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Sample records for phosphatidyl serine ps

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

    PubMed

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

    2009-05-01

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

  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 naïve 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.

  3. Phosphatidyl glycerophosphate phosphatase.

    PubMed

    Chang, Y Y; Kennedy, E P

    1967-09-01

    An enzyme (phosphatidyl glycerophosphate phosphatase) that catalyzes the formation of phosphatidyl glycerol from phosphatidyl glycerophosphate has been rendered soluble by treatment of the particulate fraction of E. coli with Triton X-100 in the presence of EDTA, and has been partially purified. The enzyme is specific for phosphatidyl glycerophosphate and does not catalyze the hydrolysis of other simple phosphomonoesters. It requires Mg(++) for activity and is inhibited by sulfhydryl agents. Some other properties of the enzyme are also described.

  4. Autoradiography of phosphatidyl choline

    SciTech Connect

    Saffitz, J.E.; Gross, R.W.; Williamson, J.R.; Sobel, B.E.

    1981-03-01

    Saturated choline phosphatides are extracted during conventional tissue processing for electron microscopy. To facilitate autoradiographic subcellular localization of arrhythmogenic myocardial phospholipids, we evaluated tissue processing procedures for preservation of saturated phosphatidyl choline (PC). Suspensions, of a murine plasmacytoma were incubated with negative, unilamellar liposomes containing 14C-choline-labeled PC or 14C-1-palmitate dipalmitoyl PC. Extraction of radioactivity was monitored at each processing step by liquid scintillation spectrometry. Conventional fixation with glutaraldehyde and osmium tetroxide followed by acetone dehydration and Spurr's plastic embedding led to extraction of nearly all radioactivity. However, treatment of cells with 1.5% tannic acid after glutaraldehyde but before osmium tetroxide fixation preserved 93.1 +/- .6% of 14C-choline-labeled PC. Virtually identical results were obtained with dipalmitoyl PC. Autoradiography demonstrated no significant translocation of labeled PC from plasmacytoma cells to unlabeled avian erythrocytes, mixed in equal proportions after fixation but before dehydration and embedding.

  5. Phosphatidyl-hydroxytyrosol and phosphatidyl-tyrosol bilayer properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroxytyrosol and tyrosol phospholipids were enzymatically synthesized and investigated for their bilayer properties. Dynamic light scattering demonstrated that hand extrusion at 100 nm consistently resulted in liposomes of nearly 85 nm diameter for both phosphatidyl-hydroxytyrosol (DOPHT) and phos...

  6. Phosphatidyl-hydroxytyrosol and phosphatidyl-tyrosol bilayer properties.

    PubMed

    Evans, Kervin O; Compton, David L

    2017-01-01

    Hydroxytyrosol and tyrosol phospholipids were enzymatically synthesized and investigated for their bilayer properties. Dynamic light scattering demonstrated that hand extrusion at 100nm consistently resulted in liposomes of nearly 85nm diameter for both phosphatidyl-hydroxytyrosol (DOPHT) and phosphatidyl-tyrosol (DOPT). Transmission electron microscopy showed DOPT and DOPHT liposomes extruded at 100-nm to be spherical and non-distinctive from one another. Zeta potential measurements resulted in surface charges<-25mV, demonstrating both DOPT and DOPHT form highly stable liposomes. Quartz crystal microbalance with dissipation monitoring measurements demonstrated that liposomal adsorption was dependent on a combination of DOPT (or DOPHT) mole-percent and calcium ions concentration. Fluorescence anisotropy measurements indicated that melting temperatures of DOPT and DOPHT were below 4°C, suggesting that adsorption behavior and liposome formation was limited by electrostatic interactions and not gel-state formation.

  7. The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

    PubMed Central

    Kalina, M; Pease, DC

    1977-01-01

    The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains. PMID:71301

  8. Transfer of the phosphatidyl moiety of phosphatidylglycerol to phosphatidylethanolamine in Escherichia coli.

    PubMed Central

    Yokota, K; Kito, M

    1982-01-01

    Phosphatidylglycerol was pulse-labeled with radioactive lipid precursors in a serine auxotroph of Escherichia coli. Most of the radioactivity of phosphatidylglycerol labeled in a serine-depleted medium was transferred to phosphatidylethanolamine during a chase in the presence of L-serine, but not in its absence. Metabolism of fatty acyl moieties labeled with [1-14C]acetate, acylated glycerol moieties labeled with [2-3H]glycerol, and phosphate moieties labeled with 32Pi, followed by a chase in the presence of cerulenin, showed that the intact phosphatidyl moiety of phosphatidylglycerol was transferred to phosphatidylethanolamine. The composition of phosphatidylethanolamine molecular species was unaltered and not perturbed by the transfer of the phosphatidyl moiety of phosphatidylglycerol. The increase of phosphatidylethanolamine with a concomitant decrease of phosphatidylglycerol was not coupled with the postulated turnover of phosphatidylglycerol to membrane-derived oligosaccharides and lipoprotein. It is suggested that phosphatidylglycerol is capable of providing its phosphatidyl moiety for the production of phosphatidylethanolamine in response to the relief of serine limitation by addition of L-serine. PMID:6807966

  9. Biosynthesis of phosphatidyl glycerophosphate in Escherichia coli.

    PubMed

    Chang, Y Y; Kennedy, E P

    1967-09-01

    An enzyme (L-glycerol 3-phosphate: CMP phosphatidyltransferase) catalyzing the synthesis of phosphatidyl glycerophosphate from CDP-diglyceride and L-glycerol 3-phosphate has been rendered soluble by treatment of the particulate, membrane-containing fraction of E. coli with Triton X-100 and has been partially purified. The enzyme, devoid of phosphatidyl glycerophosphatase activity, is specific for L-glycerol 3-phosphate and is completely dependent upon added Mg(++) or Mn(++) for activity. It has high affinity for CDP-diglyceride and can be used for the assay of this nucleotide. Other properties of the enzyme are also described.

