojuan; Ma, Qingjun; Jiang, Zhengqiang

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5'-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0-9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering.

  10. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase

    PubMed Central

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-01-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE. PMID:26790714

  11. Protein phosphatase 2C (PP2C) is responsible for VP-induced dephosphorylation of AQP2 serine 261.

    PubMed

    Cheung, Pui W; Ueberdiek, Lars; Day, Jack; Bouley, Richard; Brown, Dennis

    2017-04-05

    AQP2 trafficking is regulated by phosphorylation and dephosphorylation of serine residues in the AQP2 c-terminus. Vasopressin (VP) binding to its receptor (V2R) leads to a cascade of events that result in the phosphorylation of serine 256 (S256), S264 and S269, but dephosphorylation of S261. To identify which phosphatase is responsible for VP-induced S261 dephosphorylation, we pretreated cells with different phosphatase inhibitors before VP stimulation. Only sanguinarine, a specific protein phosphatase 2C (PP2C) inhibitor, abolished VP-induced S261 dephosphorylation, but not inhibitors of PP1, PP2A (okadaic acid) or PP2B (cyclosporine). However, both sanguinarine and VP significantly increased phosphorylation of ERK, a kinase that can phosphorylate S261; inhibition of ERK by PD98059 partially decreased baseline S261 phosphorylation. These data support a role of ERK in S261 phosphorylation, but suggest that upon VP treatment, increased phosphatase activity overcomes the increase in ERK activity, resulting in overall dephosphorylation of S261. We also found that sanguinarine abolished VP-induced S261 dephosphorylation in cells expressing mutated AQP2 S256A, suggesting that the phosphorylation state of S261 is independent of S256. Sanguinarine alone did not induce AQP2 membrane trafficking, nor did it inhibit VP-induced AQP2 membrane accumulation in cells and kidney tissues, suggesting that S261 does not play an observable role in acute AQP2 membrane accumulation. In conclusion, PP2C activity is required for S261 AQP2 dephosphorylation upon VP stimulation, and this occurs independent of S256 phosphorylation. Understanding the pathways involved in modulating PP2C will help obtain a deeper understanding of the role of S261 in cellular events involving AQP2.

  12. Serine proteinases from barley malt may degrade beta-amylase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley seed proteinases are critically important to seed germination and malting in that they generate amino acids from seed N reserves, supporting embryo growth during germination and yeast fermentation during brewing. However, relatively little is known regarding the endogenous protein substrate ...

  13. Identification and functional characterization of a novel antistasin/WAP-like serine protease inhibitor from the tropical sea cucumber, Stichopus monotuberculatus.

    PubMed

    Yan, Aifen; Ren, Chunhua; Chen, Ting; Jiang, Xiao; Sun, Hongyan; Hu, Chaoqun

    2016-10-27

    A novel antistasin/WAP-like serine protease inhibitor, named as StmAW-SPI, was identified from sea cucumber (Stichopus monotuberculatus) and functionally characterized in this study. The full-length cDNA of StmAW-SPI is 1917 bp in length with a 72 bp 5'-untranslated region (UTR), a 294 bp 3'-UTR and a 1551 bp open reading frame (ORF) encoding a protein of 516 amino acids with a deduced molecular weight of 54.56 kDa. The StmAW-SPI protein has 5-fold internal repeats (IRs) of antistasin domain and 6-fold IRs of WAP domain. For the gene structure, StmAW-SPI contains 10 exons separated by 9 introns. The StmAW-SPI mRNA expression pattern was determined using quantitative real-time PCR. The highest level of StmAW-SPI was found in the intestine, followed by coelomocytes, gonad, body wall and respiratory tree. The StmAW-SPI expressions were significantly up-regulated after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge in in vitro experiments performed in primary coelomocytes. In addition, the serine protease inhibitory activity and bacterial protease inhibitory activity of StmAW-SPI were examined, and the antibacterial activity was also demonstrated in this study. Our study, as a whole, suggested that StmAW-SPI might play a critical role in the innate immune defense of sea cucumber against microbial infections, by not only inactivating the serine protease but also inhibiting the growth of pathogens.

  14. Pump apparatus including deconsolidator

    DOEpatents

    Sonwane, Chandrashekhar; Saunders, Timothy; Fitzsimmons, Mark Andrew

    2014-10-07

    A pump apparatus includes a particulate pump that defines a passage that extends from an inlet to an outlet. A duct is in flow communication with the outlet. The duct includes a deconsolidator configured to fragment particle agglomerates received from the passage.

