Sample records for phosphatidyl serine ps

  1. The separation of phosphatidyl ethanolamine and phosphatidyl serine by column chromatography

    Microsoft Academic Search

    George Rouser; John O'Brien; Dorothy Heller

    1961-01-01

    A method for the complete separation and apparent quantitative recovery of both phosphatidyl ethanolamine and phosphatidyl\\u000a serine from lipid extracts of beef brain is presented. Evidence for the purity, quantitative recovery, and the suitability\\u000a of the technique for the subsequent analysis of the fatty acid and fatty aldehyde composition of the phospholipids is described.

  2. Exposure of FVIII in the Presence of Phosphatidyl Serine Reduces Generation of Memory B-Cells and Induces Regulatory T-Cell-Mediated Hyporesponsiveness in Hemophilia A Mice.

    PubMed

    Ramakrishnan, Radha; Davidowitz, Andrew; Balu-Iyer, Sathy V

    2015-08-01

    A major complication of replacement therapy with Factor VIII (FVIII) for hemophilia A (HA) is the development of unwanted immune responses. Previous studies showed that administration of FVIII in the presence of phosphatidyl serine (PS) reduced the development of anti-FVIII antibodies in HA mice. However, the impact of PS-mediated effects on immunological memory, such as generation of memory B-cells, is not clear. The effect of PS on memory B-cells was therefore investigated using adoptive transfer approach in FVIII(-/-) HA mice. Adoptive transfer of memory B-cells from a PS-FVIII-treated group to naďve mice followed by challenge of the recipient mice with FVIII showed a significantly reduced anti-FVIII antibody response in the recipient mice, compared with animals that received memory B-cells from free FVIII and FVIII-charge matched phosphatidyl glycerol (PG) group. The decrease in memory B-cell response is accompanied by an increase in FoxP3 expressing regulatory T-cells (Tregs). Flow cytometry studies showed that the generation of Tregs is higher in PS-treated animals as compared with FVIII and FVIII-PG treated animals. The PS-mediated hyporesponsiveness was found to be antigen-specific. The PS-FVIII immunization showed hyporesponsiveness toward FVIII rechallenge but not against ovalbumin (OVA) rechallenge, an unrelated antigen. This demonstrates that PS reduces immunologic memory of FVIII and induces antigen-specific peripheral tolerance in HA mice. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2451-2456, 2015. PMID:26038127

  3. Docosahexaenoic acid-decient phosphatidyl serine and high K-tocopherol in a fetal mouse brain over-expressing

    E-print Network

    Groner, Yoram

    oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg; LPO, lipid peroxide; EPG, phosphatidyl- ethanolamine; IPG, phosphatidylinositol; PL, phospholipid; SPG

  4. Molecular species of phosphatidyl ethanolamine from egg yolk

    Microsoft Academic Search

    B. J. Holub; A. Kuksis

    1969-01-01

    Phosphatidyl ethanolamine was isolated from total egg yolk lipids by preparative thin layer chromatography (TLC). The purified\\u000a phosphatide contained 3% of the alkoxy derivative. It was degraded to diglycerides in the presence of purified sphingomyelin\\u000a by phospholipase C fromClostridium welchii. The diglycerides were acetylated and resolved on the basis of unsaturation by argentation TLC. The fatty acid composition\\u000a of the

  5. SERVER PS FACULTY PS PRACTICE

    E-print Network

    Bermúdez, José Luis

    TELECOM PS GRAD STUDENT PS BREAKROOM ELEVATOR CONTROL PS GRAD STUDENT PS GRAD STUDENT PS GRAD STUDENT / Shared Space 3.1 Interdisciplinary Spaces (Classrooms) 3.2 Interdisciplinary Spaces (Shared Spaces

  6. Facile Synthesis of Phosphatidyl Saccharides for Preparation of Anionic Nanoliposomes with Enhanced Stability

    PubMed Central

    Song, Shuang; Cheong, Ling-Zhi; Falkeborg, Mia; Liu, Lei; Dong, Mingdong; Jensen, Henrik Max; Bertelsen, Kresten; Thorsen, Michael; Tan, Tianwei; Xu, Xuebing; Guo, Zheng

    2013-01-01

    Physical stability during storage and against processing such as dehyration/rehydration are the cornerstone in designing delivery vehicles. In this work, mono-, di- and tri-saccharides were enzymatically conjugated to phosphatidyl group through a facile approach namely phospholipase D (PLD) mediated transphosphatidylation in a biphasic reaction system. The purified products were structurally identified and the connectivities of carbohydrate to phosphatidyl moiety precisely mapped by 1H, 31P, 13C NMR pulse sequences and LC-ESI-FTMS. The synthetic phosphatidyl saccharides were employed as the sole biomimetic component for preparation of nanoliposomes. It was found that the critical micelle concentration (CMC) of phosphatidyl saccharides increases as more bulky sugar moiety (mono- to tri-) is introduced. Phosphatidyl di-saccharide had the largest membrane curvature. In comparison to the zwitterionic phosphatidylcholine liposome, all phosphatidyl saccharides liposomes are anionic and demonstrated significantly enhanced stability during storage. According to the confocal laser scan microscopy (CLSM) and atom force microscopy (AFM) analyses, the nanoliposomes formed by the synthetic phosphatidyl saccharides also show excellent stability against dehydration/rehydration process in which most of the liposomal structures remained intact. The abundance hydroxyl groups in the saccharide moieties might provide sufficient H-bondings for stabilization. This work demonstrated the synthesized phosphatidyl saccharides are capable of functioning as enzymatically liable materials which can form stable nanoliposomes without addition of stabilizing excipients. PMID:24069243

  7. PHYSICAL STUDIES OF PHOSPHOLIPIDS: III. Electron Microscope Studies of Some Pure Fully Saturated 2,3-Diacyl-DL-Phosphatidyl-Ethanolamines and Phosphatidyl-Cholines

    Microsoft Academic Search

    D. Chapman; D. J. FLUCK

    1966-01-01

    On heating pure, fully saturated 2,3-diacyl-DL-phosphatidyl-ethanolamines and 2,3-dia- cylphosphatidyl-cholines (lecithins) in water to the transition temperature at which large endothermic heat changes occur, they are observed, by light microscopy, to form myelin figures. This result is discussed in terms of the large difference in the transition temperature for \\

  8. Early increase in phosphatidyl choline synthesis by choline and transmethylation pathways in spreading fibroblasts

    SciTech Connect

    Maziere, C.; Maziere, J.C.; Mora, L.; Polonovski, J.

    1986-11-01

    Phosphatidyl choline (PC) synthesis in trypsinized and reattaching fibroblasts during the spreading state was studied by incorporation of (/sup 14/C)choline and (methyl-/sup 14/C)methionine. The choline and phosphatidyl-ethanolamine (PE) transmethylation pathways were both transiently increased about 2-fold during the first 2 h after replating. Maximum increase appeared to be simultaneous with maximum spreading. Incorporation of (/sup 32/P)orthophosphate showed that the increase in PC synthesis was specific and most probably related to establishment of cell-substrate adhesion sites.

  9. Molecular Structure of Serine

    NSDL National Science Digital Library

    2002-08-21

    Serine is required for the metabolism of fat, tissue growth, and a healthy immune system. It assists in the production of immunoglobulins and antibodies. Some of its derivatives such as ethanolamine are important components of the phospholipids found in biological membranes. Serine is found in meat and dairy products, wheat gluten, peanuts, and soy products. It is not clear how toxic this substance can be, although it is known that high levels of serine may cause immune suppression and psychological symptoms such as cerebral allergies.

  10. The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer

    Microsoft Academic Search

    R L Dillon; D E White; W J Muller

    2007-01-01

    The phosphatidyl inositol 3-kinase (PI3K)\\/Akt pathway is activated downstream of a variety of extracellular signals and activation of this signaling pathway impacts a number of cellular processes including cell growth, proliferation and survival. The alteration of components of this pathway, through either activation of oncogenes or inactivation of tumor suppressors, disrupts a signaling equilibrium and can thus lead to cellular

  11. Phosphatidyl ethanolamine as a synergist for primary antioxidants in edible oils

    Microsoft Academic Search

    S. Z. Dziedzic; B. J. F. Hudson

    1984-01-01

    Dipalmitoyl phosphatidyl ethanolamine (DPE) is a potent synergist for a wide range of primary antioxidants in edible oils\\u000a at elevated temperature, i.e., above 80 C. At lower temperatures it has very little synergistic action. At 120 C the synergistic\\u000a effect increases progressively as the concentration of synergist increases from 0.025% to 0.25%. At a given level of synergist,\\u000a its effect

  12. Fluorescent products from reaction of peroxidizing polyunsaturated fatty acids with phosphatidyl ethanolamine and phenylalanine

    Microsoft Academic Search

    C. J. Dillard; A. L. Tappel

    1973-01-01

    Fluorescent chromophores produced by reaction of peroxidizing arachidonic acid or methyl docosahexaenoate with synthetic dipalmityl\\u000a phosphatidyl ethanolamine were lipid soluble, and those from reaction with phenylalanine were water soluble. In all reaction\\u000a systems that contained polyunsaturated fatty acid and only one amine compound, the development of fluorescence was linearly\\u000a related to oxygen absorption for 12–24 hr (p<0.001) and to the

  13. Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278)

    Microsoft Academic Search

    Yenny Y. Acosta; Maria Paz Zafra; Gloria Ojeda; Ilaria Seren Bernardone; Umberto Dianzani; Pilar Portolés; Jose M. Rojo

    To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding\\u000a to ICOS peptides phosphorylated at the Y191MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85?, p50-55? and p85? regulatory subunits\\u000a and p110?, p110? and p110? catalytic subunits. Intriguingly, T cells expressed high levels of both p110? or p110? catalytic\\u000a subunits, yet

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

    PubMed Central

    Madeira, C; Lourenco, M V; Vargas-Lopes, C; Suemoto, C K; Brandăo, 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-? 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-?/tau AD biomarkers. d-serine levels were higher in the hippocampus and parietal cortex of AD patients than in control subjects. Levels of both d-serine and serine racemase, the enzyme responsible for d-serine production, were elevated in experimental models of AD. Significantly, d-serine levels were higher in the CSF of probable AD patients than in non-cognitively impaired subject groups. Combining d-serine levels to the amyloid/tau index remarkably increased the sensitivity and specificity of diagnosis of probable AD in our cohort. Our results show that increased brain and CSF d-serine levels are associated with AD. CSF d-serine levels discriminated between nondemented and AD patients in our cohort and might constitute a novel candidate biomarker for early AD diagnosis. PMID:25942042

  15. Phosphatidylcholine synthesis in castor bean endosperm. I. Metabolism of L-serine. [Ricinus communis

    SciTech Connect

    Kinney, A.J.; Moore, T.S. Jr.

    1987-05-01

    Endosperm halves from 3-day-old castor bean (Ricinus communis var Hale) were incubated for 30 minutes with L(/sup 14/C)serine, after which label was observed in ethanolamine, choline, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, ethanolaminephosphate, and CDPethanolamine, but not in cholinephosphate or CDPcholine. Only later did significant amounts of isotope become incorporated into cholinephosphate and CDPcholine. The choline kinase inhibitor hemicholinium-3 prevented the incorporation of label from serine into choline-phosphate and CDPcholine, reduced the incorporation of (/sup 14/C)choline into phosphatidylcholine by 65%, but inhibited the incorporation of label into phosphatidylcholine from serine by only 15%. The inhibitor did not prevent the incorporation of labeled methyl groups from S-adenosyl-L-methionine into phosphatidyldimethylethanolamine plus phosphatidyl-choline. The amount of incorporation of label from the methyl donor was only 8% of that from choline into phosphatidylcholine. The implications of these results for the pathway and regulation of phosphatidylcholine synthesis from the water-soluble precursors are discussed.

  16. A prognostic role for anti-phosphatidyl choline antibodies in human cerebral malaria

    PubMed Central

    DAS, B. K.; PARIDA, S.; RAVINDRAN, B.

    1996-01-01

    Anti-phosphatidyl choline antibodies (?PC) have been measured in adult patients from Orissa, India with Plasmodium falciparum infection of varying clinical severity. Significantly raised levels of ?PC were observed in infected individuals in comparison with controls. The IgG ?PC were found to be generally more than IgM ?PC in most cases. The IgG ?PC levels were significantly more in those cases of cerebral malaria who recovered fully after quinine administration in comparison with fatal cases not responding to quinine therapy, indicating a role for ?PC in prognosis of adult cerebral malaria. There was no significant difference in levels of ?PC IgG between non-cerebral and fatal cerebral malaria patients, indicating an absence of a direct protective role in the development of cerebral manifestations. Subgroup typing of IgG with ?PC activity indicated IgG3 to be the predominant type, followed by IgG2. IgG1 and IgG4. A significant inverse relationship between serum tumour necrosis factor-alpha (TNF-?) levels and IgG1 antibodies with ?PC activity was found, emphasizing the importance of ?PC in modifying disease severity. These observations appear to give credence to recent reports in the literature indicating that toxic malarial antigens consist of phospholipids and that antibodies to phospholipids (?PL) inhibit such antigens in experimental systems. PMID:8608644

  17. Enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells

    SciTech Connect

    Castle, J.D.; Castle, A.M.; Ma, A.K.; Stukenbrok, H.

    1984-01-01

    Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24-25 degrees C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17-37 degrees C. Both radioactive linoleic and palmitic acids were incorporated selectively at the beta-position of the glycerol backbone of PC. PC labeling by (3H)choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced beta-acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A2 that favors PC as a substrate. Other peritoneal cell types exhibit a very similar A23187-stimulat

  18. 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

  19. A Fully Synthetic and Biochemically Validated Phosphatidyl Inositol-3-Phosphate Hapten via Asymmetric Synthesis and Native Chemical Ligation

    PubMed Central

    Chandler, Brent D.; Burkhardt, Anne L.; Foley, Klaudia; Cullis, Courtney; Driscoll, Denise; D’Amore, Natalie Roy; Miller, Scott J.

    2014-01-01

    We report the synthesis and biochemical validation of a phosphatidyl inositol-3 phosphate (PI3P) immunogen. The inositol stereochemistry was secured through peptide-catalyzed asymmetric phosphorylation catalysis, and the subsequent incorporation of a cysteine residue was achieved by native chemical ligation (NCL). Conjugation of the PI3P hapten to maleimide activated keyhole limpet hemocyanin (KLH) provided a PI3P immunogen, which was successfully used to generate selective PI3P antibodies. The incorporation of a sulfhydryl nucleophile into a phosphoinositide hapten demonstrates a general strategy to reliably access phosphoinositide immunogens. PMID:24344932

  20. Overview of protein serine\\/threonine phosphatases

    Microsoft Academic Search

    Patricia T. W. Cohen

    Protein phosphatases that cleave phosphate from phosphorylated serine and threonine in proteins are crucial for the regulation of most cellular processes. Three structurally distinct families of protein serine\\/threonine phosphatases (PPP, PPM, and FCP) are described and the phylogenetic relationships in the PPP and PPM families of Homo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae are examined. An overview of the functions

  1. The Sunflower High-oleic Mutant Ol Carries Variable Tandem Repeats of FAD2-1 , a Seed-specific Oleoyl-phosphatidyl Choline Desaturase

    Microsoft Academic Search

    Gunnar F. Schuppert; Shunxue Tang; Mary B. Slabaugh; Steven J. Knapp

    2006-01-01

    Ol, a chemically induced, incompletely dominant mutation, greatly increases oleic acid and is correlated with greatly reduced\\u000a expression of a seed-specific oleoyl-phosphatidyl choline desaturase (FAD2-1) in developing seeds of sunflower (Helianthus  annuus L.). FAD2-1 is duplicated in high-oleic (mutant) strains and cosegregates with Ol. Codominant RFLP markers have been developed for FAD2-1 and are diagnositic for the Ol mutation; however,

  2. Studies on dodecyl betainate in combination with its degradation products or with phosphatidyl choline-phase behavior and hemolytic activity.

    PubMed

    Lundberg, D; Ljusberg-Wahren, H; Norlin, A; Holmberg, K

    2004-10-15

    Surface active betaine esters contain a hydrolysable bond and give naturally occurring products (fatty alcohol and the amino acid betaine) on degradation. They are therefore interesting candidates for use as cationic surfactants in pharmaceutical applications. In this work the phase behavior of two systems of relevance for the utilization of dodecyl betainate as a pharmaceutical excipient is studied, namely dodecyl betainate/dodecanol/betaine hydrochloride/D2O and dodecyl betainate/phosphatidyl choline (PC)/ethanol/D2O. The techniques used for phase characterisation were 2H NMR measured on the solvent, small angle X-ray spectroscopy and optical microscopy. Dilute dodecyl betainate/PC dispersions were characterized using laser diffraction. It is shown that introduction of relatively small amounts of the hydrolysis products of dodecyl betainate, i.e., dodecanol and betaine (used in the form of betaine hydrochloride), has a strong effect on the phase behavior of the binary dodecyl betainate/D2O system. The degradation products change the average curvature of the surfactant film so that, instead of a hexagonal phase at concentrations above the micellar phase, a probably defective, lamellar phase seems to form. The dodecyl betainate/PC/ethanol/D2O system shows a large region of a highly swelling lamellar phase. Dispersions of dodecyl betainate/PC/ethanol in water can be prepared with low energy input; i.e., the preconcentrate can be regarded as a self-dispersing solution. Introduction of dodecyl betainate and its degradation products does not impair the ability of PC to form vesicles. Experiments for evaluating the toxicity of surface active betaine esters to erythrocytes were also performed. There are indications that the hemolytic activity of dodecyl betainate is lower than that of the stable surfactant tetradecyltrimethylammonium chloride, which has similar critical micelle concentration. A combination of dodecyl betainate and PC gives very low hemolytic activity. PMID:15450470

  3. Radiotracer evidence implicating phosphoryl and phosphatidyl bases as intermediates in betaine synthesis by water-stressed barley leaves

    SciTech Connect

    Hitz, W.D.; Rhodes, D.; Hanson, A.

    1981-10-01

    In pulse-chase experiments with barley wilted leaves, label from (/sup 14/C)-ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When (/sup 14/C)monomethylethanolamine was supplied to wilted leaves, phosphatidylcholine was initially more heavily labeled than betaine. These results are qualitatively consistent with a precursor-to-product relationship between phosphatidylcholine and betaine. The following experiments, in which tracer amounts of (/sup 14/C)ethanolamine or (/sup 14/C)formate were supplied to wilted barley leaves, implicated phosphoryl and phosphatidyl bases as intermediates in the methylation steps between ethanolamine and phosphatidylcholine. Label from both (/sup 14/C)ethanolamine and (/sup 14/C)formate entered phosphorylmonomethylethanolamine and phosphorylcholine very rapidly; these phosphoryl bases were the most heavily labeled products at 15 to 30 minutes after label addition and lost label rapidly as the fed /sup 14/C-labeled precursor was depleted. Phosphatidylmonomethylethanolamine and phosphatidylcholine were also significantly labeled from (/sup 14/C)ethanolamine and (/sup 14/)formate at early times; the corresponding free bases and nucleotide bases were not. Addition of a trapping pool of phosphorylcholine reduced (/sup 14/C)ethanolamine conversion to both phosphatidylcholine and betaine, and resulted in accumulation of labe in the trap. A computer model of the synthesis of betaine via phosphatidylcholine was developed from /sup 14/C kinetic data. The model indicates that about 20% of the total leaf phosphatidylcholine behaves as an intermediate in betaine biosynthesis and that a marked decrease (greater than or equal to2-fold) in the half-life of this metabolically active phosphatidylcholine fraction accompanies wilting.

  4. Abnormal serine phosphorylation of insulin receptor substrate 1 is associated with tau pathology in Alzheimer's disease and tauopathies.

    PubMed

    Yarchoan, Mark; Toledo, Jon B; Lee, Edward B; Arvanitakis, Zoe; Kazi, Hala; Han, Li-Ying; Louneva, Natalia; Lee, Virginia M-Y; Kim, Sangwon F; Trojanowski, John Q; Arnold, Steven E

    2014-11-01

    Neuronal insulin signaling abnormalities have been associated with Alzheimer's disease (AD). However, the specificity of this association and its underlying mechanisms have been unclear. This study investigated the expression of abnormal serine phosphorylation of insulin receptor substrate 1 (IRS1) in 157 human brain autopsy cases that included AD, tauopathies, ?-synucleinopathies, TDP-43 proteinopathies, and normal aging. IRS1-pS(616), IRS1-pS(312) and downstream target Akt-pS(473) measures were most elevated in AD but were also significantly increased in the tauopathies: Pick's disease, corticobasal degeneration and progressive supranuclear palsy. Double immunofluorescence labeling showed frequent co-expression of IRS1-pS(616) with pathologic tau in neurons and dystrophic neurites. To further investigate an association between tau and abnormal serine phosphorylation of IRS1, we examined the presence of abnormal IRS1-pS(616) expression in pathological tau-expressing transgenic mice and demonstrated that abnormal IRS1-pS(616) frequently co-localizes in tangle-bearing neurons. Conversely, we observed increased levels of hyperphosphorylated tau in the high-fat diet-fed mouse, a model of insulin resistance. These results provide confirmation and specificity that abnormal phosphorylation of IRS1 is a pathological feature of AD and other tauopathies, and provide support for an association between insulin resistance and abnormal tau as well as amyloid-?. PMID:25107476

  5. Enzymic imbalance in serine metabolism in rat hepatomas.

    PubMed Central

    Snell, K; Weber, G

    1986-01-01

    The activity of 3-phosphoglycerate dehydrogenase was high in tissues of high cell-renewal capacity, and was increased in neonatal and regenerating liver and, more markedly, in hepatomas. Serine hydroxymethyltransferase activity was present in hepatomas, whereas other enzymes of serine utilization (serine dehydratase and serine aminotransferase) were absent. This enzymic imbalance couples serine biosynthesis preferentially to nucleotide precursor formation in cancer cells. PMID:3082329

  6. STAT3 Phosphorylation at Tyrosine 705 and Serine 727 Differentially Regulates Mouse ESC Fates

    PubMed Central

    Huang, Guanyi; Yan, Hexin; Ye, Shoudong; Tong, Chang; Ying, Qi-Long

    2014-01-01

    STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine 727 residue. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF) signaling maintains pluripotency by inducing JAK-mediated phosphorylation of STAT3 Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in regulating mESC identities, using a small molecule-based system to post-translationally modulate the quantity of transgenic STAT3 in STAT3?/? mESCs. We demonstrated that pY705 is absolutely required for STAT3-mediated mESC self-renewal, while pS727 is dispensable, serving only to promote proliferation and optimal pluripotency. S727 phosphorylation is regulated directly by fibroblast growth factor/Erk signaling and crucial in the transition of mESCs from pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in significantly reduced neuronal differentiation potential, which could be recovered by a S727 phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem cells to naďve pluripotency, suggesting a dynamic equilibrium of STAT3 pY705 and pS727 in the control of mESC fate. PMID:24302476

  7. Serine:glyoxylate aminotransferase mutant of barley

    SciTech Connect

    Blackwell, R.; Murray, A.; Joy, K.; Lea, P.

    1987-08-01

    A photorespiratory mutant of barley (LaPr 85/84), deficient in both of the major peaks of serine:glyoxylate aminotransferase activity detected in the wild type, also lacks serine:pyruvate and asparagine:glyoxylate aminotransferase activities. Genetic analysis of the mutation demonstrated that these three activities are all carried on the same enzyme. The mutant, when placed in air, accumulated a large pool of serine, showed the expected rate (50%) of ammonia release during photorespiration but produced CO/sub 2/ at twice the wild type rate when it was fed (/sup 14/C) glyoxylate. Compared with the wild type, LaPr 85/84 exhibited abnormal transient changes in chlorophyll a fluorescence when the CO/sub 2/ concentration of the air was altered, indicating that the rates of the fluorescence quenching mechanisms were affected in vivo by the lack of this enzyme.

  8. Serine proteinase from Cucurbita ficifolia seeds.

    PubMed

    Dryja?ski, M; Otlewski, J; Wilusz, T

    1990-01-01

    A new serine proteinase was isolated from Cucurbita ficifolia seeds by the purification procedure, which includes: extraction, salting out with ammonium sulphate, chromatography on CM-cellulose. Sephacryl S-300 gel filtration and h.p.l.c. on DEAE-2SW TSK column. The enzyme was homogeneous both in native and SDS PAGE. Three independent methods showed its molecular mass to be approximately 77 kDa. The enzyme was inhibited by specific serine proteinase organic inhibitors, and was active in the presence of inhibitors specific for other proteinase classes. Surprisingly, squash proteinase exhibited a very high and broad pH optimum with a maximum at 10.7. It hydrolysed many different peptide bonds in B-chain of insulin and was able to cleave four bonds in endogenous serine proteinase inhibitor (CMTI). PMID:2087907

  9. Viral Serine/Threonine Protein Kinases ?

    PubMed Central

    Jacob, Thary; Van den Broeke, Céline; Favoreel, Herman W.

    2011-01-01

    Phosphorylation represents one the most abundant and important posttranslational modifications of proteins, including viral proteins. Virus-encoded serine/threonine protein kinases appear to be a feature that is unique to large DNA viruses. Although the importance of these kinases for virus replication in cell culture is variable, they invariably play important roles in virus virulence. The current review provides an overview of the different viral serine/threonine protein kinases of several large DNA viruses and discusses their function, importance, and potential as antiviral drug targets. PMID:21084474

  10. Serine proteases as mediators of mosquito immune responses

    Microsoft Academic Search

    Maureen J. Gorman; Susan M. Paskewitz

    2001-01-01

    Serine proteases regulate several invertebrate defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization of pathogen surfaces. These processes require the presence of serine proteases in the hemolymph where they can rapidly activate immune pathways in response to pathogen detection. Hemolymph coagulation in the horseshoe crab is controlled by several serine proteases, including two that are pathogen recognition molecules

  11. 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

  12. Vanadium inhibition of serine and cysteine proteases.

    PubMed

    Guerrieri, N; Cerletti, P; De Vincentiis, M; Salvati, A; Scippa, S

    1999-03-01

    A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain. PMID:10356762

  13. Neuroserpin, an axonally secreted serine protease inhibitor.

    PubMed Central

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

    1996-01-01

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

  14. Modulating the function of human serine racemase and human serine dehydratase by protein engineering.

    PubMed

    Wang, Cyong-Yi; Ku, Shan Chi; Lee, Cheng-Chung; Wang, Andrew H-J

    2012-11-01

    D-Serine is a co-agonist of N-methyl D-aspartate, a glutamate receptor, which is a major excitatory neurotransmitter receptor in the brain. Human serine racemase (hSR) and serine dehydratase (hSDH) are two important pyridoxal-5'-phosphate-dependent enzymes that synthesize and degrade D-serine, respectively. hSR and hSDH have significant sequence homology (28% identity) and are similar in their structural folds (root-mean-square deviation, 1.12 Ĺ). Sequence alignment and structural comparison between hSR and hSDH reveal that S84 in hSR and A65 in hSDH play important roles in their respective enzyme activities. We surmise that exchange of these two amino acids by introducing S84A hSR and A65S hSDH mutants may result in switching their protein functions. To understand the modulating mechanism of the key residues, mutants S84A in hSR and A65S in hSDH were constructed to monitor the change of activities. The structure of A65S hSDH mutant was determined at 1.3 Ĺ resolution (PDB 4H27), elucidating the role of this critical amino acid. Our study demonstrated S84A hSR mutant behaved like hSDH, whereas A65S hSDH mutant acquired an additional function of using D-serine as a substrate. PMID:23112234

  15. Characterisation of a serine proteinase from Penaeus vannamei haemocytes

    Microsoft Academic Search

    Florinda Jiménez-Vega; Francisco Vargas-Albores; Kenneth Söderhäll

    2005-01-01

    Serine proteinases are involved, besides digestive role, in immune response processes. In addition to the typical serine proteinase domain, proteinases from arthropod haemocytes contain so-called clip domains which are believed to exert regulatory functions. Clones coding for clip domain-containing serine proteinases were isolated from both Penaeus vannamei and Penaeus monodon haemocyte cDNA libraries. These proteins have most of the structural

  16. Metabolism and decarboxylation of glycollate and serine in leaf peroxisomes

    Microsoft Academic Search

    Nicholas J. Walton; Vernon S. Butt

    1981-01-01

    The linked utilization of glycollate and L-serine has been studied in peroxisomal preparations from leaves of spinach beet (Beta vulgaris L.). The generation of glycine from glycollate was found to be balanced by the production of hydroxypyruvate from serine and similarly by 2-oxoglutarate when L-glutamate was substituted for L-serine. In the presence of L-malate and catalytic quantities of NAD+, about

  17. The `catalytic triad' mechanism, which involves a serine, aspartic acid, has become synonymous with serine pr

    E-print Network

    Paetzel, Mark

    The `catalytic triad' mechanism, which involves a serine, aspartic acid, has become synonymous, aspartic acid an metal10proteases. ineproteases,whichhaveevolvedboth byconvergentanddivergent involvesthe. The third playerin the triad,an acidic resi- due,acts to orient the histidineresi and neutralize the charged

  18. The pharmacological landscape and therapeutic potential of serine hydrolases

    Microsoft Academic Search

    Daniel A. Bachovchin; Benjamin F. Cravatt

    2012-01-01

    Serine hydrolases perform crucial roles in many biological processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiological substrates and functions, and the vast

  19. 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

  20. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Microsoft Academic Search

    Lokesh P Tripathi; R Sowdhamini

    2006-01-01

    BACKGROUND: Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa) genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. RESULTS: Despite the differences in

  1. D-Serine Regulation of NMDA Receptor Activity

    NSDL National Science Digital Library

    Herman Wolosker (Technion-Israel Institute of Technology; Department of Biochemistry REV)

    2006-10-10

    The N-Methyl-D-aspartate–type glutamate receptor (NMDAR) plays a key role in several important processes involving the nervous system, including brain development, synaptic plasticity, and learning. Unlike other neurotransmitter receptors, which are activated by individual neurotransmitters, activation of NMDARs requires the binding of a coagonist (D-serine or glycine) in addition to glutamate. Although previously considered an "unnatural" amino acid, D-serine is a key regulator of NMDAR activity and may be the main physiological ligand at the coagonist site. D-Serine is synthesized in the mammalian brain and is enriched in astrocytes, a class of glial cells that ensheath synapses in the brain. Astrocytes physiologically affect NMDAR neurotransmission by releasing D-serine, suggesting that D-serine acts as a gliotransmitter. However, recent findings indicate that D-serine signaling does not depend solely on glia, because D-serine and its biosynthetic enzyme are also present in substantial amounts in neurons. Here, we discuss these new findings, which begin to shed light on the relative roles of glia and neurons in D-serine signaling.

  2. CHARACTERIZATION OF SERINE PROTEASES IN DEVELOPMENTAL STAGES OF EIMERIA TENELLA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Because of the importance of enzymes of the serine class as targets for novel controls of human diseases, the current study investigates the occurrence and function of serine proteases in Emeria tenella developmental stages. Using gel electrophoresis with casein imbedded gels (zymograms), bands of ...

  3. Regulation of hepatic l-serine dehydratase and l-serine-pyruvate aminotransferase in the developing neonatal rat

    PubMed Central

    Snell, Keith; Walker, Deryck G.

    1974-01-01

    1. The activities of l-serine dehydratase and l-serine–pyruvate aminotransferase were determined in rat liver during foetal and neonatal development. 2. l-Serine–pyruvate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. l-Serine dehydratase activity is very low prenatally, but increases rapidly after birth to a transient peak. After a second transient peak around the time weaning begins, activity gradually rises to the adult value. Both of these peaks have similar isoenzyme compositions. 4. In foetal liver both l-serine dehydratase and l-serine–pyruvate aminotransferase activities are increased after injection in utero of glucagon or dibutyryl cyclic AMP. Cycloheximide or actinomycin D inhibited the prenatal induction of both enzymes and actinomycin D blocked the natural increase of l-serine dehydratase immediately after birth. Glucose or insulin administration also blocked the perinatal increase of l-serine dehydratase. 5. After the first perinatal peak of l-serine dehydratase, activity is increased by cortisol and this is inhibited by actinomycin D. After the second postnatal peak, activity is increased by amino acids or cortisol and this is insensitive to actinomycin D inhibition. Glucose administration blocks the cortisol-stimulated increase in l-serine dehydratase and also partially lowers the second postnatal peak of activity. 6. The developmental patterns of the enzymes are discussed in relation to the pathways of gluconeogenesis from l-serine. The regulation of enzyme activity by hormonal and dietary factors is discussed with reference to the changes in stimuli that occur during neonatal development and to their possible mechanisms of action. PMID:4377655

  4. Straightforward isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) and ubiquitin from bovine testis by hydrophobic-interaction chromatography (HIC).

    PubMed

    Haj Hassan, Maya; Klett, Daničle; Cahoreau, Claire; Combarnous, Yves

    2011-10-01

    Isolation of phosphatidyl-ethanolamine-binding protein-1 (PEBP-1) from bovine brain was described almost three decades ago but it required a large number of steps to reach high purity. After the fractionation of bovine testis proteins by ammonium sulfate precipitation we found that PEBP-1, detected by Western blotting, was among the very few proteins still soluble at 80% ammonium sulfate saturation (3.2M). This soluble fraction (S80) was directly loaded onto a phenyl sepharose column equilibrated at the same ammonium sulfate concentration (3.2M). A stepwise elution of the retained material at 1.0, 0.5, 0.2, 0.1M ammonium sulfate in ammonium hydrogen carbonate was performed and then with ammonium hydrogen carbonate alone and finally with 50% ethylene glycol. All fractions were analyzed by SDS-PAGE and Western blotting and the fractions containing PEBP-1 was further fractionated by size exclusion chromatography on a HR75 Superdex column permitting the isolation of ubiquitin in addition to PEBP-1 as demonstrated by Western blotting and mass spectrometry. This study shows the feasibility of hydrophobic interaction chromatography (HIC) on phenyl sepharose at a very high ammonium sulfate concentration (3.2M; 80% saturation) to efficiently purify the proteins that are still soluble in these extreme conditions. PMID:21917533

  5. Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-Kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity

    SciTech Connect

    Xie,P.; Williams, D.; Atilla-Gokcumen, G.; Milk, L.; Xiao, M.; Smalley, K.; Herlyn, M.; Meggers, E.; Marmorstein, R.

    2008-01-01

    Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3K?/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.

  6. Serine catabolism regulates mitochondrial redox control during hypoxia

    PubMed Central

    Ye, Jiangbin; Fan, Jing; Venneti, Sriram; Wan, Ying-Wooi; Pawel, Bruce R.; Zhang, Ji; Finley, Lydia W.S.; Lu, Chao; Lindsten, Tullia; Cross, Justin; Qing, Guoliang; Liu, Zhandong; Simon, M. Celeste; Rabinowitz, Joshua D.; Thompson, Craig B.

    2014-01-01

    The de novo synthesis of the non-essential amino acid serine is often upregulated in cancer. In this study we demonstrate that the serine catabolic enzyme, mitochondrial serine hydroxymethyltransferase (SHMT2) is induced when Myc-transformed cells are subjected to hypoxia. In mitochondria, SHMT2 can initiate the degradation of serine to CO2 and NH4+ resulting in net production of NADPH from NADP+. Knockdown of SHMT2 in Myc-dependent cells reduced cellular NADPH/NADP+ ratio, increased cellular reactive oxygen species (ROS) and triggered hypoxia-induced cell death. In vivo, SHMT2 suppression led to impaired tumor growth. In myc-amplified neuroblastoma patient samples, there was a significant correlation between SHMT2 and Hypoxia-inducible factor-1 ? (HIF-1?) and SHMT2 expression correlated with unfavorable patient prognosis. Together these data demonstrate that mitochondrial serine catabolism supports tumor growth by maintaining mitochondrial redox balance and cell survival. PMID:25186948

  7. 3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis.

    PubMed Central

    Jaeken, J; Detheux, M; Van Maldergem, L; Foulon, M; Carchon, H; Van Schaftingen, E

    1996-01-01

    Serine concentrations were markedly decreased in the cerebrospinal fluid of two brothers with congenital microcephaly, profound psychomotor retardation, hypertonia, epilepsy, growth retardation, and hypogonadism. The youngest boy also had congenital bilateral cataract. Magnetic resonance imaging of the brain showed evidence of dysmyelination. Plasma serine as well as plasma and cerebrospinal fluid glycine concentrations were also decreased but to a lesser extent. Treatment with oral serine in the youngest patient significantly increased cerebrospinal fluid serine and abolished the convulsions. In fibroblasts of both patients, a decreased activity was demonstrated of 3-phosphoglycerate dehydrogenase, the first step of serine biosynthesis (22% and 13% of the mean control value). This is an unusual disorder as the great majority of aminoacidopathies are catabolic defects. It is a severe but potentially treatable inborn error of metabolism that has not been previously reported in man. PMID:8758134

  8. Neutrophil serine proteases fine-tune the inflammatory response

    PubMed Central

    Pham, Christine T. N.

    2008-01-01

    Neutrophil serine proteases are granule-associated enzymes known mainly for their function in the intracellular killing of pathogens. Their extracellular release upon neutrophil activation is traditionally regarded as the primary reason for tissue damage at the sites of inflammation. However, studies over the past several years indicate that neutrophil serine proteases may also be key regulators of the inflammatory response. Neutrophil serine proteases specifically process and release chemokines, cytokines, and growth factors, thus modulating their biological activity. In addition, neutrophil serine proteases activate and shed specific cell surface receptors, which can ultimately prolong or terminate cytokine-induced responses. Moreover, it has been proposed that these proteases can impact cell viability through their caspase-like activity and initiate the adaptive immune response by directly activating lymphocytes. In summary, these studies point to neutrophil serine proteases as versatile mediators that fine-tune the local immune response and identify them as potential targets for therapeutic interventions. PMID:18180196

  9. Structural characterization of phosphatidyl- myo -inositol mannosides from Mycobacterium bovis bacillus calmette guérin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. I. PIMs and lyso-PIMs

    Microsoft Academic Search

    Fong-Fu Hsu; John Turk; Róisín M. Owens; Elizabeth R. Rhoades; David G. Russell

    2007-01-01

    We described a multiple-stage ion-trap mass spectrometric approach to characterize the structures of phosphatidylinositol\\u000a and phosphatidyl-myoinositol mannosides (PIMs) in a complex mixture isolated from Mycobacterium bovis Bacillus Calmette Guérin. The positions of the fatty acyl substituents of PIMs at the glycerol backbone can be easily assigned,\\u000a based on the findings that the ions arising from losses of the fatty acid

  10. Inhibition of serine proteinases by squash inhibitors.

    PubMed

    Otlewski, J; Zbyryt, T; Krokoszy?ska, I; Wilusz, T

    1990-07-01

    The squash inhibitors of serine proteinases have been discovered as proteins, which inhibit the catalytic activity of bovine trypsin. In this report we show, that three human enzymes of trypsin-like specificity - i.e. plasmin, plasma kallikrein and thrombin - are also inhibited by squash inhibitors. Moreover, rather strong inhibition was demonstrated for human cathepsin G. Lower association constants were found for Streptomyces griseus proteinase B (SGPB) and subtilisin BPN'. No association was detected for bovine chymotrypsin, even at millimolar concentrations of the inhibitors. Porcine pancreatic elastase showed extremely weak inhibition by squash inhibitors. Most of the enzymes examined did not exhibit a clear discrimination between P1 Arg and P1 Lys inhibitors. However, human plasma kallikrein and human thrombin formed much stronger complexes with CMTI I (P1-Arg) than with CPTI II (P1-Lys). PMID:2145863

  11. d-Serine in Glia and Neurons Derives from 3-Phosphoglycerate Dehydrogenase

    PubMed Central

    Ehmsen, Jeffrey T.; Ma, Ting Martin; Sason, Hagit; Rosenberg, Dina; Ogo, Tadashi; Furuya, Shigeki

    2013-01-01

    d-Serine is an endogenous ligand for NMDARs generated from l-serine by the enzyme serine racemase (Srr). Both neuronal and glial localizations have been reported for d-serine and Srr. 3-Phosphoglycerate dehydrogenase is an exclusively astrocytic enzyme that catalyzes the first committed step of l-serine biosynthesis. Using transgenic mice expressing enhanced green fluorescent protein under the Srr promoter and mice with targeted deletion of Srr or 3-Phosphoglycerate dehydrogenase, we demonstrate predominantly neuronal sources of d-serine dependent on astrocytic supply of l-serine. These findings clarify the cellular basis for the regulation of NMDAR neurotransmission by d-serine. PMID:23884950

  12. Effects of Halothane on Phosphatidylcholine, -ethanolamine, -glycerol, and -serine Monolayer Order at a

    E-print Network

    Richmond, Geraldine L.

    Effects of Halothane on Phosphatidylcholine, -ethanolamine, -glycerol, and -serine Monolayer Order headgroups (choline, ethanolamine, glycerol, and serine) were studied. Each monolayer exhibits significant

  13. D-serine in glia and neurons derives from 3-phosphoglycerate dehydrogenase.

    PubMed

    Ehmsen, Jeffrey T; Ma, Ting Martin; Sason, Hagit; Rosenberg, Dina; Ogo, Tadashi; Furuya, Shigeki; Snyder, Solomon H; Wolosker, Herman

    2013-07-24

    d-Serine is an endogenous ligand for NMDARs generated from l-serine by the enzyme serine racemase (Srr). Both neuronal and glial localizations have been reported for d-serine and Srr. 3-Phosphoglycerate dehydrogenase is an exclusively astrocytic enzyme that catalyzes the first committed step of l-serine biosynthesis. Using transgenic mice expressing enhanced green fluorescent protein under the Srr promoter and mice with targeted deletion of Srr or 3-Phosphoglycerate dehydrogenase, we demonstrate predominantly neuronal sources of d-serine dependent on astrocytic supply of l-serine. These findings clarify the cellular basis for the regulation of NMDAR neurotransmission by d-serine. PMID:23884950

  14. LCD display Keyboard (PS/2)

    E-print Network

    Kalla, Priyank

    1 Other I/O LCD display Flash ROM SPI EPROM Keyboard (PS/2) UART connectors DAC ADC LCD Display 2-line, 16 character LCD display 4-bit interface Relatively easy to use once you have it mapped! Scrolling at half-second intervals is about as fast as you can go and still have a clear display LCD LCD

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

    SciTech Connect

    Qiu Yuling [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China); Choo, Young Moo [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Yoon, Hyung Joo [Department of Agricultural Biology, National Academy of Agricultural Science, Suwon (Korea, Republic of); Jia Jingming; Cui Zheng; Wang Dong [Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China); Kim, Doh Hoon [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China); Sohn, Hung Dae [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Jin, Byung Rae, E-mail: brjin@dau.ac.kr [College of Natural Resources and Life Science, Dong-A University, Busan 604-714 (Korea, Republic of); Joint Laboratory between Dong-A University and Shenyang Pharmaceutical University, Shenyang Pharmaceutical University, Shenyang (China)

    2011-09-01

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

  16. Functional evaluation of serine 252 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

    PubMed

    Castillo, Daniel; Sepúlveda, Carolina; Cardemil, Emilio; Jabalquinto, Ana M

    2009-02-01

    Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase mutant Ser252Ala, affecting the conserved Walker A serine residue, was characterized to elucidate the role of this serine residue. The substitution did not result in changes in the protein structure, as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analysis of the mutated enzyme in both directions of the main reaction and in the two secondary reactions showed an approximately 50-fold increase in apparent K(m) for oxaloacetate with minor alterations in the other kinetic parameters. These results show that the hydroxyl group of serine 252 is required for proper oxaloacetate interaction. PMID:18996167

  17. 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

  18. 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...

  19. Crystal structure of D-serine dehydratase from Escherichia coli.

    PubMed

    Urusova, Darya V; Isupov, Michail N; Antonyuk, Svetlana; Kachalova, Galina S; Obmolova, Galina; Vagin, Alexei A; Lebedev, Andrey A; Burenkov, Gleb P; Dauter, Zbigniew; Bartunik, Hans D; Lamzin, Victor S; Melik-Adamyan, William R; Mueller, Thomas D; Schnackerz, Klaus D

    2012-03-01

    D-Serine dehydratase from Escherichia coli is a member of the ?-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Ĺ-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Ĺ resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the ?-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed. PMID:22197591

  20. d-Serine-induced nephrotoxicity: possible interaction with tyrosine metabolism

    Microsoft Academic Search

    R. E Williams; E. A Lock

    2004-01-01

    d-Serine selectively damages renal proximal tubule cells in rats by a mechanism that is not fully understood. Recent proteomic analysis identified that d-serine elevated plasma fumarylacetoacetate hydrolase (FAH). FAH is involved in tyrosine catabolism; hence, this pathway may be involved in mediating the toxicity. This work examines whether 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC), a potent inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) located

  1. D-serine-induced nephrotoxicity: possible interaction with tyrosine metabolism.

    PubMed

    Williams, R E; Lock, E A

    2004-09-01

    D-serine selectively damages renal proximal tubule cells in rats by a mechanism that is not fully understood. Recent proteomic analysis identified that D-serine elevated plasma fumarylacetoacetate hydrolase (FAH). FAH is involved in tyrosine catabolism; hence, this pathway may be involved in mediating the toxicity. This work examines whether 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC), a potent inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) located upstream of FAH, modulates D-serine-induced nephrotoxicity. Rats were pretreated with NTBC (0.5 mg/kg p.o.) or corn oil and then 30 min later given either D-serine (250 mg/kg i.p.) or water. Urine was collected every 12 h until termination (48 h) and analysed by 1H NMR spectroscopy and principal component analysis (PCA). Markers of proximal tubule injury were evident in urine following treatment with D-serine and NTBC + D-serine. PCA could not distinguish between these urine samples suggesting that NTBC does not effect the development of nephrotoxicity. Clinical chemistry analysis of urine and terminal plasma samples and histopathological examination of the kidneys confirmed this. NTBC alone caused a marked increase in the excretion of 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA); however, HPPA and HPLA excretion was minimal following NTBC + D-serine. Instead marked tyrosinuria was observed suggesting that D-serine-induced renal damage markedly affects the handling of increased levels of HPPA and HPLA resulting from the inhibition of HPPD. PMID:15297036

  2. Liver enzymes of serine metabolism during neonatal development of the rat.

    PubMed Central

    Snell, K

    1980-01-01

    The developmental patterns of L-serine hydroxymethyltransferase, L-phosphoserine aminotransferase, L-serine aminotransferase and L-serine dehydratase were determined in rat liver. The results point to an increased capacity for serine biosynthesis de novo in the perinatal period. It is suggested that serine at this time, and also at weaning, may serve as a precursor, via the serine hydroxymethyltransferase reaction, for nucleotide biosynthesis to support the rapid phases of liver growth. The role of the alternative pathways of serine metabolism during neonatal development is discussed. PMID:6781481

  3. ps

    E-print Network

    2003-05-30

    May 30, 2003 ... (ii) exp ? is the restriction of an entire function of exponential type to the. real line. ..... be a Gaussian stationary random process, normalized by E?(0) = 0 and ...... A serious modification is needed in our final argument where.

  4. ps

    E-print Network

    2010-02-14

    Feb 14, 2010 ... are disjoint such that the union of all ?? has Hausdorff dimension 1. ... iterates fk of f do not form a normal family. ... McMullen [15, Theorem 1.1] and Karpinska [12, Theorem 3] also considered the case of .... d | + M ? 5|xk+1. d.

  5. ps

    E-print Network

    2006-08-24

    Aug 24, 2006 ... that this result still holds for entire functions of genus 0 or 1, but in general ..... On the other hand, we show that a harmonic function u satisfying (5) in a. Stolz angle cannot have ... As our version of this factorization is different from [12, ..... Fk(z) := z ?rk(z), and t = |rk(z)|, where we have set rk := 1/qk to simplify.

  6. ps

    E-print Network

    2014-09-07

    Sep 7, 2014 ... Smirnov proved that for all sets of data satisfying (2.9), there exists. a sequence of ... |k| times, and the other two sides are proper subsets of their corresponding. circles. ..... whose power series begins with the term (z ? 1)m+1. ...... a missing pair can be added to T . Finally, we can assume inductively that.

  7. Serine tRNA complementary to the nonuniversal serine codon CUG in Candida cylindracea: evolutionary implications.

    PubMed Central

    Yokogawa, T; Suzuki, T; Ueda, T; Mori, M; Ohama, T; Kuchino, Y; Yoshinari, S; Motoki, I; Nishikawa, K; Osawa, S

    1992-01-01

    In the asporogenic yeast Candida cylindracea, the codon CUG is read as serine instead of leucine. This is an unusual instance in which the amino acid assignment of a codon deviates from the universal code. To infer the evolutionary process of this change, the tRNA with the anticodon sequence CAG, which is complementary to and thus responsible for translation of the codon CUG, has been identified. Indeed, this tRNA translates an in-frame CUG codon in a synthetic mRNA as serine in an in vitro translation system. The gene for the tRNA is interrupted by an intron in the anticodon loop. Sequence comparisons of the tRNA and its gene suggest that a single cytidine was inserted into the anticodon loop of the gene for tRNA(Ser)IGA during evolution to produce tRNA(Ser)CAG. The tRNA(Ser)CAG may be produced from its precursor molecule containing the cytidine insertion by splicing. PMID:1502151

  8. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

    Microsoft Academic Search

    Lokesh P Tripathi; R Sowdhamini

    2008-01-01

    BACKGROUND: Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown.