  10. Isolation and Characterization of Phosphatidyl Choline from Spinach Leaves.

    ERIC Educational Resources Information Center

    Devor, Kenneth A.

    1979-01-01

    This inexpensive but informative experiment for undergraduate biochemistry students involves isolating phosphatidyl choline from spinach leaves. Emphasis is on introducing students to techniques of lipid extraction, separation of lipids, identification using thin layer chromatography, and identification of fatty acids. Three periods of three hours…

  11. Detection of related substances in polyene phosphatidyl choline extracted from soybean and in its commercial capsule by comprehensive supercritical fluid chromatography with mass spectrometry compared with HPLC with evaporative light scattering detection.

    PubMed

    Jiang, Qikun; Liu, Wanjun; Li, Xiaoting; Zhang, Tianhong; Wang, Yongjun; Liu, Xiaohong

    2016-01-01

    Supercritical fluid chromatography with tandem mass spectrometry was used to comprehensively profile polyene phosphatidyl choline (PPC) extracted from soybean. We achieved an efficient chromatographic analysis using a BEH-2EP column (3 × 100 mm(2) , 1.7 μm) with a mobile phase consisting of CO2 and a cosolvent in gradient combination at a flow rate of 1.0 mL/min. The cosolvent consisted of methanol, acetonitrile, and water (containing 10 mM ammonium acetate and 0.2% formic acid). The total single-run time was 7 min. We used this method to accurately detect ten different phospholipids (PLs) during extraction. The limits of quantification for phosphatidyl choline, lyso-phosphatidylcholine (LPC), phosphatidic acid (PA), sphingomyelin, phosphatidyl glycerol, phosphatidyl inositol (PI), cholesterol, cardiolipin, phosphatidyl serine, and phosphatidyl ethanolamine (PE) were 20.6, 19.52, 1.21, 2.38, 0.50, 2.28, 54.3, 0.60, 0.65, and 4.85 ng/mL, respectively. However, adopting the high-performance liquid chromatography with evaporative light scattering detection method issued by the China Food and Drug Administration, only PA, LPC, PE, PI, and PPC could be analyzed accurately, and the limits of quantification were 33.89, 60.5, 30.3, 10.9, and 61.79 μg/mL, respectively. The total single-run time was at the least 20 min. Consequently, the supercritical fluid chromatography with tandem mass spectrometry method was more suitable for the analysis of related PLs.

  12. 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

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

    PubMed

    Gilbert, G E; Drinkwater, D

    1993-09-21

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

  14. Serine proteases inhibiting cyanopeptides.

    PubMed

    Radau, G

    2000-08-01

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

  15. Pathogenic PS1 phosphorylation at Ser367

    PubMed Central

    Maesako, Masato; Horlacher, Jana; Zoltowska, Katarzyna M; Kastanenka, Ksenia V; Kara, Eleanna; Svirsky, Sarah; Keller, Laura J; Li, Xuejing; Hyman, Bradley T; Bacskai, Brian J; Berezovska, Oksana

    2017-01-01

    The high levels of serine (S) and threonine (T) residues within the Presenilin 1 (PS1) N-terminus and in the large hydrophilic loop region suggest that the enzymatic function of PS1/γ-secretase can be modulated by its ‘phosphorylated’ and ‘dephosphorylated’ states. However, the functional outcome of PS1 phosphorylation and its significance for Alzheimer’s disease (AD) pathogenesis is poorly understood. Here, comprehensive analysis using FRET-based imaging reveals that activity-driven and Protein Kinase A-mediated PS1 phosphorylation at three domains (domain 1: T74, domain 2: S310 and S313, domain 3: S365, S366, and S367), with S367 being critical, is responsible for the PS1 pathogenic ‘closed’ conformation, and resulting increase in the Aβ42/40 ratio. Moreover, we have established novel imaging assays for monitoring PS1 conformation in vivo, and report that PS1 phosphorylation induces the pathogenic conformational shift in the living mouse brain. These phosphorylation sites represent potential new targets for AD treatment. DOI: http://dx.doi.org/10.7554/eLife.19720.001 PMID:28132667

  16. Phosphatidyl derivative of hydroxytyrosol. In vitro intestinal digestion, bioaccessibility, and its effect on antioxidant activity.

    PubMed

    Martin, Diana; Moran-Valero, Maria I; Casado, Víctor; Reglero, Guillermo; Torres, Carlos F

    2014-10-08

    Intestinal digestion of phosphatidyl derivatives of HT (PHT) and its bioaccessibility under in vitro conditions was performed. First, an in vitro intestinal digestion model for phospholipids was developed. The impact of digestion in the antioxidant ability of PHT was also assayed. PHT was progressively hydrolyzed to lyso-PHT. However, digestion was slower than the phospholipid control. Nevertheless, most hydrolysis products were found at the micellar phase fraction, meaning a high bioaccessibility. Either PHT or digested PHT showed lower antioxidant activity than HT. However, PHT improved its antioxidant ability after digestion, likely related to lyso-PHT. As a summary, the synthetic phosphatidyl derivative of HT as PHT is recognized by phospholipases during simulation of intestinal digestion, although less efficiently than analogous phospholipids. Nevertheless, taking into account the bioaccessibility and the antioxidant activity of digested PHT, the potential of carriers of HT under the form of phospholipids might be of interest.

  17. Radiotracer Evidence Implicating Phosphoryl and Phosphatidyl Bases as Intermediates in Betaine Synthesis by Water-Stressed Barley Leaves 12

    PubMed Central

    Hitz, William D.; Rhodes, David; Hanson, Andrew D.