  15. Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex

    PubMed Central

    Drobic, Bojan; Pérez-Cadahía, Beatriz; Yu, Jenny; Kung, Sam Kam-Pun; Davie, James R.

    2010-01-01

    Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and sequential chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 the ATPase subunit of the SWI/SNF remodeler, is recruited to the promoter of target genes by transcription factors such as Elk-1 or NF-κB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of IE genes enabling the binding of transcription factors like JUN and the onset of transcription. PMID:20129940

  16. Lipopolysaccharide-induced activation of NF-{kappa}B non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations

    SciTech Connect

    Bhattacharyya, Sumit; Borthakur, Alip; Dudeja, Pradeep K.; Tobacman, Joanne K.

    2010-11-15

    Background and aims: B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-{kappa}B (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods: Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-{kappa}B activation, including nuclear RelB and p52 and phospho- and total NF-{kappa}B inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results: By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-I{kappa}B{alpha} of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions: LPS induces activation of both canonical and non-canonical pathways of NF-{kappa}B in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.

  17. On the roles of the alanine and serine in the β-sheet structure of fibroin.

    PubMed

    Carrascoza Mayen, Juan Francisco; Lupan, Alexandru; Cosar, Ciprian; Kun, Attila-Zsolt; Silaghi-Dumitrescu, Radu

    2015-02-01

    In its silk II form, fibroin is almost exclusively formed from layers of β-sheets, rich in glycine, alanine and serine. Reported here are computational results on fibroin models at semi-empirical, DFT levels of theory and molecular dynamics (MD) for (Gly)10, (Gly-Ala)5 and (Gly-Ser)5 decapeptides. While alanine and serine introduce steric repulsions, the alanine side-chain adds to the rigidity of the sheet, allowing it to maintain a properly pleated structure even in a single β-sheet, and thus avoiding two alternative conformations which would interfere with the formation of the multi-layer pleated-sheet structure. The role of the serine is proposed to involve modulation of the hydrophobicity in order to construct the supramolecular assembly as opposed to random precipitation due to hydrophobicity.

  18. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms.

    PubMed

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M

    2014-08-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding.

  19. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms

    PubMed Central

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M.

    2014-01-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding. PMID:24878526

  20. Adaptation of caddisfly larval silks to aquatic habitats by phosphorylation of h-fibroin serines.

    PubMed

    Stewart, Russell J; Wang, Ching Shuen

    2010-04-12

    Aquatic caddisflies diverged from a silk-spinning ancestor shared with terrestrial moths and butterflies. Caddisfly larva spin adhesive silk underwater to construct protective shelters with adventitiously gathered materials. A repeating (SX)(n) motif conserved in the H-fibroin of several caddisfly species is densely phosphorylated. In total, more than half of the serines in caddisfly silk may be phosphorylated. Major molecular adaptations allowing underwater spinning of an ancestral dry silk appear to have been phosphorylation of serines and the accumulation of basic residues in the silk proteins. The amphoteric nature of the silk proteins could contribute to silk fiber assembly through electrostatic association of phosphorylated blocks with arginine-rich blocks. The presence of Ca(2+) in the caddisfly larval silk proteins suggest phosphorylated serines could contribute to silk fiber periodic substructure through Ca(2+) crossbridging.

  1. TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases.

    PubMed

    Busch, Florian; Rajendran, Chitra; Mayans, Olga; Löffler, Patrick; Merkl, Rainer; Sterner, Reinhard

    2014-09-30

    The rapid increase of the number of sequenced genomes asks for the functional annotation of the encoded enzymes. We used a combined computational-structural approach to determine the function of the TrpB2 subgroup of the tryptophan synthase β chain/β chain-like TrpB1-TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases, whereas TrpB1 enzymes catalyze the l-serine dependent synthesis of tryptophan. We found a single residue being responsible for the different substrate specificities of TrpB1 and TrpB2 and confirmed this finding by mutagenesis studies and crystallographic analysis of a TrpB2 enzyme with bound O-phospho-l-serine.

  2. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Arasappan, Ashok; Venkatraman, Srikanth; Vibulbhan, Bancha; Yang, Weiying; Parekh, Tejal N.; Pichardo, John; Prongay, Andrew; Cheng, Kuo-Chi; Butkiewicz, Nancy; Yao, Nanhua; Madison, Vincent; Girijavallabhan, Viyyoor

    2008-06-30

    The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic {alpha}-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K*{sub i}). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K*{sub i} = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K*{sub i} = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC{sub 50} of 130 nM in a cellular replicon assay, while IC{sub 50} for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the