  9. Endothelin-1 stimulates catalase activity through the PKC? mediated phosphorylation of Serine 167

    PubMed Central

    Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Hou, Yali; Kangath, Archana; Pardo, Daniel; Fineman, Jeffrey R.; Black, Stephen M.

    2013-01-01

    Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells (PAEC) to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine phosphorylated catalase. Using the inhibitory peptide ?V1.1, this phosphorylation was shown to be PKC? dependent. Mass spectrometry identified serine167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from E.coli or transiently transfected COS-7 cells, demonstrated that S167D-catalase had an increased ability to degrade H2O2 compared to the wildtype enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist, tezosentan. S167 is being located on the dimeric interface suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel-filtration to examine the multimeric structure of recombinant wildtype- and S167D-catalase. We found that recombinant wildtype catalase was present as a mixture of monomers and dimers while S167D catalase was primarily tetrameric. Further, the incubation of wildtype catalase with PKC? was sufficient to convert wildtype catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity. PMID:24211614

  10. 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

  11. An improved method to determine serine palmitoyltransferase activity

    PubMed Central

    Rütti, Markus F.; Richard, Stéphane; Penno, Anke; von Eckardstein, Arnold; Hornemann, Thorsten

    2009-01-01

    Serine palmitoyltransferase (SPT) catalyzes the condensation of l-serine and palmitoyl-CoA, which is the rate-limiting step in the de novo synthesis of sphingolipids. SPT activity is commonly measured by monitoring the incorporation of radiolabeled l-serine into 3-ketodihydrosphingosine. In this article, we introduce several adaptations of the established protocol to improve sensitivity, reproducibility, and practicability of the assay. A significant improvement of this new protocol is the possibility to measure SPT activity in total cell lysate instead of microsomes. The assay is furthermore extended by the introduction of a nonradioactive, HPLC-based detection protocol. The suggested HPLC method offers several advantages, most importantly, a 20-fold lower detection limit compared with the radioactive assay and the possibility to use an internal standard to correct for variation in the extraction. PMID:19181628

  12. Tetrameric structure of a serine integrase catalytic domain.

    PubMed

    Yuan, Peng; Gupta, Kushol; Van Duyne, Gregory D

    2008-08-01

    The serine integrases have recently emerged as powerful new chromosome engineering tools in various organisms and show promise for therapeutic use in human cells. The serine integrases are structurally and mechanistically unrelated to the bacteriophage lambda integrase but share a similar catalytic domain with the resolvase/invertase enzymes typified by the resolvase proteins from transposons Tn3 and gammadelta. Here we report the crystal structure and solution properties of the catalytic domain from bacteriophage TP901-1 integrase. The protein is a dimer in solution but crystallizes as a tetramer that is closely related in overall architecture to structures of activated gammadelta-resolvase mutants. The ability of the integrase tetramer to explain biochemical experiments performed in the resolvase and invertase systems suggests that the TP901 integrase tetramer represents a unique intermediate on the recombination pathway that is shared within the serine recombinase superfamily. PMID:18682229

  13. Negative regulation of Raf-1 by phosphorylation of serine 621.

    PubMed Central

    Mischak, H; Seitz, T; Janosch, P; Eulitz, M; Steen, H; Schellerer, M; Philipp, A; Kolch, W

    1996-01-01

    The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation. PMID:8816453

  14. Franklin PS-2 (XPS-2) Glider

    NASA Technical Reports Server (NTRS)

    1938-01-01

    This Franklin PS-2 training glider is about to be towed aloft by the specially modified car in front. NACA researchers used the PS-2 in a study of ground effect on a towed glider. Langley flew two of the Franklin gliders in the late 1930s, but the Navy never really found a good use for training gliders.

  15. Console Hacking 2010 PS3 Epic Fail

    E-print Network

    Touretzky, David S.

    Console Hacking 2010 PS3 Epic Fail bushing, marcan, segher, sven 27th Chaos Communication Congress Hack Homebrew Channel Drivechips Bannerbomb Bannerbomb for 4.2 latest update broken Indiana Pwns t Wii Xbox 360 PS3 2006 2011 2010 2009 2008 2007 Mittwoch, 29. Dezember 2010 #12;Twiizer Attack Twilight Hack

  16. Franklin PS-2 (XPS-2) Glider

    NASA Technical Reports Server (NTRS)

    1938-01-01

    Franklin PS-2 (XPS-2) Glider: This Franklin PS-2 training glider is about to be towed aloft by the specially modified car in front. NACA researchers used the PS-2 in a study of ground effect on a towed glider. Langley flew two of the Franklin gliders in the late 1930s, but the Navy never really found a good use for training gliders.: This Franklin PS-2 training glider is about to be towed aloft by the specially modified car in front. NACA researchers used the PS-2 in a study of ground effect on a towed glider. Langley flew two of the Franklin gliders in the late 1930s, but the Navy never really found a good use for training gliders.

  17. 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 between before and after clozapine treatment. This study firstly demonstrated changes of d-/l-serine and glycine/l-serine ratio between before and after clozapine treatment, suggesting that the plasma d-/l-serine ratio and glycine/l-serine ratio could be markers of therapeutic efficacy or clinical state in treatment-resistant schizophrenia. PMID:25218715

  18. Solid-phase synthesis of benzisothiazolones as serine protease inhibitors.

    PubMed

    Yu, K L; Civiello, R; Roberts, D G; Seiler, S M; Meanwell, N A

    1999-03-01

    An efficient solid-phase synthesis of benzisothiazolone-1,1-dioxide-based serine protease inhibitors involving alkylation of carboxylic acids with N-(bromomethyl)benzisothiazolone-1,1-dioxide has been developed. An example using this procedure for preparation of a library of human mast cell tryptase inhibitors is described. PMID:10201825

  19. Outer membrane-associated serine protease of intestinal spirochetes

    Microsoft Academic Search

    Nagaraja Muniappa; Gerald E Duhamel

    1997-01-01

    Pathogenic intestinal spirochetes cause damage to the intestinal mucosa of humans and animals by an unknown mechanism. The purpose of this study was to assess the pathogenic intestinal spirochetes Serpulina hyodysenteriae, Serpulina pilosicoli, and Brachyspira aalborgi and the non-pathogenic commensal intestinal spirochetes Serpulina innocens and Treponema succinifaciens for protease activity. A partially heat stable, subtilisin-like, serine protease was identified in

  20. A serine sensor for multicellularity in a bacterium

    PubMed Central

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

    2013-01-01

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

  1. 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. PMID:18564178

  2. PP/PS anisotropic stereotomography

    NASA Astrophysics Data System (ADS)

    Nag, Steinar; Alerini, Mathias; Ursin, Bjřrn

    2010-04-01

    Stereotomography is a slope tomographic method which gives good results for background velocity model estimation in 2-D isotropic media. We develop here the extension of the method to 3-D general anisotropic media for PP and PS events. We do not take into account the issue of shear wave degeneracy. As in isotropic media, the sensitivity matrix of the inversion can be computed by paraxial ray tracing. We introduce a `constant Z stereotomography' approach, which can reduce the size of the sensitivity matrix. Based on ray perturbation theory, we give all the derivatives of stereotomography data parameters with respect to model parameters in a 3-D general anisotropic medium. These general formulas for the derivatives can also be used in other applications that rely on anisotropic ray perturbation theory. In particular, we obtain derivatives of the phase velocity with respect to position, phase angle and elastic medium parameters, all for general anisotropic media. The derivatives are expressed using the Voigt notation for the elastic medium parameters. We include a Jacobian that allows to change the model parametrization from Voigt to Thomsen parameters. Explicit expressions for the derivatives of the data are given for the case of 2-D tilted transversely isotropic (TTI) media. We validate the method by single-parameter estimation of each Thomsen parameter field of a 2-D TTI synthetic model, where data are modelled by ray tracing. For each Thomsen parameter, the estimated velocity field fits well with the true velocity field.

  3. Separate mechanisms for age-related truncation and racemisation of peptide-bound serine.

    PubMed

    Lyons, Brian; Jamie, Joanne F; Truscott, Roger J W

    2014-01-01

    Some amino acids are particularly susceptible to degradation in long-lived proteins. Foremost among these are asparagine, aspartic acid and serine. In the case of serine residues, cleavage of the peptide bond on the N-terminal side, as well as racemisation, has been observed. To investigate the role of the hydroxyl group, and whether cleavage and racemisation are linked by a common mechanism, serine peptides with a free hydroxyl group were compared to analogous peptides where the serine hydroxyl group was methylated. Peptide bond cleavage adjacent to serine was increased when the hydroxyl group was present, and this was particularly noticeable when it was present as the hydroxide ion. Adjacent amino acid residues also had a pronounced affect on cleavage at basic pH, with the SerPro motif being especially susceptible to scission. Methylation of the serine hydroxyl group abolished truncation, as did insertion of a bulky amino acid on the N-terminal side of serine. By contrast, racemisation of serine occurred to a similar extent in both O-methylated and unmodified peptides. On the basis of these data, it appears that racemisation of Ser, and cleavage adjacent to serine, occur via separate mechanisms. Addition of water across the double bond of dehydroalanine was not detected, suggesting that this mechanism was unlikely to be responsible for conversion of L-serine to D-serine. Abstraction of the alpha proton may account for the majority of racemisation of serine in proteins. PMID:24306455

  4. Determination of the PS I content of PS II core preparations using selective emission: a new emission of PS II at 780nm.

    PubMed

    Morton, Jennifer; Hall, Jeremy; Smith, Paul; Akita, Fusamichi; Koua, Faisal Hammad Mekky; Shen, Jian-Ren; Krausz, Elmars

    2014-01-01

    Routinely prepared PS II core samples are often contaminated by a significant (~1-5%) fraction of PS I, as well as related proteins. This contamination is of little importance in many experiments, but masks the optical behaviour of the deep red state in PS II, which absorbs in the same spectral range (700-730nm) as PS I (Hughes et al. 2006). When contamination levels are less than ~1%, it becomes difficult to quantify the PS I related components by gel-based, chromatographic, circular dichroism or EPR techniques. We have developed a fluorescence-based technique, taking advantage of the distinctively different low-temperature emission characteristics of PS II and PS I when excited near 700nm. The approach has the advantage of providing the relative concentration of the two photosystems in a single spectral measurement. A sensitivity limit of 0.01% PS I (or better) can be achieved. The procedure is applied to PS II core preparations from spinach and Thermosynechococcus vulcanus. Measurements made of T. vulcanus PS II preparations prepared by re-dissolving crystallised material indicate a low but measurable PS I related content. The analysis provides strong evidence for a previously unreported fluorescence of PS II cores peaking near 780nm. The excitation dependence of this emission as well as its appearance in both low PS I cyanobacterial and plant based PS II core preparations suggests its association with the deep red state of PS II. PMID:24055633

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

    PubMed Central

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

    2014-01-01

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

  6. First Experiments with MePS

    NASA Astrophysics Data System (ADS)

    Jungmann, M.; Haeberle, J.; Krause-Rehberg, R.; Anwand, W.; Butterling, M.; Wagner, A.; Johnson, J. M.; Cowan, T. E.

    2013-06-01

    MePS (Mono-energetic Positron System) is part of the EPOS system (ELBE Positron Source) in the HZDR (Helmholtz-Zentrum Dresden-Rossendorf). It is one of the installations at ELBE (Electron LINAC for beams with high Brilliance and low Emittance), which supplies a 40-MeV electron beam. MePS makes use of the excellent time structure of the primary electron beam of ELBE (repetition frequency up to 26 MHz; bunch length < 5ps) to produce a pulsed, intense slow positron beam to allow positron lifetime spectroscopy. In order to avoid spurious signals, which, in other systems, are often obtained by positrons reflecting from the sample surface, a bent tube (45°) was added between accelerator and sample chamber. The MePS system has been used to study the pore system of a series of low-k dielectric layers.

  7. Franklin PS-2 (XPS-2) Glider

    NASA Technical Reports Server (NTRS)

    1936-01-01

    Franklin PS-2 (XPS-2) Glider: This beefy-looking glider is a Franklin PS-2, a pair of which were operated by the NACA at Langley beginning in April 1936. The Navy only ordered half a dozen of these training gliders, which had a glide ratio of 15 feet forward for every foot down. The devices above the pilot's seat are venturi tubes, which gathered data for the instruments. Airfield lights are seen above the wing.

  8. Characterization of EntF as a serine-activating enzyme.

    PubMed Central

    Reichert, J.; Sakaitani, M.; Walsh, C. T.

    1992-01-01

    EntF is the enzyme responsible for serine activation during the biosynthesis of enterobactin (a cyclic trimer of N-dihydroxybenzoyl serine) in Escherichia coli. EntF has been overexpressed and purified to > 90% homogeneity. The enzyme has been shown to complement the entF- MK1 strain in the synthesis of 2,3-dihydroxybenzoyl serine derivatives and exhibits L-serine-dependent ATP[32P] pyrophosphate exchange activity with a Km for serine of 260 mM and a turnover number of 760 min-1. Release of PPi during incubation of EntF with serine and ATP was observed, but with a low turnover number of 1.0 min-1. These results suggested the presence of an enzyme-bound intermediate, which has been shown by gel filtration analysis to be (L-serine)adenylate. PMID:1338974

  9. P27Kip1 serine 10 phosphorylation determines its metabolism and interaction with cyclin-dependent kinases.

    PubMed

    Bencivenga, Debora; Tramontano, Annunziata; Borgia, Alessia; Negri, Aide; Caldarelli, Ilaria; Oliva, Adriana; Perrotta, Silverio; Della Ragione, Fulvio; Borriello, Adriana

    2014-01-01

    p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its "adaptability" to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase. PMID:25483085

  10. Calcium-induced inactivation of alamethicin in asymmetric lipid bilayers

    PubMed Central

    1982-01-01

    This paper discusses a calcium-dependent inactivation of alamethicin- induced conductance in asymmetric lipid bilayers. The bilayers used were formed with one leaflet of phosphatidyl ethanolamine (PE) and one of phosphatidyl serine (PS). Calcium, initially confined to the neutral lipid (PE) side, can pass through the open alamethicin channel to the negative lipid (PS) side, where it can bind to the negative lipid and reduce the surface potential. Under appropriate circumstances, the voltage-dependent alamethicin conductance is thereby inactivated. We have formulated a model for this process based on the diffusion of calcium in the aqueous phases and we show that the model describes the kinetic properties of the alamethicin conductance under various circumstances. EGTA on the PS side of the membrane reduces the effects of calcium dramatically as predicted by the model. PMID:7077290

  11. Voluntary Product Standard PS 20-05 American Softwood Lumber

    E-print Network

    #12;Voluntary Product Standard PS 20-05 American Softwood Lumber Standard Supersedes Voluntary Product Standard PS 20-99 September 2005 U.S. Department of Commerce Carlos M. Gutierrez, Secretary & Technology Voluntary Product Standard PS 20-05 Natl. Inst. Stand. Technol. Prod. Stand. PS 20-05, 50 pages

  12. Protein chemical synthesis by serine and threonine ligation

    PubMed Central

    Zhang, Yinfeng; Lam, Hiu Yung; Lee, Chi Lung; Li, Xuechen

    2013-01-01

    An efficient method has been developed for the salicylaldehyde ester-mediated ligation of unprotected peptides at serine (Ser) or threonine (Thr) residues. The utility of this peptide ligation approach has been demonstrated through the convergent syntheses of two therapeutic peptides––ovine-corticoliberin and Forteo––and the human erythrocyte acylphosphatase protein (?11 kDa). The requisite peptide salicylaldehyde ester precursor is prepared in an epimerization-free manner via Fmoc–solid-phase peptide synthesis. PMID:23569249

  13. 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. (Glasgow); (UC)

    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.

  14. Rubidium penta­aqua­(l-serine)cobalt(II) hexa­hydrogenhexa­molybdocobaltate(III) l-serine monosolvate deca­hydrate

    PubMed Central

    Iijima, Jun; Naruke, Haruo; Takiyama, Hiroshi

    2013-01-01

    The Co2+ ion in the title compound, Rb[Co(C3H7NO3)(H2O)5][H6CoMo6O24]·C3H7NO3·10H2O, is coordinated by five water mol­ecules 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 [H6CoIIIMo6O24]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 mol­ecules. PMID:24454041

  15. L-Serine Catabolism via an Oxygen-Labile L-Serine Dehydratase Is Essential for Colonization of the Avian Gut by Campylobacter jejuni

    Microsoft Academic Search

    Jyoti Velayudhan; Michael A. Jones; Paul A. Barrow; David J. Kelly

    2004-01-01

    Campylobacter jejuni is a microaerophilic, asaccharolytic bacterium. The identity of the carbon and energy sources used by C. jejuni in vivo is unknown, but the genome sequence of strain NCTC11168 indicates the presence of genes for catabolism of a limited range of amino acids, including serine. Specific omission of L-serine from a defined medium containing a mixture of amino acids

  16. Final results for ?± production in the HARP/PS214 experiment at CERN PS

    NASA Astrophysics Data System (ADS)

    Bonesini, M.; HARP/PS214 Collaboration

    2012-08-01

    The final results on ?± production in proton nucleus or ?± nucleus interactions for incident particle momenta between 1.5 GeV/c and 15 GeV/c as measured in the HARP/PS214 experiment at CERN PS are presented.

  17. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

    PubMed Central

    Tripathi, Lokesh P; Sowdhamini, R

    2008-01-01

    Background Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms. Results A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc.) were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes. Conclusion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes, suggesting distinct roles for serine proteases in prokaryotes. Many domain combinations were found unique to specific prokaryotic species, suggesting functional specialisation in various cellular and physiological processes. PMID:19019219

  18. Increased liver l-serine–pyruvate aminotransferase activity under gluconeogenic conditions (Short Communication)

    PubMed Central

    Rowsell, Edward V.; Al-Tai, Ali H.; Carnie, John A.; Rowsell, Kathleen V.

    1973-01-01

    Rat liver l-serine–pyruvate aminotransferase activity exceeds markedly the normal adult value (a) in the neonatal period, (b) after glucagon injection and (c) after alloxan injection, observations that reinforce the suggestion from comparative findings that the aminotransferase has a role in gluconeogenesis. Some findings, however, argue in favour of l-serine dehydratase as the enzyme of gluconeogenesis from l-serine. PMID:4723229

  19. New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina

    Microsoft Academic Search

    Juan Li; Li Yu; Jinkui Yang; Linqian Dong; Baoyu Tian; Zefen Yu; Lianming Liang; Ying Zhang; Xu Wang; Keqin Zhang

    2010-01-01

    BACKGROUND: Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors.

  20. Transcribing RNA Polymerase II Is Phosphorylated at CTD Residue Serine7

    Microsoft Academic Search

    Rob D. Chapman; Martin Heidemann; Thomas K. Albert; Reinhard Mailhammer; Andrew Flatley; Michael Meisterernst; Elisabeth Kremmer; Dirk Eick

    2007-01-01

    RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5' and 3' regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes.

  1. Molecular Cloning and Characterization of Two Serine Proteinase Genes from the Crayfish Plague Fungus, Aphanomyces astaci

    Microsoft Academic Search

    Eakaphun Bangyeekhun; Lage Cerenius; Kenneth Söderhäll

    2001-01-01

    Two novel genes encoding the serine proteinases, subtilisin (AaSP1) and trypsin (AaSP2), from Aphanomyces astaci were identified. Based on the amino acidconsensus sequences around the catalytic triad of these serine proteinases, degenerated oligonucleotides were designed for isolation of serine proteinase genes from a genomic DNA library. The AaSP1 gene encodes a full-length protein of 515 amino acids as a large

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

    PubMed

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

    2015-06-01

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

  3. 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

  4. Insulin resistance and muscle insulin receptor substrate-1 serine hyperphosphorylation.

    PubMed

    Stuart, Charles A; Howell, Mary E A; Cartwright, Brian M; McCurry, Melanie P; Lee, Michelle L; Ramsey, Michael W; Stone, Michael H

    2014-12-01

    Insulin resistance in metabolic syndrome subjects is profound in spite of muscle insulin receptor and insulin-responsive glucose transporter (GLUT4) expression being nearly normal. Insulin receptor tyrosine kinase phosphorylation of insulin receptor substrate-1 (IRS-1) at Tyr896 is a necessary step in insulin stimulation of translocation of GLUT4 to the cell surface. Serine phosphorylation of IRS-1 by some kinases diminishes insulin action in mice. We evaluated the phosphorylation status of muscle IRS-1 in 33 subjects with the metabolic syndrome and seventeen lean controls. Each underwent euglycemic insulin clamps and a thigh muscle biopsy before and after 8 weeks of either strength or endurance training. Muscle IRS-1 phosphorylation at six sites was quantified by immunoblots. Metabolic syndrome muscle IRS-1 had excess phosphorylation at Ser337 and Ser636 but not at Ser307, Ser789, or Ser1101. Ser337 is a target for phosphorylation by glycogen synthase kinase 3 (GSK3) and Ser636 is phosphorylated by c-Jun N-terminal kinase 1 (JNK1). Exercise training without weight loss did not change the IRS-1 serine phosphorylation. These data suggest that baseline hyperphosphorylation of at least two key serines within muscle IRS-1 diminishes the transmission of the insulin signal and thereby decreases the insulin-stimulated translocation of GLUT4. Excess fasting phosphorylation of muscle IRS-1 at Ser636 may be a major cause of the insulin resistance seen in obesity and might prevent improvement in insulin responsiveness when exercise training is not accompanied by weight loss. PMID:25472611

  5. Intermediates in serine recombinase-mediated site-specific recombination.

    PubMed

    Marshall Stark, W; Boocock, Martin R; Olorunniji, Femi J; Rowland, Sally-J

    2011-04-01

    Site-specific recombinases are enzymes that promote precise rearrangements of DNA sequences. They do this by cutting and rejoining the DNA strands at specific positions within a pair of target sites recognized and bound by the recombinase. One group of these enzymes, the serine recombinases, initiates strand exchange by making double-strand breaks in the DNA of the two sites, in an intermediate built around a catalytic tetramer of recombinase subunits. However, these catalytic steps are only the culmination of a complex pathway that begins when recombinase subunits recognize and bind to their target sites as dimers. To form the tetramer-containing reaction intermediate, two dimer-bound sites are brought together by protein dimer-dimer interactions. During or after this initial synapsis step, the recombinase subunit and tetramer conformations change dramatically by repositioning of component subdomains, bringing about a transformation of the enzyme from an inactive to an active configuration. In natural serine recombinase systems, these steps are subject to elaborate regulatory mechanisms in order to ensure that cleavage and rejoining of DNA strands only happen when and where they should, but we and others have identified recombinase mutants that have lost dependence on this regulation, thus facilitating the study of the basic steps leading to catalysis. We describe how our studies on activated mutants of two serine recombinases, Tn3 resolvase and Sin, are providing us with insights into the structural changes that occur before catalysis of strand exchange, and how these steps in the reaction pathway are regulated. PMID:21428950

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

    PubMed Central

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

    1995-01-01

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

  7. 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. PMID:25037918

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

    PubMed

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

    2009-03-01

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

  9. -Standard -PS 3.11-2008

    E-print Network

    Rumolo, Giovanni

    - Standard - PS 3.11-2008 Digital Imaging and Communications in Medicine (DICOM) Part 11: Media and it does not independently test, evaluate, or verify the accuracy or completeness of any information, as to the accuracy or completeness of any information published herein, and disclaims and makes no warranty

  10. -Standard -PS 3.12-2008

    E-print Network

    Rumolo, Giovanni

    - Standard - PS 3.12-2008 Digital Imaging and Communications in Medicine (DICOM) Part 12: Media Formats and Physical Media for Media Interchange Published by National Electrical Manufacturers and it does not independently test, evaluate, or verify the accuracy or completeness of any information

  11. Comparing FB and PS scheduling policies

    Microsoft Academic Search

    Patrick Brown

    2006-01-01

    In this paper we obtain new results concerning the expected response time of the foreground-background (FB) scheduling discipline and its comparison with processor sharing (PS). Some results previously derived for job sizes with finite second moment or bounded sizes, are extended to infinite second moments. New bounds and asymptotic results are also derived. We show that for job sizes with

  12. Positron Annihilation in the Bipositronium Ps2

    SciTech Connect

    Bailey, David H.; Frolov, Alexei M.

    2005-07-01

    The electron-positron-pair annihilation in the bipositronium PS2 is considered. In particular, the two-, three-, one- and zero-photon annihilation rates are determined to high accuracy. The corresponding analytical expressions are also presented. Also, a large number of bound state properties have been determined for this system.

  13. l-Serine Catabolism via an Oxygen-Labile l-Serine Dehydratase Is Essential for Colonization of the Avian Gut by Campylobacter jejuni

    PubMed Central

    Velayudhan, Jyoti; Jones, Michael A.; Barrow, Paul A.; Kelly, David J.

    2004-01-01

    Campylobacter jejuni is a microaerophilic, asaccharolytic bacterium. The identity of the carbon and energy sources used by C. jejuni in vivo is unknown, but the genome sequence of strain NCTC11168 indicates the presence of genes for catabolism of a limited range of amino acids, including serine. Specific omission of l-serine from a defined medium containing a mixture of amino acids led to a dramatic decrease in cell yields. As C. jejuni does not have a biosynthetic serine requirement, this supports earlier suggestions that l-serine is a preferentially catabolized amino acid. Serine transport was found to be mediated by at least two systems in strain 11168; a high-capacity, low-affinity l-serine-specific system encoded by Cj1625c (sdaC) and a higher-affinity l-serine/l-threonine system responsible for residual l-serine transport in an sdaC mutant. Catabolism of l-serine to pyruvate and ammonia is carried out by SdaA (encoded by Cj1624c), which was overexpressed, purified, and shown to be an oxygen-labile iron-sulfur enzyme. l-Serine dehydratase activity in an sdaA mutant was reduced 10-fold compared to that in the wild type, but the residual activity (due to the anabolic l-threonine dehydratase) could not support either growth on or utilization of l-serine in defined media. However, although sdaA mutants showed no obvious growth defect in complex media, they completely failed to colonize 3-week-old chickens as assayed both by cloacal swabs taken over a 6-week period and by cecal colony counts postmortem. In contrast, the isogenic parent strain colonized chickens to high levels within 1 week of inoculation. The results show that an active SdaA is essential for colonization of the avian gut by C. jejuni and imply that catabolism of l-serine is crucially important for the growth of this bacterium in vivo. PMID:14688104

  14. AVO approximation for PS-wave and its application in PP/PS joint inversion

    NASA Astrophysics Data System (ADS)

    Wang, Pu; Hu, Tian-Yue

    2011-09-01

    Multi-component exploration has many advantages over ordinary P-wave exploration. PP/PS joint AVO analysis and inversion are useful and powerful methods to discriminate between reservoir and non-productive lithology. In this paper, we derive a new PS-wave reflection coefficient approximation equation which is more accurate at larger incidence angles. The equation is simplified for small incidence angles, which makes AVO analysis clearer and easier for angles less than 30 degrees. Based on this approximation, a PP/PS joint inversion is introduced. A real data example shows that oil sands, brine sands and shales can be differentiated based on the P- to S-wave velocity ratio from the PP/PS joint inversion. Fluid factors and Poisson's ratio also indicate an anomaly in the target zone at the oil well location.

  15. 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. PMID:25686955

  16. Inhibition of pan-class I phosphatidyl-inositol-3-kinase by NVP-BKM120 effectively blocks proliferation and induces cell death in diffuse large B-cell lymphoma.

    PubMed

    Zang, Chuanbing; Eucker, Jan; Liu, Hongyu; Coordes, Annekatrin; Lenarz, Minoo; Possinger, Kurt; Scholz, Christian Wilfried

    2014-02-01

    Diffuse large B-cell lymphoma (DLBCL) is the most frequent aggressive lymphoma, with a great demand for novel treatments for relapsing and refractory disease. Constitutive activation of the phosphatidyl-inositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is often detected in this lymphoma. Inhibition of this signaling cascade with the pan-class I PI3K inhibitor NVP-BKM120 decreased cell proliferation and increased apoptotic cell death. DLBCL proliferation was further decreased if NVP-BKM120-induced autophagy was blocked. Treatment with NVP-BKM120 was associated with an increase of the pro-apoptotic BH3-only proteins Puma and Bim and down-regulation of the anti-apoptotic Bcl-xL and Mcl-1. Translation of Bcl-xL and Mcl-1 is facilitated by cap-dependent mRNA translation, a process that was partially inhibited by NVP-BKM120. Overall, we demonstrated here the potential of NVP-BKM120 for the treatment of DLBCL. PMID:23721513

  17. College of Arts and Sciences PS Political Science

    E-print Network

    MacAdam, Keith

    College of Arts and Sciences PS Political Science KEY: # = new course * = course changed = course TO POLITICAL ANALYSIS. (3) Introduction to the basic knowledge of research methodology in political science. Prereq: UN2 status; PS majors only. PS 391 SPECIAL TOPICS IN POLITICAL SCIENCE (Subtitle required). (3

  18. Serine racemase: a key player in neuron activity and in neuropathologies.

    PubMed

    Campanini, Barbara; Spyrakis, Francesca; Peracchi, Alessio; Mozzarelli, Andrea

    2013-01-01

    Serine racemase is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes L-serine racemisation to D-serine, and L- and D-serine beta-elimination in mammalian brain. D-serine is the essential co-agonist of the N-methyl-D-aspartate receptor, that mediates neurotransmission, synaptic plasticity, cell migration and long term potentiation. High and low D-serine levels have been associated with distinct neuropathologies, aging-related deficits and psychiatric disorders due to either hyper- or hypo-activation of the receptor. Serine racemase dual activity is regulated by ATP, divalent cations, cysteine nitrosylation, post-translational modifications, and interactions with proteins that bind either at the N- or C-terminus. A detailed elucidation of the molecular basis of catalysis, regulation and conformational plasticity, as well as enzyme and D-serine localization and neurons and astrocytes cross-talk, opens the way to the development of enzyme inhibitors and effectors for tailored therapeutic treatments. PMID:23747871

  19. Characteristics of hepatic serine-pyruvate aminotransferase in different mammalian species.

    PubMed Central

    Noguchi, T; Takada, Y; Kido, R

    1977-01-01

    1. Serine-pyruvate aminotransferase was purified from mouse, rat, dog and cat liver. Each enzyme preparation was homogeneous as judged by polyacrylamide-disc-gel electrophoresis in the presence of sodium dodecyl sulphate. However, isoelectric focusing resulted in the detection of two or more active forms from enzyme preparations from dog, cat and mouse. A single active form was obtained with the rat enzyme. All four enzyme preparations had similar pH optima and molecular weights. 2. Both mouse and rat preparations catalysed transamination between a number of L-amino acids (serine, leucine, asparagine, methionine, glutamine, ornithine, histidine, phenylalanine or tyrosine) and pyruvate. Effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine as amino donor. The reverse transamination activity, with hydroxypyruvate and alanine as subtrates, was lower than with serine and pyruvate for both species. Serine-pyruvate aminotransferase activities were inhibited by isonicotinic acid hydrazide. 3. In contrast, both dog and cat enzyme preparations were highly specific for serine as amino donor with pyruvate, and utilized pyruvate and glyoxylate as effective amino acceptors. A little activity was detected with phenylpyruvate. The reverse activity was higher than with serine and pyruvate for both species. Serine-pyruvate amino-transferase activities were not inhibited by isonicotinic acid hydrazide. PMID:851432

  20. The structure of mammalian serine racemase: evidence for conformational changes upon inhibitor binding.

    PubMed

    Smith, Myron A; Mack, Volker; Ebneth, Andreas; Moraes, Isabel; Felicetti, Brunella; Wood, Michael; Schonfeld, Dorian; Mather, Owen; Cesura, Andrea; Barker, John

    2010-04-23

    Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5'-phosphate-dependent enzyme is involved both in the reversible conversion of L- to D-serine and serine catabolism by alpha,beta-elimination of water, thereby regulating D-serine levels. Because D-serine affects NMDAR signaling throughout the brain, serine racemase is a promising target for the treatment of disorders related to NMDAR dysfunction. To provide a molecular basis for rational drug design the x-ray crystal structures of human and rat serine racemase were determined at 1.5- and 2.1-A resolution, respectively, and in the presence and absence of the orthosteric inhibitor malonate. The structures revealed a fold typical of beta-family pyridoxal 5'-phosphate enzymes, with both a large domain and a flexible small domain associated into a symmetric dimer, and indicated a ligand-induced rearrangement of the small domain that organizes the active site for specific turnover of the substrate. PMID:20106978

  1. A novel serine protease inhibitor acts as an immunomodulatory switch while maintaining homeostasis.

    PubMed

    Jiang, N; Thangamani, S; Chor, C F; Wang, S Y; Winarsih, I; Du, R J; Sivaraman, J; Ho, B; Ding, J L

    2009-01-01

    Serine protease cascades boost immune responses while maintaining homeostasis. These crucial actions are intricately regulated by cognate serine protease inhibitors. However, the mechanism underlying such a dynamic immunomodulation during acute phase infection remains obscure, particularly where the pathogen's serine protease adds a new challenge to the host. Here, we found that infection of horseshoe crab, Carcinoscorpius rotundicauda, induced reciprocal profiles of CrSPI (serine protease inhibitor) and CrFurin (serine protease) with respect to their transcription and protein activities. Using recombinant rCrSPI, we explored its inhibitory activity against various microbial proteases and found it most efficacious against a model serine protease, subtilisin A. rCrSPI inhibited subtilisin at Ki 10(-9)M with a molar ratio of 1 rCrSPI:2 subtilisin. The rCrSPI also inhibited plasma CrFurin, suppressed subtilisin-mediated activation of prophenoloxidase (PPO) and interacted with complement C3. Taken together, CrSPI acts as a key immunomodulatory 'on-off' switch in a 2-way regulation of serine protease microbial subtilisin and host serine proteases (CrFurin and CrC3), thereby controlling immune responses involving the complements and the PPO-mediated antimicrobial activities, while maintaining homeostasis. PMID:20375604

  2. Intrinsic Viscosity Characterization of PS and PMMA

    NSDL National Science Digital Library

    DeRosa, Rebecca L.

    2008-09-26

    In this experiment, you will use intrinsic viscosity measurements to determine the molecular weight of polystyrene, PS, or poly(methyl methacrylate), PMMA. After in-class presentation, completion of hands-on laboratory experiment and review of the information provided, you should be able to: • Identify several laboratory methods for molecular weight analysis of polymers. • Confidently discuss the differences between the methods of analysis for polymer molecular weight. • Discuss how polymer solution behavior affects molecular weight measurements.

  3. Low-Molecular-Weight O-Acetylserine Sulfhydrylase and Serine Sulfhydrylase of Saccharomyces cerevisiae Are the Same Protein

    PubMed Central

    Yamagata, Shuzo

    1981-01-01

    Low-molecular-weight O-acetyl-l-serine sulfhydrylase was purified from a cysteine auxotroph of Saccharomyces cerevisiae and was demonstrated to be identical with l-serine sulfhydrylase. Images PMID:7021536

  4. L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum.

    PubMed

    Zhu, Qinjian; Zhang, Xiaomei; Luo, Yuchang; Guo, Wen; Xu, Guoqiang; Shi, Jinsong; Xu, Zhenghong

    2015-02-01

    The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the L-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of L-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity. PMID:25434811

  5. Comparative Immunohistochemical Analysis of Ochratoxin A Tumourigenesis in Rats and Urinary Tract Carcinoma in Humans; Mechanistic Significance of p-S6 Ribosomal Protein Expression

    PubMed Central

    Gazinska, Patrycja; Herman, Diana; Gillett, Cheryl; Pinder, Sarah; Mantle, Peter

    2012-01-01

    Ochratoxin A (OTA) is considered to be a possible human urinary tract carcinogen, based largely on a rat model, but no molecular genetic changes in the rat carcinomas have yet been defined. The phosphorylated-S6 ribosomal protein is a marker indicating activity of the mammalian target of rapamycin, which is a serine/threonine kinase with a key role in protein biosynthesis, cell proliferation, transcription, cellular metabolism and apoptosis, while being functionally deregulated in cancer. To assess p-S6 expression we performed immunohistochemistry on formalin-fixed and paraffin-embedded tumours and normal tissues. Marked intensity of p-S6 expression was observed in highly proliferative regions of rat renal carcinomas and a rare angiosarcoma, all of which were attributed to prolonged exposure to dietary OTA. Only very small OTA-generated renal adenomas were negative for p-S6. Examples of rat subcutaneous fibrosarcoma and testicular seminoma, as well as of normal renal tissue, showed no or very weak positive staining. In contrast to the animal model, human renal cell carcinoma, upper urinary tract transitional cell carcinoma from cases of Balkan endemic nephropathy, and a human angiosarcoma were negative for p-S6. The combined findings are reminiscent of constitutive changes in the rat tuberous sclerosis gene complex in the Eker strain correlated with renal neoplasms, Therefore rat renal carcinogenesis caused by OTA does not obviously mimic human urinary tract tumourigenesis. PMID:23105973

  6. A Self-compartmentalizing Hexamer Serine Protease from Pyrococcus Horikoshii

    PubMed Central

    Menyhárd, Dóra K.; Kiss-Szemán, Anna; Tichy-Rács, Éva; Hornung, Balázs; Rádi, Krisztina; Szeltner, Zoltán; Domokos, Klarissza; Szamosi, Ilona; Náray-Szabó, Gábor; Polgár, László; Harmat, Veronika

    2013-01-01

    Oligopeptidases impose a size limitation on their substrates, the mechanism of which has long been under debate. Here we present the structure of a hexameric serine protease, an oligopeptidase from Pyrococcus horikoshii (PhAAP), revealing a complex, self-compartmentalized inner space, where substrates may access the monomer active sites passing through a double-gated “check-in” system, first passing through a pore on the hexamer surface and then turning to enter through an even smaller opening at the monomers' domain interface. This substrate screening strategy is unique within the family. We found that among oligopeptidases, a residue of the catalytic apparatus is positioned near an amylogenic ?-edge, which needs to be protected to prevent aggregation, and we found that different oligopeptidases use different strategies to achieve such an end. We propose that self-assembly within the family results in characteristically different substrate selection mechanisms coupled to different multimerization states. PMID:23632025

  7. D-Serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor

    Microsoft Academic Search

    Jean-Pierre Mothet; Angčle T. Parent; Herman Wolosker; Roscoe O. Brady Jr.; David J. Linden; Christopher D. Ferris; Michael A. Rogawski; Solomon H. Snyder

    2000-01-01

    Functional activity of N-methyl-D-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although D-serine is a more potent agonist. Localizations of D-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of D-serine with D-amino

  8. Regulated Proteolysis by Cortical Granule Serine Protease 1 at Fertilization

    PubMed Central

    Haley, Sheila A.; Wessel, Gary M.

    2004-01-01

    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 ?-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. PMID:14978210

  9. Plasma motion during the formation phase of the PS-3 and PS-3. 5 spheromaks

    SciTech Connect

    Peyser, T.A.

    1987-01-01

    Spectroscopic Doppler-shift measurements of C III impurity ion in the ultraviolet are reported from the University of Maryland spheromak experiments PS-3 ad PS-3.5. A new time- and space-resolved Doppler-shift diagnostic with absolute wavelength calibration to +/- 0.02 A is described. Large-velocity fields (on the order of the Alfven speed) observed in both the PS-3 and PS-3.5 experiments during the formation phase, but not during the equilibrium, suggest that flow fields may play an important role in the reconnection and relaxation process. The standard theory of Taylor relaxation may need to be modified in order to include the effects of bulk plasma motion. Doppler-shift measurements of the C III 2296.87-A impurity-emission line in the PS-3.5 spheromak were made side-on above and below the machine axis. During the formation phase, blue shifts are observed below and red shifts observed above the axis suggesting a toroidal rotation in the direction of the confined-plasma diamagnetic drift. The plasma emissivity near the C III wavelength was obtained from Abel inversion of the line-integrated intensities. Computer modeling of velocity fields, emissivity profiles, and line shapes is used to interpret the experimental data.

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

    PubMed

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

    2015-07-01

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

  11. Serine integrase chimeras with activity in E. coli and HeLa cells

    PubMed Central

    Farruggio, Alfonso P.; Calos, Michele P.

    2014-01-01

    ABSTRACT In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC) and phiC31-TG1 (CT) hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{?1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies. PMID:25217617

  12. Uptake of the neutral amino acids glutamine, leucine, and serine by Pneumocystis carinii.

    PubMed

    Basselin, M; Qiu, Y H; Lipscomb, K J; Kaneshiro, E S

    2001-07-01

    Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period. PMID:11414689

  13. Enzymatic Synthesis of Galactosylated Serine/Threonine Derivatives by ?-Galactosidase from Escherichia coli.

    PubMed

    Seo, Sooyoun; Rebehmed, Joseph; de Brevern, Alexandre G; Karboune, Salwa

    2015-01-01

    The transgalactosylations of serine/threonine derivatives were investigated using ?-galactosidase from Escherichia coli as biocatalyst. Using ortho-nitrophenyl-?-D-galactoside as donor, the highest bioconversion yield of transgalactosylated N-carboxy benzyl L-serine benzyl ester (23.2%) was achieved in heptane:buffer medium (70:30), whereas with the lactose, the highest bioconversion yield (3.94%) was obtained in the buffer reaction system. The structures of most abundant galactosylated serine products were characterized by MS/MS. The molecular docking simulation revealed that the binding of serine/threonine derivatives to the enzyme's active site was stronger (-4.6~-7.9 kcal/mol) than that of the natural acceptor, glucose, and mainly occurred through interactions with aromatic residues. For N-tert-butoxycarbonyl serine methyl ester (6.8%) and N-carboxybenzyl serine benzyl ester (3.4%), their binding affinities and the distances between their hydroxyl side chain and the 1'-OH group of galactose moiety were in good accordance with the quantified bioconversion yields. Despite its lower predicted bioconversion yield, the high experimental bioconversion yield obtained with N-carboxybenzyl serine methyl ester (23.2%) demonstrated the importance of the thermodynamically-driven nature of the transgalactosylation reaction. PMID:26084049

  14. Enzymatic Synthesis of Galactosylated Serine/Threonine Derivatives by ?-Galactosidase from Escherichia coli

    PubMed Central

    Seo, Sooyoun; Rebehmed, Joseph; de Brevern, Alexandre G.; Karboune, Salwa

    2015-01-01

    The transgalactosylations of serine/threonine derivatives were investigated using ?-galactosidase from Escherichia coli as biocatalyst. Using ortho-nitrophenyl-?-d-galactoside as donor, the highest bioconversion yield of transgalactosylated N-carboxy benzyl l-serine benzyl ester (23.2%) was achieved in heptane:buffer medium (70:30), whereas with the lactose, the highest bioconversion yield (3.94%) was obtained in the buffer reaction system. The structures of most abundant galactosylated serine products were characterized by MS/MS. The molecular docking simulation revealed that the binding of serine/threonine derivatives to the enzyme’s active site was stronger (?4.6~?7.9 kcal/mol) than that of the natural acceptor, glucose, and mainly occurred through interactions with aromatic residues. For N-tert-butoxycarbonyl serine methyl ester (6.8%) and N-carboxybenzyl serine benzyl ester (3.4%), their binding affinities and the distances between their hydroxyl side chain and the 1?-OH group of galactose moiety were in good accordance with the quantified bioconversion yields. Despite its lower predicted bioconversion yield, the high experimental bioconversion yield obtained with N-carboxybenzyl serine methyl ester (23.2%) demonstrated the importance of the thermodynamically-driven nature of the transgalactosylation reaction. PMID:26084049

  15. Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5'-Phosphate-Dependent Enzyme.

    PubMed

    Sukanya, N; Vijaya, M; Savithri, H S; Radhakrishnan, A N; Rao, N A

    1991-02-01

    Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5'-phosphate-containing enzyme, but the requirement of pyridoxal-5'-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5'-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5'-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5'-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5'-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5'-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5' phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5'-phosphate that is present in the mammalian and bacterial enzymes. PMID:16667990

  16. The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

    Microsoft Academic Search

    V. Würtz Jürgensen; K. Jalkanen

    2006-01-01

    The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory with the Becke 3LYP hybrid exchange correlation (XC) functional and the PW91 GGA XC functional and the 6-31G* and aug-cc-pVDZ basis

  17. Ultrafast Glycerophospholipid-selective Transbilayer Motion Mediated by a Protein in the Endoplasmic Reticulum Membrane*

    E-print Network

    - dylserine, and phosphatidylethanolamine. Such trans- port is nonvectorial and leads to a symmetric trans phosphatidylcho- line, phosphatidylethanolamine, and in part phosphatidyl- serine. These synthetic activities

  18. Characterization of the Yeast DGK1-encoded CTP-dependent Diacylglycerol Kinase*

    E-print Network

    Chen, Kuang-Yu

    phosphatidylinositol, phosphatidyl- serine, phosphatidylethanolamine, and phosphatidylcholine are derived from PA via are similarly derived from PA via CDP-DAG (not shown in Fig. 1) (6). Alternatively, phosphatidylethanolamine

  19. d-Amino acid oxidase and serine racemase in human brain: normal distribution and altered expression in schizophrenia

    PubMed Central

    Verrall, Louise; Walker, Mary; Rawlings, Nancy; Benzel, Isabel; Kew, James N. C.; Harrison, Paul J.; Burnet, Philip W. J.

    2007-01-01

    The N-methyl-d-aspartate receptor co-agonist d-serine is synthesized by serine racemase and degraded by d-amino acid oxidase. Both d-serine and its metabolizing enzymes are implicated in N-methyl-d-aspartate receptor hypofunction thought to occur in schizophrenia. We studied d-amino acid oxidase and serine racemase immunohistochemically in several brain regions and compared their immunoreactivity and their mRNA levels in the cerebellum and dorsolateral prefrontal cortex in schizophrenia. d-Amino acid oxidase immunoreactivity was abundant in glia, especially Bergmann glia, of the cerebellum, whereas in prefrontal cortex, hippocampus and substantia nigra, it was predominantly neuronal. Serine racemase was principally glial in all regions examined and demonstrated prominent white matter staining. In schizophrenia, d-amino acid oxidase mRNA was increased in the cerebellum, and as a trend for protein. Serine racemase was increased in schizophrenia in the dorsolateral prefrontal cortex but not in cerebellum, while serine racemase mRNA was unchanged in both regions. Administration of haloperidol to rats did not significantly affect serine racemase or d-amino acid oxidase levels. These findings establish the major cell types wherein serine racemase and d-amino acid oxidase are expressed in human brain and provide some support for aberrant d-serine metabolism in schizophrenia. However, they raise further questions as to the roles of d-amino acid oxidase and serine racemase in both physiological and pathophysiological processes in the brain. PMID:17880399

  20. 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

  1. LBNL-61925-Update ECLOUD in PS2, PS+, SPS+: AN UPDATE

    E-print Network

    Furman, Miguel

    recall that the labels "copper" and "stainless steel" refer here to a Work supported by the U.S. DOE, respectively, plotted as a function of peak SEY max. Numerical conver- gence is clear at Nk = 501 for tb = 25 of the heat load for the PS50, for copper vs. stainless steel chamber surface. The simu- lated results

  2. Timing between streak cameras with a precision of 10 ps

    SciTech Connect

    Lerche, R.A.

    1990-12-07

    The laser beams irradiating a target at the Nova laser facility comprise a set of ten simultaneous events. Two streak cameras, whose resolutions are 40 ps, record the power history for each beam, five beams to a camera; their time bases are cross-timed with a fiducial pulse. Analysis of data recorded for target experiments conducted over a six month period show the precision for cross-timing signals between two streak cameras to be {plus minus}9 ps and for characterizing a single temporal feature of a pulse to be {plus minus}5 ps. Beam synchronization at the end of six months was within 20 ps of the synchronization at the beginning of the experiments. A beam timing shift greater than 25 ps can be detected on a single laser shot; shifts of 10 to 20 ps require several shots to detect. 2 refs., 6 figs.