    1981-01-01

    In barley, glycine betaine is a metabolic end product accumulated by wilted leaves; betaine accumulation involves acceleration of de novo synthesis from serine, via ethanolamine, N-methylethanolamines, choline, and betaine aldehyde (Hanson, Scott 1980 Plant Physiol 66: 342-348). Because in animals and microorganisms the N-methylation of ethanolamine involves phosphatide intermediates, and because in barley, wilting markedly increases the rate of methylation of ethanolamine to choline, the labeling of phosphatides was followed after supplying [14C]ethanolamine to attached leaf blades of turgid and wilted barley plants. The kinetics of labeling of phosphatidylcholine and betaine showed that phosphatidylcholine became labeled 2.5-fold faster in wilted than in turgid leaves, and that after short incubations, phosphatidylcholine was always more heavily labeled than betaine. In pulse-chase experiments with wilted leaves, label from [14C]ethanolamine continued to accumulate in betaine as it was being lost from phosphatidylcholine. When [14C]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 [14C]ethanolamine or [14C]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 [14C]ethanolamine and [14C]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 14C-labeled precursor was depleted. Phosphatidylmonomethylethanolamine and phosphatidylcholine were also significantly labeled from [14C]ethanolamine and [14C]formate at early

  18. Conformers of Gaseous Serine.

    PubMed

    He, Kedan; Allen, Wesley D

    2016-08-09

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

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

    PubMed Central

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

    2015-01-01

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

  20. The phosphatidyl-myo-inositol mannosyltransferase PimA is essential for Mycobacterium tuberculosis growth in vitro and in vivo.

    PubMed

    Boldrin, Francesca; Ventura, Marcello; Degiacomi, Giulia; Ravishankar, Sudha; Sala, Claudia; Svetlikova, Zuzana; Ambady, Anisha; Dhar, Neeraj; Kordulakova, Jana; Zhang, Ming; Serafini, Agnese; Vishwas, K G; Vishwas, V G; Kolly, Gaëlle S; Kumar, Naveen; Palù, Giorgio; Guerin, Marcelo E; Mikusova, Katarina; Cole, Stewart T; Manganelli, Riccardo

    2014-10-01

    The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.

  1. The Phosphatidyl-myo-Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

    PubMed Central

    Boldrin, Francesca; Ventura, Marcello; Degiacomi, Giulia; Ravishankar, Sudha; Sala, Claudia; Svetlikova, Zuzana; Ambady, Anisha; Dhar, Neeraj; Kordulakova, Jana; Zhang, Ming; Serafini, Agnese; Vishwas, V. G.; Kolly, Gaëlle S.; Kumar, Naveen; Palù, Giorgio; Guerin, Marcelo E.; Mikusova, Katarina; Cole, Stewart T.

    2014-01-01

    The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs. PMID:25049093

  2. 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.

  3. Serine Proteases of Parasitic Helminths

    PubMed Central

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

    2015-01-01

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

  4. The ps in therapeutics.

    PubMed

    Newton, David W

    2012-01-01

    The decline of ps (pharmaceutical sciences) content and emphasis, especially pharmaceutics in pharmacy education, has been followed by pharmacy practice journals since the final BS to PharmD degree transition of 1990-2000. The particular deficit of drug compatibility, compounding, packaging, reactivity, solubility, stability and storage instruction, and information was a major impetus for the 1996 premier of the International journal of pharmaceutical compounding and the 2011 introduction of the Science and technology for the hospital pharmacist electronic newsletter. The four ps examples provided in this article to corroborate the introduction to Vol. 1, No. 2 of the Science and technology for the hospital pharmacist, which featured Dr. Richard Penna's prudent reflection that biological, chemical, and physical ps facts and knowledge are vital to patient care.

  5. 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

  6. Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu.

    PubMed

    Matthews, S A; Rozengurt, E; Cantrell, D

    1999-09-10

    Activation of the serine kinase protein kinase D (PKD)/PKCmicro is controlled by the phosphorylation of two serine residues within its activation loop via a PKC-dependent signaling cascade. In this study we have identified the C-terminal serine 916 residue as an in vivo phosphorylation site within active PKD/PKCmu. An antibody that recognized PKD/PKCmu proteins specifically phosphorylated on the serine 916 residue was generated and used to show that phosphorylation of Ser-916 is induced by phorbol ester treatment of cells. Thus, the pS916 antibody is a useful tool to study the regulation of PKD/PKCmu activity in vivo. Antigen receptor ligation of T and B lymphocytes also induced phosphorylation of the serine 916 residue of PKD/PKCmu. Furthermore the regulatory FcgammaRIIB receptor, which mediates vital negative feedback signals to the B cell antigen receptor complex, inhibited the antigen receptor-induced activation and serine 916 phosphorylation of PKD/PKCmu. The degree of serine 916 phosphorylation during lymphocyte activation and inhibition exactly correlated with the activation status of PKD/PKCmu. Moreover, using different mutants of PKD/PKCmu, we show that serine 916 is not trans-phosphorylated by an upstream kinase but is rather an autophosphorylation event that occurs following activation of PKD/PKCmu.

  7. L-serine in disease and development.

    PubMed Central

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

    2003-01-01

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

  8. Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

    SciTech Connect

    Johnsson, Anna-Karin; Karlsson, Roger

    2012-01-15

    Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

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

    PubMed

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

    1999-06-04

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

  10. Serine recombinases as tools for genome engineering.

    PubMed

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

    2011-04-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Viscoelastic and Transport Properties of Sulfonated PS-PIB-PS Block Copolymers

    DTIC Science & Technology

    2001-05-01

    000 1200 Time (min) Figure 13. IR intensity vs. time for ethanol through three sulfonated P5-PIB- PS membranes. Figure 14 shows a comparison of four ...sulfonated PS -PIB- PS membrane. Table 1 shows a comparison of the equilibrium sorbtion values for the four alcohols in the sulfonated PS -PIB- PS . Table 1...Army Research Laboratory Aberdeen Proving Ground, MD 21005-5069 ARL-TR-2482 May2001 Viscoelastic and Transport Properties of Sulfonated PS -PIB- PS

  13. Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: D-phosphatidyl-myo-inositol 3-phosphate.