  3. The biological functions of MBL-associated serine proteases (MASPs).

    PubMed

    Hajela, Krishnan; Kojima, Mayumi; Ambrus, Geza; Wong, K H Nicky; Moffatt, Beryl E; Ferluga, Janez; Hajela, Sumati; Gál, Peter; Sim, Robert B

    2002-09-01

    The Mannose-binding lectin-associated serine proteases (MASPs) have been the subject of intensive research particularly over the past 10 years. First one, then two, and currently 3 MASPs have been characterized. Initially it was thought likely that the MBL + MASPs system would resemble very closely the C1 complex of the complement classical pathway, and that MASP1 and MASP2 would have similar activities to their classical pathway homologues C1r and C1s. MASP2 does certainly have similar activities to C1s, but MASP1 does not have the activities of either C1r or C1s. MASP1 has been thought to act on the complement system by cleaving C3 directly, but work with recombinant and purified native MASP1 shows that direct C3 cleavage by this protease is very slow, and may not be biologically significant. MASP1 and MASP2 appear not to have such a narrow specificity as C1r and C1s, and may have significant substrates other than complement proteins. As an example, MASP1 does cleave fibrinogen, releasing fibrinopeptide B (a chemotactic factor) and also cleaves and activates plasma transglutaminase (Factor XIII). These reactions are also relevant to defence against microorganisms, and may represent a biologically significant action of MASP1. PMID:12396008

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

    PubMed

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

    2015-01-01

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

  5. Structure-guided reprogramming of serine recombinase DNA sequence specificity.

    PubMed

    Gaj, Thomas; Mercer, Andrew C; Gersbach, Charles A; Gordley, Russell M; Barbas, Carlos F

    2011-01-11

    Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences > 10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with > 80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins. PMID:21187418

  6. Novel aspects and new roles for the serine protease plasmin.

    PubMed

    Syrovets, T; Simmet, Th

    2004-04-01

    The serine protease plasmin is distributed throughout the human body in the form of the zymogen plasminogen. The plasminogen activation system is mostly recognized for its fibrinolytic activity but is also upregulated in chronic inflammatory diseases, including atherosclerosis and arthritis. Plasmin can bind to a variety of cells, including monocytes, through low-affinity binding sites and triggers aggregation of neutrophils, platelet degranulation and arachidonate release from endothelial cells. In monocytes, plasmin elicits full-scale proinflammatory activation, including lipid mediator release, chemotaxis and cytokine expression, as well as induction of other proinflammatory genes. The effects of plasmin are specific, require the active catalytic center and can be antagonized by lysine analogues, implying binding of the plasmin molecule to the cell membrane through its lysine binding sites. In view of the upregulation of the fibrinolytic genes in chronic inflammatory diseases, cell activation by plasmin is likely to play a major pathophysiological role, a view that is further supported by data from transgenic mice. PMID:15095009

  7. Pramipexole reduces phosphorylation of ?-synuclein at serine-129.

    PubMed

    Chau, Kai-Yin; Cooper, J Mark; Schapira, Anthony Henry V

    2013-10-01

    ?-Synuclein is a central component of the pathogenesis of Parkinson's disease (PD). Phosphorylation at serine-129 represents an important post-translational modification and constitutes the major form of the protein in Lewy bodies. Several kinases have been implicated in the phosphorylation of ?-synuclein. The targeting of kinase pathways as a potential to influence the pathogenesis of PD is an important focus of attention, given that mutations of specific kinases (LRRK2 and PINK1) are causes of familial PD. Pramipexole (PPX) is a dopamine agonist developed for the symptomatic relief of PD. Several in vitro and in vivo laboratory studies have demonstrated that PPX exerts neuroprotective properties in model systems of relevance to PD. The present study demonstrates that PPX inhibits the phosphorylation of ?-synuclein and that this is independent of dopamine receptor activation. PPX blocks the increase in phosphorylated ?-synuclein induced by inhibition of the ubiquitin proteasomal system. The phosphorylation of ?-synuclein occurs in part at least through casein kinase 2, and PPX in turn reduces the phosphorylation of this enzyme, thereby inhibiting its activity. Thus, PPX decreases the phosphorylation of ?-synuclein, and this mechanism may contribute to its protective properties in PD models. PMID:23681749

  8. Structural insights into the enzymes of the serine and biotin biosynthetic pathways in mycobacterium tuberculosis 

    E-print Network

    Dey, Sanghamitra

    2009-05-15

    Mycobacterium tuberculosis (Mtb) utilizes different metabolic pathways for its survival during infection. Enzymes of these pathways are often targets for antibiotic development. Genetic studies indicate the importance of the serine and biotin...

  9. A cocrystal of pyridine-2,4-dicarboxylic acid and serine

    PubMed Central

    Liang, Peng

    2008-01-01

    The title compound, pyridine-2,4-dicarboxylic acid–S-serine (1/1), C7H5NO4·C3H7NO3, has serine in its zwitterionic form. The crystal structure is stabilized by an extensive series of inter­molecular O—H?O, N—H?N and N—H?O hydrogen bonds, forming a three-dimensional network. PMID:21200918

  10. The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum

    Microsoft Academic Search

    Verena Pöll; Ursula Denk; Horng-Der Shen; Raphael C. Panzani; Oliver Dissertori; Peter Lackner; Wolfgang Hemmer; Adriano Mari; Reto Crameri; Friedrich Lottspeich; Raphaela Rid; Klaus Richter; Michael Breitenbach; Birgit Simon-Nobbe

    2009-01-01

    Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in

  11. Evolutionary Analysis of Novel Serine Proteases in the Venom Gland Transcriptome of Bitis gabonica rhinoceros

    PubMed Central

    Vaiyapuri, Sakthivel; Wagstaff, Simon C.; Harrison, Robert A.; Gibbins, Jonathan M.; Hutchinson, E. Gail

    2011-01-01

    Background Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. Methodology and Principal Findings Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. Conclusions and Significance Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites. PMID:21731776

  12. Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis.

    PubMed

    Pollari, Sirkku; Käkönen, Sanna-Maria; Edgren, Henrik; Wolf, Maija; Kohonen, Pekka; Sara, Henri; Guise, Theresa; Nees, Matthias; Kallioniemi, Olli

    2011-01-01

    Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, an understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidences for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L: -serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L: -serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L: -serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L: -serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions. PMID:20352489

  13. Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine.

    PubMed Central

    Stern, D F; Zheng, P; Beidler, D R; Zerillo, C

    1991-01-01

    A Saccharomyces cerevisiae lambda gt11 library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-S-transferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on serine and threonine. The gene was designated SPK1 for serine-protein kinase. Expression of the Spk1 fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spk1 is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases. Images PMID:1899289

  14. 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.; (Monash); (Queensland Inst. of Med. Rsrch.)

    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.

  15. Molecular cloning of a serine proteinase inhibitor from Brugia malayi.

    PubMed Central

    Yenbutr, P; Scott, A L

    1995-01-01

    The antigens produced by the infective-stage larvae of filarial parasites are potentially important targets for a protective immune response. A major impediment to studies on the biochemistry and molecular biology of antigens from infective larvae is a lack of parasite material. By employing a reverse transcription PCR-based strategy which exploited the presence of a conserved 22-nucleotide spliced leader sequence present at the 5' end of a proportion of nematode transcripts, spliced leader-containing cDNAs were amplified from the late-vector-stage larvae of the filarial nematode Brugia malayi. A major 1.4-kb PCR product was cloned into pBluescript. One of the PCR cDNA clones (BmY8) contained a 1,287-bp insert that encoded the first member of the serine proteinase inhibitor (serpin) superfamily to be described from nematodes. Reverse transcription PCR analysis of RNA isolated from different developmental stages of the parasite showed that transcription of the B. malayi serpin (Bmserpin) begins between days 8 and 9 of larval development within the insect vector and continues through to the adult and microfilarial stages. In immunoblot analyses of B. malayi somatic extracts, the native protein was estimated to have a molecular weight of 44,000. In immunoblots using excretory-secretory products from infective- and fourth-stage larvae, a single band with an estimated molecular weight of 75,000 was detected. A quantitative analysis of somatic extracts demonstrated that infective-stage larvae contained 10- to 16-fold-more Bmserpin than adults or microfilariae. Bmserpin was immunogenic in gerbils and was recognized strongly by sera from immunized animals. Bmserpin, which has the potential for modifying host defense responses, may play an important role in parasite survival during the early phase of vertebrate-stage development. PMID:7729881

  16. Serine proteases in the spiny lobster olfactory organ: Their functional expression along a developmental axis, and the contribution of a CUB-serine protease

    Microsoft Academic Search

    Malcolm E. Johns; Phang C. Tai; Charles D. Derby

    2004-01-01

    Several serine proteases and protease inhibitors have been identified in the crustacean olfac- tory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or

  17. Effect of different serine protease inhibitors in validating the 115 kDa Leishmania donovani secretory serine protease as chemotherapeutic target.

    PubMed

    Chakraborti, Tapati; Das, Partha; Choudhury, Rajdeep; De, Tripti

    2015-02-01

    Proteases have been considered as an important group of targets for development of antiprotozoal drugs due to their essential roles in host-parasite interactions, parasite immune evasion, life cycle transition and pathogenesis of parasitic diseases. The development of potent and selective serine protease inhibitors targeting L. donovani secretory serine protease (pSP) could pave the way to the discovery of potential antileishmanial drugs. Here, we employed different classical serine protease inhibitors (SPIs), such as aprotinin, N-tosyl-1-phenylalanine chloromethyl ketone (TPCK), N-tosyl-lysine chloromethyl ketone (TLCK), benzamidine (Bza) and pSP-antibody to determine the role of the protease in parasitic survival, growth and infectivity. Among the different classical SPIs, aprotinin appeared to be more potent in arresting L. donovani promastigotes growth with significant morphological alterations. Furthermore, aprotinin and anti-pSP treated parasites significantly decreased the intracellular parasites and percentage of infected macrophages. These results suggest that SPIs may reduce the infectivity by targeting the serine protease activity and may prove useful to elucidate defined molecular mechanisms of pSP, as well as for the development of novel antileishmanial drugs in future. PMID:26040107

  18. Policy Statement Number: PS-49 Title/Topic: Building Coordinator

    E-print Network

    Harms, Kyle E.

    , to Facility Services Work Control. 11. Report building maintenance requirements in building public areasPolicy Statement Number: PS-49 Title/Topic: Building Coordinator Effective Date: 12/07/2007 Revision Number: PS0049.R05 BUILDING COORDINATOR PURPOSE To set forth duties and responsibilities

  19. Safeguarding Nonhuman Primate iPS Cells With Suicide Genes

    PubMed Central

    Zhong, Bonan; Watts, Korashon L; Gori, Jennifer L; Wohlfahrt, Martin E; Enssle, Joerg; Adair, Jennifer E; Kiem, Hans-Peter

    2011-01-01

    The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, resulting in the formation of teratomas, have remained considerable obstacles. Here, we demonstrate the utility of suicide genes to safeguard iPS cells and their derivatives. We found suicide genes can control the cell fate of iPS cells in vitro and in vivo without interfering with their pluripotency and self-renewal capacity. This study will be useful to evaluate the safety of iPS cell technology in a clinically highly relevant, large animal model and further benefit the clinical use of human iPS cells. PMID:21587213

  20. Improving QoS for UGS, rtPS, nrtPS, BE in WIMAX networks

    Microsoft Academic Search

    Jalel Ben-Othman; Lynda Mokdad

    2011-01-01

    In this paper we present a new admission control (AC) for IEEE 802.16. The AC aims to accept new connections according to the negotiated service class (UGS, rtPS, nrTPS, and BE). To achieve this goal we propose to use the token bucket concept that provide QoS for real time traffics without degrading the QoS of non real time traffic. To

  1. High-precision calculation of loosely bound states of LiPs+ and NaPs+

    NASA Astrophysics Data System (ADS)

    Yamashita, Takuma; Kino, Yasushi

    2015-06-01

    A positronic alkali atom would be the first step to investigate behavior of a positronium(Ps) in an external field from atoms/molecules because the system can be regarded as a simple three-body system using model potentials reflecting electron orbitals of the ion core. In order to precisely determine binding energies and structures of positronic alkali atoms (LiPs+ and NaPs+), we improve the model potential so as to reproduce highly excited atomic energy levels of alkali atoms (Li and Na). The polarization potential included by the model potential is expanded in terms of Gaussian functions to finely determine a short range part of the potential which has been assumed to be a simple form. We find better reproducibility not only of atomic levels of the alkali atoms but also of the dipole polarizability of the core ion than previous works. We construct a model potential between a positron and an ion core based on the model potential between the valence electron and ion core. Binding energies associated with a dissociation of the alkali ion core and positronium, and interparticle distances are recalculated. Our results show slightly deeper bound than other previous studies.

  2. Functional Myogenic Engraftment from Mouse iPS Cells

    PubMed Central

    Darabi, Radbod; Pan, Weihong; Bosnakovski, Darko; Baik, June; Kyba, Michael

    2015-01-01

    Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies. PMID:21461712

  3. Functional myogenic engraftment from mouse iPS cells.

    PubMed

    Darabi, Radbod; Pan, Weihong; Bosnakovski, Darko; Baik, June; Kyba, Michael; Perlingeiro, Rita C R

    2011-11-01

    Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies. PMID:21461712

  4. Telomere Reprogramming and Maintenance in Porcine iPS Cells

    PubMed Central

    Ji, Guangzhen; Ruan, Weimin; Liu, Kai; Wang, Fang; Sakellariou, Despoina; Chen, Jijun; Yang, Yang; Okuka, Maja; Han, Jianyong; Liu, Zhonghua; Lai, Liangxue; Gagos, Sarantis; Xiao, Lei; Deng, Hongkui; Li, Ning; Liu, Lin

    2013-01-01

    Telomere reprogramming and silencing of exogenous genes have been demonstrated in mouse and human induced pluripotent stem cells (iPS cells). Pigs have the potential to provide xenotransplant for humans, and to model and test human diseases. We investigated the telomere length and maintenance in porcine iPS cells generated and cultured under various conditions. Telomere lengths vary among different porcine iPS cell lines, some with telomere elongation and maintenance, and others telomere shortening. Porcine iPS cells with sufficient telomere length maintenance show the ability to differentiate in vivo by teratoma formation test. IPS cells with short or dysfunctional telomeres exhibit reduced ability to form teratomas. Moreover, insufficient telomerase and incomplete telomere reprogramming and/or maintenance link to sustained activation of exogenous genes in porcine iPS cells. In contrast, porcine iPS cells with reduced expression of exogenous genes or partial exogene silencing exhibit insufficient activation of endogenous pluripotent genes and telomerase genes, accompanied by telomere shortening with increasing passages. Moreover, telomere doublets, telomere sister chromatid exchanges and t-circles that presumably are involved in telomere lengthening by recombination also are found in porcine iPS cells. These data suggest that both telomerase-dependent and telomerase-independent mechanisms are involved in telomere reprogramming during induction and passages of porcine iPS cells, but these are insufficient, resulting in increased telomere damage and shortening, and chromosomal instability. Active exogenes might compensate for insufficient activation of endogenous genes and incomplete telomere reprogramming and maintenance of porcine iPS cells. Further understanding of telomere reprogramming and maintenance may help improve the quality of porcine iPS cells. PMID:24098638

  5. D-Serine-induced nephrotoxicity: a HPLC-TOF/MS-based metabonomics approach.

    PubMed

    Williams, R E; Major, H; Lock, E A; Lenz, E M; Wilson, I D

    2005-02-14

    HPLC-MS-based metabonomic analysis was used to investigate urinary metabolic perturbations associated with D-serine-induced nephrotoxicity. D-Serine causes selective necrosis of the proximal straight tubules in the rat kidney accompanied by aminoaciduria, proteinuria and glucosuria. Alderely Park (Wistar-derived) rats were dosed with either D-serine (250 mg/kg ip) or vehicle (deionised water) and urine was collected at 0-12, 12-24, 24-36 and 36-48 h post-dosing. Samples were analysed using a Waters Alliance HT 2795 HPLC system coupled to a Waters Micromass Q-ToF-micro equipped with an electrospray source operating in either positive or negative ion mode. Changes to the urinary profile were detected at all time points compared to control. In negative ion mode, increases were observed in serine (m/z=103.0077), m/z=104.0376 (proposed to be hydroxypyruvate) and glycerate (m/z=105.0215), the latter being metabolites of D-serine. Furthermore, an increase in tryptophan, phenylalanine and lactate and decreases in methylsuccinic acid and sebacic acid were observed. Positive ion analysis revealed a decrease in xanthurenic acid, which has previously been assigned and reported using HPLC-MS following exposure to mercuric chloride and cyclosporine A. A general aminoaciduria, including proline, methionine, leucine, tyrosine and valine was also observed as well as an increase in acetyl carnitine. Investigation of additional metabolites altered as a result of exposure to D-serine is on-going. Thus, HPLC-MS-based metabonomic analysis has provided information concerning the mechanism of D-serine-induced renal injury. PMID:15596249

  6. 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

  7. Identity of isoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase.

    PubMed Central

    Noguchi, T; Okuno, E; Kido, R

    1976-01-01

    After glucagon injection, rats showed virtually identical percentage increases in hepatic histidine-pyruvate aminotransferase and serine-pyruvate aminotransferase activities, both in the mitochondria and in the cytosol. Histidine-pyruvate aminotransferase isoenzyme 1, with pI8.0, was purified to homogeneity from the mitochondrial fraction of liver from glucagon-injected rats. The purified enzyme catalysed transamination between a number of amino acids and pyruvate or phenylpyruvate. For transamination with pyruvate, the activity with serine reached a constant ratio to that with histidine during purification, which was unchanged by a variety of treatments of the purified enzyme. Serine was found to act as a competitive inhibitor of histidine transamination, and histidine of serine transamination. These results suggest that histidine-pyruvate amino-transferase isoenzymes 1 is identical with serine-pyruvate aminotransferase. The enzyme is probably composed of two identical subunits with mol. wt. approx. 38000. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents strongly indicate the presence of pyridoxal phosphate. PMID:12742

  8. Control analysis of mammalian serine biosynthesis. Feedback inhibition on the final step.

    PubMed Central

    Fell, D A; Snell, K

    1988-01-01

    The flux of serine biosynthesis in the liver of the normal rabbit, and of the rat on a low protein diet, is most sensitive to the activity of phosphoserine phosphatase (flux control coefficient up to 0.97), the last of the three enzymes in the pathway after it branches from glycolysis. The concentration of the pathway product, serine, has a strong controlling influence on the flux (response coefficient up to -0.64) through feedback inhibition at this step. The pathway is therefore controlled primarily by the demand for serine rather than the supply of the pathway precursor, 3-phosphoglycerate. Under conditions where there is a lower biosynthetic flux, the flux control coefficients of the first two enzymes of the pathway are increased, and are probably dominant in the rat on a normal diet. In rabbit liver, when ethanol is used to inhibit serine biosynthesis, control can be distributed between the three enzymes, even though the reactions catalysed by the first two remain close to equilibrium. Apart from their intrinsic value in aiding the understanding of the regulation of mammalian serine metabolism, our findings illustrate the danger of assuming that there are invariant design principles in the regulation of metabolic pathways, such as feedback control on the first step after a branch. PMID:2851987

  9. Genetic and Physiological Control of Serine and Glycine Biosynthesis in Saccharomyces1

    PubMed Central

    Ulane, Rodney; Ogur, Maurice

    1972-01-01

    Two of the three known metabolic pathways to serine and glycine have been shown to be present in prototrophic yeast strains, i.e., the phosphorylated pathway from glycolytic intermediates and the glyoxylate pathway from tricarboxylic acid cycle intermediates. Two serine-glycine auxotrophs (ser1 and ser2) were found to be blocked in the phosphoglycerate pathway. The ser1 gene controls l-glutamate:phosphohydroxypyruvate transaminase biosynthesis, and the ser2 gene controls phosphoserine phosphatase biosynthesis. The other pathway to glycine, from isocitrate, is repressed by growth in glucose media, specifically, at isocitrate lyase and at the alanine:glyoxylate transaminase. This pathway is derepressed by growth to stationary phase in glucose media yielding high activity of these enzymes. The phosphorylated pathway appears to be the principal biosynthetic pathway to serine and glycine during growth on sugar media. Strains which are serine-glycine dependent in glucose media became capable of serine-glycine independent growth on acetate media. These results describe a method of physiological control of a secondary metabolic pathway allowing a single lesion in the principal biosynthetic pathway to produce auxotrophy. This may be termed conditional auxotrophy. Images PMID:4333378

  10. Trypsin/alpha-amylase inhibitors inactivate the endogenous barley/malt serine endoproteinase SEP-1.

    PubMed

    Jones, Berne L; Fontanini, Debora

    2003-09-10

    Barley (Hordeum vulgare L.) malt contains endoproteinases belonging to all four of the commonly occurring classes, including serine proteinases. It also contains low molecular weight proteins that inhibit the activities of many of these endoproteinases, but it had never been shown that any barley or malt serine proteinases could be inhibited by any of these endogenous proteins. It is now reported that some proteins that were concentrated using an "affinity" method inhibited the activity of a malt serine endoproteinase. Two-dimensional electrophoretic and in vitro analyses showed that the inhibited enzyme was serine endoproteinase 1 (SEP-1) and that the inhibition could be quantified using a semipurified preparation of this enzyme. Amino acid sequencing and MALDI-TOF MS were used to identify the components of the partially purified inhibiting fractions. Only the "trypsin/alpha-amylase inhibitors" or chloroform/methanol (CM) proteins, most of which had truncated N and C termini, and one fragment of beta-amylase were present in the inhibitory fractions. When a CM protein fraction was prepared from barley according to traditional methods, some of its component proteins inhibited the activity of SEP-1 and some did not. This is the first report of the purification and identification of barley malt proteins that can inhibit an endogenous serine proteinase. It shows that some of the CM proteins probably play a role in controlling the activity of barley proteinases during germination, as well as possibly protecting the seed and young plant from microbes or pests. PMID:12952437

  11. Astrocyte-induced cortical vasodilation is mediated by D-serine and endothelial nitric oxide synthase

    PubMed Central

    Stobart, Jillian L. LeMaistre; Lu, Lingling; Mori, Hisashi; Anderson, Christopher M.

    2013-01-01

    Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D-serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca2+ produced dilation of penetrating arterioles in a manner attenuated by scavenging D-serine with D-amino acid oxidase, deleting the enzyme responsible for D-serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D-serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling. PMID:23386721

  12. Neuropathogenesis of HIV-1-associated neurocognitive disorders: a possible involvement of D-serine

    PubMed Central

    Xia, Jianxun; Xiong, Huangui

    2013-01-01

    A unique feature of N-methyl-D-aspartate receptors (NMDARs) that distinguishes them from other ionic receptors is that their activation requires more than one agonist to bind simultaneously to distinct binding sites on the receptor. D-serine, a co-agonist binding to the glycine site of NMDARs, has been implicated in several NMDAR-dependent physiological processes, and altered D-serine levels under certain pathophysiological conditions contribute to neural dysfunction via NMDARs in the central nervous system. Entry of HIV-1 in the brain causes neuronal injury leading to cognitive, behavioral and motor impairments known as HIV-associated neurocognitive disorders (HAND). As HIV-1 does not infect neurons, neuronal injury is believed to be primarily mediated by an indirect mechanism,that is, HIV-1-infected and/or immune-activated macrophages and microglial cells release soluble molecules leading to neuronal injury or death. Among the soluble factors is D-serine. In this article we try to address recent progresses on the role D-serine might play in the pathogenesis of neurodegenerative disorders with a particular emphasis of the involvement of D-serine in HIV-1-associated neurotoxicity. PMID:24044033

  13. iPS cells: a source of cardiac regeneration.

    PubMed

    Yoshida, Yoshinori; Yamanaka, Shinya

    2011-02-01

    For the treatment of heart failure, a new strategy to improve cardiac function and inhibit cardiac remodeling needs to be established. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent cells that can differentiate into cell types from all three germ layers both in vitro and in vivo. The therapeutic effect of ES/iPS cell-derived progeny was reported in animal model. Mouse and human somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by the transduction of four transcription factors, Oct 3/4, Sox2, Klf4, and c-Myc. However, the low induction efficiency hinders the clinical application of iPS technology, and efforts have been made to improve the reprogramming efficiency. There are variations in the characteristics in ES/iPS cell lines, and the further understanding is necessary for the applications of ES/iPS cell technology. Some improvements were also made in the methods to induce cardiomyocytes from ES/iPS cells efficiently. This review article is focused on generation of iPS cells, cardiomyocyte differentiation from ES/iPS cells, and transplantation of derived cardiomyocytes.This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited". PMID:21040726

  14. A wavelength tunable 2-ps pulse VECSEL

    NASA Astrophysics Data System (ADS)

    Morris, Oliver J.; Wilcox, Keith G.; Head, C. Robin; Turnbull, Andrew P.; Mosley, Peter J.; Quarterman, Adrian H.; Kbashi, Hani J.; Farrer, Ian; Beere, Harvey E.; Ritchie, David A.; Tropper, Anne C.

    2012-03-01

    We report a mode-locked Vertical-External-Cavity Surface-Emitting Laser (VECSEL) that exhibits 13.7 nm of tuning around a centre wavelength of 1042 nm. The wavelength tuning is achieved by incorporating an uncoated, 25 ?m thick, fused silica etalon into the cavity of the laser at Brewster's angle. The etalon is then tilted with respect to the cavity axis. The etalon has a calculated free spectral range of 14 nm at normal incidence. The repetition rate of the laser is measured to be 1.88 GHz. The pulse duration, averaged over the tuning range, is 1.9 ps corresponding to a mean time bandwidth product of 0.46. For a sech2 pulse this is 1.46 times larger than the transform limit. The average power of the laser does not fall below 2.6 mW and, over the tuning range, averages 3.5 mW. With appropriate amplification, such a laser would be highly suited to the generation of heralded single photons in photonic crystal fibre.

  15. Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets

    PubMed Central

    2014-01-01

    Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity. Widespread drug resistance against currently available antimalarials warrants the identification of novel drug targets and development of new drugs. Malarial proteases are a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibility of designing specific inhibitors against them. Proteases belonging to various mechanistic classes are found in P. falciparum, of which serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion. In P. falciparum, a number of serine proteases belonging to chymotrypsin, subtilisin, and rhomboid clans are found. This review focuses on the potential of P. falciparum serine proteases as antimalarial drug targets. PMID:24799897

  16. Function of the active-site lysine in Escherichia coli serine hydroxymethyltransferase.

    PubMed

    Schirch, D; Delle Fratte, S; Iurescia, S; Angelaccio, S; Contestabile, R; Bossa, F; Schirch, V

    1993-11-01

    Serine hydroxymethyltransferase has a conserved lysine residue (Lys-229) that forms the internal aldimine with pyridoxal 5'-phosphate. In other pyridoxal 5'-phosphate enzymes investigated so far, this conserved lysine residue also plays a catalytic role as a base that removes the alpha-proton from the amino acid substrate. Three mutant forms of Escherichia coli serine hydroxymethyltransferase (K229Q, K229R, and K229H) were constructed, expressed, and purified. The absorbance spectra, rapid reaction kinetics, and thermal denaturation of the mutant analogs were studied. Only the K229Q mutant serine hydroxymethyltransferase resembled the wild-type enzyme. The results indicate that Lys-229 plays a critical role in expelling the product by converting the external aldimine to an internal aldimine. In the absence of Lys-229, ammonia can also catalyze the same function at a much slower rate. However, Lys-229 apparently is not the base that removes the alpha-proton from the amino acid substrate. The K229Q mutant enzyme could catalyze one turnover of either serine to glycine or glycine to serine at rates approaching those of the wild-type enzyme. After one turnover, the mutant enzyme could not expel the product and bind new substrate. The K229Q mutant enzyme can also transaminate D-alanine, which, like the hydroxymethyltransferase activity, also requires removing the alpha-proton from the substrate. The absorbance spectra of the K229R and K229H serine hydroxymethyltransferases showed that their pyridoxal 5'-phosphate could not readily form an external aldimine with substrates, suggesting that Lys-229 in the wild-type enzyme may never bear a positive charge, further evidence that it is not the base that removes the alpha-proton. PMID:8226831

  17. Transient dephosphorylation of p53 serine 376 as an early response to ionizing radiation.

    PubMed

    Warters, Raymond L; Gaffney, David K; Kramer, Gwen F; Martinez, Jesse D; Cress, Anne E

    2009-06-01

    In a previous paper we reported that the cytoplasmic sequestered p53 in cells of the SK-N-SH neuroblastoma cell line could be induced to translocate to the nucleus by exposure to ionizing radiation. We have extended these studies to determine the fate of p53 in HCT116 colorectal carcinoma cells where constitutive p53 protein resides in the nucleus. A continuous increase in the nuclear p53 protein was observed in irradiated cells beginning 1 h after irradiation that persisted for 8 h. Surprisingly, immunofluorescence microscopy revealed a transient, rapid and sensitive increase in a radiation-induced nuclear dephosphorylated p53 using antibody PAb421, which detects p53 when serine 376 is dephosphorylated. The PAb421 epitope was detectable after exposure to radiation doses as low as 0.5 cGy and was 10 to 20 times more sensitive compared to detection of p53 protein levels. The results are consistent with a radiation-induced, sensitive and rapid dephosphorylation of p53 at serine 376. The rapid increase in the nuclear PAb421 epitope was blocked by the protein serine phosphatase inhibitor calyculin A but was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that serine 376 was dephosphorylated by protein serine phosphatase 1 or 2A acting on pre-existing p53 protein. The data suggest that dephosphorylation of serine 376 on constitutive nuclear p53 is a sensitive and early signaling event in the response of cells to DNA damage induced by ionizing radiation. PMID:19580479

  18. LLNL PuPS Weld Qualification Plan

    SciTech Connect

    Dodson, K E; Riley, D

    2001-08-24

    This plan ensures the quality of the Lawrence Livermore National Laboratory (LLNL) DOE 3013 Standard Plutonium Packaging System (PuPS) can welds meet the requirements stipulated in the DOE Standard 3013-00 ''Stabilization, Packaging, and Storage of Plutonium-Bearing Materials'' (Reference 1) and G-ESR-G-00035, Revision 1 dated July 26, 2000, ''Savannah River Site Stabilization and Packaging Requirements for Plutonium Bearing Materials for Storage.'' (Reference 2) This plan also meets the requirements for a weld qualification plan as stipulated in the G-ESR-G-00035. The Outer Can weld must meet ASME VIII & IX. The Outer Can welds will be evaluated initially and during production. The initial evaluation will be done by performing the following: ASME IX welding procedure qualification, ASME IX operator qualification, and a 25 can Dummy Outer Can (DOC) verification run. During production, product cans and DOCs will be evaluated. Product cans will be evaluated by a combination of visual examination of the weld faces and the use of helium leak checking. The DOCs will be examined by visual examination, leak check, radiographic examination and metallographic examination. Appendix 2 summarizes the requirements of each of these evaluations. The Inner Can weld must meet the leak tightness requirements of DOE 3013. The Inner Can weld, while not required to meet ASME requirements, will be controlled as described in this plan to ensure a reliable leak path barrier and consistent production processing behavior. The product Inner Cans will be evaluated by a combination of visual examination of the weld faces and the use of helium leak checking.

  19. Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom.

    PubMed

    Samel, Mari; Subbi, Juhan; Siigur, Jüri; Siigur, Ene

    2002-01-01

    Two glycosylated serine fibrinogenases isolated from Vipera lebetina venom have homologous N-terminal sequences and antigenic determinants but can be clearly differentiated according to substrate specificity, glycosylation levels, molecular mass and fibrinogen degradation. alpha-Fibrinogenase has no homolog among known serine proteinases. It has N-terminal similarity with snake venom arginine esterases but does not hydrolyze the esters of arginine, lysine and tyrosine. The enzyme has strong proteolytic activity and degrades alpha-chain of fibrinogen altering its clottability by thrombin. beta-Fibrinogenase is a typical arginine esterase which hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen. PMID:11602278

  20. Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

    PubMed Central

    Radisky, Evette S.; Lee, Justin M.; Lu, Chia-Jung Karen; Koshland, Daniel E.

    2006-01-01

    Atomic resolution structures of trypsin acyl-enzymes and a tetrahedral intermediate analog, along with previously solved structures representing the Michaelis complex, are used to reconstruct events in the catalytic cycle of this classic serine protease. Structural comparisons provide insight into active site adjustments involved in catalysis. Subtle motions of the catalytic serine and histidine residues coordinated with translation of the substrate reaction center are seen to favor the forward progress of the acylation reaction. The structures also clarify the attack trajectory of the hydrolytic water in the deacylation reaction. PMID:16636277

  1. A short synthesis of d-[1-(14) C]-serine of high enantiomeric purity.

    PubMed

    Song, Fengbin; Salter, Rhys; Weaner, Larry E

    2015-04-01

    Herein, we report a short, three-step synthesis of d-[1-(14) C]-serine (4) in high enantiomeric purity. Starting from [(14) C]-KCN and 2-(benzyloxy)acetaldehyde, Strecker reaction using (R)-1-phenylethylamine as the chiral auxiliary gave two diastereomeric aminonitriles 1 and 2 in the ratio of 4:3, which were conveniently separated and purified chromatographically. Following hydrolysis and subsequent hydrogenolysis, the purified major diastereomer 1, was smoothly converted to d-[1-(14) C]-serine (4) in an enantiomeric excess of >99%, thus circumventing time intensive chiral HPLC enantiomeric resolution. PMID:25728900

  2. KNb1.75V0.25PS10

    PubMed Central

    Yu, Jaemin; Yun, Hoseop

    2011-01-01

    The title compound, potassium diniobium vanadium phospho­rus deca­sulfide, KNb1.75V0.25PS10, was obtained by reaction of the elements with a eutectic mixture of KCl/LiCl. It is isostructural with the quaternary KNb2PS10, but the Nb sites are occupied by statistically disordered Nb (87.5%) and V (12.5%) atoms. The structure is composed of anionic ? 1[M 2PS10]? chains (M = Nb/V) separated from each other by K+ ions. The chain is composed of [MS8] distorted bicapped trigonal prisms and [PS4] tetra­hedra. There are no inter­chain bonding inter­actions. The crystal used for the X-ray analysis was a racemic twin. PMID:21522232

  3. Development and operating characteristics of the PS132 thermal battery

    NASA Astrophysics Data System (ADS)

    Krieger, F. C.

    1986-01-01

    The development and operating characterisitics of the PS132 thermal battery are described. The PS132 uses the electrochemical system Ca/LiCl-KCl eutectic-SiO2CACRO4, and it is one of the smallest batteries of its type ever built that can meet its electrical and envronmental requirements. A development program that included construction of approximately 400 PS132-like batteries showed that obtaining acceptable DEB electrolyte-cathode powders was a major problem. Most commercial DEB powders caused excessive amounts of CaLi2 molten metal to form in the operating thermal cells of the PS132. This molten metal then flowed from the cells and caused electrical short circuits.

  4. Hierarchy of scales in B{yields}PS decays

    SciTech Connect

    Delepine, D.; Lucio M, J. L. [Instituto de Fisica, Universidad de Guanajuato Loma del Bosque no 103, Lomas del Campestre, 37150 Leon, Guanajuato (Mexico); Mendoza S, J. A. [Depto. de Fisica-Matematicas, Universidad de Pamplona Pamplona, Norte de Santander (Colombia); Ramirez, Carlos A. [Escuela de Fisica, Universidad Industrial de Santander, A.A. 678, Bucaramanga (Colombia)

    2008-08-31

    We show that the naive factorization approach can accommodate the existence of the observed hierarchy of branching ratios for the B{yields}PS decays (P stands for pseudoscalar and S for scalar mesons respectively.

  5. ES and iPS cell research for cardiovascular regeneration.

    PubMed

    Yamashita, Jun K

    2010-10-01

    Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, which are ES-like stem cells induced from adult tissues, are twin stem cells with currently (with the exception of fertilized eggs) the broadest differentiation potentials. These two stem cells show various similarities in appearance, maintenance methods, growth and differentiation potentials, i.e. theoretically, those cells can give rise to all kinds of cells including germ-line cells. Generation of human ES and iPS cells is further facilitating the researches towards the realization of regenerative medicine. The following three issues are important purposes of ES and iPS cell researches for regenerative medicine: (1) dissection of differentiation mechanisms, (2) application to cell transplantation, and (3) drug discovery. In this review, the current status of cardiovascular regenerative trials using ES and iPS cells is briefly discussed. PMID:20385126

  6. Preliminary Tuft Testing of Metallic Bristles Versus PS212, PS300, and HVOF300

    NASA Technical Reports Server (NTRS)

    Fellenstein, James A.; DellaCorte, Christopher

    1998-01-01

    Turbine engine brush seals are designed with sacrificial brushes and hard shaft coatings to minimize shaft wear and reduce the cost of engine overhauls. Replacing a worm seal is more cost and time effective than refinishing an engine shaft. However, this tribological design causes excessive brush wear and reduces long term seal efficiency. An alternative approach is to coat the shaft with a solid lubricant and allow the bristles to wear into the shaft coating similar to traditional abradable labyrinth seals. This approach can result in reduced seal leakage by forcing the leakage to flow through the seal bristle pack or through a more tortuous shaft wear track. Key to this approach is limiting the shaft wear to an acceptable level were surface refinishing would not be required during every engine overhaul. Included in this paper are brush seal tuft test results for four metallic bristles (nickel-chrome or cobalt-chrome based superalloys) tested against three solid lubricant coatings (NASA's PS212, PS300, and HVOF300). These test results are also compared to previous baseline tests conducted with plasma sprayed chrome carbide. Compared to the baseline results, no tribological benefit was achieved with the metallic bristle/solid lubricant tribopairs tested. To improve the performance of the solid lubricant coatings, issues regarding lubricant phase sizes (homogeneity), and composition need to be addressed.

  7. The PS1 Science Mission - Status and Results

    NASA Astrophysics Data System (ADS)

    Chambers, Kenneth C.

    2013-06-01

    PS1, the Pan-STARRS1 Telescope is in its last year of the PS1 Science Mission. Operations of the PS1 System include the Observatory, Telescope, 1.4 Gigapixel Camera, Image Processing Pipeline , PSPS relational database and reduced science product software servers. The PS1 Surveys include: (1) A 3pi Steradian Survey, (2) A Medium Deep survey of 10 PS1 footprints spaced around the sky; (3) A solar system survey optimized for Near Earth Objects, (4) a Stellar Transit Survey; and (5) a Deep Survey of M31. The PS1 3pi Survey has now covered the sky north of dec=-30 with 8 to 12 visits in five bands: g,r,i,z and y or over ~45 epochs per point on sky. The performance of the PS1 system, sky coverage, cadence, and data quality of the surveys will be presented as well as progress in reprocessing of the data taken to date and plans for serving the data to the public. A summary of science highlights will be included. The PS1 Science Consortium consists of The Institute for Astronomy at the University of Hawai'i in Manoa, the Max Planck Institute for Astronomy, Heidelberg and the Max Planck Institute for Extraterrestrial Physics, Garching, The Johns Hopkins University, the University of Durham, the University of Edinburgh, the Queen's University Belfast, the Harvard-Smithsonian Center for Astrophysics, the Los Cumbres Observatory Global Telescope Network Incorporated, and the National Central University of Taiwan, NASA, and NSF.

  8. Regulation of enzymes of serine and one-carbon metabolism by testosterone in rat prostate, liver, and kidney

    Microsoft Academic Search

    T. A. Sanborn; R. L. Kowle; H. J. Sallach

    1975-01-01

    A significant decrease in the specific activity of 3 enzymes of serine and one-carbon metabolism (3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, and serine hydroxymethyltransferase) was found in the rat prostate gland with castration. A single injection of testosterone propionate to rats 3 days after castration resulted in a significant increase in the 3 enzyme activities within 24 h. This increase in specific

  9. Mutational Analysis of the Connector Segment in the HAMP Domain of Tsr, the Escherichia coli Serine Chemoreceptor

    Microsoft Academic Search

    Peter Ames; Qin Zhou; John S. Parkinson

    2008-01-01

    HAMP domains are 50-residue motifs, found in many bacterial signaling proteins, that consist of two amphiphilic helices joined by a nonhelical connector segment. The HAMP domain of Tsr, the serine chemo- receptor of Escherichia coli, receives transmembrane input signals from the periplasmic serine binding domain and in turn modulates output signals from the Tsr kinase control domain to elicit chemotactic

  10. A serine elastase inhibitor reduces inflammation and fibrosis and preserves cardiac function after experimentally-induced murine myocarditis

    Microsoft Academic Search

    Jong K. Lee; Syed H. E. Zaidi; Peter Liu; Fayez Dawood; Alexander Y. L. Cheah; Wen-Hu Wen; Yuriko Saiki; Marlene Rabinovitch

    1998-01-01

    In viral myocarditis, inflammation and destruction of cardiac myocytes leads to fibrosis, causing progressive impairment in cardiac function. Here we show the etiologic importance of serine elastase activity in the pathophysiology of acute viral myocarditis and the therapeutic efficacy of an elastase inhibitor. In DBA\\/2 mice inoculated with the encephalomyocarditis virus, a more than 150% increase in myocardial serine elastase

  11. A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro

    PubMed Central

    Herbert, Jenny A.; Mitchell, Andrea M.; Mitchell, Timothy J.

    2015-01-01

    The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin. PMID:26090876

  12. [Effects of cucumber leaf's PS II activity and electron transfer on its PS I activity in recovery process after chilling-induced photoinhibition].

    PubMed

    Zhang, Zi-Shan; Yang, Cheng; Gao, Hui-Yuan; Wang, Wei-Wei; Sun, Xue-Juan; Meng, Xiang-Long; Meng, Qing-Wei

    2012-04-01

    Taking Cucumis sativus L. (Jinchun No. 4) as test material, through the determination of chlorophyll-a fluorescence transient and light absorbance at 820 nm, and in combining with chlorophyll quenching, this paper studied the recovery of cucumber leaf' s PS I and PS I activities and the interactions between PS I and PS II in the recovery process at room temperature (25 degrees C) and under different light intensities (0, 15, and 200 micromol x m(-2) x s(-1)) after six hours of low temperature (4 degrees C) and strong light (200 micromol x m(-2) x s(-1)) stress. Different extent of photoinhibition of the PS II and PS I occurred after the stress. During the recovery process at room temperature, the PS II activity recovered quickly and was insensitive to light intensity, while the PS I activity recovered quickly under weak light intensity (15 micromol x m(-2) x s(-1)) but slowly under strong light intensity (200 micromol x m(-2) x s(-1)), suggesting that after the chilling-induced photoinhibition, the reduced electron transfer from PS II to PS I protected the PS I from further inhibition, accelerating the recovery of PS I activity. In the breeding of chilling-resistant species of cucumber, it should not only pursue the higher chilling-resistance of PS II and faster recovery of PS II after chilling-induced photoinhibition, but also pay more attention to the coordinating of PS I and PS II during and after the chilling-induced photoinhibition. In the culture of cucumber, after chilling happened, a practical method to reduce light intensity would help the recovery of cucumber leaf PS I activity to protect the photosynthetic apparatus against photoinhibition. PMID:22803473

  13. Voluntary Product Standard PS 20-10 American Softwood Lumber Standard

    E-print Network

    #12;Voluntary Product Standard PS 20-10 American Softwood Lumber Standard Supersedes Voluntary Product Standard PS 20-05 June 2010 U.S. Department of Commerce Gary Locke, Secretary National Institute Voluntary Product Standard PS 20-10 Natl. Inst. Stand. Technol. Prod. Stand. PS 20-10, 50 pages (June 2010

  14. Activities, localizations, and roles of serine proteases and their inhibitors in human brain tumor progression

    Microsoft Academic Search

    Masaaki Yamamoto; Raymond Sawaya; Sanjeeva Mohanam; Velidi H. Rao; Janet M. Bruner; Garth L. Nicolson; Kohichi Ohshima; Jasti S. Rao

    1994-01-01

    Summary The plasminogen activation system consists of plasminogen activators and their inhibitors, serine proteases, and serpins. The proteases and inhibitors regulate a variety of processes in tissue morphogenesis, differentiation, cell migration, and cancer cell invasiveness and metastasis. One of the plasminogen activators, urokinase-type plasminogen activator (uPA), binds to a specific surface and provides a localized cell surface proteolytic activity required

  15. The subcellular distribution of alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase in dog liver.

    PubMed Central

    Okuno, E; Minatogawa, Y; Nakanishi, J; Nakamura, M; Kamoda, N; Makino, M; Kido, R

    1979-01-01

    The subcellular distributions of alanine-glyoxylate aminotransferase and serine-pyruvate aminotransferase in the particulate fraction of dog liver were examined by centrifugation in a sucrose density gradient. Most of both enzyme activities in the particulate fraction were localized in the mitochondria, but not in the peroxisomes. PMID:518570

  16. A Single-Copy Gene Encodes Kex1, a Serine Endoprotease of Pneumocystis jiroveci

    PubMed Central

    Kutty, Geetha; Kovacs, Joseph A.

    2003-01-01

    We have cloned and characterized the kex1 gene of Pneumocystis jiroveci. Unlike the case for Pneumocystis carinii, in which the homologous PRT-1 genes are multicopy, kex1 is a single-copy gene encoding a protein homologous to fungal serine endoproteases, which localize to the Golgi apparatus. Thus, substantial biological differences can be seen among Pneumocystis species. PMID:12496214

  17. The Serine\\/Threonine\\/Tyrosine Phosphoproteome of the Model Bacterium Bacillus subtilis

    Microsoft Academic Search

    Boris Macek; Ivan Mijakovic; Jesper V. Olsen; Florian Gnad; Chanchal Kumar; Peter R. Jensen; Matthias Mann

    2007-01-01

    Protein phosphorylation on serine, threonine, and tyrosine (Ser\\/Thr\\/Tyr) is well established as a key regulatory post- translational modification in eukaryotes, but little is known about its extent and function in prokaryotes. Although pro- tein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical bio- chemical approaches have so far revealed only a few sub-

  18. An improved synthesis of Fmoc- N-methyl serine and threonine

    Microsoft Academic Search

    Rajesh H. Bahekar; Pradip A. Jadav; Dipam N. Patel; Vijay M. Prajapati; Arun A. Gupta; Mukul R. Jain; Pankaj R. Patel

    2007-01-01

    An improved method for the synthesis of Fmoc-N-methyl serine and threonine has been developed, which involves formation and subsequent reduction of the corresponding oxazolidinone with a Lewis acid under mild conditions, with improved yields and shorter reaction times.

  19. A Broad Spectrum Kunitz Type Serine Protease Inhibitor Secreted by the Hookworm Ancylostoma ceylanicum

    Microsoft Academic Search

    Aaron M. Milstone; Lisa M. Harrison; Richard D. Bungiro; Petr Kuzmic; Michael Cappello

    2000-01-01

    Although blood-feeding hookworms infect over a bil- lion people worldwide, little is known about the molec- ular mechanisms through which these parasitic nema- todes cause gastrointestinal hemorrhage and iron deficiency anemia. A cDNA corresponding to a secreted Kunitz type serine protease inhibitor has been cloned from adult Ancylostoma ceylanicum hookworm RNA. The translated sequence of the A. ceylanicum Kunitz type

  20. Glutamate-dependent glutamine, aspartate and serine release from rat cortical glial cell cultures

    Microsoft Academic Search

    Tadimeti S. Rao; Karen D. Lariosa-Willingham; Naichen Yu

    2003-01-01

    Glia play a pivotal role in glutaminergic excitatory neurotransmission in the central nervous system by regulating synaptic levels of glutamate and by providing glutamine as the sole precursor for the neurotransmitter pool glutamate to neurons through the glutamate–glutamine cycle. In the present investigation, we examined the influence of glutamate application on glutamine, serine and aspartate release from rat cortical glial

  1. Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain

    PubMed Central

    Barbato, G.; Cicero, D.O.; Cordier, F.; Narjes, F.; Gerlach, B.; Sambucini, S.; Grzesiek, S.; Matassa, V.G.; De Francesco, R.; Bazzo, R.

    2000-01-01

    Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N–terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His–Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His–Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes. PMID:10716920

  2. Loss of hippocampal serine protease BSP1/neuropsin predisposes to global seizure activity.

    PubMed

    Davies, B; Kearns, I R; Ure, J; Davies, C H; Lathe, R

    2001-09-15

    Serine proteases in the adult CNS contribute both to activity-dependent structural changes accompanying learning and to the regulation of excitotoxic cell death. Brain serine protease 1 (BSP1)/neuropsin is a trypsin-like serine protease exclusively expressed, within the CNS, in the hippocampus and associated limbic structures. To explore the role of this enzyme, we have used gene targeting to disrupt this gene in mice. Mutant mice were viable and overtly normal; they displayed normal hippocampal long-term synaptic potentiation (LTP) and exhibited no deficits in spatial navigation (water maze). Nevertheless, electrophysiological studies revealed that the hippocampus of mice lacking this specifically expressed protease possessed an increased susceptibility for hyperexcitability (polyspiking) in response to repetitive afferent stimulation. Furthermore, seizure activity on kainic acid administration was markedly increased in mutant mice and was accompanied by heightened immediate early gene (c-fos) expression throughout the brain. In view of the regional selectivity of BSP1/neuropsin brain expression, the observed phenotype may selectively reflect limbic function, further implicating the hippocampus and amygdala in controlling cortical activation. Within the hippocampus, our data suggest that BSP1/neuropsin, unlike other serine proteases, has little effect on physiological synaptic remodeling and instead plays a role in limiting neuronal hyperexcitability induced by epileptogenic insult. PMID:11549709

  3. The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

    NASA Astrophysics Data System (ADS)

    Jürgensen, V. Würtz; Jalkanen, K.

    2006-03-01

    The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory with the Becke 3LYP hybrid exchange correlation (XC) functional and the PW91 GGA XC functional and the 6-31G* and aug-cc-pVDZ basis sets. Subsequently, the vibrational absorption, vibrational circular dichroism, Raman and Raman optical activity spectra were simulated in order to compare them to experimentally measured spectra. In addition, we compare to previously reported studies for both structural determination and spectral simulations and measurements. A comparison of the various ways to treat the effects of the environment and solvation on both the structure and the spectral properties is thoroughly investigated for one conformer, with the goal to determine which level of theory is appropriate to use in the systematic search of the conformational space. In addition, the effects of the counterion, here Cl- anion, are also investigated. Here we present the current state of the art in nanobiology, where the latest methods in experimental and theoretical vibrational spectroscopy are used to gain useful information about the coupling of the nuclear, electronic and magnetic degrees of freedom and structure of tri-L-serine and its capped peptide analog with the environment.

  4. Contribution of astrocytes to hippocampal long-term potentiation through release of D-serine

    E-print Network

    Newman, Eric A.

    in the brain, are found to associate closely with neuronal synapses (1, 2). Although astrocytes lack­11). There is evidence that astrocytes may also contribute to high-level brain functions such as motivation, learningContribution of astrocytes to hippocampal long-term potentiation through release of D-serine Yunlei

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

    PubMed

    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

    2014-05-01

    A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, 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 that the designs could provide the basis for a new class of organophosphate capture agents. PMID:24705591

  6. Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin.

    PubMed

    Petrillo, Teodolinda; O'Donohoe, Catrina A; Howe, Nicole; Malthouse, J Paul G

    2012-08-01

    Two new inhibitors in which the terminal ?-carboxyl groups of Z-Ala-Ala-Phe-COOH and Z-Ala-Pro-Phe-COOH have been replaced with a proton to give Z-Ala-Ala-Phe-H and Z-Ala-Pro-Phe-H, respectively, have been synthesized. Using these inhibitors, we estimate that for ?-chymotrypsin and subtilisin Carlsberg the terminal carboxylate group decreases the level of inhibitor binding 3-4-fold while a glyoxal group increases the level of binding by 500-2000-fold. We show that at pH 7.2 the effective molarities of the catalytic hydroxyl group of the active site serine are 41000-229000 and 101000-159000 for ?-chymotrypsin and subtilisin Carlsberg, respectively. It is estimated that oxyanion stabilization and the increased effective molarity of the catalytic serine hydroxyl group can account for the catalytic efficiency of the reaction. We argue that substrate binding induces the formation of a strong hydrogen bond or low-barrier hydrogen bond between histidine-57 and aspartate-102 that increases the pK(a) of the active site histidine, allowing it to be an effective general base catalyst for the formation of the tetrahedral intermediate and increasing the effective molarity of the catalytic hydroxyl group of serine-195. A catalytic mechanism for acyl intermediate formation in the serine proteases is proposed. PMID:22757750

  7. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi.

    PubMed

    Sigle, Leah Theresa; Ramalho-Ortigăo, Marcelo

    2013-09-01

    Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited ?-chymotrypsin to 9.4% residual activity and also inhibited ?-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania. PMID:24037187

  8. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases

    Microsoft Academic Search

    Steffen Massberg; Lenka Grahl; Marie-Luise von Bruehl; Davit Manukyan; Susanne Pfeiler; Christian Goosmann; Volker Brinkmann; Michael Lorenz; Kiril Bidzhekov; Avinash B Khandagale; Ildiko Konrad; Elisabeth Kennerknecht; Katja Reges; Stefan Holdenrieder; Siegmund Braun; Christoph Reinhardt; Michael Spannagl; Klaus T Preissner; Bernd Engelmann

    2010-01-01

    Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases

  9. 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.

  10. Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarrays containing Tribolium castaneum whole-genome sequences were developed to study the transcriptome response of T. castaneum larvae to dietary protease inhibitors. In larvae fed diets containing 0.1% of the cysteine protease inhibitor E-64 alone or in combination with 5.0% of the serine pro...

  11. VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF ENOS

    EPA Science Inventory

    VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS. Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and NHEERL, US EPA, Chapel Hill, North Ca...

  12. VANADL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF ENOS

    EPA Science Inventory

    VANADYL SULFATE INHIBITS NO PRODUCTION BY DIFFERENTIALLY REGULATING SERINE/THREONINE PHOSPHORYLATION OF eNOS. Zhuowei Li, Jacqueline D. Carter, Lisa A. Dailey, Joleen Soukup, Yuh-Chin T. Huang. CEMALB, University of North Carolina and ORD, US EPA, Chapel Hill, North Carolina V...

  13. Serine\\/threonine protein phosphatase 6 modulates the radiation sensitivity of glioblastoma

    Microsoft Academic Search

    Y Shen; Y Wang; K Sheng; X Fei; Q Guo; J Larner; X Kong; Y Qiu; J Mi

    2011-01-01

    Increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma multiforme (GBM). This study aims to determine if serine\\/threonine phosphatase (protein phosphatase 6 (PP6)) is a molecular target for GBM radiosensitization treatment. The GBM orthotopic xenograft mice model was used in this study. Our data demonstrated that the protein level of

  14. Phosphorylation of Serine 186 of bHLH Transcription Factor SPEECHLESS Promotes Stomatal Development in Arabidopsis.

    PubMed

    Yang, Ke-Zhen; Jiang, Min; Wang, Ming; Xue, Shan; Zhu, Ling-Ling; Wang, Hong-Zhe; Zou, Jun-Jie; Lee, Eun-Kyoung; Sack, Fred; Le, Jie

    2015-05-01

    The initiation of stomatal lineage and subsequent asymmetric divisions in Arabidopsis require the activity of the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). It has been shown that SPCH controls entry into the stomatal lineage as a substrate either of the MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade or GSK3-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2). Here we show that three serine residues of SPCH appear to be the primary phosphorylation targets of Cyclin-Dependent Kinases A;1 (CDKA;1) in vitro, and among them Serine 186 plays a crucial role in stomatal formation. Expression of an SPCH construct harboring a mutation that results in phosphorylation deficiencies on Serine 186 residue failed to rescue stomatal defects in spch null mutants. Expression of a phosphorylation-mimic mutant SPCH(S186D) complemented stomatal production defects in the transgenic lines harboring the targeted expression of dominant-negative CDKA;1.N146. Therefore, in addition to MAPK- and BIN2-mediated phosphorylation on SPCH, phosphorylation at Serine 186 is positively required for SPCH function in regulating stomatal development. PMID:25680231

  15. Rat NTE-related esterase is a membrane-associated protein, hydrolyzes phenyl valerate, and interacts with diisopropylfluorophosphate through a serine catalytic machinery

    Microsoft Academic Search

    Mingxing Xie; Dongfang Yang; Lynn Matoney; Bingfang Yan

    2003-01-01

    The serine hydrolases constitute multi-families of proteins that include lipases, esterases, and proteases. These enzymes contain a signature motif GXSXG, in which the serine residue acts as the nucleophile and initiates catalysis. This report describes the characterization of a novel serine hydrolase from rat. This enzyme exhibits a moderate sequence identity with the neuropathy target esterase (NTE), thus is designated

  16. Processing of Neutrophil ?-Defensins Does Not Rely on Serine Proteases In Vivo.

    PubMed

    Glenthřj, Andreas; Nickles, Katrin; Cowland, Jack; Borregaard, Niels

    2015-01-01

    The ?-defensins, human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed in promyelocytes and stored as mature HNPs in azurophil granules. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme that processes proHNPs nor the localization of processing has been identified. It has been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which are able to process recombinant proHNP into HNP in vitro. We investigated whether serine proteases are in fact responsible for processing of proHNP in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing in primary human bone marrow cells. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not enhance the proHNP processing capability. Finally, two patients suffering from Papillon-Lefčvre syndrome, who lack active neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils. Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s). PMID:25945506

  17. Roscovitine inhibits EBNA1 serine 393 phosphorylation, nuclear localization, transcription, and episome maintenance.

    PubMed

    Kang, Myung-Soo; Lee, Eun Kyung; Soni, Vishal; Lewis, Timothy A; Koehler, Angela N; Srinivasan, Viswanathan; Kieff, Elliott

    2011-03-01

    Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and (486)RALL(489) and (580)KDLVM(584) as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence. PMID:21209116

  18. Breast Cancer-Associated pS2 Protein: Synthesis and Secretion by Normal Stomach Mucosa

    Microsoft Academic Search

    M. C. Rio; J. P. Bellocq; J. Y. Daniel; C. Tomasetto; R. Lathe; M. P. Chenard; A. Batzenschlager; P. Chambon

    1988-01-01

    The human pS2 gene is specifically expressed under estrogen transcriptional control in a subclass of estrogen receptor-containing human breast cancer cells. The pS2 gene encodes an 84-amino acid protein that is secreted after signal peptide cleavage. The distribution of pS2 protein in normal human tissues was studied with antibodies to pS2; pS2 was specifically expressed and secreted by mucosa cells

  19. Cs0.49NbPS6

    PubMed Central

    Lee, Eunsil; Lee, Yonghee; Yun, Hoseop

    2011-01-01

    The quaternary thio­phosphate, Cs0.49NbPS6, caesium hexa­thio­niobiophosphate(V), has been synthesized by the reactive halide flux method. The title compound is isotypic with Rb0.46TaPS6 and is made up of a bicapped trigonal–biprismatic [Nb2S12] unit and a tetra­hedral [PS4] group. The [Nb2S12] units linked by the [PS4] tetra­hedra form infinite chains, yielding a three-dimensional network with rather large van der Waals gaps along the c axis in which the disordered Cs+ ions reside. The electrons released by the Cs atoms are transferred to the pairwise niobium metal site and there are substantial inter­metallic Nb—Nb bonding inter­actions. This leads to a significant decrease of the inter­metallic distance in the title compound compared to that in TaPS6. The classical charge balance of the title compound may be represented as [Cs+]0.49[Nb4.51+][P5+][S2?]4[S2 2?]. PMID:21522512

  20. High-resolution PS-OCT of Enamel Remineralization

    PubMed Central

    Can, Anna M.; Darling, Cynthia L.; Fried, Daniel

    2011-01-01

    Previous studies have demonstrated that Polarization Sensitive Optical Coherence Tomography (PS-OCT) can be used to image the remineralization of early artificial caries lesions. However, the depth resolution of the imaging system employed in those previous studies was limited and the outer surface structure of the lesions were not resolved as clearly as desired. The purpose of this study was to repeat the earlier remineralization study using a broadband light-source of higher resolution to determine if there can be improved resolution of the remineralized surface zones of the lesions. An all polarization-maintaining fiber based PS-OCT system operating at 1310-nm was used to acquire polarization resolved images of bovine enamel surfaces exposed to a demineralizing solution at pH-4.9 followed by a fluoride containing remineralizing solution at pH-7.0 containing 2-ppm fluoride. The structure of the surface zones could be clearly resolved using PS-OCT in the samples that underwent remineralization. The PS-OCT measurements indicated a significant (p<0.05) reduction in the integrated reflectivity between the severity of the lesions that were exposed to the remineralization solution and those that were not. The lesion depth and mineral loss were also measured with polarized light microscopy and transverse microradiography after sectioning the enamel blocks. These results show that PS-OCT can be used to non-destructively monitor the remineralization potential of anti-caries agents. PMID:21909226

  1. Characterization of crosslinked polystyrene(PS) beads in SBR matrix

    SciTech Connect

    Cha, Yoon-Jong; Choe, Soonja [Inha Univ. (Korea, Republic of)

    1995-12-01

    Monodisperse sized crosslinked polystyrene(PS) beads were prepared by a reaction of semibatch emulsion polymerization with styrene monomer, divinylbenzene(DVB) crosslinking agent and potassium persulfate(K{sub 2}S{sub 2}O{sub 9}) initiator in the absence of emulsifier. The glass transition temperature(T{sub g}) and the mean diameter of the beads were increased from 100{degrees}C to 135{degrees}C and from 402 nm to 532 nm, respectively, for an incorporation of 2 to 10 mol% DVB. Crosslinking density was also linearly increased with DVB content. SEM microphotographs of SBR composite filled with various contents of PS beads revealed that PS beads are relatively well dispersed without changing the spherical shape of the beads in all range of compositions. In stress-strain analysis, elongation at break and tensile strength of SBR composite were increased with the bead content. Applicability of the PS beads as a filler in SBR matrix is tested by plotting Mooney-Rivlin or Guth-Smallwood equations. However, mechanical properties of the composite with the beads were not so excellent as those of the composite with carbon black. Crosslinked PS beads are still tentative as a white color reinforcing filler on SBR matrix.

  2. Screening Tests of Reproductive Immunology in Systemic Lupus Erythematosus

    PubMed Central

    Ulcova-Gallova, Zdenka; Mockova, Alice; Cedikova, Miroslava

    2012-01-01

    Female patients in reproductive age with systemic lupus erythematosus and fertility complications together are observed by rheumatologists, gynecologists, and reproductive immunologists. The paper notes the presence of autoantibodies to zona pellucida, to phospholipids (phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidic acid, annexin V, beta-2 glycoprotein I, and cardiolipin) and of isoantibodies to sperm cells. Isoantibodies to sperm cells are not significantly predominant, but autoimmunity is well expressed in IgG positivity against phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl serine, cardiolipin, and beta-2 glycoprotein I, as well as antizona pellucida antibodies in IgG isotype. According to the levels of autoantibodies we have to choose preventive treatment to protect mother and her foetus. PMID:23150811

  3. Reduction of inflammatory responses by l-serine treatment leads to neuroprotection in mice after traumatic brain injury.

    PubMed

    Zhai, Pei-Pei; Xu, Li-Hua; Yang, Juan-Juan; Jiang, Zheng-Lin; Zhao, Guang-Wei; Sun, Li; Wang, Guo-Hua; Li, Xia

    2015-08-01

    This study was designed to evaluate the neuroprotective effect of l-serine and the underlying mechanisms in mice after traumatic brain injury (TBI) induced using a weight drop model. The mice were intraperitoneally injected with l-serine 3 h after TBI and then injected twice each day for 7 days or until the end of the experiment. The neurological severity score, brain water content, lesion volume, and neurone loss were determined. The levels of TNF-?, IL-1?, IL-6, and IL-10 and the number of GFAP- and Iba-1-positive cells and activated caspase-3-positive neurones in the brain tissue ipsilateral to TBI were also measured. Simultaneously, the influences of l-serine on these variables were observed. In addition, the expression of glycine receptors and l-serine-induced currents were measured. We found l-serine treatment: 1) decreased the neurological deficit score, brain water content, lesion volume, and neurone loss; 2) inhibited activated caspase-3; and 3) reduced the levels of TNF-?, IL-1? and IL-6 and the number of GFAP- and Iba-1-positive cells. The effects of l-serine were antagonised by the administration of strychnine, an antagonist of glycine receptors. In addition, we found that glycine receptors were expressed mainly in the cortical neurones but less in the astrocytes or microglial cells, and l-serine activated these receptors and induced strychnine-sensitive currents in these neurones. In conclusion, l-serine induces the activation of glycine receptors, which alleviates neuronal excitotoxicity, a secondary brain injury process, thereby reduces the activation of astrocytes and microglial cells and secretion of proinflammatory cytokines and inhibits neuronal apoptosis. Thus, l-serine treatment leads to neuroprotection of brain tissue through reducing inflammatory responses and improves recovery of the neurological functions in mice after traumatic brain injury. PMID:25747604

  4. Positron annihilation in the MuPs system

    E-print Network

    Alexei M. Frolov

    2012-07-25

    The life-time of the four-body atomic system MuPs ($\\mu^{+} e^{-}_2 e^{+}$ or muonium-positronium) against positron annihilation has been evaluated as $\\tau = \\frac{1}{\\Gamma} \\approx 4.076453 \\cdot 10^{-10}$ $sec$. Various annihilation rates for MuPs are determined to a good numerical accuracy, e.g., $\\Gamma_{2 \\gamma} \\approx$ 2.446485$\\cdot 10^{9}$ $sec^{-1}$, $\\Gamma_{3 \\gamma} \\approx$ 6.62798$\\cdot 10^{6}$ $sec^{-1}$, $\\Gamma_{4 \\gamma} \\approx$ 3.61680$\\cdot 10^{3}$ $sec^{-1}$, $\\Gamma_{5 \\gamma} \\approx$ 6.32973 $sec^{-1}$. The hyperfine structure splitting for the ground state in the MuPs system has also been evaluated as $\\Delta$ = 23.078 $MHz$.

  5. Local order in layered NiPS3 and Ni0.7Mg0.3PS3

    NASA Astrophysics Data System (ADS)

    Goossens, D. J.; James, D.; Dong, J.; Whitfield, R. E.; Norén, L.; Withers, R. L.

    2011-02-01

    The family of two-dimensional magnetic materials MIIPS3 where M = Mn, Fe, Ni, Mg, Zn, etc shows a wide range of fascinating magnetic behaviour. It also shows potentially useful chemical properties including intercalation of nonlinear optical molecules and lithium ions. These properties are due to a crystal structure in which the ab planes are well-ordered in the plane but poorly correlated along c. Here, the short-range ordering is modelled in NiPS3 and Ni1 - xMgxPS3 (x = 0.3). X-ray diffuse scattering from NiPS3 shows pronounced streaking along c*, indicative of stacking faulting in these layered compounds. Electron diffraction from Ni1 - xMgxPS3 (x = 0.3) shows substantial diffuse scattering due to short-range order within the ab plane, and this can be modelled by allowing the metal species to cluster. The possibility of clustering has implications for interpretation of the magnetic behaviour of the family, including the glassiness observed in Fe1 - xMnxPS3.

  6. PS-b-PAA as a high ? polymer for directed self-assembly: a study of solvent and thermal annealing processes for PS-b-PAA

    NASA Astrophysics Data System (ADS)

    Cheng, Jing; Lawson, Richard A.; Yeh, Wei-Ming; Jarnagin, Nathan D.; Tolbert, Laren M.; Henderson, Clifford L.

    2013-03-01

    Poly(styrene)-b-poly(acrylic acid) copolymers (PS-b-PAA) was shown to be one promising material for achieving substantially smaller pitch patterns than PS-b-PMMA while still retaining high etch contrast and application for chemoepitaxy. Phase separation of acetone vapor annealed PS-b-PAA (Mw=16,000 g/mol with 50:50 volume ratio of PS: PAA) on PS brush achieved a lamellar morphology with a pattern pitch size (L0) of 30 nm. However the thermal annealing of the same PS-b-PAA generated a dramatically larger pitch size of 43 nm. SEM and GPC analysis revealed that the intermolecular crosslinking during thermal annealing process has increased the effective N (degree of polymerization), which suggests that even a small amount of crosslinking would lead to big pitch change. Thus, PS-b-PAA is not suitable for fast thermal annealing process as it loses pitch size control due to PAA crosslinking.

  7. Syntheses, crystal structures, optical and theoretical studies of the actinide thiophosphates SrU(PS4)2, BaU(PS4)2, and SrTh(PS4)2.

    PubMed

    Mesbah, Adel; Prakash, Jai; Beard, Jessica C; Lebčgue, Sébastien; Malliakas, Christos D; Ibers, James A

    2015-03-16

    Three new actinide thiophosphates, SrU(PS4)2, BaU(PS4)2, and SrTh(PS4)2, have been synthesized by high-temperature solid-state methods, and their crystal structures were determined from single-crystal X-ray diffraction studies. These three isostructural compounds crystallize in a new structure type in space group D4h13-P42/mbc of the tetragonal system. Their structure features infinite one-dimensional chains of ?1[An(PS4)2(2–)] anions (An = U or Th). Each An atom is coordinated by eight S atoms in a bicapped trigonal prism, and each P atom is tetrahedrally bonded to four S atoms. The compounds are readily charge balanced as Ak2+An4+(P5+(S2–)4)2. Optical studies on single crystals of SrU(PS4)2 and BaU(PS4)2 as well as ground single crystals of SrTh(PS4)2 revealed a direct band gap of 2.13(2) eV and an indirect band gap value of 1.99(2) eV for SrU(PS4)2 and a direct and indirect gap of about 2.28(2) eV for BaU(PS4)2. SrTh(PS4)2 has a relatively large band gap of 3.02(2) eV. DFT calculations for SrU(PS4)2 and BaU(PS4)2 using the HSE functional predict both compounds to be antiferromagnetic and have very similar electronic structures with band gaps of 2.7 eV. The band gap calculated for SrTh(PS4)2 is 3.2 eV. PMID:25714855

  8. QSAR and docking studies of HCV NS3 serine protease inhibitors.

    PubMed

    da Cunha, Elaine F F; Matos, Karina S; Ramalho, Teodorico C

    2013-09-01

    Hepatitis C virus (HCV) is a Hepacivirus that causes chronic liver disease, leading to hepatocellular carcinoma, cirrhosis, and chronic hepatitis in about 3% of the world population. In this study, novel HCV NS3 serine protease inhibitors based on 93 boceprevir analogs were studied by QSAR analyses using thermodynamic, structural and topological descriptors, including E-state descriptors. Novel compounds were proposed using the QSAR models. Both models were highly predictive, with calibration, leave-one-out validation and external validation R2 of 0.66, 0.65 and 0.52, respectively. The most promising structures were docked into the HCV NS3 serine protease active site demonstrating, then, the high affinity of some new structures. PMID:23140577

  9. The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

    NASA Technical Reports Server (NTRS)

    Rojas, Ana; Doolittle, Russell F.

    2003-01-01

    Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

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

    PubMed Central

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

    2015-01-01

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

  11. Feedback inhibition of fully unadenylylated glutamine synthetase from Salmonella typhimurium by glycine, alanine, and serine.

    PubMed Central

    Liaw, S H; Pan, C; Eisenberg, D

    1993-01-01

    Bacterial glutamine synthetase (GS; EC 6.3.1.2) was previously shown to be inhibited by nine end products of glutamine metabolism. Here we present four crystal structures of GS, complexed with the substrate Glu and with each of three feedback inhibitors. The GS of the present study is from Salmonella typhimurium, with Mn2+ ions bound, and is fully unadenylylated. From Fourier difference maps, we find that L-serine, L-alanine, and glycine bind at the site of the substrate L-glutamate. In our model, these four amino acids bind with the atoms they share in common (the "main chain" +NH3-CH-COO-) in the same positions. Thus on the basis of our x-ray work, glycine, alanine, and serine appear to inhibit GS-Mn by competing with the substrate glutamate for the active site. Images Fig. 1 PMID:8099447

  12. Preliminary characterization of a Kazal-type serine protease inhibitor from Caiman crocodilus yacare plasma.

    PubMed

    Araujo, M S; Nunes, V A; Gozzo, A J; Sampaio, M U; Auerswald, E; Ura, N; Shimamoto, K; Sampaio, C A

    1999-12-01

    Blood serine protease inhibitors are becoming better understood and increasingly applied in blood clotting, cancer and other diseases. Reptiles are suitable models for blood coagulation and related processes, moreover, caiman is a good comparative model of a non-poisonous reptile. Recently, we reported the purification of a kininogen, the presence of proteases involved in blood clotting, and a serine protease inhibitor in Caiman crocodilus yacare plasma. In this paper, we described the partial sequence of an inhibitor (CcTI). The inhibitor is an 80-kDa protein, and it inactivates trypsin and chymotrypsin the hydrolysis of specific chromogenic substrates and in the degradation of gelatin. The inhibitor is member of Kazal-type inhibitor family and consists of several domains, its putative reactive site is Arg-His. PMID:10615009

  13. Biosynthesis of a defensive insect alkaloid: epilachnene from oleic acid and serine.

    PubMed Central

    Attygalle, A B; Blankespoor, C L; Eisner, T; Meinwald, J

    1994-01-01

    The biosynthesis of the azamacrolide epilachnene by the coccinellid beetle Epilachna varivestis has been studied with 2H-labeled oleic acid, 2H-labeled L-serine, and 13C,15N-labeled L-serine. The incorporation of these precursors into epilachnene defines the origin of the alkaloid's entire carbon/nitrogen skeleton. GC/MS and GC/IR studies of alkaloid produced by Epilachna fed with deuteriated oleic acid show that oleic acid loses four carbon atoms from its carboxyl end during the biosynthesis. Other details, including the mechanism of carbon-nitrogen bond formation between the fatty acid and amino acid moieties, remain to be established. PMID:7809122

  14. Molecular evolution of serine/arginine splicing factors family (SR) by positive selection.

    PubMed

    Escobar, Andrés J Gutiérrez; Arenas, Aylan Farid; Gómez-Marin, Jorge Enrique

    2006-01-01

    The serine-rich (SR) protein family is involved in the pre-mRNA splicing process and the DNA sequences of the corresponding genes are highly conserved in the metazoan organisms. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD and a RS (arginine/serine-rich) domain. We used the amino acid and nucleotide sequences deposited in GenBank and Swiss-Prot databases to perform a phylogenetic analysis using bioinformatics tools. The results of the phylogenetic trees suggest that this family has evolved by several gene duplication events as a result of a positive selection mechanism. PMID:16922697

  15. Phosphorylation of adenylyl cyclase III at serine1076 does not attenuate olfactory response in mice

    PubMed Central

    Cygnar, Katherine D; Collins, Sarah Ellen; Ferguson, Christopher H; Bodkin-Clarke, Chantal; Zhao, Haiqing

    2012-01-01

    Feedback inhibition of adenylyl cyclase III (ACIII) via Ca2+-induced phosphorylation has long been hypothesized to contribute to response termination and adaptation of olfactory sensory neurons (OSNs). To directly determine the functional significance of this feedback mechanism for olfaction in vivo, we genetically mutated serine1076 of ACIII, the only residue responsible for Ca2+-induced phosphorylation and inhibition of ACIII (Wei et al., 1996; Wei et al., 1998), to alanine in mice. Immunohistochemistry and Western blot analysis showed that the mutation affects neither the cilial localization nor the expression level of ACIII in OSNs. Electroolfactogram analysis showed no differences in the responses between wildtype and mutant mice to single-pulse odorant stimulations or in several stimulation paradigms for adaptation. These results suggest that phosphorylation of ACIII on serine1076 plays a far less important role in olfactory response attenuation than previously thought. PMID:23077041

  16. Expression and Characterization of the Mycobacterium tuberculosis Serine\\/Threonine Protein Kinase PknB

    Microsoft Academic Search

    YOSSEF AV-GAY; SARWAT JAMIL; STEVEN J. DREWS

    1999-01-01

    PknB is a member of the newly discovered eukaryotic-like protein serine\\/threonine kinase (PSTK) family of proteins. The pknB gene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser\\/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and

  17. The mitochondrial serine protease HtrA2\\/Omi: an overview

    Microsoft Academic Search

    L Vande Walle; M Lamkanfi; P Vandenabeele

    2008-01-01

    The HtrA family refers to a group of related oligomeric serine proteases that combine a trypsin-like protease domain with at least one PDZ interaction domain. Mammals encode four HtrA proteases, named HtrA1–4. The protease activity of the HtrA member HtrA2\\/Omi is required for mitochondrial homeostasis in mice and humans and inactivating mutations associated with neurodegenerative disorders such as Parkinson's disease.

  18. The Regulation and Activities of the Multifunctional Serine\\/Threonine Kinase Akt\\/PKB

    Microsoft Academic Search

    Eugene S. Kandel; Nissim Hay

    1999-01-01

    The serine\\/threonine kinase Akt, or protein kinase B (PKB), has recently been a focus of intense research. It appears that Akt\\/PKB lies in the crossroads of multiple cellular signaling pathways and acts as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI 3-kinase). Akt\\/PKB is particularly important in mediating several metabolic actions of insulin.

  19. Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans

    Microsoft Academic Search

    Weon-Kyoo You; Young-Doug Sohn; Ki-Yong Kim; Doo-Hong Park; Yangsoo Jang; Kwang-Hoe Chung

    2004-01-01

    A novel serine protease, named as scolonase, was purified and characterized from the tissue of the Korean centipede, Scolopendra subspinipes mutilans. Purified scolonase showed an apparent molecular weight of 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and an isoelectric point of 4.8 on isoelectric focusing gel. Scolonase was able to preferentially hydrolyze arginine over lysine at the cleavage

  20. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

    Microsoft Academic Search

    Jack A. M. Leunissen; William M. de Vos; Bauke W. Dijkstra; Roland J. Siezen

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, >50 subtilases are known, >40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues,

  1. Biotinylated aprotinin: a versatile probe for the detection of serine proteinases on western blots

    Microsoft Academic Search

    J. Melrose; P. Ghosh; M. Patel

    1995-01-01

    The present study was undertaken to provide a highly sensitive detection system for the identification and characterisation of serine proteinases separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Biotinylated aprotinin of high specific activity (88–92% active) was prepared (i) by reaction of aprotinin directly with N-hydroxy sulfosuccinimidyl-6-(biotinamido) hexanoate, and (ii) by reaction of aprotinin-trypsin complex with N-hydroxy succinimidobiotin. Both biotinylated

  2. Regulation of Interferon Regulatory Factor3 by the Hepatitis C Virus Serine Protease

    Microsoft Academic Search

    Eileen Foy; Kui Li; Chunfu Wang; Rhea Sumpter; Masanori Ikeda; Stanley M. Lemon; Michael Gale

    2003-01-01

    Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3\\/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor-3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3\\/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3

  3. Serine proteinases of the human body louse ( Pediculus humanus ): sequence characterization and expression patterns

    Microsoft Academic Search

    Peter J. Waniek; Ulrike B. Hendgen-Cotta; Pia Stock; Christoph Mayer; Astrid H. Kollien; Günter A. Schaub

    2005-01-01

    After the previous characterization of one trypsin gene (Try1) of the human body louse Pediculus humanus, genes encoding a second trypsin (Try2) and a chymotrypsin (Chy1) have been cloned using degenerate serine proteinase primers and 5?- and 3?-RACE, and sequenced. The deduced 259 and 267 amino acid sequences of Try2 and Chy1 show an identity of 33%–40% to trypsinogens and

  4. EAAT1 and d-serine expression are early features of human retinal development

    Microsoft Academic Search

    Claudia M. Diaz; Lauren T. Macnab; Susan M. Williams; Robert K. P. Sullivan; David V. Pow

    2007-01-01

    In the developing central nervous system (CNS), the activation of N-methyl-d-aspartate (NMDA) receptors is probably an important regulator of processes such as synaptogenesis and neurite growth. NMDA receptor activation is dependent upon the homeostasis of glutamate and the presence of co-agonists such as d-serine. We have investigated the expression of the glutamate transporter excitatory amino acid transporter-1 (EAAT1 or GLAST)

  5. Serine Palmitoyltransferase, the First Step Enzyme in Sphingolipid Biosynthesis, Is Involved in Nonhost Resistance

    Microsoft Academic Search

    Yoshihiro Takahashi; Thomas Berberich; Hiroyuki Kanzaki; Hideo Matsumura; Hiromasa Saitoh; Tomonobu Kusano; Ryohei Terauchi

    2009-01-01

    An overexpression screen of Nicotiana benthamiana cDNAs identified a gene for the LCB2 subunit of serine palmitoyl- transferase (SPT) as a potent inducer of hypersensitive re- sponse-like cell death. The pyridoxal 5'-phosphate binding site of NbLCB2 is required for its function as a cell death inducer. NbLCB2 mRNA is accumulated after infection by nonhost pathogen Pseudomonas cichorii. Resistance of N.

  6. Three-dimensional modeling of squamous cell carcinoma antigen 2 and its interactions with serine protease

    Microsoft Academic Search

    Xue-feng Gao; Xu-ri Huang; Jian-kang Yu; Xi Zhao; Chia-chung Sun

    2004-01-01

    An attempt is made to build up a three-dimensional model of squamous cell carcinoma antigen 2 (SCCA2) by means of the homology module of Insight II, where SCCA2 contains the stressed and relaxed forms, i.e. SCCA2(S) and SCCA2(R). The docking of SCCA2(S) with two different target serine proteinases, that are the cathepsin G (Cat G) and the human mast cell

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

    PubMed

    Vorlová, Barbora; Nachtigallová, Dana; Jirásková-Vaní?ková, Jana; Ajani, Haresh; Jansa, Petr; Rezá?, Jan; Fanfrlík, Jind?ich; Otyepka, Michal; Hobza, Pavel; Konvalinka, Jan; Lepšík, Martin

    2015-01-01

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

  8. Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration

    Microsoft Academic Search

    Jeremy Chien; Takayo Ota; Giovanni Aletti; Ravi Shridhar; Mariarosaria Boccellino; Lucio Quagliuolo; Alfonso Baldi; Viji Shridhar

    2009-01-01

    HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human

  9. Implications of the serine protease HtrA1 in amyloid precursor protein processing

    Microsoft Academic Search

    Sandra Grau; Alfonso Baldi; Rossana Bussani; Xiaodan Tian; Raluca Stefanescu; Michael Przybylski; Peter Richards; Simon A. Jones; Viji Shridhar; Tim Clausen; Michael Ehrmann

    2005-01-01

    The defining features of the widely conserved HtrA (high temperature requirement) family of serine proteases are the combination of a catalytic protease domain with one or more C-terminal PDZ domains and reversible zymogen activation. Even though HtrAs have previously been implicated in protein quality control and various diseases, including cancer, arthritis, and neuromuscular disorder, the biology of the human family

  10. The Serine Protease HtrA1 Is Upregulated in the Human Placenta during Pregnancy

    Microsoft Academic Search

    Antonio De Luca; Maria De Falco; Valentina Fedele; Luigi Cobellis; Annunziata Mastrogiacomo; Vincenza Laforgia; Ioana L. Tuduce; Mara Campioni; Domenico Giraldi; Marco G. Paggi; Alfonso Baldi

    2004-01-01

    The placenta has a dynamic and continuous capacity for self-renewal. The molecular mechanisms responsible for controlling trophoblast proliferation are still unclear. It is generally accepted that the simultaneous activity of proteins involved in cell proliferation, apoptosis, and extracellular matrix degradation plays an important role in correct placental development. We investigated in depth the expression of the serine protease HtrA1 during

  11. Alternaria-derived serine protease activity drives IL-33–mediated asthma exacerbations

    PubMed Central

    Snelgrove, Robert J.; Gregory, Lisa G.; Peiró, Teresa; Akthar, Samia; Campbell, Gaynor A.; Walker, Simone A.; Lloyd, Clare M.

    2014-01-01

    Background The fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear. Objective We sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset allergic inflammation. Methods IL-33 levels were quantified in wild-type and ST2?/? mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later. Results Alternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease–IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model. Conclusion Alternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease. PMID:24636086

  12. Macluralisin — a serine proteinase from fruits of Maclura pomifera (Raf.) Schneid

    Microsoft Academic Search

    G. N. Rudenskaya; E. A. Bogdanova; L. P. Revina; B. N. Golovkin; V. M. Stepanov

    1995-01-01

    A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that

  13. The distribution of the serin ( Serinus serinus L., 1766) in the 16th century

    Microsoft Academic Search

    Ragnar K. Kinzelbach

    2004-01-01

    All the sources of records of the serin ( Serinus serinus) in 16th century Europe are (re-)examined, both those already known and some that have been newly discovered. Interpretation of this more detailed information confirms the results which were published by Ernst Mayr in 1926 in his doctoral thesis: north of 48°N there were no free-living populations of Serinus serinus

  14. Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans

    Microsoft Academic Search

    Leon D. Kluskens; Wilfried G. Voorhorst; Roland J. Siezen; Ruth M. Schwerdtfeger; Garabed Antranikian; John van der Oost; Willem M. de Vos

    2002-01-01

    The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction. Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases. It was cloned into a pET9d vector,

  15. Taraxalisin – a serine proteinase from dandelion Taraxacum officinale Webb s.l

    Microsoft Academic Search

    G. N. Rudenskaya; A. M. Bogacheva; A. Preusser; A. V. Kuznetsova; Ya. E. Dunaevsky; B. N. Golovkin; V. M. Stepanov

    1998-01-01

    Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-Ala-Leu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-Ala-Leu-mrp

  16. Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate

    Microsoft Academic Search

    Priya Pillai; G. Archana

    2008-01-01

    Keratinases play an important role in biotechnological applications such as improvement of feather meal, enzymatic dehairing\\u000a and production of amino acids or peptides from high molecular weight substrates. Bacillus subtilis P13, isolated from Vajreshwari hot spring (45–50°C) near Mumbai, India, produces a neutral serine protease and has an optimum\\u000a temperature of 65°C. This enzyme preparation was keratinolytic in nature and

  17. Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

    PubMed Central

    Hasnain, Sumaira Z.; McGuckin, Michael A.; Grencis, Richard K.; Thornton, David J.

    2012-01-01

    The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a TH2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s). PMID:23071854

  18. Inhibitory effect of serine protease inhibitors on neutrophil- mediated endothelial cell injury

    Microsoft Academic Search

    Keigo Nakatani; Seiichiro Takeshita; Hiroshi Tsujimoto; Youichi Kawamura; Isao Sekine

    To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil-me- diated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbil- ical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose-dependently by urinary trypsin inhibitor (uli- nastatin, UTI) and ONO-5046, which have the ability to inactivate

  19. Purification, characterization and identification of a senescence related serine protease in dark-induced senescent wheat leaves.

    PubMed

    Wang, Renxian; Liu, Shaowei; Wang, Jin; Dong, Qiang; Xu, Langlai; Rui, Qi

    2013-11-01

    Senescence-related proteases play important roles in leaf senescence by regulating protein degradation and nutrient recycling. A 98.9kDa senescence-related protease EP3 in wheat leaves was purified by ammonium sulfate precipitation, Q-Sepharose fast flow anion exchange chromatography and gel slicing after gel electrophoresis. Due to its relatively high thermal stability, its protease activity did not decrease after incubation at 40°C for 1-h. EP3 protease was suggested to be a metal-dependent serine protease, because its activity was inhibited by serine protease inhibitors PMSF and AEBSF and metal related protease inhibitor EGTA. It was identified as a subtilisin-like serine protease of the S8A family based on data from both mass spectrometry and the cloned cDNA sequence. Therefore, these data suggest that a serine protease of the S8A subfamily with specific biochemical properties is involved in senescence-associated protein degradation. PMID:23910959

  20. 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. PMID:23274980

  1. (PS)2: protein structure prediction server version 3.0

    PubMed Central

    Huang, Tsun-Tsao; Hwang, Jenn-Kang; Chen, Chu-Huang; Chu, Chih-Sheng; Lee, Chi-Wen; Chen, Chih-Chieh

    2015-01-01

    Protein complexes are involved in many biological processes. Examining coupling between subunits of a complex would be useful to understand the molecular basis of protein function. Here, our updated (PS)2 web server predicts the three-dimensional structures of protein complexes based on comparative modeling; furthermore, this server examines the coupling between subunits of the predicted complex by combining structural and evolutionary considerations. The predicted complex structure could be indicated and visualized by Java-based 3D graphics viewers and the structural and evolutionary profiles are shown and compared chain-by-chain. For each subunit, considerations with or without the packing contribution of other subunits cause the differences in similarities between structural and evolutionary profiles, and these differences imply which form, complex or monomeric, is preferred in the biological condition for the subunit. We believe that the (PS)2 server would be a useful tool for biologists who are interested not only in the structures of protein complexes but also in the coupling between subunits of the complexes. The (PS)2 is freely available at http://ps2v3.life.nctu.edu.tw/. PMID:25943546

  2. The upper mantle discontinuities in western Canada from Ps conversions

    Microsoft Academic Search

    M. G. Bostock; J. F. Cassidy

    1995-01-01

    We have investigated variations in the travel times ofPs converted phases from the upper mantle 410 and 660 km discontinuities recorded on the western stations of the Canadian National Seismograph Network using a variant of the technique introduced byVinnik (1977). Clear and unambiguous signals for both discontinuities are observed at 8 of the 11 stations considered and exhibit variations which

  3. The upper mantle discontinuities in western Canada from Ps conversions

    Microsoft Academic Search

    M. G. Bostock; J. F. Cassidy

    1995-01-01

    We have investigated variations in the travel times of Ps converted phases from the upper mantle 410 and 660 km discontinuities recorded on the western stations of the Canadian National Seismograph Network using a variant of the technique introduced by Vinnik (1977). Clear and unambiguous signals for both discontinuities are observed at 8 of the 11 stations considered and exhibit

  4. Voluntary Product Standard PS 1-09 Structural Plywood

    E-print Network

    #12;Voluntary Product Standard PS 1-09 Structural Plywood May 2010 U.S. Department of Commerce Gary Page left blank on purpose #12;iii DEPARTMENT OF COMMERCE (DOC) VOLUNTARY PRODUCT STANDARDS DOC Voluntary Product Standards are developed under procedures published by the Department of Commerce in Title

  5. PS145A Making Democracy Work: Lessons from India

    E-print Network

    Jacobs, Lucia

    PS145A Making Democracy Work: Lessons from India Course Information Course Instructor Dr. Pradeep has been written and said about the link between democracy and development, religious and ethnic of the world's most thriving democracies for over 60 years, and, in doing so offered a puzzle for many

  6. T-PS-P Task Force Minutes April 25, 2012

    E-print Network

    Jawitz, James W.

    the peer review guidelines developed by Kirby Barrick and obtain feedback from the Faculty Assembly for Extension. · Hints should be procedural, not involving T-PS-P criteria. · The process of developing new teaching guidelines is as important as the final product. · The final course peer evaluation guidelines

  7. BioMaPS: A Roadmap for Success

    ERIC Educational Resources Information Center

    McCarthy, Maeve L.; Fister, K. Renee

    2010-01-01

    The manuscript outlines the impact that our National Science Foundation Interdisciplinary Training for Undergraduates in Biological and Mathematical Sciences program, BioMaPS, has had on the students and faculty at Murray State University. This interdisciplinary program teams mathematics and biology undergraduate students with mathematics and…

  8. BiiPS software: statistical learning in finance

    E-print Network

    Del Moral , Pierre

    BiiPS software: statistical learning in finance ALEA project-team, Inria Bordeaux ­ Sud-Ouest 3. 3 avril 2012ALEA project-team, Inria Bordeaux ­ Sud-Ouest - 2 #12;Problem Modern finance is becoming project-team, Inria Bordeaux ­ Sud-Ouest 3 avril 2012 - 12 More finance applications 3 #12;Pricing

  9. Consistent scenario for B{yields}PS decays

    SciTech Connect

    Delepine, D.; Lucio M, J. L.; Mendoza S, J. A.; Ramirez, Carlos A. [Instituto de Fisica, Universidad de Guanajuato Loma del Bosque 103, Lomas del Campestre, 37150 Leon, Guanajuato (Mexico); Depto. de Fisica-Matematicas, Universidad de Pamplona Pamplona, Norte de Santander (Colombia); Escuela de Fisica, Universidad Industrial de Santander, A.A. 678, Bucaramanga (Colombia)

    2008-12-01

    We consider B{yields}PS decays where P stands for pseudoscalar and S for a heavy (1500 MeV) scalar meson. We achieve agreement with available experimental data, which includes two orders of magnitude hierarchy, assuming the scalars mesons are two quark states. The contribution of the dipolar penguin operator O{sub 11} is quantified.

  10. Transcriptional upregulation of the human MRP2 gene expression by serine/threonine protein kinase inhibitors.

    PubMed

    Pu?aski, L; Szemraj, J; Uchiumi, T; Kuwano, M; Bartosz, G

    2005-01-01

    Transcriptional regulation by cellular signalling pathways of multidrug resistance proteins that pump anticancer drugs out of cells is one of key issues in the development of the multidrug resistance phenotype. In our study, we have used the reporter gene approach as well as determination of mRNA levels in two cancer cell lines of human origin, MCF-7 and A549, to study the regulation of multidrug resistance proteins 2 and 3 (MRP2 AND MRP3) by serine/threonine protein kinases. Since a prototypic PKC inducer, PMA, caused a marked upregulation of transcription from both human MRP2 and MRP3 promoters, a role for PKC isoforms in positive control of expression of these proteins could be postulated. Interestingly, broad-spectrum serine-threonine protein kinase inhibitors which also inhibit PKC, staurosporine and H-7, stimulated expression from the MRP2 promoter instead of inhibiting it. This effect was not seen for MRP3. MRP2 induction by staurosporine and H-7 was shown to have phenotypic consequences in whole cells, rendering them more resistant to etoposide and increasing their ability to export calcein through the plasma membrane. These results point to the involvement of serine/threonine protein kinases in negative regulation of the human MRP2 gene and to the necessity of testing novel anti-cancer drugs acting as protein kinase inhibitors with regard to their potential ability to induce multidrug resistance. PMID:16602625

  11. Serine protease inhibitors modulate smoke-induced chemokine release from human lung fibroblasts.

    PubMed

    Numanami, Hiroki; Koyama, Sekiya; Nelson, Dan K; Hoyt, Jeffrey C; Freels, Jon L; Habib, Michael P; Amano, Jun; Haniuda, Masayuki; Sato, Etsuro; Robbins, Richard A

    2003-11-01

    Smoking is associated with lung inflammation and a protease-antiprotease imbalance. We previously reported that cigarette smoke extract (CSE) stimulates human lung fibroblasts to release chemotactic cytokines. We hypothesized that serine protease inhibitors might modulate lung fibroblast release of chemotactic cytokines in response to CSE. To test this hypothesis, serine protease inhibitors (FK706, alpha1-antitrypsin, methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone, or Nalpha-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) from human fetal lung fibroblasts by the blind-well chemotactic chamber. Metalloproteinases and cysteine proteinases were not examined in this study. Similarly, the release and gene expression of chemokines and nuclear factor-kappaB (NF-kappaB) activation were measured by means of enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Release of NCA, MCA, chemotactic chemokines including interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor, and the expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA were attenuated by FK706. Furthermore, FK706 suppressed NF-kappaB activation. These data suggest that serine protease inhibitors attenuate the CSE-induced release of NCA and MCA from human fetal lung fibroblasts and that the inhibitory action of antiproteases might depend on NF-kappaB signaling pathway. PMID:12738688

  12. Proton spin-lattice relaxation in silkworm cocoons: physisorbed water and serine side-chain motions.

    PubMed

    Geppi, Marco; Mollica, Giulia; Borsacchi, Silvia; Cappellozza, Silvia

    2010-03-01

    The molecular dynamic behavior of silkworm cocoons produced by a single Bombyx mori strain was investigated by means of high- and low-resolution solid-state NMR experiments. Cocoons with different moisture content were prepared to study the effects of physisorbed water on their molecular dynamics in the MHz regime, which was probed through the measurement of (1)H T(1) relaxation times at 25 MHz in the 25-95 degrees C temperature range. The water content of the different samples was determined from the analysis of (1)H free-induction decays. In addition to the rotation of methyl groups, mostly from alanine, and to the reorientation of physisorbed water molecules, already identified in previous works as relaxation sinks, the reorientation of serine side-chains was here found to contribute to (1)H T(1) above room temperature. The analysis of the trends of (1)H T(1) versus temperature was carried out in terms of semiempirical models describing the three main motional processes, and indicated that methyl rotation, water reorientation and serine side-chain motions are the most efficient relaxation mechanisms below 0 degrees C, between 0 and 60 degrees C, and above 60 degrees C, respectively. The activation energies were found to decrease passing from serine to water to methyl motions. PMID:20136080

  13. Serine/threonine/tyrosine protein kinase phosphorylates oleosin, a regulator of lipid metabolic functions.

    PubMed

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

    2012-05-01

    Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A(2) activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A(2) activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca(2+) inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation. PMID:22434039

  14. Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

    PubMed Central

    Di Salvo, Martino L.; Scarsdale, J. Neel; Kazanina, Galina; Contestabile, Roberto; Schirch, Verne; Wright, H. Tonie

    2013-01-01

    Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5?-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the ?-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs. PMID:23956983

  15. Role of pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase.

    PubMed

    Talwar, R; Leelavathy, V; Krishna Rao, J V; Appaji Rao, N; Savithri, H S

    2000-09-15

    Serine hydroxymethyltransferase belongs to the alpha class of pyridoxal-5'-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the gamma-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85-90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5'-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with L-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase. PMID:10970801

  16. The host metabolite D-serine contributes to bacterial niche specificity through gene selection.

    PubMed

    Connolly, James P R; Goldstone, Robert J; Burgess, Karl; Cogdell, Richard J; Beatson, Scott A; Vollmer, Waldemar; Smith, David G E; Roe, Andrew J

    2015-04-01

    Escherichia coli comprise a diverse array of both commensals and niche-specific pathotypes. The ability to cause disease results from both carriage of specific virulence factors and regulatory control of these via environmental stimuli. Moreover, host metabolites further refine the response of bacteria to their environment and can dramatically affect the outcome of the host-pathogen interaction. Here, we demonstrate that the host metabolite, D-serine, selectively affects gene expression in E. coli O157:H7. Transcriptomic profiling showed exposure to D-serine results in activation of the SOS response and suppresses expression of the Type 3 Secretion System (T3SS) used to attach to host cells. We also show that concurrent carriage of both the D-serine tolerance locus (dsdCXA) and the locus of enterocyte effacement pathogenicity island encoding a T3SS is extremely rare, a genotype that we attribute to an 'evolutionary incompatibility' between the two loci. This study demonstrates the importance of co-operation between both core and pathogenic genetic elements in defining niche specificity. PMID:25526369

  17. RSK2 regulates endocytosis of FGF receptor 1 by phosphorylation on serine 789.

    PubMed

    Nadratowska-Wesolowska, B; Haugsten, E M; Zakrzewska, M; Jakimowicz, P; Zhen, Y; Pajdzik, D; Wesche, J; Wiedlocha, A

    2014-10-01

    FGFR1 (fibroblast growth factor receptor 1) regulates many key cellular responses including proliferation, migration and differentiation through activation of signaling pathways. Irregularities in FGFR1 signaling have been implicated in several pathological conditions, including human cancer. In order to discover novel regulators of FGFR1 signaling, we performed yeast two-hybrid screens and identified RSK2 (p90 ribosomal S6 kinase 2) as a potential FGFR1 interaction partner. RSK2 belongs to the family of serine/threonine kinases that are activated through the Ras-MAPK signal transduction pathway. Both in vitro and in vivo experiments confirmed the interaction and we show that phosphorylated RSK2 binds to and phosphorylates serine 789 in the C-terminal tail of FGFR1. Inhibition of RSK2 activity led to prolonged tyrosine transphosphorylation of FGFR1. Furthermore, prevention of FGFR1 phosphorylation by inhibition of RSK2 activity or mutation of serine 789 to alanine reduced FGFR1 endocytosis and ubiquitination explaining mechanistically the prolonged signaling activity. We propose a novel regulatory mechanism whereby activated RSK2 directly interacts with and phosphorylates FGFR1, thereby modulating receptor signaling through regulation of endocytosis. PMID:24141780

  18. Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila.