    PubMed Central

    Stephens, L; Hawkins, P T; Downes, C P

    1989-01-01

    When human 1321 N1 astrocytoma cells were labelled to steady state with [3H]inositol and briefly with [32P]orthophosphate, a compound which contained both radiotracers and which co-migrated with phosphatidylinositol-myo-inositol 4-phosphate during t.l.c. could be extracted in acidic chloroform/methanol. Treatment with methylamine under conditions which lead to deacylation of conventional glycerophospholipids yielded a water-soluble moiety which was labelled with both radioisotopes and was eluted from an anion-exchange h.p.l.c. column with a retention time similar to, but distinct from, that of glycerophosphoinositol 4-phosphate. Experiments using sodium periodate and selective phosphatase enzymes to degrade this compound systematically generated a series of products which suggested the structure of the parent phospholipid was phosphatidyl-myo-inositol 3-phosphate (PtdIns3P). PtdIns3P is metabolically closely related to the pool(s) of inositol phospholipid(s) that serves as substrate(s) for an agonist-sensitive phosphoinositidase C, as the levels of PtdIns3P fell significantly when 1321 N1 cells were stimulated with carbachol. The relative rate of turnover of the inositol moiety of PtdIns3P is similar to that of both of the major polyphosphoinositides and significantly higher than that of total cellular phosphatidyl-myo-inositol. This suggests that all three polyphosphoinositides are synthesized from a common, rapidly metabolized, pool of phosphatidyl-myo-inositol. PMID:2541684

  14. G/sub o/ protein of fat cells: role in hormonal regulation of agonist-stimulated phosphatidyl inositol breakdown

    SciTech Connect

    Rapiejko, P.J.; Northup, J.K.; Malbon, C.C.

    1986-05-01

    Incubating rat fat cell membranes in the presence of (/sup 32/P)NAD/sup +/ and pertussis toxin (PT) results in the ADP-ribosylation of two peptides (M/sub r/ = 41,000 and 40,000). The 41,000-M/sub r/ peptide is the inhibitory G-protein of adenylate cyclase (G/sub i/). The 40,000-M/sub r/ peptide radiolabeled in the presence of (/sup 32/P)NAD/sup +/ and PT has been purified from rabbit heart and bovine brain, but has not been identified uniformly in membranes of fat cells. Two rabbit polyclonal antisera raised against the alpha-subunit of bovine brain G/sub o/ were used to probe the nature of the 40,000-M/sub r/ peptide in rat fat cell membranes that had been separated by gel electrophoresis in the presence of sodium dodecyl sulfate and transferred electrophoretically to nitrocellulose. Both antisera specific for the alpha-subunit of G/sub o/ recognized the M/sub r/ = 40,000 peptide of fat cells that is ADP-ribosylated in the presence of PT. PT treatment of rat fat cells blocks epinephrine-stimulated inositol 1,4,5 trisphosphate (IP/sub 3/) generation. The inhibition of IP/sub 3/ generation by PT suggests a role for either G/sub i/ or G/sub o/ in receptor-mediated phosphatidyl inositol breakdown in the rat fat cell.

  15. Restricted immunoglobulin variable region gene usage by normal Ly-1 (CD5+) B cells that recognize phosphatidyl choline

    PubMed Central

    1989-01-01

    5-15% of lymphocytes in the peritoneums of normal adult B10.H-2aH- 4bp/Wts (2a4b) mice are CD5+ (Ly-1) B cells that recognize phosphatidyl choline (PtC), a phospholipid component of all mammalian cells. We produced a set of IgM-secreting hybridomas from the peritoneal cells of normal, adult 2a4b mice. We found that this set of hybridomas shows a similarly high frequency of antibodies specific for PtC (21 of 86) that also react with bromelain-treated mouse erythrocytes. Restriction fragment analysis of Ig gene rearrangements and analysis of expressed Ig idiotypes reveal that these cells use a restricted set of variable region genes to generate the PtC-specific antibodies. The Ig genes used by the PtC-specific hybridomas appear to be the same as those found in the PtC-specific Ly-1 B cell lymphomas, CH27 and CH34. PMID:2499651

  16. MUW Approach of PS OCT

    NASA Astrophysics Data System (ADS)

    Hitzenberger, Christoph K.; Pircher, Michael

    Polarization sensitive (PS) OCT is a functional extension of OCT that exploits the light's polarization state to generate intrinsic, tissue specific contrast and enables quantitative measurements of tissue parameters. This chapter explains the technique, discusses polarization-changing light-tissue interactions and demonstrates the application of PS-OCT to retinal imaging. Two polarization-changing light-tissue interactions are discussed and their use for retinal diagnostics are demonstrated: (i) birefringence, which is found in fibrous tissues like the retinal nerve fiber layer and can be used for glaucoma diagnostics; and (ii) depolarization, which is observed in the retinal pigment epithelium (RPE) and can be used to segment the RPE and associated lesions like drusen or geographic atrophies in age related macular degeneration.

  17. Results from experiment PS199

    NASA Astrophysics Data System (ADS)

    Agnello, M.; Ahmidouch, A.; Arvieux, J.; Bertini, R.; Birsa, R.; Bradamante, F.; Bressan, A.; Bressani, T.; Chiavassa, E.; Torre-Colautti, S. Dalla; De Marco, N.; Faivre, J. C.; Giorgi, M.; Heer, E.; Hess, R.; Iazzi, F.; Kunne, R. A.; Lamanna, M.; Luc, C. Lechanoine-Le; Macciotta, M. P.; Martin, A.; Mascarini, C.; Masoni, A.; Minetti, B.; Musso, A.; Penzo, A.; Perrot-Kunne, F.; Piccotti, A.; Puddu, G.; Rapin, D.; Serci, S.; Schiavon, P.; Tessarotto, F.