    PubMed

    Yung, Philip Yuk Kwong; Stuetzer, Alexandra; Fischle, Wolfgang; Martinez, Anne-Marie; Cavalli, Giacomo

    2015-06-01

    Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis. PMID:26004180

  19. Serine Protease MP2 Activates Prophenoloxidase in the Melanization Immune Response of Drosophila melanogaster

    PubMed Central

    Chu, Yuan; Zhao, Zhangwu

    2013-01-01

    In arthropods, melanization plays a major role in the innate immune response to encapsulate and kill the invasive organisms. It is mediated by a serine protease cascade and is regulated by serpins. The identification of the molecular components of melanization and the regulation of those components are still unclear in Drosophila melanogaster, although some genetic research on the activation of melanization has been reported. Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. Overexpression or repression of MP2 in flies resulted in increased and decreased rates of cleavage, respectively, of prophenoloxidase-1. Moreover, serine protease inhibitor Spn27A formed SDS-stable complexes with MP2, both in vitro and in vivo. The amidase activity of MP2 was inhibited efficiently by Spn27A. Spn27A also prevented MP2 from cleaving prophenoloxidase-1. Taken together, these results indicate that under our experimental conditions MP2 functions as a prophenoloxidase-activating protease, and that this function is inhibited by Spn27A. MP2 and Spn27A thus constitute a regulatory unit in the prophenoloxidase activation cascade in Drosophila. The combination of genetic, molecular genetic and biochemical approaches should allow further advances in our understanding of the prophenoloxidase-activating cascade in insects and indirectly shed further light on protease-cascades in humans and other vertebrates. PMID:24260243

  20. Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia.

    PubMed

    Ksiazek, Miroslaw; Karim, Abdulkarim Y; Bryzek, Danuta; Enghild, Jan J; Thřgersen, Ida B; Koziel, Joanna; Potempa, Jan

    2015-03-01

    The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37. PMID:25391881

  1. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan

    2014-01-01

    Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935

  2. Positive correlation between expression level of mitochondrial serine hydroxymethyltransferase and breast cancer grade

    PubMed Central

    Yin, Ke

    2015-01-01

    Metabolic reprogramming plays an essential role in supporting the survival and proliferation of cancer cells. Serine hydroxymethyltransferase (SHMT) directs serine to the metabolism of one-carbon unit and the synthesis of thymidilate as a key factor in this metabolic shift. Although the mitochondrial isoform of SHMT (SHMT2) has been proven to be a crucial factor in the serine/glycine metabolism in several cancer cell types, the expression pattern of SHMT2 and the correlation of expression level of SHMT2 and other clinicopathological parameters in clinical breast cancer remain to be explored. In this research, 76 breast cancer patients who underwent modified radical mastectomy were enrolled for immunohistochemical analysis of the expression level of SHMT2 in their cancerous breast tissues for comparison with that in matching, distant noncancerous tissues. The results showed that SHMT2 was not expressed in the distant noncancerous cells. In contrast, SHMT2 protein could be stained in all breast cancer samples at varying degrees. Higher level of SHMT2 was expressed in grade III breast cancer cells than that those in grade I–II (P<0.05). In conclusion, SHMT2 was highly expressed in breast cancer cells, and the expression level of SHMT2 was positively correlated with breast cancer grade, suggesting that SHMT2 could be a target for anticancer therapies. PMID:25999742

  3. Purification and Functional Characterisation of Rhinocerase, a Novel Serine Protease from the Venom of Bitis gabonica rhinoceros

    PubMed Central

    Vaiyapuri, Sakthivel; Harrison, Robert A.; Bicknell, Andrew B.; Gibbins, Jonathan M.; Hutchinson, Gail

    2010-01-01

    Background Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. Methodology/Principal Findings In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading ? and ? chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. Conclusions/Significance A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes. PMID:20300193

  4. Construction of hepatitis C-SIN virus recombinants with replicative dependency on hepatitis C virus serine protease activity

    Microsoft Academic Search

    Young-Gyu Cho; Hyun-Sun Moon; Young-Chul Sung

    1997-01-01

    An in vivo assay system was developed for the serine protease of hepatitis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication of the HCV-SIN chimeric virus. Two chimeric viral cDNA clones were constructed by inserting the NS3\\/4A region and NS3\\/4A region with the putative helicase deleted, into

  5. Neutrophil elastase, an acid-independent serine protease, facilitates reovirus uncoating and infection in U937 promonocyte cells

    Microsoft Academic Search

    Joseph W Golden; Leslie A Schiff

    2005-01-01

    Background  Mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. During enteric infections, proteolysis\\u000a of the reovirus outer capsid protein ?3 is mediated by pancreatic serine proteases. In contrast, the proteases critical for\\u000a reovirus replication in the lung are unknown. Neutrophil elastase (NE) is an acid-independent, inflammatory serine protease\\u000a predominantly expressed by neutrophils. In addition to its normal

  6. d-serine enhances extinction of auditory cued fear conditioning via ERK1/2 phosphorylation in mice.

    PubMed

    Matsuda, Shingo; Matsuzawa, Daisuke; Nakazawa, Ken; Sutoh, Chihiro; Ohtsuka, Hiroyuki; Ishii, Daisuke; Tomizawa, Haruna; Iyo, Masaomi; Shimizu, Eiji

    2010-08-16

    Several lines of evidence suggest that the N-methyl-D-aspartate (NMDA) receptor plays a significant role in fear conditioning and extinction. However, our knowledge of the role of D-serine, an endogenous ligand for the glycine site of the NMDA receptor, in fear extinction is quite limited compared to that of D-cycloserine, an exogenous partial agonist for the same site. In the current study, we examined the effects of D-serine on fear extinction and phosphorylation of extracellular signal-regulated kinase (ERK) in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC) during the process of fear extinction. Systemic administrations of D-serine (2.7 g/kg, i.p.) with or without the ERK inhibitor SL327 (30 mg/kg, i.p.) to C57BL/6J mice were performed before fear extinction in a cued fear conditioning and extinction paradigm. Cytosolic and nuclear ERK 1/2 phosphorylation in the hippocampus, BLA, and mPFC were measured 1h after extinction (E1h), 24h after extinction (E24h), and 1h after recall (R1h) by Western blotting. We found that D-serine enhanced the extinction of fear memory, and the effects of D-serine were reduced by the ERK phosphorylation inhibitor SL327. The Western blot analyses showed that D-serine significantly increased cytosolic ERK 2 phosphorylation at E1h in the hippocampus and cytosolic ERK 1/2 phosphorylation at R1h in the BLA. The present study suggested that D-serine might enhance fear extinction through NMDA receptor-induced ERK signaling in mice, and that D-serine has potential clinical importance for the treatment of anxiety disorders. PMID:20416352

  7. Homologous IRCM-Serine Protease 1 from pituitary, heart atrium and ventricle: A common pro-hormone maturation enzyme?

    Microsoft Academic Search

    Nabil G. Seidah; James A. Cromlish; Josée Hamelin; Gaétan Thibault; Michel Chrétien

    1986-01-01

    IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem.261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem.261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a

  8. Regulation of enzymes of serine and one-carbon metabolism by testosterone in rat prostate, liver, and kidney.

    PubMed

    Sanborn, T A; Kowle, R L; Sallach, H J

    1975-10-01

    A significant decrease in the specific activity of 3 enzymes of serine and one-carbon metabolism (3-phosphoglycerate dehydrgenase, phosphoserine phosphatase, and serine hydroxy-methyltransferase) was found in the rat prostate gland with castration. A single injection of testosterone propionate to rats 3 days after castration resulted in a significant increase in the 3 enzyme activities within 24 h. This increase in specific activity was maximal 72 h after injection of testosterone in the case of 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase. When cycloheximide was administered in conjunction with testosterone, the increase in 3-phosphoglycerate dehydrogenase and serine hydroxy-methyltransferase activity was significantly reduced compared to injection of testosterone alone. N6, O2'-dibutyryl adenosine-3',5'-cyclic monophosphate [(Bu)2 cAMP] and theophylline injected at 8 h intervals to rats 3 days after castration failed to mimic the action of testosterone on these 3 enzymes 24 and 72 h after beginning injections. Castration had no effect on the specific activity of these enzymes in the kidney; however, 3-phosphoglycerate dehydrogenase was significantly diminished in the liver 6 days after castration. A single injection of testosterone to rats 3 days after castration restored the activity to sham-operated levels. Serine hydroxy-methyltransferase and phosphoserine phosphatase activity in the liver were unaffected 6 days after castration. Thus testosterone exerts a regulatory role on serine and one-carbon metabolism in the prostate and liver which (Bu), cAMP is unable to minic. PMID:172314

  9. 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. PMID:25986610

  10. Expression profiling and comparative analyses of seven midgut serine proteases from the yellow fever mosquito, Aedes aegypti

    PubMed Central

    Brackney, Doug E.; Isoe, Jun; Black, W.C.; Zamora, Jorge; Foy, Brian D.; Miesfeld, Roger L.; Olson, Ken E.

    2010-01-01

    Aedes aegypti utilizes blood for energy production, egg maturation and replenishment of maternal reserves. The principle midgut enzymes responsible for bloodmeal digestion are endoproteolytic serine-type proteases within the S1.A subfamily. While there are hundreds of serine protease-like genes in the A. aegypti genome, only five are known to be expressed in the midgut. We describe the cloning, sequencing and expression profiling of seven additional serine proteases and provide a genomic and phylogenetic assessment of these findings. Of the seven genes, four are constitutively expressed and three are transcriptionally induced upon blood feeding. The amount of transcriptional induction is strongly correlated among these genes. Alignments reveal that, in general, the conserved catalytic triad, active site and accessory catalytic residues are maintained in these genes and phylogenetic analysis shows that these genes fall within three distinct clades; trypsins, chymotrypsins and serine collagenases. Interestingly, a previously described trypsin consistently arose with other serine collagenases in phylogenetic analyses. These results suggest that multiple gene duplications have arisen within the S1.A subfamily of midgut serine proteases and/or that A. aegypti has evolved an array of proteases with a broad range of substrate specificities for rapid, efficient digestion of bloodmeals. PMID:20100490

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

    PubMed Central

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

    2013-01-01

    Most of pyruvoyl-dependent proteins observed in prokaryotes and eukaryotes are critical regulatory enzymes, which are primary targets of inhibitors for anti-cancer and anti-parasitic therapy. These proteins undergo an autocatalytic, intramolecular self-cleavage reaction in which a covalently bound pyruvoyl group is generated on a conserved serine residue. Traditional detections of the modified serine sites are performed by experimental approaches, which are often labor-intensive and time-consuming. In this study, we initiated in an attempt for the computational predictions of such serine sites with Feature Selection based on a Random Forest. Since only a small number of experimentally verified pyruvoyl-modified proteins are collected in the protein database at its current version, we only used a small dataset in this study. After removing proteins with sequence identities >60%, a non-redundant dataset was generated and was used, which contained only 46 proteins, with one pyruvoyl serine site for each protein. Several types of features were considered in our method including PSSM conservation scores, disorders, secondary structures, solvent accessibilities, amino acid factors and amino acid occurrence frequencies. As a result, a pretty good performance was achieved in our dataset. The best 100.00% accuracy and 1.0000 MCC value were obtained from the training dataset, and 93.75% accuracy and 0.8441 MCC value from the testing dataset. The optimal feature set contained 9 features. Analysis of the optimal feature set indicated the important roles of some specific features in determining the pyruvoyl-group-serine sites, which were consistent with several results of earlier experimental studies. These selected features may shed some light on the in-depth understanding of the mechanism of the post-translational self-maturation process, providing guidelines for experimental validation. Future work should be made as more pyruvoyl-modified proteins are found and the method should be evaluated on larger datasets. At last, the predicting software can be downloaded from http://www.nkbiox.com/sub/pyrupred/index.html. PMID:23805260

  12. Particle Swarm Inspired Evolutionary Algorithm (PS-EA) for Multiobjective Optimization Problems

    E-print Network

    Coello, Carlos A. Coello

    to extend PSO algorithm to effectively search in multiconstrained solution spaces, due to the constaints rigidly imposed by the PSO equations. To overcome the constraints, PS-EA replaces the PSO equations is performed between PS-EA with Genetic Algorithm (GA) and PSO and it is found that PS-EA provides an advantage

  13. SENTENCING MEMORANDUM -1 Waldo, Schweda & Montgomery, P.S. 2206 North Pines Road

    E-print Network

    Gollin, George

    Waldo, Schweda & Montgomery, P.S. 2206 North Pines Road Spokane, WA 99206 509/924-3686 Fax: 509/922-2196 Peter S. Schweda Waldo, Schweda & Montgomery, P.S. 2206 North Pines Road Spokane, WA 99206 509 - 2 Waldo, Schweda & Montgomery, P.S. 2206 North Pines Road Spokane, WA 99206 509/924-3686 Fax: 509

  14. The PS/PDI: a high accuracy development tool for diffraction limited short-wavelength optics

    E-print Network

    The PS/PDI: a high accuracy development tool for diffraction limited short-wavelength optics ultraviolet (EUV) phase-shifting point diffraction interferometer (PS/PDI) was developed and implemented projection lithography optics. The PS/PDI has been in continuous use and under ongoing development since 1996

  15. PS Integrins and Laminins: Key Regulators of Cell Migration during Drosophila Embryogenesis

    PubMed Central

    Urbano, Jose M.; Domínguez-Giménez, Paloma; Estrada, Beatriz; Martín-Bermudo, María D.

    2011-01-01

    During embryonic development, there are numerous cases where organ or tissue formation depends upon the migration of primordial cells. In the Drosophila embryo, the visceral mesoderm (vm) acts as a substrate for the migration of several cell populations of epithelial origin, including the endoderm, the trachea and the salivary glands. These migratory processes require both integrins and laminins. The current model is that ?PS1?PS (PS1) and/or ?PS3?PS (PS3) integrins are required in migrating cells, whereas ?PS2?PS (PS2) integrin is required in the vm, where it performs an as yet unidentified function. Here, we show that PS1 integrins are also required for the migration over the vm of cells of mesodermal origin, the caudal visceral mesoderm (CVM). These results support a model in which PS1 might have evolved to acquire the migratory function of integrins, irrespective of the origin of the tissue. This integrin function is highly specific and its specificity resides mainly in the extracellular domain. In addition, we have identified the Laminin ?1,2 trimer, as the key extracellular matrix (ECM) component regulating CVM migration. Furthermore, we show that, as it is the case in vertebrates, integrins, and specifically PS2, contributes to CVM movement by participating in the correct assembly of the ECM that serves as tracks for migration. PMID:21949686

  16. Rapidly metabolizing pool of phosphatidylglycerol as a precursor for phosphatidylethanolamine and diglyceride in Bacillus megaterium

    SciTech Connect

    Lombardi, F.J.; Chen, S.L.; Fulco, A.J.

    1980-02-01

    Pulse-chase experiments in Bacillus megaterium ATCC 14581 with (U-/sup 14/C)-palmitate, L-(U-/sup 14/C)serine, and (U-/sup 14/C)glycerol showed that a large pool of phosphatidylglycerol (PG) which exhibited rapid turnover in the phosphate moiety (PG/sub t/) underwent very rapid interconversion with the large diglyceride (DG) pool. Kinetics of DG labeling indicated that the fatty acyl and diacylated glycerol moieties of PG/sub t/ were also utilized as precursors for net DG formation. The (U-/sup 14/C)glycerol pulse-chase results also confirmed the presence of a second, metabolically stable pool of PG (PG/sub s/), which was deduced from (/sup 32/P)phosphate studies. The other major phospholipid, phosphatidylethanolamine (PE), exhibited pronounced lags relative to PG and DG in /sup 14/C-fatty acid, (/sup 14/C)glycerol, and (/sup 32/P)-phosphate incorporation, but not for incorporation of L-(U-/sup 14/C)serine into the ethanolamine group of PE or into the serine moiety of the small phosphatidylserine (PS) pool. Furthermore, initial rates of L-(U-/sup 14/C)serine incorporation into the serine and ethanolamine moieties of PS and PE were unaffected by cerulenin. The results provided compelling in vivo evidence that de novo PG/sub t/, PS, and PE syntheses in this organism proceed for the most part sequentially in the order PG/sub t/ ..-->.. PS ..-->.. PE rather than via branching pathways from a common intermediate and that the phosphatidyl moiety in PS and PE is derived largely from the corresponding moiety in PG/sub t/, whereas the DG pool indirectly provides an additional source for this conversion by way of the facile PG/sub t/ in equilibrium DG interconversion.

  17. Allosteric modulation of NMDA receptor via elevation of brain glycine and D-serine: the therapeutic potentials for schizophrenia.

    PubMed

    Yang, Charles R; Svensson, Kjell A

    2008-12-01

    Ionotropic AMPA and NMDA glutamate receptors are ligand-gated ion channels that mediate fast synaptic transmission in the brain and play a crucial role in learning and memory. Dysfunction of these receptors is believed to be associated with a number of neuropsychiatric disorders, including schizophrenia. As direct activation of these ionotropic receptors can lead to excitoxicity, allosteric modulation of these receptors could minimize side-effects to achieve better therapeutic efficacy. Our review here focuses on the allosteric modulation of the NMDA receptor. Endogenous glycine and D-serine both act as co-agonists on the strychnine-insensitive GlyB site on the NMDA receptor, and along with glutamate, co-activate the NMDA receptor. Forebrain synaptic glycine and d-serine levels are regulated by the Glycine Transporter-1 (GlyT1) and the arginine-serine-cysteine transporter-1 (Asc-1), respectively; in addition to D-serine metabolism by D-Amino Acid Oxidase (DAAO). Together, these processes prevent the GlyB site from being saturated by the high extracellular levels of brain glycine, and perhaps d-serine, in vivo. Blockade of NMDA receptors by phencyclidine induces schizophrenia-like symptoms with the associated cognitive deficits. It was proposed that: a) blockade of GlyT1 mediated reuptake of glycine, or b) inhibition of D-amino Acid Oxidase, or Asc-1 will elevate brain glycine, and D-serine to upregulate NMDA receptor functions via glycine and D-serine co-agonistic allosteric modulation of the GlyB sites on the NMDA receptor. These approaches may provide novel treatments to schizophrenia, provided that some of the known adverse effects associated with existing GlyT1 agents can be safely and adequately dealt with. PMID:18805436

  18. DP-PS: A safety program for DP operations

    SciTech Connect

    Cordeiro, A.; Paschoalin, R.; Fartes, E. [Petrobras, Rio de Janeiro (Brazil)

    1996-12-31

    As Petrobras develops its deepwater projects, more and more DP units are used on different applications like drilling, completion, intervention and pipeline handling. The increasing complexity of the operations has been considered in a program entitled DP-PS (Safety Program For Dynamic Positioning Operations) in order to minimize the risks of failures which could lead to serious accidents on subsea and surface installations as well as to the DP vessels. This paper briefly presents the DP-PS, consisting of a group of related projects considering different aspects of DP operations, like: Vessels Incidents Databank, Emergency Disconnection procedures, Risk Comparison, DGPS (Differential Global Positioning System) application, Well Control Equipment and Standard procedures, Testing Programs and Guidelines for DP Operations. The high number of DP contractors operating in Brazil and the atmosphere of cooperation in which the program has been developed was a key factor for its success. As a result, the number of DP incidents has remarkably decreased.

  19. Laboratory spectrum of the PS radical and related astronomical search

    SciTech Connect

    Ohishi, M.; Yamamoto, S.; Saito, S.; Kawaguchi, K.; Suzuki, H.

    1988-06-01

    The millimeter-wave rotational spectrum of the PS radical (X 2Pi r) was observed in the laboratory for the first time in the frequency region of 79-293 GHz by discharging the mixture of PSCl3 and He. Some 44 lines were measured, and the rotational constant, the centrifugal distortion constant, the centrifugal distortion term of the spin-orbit coupling constant, the Lambda-type doubling constants, and the hyperfine coupling constants were determined. Based on the measured and calculated frequencies, an astronomical search for the interstellar and circumstellar PS radical was made without success in Orion KL, Sgr B2, L134N,IRC + 10216, VY CMa, and OH 231.8 + 4.2. 29 references.

  20. Opportunities for Human iPS Cells in Predictive Toxicology

    PubMed Central

    Anson, Blake D.; Kolaja, Kyle; Kamp, Timothy J.

    2013-01-01

    Toxicity assessment is a major challenge for cost-effective drug development, and there is great need for better tools to accurately predict adverse drug reactions. Technological advances are empowering new cell-based assays for predictive toxicology, and these assays are critically dependent on the cell source. Here we describe the properties of human induced pluripotent stem (iPS) cells that make them a promising cell source for toxicity assessment and highlight progress to date and important roadblocks remaining. PMID:21430658

  1. Optimized drivers for PS\\/2 and VGA using HDL

    Microsoft Academic Search

    Punj Pokharel; Binod Bhatta; Anand D. Darji

    2011-01-01

    FPGA is increasingly popular as it has reliable prototyping, low cost and high speed. Input\\/output interfacing is inevitable for any system. So, the necessary signals for VGA and PS\\/2 interface are generated using HDL. The design is synthesized and implemented in Xilinx Virtex-II Pro. The mapped signals in FPGA Board are optimized to minimize resource utilization. The entire design is

  2. iPS cells: A source of cardiac regeneration

    Microsoft Academic Search

    Yoshinori Yoshida; Shinya Yamanaka

    2011-01-01

    For the treatment of heart failure, a new strategy to improve cardiac function and inhibit cardiac remodeling needs to be established. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent cells that can differentiate into cell types from all three germ layers both in vitro and in vivo. The therapeutic effect of ES\\/iPS cell-derived progeny was reported

  3. Performance analysis of CSMA\\/CD?PS systems

    Microsoft Academic Search

    Masahiro Ishigaki; Yutaka Takahashi; Toshiharu Hasegawa

    1998-01-01

    Recently, the interest in wireless LANs has been increasing. For wireless LANs, light and simple terminals are desirable,\\u000a and random access protocols such as CSMA and CDMA are preferable. In order to improve the performance of these protocols,\\u000a some variations have been proposed. Among them is the CSMA\\/CD?PS (Carrier Sense Multiple Access with Collision Detection and\\u000a Packet Segmentation) protocol, in

  4. Research of beam smoothing technologies using CPP, SSD, and PS

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Su, Jingqin; Hu, Dongxia; Li, Ping; Yuan, Haoyu; Zhou, Wei; Yuan, Qiang; Wang, Yuancheng; Tian, Xiaocheng; Xu, Dangpeng; Dong, Jun; Zhu, Qihua

    2015-02-01

    Precise physical experiments place strict requirements on target illumination uniformity in Inertial Confinement Fusion. To obtain a smoother focal spot and suppress transverse SBS in large aperture optics, Multi-FM smoothing by spectral dispersion (SSD) was studied combined with continuous phase plate (CPP) and polarization smoothing (PS). New ways of PS are being developed to improve the laser irradiation uniformity and solve LPI problems in indirect-drive laser fusion. The near field and far field properties of beams using polarization smoothing were studied and compared, including birefringent wedge and polarization control array. As more parameters can be manipulated in a combined beam smoothing scheme, quad beam smoothing was also studies. Simulation results indicate through adjusting dispersion directions of one-dimensional (1-D) SSD beams in a quad, two-dimensional SSD can be obtained. Experiments have been done on SG-III laser facility using CPP and Multi-FM SSD. The research provides some theoretical and experimental basis for the application of CPP, SSD and PS on high-power laser facilities.

  5. Effects of Preplasma in 10-ps Relativistic Laser Matter Interaction

    NASA Astrophysics Data System (ADS)

    Wei, M. S.; Stephens, R. B.; Peebles, J.; McGuffey, C.; Qiao, B.; Beg, F.; Sentoku, Y.; Link, A.; Chen, H.; McLean, H.; Theobald, W.; Haberberger, D.; Davies, A.

    2014-10-01

    Experiments were performed using the kJ 10-ps OMEGA EP laser to study the effect of preplasma on fast electron generation and energy coupling in relativistic laser plasma interaction (LPI) with a controlled preplasma at various scalelength created by a 1-ns UV laser. Targets were multilayered planar foil consisting of an Al substrate, a buried Cu layer and a thick conductive CH layer. Preplasma density profile and relativistic LPI generated fields were characterized using a 10-ps 4 ? optical probe (angular filter refractometry and polarimetry) together with radiography using a high-energy proton beam produced by the second kJ 10-ps EP beam. Fast electrons were diagnosed by measuring Cu K-shell fluorescence emission and bremsstrahlung radiation. Electron energy spectrum was monitored by a magnetic spectrometer. Preliminary results showed nonlinear interaction instabilities and a reduced electron temperature with increasing preplasma scalelength. Dynamics of the relativistic LPI and the resultant fast electron beam characteristics and energy coupling will be presented. Supported by the US DOE under DE-NA0002026 and DE-FC02-04ER54789.

  6. Molecular cloning and nucleotide sequence of the 90k serine protease gene, hspK , from Bacillus subtilis (natto) No. 16

    Microsoft Academic Search

    Youhei Yamagata; Rei Abe; Yasunori Fujita; Eiji Ichishima

    1995-01-01

    We previously reported purification and characterization of a 90k serine protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed different and unique substrate specificity towards the oxidized B-chain of insulin from those of well-known bacterial serine proteases from Bacillus subtilisins. The structural gene, hspK, for the 90k serine

  7. Phosphorylation of UBF at serine 388 is required for interaction with RNA polymerase I and activation of rDNA transcription

    Microsoft Academic Search

    Renate Voit; Ingrid Grummt

    2001-01-01

    Modulation of the activity of the upstream binding factor (UBF) plays a key role in cell cycle-dependent regulation of rRNA synthesis. Activation of rDNA transcription on serum stimulation requires phosphorylation of UBF at serine 484 by G1-specific cyclin-dependent kinase (cdk)\\/cyclin complexes. After G1 progression UBF is phosphorylated at serine 388 by cdk2\\/cyclin E and cdk2\\/cyclin A. Conversion of serine 388

  8. How pyridoxal 5'-phosphate differentially regulates human cytosolic and mitochondrial serine hydroxymethyltransferase oligomeric state.

    PubMed

    Giardina, Giorgio; Brunotti, Paolo; Fiascarelli, Alessio; Cicalini, Alessandra; Costa, Mauricio G S; Buckle, Ashley M; di Salvo, Martino L; Giorgi, Alessandra; Marani, Marina; Paone, Alessio; Rinaldo, Serena; Paiardini, Alessandro; Contestabile, Roberto; Cutruzzolŕ, Francesca

    2015-04-01

    Adaptive metabolic reprogramming gives cancer cells a proliferative advantage. Tumour cells extensively use glycolysis to sustain anabolism and produce serine, which not only refuels the one-carbon units necessary for the synthesis of nucleotide precursors and for DNA methylation, but also affects the cellular redox homeostasis. Given its central role in serine metabolism, serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, is an attractive target for tumour chemotherapy. In humans, the cytosolic isoform (SHMT1) and the mitochondrial isoform (SHMT2) have distinct cellular roles, but high sequence identity and comparable catalytic properties, which may complicate development of successful therapeutic strategies. Here, we investigated how binding of the cofactor PLP controls the oligomeric state of the human isoforms. The fact that eukaryotic SHMTs are tetrameric proteins while bacterial SHMTs function as dimers may suggest that the quaternary assembly in eukaryotes provides an advantage to fine-tune SHMT function and differentially regulate intertwined metabolic fluxes, and may provide a tool to address the specificity problem. We determined the crystal structure of SHMT2, and compared it to the apo-enzyme structure, showing that PLP binding triggers a disorder-to-order transition accompanied by a large rigid-body movement of the two cofactor-binding domains. Moreover, we demonstrated that SHMT1 exists in solution as a tetramer, both in the absence and presence of PLP, while SHMT2 undergoes a dimer-to-tetramer transition upon PLP binding. These findings indicate an unexpected structural difference between the two human SHMT isoforms, which opens new perspectives for understanding their differing behaviours, roles or regulation mechanisms in response to PLP availability in vivo. PMID:25619277

  9. Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila

    PubMed Central

    Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William

    2013-01-01

    After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905

  10. Complement 1s is the Serine Protease that Cleaves IGFBP-5 in Human Osteoarthritic Joint Fluid

    PubMed Central

    Busby, Walker H.; Yocum, Sue A.; Rowland, Michael; Kellner, Debra; Lazerwith, Scott; Sverdrup, Francis; Yates, Matthew; Radabaugh, Melissa; Clemmons, David R.

    2010-01-01

    Insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) are trophic factors for cartilage and have been shown to be chondroprotective in animal models of osteoarthritis. IGFBP-5 is degraded in joint fluid and inhibition of IGFBP-5 degradation has been shown to enhance the trophic effects of IGF-I. Objective To determine the identity of IGFBP-5 protease activity in human osteoarthritic (OA) joint fluid. Method OA joint fluid was purified and the purified material analyzed by IGFBP-5 zymography. Results Both crude joint fluid and purified material contained a single band of proteolytic activity that cleaved IGFBP-5. Immunoblotting of joint fluid for complement 1s (C1s) showed a band that had the same Mr estimate, e.g. 88 kDa. In gel tryptic digestion and subsequent peptide analysis by LC-MS/MS showed that the band contained human complement 1s. A panel of protease inhibitors was tested for their ability to inhibit IGFBP-5 cleavage by the purified protease. Three serine protease inhibitors, FUT175 and CP 143217 and CB-349547 had IC50’s between 1and 6 uM. Two other serine protease inhibitors had intermediate activity (e.g. IC50’s 20–40 uM) and MMP inhibitors had no detectible activity at concentrations up to 300 uM. Conclusion Human OA fluid contains a serine protease that cleaves IGFBP-5. Zymography, immunoblotting and LCMS/MS analysis indicate that complement 1s is the protease that accounts for this activity. PMID:18930415

  11. Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10

    Microsoft Academic Search

    Sreeja Chellappan; C. Jasmin; Soorej M. Basheer; Archana Kishore; K. K. Elyas; Sarita G. Bhat; M. Chandrasekaran

    2011-01-01

    An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications.\\u000a Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated\\u000a as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin\\u000a confirmed the serine protease nature of the

  12. Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

    PubMed Central

    Canova, Marc J.; Molle, Virginie

    2014-01-01

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

  13. Characterization of Pyridoxine Auxotrophs of Escherichia coli: Serine and PdxF Mutants

    PubMed Central

    Dempsey, Walter B.; Itoh, Hajime

    1970-01-01

    At least six phenotypically distinct classes of mutants of Escherichia coli which require serine or pyridoxine or both can be isolated. Three of the six classes lack 3-phosphoserine-2-oxoglutarate aminotransferase. One of these classes contains WG5, a mutant previously characterized as containing the pdxF5 allele. The aminotransferase isolated from this mutant has been compared to that isolated from wild-type E. coli and found to have apparently normal affinity for pyridoxal 5?-phosphate, but reduced affinity for pyridoxamine 5?-phosphate. PMID:4321330

  14. 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 ?-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. PMID:25790104

  15. Characterization of a novel, potent, and specific inhibitor of serine palmitoyltransferase.

    PubMed

    Zweerink, M M; Edison, A M; Wells, G B; Pinto, W; Lester, R L

    1992-12-15

    We have examined the mechanism of action of two natural products identified as broad spectrum antifungal agents (VanMiddlesworth, F., Dufresne, C., Wincott, F. E., Mosley, R. T., and Wilson, K. E. (1992) Tetrahedron Lett., in press; VanMiddlesworth, F., Giacobbe, R. A., Lopez, M. Garrity, G., Bland, J. A., Bartizal, K., Fromtling, R. A., Polishook, J., Zweerink, M. M., Edison, A. M., Rozdilsky, W., Wilson, K. E., and Monaghan, R. L. (1992) J. Antibiot. (Tokyo) 45, 861-867), designated sphingofungin B (2S-amino-3R,4R,5S,14-tetrahydroxyeicos-6-enoic acid) and sphingofungin C (2S-amino-5S-acetoxy-3R,4R,14-trihydroxyeicos-6-enoic acid), and find they are potent specific inhibitors of serine palmitoyltransferase, which catalyze the committed step of sphingolipid biosynthesis. We used Saccharomyces cerevisiae as a model to investigate the mechanism of the antifungal activity of these compounds. Macromolecular synthesis was not immediately affected by either sphingofungin B or C, synthesis continued for 60-90 min following the addition of drug to growing cultures. Significant loss of viability with sphingofungins required growing cultures and began only after several hours, with greater than 99.9% of drug-treated cells non-viable after 24 h. No lysis or other gross changes in cell morphology were observed in drug-treated cells. The structural similarity of sphingofungin B and C to sphingosine and phytosphingosine prompted us to investigate their effects on sphingolipid synthesis. Nanomolar levels of the drugs inhibited the incorporation of [3H]inositol into sphingolipid before incorporation into the sphingolipid precursor, phosphatidylinositol was affected, suggesting specific inhibition of sphingolipid synthesis. This hypothesis was confirmed by experiments in which the growth inhibitory activity of both drugs was completely ablated by the addition of phytosphingosine, dihydrosphingosine, or ketodihydrosphingosine to the culture medium. Reversal of antifungal activity by ketodihydrosphingosine suggested that serine palmitoyltransferase could be the actual target of these compounds. Direct evidence for this hypothesis was the observation of inhibition of serine palmitoyltransferase activity in crude membrane preparations at nanomolar concentrations of each drug. The potent inhibition of serine palmitoyltransferase coupled with the apparent lack of effect of these compounds on other cellular functions suggests that sphingofungin B and C will prove to be important new tools for studying the role of sphingolipids in yeast and perhaps in other organisms. PMID:1460005

  16. Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment

    NASA Technical Reports Server (NTRS)

    Stroud, Jason D.; Baicu, Catalin F.; Barnes, Mary A.; Spinale, Francis G.; Zile, Michael R.

    2002-01-01

    To determine whether and to what extent one component of the extracellular matrix, fibrillar collagen, contributes causally to abnormalities in viscoelasticity, collagen was acutely degraded by activation of endogenous matrix metalloproteinases (MMPs) with the serine protease plasmin. Papillary muscles were isolated from normal cats and cats with right ventricular pressure overload hypertrophy (POH) induced by pulmonary artery banding. Plasmin treatment caused MMP activation, collagen degradation, decreased the elastic stiffness constant, and decreased the viscosity constant in both normal and POH muscles. Thus, whereas many mechanisms may contribute to the abnormalities in myocardial viscoelasticity in the POH myocardium, changes in fibrillar collagen appear to play a predominant role.

  17. Enthalpies and constants of dissociation for D,L-Alanyl-D,L-Serine at 298 K

    NASA Astrophysics Data System (ADS)

    Gridchin, S. N.; Pyreu, D. F.

    2015-01-01

    Protolytic equilibria in aqueous solutions of D,L-alanyl-D,L-serine are studied by means of potentiometry and calorimetry. The dissociation constants and thermal effects of this reaction of the dipeptide are determined at 298.15 K and ionic strengths of 0.1, 0.3, 0.5, and 1.0 (KNO3). The standard thermodynamic characteristics (p K°, ?r G°, ?r H°, and ?r S°) of the studied equilibria are calculated. The final results are compared with the corresponding data on the related compounds.

  18. Phosphorylation of insulin receptor substrate-1 serine 307 correlates with JNK activity in atrophic skeletal muscle

    NASA Technical Reports Server (NTRS)

    Hilder, Thomas L.; Tou, Janet C L.; Grindeland, Richard E.; Wade, Charles E.; Graves, Lee M.

    2003-01-01

    c-Jun NH(2)-terminal kinase (JNK) has been shown to negatively regulate insulin signaling through serine phosphorylation of residue 307 within the insulin receptor substrate-1 (IRS-1) in adipose and liver tissue. Using a rat hindlimb suspension model for muscle disuse atrophy, we found that JNK activity was significantly elevated in atrophic soleus muscle and that IRS-1 was phosphorylated on Ser(307) prior to the degradation of the IRS-1 protein. Moreover, we observed a corresponding reduction in Akt activity, providing biochemical evidence for the development of insulin resistance in atrophic skeletal muscle.

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

    PubMed Central

    Locasale, Jason W

    2013-01-01

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

  20. Purification and characterization of a high molecular mass serine carboxypeptidase from Monascus pilosus

    Microsoft Academic Search

    Fang Liu; Shinjiro Tachibana; Toki Taira; Masanobu Ishihara; Fumio Kato; Masaaki Yasuda

    2004-01-01

    Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases

  1. Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity.

    PubMed

    Senthilkumar, Velusamy; Busby, Stephen J W; Gunasekaran, Paramasamy; Senthikumar, Velusamy; Bushby, Stephen J W

    2003-10-01

    Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues. PMID:14584923

  2. [The role of calcium/calmodulin dependent serine protein kinase in embryonic development and related diseases].

    PubMed

    Wang, Yaqing; Yuan, Li

    2015-06-10

    Calcium/calmodulin dependent serine protein kinase (CASK), which belongs to the family of membrane associated guanylate kinase (MAGUK) proteins, has several isoforms. CASK expresses differently in embryonic tissues and adult tissues. It can be modulated by phosphorylation and SUMOylation. CASK plays an important role in neural development, spermatozoal development and renal development. Dysfunction of CASK may lead to diseases. CASK is distributed extensively in the brain, regulating synapse formation. Mutation of CASK can lead to several neurologic diseases. CASK is also involved in the development and maturation of sperm and fertilization. It also can influence renal development through interaction with DLG1. PMID:26037366

  3. 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 assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulfate assimilation. PMID:16668378

  4. Water-Soluble Poly(L-serine)s with Elongated and Charged Side-Chains: Synthesis, Conformations and Cell-Penetrating Properties

    PubMed Central

    Tang, Haoyu; Yin, Lichen; Lu, Hua; Cheng, Jianjun

    2012-01-01

    Water-soluble poly(L-serine)s bearing long side-chain with terminal charge groups were synthesized via ring-opening polymerization of O-pentenyl-L-serine N-carboxyanhydride followed by thiol-ene reactions. These side-chain modified poly(L-serine)s adopt ?-sheet conformation in aqueous solution with excellent stability against changes in pH and temperature. These water-soluble poly(L-serine) derivatives with charged side-chain functional groups and stable ?-sheet conformations showed membrane-penetrating capabilities in different cell lines with low cytotoxicity. PMID:22853191

  5. Synthesis of water-soluble poly(?-hydroxy acids) from living ring-opening polymerization of O-benzyl-L-serine carboxyanhydrides.

    PubMed

    Lu, Yanbing; Yin, Lichen; Zhang, Yanfeng; Zhonghai, Zhang; Xu, Yunxiang; Tong, Rong; Cheng, Jianjun

    2012-04-17

    O-benzyl-L-serine carboxyanhydrides were synthesized via diazotization of O-benzyl-L-serine with sodium nitrite in aqueous sulfuric acid solution followed by cyclization of the resulting serine-based ?-hydroxy acid with phosgene. Degradable, water-soluble poly(?-hydroxy acids) bearing pendant hydroxyl groups were readily prepared under mild conditions via ring-opening polymerization of O-benzyl-L-serine carboxyanhydrides followed by removal of the benzyl group and showed excellent cell compatibility, suggesting their potential being used as novel materials in constructing drug delivery systems and as hydrogel scaffolds for tissue engineering applications. PMID:23359651

  6. 'Heat-treatment aqueous two phase system' for purification of serine protease from Kesinai (Streblus asper) leaves.

    PubMed

    Mehrnoush, Amid; Mustafa, Shuhaimi; Yazid, Abdul Manap Mohd

    2011-01-01

    A 'Heat treatment aqueous two phase system' was employed for the first time to purify serine protease from kesinai (Streblus asper) leaves. In this study, introduction of heat treatment procedure in serine protease purification was investigated. In addition, the effects of different molecular weights of polyethylene glycol (PEG 4000, 6000 and 8000) at concentrations of 8, 16 and 21% (w/w) as well as salts (Na-citrate, MgSO? and K?HPO?) at concentrations of 12, 15, 18% (w/w) on serine protease partition behavior were studied. Optimum conditions for serine protease purification were achieved in the PEG-rich phase with composition of 16% PEG6000-15% MgSO?. Also, thermal treatment of kesinai leaves at 55 °C for 15 min resulted in higher purity and recovery yield compared to the non-heat treatment sample. Furthermore, this study investigated the effects of various concentrations of NaCl addition (2, 4, 6 and 8% w/w) and different pH (4, 7 and 9) on the optimization of the system to obtain high yields of the enzyme. The recovery of serine protease was significantly enhanced in the presence of 4% (w/w) of NaCl at pH 7.0. Based on this system, the purification factor was increased 14.4 fold and achieved a high yield of 96.7%. PMID:22158589

  7. Interactome Analyses of Mature ?-Secretase Complexes Reveal Distinct Molecular Environments of Presenilin (PS) Paralogs and Preferential Binding of Signal Peptide Peptidase to PS2*

    PubMed Central

    Jeon, Amy Hye Won; Böhm, Christopher; Chen, Fusheng; Huo, Hairu; Ruan, Xueying; Ren, Carl He; Ho, Keith; Qamar, Seema; Mathews, Paul M.; Fraser, Paul E.; Mount, Howard T. J.; St George-Hyslop, Peter; Schmitt-Ulms, Gerold

    2013-01-01

    ?-Secretase plays a pivotal role in the production of neurotoxic amyloid ?-peptides (A?) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature ?-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the ?-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H+-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing ?-secretase complexes and was observed to influence A? production. PMID:23589300

  8. Age-dependent racemization of serine residues in a human chaperone protein

    PubMed Central

    Hooi, Michelle Y S; Raftery, Mark J; Truscott, Roger J W

    2013-01-01

    Racemization is one of the most abundant modifications in long-lived proteins. It has been proposed that the accumulation of such modifications over time could lead to changes in tissues and ultimately human age-related diseases. Serine is one of the main amino acids involved in racemization; however, the site of D-Ser in any aged protein has yet to be reported. In this study, racemization of two residues, Ser 59 and Ser 62, has been demonstrated in an unstructured region of the small heat shock protein, ?A-crystallin. ?A-crystallin is also the most abundant structural protein in the human lens. D-Ser increased linearly with age in normal lenses, until it accounted for approximately 35% of the Ser at both sites by the age of 75 years. In agreement with a possible role in human age-related disease, levels were significantly higher in cataract lenses. It is likely that such prevalent age-related changes contribute to the denaturation of ?-crystallin, and therefore its ability to act as a chaperone. Racemization of amino acids, such as serine, in flexible regions of long-lived proteins, could be associated with the development of human age-related conditions such as cataract. PMID:23139182

  9. Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome

    SciTech Connect

    Elsea, S.H.; Juyal, R.C.; Jiralerspong, S. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-12-01

    Folate-dependent one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth, and serine hydroxymethyltransferase (SHMT) is central to this process. Our studies reveal that the gene for cytosolic SHMT (cSHMT) maps to the critical interval for Smith-Magenis syndrome (SMS) on chromosome 17p11.2. The basic organization of the cSHMT locus on chromosome 17 was determined and was found to span{approximately}40 kb. The gene for cSHMT was found to be deleted in all 26 SMS patients examined by PCR, FISH, and/or Southern analysis. Furthermore, with respect to haploinsufficiency, cSHMT enzyme activity in patient lymphoblasts was determined to be {approximately}50% that of unaffected parent lymphoblasts. Serine, glycine, and folate levels were also assessed in three SMS patients and were found to be within normal ranges. The possible effects of cSHMT hemizygosity on the SMS phenotype are discussed. 40 refs., 3 figs., 21 tabs.

  10. Initial characterization of histone H3 serine 10 O-acetylation

    PubMed Central

    Britton, Laura-Mae P; Newhart, Alyshia; Bhanu, Natarajan V; Sridharan, Rupa; Gonzales-Cope, Michelle; Plath, Kathrin; Janicki, Susan M; Garcia, Benjamin A

    2013-01-01

    In eukaryotic organisms, histone posttranslational modifications (PTMs) are indispensable for their role in maintaining cellular physiology, often through their mediation of chromatin-related processes such as transcription. Targeted investigations of this ever expanding network of chemical moieties continue to reveal genetic, biochemical, and cellular nuances of this complex landscape. In this study, we present our findings on a novel class of histone PTMs: Serine, Threonine, and Tyrosine O-acetylation. We have combined highly sensitive nano-LC-MS/MS experiments and immunodetection assays to identify and validate these unique marks found only on histone H3. Mass spectrometry experiments have determined that several of these O-acetylation marks are conserved in many species, ranging from yeast to human. Additionally, our investigations reveal that histone H3 serine 10 acetylation (H3S10ac) is potentially linked to cell cycle progression and cellular pluripotency. Here, we provide a glimpse into the functional implications of this H3-specific histone mark, which may be of high value for further studies of chromatin. PMID:23949383

  11. 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

  12. Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans.

    PubMed

    You, Weon-Kyoo; Sohn, Young-Doug; Kim, Ki-Yong; Park, Doo-Hong; Jang, Yangsoo; Chung, Kwang-Hoe

    2004-03-01

    A novel serine protease, named as scolonase, was purified and characterized from the tissue of the Korean centipede, Scolopendra subspinipes mutilans. Purified scolonase showed an apparent molecular weight of 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and an isoelectric point of 4.8 on isoelectric focusing gel. Scolonase was able to preferentially hydrolyze arginine over lysine at the cleavage site among the several synthetic peptide substrates. Scolonase has also a potent fibrinolytic activity by converting human Glu-plasminogen to activated plasmin due to the specific cleavage of the molecule at the peptide bond Arg(561)-Val(562). The enzyme activity of scolonase was completely inhibited by phenylmethanesulfonyl fluoride and difluorophosphate. The cDNA encoding scolonase was cloned from the cDNA library of the centipede constructed with oligonucleotide probe, which was designed on the basis of the N-terminal amino acid sequence of scolonase. The deduced complete amino acid sequence of scolonase demonstrated that the protein is composed of 277 amino acids including 33 amino acids as a leader sequence, and that it has significant sequence homology with other serine proteases. PMID:14871620

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

    PubMed Central

    2015-01-01

    Production of ?-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight ?-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-?-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

  14. Proteolytic Cleavage of Human Acid-sensing Ion Channel 1 by the Serine Protease Matriptase*

    PubMed Central

    Clark, Edlira B.; Jovov, Biljana; Rooj, Arun K.; Fuller, Catherine M.; Benos, Dale J.

    2010-01-01

    Acid-sensing ion channel 1 (ASIC1) is a H+-gated channel of the amiloride-sensitive epithelial Na+ channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases. The type II transmembrane serine protease matriptase activates the prototypical ???ENaC channel, but we found that matriptase is expressed in glioma cells and its expression is higher in glioma compared with normal astrocytes. Therefore, the goal of this study was to test the hypothesis that matriptase regulates ASIC1 function. Matriptase decreased the acid-activated ASIC1 current as measured by two-electrode voltage clamp in Xenopus oocytes and cleaved ASIC1 expressed in oocytes or CHO K1 cells. Inactive S805A matriptase had no effect on either the current or the cleavage of ASIC1. The effect of matriptase on ASIC1 was specific, because it did not affect the function of ASIC2 and no matriptase-specific ASIC2 fragments were detected in oocytes or in CHO cells. Three matriptase recognition sites were identified in ASIC1 (Arg-145, Lys-185, and Lys-384). Site-directed mutagenesis of these sites prevented matriptase cleavage of ASIC1. Our results show that matriptase is expressed in glioma cells and that matriptase specifically cleaves ASIC1 in heterologous expression systems. PMID:20601429

  15. Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis.

    PubMed

    Takagi, Hiroshi; Yoshioka, Kenji; Awano, Naoki; Nakamori, Shigeru; Ono, Bun ichiro

    2003-01-28

    Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine/O-acetyl-L-homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine beta-synthase and cystathionine gamma-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiae L-cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L-cysteine, those expressing OAS-SHLase did not grow at all. The cells expressing both enzymes grew very well without L-cysteine. These results indicate that S. cerevisiae SATase cannot support L-cysteine biosynthesis and that S. cerevisiae OAS/OAH-SHLase produces L-cysteine if enough OAS is provided by E. coli SATase. It appears as if S. cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization. For example, S. cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L-cysteine. PMID:12586406

  16. Serine protease inhibitors modulate chemotactic cytokine production by human lung fibroblasts in vitro.

    PubMed

    Numanami, Hiroki; Koyama, Sekiya; Sato, Esturo; Haniuda, Masayuki; Nelson, Dan K; Hoyt, Jeffrey C; Freels, Jon L; Habib, Michael P; Robbins, Richard A

    2003-05-01

    Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha(1)-antitrypsin, or N(alpha)-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release. PMID:12676771

  17. Crystal Structure of a Novel Viral Protease with a Serine/Lysine Catalytic Dyad Mechanism

    SciTech Connect

    Feldman,A.; Lee, J.; Delmas, B.; Paetzel, M.

    2006-01-01

    The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.

  18. Serine Hydroxymethyltransferase 1 and 2: Gene Sequence Variation and Functional Genomic Characterization

    PubMed Central

    Hebbring, Scott J.; Chai, Yubo; Ji, Yuan; Abo, Ryan P.; Jenkins, Gregory D.; Fridley, Brooke; Zhang, Jianping; Eckloff, Bruce W.; Wieben, Eric D.; Weinshilboum, Richard M.