    1993-06-01

    The aim of the experiment PS199 was to study the spin structure of the charge-exchange overlinepp → overlinenn channel at LEAR. During 1990 we collected data at several energies to measure the analyzing power Aon and the spin depolarization parameter Donon.Results for Aon at all energies and first results for Donon at 905 MeV/c incident antiproton momentum are presented.

  18. Muramyl tripeptide-phosphatidyl ethanolamine encapsulated in liposomes (L-MTP-PE) in the treatment of osteosarcoma.

    PubMed

    Meyers, Paul A; Chou, Alexander J

    2014-01-01

    Bacille Calmette-Guerin (BCG) has been used for decades as an immune stimulant to treat cancer. Early work by Fidler and Kleinerman identified muramyl dipeptide (MDP) as a critical component of the BCG cell wall which retained most of the immunostimulatory properties of the native BCG. Addition of a peptide to MDP resulted in muramyl tripeptide (MTP) which allowed incorporation into liposomal membranes. The resulting pharmaceutical, liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE or mifamurtide) showed activity in preclinical models of human cancers. Phase I studies documented the safety of the compound for human administration. These trials did not reach a maximally tolerated dose (MTD), and the dose chosen for phase II trials was a biologically optimized dose, not an MTD. Phase II studies showed decreased risk of further recurrence in patients who received mifamurtide after surgical ablation of metastatic osteosarcoma. A phase III prospective randomized trial demonstrated a statistically significant reduction in the risk of death from osteosarcoma when MTP was added to systemic chemotherapy for the treatment of localized osteosarcoma. The same trial allowed treatment of patients who presented with initially metastatic disease. While the overall and event-free survival was improved in patients with metastatic osteosarcoma who received L-MTP-PE, the sample size was small and the improvement did not achieve conventional statistical significance. From 2008 to 2012, patients with metastatic and recurrent osteosarcoma were given L-MTP-PE in an expanded access trial, and the results suggest a decreased risk of subsequent recurrence and death with the inclusion of L-MTP-PE in the treatment strategy for these high-risk patients.

  19. 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.

  20. Electrophysiological and Electrochemical Methods Development for the Detection of Biologically Active Chemical Agents

    DTIC Science & Technology

    1988-11-01

    Hexane +/- 90-130 Very good Phosphatidic acid * Sigma Hexane ++ 60-90 Good PA:lecithin 4:1 Sigma Pentane +++ >90 Good Cholesterol Sigma Hexane - None...to chamber in a concentration of 5-15 mg/ml of solvent. P’tdyl, phosphatidyl; PA, phosphatidic acid ; PC, phosphatidyl choline; PS, phosphatidyl serine...in Figure 15 were from a lecithin:sphingomyelin (5:1) bilayer. The records in Figure 16 were from a phosphatidic acid bilayer. Amphotericin B inserts

  1. Production Ratio for Para- and Ortho-Ps in Photodetachment of Ps^-

    NASA Astrophysics Data System (ADS)

    Igarashi, Akinori

    2017-01-01

    Para- and ortho-Ps atoms are formed in the photodetachment of positronium negative ion. Since the lifetime against the pair annihilation is much shorter for para-Ps( ns) than for ortho-Ps( ns), the production ratio of para- and ortho-Ps atoms is important for the photodetachment experiments. We have derived the ratio explicitly.

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

    PubMed

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

    2013-12-01

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

  3. Polarizabilities of the Ps Negative Ion

    NASA Technical Reports Server (NTRS)

    Bhatia, A. K.; Drachman, Richard J.

    2007-01-01

    We have calculated polarizabilities (alpha(sub l), beta(sub 1), gamma(sub l), alpha(sub 2), beta(sub 2), and gamma(sub 2)) of Ps(sup -) by the pseudostate method. These parameters can be used to calculate Rydberg states of Ps(sup -) in the presence of an external electron with high quantum numbers N and L. They are also of importance in a system containing Ps(sup -) bound to a proton [PsH], and also Rydberg states of Ps(sub 2).

  4. Revealing the multiple structures of serine

    PubMed Central

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

    2007-01-01

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

  5. Molecular markers of serine protease evolution

    PubMed Central

    Krem, Maxwell M.; Di Cera, Enrico

    2001-01-01

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

  6. 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

  7. Inhibition of neointimal formation by trans-resveratrol: role of phosphatidyl inositol 3-kinase-dependent Nrf2 activation in heme oxygenase-1 induction.

    PubMed

    Kim, Jung Woo; Lim, Sung Chul; Lee, Moo Yeol; Lee, Jeong Woon; Oh, Won Keun; Kim, Sang Kyum; Kang, Keon Wook

    2010-10-01

    Neointima, defined as abnormal growth of the intimal layer of blood vessels, is believed to be a critical event in the development of vascular occlusive disease. Although resveratrol's inhibitory effects on proliferation and migration of vascular smooth muscle cells has been reported, its activity on neointimal formation is still unclear. Oral administration of trans-resveratrol significantly suppressed intimal hyperplasia in a wire-injured femoral artery mouse model. In cultured vascular smooth muscle cells, trans-resveratrol inhibited platelet-derived growth factor-stimulated DNA synthesis and cell proliferation with down-regulation of cyclin D and pRB. Moreover, platelet-derived growth factor-induced production of reactive oxygen species was inhibited by trans-resveratrol and the compound induced heme oxygenase-1 (HO-1). The anti-proliferative activity of trans-resveratrol was reversed by an HO-1 inhibitor, ZnPPIX. Subcellular fractionation and reporter gene analyses revealed that trans-resveratrol increased the level of nuclear Nrf2 and antioxidant response element reporter activity, and that these were essential for the induction of HO-1. Trans-resveratrol also enhanced the activities of phosphatidyl inositol 3-kinase and extracellular signal regulated kinase, and phosphatidyl inositol 3-kinase was required for Nrf2/antioxidant response element-dependent HO-1 induction. These data have significant implications for the elucidation of the pharmacological mechanism by which trans-resveratrol prevents vascular occlusive diseases.