    2012-01-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the transfer of a beta carbon from serine to tetrahydrofolate (THF) to form glycine and 5,10-methylene-THF. This reaction plays an important role in neurotransmitter synthesis and metabolism. We set out to resequence SHMT1 and SHMT2, followed by functional genomic studies. We identified 87 and 60 polymorphisms in SHMT1 and SHMT2, respectively. We observed no significant functional effect of the 13 nonsynonymous SNPs in these genes, either on catalytic activity or protein quantity. We imputed additional variants across the two genes using “1000 Genomes” data, and identified 14 variants that were significantly associated (p-value < 1.0E-10) with SHMT1 mRNA expression in lymphoblastoid cell lines. Many of these SNPs were also significantly correlated with basal SHMT1 protein expression in 268 human liver biopsy samples. Reporter gene assays suggested that the SHMT1 promoter SNP, rs669340, contributed to this variation. Finally, SHMT1 and SHMT2 expression were significantly correlated with those of other Folate and Methionine Cycle genes at both the mRNA and protein levels. These experiments represent a comprehensive study of SHMT1 and SHMT2 gene sequence variation and its functional implications. In addition, we obtained preliminary indications that these genes may be co-regulated with other Folate and Methionine Cycle genes. PMID:22220685

  19. A Blood Fluke Serine Protease Inhibitor Regulates an Endogenous Larval Elastase*

    PubMed Central

    Quezada, Landys A. Lopez; Sajid, Mohammed; Lim, Kee C.; McKerrow, James H.

    2012-01-01

    The larvae of Schistosoma mansoni invade their mammalian host by utilizing a serine protease, cercarial elastase (SmCE), to degrade macromolecular proteins in host skin. The catalytic activity of serine and cysteine proteases can be regulated after activation by serpins. SmSrpQ, one of two S. mansoni serpins found in larval secretions, is only expressed during larval development and in the early stages of mammalian infection. In vitro, 35S-SmSrpQ was able to form an SDS-stable complex with a component of the larval lysate, but no complex was detected when 35S-SmSrpQ was incubated with several mammalian host proteases. Formation of a complex was sensitive to the protease active site inhibitors PMSF, Z-AAPF-CMK, and Z-AAPL-CMK. Western blot analysis of parasite lysates from different life stages detected a complex of comparable size to SmCE bound to SmSrpQ using anti-SmSrpQ or anti-SmCE antibodies. SmSrpQ and SmCE are located in adjacent but discrete compartments in the secretion glands of the parasite. Fluorescence immunohistochemical analysis of simulated infection showed co-localization of SmCE and SmSrpQ in host tissue suggesting a post release regulation of parasite protease activity during skin transversal. The results of this study suggest that cercarial elastase degradation of skin tissue is carefully regulated by SmSrpQ. PMID:22174417

  20. Interaction of Protein C Inhibitor with the Type II Transmembrane Serine Protease Enteropeptidase

    PubMed Central

    Furtmüller, Margareta; Geiger, Margarethe

    2012-01-01

    The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48×104 M?1 s?1. Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation. PMID:22723979

  1. Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

    PubMed

    Prohaska, Thomas A; Wahlmüller, Felix C; Furtmüller, Margareta; Geiger, Margarethe

    2012-01-01

    The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4) M(-1) s(-1). Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation. PMID:22723979

  2. Neutral Sphingomyelinase 2 Activity and Protein Stability Are Modulated by Phosphorylation of Five Conserved Serines*

    PubMed Central

    Filosto, Simone; Ashfaq, Majid; Chung, Samuel; Fry, William; Goldkorn, Tzipora

    2012-01-01

    We previously presented that the neutral sphingomyelinase 2 (nSMase2) is the only SMase activated in human airway epithelial (HAE) cells following exposure to oxidative stress (ox-stress), yielding ceramide accumulation and thereby inducing apoptosis. Furthermore, we reported that nSMase2 is a phospho-protein in which the level of phosphorylation controls nSMase2 activation induced by ox-stress. Here we identify five specific serines that are phosphorylated in nSMase2 and demonstrate that their phosphorylation controls the nSMase2 activity upon ox-stress exposure in an interdependent manner. Furthermore, we show that the nSMase2 protein stability and thus its level of expression is also post-translationally regulated by these five serine phosphorylation sites. This study provides initial structure/function insights regarding nSMase2 phosphorylation sites and offers some new links for future studies aiming to fully elucidate nSMase2 regulatory machinery. PMID:22074919

  3. A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase.

    PubMed

    Acharya, J K; Rao, N A

    1992-09-25

    An unusual intermediate bound to the enzyme was detected in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. This intermediate had absorbance maxima at 464 and 440 nm. Such spectra are characteristic of resonance stabilized intermediates detected in the interaction of substrates and quasi-substrates with pyridoxal phosphate enzymes. An intermediate of this kind has not been detected in the interaction of thiosemicarbazide with other pyridoxal phosphate enzymes. This intermediate was generated slowly (t 1/2 = 4 min) following the addition of thiosemicarbazide (200 microM) to sheep liver serine hydroxymethyltransferase (5 microM). It was bound to the enzyme as evidenced by circular dichroic bands at 464 and 440 nm and the inability to be removed upon Centricon filtration. The kinetics of interaction revealed that thiosemicarbazide was a slow binding reversible inhibitor in this phase with a k(on) of 11 M-1 s-1 and a k(off) of 5 x 10(-4) s-1. The intermediate was converted very slowly (k = 4 x 10(-5) s-1) to the final products, namely the apoenzyme and the thiosemicarbazone of pyridoxal phosphate. A minimal kinetic mechanism involving the initial conversion to the intermediate absorbing at longer wavelengths and the conversion of this intermediate to the final product, as well as, the formation of pyridoxal phosphate-thiosemicarbazone directly by an alternate pathway is proposed. PMID:1527031

  4. Characterization of a Novel Serine Protease Inhibitor Gene from a Marine Metagenome

    PubMed Central

    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 ?-chymotrypsin and trypsin with Ki 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. PMID:22131953

  5. Portulaca oleracea L. as a Prospective Candidate Inhibitor of Hepatitis C Virus NS3 Serine Protease.

    PubMed

    Noreen, Sobia; Hussain, Ishtiaq; Tariq, Muhammad Ilyas; Ijaz, Bushra; Iqbal, Shahid; Qamar-Ul-Zaman; Ashfaq, Usman Ali; Husnain, Tayyab

    2015-06-01

    Hepatitis C virus (HCV) infection is a worldwide health problem affecting about 300 million individuals. HCV causes chronic liver disease, liver cirrhosis, hepatocellular carcinoma, and death. Many side effects are associated with the current treatment options. Natural products that can be used as anti-HCV drugs are thus of considerable potential significance. NS3 serine protease (NS3-SP) is a target for the screening of antiviral activity against HCV. The present work explores plants with anti-HCV potential, isolating possible lead compounds. Ten plants, used for medicinal purposes against different infections in rural areas of Pakistan, were collected. The cellular toxicity effects of methanolic extracts of the plants on the viability of Huh-7 cells were studied through the Trypan blue dye exclusion method. Following this, the anti-HCV potential of phytoextracts was assessed by infecting liver cells with HCV-3a-infected serum inoculum. Only the methanolic extract of Portulaca oleracea L. (PO) exhibited more than 70% inhibition. Four fractions were obtained through bioassay-guided extraction of PO. Subsequent inhibition of all organic extract fractions against NS3 serine protease was checked to track the specific target in the virus. The results showed that the PO methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. Hence, PO extract and its constituents either alone or with interferon could offer a future option to treat chronic HCV. PMID:25871297

  6. Realizing Serine/Threonine Ligation: Scope and Limitations and Mechanistic Implication Thereof

    NASA Astrophysics Data System (ADS)

    Wong, Clarence; Li, Tianlu; Lam, Hiu Yung; Zhang, Yinfeng; LI, Xuechen

    2014-05-01

    Serine/Threonine ligation (STL) has emerged as an alternative tool for protein chemical synthesis, bioconjugations as well as macrocyclization of peptides of various sizes. Owning to the high abundance of Ser/Thr residues in natural peptides and proteins, STL is expected to find a wide range of applications in chemical biology research. Herein, we have fully investigated the compatibility of the serine/threonine ligation strategy for X-Ser/Thr ligation sites, where X is any of the 20 naturally occurring amino acids. Our studies have shown that 17 amino acids are suitable for ligation, while Asp, Glu, and Lys are not compatible. Among the working 17 C-terminal amino acids, the retarded reaction resulted from the bulky ?-branched amino acid (Thr, Val and Ile) is not seen under the current ligation condition. We have also investigated the chemoselectivity involving the amino group of the internal lysine which may compete with the N-terminal Ser/Thr for reaction with the C-terminal salicylaldehyde (SAL) ester aldehyde group. The result suggested that the free internal amino group does not adversely slow down the ligation rate.

  7. Amino acid-based zwitterionic poly(serine methacrylate) as an antifouling material.

    PubMed

    Liu, Qingsheng; Singh, Anuradha; Liu, Lingyun

    2013-01-14

    A serine-based zwitterionic poly(serine methacrylate) (pSerMA) was developed in this work to be used as a potential antifouling material. A surface-initiated photoiniferter-mediated polymerization (SI-PIMP) method was used to graft polymer brushes on gold surfaces. The pSerMA-grafted samples with different polymer film thicknesses were readily prepared by varying the UV-irradiation time. With the optimal film thickness, the adsorptions from bovine serum albumin, human serum, and human plasma onto the pSerMA-grafted surfaces, as evaluated by a surface plasmon resonance (SPR) biosensor, were 1.8, 9.2, and 12.9 ng/cm(2), respectively, comparable to the traditional antifouling material such as poly(ethylene glycol). The pSerMA-grafted surfaces also strongly resisted adhesion from bovine aortic endothelial cells. This is the first work to develop an amino acid-based zwitterionic polymer as an antifouling material, demonstrating that pSerMA is a promising alternative to the traditional ethylene glycol-based antifouling materials. PMID:23228182

  8. New experimental evidence for in-chain amino acid racemization of serine in a model peptide.

    PubMed

    Demarchi, Beatrice; Collins, Matthew; Bergström, Ed; Dowle, Adam; Penkman, Kirsty; Thomas-Oates, Jane; Wilson, Julie

    2013-06-18

    The facile racemization of protein-bound amino acids plays an important role in the aging and pathologies of living tissues, and it can be exploited for protein geochronological studies in subfossil biominerals. However, the in-chain degradation pathways of amino acids are complex and difficult to elucidate. Serine has proven to be particularly elusive, and its ability to racemize as a peptide-bound residue (like asparagine and aspartic acid) has not been demonstrated. This study investigates the patterns of degradation of a model peptide (WNSVWAW) at elevated temperatures, quantifying the extent of racemization and peptide bond hydrolysis using reverse-phase high-performance liquid chromatography (RP-HPLC) and tracking the presence of degradation products by MALDI-MS. We provide direct evidence that, under these experimental conditions, both serine and asparagine are able to undergo racemization as internally bound residues, which shows their potential for initiating protein breakdown and provides an explanation for the presence of d-enantiomers in living mammalian tissues. PMID:23705982

  9. Abrogation of IFN-? mediated epithelial barrier disruption by serine protease inhibition

    PubMed Central

    Willemsen, LEM; Hoetjes, JP; Van Deventer, SJH; Van Tol, EAF

    2005-01-01

    The intestinal barrier function is often impaired in a variety of diseases including chronic inflammatory bowel disease. Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex. IFN-? has been widely studied for its effect on barrier function and tight junction structures but its mode of action remains unclear. Since the claudin family of tight junction proteins is proposed to be involved in barrier maintenance we studied the effect of IFN-? on claudin expression in relation to epithelial barrier function. Cycloheximide and protease inhibitors were used to study mechanisms of IFN-? mediated barrier disruption. Intestinal epithelial cells were exposed to IFN-? and permeability was evaluated by horse radish peroxidase (HRP) and 4 kD FITC-dextran fluxes. Occludin and claudin-1, -2, -3, and -4 tight junction protein expression was determined by Western blotting. Occludin and claudin-2 protein expression was dramatically reduced after IFN-? exposure, which correlated with increased permeability for HRP and FITC-dextran. Interestingly, cleavage of claudin-2 was observed after incubation with IFN-?. Serine protease inhibitor AEBSF completely abrogated IFN-? mediated barrier disruption which was associated with preservation of claudin-2 expression. Moreover, IFN-? induced loss of barrier integrity was found to affect claudin-2 and occludin expression through different mechanisms. Since inhibition of serine protease activity abrogates IFN-? mediated barrier disruption this may be an important target for therapeutic intervention. PMID:16232214

  10. Staphylococcal serine proteinase increases intracellular free calcium concentration in human polymorphonuclear leukocytes.

    PubMed

    Nowak, D; Krol, M; Miedzobrodzki, J; Bialasiewicz, P

    1997-10-01

    Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 micrograms/ml. The highest Ca2+ rise (104 +/- 47 nM) was observed for 10 micrograms/ml SSP. It was mainly dependent (81 +/- 11%) on extracellular calcium influx, however, SSP mobilized 68 +/- 7% of Ca2+ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca2+ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca2+ concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways. PMID:9403110

  11. Memory impairment in rats by hippocampal administration of the serine protease subtilisin.

    PubMed

    Kornisiuk, Edgar; Snitcofsky, Marina; Blanco, Carlos; Harvey, Alan L; Stone, Trevor W; Jerusalinsky, Diana

    2011-05-16

    Since the serine protease subtilisin has been reported to generate a novel form of long-term depression (LTD) in rat hippocampal slices, the present work was designed to determine whether it has any effect on learning and memory processes. Rats were used to examine the effects of subtilisin, injected directly into the dorsal hippocampus, on task performance in a step-through inhibitory avoidance of a mild footshock. The administration of 100 ng of subtilisin into each hippocampus, immediately after training, was sufficient to induce a detectable learning deficit with a footshock stimulus of 0.5 mA. Higher doses produced dose-related impairments in memory consolidation. These effects were not the result of irreversible toxicity, since rats trained with a higher amplitude footshock (0.75 mA) were able to perform as control animals; therefore, the amnesic effect was not further evident. Furthermore, the administration of subtilisin before avoidance training did not produce any detectable effect on performance during the training or test sessions, indicating that neither acquisition nor consolidation was affected. It is concluded that the post-training administration of a serine protease inhibitor is able to produce robust deficits of memory consolidation consistent with its ability to generate LTD, raising the possibility that related molecules could play physiological or pathological roles in the modulation of learning and memory. PMID:21185873

  12. The serine protease subtilisin suppresses epileptiform activity in rat hippocampal slices and neocortex in vivo.

    PubMed

    Addae, J I; Stone, T W

    2011-12-29

    Serine proteases of the S8A family and those belonging to the subtilase group generate a long-lasting inhibition of hippocampal evoked potentials, which shows little recovery and resembles long-term depression. The present work investigates the effects of subtilisin A on epileptiform activity induced in hippocampal slices. Interictal bursts were generated by perfusion with 4-aminopyridine in magnesium-free medium, whereas ictal bursts were produced by the addition of baclofen. Subtilisin A superfused for 10 min at concentrations of 50 nM and above reduced the duration of ictal bursts, whereas higher concentrations reduced the frequency of interictal activity with little or no recovery, indicating similarity with the long-term depression reported previously. The anti-epileptiform activity was not prevented by inhibitors of phosphatases or several kinases, but the inhibition of ictal activity was selectively reduced by the tyrosine kinase inhibitor genistein. The rho-activated coiled-coil kinase (ROCK) inhibitor Y-27632 had no effect on the suppression of ictal or interictal bursts. Subtilisin applied at nanomolar concentrations to the surface of the cerebral cortex in vivo also suppressed epileptiform spikes induced by bicuculline. It is concluded that serine proteases of the subtilase group are highly potent inhibitors of epileptiform activity, especially ictal bursts, and that tyrosine kinases may be involved in that inhibition. The mechanism of inhibition is different from the long-lasting depression of evoked potentials, which is partly mediated via ROCK. PMID:22033457

  13. Neu-Laxova Syndrome, an Inborn Error of Serine Metabolism, Is Caused by Mutations in PHGDH

    PubMed Central

    Shaheen, Ranad; Rahbeeni, Zuhair; Alhashem, Amal; Faqeih, Eissa; Zhao, Qi; Xiong, Yong; Almoisheer, Agaadir; Al-Qattan, Sarah M.; Almadani, Halima A.; Al-Onazi, Noufa; Al-Baqawi, Badi S.; Saleh, Mohammad Ali; Alkuraya, Fowzan S.

    2014-01-01

    Neu-Laxova syndrome (NLS) is a rare autosomal-recessive disorder characterized by severe fetal growth restriction, microcephaly, a distinct facial appearance, ichthyosis, skeletal anomalies, and perinatal lethality. The pathogenesis of NLS remains unclear despite extensive clinical and pathological phenotyping of the >70 affected individuals reported to date, emphasizing the need to identify the underlying genetic etiology, which remains unknown. In order to identify the cause of NLS, we conducted a positional-mapping study combining autozygosity mapping and whole-exome sequencing in three consanguineous families affected by NLS. Surprisingly, the NLS-associated locus identified in this study was solved at the gene level to reveal mutations in PHGDH, which is known to be mutated in individuals with microcephaly and developmental delay. PHGDH encodes the first enzyme in the phosphorylated pathway of de novo serine synthesis, and complete deficiency of its mouse ortholog recapitulates many of the key features of NLS. This study shows that NLS represents the extreme end of a known inborn error of serine metabolism and highlights the power of genomic sequencing in revealing the unsuspected allelic nature of apparently distinct clinical entities. PMID:24836451

  14. Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin)

    PubMed Central

    Zhao, Yanmei; Sun, Wei; Zhang, Pan; Chi, Hao; Zhang, Mei-Jun; Song, Chun-Qing; Ma, Xuan; Shang, Yunlong; Wang, Bin; Hu, Youqiao; Hao, Zhiqi; Hühmer, Andreas F.; Meng, Fanxia; L'Hernault, Steven W.; He, Si-Min; Dong, Meng-Qiu; Miao, Long

    2012-01-01

    Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated. PMID:22307610

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

    PubMed

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

    2014-06-26

    Production of ?-lactamases (BLs) is the most widespread resistance mechanism adopted by bacteria to fight ?-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-?-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

  16. Characterization of a Family of Novel Cysteine- Serine-Rich Nuclear Proteins (CSRNP)

    PubMed Central

    Gingras, Sébastien; Pelletier, Stéphane; Boyd, Kelli; Ihle, James N.

    2007-01-01

    Gene array analysis has been widely used to identify genes induced during T cell activation. Our studies identified an immediate early gene that is strongly induced in response to IL-2 in mouse T cells which we named cysteine- serine-rich nuclear protein-1 (CSRNP-1). The human ortholog was previously identified as an AXIN1 induced gene (AXUD1). The protein does not contain sequence defined domains or motifs annotated in public databases, however the gene is a member of a family of three mammalian genes that share conserved regions, including cysteine- and serine-rich regions and a basic domain, they encode nuclear proteins, possess transcriptional activation domain and bind the sequence AGAGTG. Consequently we propose the nomenclature of CSRNP-1, -2 and -3 for the family. To elucidate the physiological functions of CSRNP-1, -2 and -3, we generated mice deficient for each of these genes by homologous recombination in embryonic stem cells. Although the CSRNP proteins have the hallmark of transcription factors and CSRNP-1 expression is highly induced by IL-2, deletion of the individual genes had no obvious consequences on normal mouse development, hematopoiesis or T cell functions. However, combined deficiencies cause partial neonatal lethality suggesting that the genes have redundant functions. PMID:17726538

  17. Coagulation, an ancestral serine protease cascade, exerts a novel function in early immune defense.

    PubMed

    Loof, Torsten G; Mörgelin, Matthias; Johansson, Linda; Oehmcke, Sonja; Olin, Anders I; Dickneite, Gerhard; Norrby-Teglund, Anna; Theopold, Ulrich; Herwald, Heiko

    2011-09-01

    Phylogenetically conserved serine protease cascades play an important role in invertebrate and vertebrate immunity. The mammalian coagulation system can be traced back some 400 million years and shares homology with ancestral serine proteinase cascades that are involved in, for example, Toll receptor signaling in insects and release of antimicrobial peptides during hemolymph clotting. In the present study, we show that the induction of coagulation by bacteria leads to immobilization and killing of Streptococcus pyogenes bacteria inside the clot. The entrapment is mediated via cross-linking of bacteria to fibrin fibers by the action of coagulation factor XIII (fXIII), an evolutionarily conserved transglutaminase. In a streptococcal skin infection model, fXIII(-/-) mice developed severe signs of pathologic inflammation at the local site of infection, and fXIII treatment of wild-type animals dampened bacterial dissemination during early infection. Bacterial killing and cross-linking to fibrin networks was also detected in tissue biopsies from patients with streptococcal necrotizing fasciitis, supporting the concept that coagulation is part of the early innate immune system. PMID:21613262

  18. The importance of serine 776 in Ataxin-1 partner selection: a FRET analysis.

    PubMed

    Menon, Rajesh P; Soong, Daniel; de Chiara, Cesira; Holt, Mark R; Anilkumar, Narayana; Pastore, Annalisa

    2012-01-01

    Anomalous expansion of a polymorphic tract in Ataxin-1 causes the autosomal dominant spinocerebellar ataxia type 1. In addition to polyglutamine expansion, requirements for development of pathology are phosphorylation of serine 776 in Ataxin-1 and nuclear localization of the protein. The phosphorylation state of serine 776 is also crucial for selection of the Ataxin-1 multiple partners. Here, we have used FRET for an in cell study of the interaction of Ataxin-1 with the spliceosome-associated U2AF65 and the adaptor 14-3-3 proteins. Using wild-type Ataxin-1 and Ser776 mutants to a phosphomimetic aspartate and to alanine, we show that U2AF65 binds Ataxin-1 in a Ser776 phosphorylation independent manner whereas 14-3-3 interacts with phosphorylated wild-type Ataxin-1 but not with the mutants. These results indicate that Ser776 acts as the molecular switch that discriminates between normal and aberrant function and that phosphomimetics is not a generally valid approach whose applicability should be carefully validated. PMID:23213356

  19. The importance of serine 776 in Ataxin-1 partner selection: A FRET Analysis

    PubMed Central

    Menon, Rajesh P.; Soong, Daniel; de Chiara, Cesira; Holt, Mark R.; Anilkumar, Narayana; Pastore, Annalisa

    2012-01-01

    Anomalous expansion of a polymorphic tract in Ataxin-1 causes the autosomal dominant spinocerebellar ataxia type 1. In addition to polyglutamine expansion, requirements for development of pathology are phosphorylation of serine 776 in Ataxin-1 and nuclear localization of the protein. The phosphorylation state of serine 776 is also crucial for selection of the Ataxin-1 multiple partners. Here, we have used FRET for an in cell study of the interaction of Ataxin-1 with the spliceosome-associated U2AF65 and the adaptor 14-3-3 proteins. Using wild-type Ataxin-1 and Ser776 mutants to a phosphomimetic aspartate and to alanine, we show that U2AF65 binds Ataxin-1 in a Ser776 phosphorylation independent manner whereas 14-3-3 interacts with phosphorylated wild-type Ataxin-1 but not with the mutants. These results indicate that Ser776 acts as the molecular switch that discriminates between normal and aberrant function and that phosphomimetics is not a generally valid approach whose applicability should be carefully validated. PMID:23213356

  20. One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity

    Microsoft Academic Search

    Patrick Vincent; Lilly Maneta-Peyret; Bénédicte Sturbois-Balcerzak; Michel Duvert; Claude Cassagne; Patrick Moreau

    1999-01-01

    In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS.

  1. Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production.

    PubMed

    Nistal-Villán, Estanislao; Gack, Michaela U; Martínez-Delgado, Gustavo; Maharaj, Natalya P; Inn, Kyung-Soo; Yang, Heyi; Wang, Rong; Aggarwal, Aneel K; Jung, Jae U; García-Sastre, Adolfo

    2010-06-25

    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. PMID:20406818

  2. Pharmacological PPAR? Activation Markedly Alters Plasma Turnover of the Amino Acids Glycine, Serine and Arginine in the Rat

    PubMed Central

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

    2014-01-01

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

  3. Pharmacological PPAR? activation markedly alters plasma turnover of the amino acids glycine, serine and arginine in the rat.

    PubMed

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

    2014-01-01

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

  4. Differential expression of GSK3? and pS9GSK3? in normal human tissues: can pS9GSK3? be an epithelial marker?

    PubMed Central

    Lee, Hojung; Ro, Jae Y

    2015-01-01

    Glycogen synthase kinase 3? (GSK3?) and phosphorylated GSK3? at Ser9 (pS9GSK3?) are crucial in cellular proliferation and metabolism. GSK3? and pS9GSK3? are deregulated in many diseases including tumors. Data on altered expression of GSK3? and pS9GSK3? are mainly limited to tumor tissues, thus the expression of GSK3? and pS9GSK3? in normal human tissue has been largely unknown. Thus, we examined the immunohistochemical localization of GSK3? and pS9GSK3? in human fetal and adult tissues, and also compared the expression pattern of GSK3? and pS9GSK3? with that of the CK7 and CK20. We found GSK3? expression in neurons of brain, myenteric plexus in gastrointestinal tract, squamous epithelium of skin, and mammary gland. The expression of pS9GSK3? was restricted to the epithelial cells of breast and pancreaticobiliary duct, distal nephron of kidney, gastrointestinal tract, fallopian tube, epididymis, secretory cell of prostatic gland, and umbrella cell of urinary tract. The staining pattern of pS9GSK3? and CK7 was overlapped in most organs except for gastrointestinal tract where CK7 was negative and CK20 was positive. Our results show that the expression of GSK3? may be associated with differentiation of ectodermal derived tissues and pS9GSK3? with that of epithelial cells of endodermal derived tissues in human. In addition, the expression of pS9GSK3? in the selective epithelial cells may indicate its association with secretory or barrier function of specific cells and may serve as another immunohistochemical marker for epithelial cells. PMID:26097594

  5. Serine transhydroxymethylase: a simplified radioactive assay; purification and stabilization of enzyme activity employing Affi-Gel Blue

    SciTech Connect

    Braman, J.C.; Black, M.J.; Mangum, J.H.

    1981-01-01

    An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the (14C) HCHO which is generated when (3- 14C)-serine is employed as the substrate. The new assay eliminated the need for a solvent extraction of a (14C) HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach. The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4 degrees indefinitely.

  6. Serine transhydroxymethylase: a simplified radioactive assay; purification and stabilization of enzyme activity employing Affi-Gel Blue

    SciTech Connect

    Braman, J.C.; Black, M.J.; Mangum, J.H.

    1981-01-01

    An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the (/sub 14/C)HCHO which is generated when (3- /sub 14/C)-serine is employed as the substrate. The new assay eliminates the need for a solvent extraction of a (/sub 14/C)HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach. The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4 degrees indefinitely.

  7. Create and Publish a Hierarchical Progressive Survey (HiPS)

    NASA Astrophysics Data System (ADS)

    Fernique, P.; Boch, T.; Pineau, F.; Oberto, A.

    2014-05-01

    Since 2009, the CDS promotes a method for visualizing based on the HEALPix sky tessellation. This method, called “Hierarchical Progressive Survey" or HiPS, allows one to display a survey progressively. It is particularly suited for all-sky surveys or deep fields. This visualization method is now integrated in several applications, notably Aladin, the SiTools/MIZAR CNES framework, and the recent HTML5 “Aladin Lite". Also, more than one hundred surveys are already available in this view mode. In this article, we will present the progress concerning this method and its recent adaptation to the astronomical catalogs such as the GAIA simulation.

  8. Temperature Dependence of Polymer Diffusion in MWCNT/PS Nanocomposites

    NASA Astrophysics Data System (ADS)

    Tung, Wei-Shao; Clarke, Nigel; Composto, Russell J.; Winey, Karen I.

    2012-02-01

    Temperature dependence of homopolymer diffusion can be explained by the WLF equation. Here, we explore whether the WLF equation applies to polymer diffusion in nanocomposites. Previously, we found the diffusion coefficient shows a minimum with increasing MWCNT concentration. By studying the temperature dependence of polymer diffusion in this system, we will investigate the relative importance of entropic barriers or enthalpy interactions between polymer chain and fillers. Our composites contain MWCNT and polystyrene and are fabricated by a coagulation method. Using forward recoil elastic scattering (FRES), we probe the depth profile of tracer polymer (dPS) and obtain the diffusion coefficients by fitting the profile with Fick's second law.

  9. ARABIDOPSIS PLANTS WITH AN INDUCED RNAI HAIRPIN OR A CONSTITUTIVELY EXPRESSED DOMINANT-NEGATIVE ALLELE OF THE SERINE PALMITOYLTRANSFERASE LCB2 GENE HAVE ALTERED SPHINGOLIPID CONTENT AND DEVELOPMENT

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The first step in sphingolipid biosynthesis is the condensation of palmitoyl-CoA and serine to form 3-ketosphinganine. This reaction is catalyzed by serine palmitoyltransferase (SPT), a pyridoxal 5’ phosphate-dependent enzyme. In yeast, SPT is a heterodimer that consists of the polypeptides LCB1 and...

  10. Protein Engineering vol.1 no.4 pp.313--318, 1987 Comparison of the solution and X-ray structures of barley serine

    E-print Network

    Clore, G. Marius

    of barley serine proteinase inhibitor 2 G.Marius Gore3 , Angela M.Gronenborn, Michael N.G.James2 , Mogens 'Authors to whom reprint requests should be sent A comparison of the solution n.m.r. structures of barley for the restrained energy minimized mean dynamics structure are 1.5 and 2.4 A, respectively Key words: barley serine

  11. Serine hydrolase organophosphate interactions: Molecular modeling. Final report, 15 June 1991-31 December 1994

    SciTech Connect

    Kovach, I.M.

    1995-02-03

    The aging reaction from the adducts of electric eel (EE) and fetal bovine serum (FBS) acetyicholinesterase (AChE) with soman P(-)C(-) and P(-)C(+) show a bell-shaped pH-rate profile. The aging of soman and sarin are specific acid catalyzed and show small (1.1 - 1.6) solvent isotope effects. These results and molecular mechanics calculations support the push-pull mechanism by Glu199 and the H-bonding array of the oxyanion hole in AChE. Semi-empirical calculations for ten phosphonate derivatives gave optimal results with the MNDO Hamiltonian. MNDO was also used for the generation of optimal - geometries and charges for phosphonate esters of Ser- 195 fragments from AChE. Full refinements were performed using program YETI with the fully solvated native Torpedo califomica - AChE, trypsin and chymotrypsm and their adducts with monoisopropyl and diisopropyl phosphate (trypsin only) diastereomers of 2-propyl methylphosphonate, pinacolyl methylphosphonate and their dealkylation products. The P(R) adducts of soman-inhibited AChE are 17-26 kcal/mol less stable than the Ps adduct. The P(R) 2-propyl methyiphosphonyl adduct of trypsin is less stable than the Ps isomer by 2 kcallmol and the product of dealkylation of the Ps is more stable by 3.7 kcal/mol.

  12. Role of Serine Racemase in Behavioral Sensitization in Mice after Repeated Administration of Methamphetamine

    PubMed Central

    Horio, Mao; Kohno, Mami; Fujita, Yuko; Ishima, Tamaki; Inoue, Ran; Mori, Hisashi; Hashimoto, Kenji

    2012-01-01

    Background The N-methyl-D-aspartate (NMDA) receptors play a role in behavioral abnormalities observed after administration of the psychostimulant, methamphetamine (METH). Serine racemase (SRR) is an enzyme which synthesizes D-serine, an endogenous co-agonist of NMDA receptors. Using Srr knock-out (KO) mice, we investigated the role of SRR on METH-induced behavioral abnormalities in mice. Methodology/Principal Findings Evaluations of behavior in acute hyperlocomotion, behavioral sensitization, and conditioned place preference (CPP) were performed. The role of SRR on the release of dopamine (DA) in the nucleus accumbens after administration of METH was examined using in vivo microdialysis technique. Additionally, phosphorylation levels of ERK1/2 proteins in the striatum, frontal cortex and hippocampus were examined using Western blot analysis. Acute hyperlocomotion after a single administration of METH (3 mg/kg) was comparable between wild-type (WT) and Srr-KO mice. However, repeated administration of METH (3 mg/kg/day, once daily for 5 days) resulted in behavioral sensitization in WT, but not Srr-KO mice. Pretreatment with D-serine (900 mg/kg, 30 min prior to each METH treatment) did not affect the development of behavioral sensitization after repeated METH administration. In the CPP paradigm, METH-induced rewarding effects were demonstrable in both WT and Srr-KO mice. In vivo microdialysis study showed that METH (1 mg/kg)-induced DA release in the nucleus accumbens of Srr-KO mice previously treated with METH was significantly lower than that of the WT mice previously treated with METH. Interestingly, a single administration of METH (3 mg/kg) significantly increased the phosphorylation status of ERK1/2 in the striatum of WT, but not Srr-KO mice. Conclusions/Significance These findings suggest first, that SRR plays a role in the development of behavioral sensitization in mice after repeated administration of METH, and second that phosphorylation of ERK1/2 by METH may contribute to the development of this sensitization as seen in WT but not Srr-KO mice. PMID:22530033

  13. Study of the crystalline structures of the syndiotactic polystyrene (sPS) under mechanical deformation

    NASA Astrophysics Data System (ADS)

    Nagasaka, Suguru; Hotta, Atsushi

    2008-03-01

    Polystyrene (PS) has become one of the important, yet complicated semi-crystalline materials since the successful synthesis of the syndiotactic PS (sPS) by Ziegler-Natta catalyst. Since then, sPS has been actively investigated and four different crystalline forms (?, ?, ? and ?), two mesomorphic forms and various clathrate forms have been found, indicating complex feature of its crystalline structures. Among the four crystalline forms, ? and ?-crystals can be obtained by different annealing processes and both crystalline structures comprise the same all-trans planar conformation. In this work, ?-crystal structure was made by high temperature annealing and the sPS with ?-crystal structure was mechanically stretched above the glass transition temperature of sPS, followed by the crystalline transition analysis studied by FT-IR.

  14. Internal structure and positron annihilation in the four-body MuPs system

    E-print Network

    Alexei M. Frolov

    2015-01-07

    A large number of bound state properties of the four-body muonium-positronium system MuPs (or $\\mu^{+} e^{-}_2 e^{+}$) are determined to high accuracy. Based on these expectation values we predict that the weakly-bound four-body MuPs system has the `two-body' cluster structure Mu + Ps. The two neutral clusters Mu ($\\mu^{+} e^{-}$) and Ps ($e^{+} e^{-}$) interact with each other by the attractive van der Waals forces. By using our expectation values of the electron-positron delta-functions we evaluated the half-life $\\tau_a$ of the MuPs system against annihilation of the electron-positron pair: $\\tau_a = \\frac{1}{\\Gamma} \\approx 4.071509 \\cdot 10^{-10}$ $sec$. The hyperfine structure splitting of the ground state in the MuPs system evaluated with our expectation values is $\\Delta \\approx$ 23.064(5) $MHz$.

  15. STS-52 PS MacLean, backup PS Tryggvason, and PI pose on JSC's CCT flight deck

    NASA Technical Reports Server (NTRS)

    1992-01-01

    STS-52 Columbia, Orbiter Vehicle (OV) 102, Canadian Payload Specialist (PS) Steven G. MacLean (left) and backup Payload Specialist Bjarni V. Tryggvason (right) take a break from a camera training session in JSC's Crew Compartment Trainer (CCT). The two Canadian Space Agency (CSA) representatives pose on the CCT's aft flight deck with Canadian scientist David Zimick, the principal investigator (PI) for the materials experiment in low earth orbit (MELEO). MELEO is a component of the CANEX-2 experiment package, manifest to fly on the scheduled October 1992 STS-52 mission. The CCT is part of the shuttle Mockup and Integration Laboratory (MAIL) Bldg 9NE.

  16. Properties of Extruded PS-212 Type Self-Lubricating Materials

    NASA Technical Reports Server (NTRS)

    Waters, W. J.; Sliney, H. E.; Soltis, R. F.

    1993-01-01

    Research has been underway at the NASA Lewis Research Center since the 1960's to develop high temperature, self-lubricating materials. The bulk of the research has been done in-house by a team of researchers from the Materials Division. A series of self-lubricating solid material systems has been developed over the years. One of the most promising is the composite material system referred to as PS-212 or PM-212. This material is a powder metallurgy product composed of metal bonded chromium carbide and two solid lubricating materials known to be self-lubricating over a wide temperature range. NASA feels this material has a wide potential in industrial applications. Simplified processing of this material would enhance its commercial potential. Processing changes have the potential to reduce processing costs, but tribological and physical properties must not be adversely affected. Extrusion processing has been employed in this investigation as a consolidation process for PM-212/PS-212. It has been successful in that high density bars of EX-212 (extruded PM-212) can readily be fabricated. Friction and strength data indicate these properties have been maintained or improved over the P.M. version. A range of extrusion temperatures have been investigated and tensile, friction, wear, and microstructural data have been obtained. Results indicate extrusion temperatures are not critical from a densification standpoint, but other properties are temperature dependent.

  17. A novel strategy to derive iPS cells from porcine fibroblasts

    Microsoft Academic Search

    WeiMin Ruan; JianYong Han; Pin Li; SuYing Cao; Yang An; Bing Lim; Ning Li

    2011-01-01

    Induced pluripotent stem (iPS) cell technology demonstrates that somatic cells can be reprogrammed to a pluripotent state\\u000a by over-expressing four reprogramming factors. This technology has created an interest in deriving iPS cells from domesticated\\u000a animals such as pigs, sheep and cattle. Moloney murine leukemia retrovirus vectors have been widely used to generate and study\\u000a mouse iPS cells. However, this retrovirus

  18. Role of serine biosynthesis and its utilization in the alternative pathway from glucose to glycogen during the response to insulin in cultured foetal-rat hepatocytes.

    PubMed Central

    Bismut, H; Plas, C

    1991-01-01

    The role of serine as a possible intermediate of the alternative pathway from glucose to glycogen was investigated under basal and insulin-stimulated conditions in 18-day cultured foetal-rat hepatocytes because these cells cannot use pyruvate-derived metabolites [Bismut & Plas (1989) Biochem. J. 263, 889-895]. Incubation of cells with [U-14C]glucose for 24 h led to a release of labelled serine in the medium concomitantly with a net serine production (100 nmol/24 h per culture). The rate of [14C]serine formation (close to 3 nmol/h per culture) indicated that a large part of newly formed serine originated from glucose. When short-term experiments were performed at day 2, glycogen labelling from [U-14C]serine or [U-14C]glycine, which was increased 3-fold by insulin after 2 h, evidenced their participation as glycogenic precursors. When a double-isotope procedure with [U-14C,3-3H]glucose was used, the direct and the alternative pathways from glucose were found to contribute to glycogenesis by 75 and 25% respectively. Cycloserine (18 mM), a transaminase inhibitor, strongly inhibited glycogen labelling from [U-14C] serine while producing a 70% increase in glucose incorporation by the alternative pathway, in both the presence and the absence of insulin. The inhibitor had no effect on the direct pathway from glucose to glycogen. Supplementation with 1 mM-hydroxypyruvate, a serine-derived metabolite, did not affect direct glucose incorporation, whereas the alternative pathway was stimulated whether insulin was present or not. These results indicate that the sequence glucose----serine----glycogen is operative in cultured foetal hepatocytes. The alternative pathway interferes with hydroxypyruvate utilization, and is likely mediated by the serine aminotransferase pathway, independently of the acute glycogenic action of insulin. PMID:1905920

  19. Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth

    SciTech Connect

    Torres-Rosado, A.; O'Shea, K.S.; Tsuji, A.; Kurachi, K. (Univ. of Michigan Medical School, Ann Arbor (United States)); Chou, S.H. (Univ. of Washington, Seattle (United States))

    1993-08-01

    Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsin-specific antisene oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology. 20 refs., 4 figs.

  20. Crassaostrea gigas oyster shell extract inhibits lipogenesis via suppression of serine palmitoyltransferase.

    PubMed

    Tran, Nguyen Khoi Song; Kwon, Jeong Eun; Kang, Se Chan; Shim, Soon-Mi; Park, Tae-Sik

    2015-02-01

    Oysters are widely consumed seafood, but their shells impose a serious environmental problem. To extend the utilization of oyster shell waste, we investigated the biological role of oyster shell extract. In this study, we verified that the ethanol extract of oyster shell (EOS) contains taurine and betaine, the major components of oyster body. EOS downregulated transcription of Sptlc1 and Sptlc2 mRNA, the subunits of serine palmitoyltransferase (SPT). Suppression of SPT subunits reduced sphinganine and sphingomyelin by inhibiting de novo sphingolipid biosynthesis. Inhibition of sphingomyelin biosynthesis resulted in downregulation of lipogenic gene expression such as ACC, FAS, SCD1, and DGAT2. Consistent with inhibition of lipogenesis, cellular triglyceride levels were diminished by EOS, but cholesterol levels were not altered. Taken together, these results suggest that EOS has a lipid-lowering effect and could be applied as either a therapeutic or preventive measure for metabolic dysfunction. PMID:25920281

  1. Regulation of a serine protease homolog by the JNK pathway during thoracic development of Drosophila melanogaster.

    PubMed

    Srivastava, Ajay; Dong, Qian

    2015-01-01

    The importance of the Jun N-terminal Kinase (JNK) pathway during normal development and tumor invasion has been well documented in Drosophila. Here, this pathway plays important roles in epithelial morphogenesis, wound healing, apoptosis, immunity and regulation of lifespan. However, which downstream molecules facilitate these effects is not very well elucidated. In this study, data are presented on a serine protease homolog (SPH), scarface. These data show that scarface is under regulatory control of the JNK pathway and that this pathway is both necessary and sufficient for its expression within the context of thoracic development. Consequently, down-regulation of scarface results in a thoracic-cleft phenotype that phenocopies the JNK pathway defect. A possible role of scarface during thoracic development in Drosophila is discussed. PMID:25737837

  2. Molecular heterogeneity of the bdellovibrios: Metallo and serine proteases unique to each species

    Microsoft Academic Search

    Louis Gloor; Brian Klubek; Ramon J. Seidler

    1974-01-01

    Summary  \\u000a \\u000a \\u000a \\u000a 1. \\u000a \\u000a \\u000a Bdellovibrio bacteriovorus, B. starrii, andB. stolpii each produce at least two or three extracellular proteolytic enzymes. One enzyme (metallo enzyme) is inhibited by 10 mM EDTA\\u000a and the other (serine protease) is inhibited by 1 mM phenylmethyl sulfonylfluoride. Separation of the two major enzymes was\\u000a achieved only by polyacrylamide gel electrophoresis.\\u000a \\u000a \\u000a \\u000a \\u000a 2. \\u000a \\u000a Bands of protease activity were developed

  3. Serine palmitoyltransferase, the first step enzyme in sphingolipid biosynthesis, is involved in nonhost resistance.

    PubMed

    Takahashi, Yoshihiro; Berberich, Thomas; Kanzaki, Hiroyuki; Matsumura, Hideo; Saitoh, Hiromasa; Kusano, Tomonobu; Terauchi, Ryohei

    2009-01-01

    An overexpression screen of Nicotiana benthamiana cDNAs identified a gene for the LCB2 subunit of serine palmitoyltransferase (SPT) as a potent inducer of hypersensitive response-like cell death. The pyridoxal 5'-phosphate binding site of NbLCB2 is required for its function as a cell death inducer. NbLCB2 mRNA is accumulated after infection by nonhost pathogen Pseudomonas cichorii. Resistance of N. benthamiana against P. cichorii was compromised by treatment with an SPT inhibitor and in NbLCB2- and NbLCB1-silenced plants. These results suggest that biosynthesis of sphingolipids is necessary for the nonhost resistance of N. benthamiana against P. cichorii. PMID:19061400

  4. Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth.

    PubMed Central

    Torres-Rosado, A; O'Shea, K S; Tsuji, A; Chou, S H; Kurachi, K

    1993-01-01

    Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsin-specific antisense oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin was observed. This reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology. Images Fig. 3 Fig. 4 PMID:8346233

  5. The Role of Serine Proteases and Antiproteases in the Cystic Fibrosis Lung

    PubMed Central

    Twigg, Matthew S.; Brockbank, Simon; Lowry, Philip; FitzGerald, S. Peter; Taggart, Clifford; Weldon, Sinéad

    2015-01-01

    Cystic fibrosis (CF) lung disease is an inherited condition with an incidence rate of approximately 1 in 2500 new born babies. CF is characterized as chronic infection of the lung which leads to inflammation of the airway. Sputum from CF patients contains elevated levels of neutrophils and subsequently elevated levels of neutrophil serine proteases. In a healthy individual these proteases aid in the phagocytic process by degrading microbial peptides and are kept in homeostatic balance by cognate antiproteases. Due to the heavy neutrophil burden associated with CF the high concentration of neutrophil derived proteases overwhelms cognate antiproteases. The general effects of this protease/antiprotease imbalance are impaired mucus clearance, increased and self-perpetuating inflammation, and impaired immune responses and tissue. To restore this balance antiproteases have been suggested as potential therapeutics or therapeutic targets. As such a number of both endogenous and synthetic antiproteases have been trialed with mixed success as therapeutics for CF lung disease. PMID:26185359

  6. Characterization of a glutenin-specific serine proteinase of Sunn bug Eurygaster integricepts Put.

    PubMed

    Konarev, Alexander V; Beaudoin, Frédéric; Marsh, Justin; Vilkova, Nina A; Nefedova, Ludmila I; Sivri, Dilek; Köksel, Hamit; Shewry, Peter R; Lovegrove, Alison

    2011-03-23

    Glutenin hydrolyzing proteinases (GHPs) have been purified, by affinity chromatography, from wheat seeds damaged by the Sunn bug Eurygaster integriceps (Hemiptera, Scutelleridae). A 28 kDa protein was partially sequenced by mass spectrometry and Edman degradation which showed homology to serine proteases from various insects. Three full length clones were obtained from cDNA isolated from Sunn bug salivary glands using degenerate PCR based on the sequences obtained. The cleavage site of the protease was determined using recombinant and synthetic peptides and shown to be between the consensus hexapeptide and nonapeptide repeat motifs present in the high molecular weight subunits of wheat glutenin (PGQGQQ?GYYPTSLQQ). Homology models were generated for the three proteinases identified in this study using the high resolution X-ray structure of a crayfish (Pontastacus leptodactylus) trypsin complexed with a peptide inhibitor as template (PDB accession 2F91). The novel specificity of this protease may find applications in both fundamental and applied studies. PMID:21323348

  7. Serine Hydroxymethyltransferase Anchors de Novo Thymidylate Synthesis Pathway to Nuclear Lamina for DNA Synthesis*

    PubMed Central

    Anderson, Donald D.; Woeller, Collynn F.; Chiang, En-Pei; Shane, Barry; Stover, Patrick J.

    2012-01-01

    The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2? (SHMT1 and SHMT2?), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2? is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks. PMID:22235121

  8. Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca.

    PubMed

    Watanabe, L; Vieira, D F; Bortoleto, R K; Arni, R K

    2002-06-01

    Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogenous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 A. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 A. PMID:12037309

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the coleopteran-specific Cry3Aa toxin from Bacillus thuringiensis (Bt). Larvae digest protein initially with cysteine peptidases in the anterior midgut and further with serine peptidases in middle and poste...

  10. Potent and Selective Inhibition of Membrane-Type Serine Protease 1 by Human Single-Chain Antibodies

    E-print Network

    Craik, Charles S.

    on cancer cells can be valuable reagents for diagnosis, prognosis, and therapy of cancer. To this end, a phage-displayed antibody library was screened against a cancer-associated serine protease, MT-SP1 in human prostate tissue samples for immunohistochemistry analysis. The mode of binding among the six

  11. In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

    Microsoft Academic Search

    Caterina Trozzi; Linda Bartholomew; Alessandra Ceccacci; Gabriella Biasiol; Laura Pacini; Sergio Altamura; Frank Narjes; Ester Muraglia; Giacomo Paonessa; Uwe Koch; Raffaele De Francesco; Christian Steinkuhler; Giovanni Migliaccio

    2003-01-01

    The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the

  12. Cloning, expression, and characterization of two novel cuticle-degrading serine proteases from the entomopathogenic fungus Cordyceps sinensis

    Microsoft Academic Search

    Yongjie Zhang; Xingzhong Liu; Mu Wang

    2008-01-01

    The entomopathogenic fungus Cordyceps sinensis has been important in traditional Chinese medicine, but is yet to be commercially cultivated. Difficulty in cultivation results in part from the low percentage of fungal infection on artificially inoculated host insects. To better understand the infection mechanism, we cloned two cuticle-degrading serine protease genes (csp1 and csp2) from C. sinensis. These enzymes are novel

  13. Structural Basis of Trypsin Inhibition and Entomotoxicity of Cospin, Serine Protease Inhibitor Involved in Defense of Coprinopsis cinerea Fruiting Bodies*

    PubMed Central

    Saboti?, Jerica; Bleuler-Martinez, Silvia; Renko, Miha; Avanzo Cagli?, Petra; Kallert, Sandra; Štrukelj, Borut; Turk, Dušan; Aebi, Markus; Kos, Janko; Künzler, Markus

    2012-01-01

    Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with Ki in the picomolar range. Determination of the crystal structure revealed that the protein has a ?-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the ?2 and ?3 strands, distinguishing cospin from other ?-trefoil-fold serine protease inhibitors in which ?4-?5 or ?5-?6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut. PMID:22167196

  14. Independent roles of methionine and O-acetyl-l-serine in the regulation of the ? subunit gene of ?-conglycinin

    Microsoft Academic Search

    Masami Yokota Hirai; Hoyeun Kim; Hiroaki Hayashi; Mitsuo Chino; Satoshi Naito; Torn Fujiwara

    2002-01-01

    The gene encoding the ? subunit of ?-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.) , is downregulated by methionine (Met) and upregulated by O-acetyl-l-serine (OAS). We examined the interaction between Met and OAS in the regulation of the expression of the ? subunit gene in in vitro-cultured immature soybean cotyledons. Application of Met

  15. Membrane anchored serine proteases: A rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer

    Microsoft Academic Search

    Sarah Netzel-Arnett; John D. Hooper; Roman Szabo; Edwin L. Madison; James P. Quigley; Thomas H. Bugge; Toni M. Antalis

    2003-01-01

    Dysregulated proteolysis is a hallmark of cancer. Malignant cells require a range of proteolytic activities to enable growth, survival, and expansion. Serine proteases of the S1 or trypsin-like family have well recognized roles in the maintenance of normal homeostasis as well as in the pathology of diseases such as cancer. Recently a rapidly expanding subgroup of S1 proteases has been

  16. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol

    Microsoft Academic Search

    WILLIAM R. MORRISON; LLOYD M. SMITH

    SUMMARY Fatty acid methyl esters and dimethylacetals suitable for gas chromatographic analysis were prepared by treatment of lipids with boron fluoride-methanol (140 g BFI per liter of methanol). This reagent is stable and easy to handle. Reaction conditions were investigated for triglycerides, di- glycerides, monoglycerides, free fatty acids, sterol esters, phos- phatidyl ethanolamines, phosphatidyl serines, phosphatidyl 'cholines, monophosphoinositides, monogalactosyl glycerides,

  17. Calibration of PS09, PS10, and PS11 trans-Alaska pipeline system strong-motion instruments, with acceleration, velocity, and displacement records of the Denali fault earthquake, 03 November 2002

    USGS Publications Warehouse

    Evans, John R.; Jensen, E. Gray; Sell, Russell; Stephens, Christopher D.; Nyman, Douglas J.; Hamilton, Robert C.; Hager, William C.

    2006-01-01

    In September, 2003, the Alyeska Pipeline Service Company (APSC) and the U.S. Geological Survey (USGS) embarked on a joint effort to extract, test, and calibrate the accelerometers, amplifiers, and bandpass filters from the earthquake monitoring systems (EMS) at Pump Stations 09, 10, and 11 of the Trans-Alaska Pipeline System (TAPS). These were the three closest strong-motion seismographs to the Denali fault when it ruptured in the MW 7.9 earthquake of 03 November 2002 (22:12:41 UTC). The surface rupture is only 3.0 km from PS10 and 55.5 km from PS09 but PS11 is 124.2 km away from a small rupture splay and 126.9 km from the main trace. Here we briefly describe precision calibration results for all three instruments. Included with this report is a link to the seismograms reprocessed using these new calibrations: http://nsmp.wr.usgs.gov/data_sets/20021103_2212_taps.html Calibration information in this paper applies at the time of the Denali fault earthquake (03 November 2002), but not necessarily at other times because equipment at these stations is changed by APSC personnel at irregular intervals. In particular, the equipment at PS09, PS10, and PS11 was changed by our joint crew in September, 2003, so that we could perform these calibrations. The equipment stayed the same from at least the time of the earthquake until that retrieval, and these calibrations apply for that interval.

  18. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases

    PubMed Central

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex with anti-proteases, at higher concentrations. Data from pharmacokinetic investigations reveals dose linearity for maximum plasma levels of free proteases not unusual for body proteases and a high inter-individual variability. There is no interference with each other after oral administration of protease combinations, and absorption follows an unusual invasion and elimination kinetic due to slow velocity of absorption and a fast 100% protein binding to anti-proteases. Oral application of proteases leads to increased proteolytic serum activity and increased plasma concentrations of the corresponding anti-proteases. Their biological activity is determined by their proteolytic activity as free proteases on soluble peptides/proteins or cell surface receptors (e.g. protease activated receptors) and their activity in the complex formed with their specific and/or unspecific anti-proteases. The anti-protease-complexes, during immune reaction and injuries often loaded with different cytokines, are cleared from body fluids and tissue by receptor mediated endocytosis on hepatocytes and/or blood cells. Oral administration of enteric coated tablets containing proteolytic enzymes of plant and animal origin may be a safe method to stabilize, positively influence or enhance physiological and immunological processes during disease processes and in healthy consumers. PMID:22461953

  19. Ligand binding promotes CDK-dependent phosphorylation of ER-alpha on hinge serine 294 but inhibits ligand-independent phosphorylation of serine 305.

    PubMed

    Held, Jason M; Britton, David J; Scott, Gary K; Lee, Elbert L; Schilling, Birgit; Baldwin, Michael A; Gibson, Bradford W; Benz, Christopher C

    2012-08-01

    Phosphorylation of estrogen receptor-? (ER?) is critical for its transcription factor activity and may determine its predictive and therapeutic value as a biomarker for ER?-positive breast cancers. Recent attention has turned to the poorly understood ER? hinge domain, as phosphorylation at serine 305 (Ser305) associates with poor clinical outcome and endocrine resistance. We show that phosphorylation of a neighboring hinge domain site, Ser294, analyzed by multiple reaction monitoring mass spectrometry of ER? immunoprecipitates from human breast cancer cells is robustly phosphorylated exclusively by ligand (estradiol and tamoxifen) activation of ER? and not by growth factor stimulation (EGF, insulin, heregulin-?). In a reciprocal fashion, Ser305 phosphorylation is induced by growth factors but not ligand activation of ER?. Phosphorylation at Ser294 and Ser305 is suppressed upon co-stimulation by EGF and ligand, respectively, unlike the N-terminal (AF-1) domain Ser118 and Ser167 sites of ER? where phosphorylation is enhanced by ligand and growth factor co-stimulation. Inhibition of cyclin-dependent kinases (CDK) by roscovitine or SNS-032 suppresses ligand-activated Ser294 phosphorylation without affecting Ser118 or Ser104/Ser106 phosphorylation. Likewise, cell-free studies using recombinant ER? and specific cyclin-CDK complexes suggest that Ser294 phosphorylation is primarily induced by the transcription-regulating and cell-cycle-independent kinase CDK7. Thus, CDK-dependent phosphorylation at Ser294 differentiates ligand-dependent from ligand-independent activation of Ser305 phosphorylation, showing that hinge domain phosphorylation patterns uniquely inform on the various ER? activation mechanisms thought to underlie the biologic and clinical diversity of hormone-dependent breast cancers. PMID:22669764

  20. Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases

    PubMed Central

    Gaillard, Erol A.; Kota, Pradeep; Gentzsch, Martina; Dokholyan, Nikolay V.; Stutts, M. Jackson

    2010-01-01

    Mammalian airways are protected from infection by a thin film of airway surface liquid (ASL) which covers airway epithelial surfaces and acts as a lubricant to keep mucus from adhering to the epithelial surface. Precise regulation of ASL volume is essential for efficient mucus clearance and too great a reduction in ASL volume causes mucus dehydration and mucus stasis which contributes to chronic airway infection. The epithelial Na+ channel (ENaC) is the rate-limiting step that governs Na+ absorption in the airways. Recent in vitro and in vivo data have demonstrated that ENaC is a critical determinant of ASL volume and hence mucus clearance. ENaC must be cleaved by either intracellular furin-type proteases or extracellular serine proteases to be active and conduct Na+, and this process can be inhibited by protease inhibitors. ENaC can be regulated by multiple pathways, and once proteolytically cleaved ENaC may then be inhibited by intracellular second messengers such as cAMP and PIP2. In the airways, however, regulation of ENaC by proteases seems to be the predominant mode of regulation since knockdown of either endogenous serine proteases such as prostasin, or inhibitors of ENaC proteolysis such as SPLUNC1, has large effects on ENaC activity in airway epithelia. In this review, we shall discuss how ENaC is proteolytically cleaved, how this process can regulate ASL volume, and how its failure to operate correctly may contribute to chronic airway disease. PMID:20401730

  1. A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs.

    PubMed

    Bonants, P J; Fitters, P F; Thijs, H; den Belder, E; Waalwijk, C; Henfling, J W

    1995-04-01

    Scanning electron micrographs of the nematode-egg-parasitic fungus Paecilomyces lilacinus infecting eggs of the root-knot nematode Meloidogyne spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N- and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode Meloidogyne hapla induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33.5 kDa, a pH optimum of 10.3, a temperature optimum of 60 degrees C and an isoelectric point above pH 10.2. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes. PMID:7773385

  2. Enzyme:substrate hydrogen bond shortening during the acylation phase of serine protease catalysis.

    PubMed

    Fodor, Krisztián; Harmat, Veronika; Neutze, Richard; Szilágyi, László; Gráf, László; Katona, Gergely

    2006-02-21

    Atomic resolution (serine protease intermediate structures revealed that the strength of the hydrogen bonds between the enzyme and the substrate changed during catalysis. The well-conserved hydrogen bonds of antiparallel beta-sheet between the enzyme and the substrate become significantly shorter in the transition from a Michaelis complex analogue (Pontastacus leptodactylus (narrow-fingered crayfish) trypsin (CFT) in complex with Schistocerca gregaria (desert locust) trypsin inhibitor (SGTI) at 1.2 A resolution) to an acyl-enzyme intermediate (N-acetyl-Asn-Pro-Ile acyl-enzyme intermediate of porcine pancreatic elastase at 0.95 A resolution) presumably synchronously with the nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. This is interpreted as an active mechanism that utilizes the energy released from the stronger hydrogen bonds to overcome the energetic barrier of the nucleophilic attack by the hydroxyl group of the catalytic serine. In the CFT:SGTI complex this hydrogen bond shortening may be hindered by the 27I-32I disulfide bridge and Asn-15I of SGTI. The position of the catalytic histidine changes slightly as it adapts to the different nucleophilic attacker during the transition from the Michaelis complex to the acyl-enzyme state, and simultaneously its interaction with Asp-102 and Ser-214 becomes stronger. The oxyanion hole hydrogen bonds provide additional stabilization for acyl-ester bond in the acyl-enzyme than for scissile peptide bond of the Michaelis complex. Significant deviation from planarity is not observed in the reactive bonds of either the Michaelis complex or the acyl-enzyme. In the Michaelis complex the electron distribution of the carbonyl bond is distorted toward the oxygen atom compared to other peptide bonds in the structure, which indicates the polarization effect of the oxyanion hole. PMID:16475800

  3. Visceral adipose tissue-derived serine protease inhibitor: A unique insulin-sensitizing adipocytokine in obesity

    PubMed Central

    Hida, Kazuyuki; Wada, Jun; Eguchi, Jun; Zhang, Hong; Baba, Masako; Seida, Aya; Hashimoto, Izumi; Okada, Tatsuo; Yasuhara, Akihiro; Nakatsuka, Atsuko; Shikata, Kenichi; Hourai, Shinji; Futami, Junichiro; Watanabe, Eijiro; Matsuki, Yasushi; Hiramatsu, Ryuji; Akagi, Shigeru; Makino, Hirofumi; Kanwar, Yashpal S.

    2005-01-01

    There is a rapid global rise in obesity, and the link between obesity and diabetes remains somewhat obscure. We identified an adipocytokine, designated as visceral adipose tissue-derived serpin (vaspin), which is a member of serine protease inhibitor family. Vaspin cDNA was isolated by from visceral white adipose tissues (WATs) of Otsuka Long-Evans Tokushima fatty (OLETF) rat, an animal model of abdominal obesity with type 2 diabetes. Rat, mouse, and human vaspins are made up of 392, 394, and 395 amino acids, respectively; exhibit ?40% homology with ?1-antitrypsin; and are related to serine protease inhibitor family. Vaspin was barely detectable in rats at 6 wk and was highly expressed in adipocytes of visceral WATs at 30 wk, the age when obesity, body weight, and insulin levels peak in OLETF rats. The tissue expression of vaspin and its serum levels decrease with worsening of diabetes and body weight loss at 50 wk. The expression and serum levels were normalized with the treatment of insulin or insulin-sensitizing agent, pioglitazone, in OLETF rats. Administration of vaspin to obese CRL:CD-1 (ICR) (ICR) mice fed with high-fat high-sucrose chow improved glucose tolerance and insulin sensitivity reflected by normalized serum glucose levels. It also led to the reversal of altered expression of genes relevant to insulin resistance, e.g., leptin, resistin, TNF?, glucose transporter-4, and adiponectin. In DNA chip analyses, vaspin treatment resulted in the reversal of expression in ?50% of the high-fat high-sucrose-induced genes in WATs. These findings indicate that vaspin exerts an insulin-sensitizing effect targeted toward WATs in states of obesity. PMID:16030142

  4. Related Arabidopsis Serine Carboxypeptidase-Like Sinapoylglucose Acyltransferases Display Distinct But Overlapping Substrate Specificities1[OA

    PubMed Central

    Fraser, Christopher M.; Thompson, Michael G.; Shirley, Amber M.; Ralph, John; Schoenherr, Jessica A.; Sinlapadech, Taksina; Hall, Mark C.; Chapple, Clint

    2007-01-01

    The Arabidopsis (Arabidopsis thaliana) genome encodes 51 proteins annotated as serine carboxypeptidase-like (SCPL) enzymes. Nineteen of these SCPL proteins are highly similar to one another, and represent a clade that appears to be unique to plants. Two of the most divergent proteins within this group have been characterized to date, sinapoyl-glucose (Glc):malate sinapoyltransferase and sinapoyl-Glc:choline sinapoyltransferase. The fact that two of the least related proteins within this clade are acyltransferases rather than true serine carboxypeptidases suggests that some or all of the remaining members of this group may have similar activities. The gene that encodes sinapoyl-Glc:malate sinapoyltransferase (sinapoyl-Glc accumulator1 [SNG1]: At2g22990) is one of five SCPL genes arranged in a cluster on chromosome 2. In this study, an analysis of deletion mutant lines lacking one or more genes in this SCPL gene cluster reveals that three of these genes also encode sinapoyl-Glc-dependent acyltransferases. At2g23000 encodes sinapoyl-Glc:anthocyanin acyltransferase, an enzyme that is required for the synthesis of the sinapoylated anthocyanins in Arabidopsis. At2g23010 encodes an enzyme capable of synthesizing 1,2-disinapoyl-Glc from two molecules of sinapoyl-Glc, an activity shared by SNG1 and At2g22980. Sequence analysis of these SCPL proteins reveals pairwise percent identities that range from 71% to 78%, suggesting that their differing specificities for acyl acceptor substrates are due to changes in a relatively small subset of amino acids. The study of these SCPL proteins provides an opportunity to examine enzyme structure-function relationships and may shed light on the role of evolution of hydroxycinnamate ester metabolism and the SCPL gene family in Arabidopsis and other flowering plants. PMID:17600138

  5. A Novel Lipid Droplet-Associated Serine Hydrolase Regulates Macrophage Cholesterol Mobilization

    PubMed Central

    Goo, Young-Hwa; Son, Se-Hee; Kreienberg, Paul B.; Paul, Antoni

    2014-01-01

    Objective Lipid-laden macrophages or foam cells are characterized by massive cytosolic lipid droplet (LD) deposition containing mostly cholesterol ester (CE) derived from the lipoproteins cleared from the arterial wall. Cholesterol efflux from foam cells is considered to be atheroprotective. Since cholesterol is effluxed as free cholesterol (FC), CE accumulation in LDs may limit FC efflux. Our objective was to identify proteins that regulate cholesterol trafficking through LDs. Approach and results In a proteomic analysis of the LD fraction of RAW 264.7 macrophages we identified an evolutionarily conserved protein with a canonical GXSXG lipase catalytic motif and a predicted ?/?-hydrolase fold, the RIKEN cDNA 1110057K04 gene, which we named lipid droplet-associated hydrolase (LDAH). LDAH association to LDs was confirmed by immunoblotting and immunocytochemistry. LDAH was labeled with a probe specific for active serine hydrolases. LDAH showed relatively weak in vitro CE hydrolase activity. However, cholesterol measurements in intact cells supported a significant role of LDAH in CE homeostasis, since LDAH upregulation and downregulation decreased and increased, respectively, intracellular cholesterol and CE in HEK293 cells and RAW 264.7 macrophages. Mutation of the putative nucleophilic serine impaired active hydrolase probe binding, in vitro CE hydrolase activity, and the cholesterol lowering effect in cells, while this mutant still localized to the LD. LDAH upregulation increased CE hydrolysis and cholesterol efflux from macrophages and, interestingly, LDAH is highly expressed in macrophage-rich areas within mouse and human atherosclerotic lesions. Conclusions The data identify a candidate target to promote reverse cholesterol transport from atherosclerotic lesions. PMID:24357060

  6. Functional analysis of a subtilisin-like serine protease gene from biocontrol fungus Trichoderma harzianum.

    PubMed

    Fan, Haijuan; Liu, Zhihua; Zhang, Rongshu; Wang, Na; Dou, Kai; Mijiti, Gulijimila; Diao, Guiping; Wang, Zhiying

    2014-02-01

    The subtilisin-like serine protease gene ThSS45 has been cloned from Trichoderma harzianum ACCC30371. Its coding region is 1302 bp in length, encoding 433 amino acids, with a predicted protein molecular weight of 44.9 kDa and pI of 5.91. ThSS45 was shown by RT-qPCR analysis to be differentially transcribed in response to eight different treatments. The transcription of ThSS45 was up-regulated when grown in mineral medium, under carbon starvation, and nitrogen starvation, and in the presence of 1% root powder, 1% stem powder, and 1% leaf powder derived from Populus davidiana × P. bolleana (Shanxin poplar) aseptic seedlings. The highest increase in transcription approached 3.5-fold that of the control at 6 h under induction with 1% poplar root powder. The transcription of ThSS45 was also slightly up-regulated by 1% Alternaria alternata cell wall and 5% A. alternata fermentation liquid. Moreover, the analyses of coding and promoter regions of ThSS45 homologs indicated that serine protease may be involved in both mycoparasitism and antibiotic secretion. ThSS45 was cloned into the pGEX-4T-2 vector and then expressed in Escherichia coli BL21. The recombinant protein, with an expected molecular weight of approximately 69 kDa, was then purified. When transformant BL21-ss was induced with 1 mM IPTG for 6 h, the purified protease activity reached a peak of 18.25 U/ml at pH 7.0 and 40°C. In antifungal assays the purified protease obviously inhibited the growth of A. alternata mycelia. PMID:24500477

  7. Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

    PubMed Central

    Giri, Pankaj Kumar; Tang, Xuhua; Thangamani, Saravanan; Shenoy, Rajesh T.; Ding, Jeak Ling; Swaminathan, Kunchithapadam; Sivaraman, J.

    2010-01-01

    Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Ĺ resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases. PMID:21188150

  8. Palmitoyl Serine: An Endogenous Neuroprotective Endocannabinoid-Like Entity After Traumatic Brain Injury.

    PubMed

    Mann, Aniv; Smoum, Reem; Trembovler, Victoria; Alexandrovich, Alexander; Breuer, Aviva; Mechoulam, Raphael; Shohami, Esther

    2015-06-01

    The endocannabinoid (eCB) system helps recovery following traumatic brain injury (TBI). Treatment with 2-arachidonoylglycerol (2-AG), a cerebral eCB ligand, was found to ameliorate the secondary damage. Interestingly, the fatty acid amino acid amide (FAAA) N-arachidonoyl-L-serine (AraS) exerts similar eCB dependent neuroprotective. The present study aimed to investigate the effects of the FAAA palmitoyl-serine (PalmS) following TBI. We utilized the TBI model in mice to examine the therapeutic potential of PalmS, injected 1 h following closed head injury (CHI). We followed the functional recovery of the injured mice for 28 days post-CHI, and evaluated cognitive and motor function, lesion volume, cytokines levels, molecular signaling, and infarct volume at different time points after CHI. PalmS treatment led to a significant improvement of the neurobehavioral outcome of the treated mice, compared with vehicle. This effect was attenuated in the presence of eCBR antagonists and in CB2-/- mice, compared to controls. Unexpectedly, treatment with PalmS did not affect edema and lesion volume, TNF? and IL1? levels, anti-apoptotic mechanisms, nor did it exert improvement in cognitive and motor function. Finally, co-administration of PalmS, AraS and 2-AG, did not enhance the effect of the individual drugs. We suggest that the neuroprotective action of PalmS is mediated by indirect activation of the eCB receptors following TBI. One such mechanism may involve receptor palmitoylation which has been reported to result in structural stabilization of the receptors and to an increase in their activity. Further research is required in order to establish this assumption. PMID:25721934

  9. Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin.

    PubMed

    Giri, Pankaj Kumar; Tang, Xuhua; Thangamani, Saravanan; Shenoy, Rajesh T; Ding, Jeak Ling; Swaminathan, Kunchithapadam; Sivaraman, J

    2010-01-01

    Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Ĺ resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC(50) of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases. PMID:21188150

  10. IrSPI, a Tick Serine Protease Inhibitor Involved in Tick Feeding and Bartonella henselae Infection

    PubMed Central

    Liu, Xiang Ye; de la Fuente, Jose; Cote, Martine; Galindo, Ruth C.; Moutailler, Sara; Vayssier-Taussat, Muriel; Bonnet, Sarah I.

    2014-01-01

    Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit. PMID:25057911

  11. Laser-Free RF-Gun as a Combined Source of Thz and Ps-Sub-Ps X-Rays

    SciTech Connect

    Agustsson, R. [RadiaBeam Technologies, Santa Monica, CA (US); Boucher, S. [RadiaBeam Technologies, Santa Monica, CA (US); Finn, O. [RadiaBeam Technologies, Santa Monica, CA (US); Hartzell, J. [RadiaBeam Technologies, Santa Monica, CA (US); Ruelas, M. [RadiaBeam Technologies, Santa Monica, CA (US); Smirnov, A.V. [RadiaBeam Technologies, Santa Monica, CA (US); Storms, S. [RadiaBeam Technologies, Santa Monica, CA (US); Ning, Z. [RadiaBeam Technologies, Santa Monica, CA (US); Murokh, A. [RadiaBeam Technologies, Santa Monica, CA (US); Campese, T. [RadiaBeam Technologies, Santa Monica, CA (US); Faillace, L. [RadiaBeam Technologies, Santa Monica, CA (US); Verma, A. [RadiaBeam Technologies, Santa Monica, CA (US); Kim, Y. [Idaho State Univ., Pocatello, ID (US); Buaphad, P. [Idaho State Univ., Pocatello, ID (US); Andrews, A. [Idaho Accelerator Center, Pocatello, ID (US); Berls, B. [Idaho Accelerator Center, Pocatello, ID (US); Eckman, C. [Idaho Accelerator Center, Pocatello, ID (US); Folkman, K. [Idaho Accelerator Center, Pocatello, ID (US); Knowles-Swingle, A. [Idaho Accelerator Center, Pocatello, ID (US); O’Neill, C. [Idaho Accelerator Center, Pocatello, ID (US); Smith, M. [Idaho Accelerator Center, Pocatello, ID (US); Grandsaert, T. [European Spalation Source, Lund (Sweden); van der Geer, B. [Pulsar Physics, Eindhoven (Netherlands); de Loos, M. [Pulsar Physics, Eindhoven (Netherlands); Berg, W.J. [Argonne National Lab., IL (US); Sereno, N.S. [Argonne National Lab., IL (US); Sun, Y. [Argonne National Lab., IL (US); Zholents, A.A. [Argonne National Lab., IL (US)

    2015-01-01

    A coherent, mm-sub-mm-wave source driven by a RF electron gun is proposed for wide research applications as well as auxiliary inspection and screening, safe imaging, cancer diagnostics, surface defectoscopy, and enhanced time-domain spectroscopy. It allows generation of high peak and average THz-sub-THz radiation power provided by beam pre-bunching and chirping in the RF gun followed by microbunching in magnetic compressor, and resonant Cherenkov radiation of an essentially flat beam in a robust, ~inch-long, planar, mm-sub-mm gap structure. The proof-of-principle has been successfully demonstrated in Phase I on a 5 MeV beam of L-band thermionic injector of Idaho Accelerator Center. The system can also deliver an intense, ps-sub-ps bursts of low-to-moderate dose of relativistic electrons and X-ray radiation produced by the same beam required for pulsed radiolysis as well as to enhance screening efficiency, throughput and safety.

  12. Laser-Free RF-Gun as a Combined Source of Thz and Ps-Sub-Ps X-Rays

    DOE PAGESBeta

    Agustsson, R.; Boucher, S.; Finn, O.; Hartzell, J.; Ruelas, M.; Smirnov, A.V.; Storms, S.; Ning, Z.; Murokh, A.; Campese, T.; et al

    2015-01-01

    A coherent, mm-sub-mm-wave source driven by a RF electron gun is proposed for wide research applications as well as auxiliary inspection and screening, safe imaging, cancer diagnostics, surface defectoscopy, and enhanced time-domain spectroscopy. It allows generation of high peak and average THz-sub-THz radiation power provided by beam pre-bunching and chirping in the RF gun followed by microbunching in magnetic compressor, and resonant Cherenkov radiation of an essentially flat beam in a robust, ~inch-long, planar, mm-sub-mm gap structure. The proof-of-principle has been successfully demonstrated in Phase I on a 5 MeV beam of L-band thermionic injector of Idaho Accelerator Center. Themore »system can also deliver an intense, ps-sub-ps bursts of low-to-moderate dose of relativistic electrons and X-ray radiation produced by the same beam required for pulsed radiolysis as well as to enhance screening efficiency, throughput and safety.« less

  13. Absolute Magnitudes of Pan-STARRS PS1 Asteroids

    NASA Astrophysics Data System (ADS)

    Veres, Peter; Jedicke, R.; Fitzsimmons, A.; Denneau, L.; Wainscoat, R.; Bolin, B.; PS1SC Collaboration

    2013-10-01

    Absolute magnitude (H) of an asteroid is a fundamental parameter describing the size and the apparent brightness of the body. Because of its surface shape, properties and changing illumination, the brightness changes with the geometry and is described by the phase function governed by the slope parameter (G). Although many years have been spent on detailed observations of individual asteroids to provide H and G, vast majority of minor planets have H based on assumed G and due to the input photometry from multiple sources the errors of these values are unknown. We compute H of ~ 180 000 and G of few thousands asteroids observed with the Pan-STARRS PS1 telescope in well defined photometric systems. The mean photometric error is 0.04 mag. Because on average there are only 7 detections per asteroid in our sample, we employed a Monte Carlo (MC) technique to generate clones simulating all possible rotation periods, amplitudes and colors of detected asteroids. Known asteroid colors were taken from the SDSS database. We used debiased spin and amplitude distributions dependent on size, spectral class distributions of asteroids dependent on semi-major axis and starting values of G from previous works. H and G (G12 respectively) were derived by phase functions by Bowell et al. (1989) and Muinonen et al. (2010). We confirmed that there is a positive systematic offset between H based on PS1 asteroids and Minor Planet Center database up to -0.3 mag peaking at 14. Similar offset was first mentioned in the analysis of SDSS asteroids and was believed to be solved by weighting and normalizing magnitudes by observatory codes. MC shows that there is only a negligible difference between Bowell's and Muinonen's solution of H. However, Muinonen's phase function provides smaller errors on H. We also derived G and G12 for thousands of asteroids. For known spectral classes, slope parameters agree with the previous work in general, however, the standard deviation of G in our sample is twice as larger, most likely due to sparse phase curve sampling. In the near future we plan to complete the H and G determination for all PS1 asteroids (500,000) and publish H and G values online. This work was supported by NASA grant No. NNX12AR65G.

  14. Preliminary Evaluation of PS300: A New Self-Lubricating High Temperature Composite Coating for Use to 800 C

    NASA Technical Reports Server (NTRS)

    Dellacorte, C.; Edmonds, B. J.

    1995-01-01

    This paper introduces PS300, a plasma sprayed, self-lubricating composite coating for use in sliding contacts at temperatures to 800 C. PS300 is a metal bonded chrome oxide coating with silver and BaF2/CaF2 eutectic solid lubricant additives. PS300 is similar to PS200, a chromium carbide based coating, which is currently being investigated for a variety of tribological applications. In pin-on-disk testing up to 650 C, PS300 exhibited comparable friction and wear properties to PS200. The PS300 matrix, which is predominantly chromium oxide rather than chromium carbide, does not require diamond grinding and polishes readily with silicon carbide abrasives greatly reducing manufacturing costs compared to PS200. It is anticipated that PS300 has potential for sliding bearing and seal applications in both aerospace and general industry.

  15. Effect of nano CdS dispersion on thermal conductivity of PS/PVC and PS/PMMA polymeric blend nanocomposites

    NASA Astrophysics Data System (ADS)

    Mathur, Vishal; Patidar, Dinesh; Sharma, Kananbala

    2015-06-01

    The effect of dispersion of CdS nano-filler particles in respective PS/PVC and PS/PMMA polymer blend matrices on the effective thermal conductivity has been studied through Hot Disk Thermal Constant Analyzer based on transient plane source (TPS) technique. The thick film samples have been prepared by dispersing nano-filler particles of CdS (6 wt%) in respective PS/PVC and PS/PMMA binary blend matrices. The nanocomposite nature of prepared samples ascertained through small angle X-ray scattering (SAXS) as well as transmission electron microscopy (TEM) measurements. It is observed that at room temperature nano CdS dispersed polymeric blend samples offer higher effective thermal conductivity.

  16. OCT/PS-OCT imaging of brachial plexus neurovascular structures

    NASA Astrophysics Data System (ADS)

    Raphael, David T.; Zhang, Jun; Zhang, Yaoping; Chen, Zhongping; Miller, Carol; Zhou, Li

    2004-07-01

    Introduction: Optical coherence tomography (OCT) allows high-resolution imaging (less than 10 microns) of tissue structures. A pilot study with OCT and polarization-sensitive OCT (PS-OCT) was undertaken to image ex-vivo neurovascular structures (vessels, nerves) of the canine brachial plexus. Methods: OCT is an interferometry-based optical analog of B-mode ultrasound, which can image through non-transparent biological tissues. With approval of the USC Animal Care and Use Committee, segments of the supra- and infraclavicular brachial plexus were excised from euthanized adult dogs, and the ex-vivo specimens were placed in cold pH-buffered physiologic solution. An OCT beam, in micrometer translational steps, scanned the fixed-position bisected specimens in transverse and longitudinal views. Two-dimensional images were obtained from identified arteries and nerves, with specific sections of interest stained with hematoxylin-eosin for later imaging through a surgical microscope. Results: with the beam scan direction transverse to arteries, the resulting OCT images showed an identifiable arterial lumen and arterial wall tissue layers. By comparison, transverse beam OCT images of nerves revealed a multitude of smaller nerve bundles contained within larger circular-shaped fascicles. PS-OCT imaging was helpful in showing the characteristic birefringence exhibited by arrayed neural structures. Discussion: High-resolution OCT imaging may be useful in the optical identification of neurovascular structures during attempted regional nerve blockade. If incorporated into a needle-shaped catheter endoscope, such a technology could prevent intraneural and intravascular injections immediately prior to local anesthetic injection. The major limitation of OCT is that it can form a coherent image of tissue structures only to a depth of 1.5 - 2 mm.

  17. Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate.

    PubMed

    Scarsdale, J N; Radaev, S; Kazanina, G; Schirch, V; Wright, H T

    2000-02-11

    Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants. PMID:10656824

  18. Role of a membrane-associated serine esterase in the oxidant activation of phospholipase A2 by t-butyl hydroperoxide.

    PubMed Central

    Chakraborti, S; Michael, J R; Gurtner, G H; Ghosh, S S; Dutta, G; Merker, A

    1993-01-01

    Exposure of bovine pulmonary-arterial endothelial cells to the oxidant lipid t-butyl hydroperoxide (t-Bu-OOH) increases cell-membrane-associated phospholipase A2 (PLA2) activity and stimulates arachidonic acid (AA) release. To test the hypothesis that a membrane-associated serine esterase plays an important role in activating PLA2, the present study was undertaken. In addition to increasing PLA2 activity and AA release, t-Bu-OOH also enhances the activity of a membrane-associated serine esterase that cleaves the synthetic substrate N alpha-p-tosyl-L-arginine methyl ester (TAME). Changes in the activity of this membrane-bound serine esterase correlate directly with changes in the activity of PLA2. Serine esterase inhibitors such as phenylmethanesulphonyl fluoride, di-isopropyl fluorophosphate and alpha 1-proteinase inhibitor, and TAME, a synthetic substrate for serine esterase, prevent the increase in serine esterase activity, PLA2 activity and AA release caused by t-Bu-OOH. Pretreatment of the endothelial cells with the antioxidant vitamin E prevents t-Bu-OOH-induced stimulation of AA release and the cell-membrane-associated serine esterase and PLA2 activities. Adding t-Bu-OOH or the serine esterase trypsin to the endothelial-cell membrane fraction also significantly augments PLA2 activity, implying that these treatments activate latent PLA2. These results suggest that t-Bu-OOH stimulates a membrane-associated serine esterase that plays a crucial role in activating PLA2 and releasing AA. PMID:8503892

  19. PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth.

    PubMed

    Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

    2015-01-01

    Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

  20. Role of propagating ionisation fronts in semiconductor generation of sub-ps THz radiation

    E-print Network

    Strathclyde, University of

    Role of propagating ionisation fronts in semiconductor generation of sub-ps THz radiation S, United Kingdom Abstract Observations of a directional asymmetry in the sub-ps THz radiation generated standing and widely accepted surface-layer current-surge description of the THz emission process. A model

  1. voluntary product standard ps 1-07 National Institute of Standards and Technology

    E-print Network

    Structural Plywood voluntary product standard ps 1-07 National Institute of Standards and Technology Technology Administration, U.S. Department of Commerce #12;Voluntary Product Standard PS 1) VOLUNTARY PRODUCT STANDARDS DOC Voluntary Product Standards are developed under procedures published

  2. Review of enigmatic MTrPs as a common cause of enigmatic musculoskeletal pain and dysfunction

    Microsoft Academic Search

    David G Simons

    2004-01-01

    This article explores how myofascial trigger points (MTrPs) may relate to musculoskeletal dysfunction (MSD) in the workplace and what might be done about it. The cause of much MSD and pain is often enigmatic to modern medicine and very costly, just as the cause of MTrPs has been elusive for the past century, despite an extensive literature that is confusing

  3. Policy Statement Number: PS-48 Title/Topic: Student Appeal Procedures

    E-print Network

    Harms, Kyle E.

    procedural guidelines which govern that appeal process. However, if the area or function under question doesPolicy Statement Number: PS-48 Title/Topic: Student Appeal Procedures Effective Date: 01/01/2002 Revision Number: PS0048.R04 GENERAL APPEAL PROCEDURE AVAILABLE TO STUDENTS PURPOSE To establish procedures

  4. 8 Research Activities 2012 CiRA: Center for iPS Cell Research and Application

    E-print Network

    Takada, Shoji

    . Shinya Yamanaka, who pioneered the research field of iPS cell technology, directs the institute. Equipped of therapeutic drugs using patient-derived iPS cells intellectual property Director Shinya Yamanaka at his office stem cell research. The announcement came just a day after Dr. Yamanaka and Sir John Gurdon were

  5. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells

    Microsoft Academic Search

    Jochen Utikal; Jose M. Polo; Matthias Stadtfeld; Nimet Maherali; Warakorn Kulalert; Ryan M. Walsh; Adam Khalil; James G. Rheinwald; Konrad Hochedlinger

    2009-01-01

    The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine

  6. SALT spectroscopic classification of PS15atx as a type-Ia supernova

    NASA Astrophysics Data System (ADS)

    Jha, S. W.; Pan, Y.-C.; Foley, R. J.; Rest, A.; Scolnic, D.; Smith, K. W.; Wright, D.; Smartt, S. J.; Huber, M.; Chambers, K. C.; Flewelling, H.; Willman, M.; Primak, N.; Schultz, A.; Gibson, B.; Magnier, E.; Waters, C.; Tonry, J.; Wainscoat, R. J.; Depagne, E.

    2015-06-01

    We obtained SALT (+RSS) spectroscopy of PS15atx on 2015 June 20.8 UT, covering the wavelength range 400-950 nm. Cross-correlation of the spectrum with a template library using SNID (Blondin & Tonry 2007, ApJ, 666, 1024) shows PS15atx to be a normal type-Ia supernova a few days before maximum light.

  7. Multimedia Authoring for CoPs Romain Deltour, Agn`es Guerraz, and Cecile Roisin

    E-print Network

    Paris-Sud XI, Université de

    Multimedia Authoring for CoPs Romain Deltour, Agn`es Guerraz, and C´ecile Roisin INRIA Rh and share information. As this information becomes more and more multimedia in nature, the challenge is to build multimedia authoring and publishing tools that meets CoPs requirements. In this paper we analyze

  8. Effect of molecular weight and shear on phase diagram of PS\\/PVME blend

    Microsoft Academic Search

    Khalil El Mabrouk; Mosto Bousmina

    2006-01-01

    The molecular weight dependence of the lower critical solution temperature of polystyrene (PS) and poly(vinyl methyl ether) blends was studied by laser light transmission. The temperature of phase separation was found to decrease with increasing PS molecular weight. In the steady shear flow conditions and near the critical temperature, shear was found to enhance fluctuations of concentration at relatively small

  9. On second-order nonlinearity and maximum algebraic immunity of some bent functions in PS + 1

    E-print Network

    On second-order nonlinearity and maximum algebraic immunity of some bent functions in PS + 1 INDIA bksingh0584@gmail.com Abstract In this paper, by modifying a subclass of bent functions in PSap, we construct another subclass of bent functions in PS + with maximum algebraic degree. We demonstrate

  10. Emission properties of porphyrin compounds in new polymeric PS:CBP host

    NASA Astrophysics Data System (ADS)

    Jafari, Mohammad Reza; Bahrami, Bahram

    2015-06-01

    In this study, a device with fundamental structure of ITO/PEDOT:PSS (60 nm)/PS:CBP (70 nm)/Al (150 nm) was fabricated. The electroluminescence spectrum of device designated a red shift rather than PS:CBP photoluminescence spectra. It can be suggested that the electroplex emission occurs at PS:CBP interface. By following this step, red light-emitting devices using porphyrin compounds as a red dopant in a new host material PS:CBP with a configuration of ITO/PEDOT:PSS (60 nm)/PS:CBP:porphyrin compounds(70 nm)/Al (150 nm) have been fabricated and investigated. The electroluminescent spectra of the porphyrin compounds were red-shifted as compared with the PS:CBP blend. OLED devices based on doping 3,4PtTPP and TPPNO2 in PS:CBP showed purer red emission compared with ZnTPP and CoTPP doped devices. We believe that the electroluminescence performance of OLED devices based on porphyrin compounds depends on overlaps between the absorption of the porphyrin compounds and the emission of PS:CBP.

  11. Proposed Policy Statement Number: PS-67 Title/Topic: Misuse of Drugs or Alcohol

    E-print Network

    Harms, Kyle E.

    603094.2 Proposed Policy Statement Number: PS-67 Title/Topic: Misuse of Drugs or Alcohol Effective Date: 01/07/2013 Revision Number: PS0067.R05 MISUSE OF DRUGS OR ALCOHOL Louisiana State University of the University. Although the University respects an employee's right to privacy, the misuse of drugs or alcohol

  12. PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth

    PubMed Central

    Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

    2015-01-01

    Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

  13. Elastomeric Capture Microparticles (ECmuPs) and Their use with Acoustophoresis to Perform Affinity Capture Assays

    NASA Astrophysics Data System (ADS)

    Cushing, Kevin Wallace

    This dissertation describes the development of elastomeric capture microparticles (ECmicroPs) and their use with acoustophoresis to perform affinity capture assays. EC?Ps that function as negative acoustic contrast particles were developed by crosslinking emulsion-based droplets composed of commercially available silicone precursors followed by functionalization with avidin/biotin reagents. The size distribution of the EC?Ps was very broad or narrow depending on the emulsion system that was used during the synthesis process. Elastomeric particles exhibited a very broad size distribution when a bulk-emulsion process was used; however, when microfluidic systems were utilized, their size distribution became comparatively narrow. The functionalization of elastomeric particles was accomplished by the non-specific adsorption of avidin protein followed by bovine serum albumin (BSA) blocking and bio-specific adsorption of a biotinylated-capture antibody. Polydisperse EC?Ps were functionalized to bind prostate specific antigen (PSA) or IgG-phycoerythrin (PE) in aqueous media (buffer, plasma, blood); whereas monodisperse EC?Ps were functionalized to bind a high density lipoprotein in the aqueous media. Polydisperse EC?Ps functionalized to bind PSA in a physiological buffer (PBS pH 7.4) demonstrated nanomolar detection using flow cytometry analysis; whereas EC?Ps functionalized to bind IgG-PE demonstrated picomolar detection in 10% porcine plasma. EC?Ps have a specific density of ~1.03 and are more compressible than their surrounding aqueous media; which allowed the EC?Ps to exhibit negative acoustic contrast properties under an ultrasonic acoustic standing wave field. The negative acoustic contrast property of EC?Ps was advantageously utilized in an IgG-PE assay conducted in 0.1% whole porcine blood. The ligand-bound EC?Ps suspended in the diluted blood sample were flowed through an acoustofluidic device where the application of an ultrasonic acoustic standing wave field focused the ligand-bound EC?Ps to pressure antinodes and the positive acoustic contrast blood cells to the central pressure node of the microchannel. As a result of laminar flow, focused ligand-bound EC?Ps and blood cells were flowed into properly aligned outlet channels at the downstream trifurcation, where they where collected separately off-chip. The cell-free fraction containing ligand-bound EC?Ps was analyzed using flow cytometry; where the detection of IgG-PE was in the picomolar range. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.

  14. [Current status and perspective on regenerative medicine for spinal cord injury using iPS cell].

    PubMed

    Nakamura, Masaya; Toyama, Yoshiaki; Okano, Hideyuki

    2013-01-01

    Stimulated by the 2012 Nobel Prize in Physiology or Medicine awarded for Shinya Yamanaka and Sir John Gurdon, there is an increasing interest in the iPS cells and reprogramming technologies in medical science. While iPS cells are expected to open new era providing enormous opportunities in the biomedical sciences in terms of cell therapies for regenerative medicine, safety-related concerns for iPS cell-based cell therapy should be resolved prior to the clinical application of iPS cells. In this symposium, the pre-clinical investigations of cell therapy for SCI using neural stem/progenitor cells derived from iPS cells, and their safety issues in vivo are outlined. PMID:24291863

  15. Self-assembled micelles of amphiphilic poly(l-phenylalanine)-b-poly(l-serine) polypeptides for tumor-targeted delivery

    PubMed Central

    Zhao, Ziming; Wang, Yu; Han, Jin; Wang, Keli; Yang, Dan; Yang, Yihua; Du, Qian; Song, Yuanjian; Yin, Xiaoxing

    2014-01-01

    The aim of this work was to design, synthesize, and characterize self-assembled micelles based on polypeptides as a potential antitumor drug carrier. Amphiphilic poly(l-phenylalanine)-b-poly(l-serine) (PFS) polypeptides were obtained through the polymerization of N-carboxyanhydride. As a novel hydrophilic segment, poly(l-serine) was utilized to enhance tumor targeting due to a large demand of tumors for serine. PFS could self-assemble into micelles with an average diameter of 110–240 nm and a slightly negative charge. PFS polypeptides adopted random coil in pH 7.4 phosphate-buffered saline and could partly transform to ?-helix induced by trifluoroethanol. PFS micelles with a low critical micelle concentration of 4.0 ?g mL?1 were stable in pH 5–9 buffers and serum albumin solution. PFS micelles had a loading capacity of 3.8% for coumarin-6 and exhibited a sustained drug release. Coumarin-6 loaded rhodamine B isothiocyanate-labeled PFS micelles were incubated with Huh-7 tumor cells to study the correlation between drugs and carriers during endocytosis. The uptake of drugs was consistent with the micelles, illustrating that the intracellular transport of drugs highly depended on the micelles. PFS micelles diffused in whole cytoplasm while coumarin-6 assumed localized distribution, suggesting that the micelles could release the loaded drugs in particular areas. The internalization mechanism of PFS micelles was involved with clathrin-mediated endocytosis and macropinocytosis. Excess serine inhibited the uptake of PFS micelles, which demonstrated that serine receptors played a positive role in the internalization of PFS. The more interesting thing was that the uptake inhibition impacted on normal cells but not on tumor cells at the physiological concentration of serine. The difference in the uptake of PFS micelles was fourfold as high between the tumor cells and the normal cells, which indicated that PFS micelles had good tumor targeting in vitro. In conclusion, PFS micelles reported in this work were a promising drug delivery system for tumor targeting therapy. PMID:25540585

  16. Self-assembly of CdSe quantum dots and colloidal titanium dioxide on copolymer microspheres (PS) for CdSe/PS and TiO2/CdSe/PS sub-microspheres with yolk-shell structure

    NASA Astrophysics Data System (ADS)

    Zhao, Qingchun

    2015-07-01

    Semiconductor nanocrystals serve as the building blocks for designing next generation solar cells, chemical/biological sensors, and metal chalcogenides (e.g., CdS, CdSe, PbS, and PbSe) are particularly useful for harnessing size-dependent optical and electronic properties in nanostructures. In this paper, relying on the interaction including van der Waals forces and hydrogen bond, CdSe/PS sub-microspheres composite and TiO2/CdSe/PS sub-microspheres with yolk-shell structure were prepared via self-assembly of CdSe quantum dots and colloidal titanium dioxide on modified PS surface. The morphology, structure and composition obtained products were investigated by scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM) and energy disperse X-ray spectroscopy (EDX). Transmission electron microscopy (TEM) investigations show the CdSe quantum dots and colloidal titanate were assembled on the surface of PS sub-microspheres. CdSe QD-polymer sub-microspheres composites in which the QDs retain their original emission efficiency can be obtained. TiO2/CdSe/PS sub-microspheres with yolk-shell structure can improve the efficiency of charge separation.

  17. Converted-wave time domain registration using the inverted pseudo-PS-wave attribute section

    NASA Astrophysics Data System (ADS)

    Chen, Shuangquan; Li, Xiang-Yang; Tang, Jianming

    2014-02-01

    The main aim of the event registration of PP- and PS-waves in multicomponent seismic exploration is to provide the same time/depth domain data for further interpretation and application to reservoir characterization and hydrocarbon detection. Most registration methods are based on waveform similarity and phase consistency by correlating the stacked records, which may cause serious errors due to differences in PP- and PS-wave reflectivity. Our motivation was to develop a time matching workflow to improve the event registration of multicomponent seismic data and enhance joint interpretation techniques. In this paper, we present a prestack inversion method to obtain a pseudo-PS-wave (PPS) section based on prestack PP-wave gathers, and establish an attributes matching workflow based on the waveform similarity comparison of PP-, PS- and PPS-wave sections. We investigate the PP-wave prestack inversion method and the waveform similarity comparison of PP-, PS- and PPS-wave sections using synthetic data. The test on synthetic data clearly shows that the PPS-wave section has much better waveform similarity to the PS-wave section than the PP-wave due to the phase consistency of the reflectivity, increasing the correlation coefficient of waveform similarity to over 90% for PPS- and PS-waves from about 50% for PP- and PS-waves. The application to field data shows a very good improvement in the event registration, especially due to the partial nonexistence of events in the PP-wave section. The benefits for the time matching are also made evident by comparing the correlation sections of PPS- and PS-waves with PP- and PS-waves. The improved accuracy in event registration leads to better joint inversion and interpretation for the stratigraphic features and hydrocarbon detection in the subsequent applications.

  18. Multipolar electrostatics based on the Kriging machine learning method: an application to serine.

    PubMed

    Yuan, Yongna; Mills, Matthew J L; Popelier, Paul L A

    2014-04-01

    A multipolar, polarizable electrostatic method for future use in a novel force field is described. Quantum Chemical Topology (QCT) is used to partition the electron density of a chemical system into atoms, then the machine learning method Kriging is used to build models that relate the multipole moments of the atoms to the positions of their surrounding nuclei. The pilot system serine is used to study both the influence of the level of theory and the set of data generator methods used. The latter consists of: (i) sampling of protein structures deposited in the Protein Data Bank (PDB), or (ii) normal mode distortion along either (a) Cartesian coordinates, or (b) redundant internal coordinates. Wavefunctions for the sampled geometries were obtained at the HF/6-31G(d,p), B3LYP/apc-1, and MP2/cc-pVDZ levels of theory, prior to calculation of the atomic multipole moments by volume integration. The average absolute error (over an independent test set of conformations) in the total atom-atom electrostatic interaction energy of serine, using Kriging models built with the three data generator methods is 11.3 kJ mol?ą (PDB), 8.2 kJ mol?ą (Cartesian distortion), and 10.1 kJ mol?ą (redundant internal distortion) at the HF/6-31G(d,p) level. At the B3LYP/apc-1 level, the respective errors are 7.7 kJ mol?ą, 6.7 kJ mol?ą, and 4.9 kJ mol?ą, while at the MP2/cc-pVDZ level they are 6.5 kJ mol?ą, 5.3 kJ mol?ą, and 4.0 kJ mol?ą. The ranges of geometries generated by the redundant internal coordinate distortion and by extraction from the PDB are much wider than the range generated by Cartesian distortion. The atomic multipole moment and electrostatic interaction energy predictions for the B3LYP/apc-1 and MP2/cc-pVDZ levels are similar, and both are better than the corresponding predictions at the HF/6-31G(d,p) level. PMID:24633774

  19. Identification and characterization of a serine protease inhibitor Esserpin from the Chinese mitten crab Eriocheir sinensis.