  8. [iPS cells in dentistry].

    PubMed

    Egusa, Hiroshi

    2012-01-01

    The discovery of the induced pluripotent stem (iPS) cell technology, which enables us to produce pluripotent stem cells by introducing a few genetic factors, commands considerable attention in the field of dentistry. These iPS cells may be of particular importance for developing innovative technologies to regenerate missing jaw bones and lost teeth, and there are expectations that several types of tissue stem cells and mucosal cells in the oral area can be used as an ideal iPS cell source. We previously reported that the gingiva, which is often resected during general dental treatments and treated as biomedical waste, is a promising source of iPS cells. In this review, the current trends in iPS cell research in dentistry are outlined, and future aspects of potential applications of the iPS cell technologies to dental treatments will be discussed.

  9. Dinaciclib potently suppresses MCL-1 and selectively induces the cell death in human iPS cells without affecting the viability of cardiac tissue

    PubMed Central

    Alsayegh, Khaled; Matsuura, Katsuhisa; Sekine, Hidekazu; Shimizu, Tatsuya

    2017-01-01

    Induced pluripotent stem (iPS) cells hold great potential for being a major source of cells for regenerative medicine. One major issue that hinders their advancement to clinic is the persistence of undifferentiated iPS cells in iPS-derived tissue. In this report, we show that the CDKs inhibitor, Dinaciclib, selectively eliminates iPS cells without affecting the viability of cardiac cells. We found that low nanomolar concentration of dinaciclib increased DNA damage and p53 protein levels in iPSCs. This was accompanied by negative regulation of the anti-apoptotic protein MCL-1. Gene knockdown experiments revealed that p53 downregulation only increased the threshold of dinaciclib induced apoptosis in iPS cells. Dinaciclib also inhibited the phosphorylation of Serine 2 of the C-terminal domain of RNA Polyemrase II through CDK9 inhibition. This resulted in the inhibition of transcription of MCL-1 and the pluripotency genes, NANOG and c-MYC. Even though dinaciclib caused a slight downregulation of MCL-1 in iPS-derived cardiac cells, the viability of the cells was not significantly affected, and beating iPS-derived cardiac cell sheet could still be fabricated. These findings suggest a difference in tolerance of MCL-1 downregulation between iPSCs and iPS-derived cardiac cells which could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues. PMID:28361959

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

    PubMed

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

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

  11. THE PCM/PS TELEMETRY SYSTEM,

    DTIC Science & Technology

    The relative transmitter power efficiency of the Pulse Code Modulation/Phase Shift (PCM/ PS ) system is com pared with other efficient telemetry...and a novel redundancy coding method are incorporated in a PCM/ PS system which in practice performs as well as the theoretical system analyzed by

  12. PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties.

    PubMed

    Leite, Natália Bueno; Aufderhorst-Roberts, Anders; Palma, Mario Sergio; Connell, Simon D; Ruggiero Neto, João; Beales, Paul A

    2015-09-01

    Polybia-MP1 (MP1) is a bioactive host-defense peptide with known anticancer properties. Its activity is attributed to excess serine (phosphatidylserine (PS)) on the outer leaflet of cancer cells. Recently, higher quantities of phosphatidylethanolamine (PE) were also found at these cells' surface. We investigate the interaction of MP1 with model membranes in the presence and absence of POPS (PS) and DOPE (PE) to understand the role of lipid composition in MP1's anticancer characteristics. Indeed we find that PS lipids significantly enhance the bound concentration of peptide on the membrane by a factor of 7-8. However, through a combination of membrane permeability assays and imaging techniques we find that PE significantly increases the susceptibility of the membrane to disruption by these peptides and causes an order-of-magnitude increase in membrane permeability by facilitating the formation of larger transmembrane pores. Significantly, atomic-force microscopy imaging reveals differences in the pore formation mechanism with and without the presence of PE. Therefore, PS and PE lipids synergistically combine to enhance membrane poration by MP1, implying that the combined enrichment of both these lipids in the outer leaflet of cancer cells is highly significant for MP1's anticancer action. These mechanistic insights could aid development of novel chemotherapeutics that target pathological changes in the lipid composition of cancerous cells.

  13. PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties

    PubMed Central

    Leite, Natália Bueno; Aufderhorst-Roberts, Anders; Palma, Mario Sergio; Connell, Simon D.; Neto, João Ruggiero; Beales, Paul A.

    2015-01-01

    Polybia-MP1 (MP1) is a bioactive host-defense peptide with known anticancer properties. Its activity is attributed to excess serine (phosphatidylserine (PS)) on the outer leaflet of cancer cells. Recently, higher quantities of phosphatidylethanolamine (PE) were also found at these cells’ surface. We investigate the interaction of MP1 with model membranes in the presence and absence of POPS (PS) and DOPE (PE) to understand the role of lipid composition in MP1’s anticancer characteristics. Indeed we find that PS lipids significantly enhance the bound concentration of peptide on the membrane by a factor of 7–8. However, through a combination of membrane permeability assays and imaging techniques we find that PE significantly increases the susceptibility of the membrane to disruption by these peptides and causes an order-of-magnitude increase in membrane permeability by facilitating the formation of larger transmembrane pores. Significantly, atomic-force microscopy imaging reveals differences in the pore formation mechanism with and without the presence of PE. Therefore, PS and PE lipids synergistically combine to enhance membrane poration by MP1, implying that the combined enrichment of both these lipids in the outer leaflet of cancer cells is highly significant for MP1’s anticancer action. These mechanistic insights could aid development of novel chemotherapeutics that target pathological changes in the lipid composition of cancerous cells. PMID:26331251

  14. D-serine transporter in Staphylococcus saprophyticus identified.

    PubMed

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

    2016-07-01

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

  15. The PS1 Gigapixel Camera

    NASA Astrophysics Data System (ADS)

    Tonry, John L.; Isani, S.; Onaka, P.