    PubMed

    Wang, Lingling; Ma, Zhaopeng; Yang, Jialong; Gai, Yunchao; Zhou, Zhi; Wang, Leilei; Yue, Feng; Song, Linsheng

    2013-06-01

    Serine protease inhibitors (serpins) represent an expanding superfamily of endogenous inhibitors that regulate proteolytic events and involve in a variety of physiological processes. A serine protease inhibitor, namely Esserpin, was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) analysis. The full-length cDNA of Esserpin was of 2367 bp, including an open reading frame (ORF) of 1371 bp encoding a polypeptide of 456 amino acids with estimated molecular mass of 49.95 kDa and theoretical isoelectric point of 6.03. A putative signal peptide of 23 amino acids and a classical serpin domain were identified in Esserpin. The deduced amino acid sequence of Esserpin shared homology with serpins from Fenneropenaeus chinensis and Pacifastacus leniusculus. The mRNA transcripts of Esserpin could be detected in all the examined tissues including heart, gill, hemocytes, muscle, gonad and hepatopancreas, and the highest expression level was present in gonad. After the crabs were challenged by Vibrio anguillarum and Pichia pastoris, the expression levels of Esserpin transcripts in hemocytes were significantly up-regulated, and peaked at 24 h (5.18-fold of blank group, P < 0.05) and 3 h (2.87-fold of blank group, P < 0.05), respectively. The functional activity of Esserpin was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombinant Esserpin (rEsserpin) could inhibit trypsin activities in a dose-dependent manner, and it could lead to 100% inhibition of trypsin activities under the concentration of 873.76 nM, while there was no evident inhibition of chymotrypsin observed with rEsserpin. Moreover, rEsserpin inhibited the growth of E. coli at the final concentration of 1747.52 nM, and it also significantly depressed (P < 0.05) the phenoloxidase activity in the plasma at the final concentration of 873.76 nM. These results indicated that Esserpin was a homologue of serpin in crab and it could be induced after immune stimulation and mediate immune response possibly via the inhibition of bacterial growth and the regulation of prophenoloxidase-activating system. PMID:23567854

  20. Phosphatidyl Inositol 3-Kinase Signaling in Hypothalamic Proopiomelanocortin Neurons

    E-print Network

    Toledo, University of

    Published Online October 9, 2009 * J.W.H. and Y.X. are joint first authors. J.J.Z. and J.K.E. are joint. Elmquist Division of Hypothalamic Research (J.W.H., Y.X., R.C., J.K.E.), Department of Internal Medicine and Pharmacology, Department of Internal Medicine and Cell Biology (Y.-R.C.), Touchstone Center for Diabetes

  1. Transgene Excision Has No Impact on In Vivo Integration of Human iPS Derived Neural Precursors

    E-print Network

    Major, Tamara

    The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo ...

  2. Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase.

    PubMed

    Bai, Hua; Sun, Mingxuan; Ghosh, Pallavi; Hatfull, Graham F; Grindley, Nigel D F; Marko, John F

    2011-05-01

    Structural and topological data suggest that serine site-specific DNA recombinases exchange duplex DNAs by rigid-body relative rotation of the two halves of the synapse, mediated by a flat protein-protein interaction surface. We present evidence for this rotational motion for a simple serine recombinase, the Bxb1 phage integrase, from a single-DNA-based supercoil-release assay that allows us to follow crossover site cleavage, rotation, religation, and product release in real time. We have also used a two-DNA braiding-relaxation experiment to observe the effect of synapse rotation in reactions on two long molecules. Relaxation and unbraiding are rapid (averaging 54 and 70 turns/s, respectively) and complete, with no discernible pauses. Nevertheless, the molecular friction associated with rotation is larger than that of type-I topoisomerases in a similar assay. Surprisingly we find that the synapse can stay rotationally "open" for many minutes. PMID:21502527

  3. Development of Mast Cells and Importance of Their Tryptase and Chymase Serine Proteases in Inflammation and Wound Healing

    PubMed Central

    Douaiher, Jeffrey; Succar, Julien; Lancerotto, Luca; Gurish, Michael F.; Orgill, Dennis P.; Hamilton, Matthew J.; Krilis, Steven A.; Stevens, Richard L.

    2014-01-01

    Mast cells (MCs) are active participants in blood coagulation and innate and acquired immunity. This review focuses on the development of mouse and human MCs, as well as the involvement of their granule serine proteases in inflammation and the connective tissue remodeling that occurs during the different phases of the healing process of wounded skin and other organs. The accumulated data suggest that MCs, their tryptases, and their chymases play important roles in tissue repair. While MCs initially promote healing, they can be detrimental if they are chronically stimulated or if too many MCs become activated at the same time. The possibility that MCs and their granule serine proteases contribute to the formation of keloid and hypertrophic scars makes them potential targets for therapeutic intervention in the repair of damaged skin. PMID:24507159

  4. Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease

    NASA Astrophysics Data System (ADS)

    Martínez-González, José Ángel; Rodríguez, Alex; Puyuelo, María Pilar; González, Miguel; Martínez, Rodrigo

    2015-01-01

    The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second-order Mřller-Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different substrates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a concerted mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated.

  5. Generation of 3.5-ps fall-time shock waves on a monolithic GaAs nonlinear transmission line

    Microsoft Academic Search

    C. J. Madden; M. J. W. Rodwell; R. A. Marsland; DAVID M. BLOOM; Y. C. Pao

    1988-01-01

    It is shown that nonlinear wave propagation on a monolithic GaAs nonlinear transmission line can form shock waves with fall time as short as 3.5 ps. An output fall time of 4.3 ps was measured for a single line driven at 15 GHz (20-ps fall time) while a cascade of two lines driven at 8 GHz (37-ps fall time) produced

  6. Endophytic Colonization of Vitis vinifera L. by Plant Growth-Promoting Bacterium Burkholderia sp. Strain PsJN

    Microsoft Academic Search

    Stephane Compant; Birgit Reiter; Angela Sessitsch; Jerzy Nowak; Christophe Clement; E. Ait Barka

    2005-01-01

    Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed

  7. Molecular dynamics of synthetic leucine-serine ion channels in a phospholipid membrane.

    PubMed

    Randa, H S; Forrest, L R; Voth, G A; Sansom, M S

    1999-11-01

    Molecular dynamics calculations were carried out on models of two synthetic leucine-serine ion channels: a tetrameric bundle with sequence (LSLLLSL)(3)NH(2) and a hexameric bundle with sequence (LSSLLSL)(3)NH(2). Each protein bundle is inserted in a palmitoyloleoylphosphatidylcholine bilayer membrane and solvated by simple point charge water molecules inside the pore and at both mouths. Both systems appear to be stable in the absence of an electric field during the 4 ns of molecular dynamics simulation. The water motion in the narrow pore of the four-helix bundle is highly restricted and may provide suitable conditions for proton transfer via a water wire mechanism. In the wider hexameric pore, the water diffuses much more slowly than in bulk but is still mobile. This, along with the dimensions of the pore, supports the observation that this peptide is selective for monovalent cations. Reasonable agreement of predicted conductances with experimentally determined values lends support to the validity of the simulations. PMID:10545343

  8. Molecular dynamics of synthetic leucine-serine ion channels in a phospholipid membrane

    PubMed Central

    Randa, HS; Forrest, LR; Voth, GA; Sansom, MS

    1999-01-01

    Molecular dynamics calculations were carried out on models of two synthetic leucine-serine ion channels: a tetrameric bundle with sequence (LSLLLSL)(3)NH(2) and a hexameric bundle with sequence (LSSLLSL)(3)NH(2). Each protein bundle is inserted in a palmitoyloleoylphosphatidylcholine bilayer membrane and solvated by simple point charge water molecules inside the pore and at both mouths. Both systems appear to be stable in the absence of an electric field during the 4 ns of molecular dynamics simulation. The water motion in the narrow pore of the four-helix bundle is highly restricted and may provide suitable conditions for proton transfer via a water wire mechanism. In the wider hexameric pore, the water diffuses much more slowly than in bulk but is still mobile. This, along with the dimensions of the pore, supports the observation that this peptide is selective for monovalent cations. Reasonable agreement of predicted conductances with experimentally determined values lends support to the validity of the simulations. PMID:10545343

  9. 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

  10. The Serine 814 of TRPC6 Is Phosphorylated under Unstimulated Conditions

    PubMed Central

    Bousquet, Simon M.; Monet, Michael; Boulay, Guylain

    2011-01-01

    TRPC are nonselective cation channels involved in calcium entry. Their regulation by phosphorylation has been shown to modulate their routing and activity. TRPC6 activity increases following phosphorylation by Fyn, and is inhibited by protein kinase G and protein kinase C. A previous study by our group showed that TRPC6 is phosphorylated under unstimulated conditions in a human embryonic kidney cells line (HEK293). To investigate the mechanism responsible for this phosphorylation, we used a MS/MS approach combined with metabolic labeling and showed that the serine at position 814 is phosphorylated in unstimulated cells. The mutation of Ser814 into Ala decreased basal phosphorylation but did not modify TRPC6 activity. Even though Ser814 is within a consensus site for casein kinase II (CK2), we showed that CK2 is not involved in the phosphorylation of TRPC6 and does not modify its activity. In summary, we identified a new basal phosphorylation site (Ser814) on TRPC6 and showed that CK2 is not responsible for the phosphorylation of this site. PMID:21448286

  11. Host Generated siRNAs Attenuate Expression of Serine Protease Gene in Myzus persicae

    PubMed Central

    Bhatia, Varnika; Bhattacharya, Ramcharan; Uniyal, Prem L.; Singh, Rajendra; Niranjan, Rampal S.

    2012-01-01

    Background Sap sucking hemipteran aphids damage diverse crop species. Although delivery of ds-RNA or siRNA through microinjection/feeding has been demonstrated, the efficacy of host-mediated delivery of aphid-specific dsRNA in developing aphid resistance has been far from being elucidated. Methodology/Principal Findings Transgenic Arabidopsis expressing ds-RNA of Myzus persicae serine protease (MySP) was developed that triggered the generation of corresponding siRNAs amenable for delivery to the feeding aphids. M. persicae when fed on the transgenic plants for different time intervals under controlled growth conditions resulted in a significant attenuation of the expression of MySP and a commensurate decline in gut protease activity. Although the survivability of these aphids was not affected, there was a noticeable decline in their fecundity resulting in a significant reduction in parthenogenetic population. Conclusions/Significance The study highlighted the feasibility of developing host based RNAi-mediated resistance against hemipteran pest aphids. PMID:23071558

  12. Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2

    PubMed Central

    Krarup, Anders; Wallis, Russell; Presanis, Julia S.; Gál, Péter; Sim, Robert B.

    2007-01-01

    The complement system is an important immune mechanism mediating both recognition and elimination of foreign bodies. The lectin pathway is one pathway of three by which the complement system is activated. The characteristic protease of this pathway is Mannan-binding lectin (MBL)-associated serine protease 2 (MASP2), which cleaves complement proteins C2 and C4. We present a novel and alternative role of MASP2 in the innate immune system. We have shown that MASP2 is capable of promoting fibrinogen turnover by cleavage of prothrombin, generating thrombin. By using a truncated active form of MASP2 as well as full-length MASP2 in complex with MBL, we have shown that the thrombin generated is active and can cleave both factor XIII and fibrinogen, forming cross-linked fibrin. To explore the biological significance of these findings we showed that fibrin was covalently bound on a bacterial surface to which MBL/MASP2 complexes were bound. These findings suggest that, as has been proposed for invertebrates, limited clotting may contribute to the innate immune response. PMID:17637839

  13. Genome-wide mapping of histone H4 serine-1 phosphorylation during sporulation in Saccharomyces cerevisiae

    PubMed Central

    Govin, Jérôme; Schug, Jonathan; Krishnamoorthy, Thanuja; Dorsey, Jean; Khochbin, Saadi; Berger, Shelley L.

    2010-01-01

    We previously showed that histone H4 serine-1 phosphorylation (H4S1ph) is evolutionarily conserved during gametogenesis, and contributes to post-meiotic nuclear compaction and to full completion of sporulation in the yeast Saccharomyces cerevisiae. Previous studies showed that H4S1ph and another modification of the same histone, H4 acetylation (H4ac), do not occur together and have opposing roles during DNA double-strand break (DSB) repair. In this study, we investigated the relationship between these marks during yeast sporulation. H4S1ph and H4ac co-exist globally during later stages of sporulation, in contrast to DSB repair. Genome-wide mapping during sporulation reveals accumulation of both marks over promoters of genes. Prevention of H4S1ph deposition delays the decline in transcription that normally occurs during spore maturation. Taken together, our results indicate that H4S1ph deposition reinforces reduced transcription that coincides with full spore compaction, without disrupting the local acetylation signature. These studies indicate distinctive features of a histone H4 modification marking system during sporulation compared with DSB repair. PMID:20375100

  14. Genome-wide mapping of histone H4 serine-1 phosphorylation during sporulation in Saccharomyces cerevisiae.

    PubMed

    Govin, Jérôme; Schug, Jonathan; Krishnamoorthy, Thanuja; Dorsey, Jean; Khochbin, Saadi; Berger, Shelley L

    2010-08-01

    We previously showed that histone H4 serine-1 phosphorylation (H4S1ph) is evolutionarily conserved during gametogenesis, and contributes to post-meiotic nuclear compaction and to full completion of sporulation in the yeast Saccharomyces cerevisiae. Previous studies showed that H4S1ph and another modification of the same histone, H4 acetylation (H4ac), do not occur together and have opposing roles during DNA double-strand break (DSB) repair. In this study, we investigated the relationship between these marks during yeast sporulation. H4S1ph and H4ac co-exist globally during later stages of sporulation, in contrast to DSB repair. Genome-wide mapping during sporulation reveals accumulation of both marks over promoters of genes. Prevention of H4S1ph deposition delays the decline in transcription that normally occurs during spore maturation. Taken together, our results indicate that H4S1ph deposition reinforces reduced transcription that coincides with full spore compaction, without disrupting the local acetylation signature. These studies indicate distinctive features of a histone H4 modification marking system during sporulation compared with DSB repair. PMID:20375100

  15. Fibrinolytic serine protease isolation from Bacillus amyloliquefaciens An6 grown on Mirabilis jalapa tuber powders.

    PubMed

    Agrebi, Rym; Hmidet, Noomen; Hajji, Mohamed; Ktari, Nawrez; Haddar, Anissa; Fakhfakh-Zouari, Nahed; Nasri, Moncef

    2010-09-01

    In this study, Mirabilis jalapa tuber powder (MJTP) was used as a new complex organic substrate for the growth and production of fibrinolytic enzymes by a newly isolated Bacillus amyloliquefaciens An6. Maximum protease activity (1,057 U/ml) with casein as a substrate was obtained when the strain was grown in medium containing (grams per liter) MJTP 30, yeast extract 6, CaCl(2) 1, K(2)HPO(4) 0.1, and K(2)HPO(4) 0.1. The strain was also found to grow and produce extracellular proteases in a medium containing only MJTP, indicating that it can obtain its carbon, nitrogen, and salts requirements directly from MJTP. The B. amyloliquefaciens An6 fibrinase (BAF1) was partially purified, and fibrinolytic activity was assayed in a test tube with an artificial fibrin clot. The molecular weight of the partially purified BAF1 fibrinolytic protease was estimated to be 30 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for the caseinolytic activity were 60 degrees C and 9.0, respectively. The enzyme was highly stable from pH 6.0 to 11.0 and retained 62% of its initial activity after 1 h incubation at 50 degrees C. However, the enzyme was inactivated at higher temperatures. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, suggesting that BAF1 is a serine protease. PMID:19842068

  16. Nucleocytoplasmic shuttling of the HSV-2 serine/threonine kinase Us3

    SciTech Connect

    Finnen, Renee L.; Johnston, Susan M.; Neron, Casey E.; Banfield, Bruce W., E-mail: bruce.banfield@queensu.ca

    2011-08-15

    The alphaherpesvirus serine/threonine kinase Us3 plays diverse roles in virus multiplication and modifies both nuclear and cytoplasmic substrates. We recently reported that treatment of HSV-2 Us3-transfected and HSV-2-infected cells with leptomycin B, an inhibitor of nuclear export mediated by interaction of chromosomal regional maintenance protein (CRM1) with leucine rich nuclear export signals (NESs), resulted in nuclear trapping of Us3. Here, we utilized fluorescence loss in photobleaching to monitor nuclear export of HSV-2 Us3 and confirm that this process proceeds solely via a CRM1-mediated mechanism. Analysis of deletion derivatives of HSV-2 Us3 fused to a nuclear export reporter protein implicated the involvement of NES-like sequences in nuclear export. However, nuclear trapping of HSV-2 Us3 proteins carrying mutations in these potential NESs was not observed, indicating that these sequences are not functional in the context of full-length protein. Our analyses also revealed previously unidentified regions of HSV-2 Us3 that contribute to its kinase activity.

  17. Generation and Analysis of Serine Protease Inhibitor Kazal Type 3-Cre Driver Mice

    PubMed Central

    Sakata, Kazuya; Ohmuraya, Masaki; Araki, Kimi; Suzuki, Chigure; Ida, Satoshi; Hashimoto, Daisuke; Wang, Jung; Uchiyama, Yasuo; Baba, Hideo; Yamamura, Ken-ichi

    2014-01-01

    Serine protease inhibitor Kazal type 1 (SPINK1; mouse homologue Spink3) was initially discovered as a trypsin-specific inhibitor in the pancreas. However, previous studies have suggested that SPINK1/Spink3 is expressed in a wide range of normal tissues and tumors, although precise characterization of its gene expression has not been described in adulthood. To further analyze Spink3 expression, we generated two mouse lines in which either lacZ or Cre recombinase genes were inserted into the Spink3 locus by Cre-loxP technology. In Spink3lacZ mice, ?-galactosidase activity was found in acinar cells of the pancreas and kidney, as well as epithelial cells of the bronchus in the lung, but not in the gastrointestinal tract or liver. Spink3cre knock-in mice were crossed with Rosa26 reporter (R26R) mice to monitor Spink3 promoter activity. In Spink3cre;R26R mice, ?-galactosidase activity was found in acinar cells of the pancreas, kidney, lung, and a small proportion of cells in the gastrointestinal tract and liver. These data suggest that Spink3 is widely expressed in endoderm-derived tissues, and that Spink3cre knock-in mice are a useful tool for establishment of a conditional knockout mice to analyze Spink3 function not only in normal tissues, but also in tumors that express SPINK1/Spink3. PMID:24521862

  18. Purification and characterization of a serine protease (CESP) from mature coconut endosperm

    PubMed Central

    Panicker, Leelamma M; Usha, Rajamma; Roy, Samir; Mandal, Chhabinath

    2009-01-01

    Background In plants, proteases execute an important role in the overall process of protein turnover during seed development, germination and senescence. The limited knowledge on the proteolytic machinery that operates during seed development in coconut (Cocos nucifera L.) prompted us to search for proteases in the coconut endosperm. Findings We have identified and purified a coconut endosperm protease (CESP) to apparent homogeneity. CESP is a single polypeptide enzyme of approximate molecular mass of 68 kDa and possesses pH optimum of 8.5 for the hydrolysis of BAPNA. Studies relating to substrate specificity and pattern of inhibition by various protease inhibitors indicated that CESP is a serine protease with cleavage specificity to peptide bonds after arginine. Purified CESP was often autolysed to two polypeptides of 41.6 kDa (CESP1) and 26.7 kDa (CESP2) and is confirmed by immunochemistry. We have shown the expression of CESP in all varieties of coconut and in all stages of coconut endosperm development with maximum amount in fully matured coconut. Conclusion Since the involvement of proteases in the processing of pre-proteins and maintenance of intracellular protein levels in seeds are well known, we suspect this CESP might play an important role in the coconut endosperm development. However this need to be confirmed using further studies. PMID:19426537

  19. Inhibition of growth hormone and prolactin secretion by a serine proteinase inhibitor

    SciTech Connect

    Rappay, G.; Nagy, I.; Makara, G.B.; Horvath, G.; Karteszi, M.; Bacsy, E.; Stark, E.

    1984-01-23

    The action of the tripeptide aldehyde t-butyloxycarbonyl-DPhe-Pro-Arg-H (boc-fPR-H), belonging to a family of serine proteinase inhibitors, on the release of immunoreactive prolactin (iPRL) and growth hormone (iGH) has been studied. In rat anterior pituitary cell cultures and pituitary quarters 1 mM boc-fPR-H inhibited basal iPRL and iGH release. Thyroliberin-induced iPRL release by cultured cells was also markedly inhibited with a concomitant accumulation of intracellular iPRL. During the short- and long-term exposure of cells to boc-fPR-H there were no changes in total cell protein contents and in activities of some lysosomal marker enzymes. The marked inhibition of basal as well as stimulated hormone release in the presence of the enzyme inhibitor might suggest that at least a portion of the hormones is released via a proteolytic enzyme-dependent process.

  20. 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. PMID:25491309

  1. Functional Domains of Fused, a Serine-Threonine Kinase Required for Signaling in Drosophila

    PubMed Central

    Therond, P.; Alves, G.; Limbourg-Bouchon, B.; Tricoire, H.; Guillemet, E.; Brissard-Zahraoui, J.; Lamour-Isnard, C.; Busson, D.

    1996-01-01

    fused (fu) is a segment-polarity gene encoding a putative serine-threonine kinase. In a wild-type context, all fu mutations display the same set of phenotypes. Nevertheless, mutations of the Suppressor of fused [Su(fu)] gene define three classes of alleles (fu0, fuI, fuII). Here, we report the molecular analysis of known fu mutations and the generation of new alleles by in vitro mutagenesis. We show that the Fused (Fu) protein functions in vivo as a kinase. The N-terminal kinase and the extreme C-terminal domains are necessary for Fu(+) activity while a central region appears to be dispensable. We observe a striking correlation between the molecular lesions of fu mutations and the phenotype displayed in their interaction with Su(fu). Indeed, fuI alleles which are suppressed by Su(fu) mutations are defined by inframe alterations of the N-terminal catalytic domain whereas the C-terminal domain is missing or altered in all fuII alleles. An unregulated FuII protein, which can be limited to the 80 N-terminal amino acids of the kinase domain, would be responsible for the neomorphic costal-2 phenotype displayed by the fuII-Su(fu) interaction. We propose that the Fu C-terminal domain can differentially regulate the Fu catalytic domain according to cell position in the parasegment. PMID:8846897

  2. Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets.

    PubMed

    Borgognone, Alessandra; Lowe, Kate L; Watson, Stephen P; Madhani, Melanie

    2014-01-01

    Cyclic guanosine-3',5'-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; e.g. atrial NP [ANP]), which activate soluble and particulate guanylyl cyclases, respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate vasodilator-stimulated phosphoprotein (VASP) at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis. PMID:23469931

  3. Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets

    PubMed Central

    Borgognone, Alessandra; Lowe, Kate L; Watson, Stephen P; Madhani, Melanie

    2013-01-01

    Cyclic guanosine-3?,5?-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; for example, atrial natriuretic peptide [ANP]), which activate soluble and particulate guanylyl cyclases (sGC and pGC), respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate VASP at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylanxthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C, NPR-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis. PMID:23469931

  4. Serine-arginine protein kinase 1 is associated with breast cancer progression and poor patient survival.

    PubMed

    Li, Xing-hua; Song, Jun-wei; Liu, Jun-ling; Wu, Shu; Wang, Le-shi; Gong, Li-yun; Lin, Xi

    2014-08-01

    Breast cancer is the most common cancer and the second leading cause of mortality for women worldwide. It is necessary to identify valuable molecular markers to predict breast cancer progression in patients and treatment effect. Serine-arginine protein kinase 1 (SRPK1), a member of SR kinase family, phosphorylates the SR splicing factors which plays essential roles in normal cell development and multiple human diseases. In the current study, we wanted to explore if there are any relationships between SRPK1 expression in breast cancer and its clinical characteristics. The results showed that SRPK1 is upregulated in breast cancer cell lines and tissues at both mRNA and protein levels, measured by quantitative reverse transcriptase PCR and Western blotting. Immunohistochemical analysis showed a high expression of SRPK1 in 132 paraffin samples of patients with breast cancer; statistical analyses demonstrated that high expression of SRPK1 significantly correlated with clinical staging of patients with breast cancer (P < 0.001), TNM classification (P < 0.05). Low expression of SRPK1 leads to longer survival time, while high expression of SRPK1 leads to shorter survival time of patients. Multivariate analysis revealed that upregulation of SRPK1 might be an independent prognostic marker for the outcomes of patients with breast cancer. In conclusion, upregulation of SRPK1 might play an important role in the progression of breast cancer and might be considered as the potential diagnostic and therapeutic target to this malignancy. PMID:24961466

  5. Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

    PubMed Central

    Kumar, Rakesh; Bhardwaj, Usha; Kumar, Pawan; Mazumdar-Leighton, Sudeshna

    2015-01-01

    Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors. PMID:25873901

  6. pknE, a serine/threonine kinase of Mycobacterium tuberculosis modulates multiple apoptotic paradigms.

    PubMed

    Kumar, Dinesh; Narayanan, Sujatha

    2012-06-01

    Mycobacterium tuberculosis, an intracellular pathogen that causes tuberculosis has developed multifactorial mechanisms to evade host signaling responses. Apoptosis, an important innate host immune response that clears the invading pathogen is suppressed by M. tuberculosis to gain persistence. Here, we examined the various apoptotic events suppressed by Protein Kinase E, (pknE) a Serine Threonine Protein Kinase (STPK) of M. tuberculosis in macrophages infected with ?pknE, a deletion mutant of pknE vs. the wild type strain H(37)Rv using microarray. The data showed increased expression of genes involved in apoptosis and chemokines with suppressed pro-inflammatory cytokines, co-stimulatory molecules, arginase1 and iNOS. The microarray data was validated using qRT-PCR, PCR array, oligoGE array, arginase assay and/or ELISA. Furthermore, we analyzed the phosphorylation of Akt that promotes cell survival using western blotting. ?pknE infected macrophages reduced the phosphorylation of Akt that correlates with the observed apoptotic responses. Experiments performed using exogenous nitrate donor, sodium nitro prusside to demonstrate the role of pknE during nitrate stress showed similar apoptotic responses to that of endogenous nitrate stress in ?pknE infected macrophages. Our data confirms the role of pknE in the intra cellular survival of M. tuberculosis by suppressing apoptosis during nitrate stress. PMID:21945589

  7. 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

  8. Arginine as a General Acid Catalyst in Serine Recombinase-mediated DNA Cleavage*

    PubMed Central

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

    2013-01-01

    Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3?O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3? phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3?O leaving group and is the prime candidate for the general acid that protonates the 3?O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin. PMID:23970547

  9. Cardiomyocyte Specific Deficiency of Serine Palmitoyltransferase Subunit 2 Reduces Ceramide but Leads to Cardiac Dysfunction*

    PubMed Central

    Lee, Su-Yeon; Kim, Jung Ran; Hu, Yunying; Khan, Raffay; Kim, Su-Jung; Bharadwaj, Kalyani G.; Davidson, Mercy M.; Choi, Cheol-Soo; Shin, Kyong-Oh; Lee, Yong-Moon; Park, Woo-Jin; Park, In-Sun; Jiang, Xian-Cheng; Goldberg, Ira J.; Park, Tae-Sik

    2012-01-01

    The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity. PMID:22493506

  10. N-arachidonoyl l-serine, an endocannabinoid-like brain constituent with vasodilatory properties

    PubMed Central

    Milman, Garry; Maor, Yehoshua; Abu-Lafi, Saleh; Horowitz, Michal; Gallily, Ruth; Batkai, Sandor; Mo, Fong-Ming; Offertaler, Laszlo; Pacher, Pal; Kunos, George; Mechoulam, Raphael

    2006-01-01

    The endocannabinoid N-arachidonoyl ethanolamine (anandamide), found both in the CNS and in the periphery, plays a role in numerous physiological systems. One might expect that the chemically related N-arachidonoyl-l-serine (ARA-S) could also be formed alongside anandamide. We have now isolated ARA-S from bovine brain and elucidated its structure by comparison with synthetic ARA-S. Contrary to anandamide, ARA-S binds very weakly to cannabinoid CB1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. However, it produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44/42 mitogen-activated protein (MAP) kinase and protein kinase B/Akt in cultured endothelial cells. ARA-S also suppresses LPS-induced formation of TNF-? in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB1 or CB2 receptors. Many of these effects parallel those reported for abnormal cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoid-type receptor. Hence, ARA-S may represent an endogenous agonist for this receptor. PMID:16467152

  11. Biphasic control logic of HAMP domain signalling in the Escherichia coli serine chemoreceptor.

    PubMed

    Zhou, Qin; Ames, Peter; Parkinson, John S

    2011-05-01

    HAMP domains mediate input-output communication in many bacterial signalling proteins. To explore the dynamic bundle model of HAMP signalling (Zhou et al., Mol. Microbiol. 73: 801, 2009), we characterized the signal outputs of 118 HAMP missense mutants of the serine chemoreceptor, Tsr, by flagellar rotation patterns. Receptors with proline or charged amino acid replacements at critical hydrophobic packing residues in the AS1 and AS2 HAMP helices had locked kinase-off outputs, indicating that drastic destabilization of the Tsr-HAMP bundle prevents kinase activation, both in the absence and presence of the sensory adaptation enzymes, CheB and CheR. Attractant-mimic lesions that enhance the structural stability of the HAMP bundle also suppressed kinase activity, demonstrating that Tsr-HAMP has two kinase-off output states at opposite extremes of its stability range. HAMP mutants with locked-on kinase outputs appeared to have intermediate bundle stabilities, implying a biphasic relationship between HAMP stability and kinase activity. Some Tsr-HAMP mutant receptors exhibited reversed output responses to CheB and CheR action that are readily explained by a biphasic control logic. The findings of this study provide strong support for a three-state dynamic bundle model of HAMP signalling in Tsr, and possibly in other bacterial transducers as well. PMID:21306449

  12. Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins.

    PubMed

    Haynes, Chad; Iakoucheva, Lilia M

    2006-01-01

    Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence. PMID:16407336

  13. Inhibition of human beta-factor XIIa by squash family serine proteinase inhibitors.

    PubMed

    Wynn, R; Laskowski, M

    1990-02-14

    Many inhibitors of trypsin and human beta-factor XIIa have been isolated from squash and related seeds and sequenced (Wieczorek et al., Biochem. Biophys. Res. Comm. (1985) 126, 646-652). The association equilibrium constants (Ka) of several of these inhibitors have now been determined with human beta-factor XIIa using a modification of the method of Green and Work (Park et al., Fed. Proc. Fed. Am. Soc. Exp. Biol. (1984) 43, 1962). The Ka's range from 7.8 x 10(4) M-1 to 3.3 x 10(8) M-1. Two isoinhibitors from Cucurbita maxima seeds, CMTI-I and CMTI-III, differ in only a single glutamate to lysine change in the P'4 position. This results in a factor of 62 increase in the Ka of the lysine inhibitor, CMTI-III (Ka = 3.3 x 10(8) M-1). To our knowledge, this is the largest effect ever seen for a residue substitution at the P'4 position of a serine proteinase inhibitor. The result is even more surprising because beta-factor XIIa's natural substrate, Factor XI, contains Gly in the P'4 position. PMID:2306254

  14. 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 ?g/g ?-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 ?-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

  15. HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

    SciTech Connect

    Devroe, Eric [Department of Systems Biology, Harvard Medical School, Boston, MA 02115 (United States); Department of Cancer Cell Biology, Dana-Farber Cancer Institute, Boston, MA 02115 (United States); Silver, Pamela A. [Department of Systems Biology, Harvard Medical School, Boston, MA 02115 (United States); Department of Cancer Cell Biology, Dana-Farber Cancer Institute, Boston, MA 02115 (United States); Engelman, Alan [Department of Pathology, Harvard Medical School, Boston, MA 02115 (United States) and Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street Boston, MA 02115 (United States)]. E-mail: alan_engelman@dfci.harvard.edu

    2005-01-05

    Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells.

  16. 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. PMID:9721213

  17. Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody.

    PubMed

    Ren, Diya; Zhang, Jian; Pritchett, Ross; Liu, Hongbin; Kyauk, Jennifer; Luo, Jun; Amanullah, Ashraf

    2011-10-01

    Sequence variants, also known as unintended amino acid substitutions in the protein primary structure, are one of the critical quality attributes needed to be monitored during process development of monoclonal antibodies (mAbs). Here we report on analytical methods for detection and identification of a sequence variant in an IgG1 mAb expressed in Chinese hamster ovary (CHO) cells. The presence of the sequence variant was detected by an imaged capillary isoelectric focusing (ICIEF) assay, showing a new basic species in mAb charge variant profile. The new basic variant was fractionated and enriched by ion-exchange chromatography, analyzed by reduced light and heavy chain mass determination, and characterized by HPLC-UV/MS/MS of tryptic and endoproteinase Lys-C peptide maps. A Serine to Arginine sequence variant was identified at the heavy chain 441 position (S441R), and confirmed by using synthetic peptides. The relative level of the S441R variant was estimated to be in the range of 0.3-0.6% for several mAb batches analyzed via extracted ion chromatogram (EIC). This work demonstrates the effectiveness of using integrated analytical methods to detect and identify protein heterogeneity and the importance of monitoring product quality during mAb bioprocess development. PMID:21900054

  18. The Serine Hydrolase ABHD6 is a Critical Regulator of the Metabolic Syndrome

    PubMed Central

    Lord, Caleb C.; Brown, Amanda L.; Marshall, Stephanie; Ferguson, Daniel; Sawyer, Janet; Davis, Matthew A.; Melchior, John T.; Blume, Lawrence C.; Howlett, Allyn C.; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Mrak, Irina; Leber, Vera; Heier, Christoph; Taschler, Ulrike; Blankman, Jacqueline L.; Cravatt, Benjamin F.; Lee, Richard G.; Crooke, Rosanne M.; Graham, Mark J.; Zimmermann, Robert; Brown, H. Alex; Brown, J. Mark

    2013-01-01

    SUMMARY The serine hydrolase ?/? hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues to identify in vivo substrates and to understand ABHD6's role in energy metabolism. Here we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high fat diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as novel therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes. PMID:24095738

  19. Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

    SciTech Connect

    Asenjo, Ana; Gonzalez-Armas, Juan C. [Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Ctra, Majadahonda-Pozuelo Km 2, Majadahonda, Madrid 28220 (Spain); Villanueva, Nieves [Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Ctra, Majadahonda-Pozuelo Km 2, Majadahonda, Madrid 28220 (Spain)], E-mail: nvilla@isciii.es

    2008-10-10

    The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) {beta} and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.

  20. Constitutive activation of Mek1 by mutation of serine phosphorylation sites.

    PubMed

    Huang, W; Erikson, R L

    1994-09-13

    A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals. PMID:8090753

  1. Photorespiratory mutants of the mitochondrial conversion of glycine to serine. [Hordeum vulgare L

    SciTech Connect

    Blackwell, R.D.; Murray, A.J.S.; Lea, P.J. (Lancaster Univ. (England))

    1990-11-01

    Two mutants of barley (Hordeum vulgare L.), LaPr 85/55 and LaPr 87/30, have been isolated that accumulate glycine, with a concomitant reduction in the aminodonors glutamate and alanine, when transferred to air. Studies have shown that these plants have wild-type levels of serine transhydroxymethylase (EC 2.1.2.1) activity. The data suggest a mutation in a glycine transport system for LaPr 85/55 and in the proteins of glycine decarboxylase for LaPr 87/30. Western blotting with antisera to the P, H, T, and L proteins of glycine decarboxylase showed cross-reaction against all four proteins for LaPr 85/55 but little cross-reaction against P or H protein for LaPr 87/30, reaffirming the possibility of a transport mutation in LaPr 85/55. We also suggest that genes for P an H proteins could be either coordinately regulated or that one protein is undetectable or unstable in the absence of the other.

  2. Activation of Protein Serine/Threonine Phosphatase PP2C? Efficiently Prevents Liver Fibrosis

    PubMed Central

    Chen, Jing; Yang, Zhengyi; Yu, Liang; Hu, Lihong; Shen, Xu

    2010-01-01

    Background Over-activation of TGF? signaling pathway and uncontrolled cell proliferation of hepatic stellate cells (HSCs) play pivotal roles in liver fibrogenesis, while the protein serine/threonine phosphatase PP2C? was reported to negatively regulate TGF? signaling pathway and cell cycle. Our study aimed to investigate the role of PP2C? in liver fibrogenesis. Methodology/Principal Findings The effects of PP2C? activation on liver fibrosis were investigated in human HSCs and primary rat HSCs in vitro using western blotting, real-time PCR, nuclear translocation, cell viability and cell cycle analyses. The antifibrogenic effects in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced mice in vivo were assessed using biochemical, histological and immunohistochemical analyses. The results demonstrated that activation of PP2C? by overexpression or the new discovered small molecular activator NPLC0393 terminated TGF?-Smad3 and TGF?-p38 signaling pathways, induced cell cycle arrest in HSCs and decreased ?-smooth muscle actin (?-SMA) expression, collagen deposition and hepatic hydroxyproline (HYP) level in CCl4- and BDL-induced mice. Conclusions/Significance Our findings suggested that PP2C? activation might be an attractive new strategy for treating liver fibrosis while the small molecular activator NPLC0393 might represent a lead compound for antifibrogenic drug development. Moreover, our study might provide the first evidence for the role of PP2C family members in the fibrotic disease. PMID:21151953

  3. 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.

  4. 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.

  5. AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at COOH-terminal serine residues.

    PubMed

    Assentoft, Mette; Larsen, Brian R; Olesen, Emma T B; Fenton, Robert A; MacAulay, Nanna

    2014-11-15

    Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 serves as a water entry site during brain edema formation, and regulation of AQP4 may therefore be of therapeutic interest. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser(321), and Ser(322). To address the role of these phosphorylation sites for AQP4 function, serine-to-alanine mutants were created to abolish the phosphorylation sites. All mutants were detected at the plasma membrane of transfected C6 cells, with the fraction of the total cellular AQP4 expressed at the plasma membrane of transfected C6 cells being similar between the wild-type (WT) and mutant forms of AQP4. Activation of protein kinases A, C, and G in primary astrocytic cultures did not affect the plasma membrane abundance of AQP4. The unit water permeability was determined for the mutant AQP4s upon heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4 appears not to be required for proper plasma membrane localization of AQP4 or to act as a molecular switch to gate the water channel. PMID:25231107

  6. Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

    PubMed

    Driesbaugh, Kathryn H; Buzza, Marguerite S; Martin, Erik W; Conway, Gregory D; Kao, Joseph P Y; Antalis, Toni M

    2015-02-01

    Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NF?B signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. PMID:25519908

  7. Effects of two serine proteases from Bothrops pirajai snake venom on the complement system and the inflammatory response.

    PubMed

    Menaldo, Danilo L; Bernardes, Carolina P; Pereira, Juliana C; Silveira, Denise S C; Mamede, Carla C N; Stanziola, Leonilda; Oliveira, Fábio de; Pereira-Crott, Luciana S; Faccioli, Lúcia H; Sampaio, Suely V

    2013-04-01

    The present study aimed to evaluate the effects of two serine proteases from Bothrops pirajai snake venom, named BpirSP27 and BpirSP41, on the complement system and the inflammatory response. The effects of these enzymes on the human complement system were assessed by kinetic hemolytic assays, evaluating the hemolysis promoted by the classical/lectin (CP/LP) and alternative (AP) pathways after incubation of normal human serum with the serine proteases. The results suggested that these enzymes were able to induce modulation of CP/LP and AP at different levels: BpirSP41 showed higher inhibitory effects on the hemolytic activity of CP/LP than BpirSP27, with inhibition values close to 40% and 20%, respectively, for the highest concentration assayed. Regarding AP, both enzymes showed percentages of inhibition of the hemolytic activity around 20% for the highest concentrations tested, indicating similar effects on this complement pathway. The proinflammatory effects of B. pirajai serine proteases were evaluated regarding their ability to induce paw edema, variations in the pain threshold and leukocyte recruitment at the site of injection. Both showed mild effects on these inflammatory processes, leading to low levels of increase of paw volumes and decrease in pain thresholds in rats up to 6 h after injection, and inducing neutrophil recruitment without significant increases in the total number of leukocytes in the inflammatory exudates after 6 and 24 h of administration into mice peritoneal cavity. These results suggest that serine proteases must present a minor role in the inflammation caused by B. pirajai snake venom. PMID:23499645

  8. The elongation defective1 mutant of Arabidopsis is impaired in the gene encoding a serine-rich secreted protein

    Microsoft Academic Search

    Kvin Lertpiriyapong; Zinmay Renee Sung

    2003-01-01

    Coordinated cell growth and differentiation is crucial for the development of higher plants. Using the elongation defective 1-1 (eld1-1) mutant, we cloned the ELD1 gene, which encodes a serine-rich protein. Genes homologous to ELD1 can be found in plants, includingArabidopsis, rice, and tobacco, but not in other organisms. Using reverse genetics, we identified a new allele, eld1-2, which is phenotypically

  9. Free energy of a potassium ion in a model of the channel formed by an amphipathic leucine-serine peptide

    Microsoft Academic Search

    Graham R. Smith; Mark S. P. Sansom

    2002-01-01

    . We use molecular dynamics simulations to investigate the position-dependent free energy of a potassium ion in a model of an ion channel formed by the synthetic amphipathic leucine-serine peptide, LS3. The channel model is a parallel bundle of six LS3 helices around which are packed 146 methane-like spheres in order to mimic a membrane. At either end of and

  10. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

    PubMed

    Pannkuk, Evan L; Risch, Thomas S; Savary, Brett J

    2015-01-01

    White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction. PMID:25785714

  11. Phosphorylation of HPV-16 E2 at Serine 243 Enables Binding to Brd4 and Mitotic Chromosomes

    PubMed Central

    Chang, Szu-Wei; Liu, Wei-Chen; Liao, Kuan-Yu; Tsao, Yeou-Ping; Hsu, Pang-Hung; Chen, Show-Li

    2014-01-01

    The papillomavirus E2 protein is involved in the maintenance of persistent infection and known to bind either to cellular factors or directly to mitotic chromosomes in order to partition the viral genome into the daughter cells. However, how the HPV-16 E2 protein acts to facilitate partitioning of the viral genome remains unclear. In this study, we found that serine 243 of HPV-16 E2, located in the hinge region, is crucial for chromosome binding during mitosis. Bromodomain protein 4 (Brd4) has been identified as a cellular binding target through which the E2 protein of bovine papillomavirus type 1 (BPV-1) tethers the viral genome to mitotic chromosomes. Mutation analysis showed that, when the residue serine 243 was substituted by glutamic acid or aspartic acid, whose negative charges mimic the effect of constitutive phosphorylation, the protein still can interact with Brd4 and colocalize with Brd4 in condensed metaphase and anaphase chromosomes. However, substitution by the polar uncharged residues asparagine or glutamine abrogated Brd4 and mitotic chromosome binding. Moreover, following treatment with the inhibitor JQ1 to release Brd4 from the chromosomes, Brd4 and E2 formed punctate foci separate from the chromosomes, further supporting the hypothesis that the association of the HPV-16 E2 protein with the chromosomes is Brd4-dependent. In addition, the S243A E2 protein has a shorter half-life than the wild type, indicating that phosphorylation of the HPV-16 E2 protein at serine 243 also increases its half-life. Thus, phosphorylation of serine 243 in the hinge region of HPV-16 E2 is essential for interaction with Brd4 and required for host chromosome binding. PMID:25340539

  12. A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent

    Microsoft Academic Search

    E. Vijay Kumar; M. Srijana; K. Kiran Kumar; N. Harikrishna; Gopal Reddy

    2011-01-01

    A serine alkaline protease from a newly isolated alkaliphilic Bacillus altitudinis GVC11 was purified and characterized. The enzyme was purified to homogeneity by acetone precipitation, DEAE-cellulose anion\\u000a exchange chromatography with 7.03-fold increase in specific activity and 15.25% recovery. The molecular weight of alkaline\\u000a protease was estimated to be 28 kDa by SDS PAGE and activity was further assessed by zymogram analysis.

  13. Serine7 of the RNA Polymerase II CTD Is Specifically Required for snRNA Gene Expression

    Microsoft Academic Search

    Sylvain Egloff; Dawn O'Reilly; Rob D. Chapman; Alice Taylor; Katrin Tanzhaus; Laura Pitts; Dirk Eick; Shona Murphy

    2007-01-01

    RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA

  14. Cloning of a novel testis specific protein serine\\/threonine phosphatase, PPN 58A, from Drosophila melanogaster

    Microsoft Academic Search

    Christopher G Armstrong; Viktor Dombradi; David J Mann; Patricia T. W Cohen

    1998-01-01

    A gene encoding a novel member of the PPP family of protein serine\\/threonine phosphatases, termed PPN 58A, was cloned from Drosophila melanogaster. The deduced amino acid sequence of PPN 58A exhibits 59–62% identity to D. melanogaster PP1 isoforms, 51% identity to D. melanogaster PPY 55A and ?40% identity to other members of the PPP family. The single copy gene PPN

  15. Serine proteases HTRA1 and HTRA3 are down-regulated with increasing grades of human endometrial cancer

    Microsoft Academic Search

    Marissa A. Bowden; Lisa A. Di Nezza-Cossens; Tom Jobling; Lois A. Salamonsen; Guiying Nie

    2006-01-01

    Objective.The high temperature requirement factor A (HTRA) family consists of serine proteases with domains homologous to those of bacterial HTRA. Four human HTRA members have been described: HTRA1–4. HTRA1 and HTRA3 share a high degree of domain homologies and may therefore share a functional similarity. HTRA1 mRNA and protein is reported to be down-regulated in SV40-transformed cells, a malignant melanoma

  16. Effects of bis (maltolato) oxovanadium (IV) on protein serine kinases in skeletal muscle of streptozotocin-diabetic rats

    Microsoft Academic Search

    Sanjay Bhanot; Jaspal Girn; Patrick Puccheret; John H. McNeill

    1999-01-01

    The in vivo effects of bis(maltolato)oxovanadium (IV) (BMOV) on the activity of protein serine kinases in skeletal muscle of STZ-diabetic Wistar rats were studied. BMOV was administered to STZ-diabetic rats at a concentration of 0.75 mg\\/ml for 8 weeks. Chronic BMOV treatment completely normalized plasma glucose levels in the diabetic animals after 8 weeks of treatment. Insulin-stimulated ERK-1 and ERK-2

  17. Serine\\/Arginine Protein-Specific Kinase 2 Promotes Leukemia Cell Proliferation by Phosphorylating Acinus and Regulating Cyclin A1

    Microsoft Academic Search

    Sung-Wuk Jang; Seung-ju Yang; Asa Ehlen; Shaozhong Dong; Hanna Khoury; Jing Chen; Jenny L. Persson; Keqiang Ye

    Serine\\/arginine (SR) protein-specific kinase (SRPK), a family of cell cycle-regulated protein kinases, phosphorylate SR domain-containing proteins in nuclear speckles and mediate the pre-mRNA splicing.However, the physiologic roles of this event in cell cycle are incompletely understood.Here, we show that SRPK2 binds and phosphorylates acinus, an SR protein essential for RNA splicing, and redistributes it from the nuclear speckles to the

  18. Involvement of a Magnaporthe grisea Serine\\/Threonine Kinase Gene, MgATG1, in Appressorium Turgor and Pathogenesis

    Microsoft Academic Search

    Xiao-Hong Liu; Jian-Ping Lu; Lei Zhang; Bo Dong; Hang Min; Fu-Cheng Lin

    2007-01-01

    We isolated an MgATG1 gene encoding a serine\\/threonine protein kinase from the rice blast fungus Magnaporthe grisea .I n theMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of

  19. Lack of the alanine-serine-cysteine transporter 1 causes tremors, seizures, and early postnatal death in mice.

    PubMed

    Xie, Xinmin; Dumas, Theodore; Tang, Lamont; Brennan, Thomas; Reeder, Thadd; Thomas, Winston; Klein, Robert D; Flores, Judith; O'Hara, Bruce F; Heller, H Craig; Franken, Paul

    2005-08-01

    The Na(+)-independent alanine-serine-cysteine transporter 1 (Asc-1) is exclusively expressed in neuronal structures throughout the central nervous system (CNS). Asc-1 transports small neutral amino acids with high affinity especially for D-serine and glycine (K(i): 8-12 microM), two endogenous glutamate co-agonists that activate N-methyl-D-aspartate (NMDA) receptors through interacting with the strychnine-insensitive glycine binding-site. By regulating D-serine (and possibly glycine) levels in the synaptic cleft, Asc-1 may play an important role in controlling neuronal excitability. We generated asc-1 gene knockout (asc-1(-/-)) mice to test this hypothesis. Behavioral phenotyping combined with electroencephalogram (EEG) recordings revealed that asc-1(-/-) mice developed tremors, ataxia, and seizures that resulted in early postnatal death. Both tremors and seizures were reduced by the NMDA receptor antagonist MK-801. Extracellular recordings from asc-1(-/-) brain slices indicated that the spontaneous seizure activity did not originate in the hippocampus, although, in this region, a relative increase in evoked synaptic responses was observed under nominal Mg(2+)-free conditions. Taken together with the known neurochemistry and neuronal distribution of the Asc-1 transporter, these results indicate that the mechanism underlying the behavioral hyperexcitability in mutant mice is likely due to overactivation of NMDA receptors, presumably resulting from elevated extracellular D-serine. Our study provides the first evidence to support the notion that Asc-1 transporter plays a critical role in regulating neuronal excitability, and indicate that the transporter is vital for normal CNS function and essential to postnatal survival of mice. PMID:16026768

  20. Molecular characterization and influence on fungal development of ALP2, a novel serine proteinase from Aspergillus fumigatus

    Microsoft Academic Search

    Utz Reichard; Garry T. Cole; Terry W. Hill; Reinhard Rüchel; Michel Monod

    2000-01-01

    A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same