    2007-12-01

    The world's largest and most advanced digital camera has been installed on the Pan-STARRS-1 (PS1) telescope on Haleakala, Maui. Built at the University of Hawaii at Manoa's Institute for Astronomy (IfA) in Honolulu, the gigapixel camera will capture images that will be used to scan the skies for killer asteroids, and to create the most comprehensive catalog of stars and galaxies ever produced. The CCD sensors at the heart of the camera were developed in collaboration with Lincoln Laboratory of the Massachusetts Institute of Technology. The image area, which is about 40 cm across, contains 60 identical silicon chips, each of which contains 64 independent imaging circuits. Each of these imaging circuits contains approximately 600 x 600 pixels, for a total of about 1.4 gigapixels in the focal plane. The CCDs themselves employ the innovative technology called "orthogonal transfer." Splitting the image area into about 4,000 separate regions in this way has three advantages: data can be recorded more quickly, saturation of the image by a very bright star is confined to a small region, and any defects in the chips only affect only a small part of the image area. The CCD camera is controlled by an ultrafast 480-channel control system developed at the IfA. The individual CCD cells are grouped in 8 x 8 arrays on a single silicon chip called an orthogonal transfer array (OTA), which measures about 5 cm square. There are a total of 60 OTAs in the focal plane of each telescope.

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

    PubMed

    Jiang, Wei; Xia, Bingzhao; Liu, Ziduo

    2013-10-01

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

  17. PS1-1000305 an AGN outburst?

    NASA Astrophysics Data System (ADS)

    Drake, A. J.; Mahabal, A. A.; Djorgovski, S. G.; Graham, M. J.; Williams, R.; Prieto, J.; Catelan, M.; Christensen, E.; Beshore, E. C.; Larson, S. M.

    2010-07-01

    Kankare et al. (2010, ATel#2716) recently reported the discovery of an AGN outburst (PS1-1000305) detected in PS1 taken data on May 19.3 UT. The redshift of the AGN is given by Kankare et al. as z=0.77 with the host galaxy SDSS J152844.16+425722.5. We have extracted the five year archival CSS/CRTS lightcurve at the location of PS1-1000305.

  18. Conserved water molecules in bacterial serine hydroxymethyltransferases.

    PubMed

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

    2015-10-01

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

  19. Enteropeptidase, a type II transmembrane serine protease.

    PubMed

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

    2009-06-01

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

  20. Highly potent fibrinolytic serine protease from Streptomyces.

    PubMed

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

    2011-01-05

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

  1. Separation of PS-PMMA block copolymers from PS precursors via selective adsorption on nanoprous silica

    NASA Astrophysics Data System (ADS)

    Ryu, Chang Yeol

    2005-03-01

    We report a simple adsorption-based separation method using nanoporous silica in solution via controlling solvent quality to remove polystyrene (PS) homopolymers from polystyrene-poly(methyl methacrylate) (PS-PMMA) diblock copolymers. In particular, the solvent quality is controlled by employing binary mixed solvents of THF (good solvent) and isooctane (nonsolvent for both PS and PMMA). The aim of this work is to qualitatively study the competitive adsorption between PS and PS-PMMA and to provide a correlative understanding of polymer adsorption in nanopores with interaction chromatography techniques. In addition, the quantitative understanding of polymer adsorption is further employed to develop a simple polymer separation scheme for manipulating polymer adsorption via solvent quality. In particular, concentration changes of PS and PS-PMMA in the supernatant solution have been quantitatively measured for the adsorption studies using solvent gradient interaction chromatography techniques. We found that the PS-PMMA (43k-32k) selectively adsorb over PS (43k) precursors at the THF composition window between 42 % and 55% in THF/IO (v/v) mixed solvents. For THF/IO solvents with composition higher than 60 % THF, polymers did not adsorb to the nanoporous silica due to the good solvent quality.

  2. 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

  3. Serine ADP-Ribosylation Depends on HPF1.

    PubMed

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

    2017-03-02

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

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

    PubMed

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

    2001-06-01

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

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

    PubMed

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

    2016-04-01

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

  6. Nanocomposites Consisting of Nanoparticles with Multidentate PS Brushes Mixed with PS Matrices

    NASA Astrophysics Data System (ADS)

    Lee, Hyemin; Wooh, Sanghyuk; Lim, Jaehoon; Zorn, Matthias; Zentel, Rudolf; Char, Kookheon

    2011-03-01

    In order to prevent massive phase separation of nanoparticles (NP) in a polymer matrix, the relevant hybridization of NPs with polymer matrices has proven to be an effective method for the high performance of nanocomposites in applications. The surface of inorganic (gold or QD) NPs of various size was modified with polystyrene (PS) polymer brushes, poly(styrene)-block-poly(cysteamine methyl disulfide), by the ligand exchange procedure. The disulfide groups in the PS brushes act as anchoring blocks for NPs. Different PS brushes were prepared with different total molecular weights and mole fractions of disulfide moieties. Compared with NPs dispersed in PS without disulfide anchoring groups, NPs anchored with PS brushes through disulfide groups were uniformly distributed within PS matrices. The dispersion of NPs within a polymer matrix was found to be influenced by the total molecular weight of PS brushes as well as the number of anchoring disulfide groups. Furthermore, the effect of the ratio between relative size of NP and the radius of gyration of a polymer brush as well as the grafting density of PS brushes anchored onto NPs on the NP distribution within a polymer matrix is discussed.

  7. 100-ps framing-camera tube.

    PubMed

    Kalibjian, R

    1978-07-01

    The optoelectronic framing-camera tube described is capable of recording two-dimensional image frames with high spatial resolution in the <100-ps range. Framing is performed by streaking a two-dimensional electron image across narrow slits. The resulting dissected electron line images from the slits are restored into framed images by a restorer deflector operating synchronously with the dissector deflector. The number of framed images on the tube's viewing screen equals the number of dissecting slits in the tube. Performance has been demonstrated in a prototype tube by recording 135-ps-duration framed images of 2.5-mm patterns at the cathode. The limitation in the framing speed is in the external drivers for the deflectors and not in the tube design characteristics. Faster frame speeds in the <100-ps range can be obtained by use of faster deflection drivers.

  8. CRTS observations of recent PS1 transients

    NASA Astrophysics Data System (ADS)

    Drake, A. J.; Mahabal, A. A.; Djorgovski, S. G.; Graham, M. J.; Williams, R.; Prieto, J.; Catelan, M.; Christensen, E.; Beshore, E. C.; Larson, S. M.

    2010-08-01

    Valenti et al. (2010, ATel#2773) recently reported the discovery of an AGN outburst (PS1-1000382) detected in PS1 taken data on June 12.23 UT with magnitude g=17.9. The redshift of the AGN is given by Valenti et al. (2010) as z=0.435 and host galaxy SDSS J160414.08+091354.0. We have extracted the five year archival CSS/CRTS lightcurve at the location of PS1-1000382 and SDSS DR7 data.

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

    SciTech Connect

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

    2011-09-01

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

  10. Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

    PubMed Central

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

    2013-01-01

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

  11. Accelerators for the PS neutrino beam

    NASA Astrophysics Data System (ADS)

    Steerenberg, R.; Calviani, M.; Gschwendtner, E.; Pardons, A.; Vincke, H.

    2013-02-01

    A recent memorandum for an experimental proposal [1] was discussed during the CERN PS and SPS experimental committee (SPSC) of April 2011 and at the Research Board of June 2011. The proposed experiment, with objective to investigate the anomalous νμ → νe oscillations, aims at re-using the discontinued CERN PS Neutrino Facility (PSNF) and experimental zones to install a 150 ton liquid argon time projection chamber (LArTPC) as near detector and a 600 ton LArTPC as far detector. This article will summarize the experimental needs, the proposed facility layout, a primary beam production scheme and the requirements for the reconstruction of the PSNF.

  12. 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

  13. Approach to knowledge of the interaction between the constituents of contact lenses and ocular tears: mixed monolayers of poly(methyl methacrylate) and dipalmitoyl phosphatidyl choline.

    PubMed

    Miñones Conde, M; Conde, O; Trillo, J M; Miñones, J

    2011-04-05

    Mixed monolayers of poly(methyl methacrylate) (PMMA), the main component of hard contact lenses, and dipalmitoyl phosphatidyl choline (DPPC), a characteristic phospholipidic constituent of ocular tear films, were selected as an in vitro model in order to observe the behavior of contact lenses on the eye. Using Langmuir monolayer and Brewster angle microscopy (BAM) techniques, the interaction between both components was analyzed from the data of surface pressure-area isotherms, compressional modulus-surface pressure, and relative film thickness versus time elapsed from the beginning of compression, together with BAM images. Regardless of the surface pressure at which the molecular/monomer areas (A(m)) were recorded, the A(m) mole fractions of PMMA (X(PMMA)) plots show that the experimental results match the theoretical values calculated from additivity rule A(m) = X(PMMA)A(PMMA) + X(DPPC)A(DPPC). The application of the Crisp phase rule to the phase diagram of the PMMA-DPPC system can explain the existence of a mixed monolayer made up of miscible components with ideal behavior at surface pressures below 25 mN/m. However, at very high surface pressures, when collapse is reached (at 60 mN/m), the single collapsed components are segregated into two independent phases. These results allows us to argue that PMMA hard contact lenses in the eye do not alter the structural characteristics of the phospholipid (DPPC) in tears.

  14. 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.

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

    PubMed

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

    2016-09-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. 10th Anniversary P.S.

    ScienceCinema

    None

    2016-07-12

    John Adams parle de la préhistoire du P.S. avec présentation des dias. Le DG B.Gregory prend la parole. Les organisateurs présentent sous la direction du "Prof.Ocktette"(?) un sketch très humoristique (p.e.existence de Quark etc.....)

  18. 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.

  19. 10th Anniversary P.S.

    SciTech Connect

    2005-10-28

    John Adams parle de la préhistoire du P.S. avec présentation des dias. Le DG B.Gregory prend la parole. Les organisateurs présentent sous la direction du "Prof.Ocktette"(?) un sketch très humoristique (p.e.existence de Quark etc.....)

  20. The 4 Ps as a Guiding Perspective

    ERIC Educational Resources Information Center

    Kalsbeek, David H.

    2013-01-01

    A 4 Ps perspective addresses immediate needs: to help institutions gain traction in their retention strategies by framing and reframing the challenges and the possible responses, by challenging some of the traditional mental models about retention that can distract or dilute those strategies, and by offering focus and coherence to institutional…

  1. Serine one-carbon catabolism with formate overflow

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2010-10-01

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

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

    EPA Science Inventory

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2013-05-01

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

  6. Biomimetic nanoassemblies of 1-O-octodecyl-2-conjugated linoleoyl-sn-glycero-3-phosphatidyl gemcitabine with phospholipase A2-triggered degradation for the treatment of cancer.

    PubMed

    Zuo, Jing; Tong, Li; Du, Lina; Yang, Ming; Jin, Yiguang

    2017-04-01

    Phospholipids are important biomolecules with strong self-assembling ability to form biomembranes or liposomes. However, biomimetic prodrugs of phospholipids are not well known, including their self-assembling behavior at the air/water interface or in aqueous media. Here we design and prepare a biomimetic phospholipid-like amphiphilic prodrug, 1-O-octodecyl-2-conjugated linoleoyl-sn-glycero-3-phosphatidyl gemcitabine (OLGPG). After spreading at the air/water interface, it formed Langmuir monolayers. Stable nanoassemblies were obtained based on molecular self-assembly after OLGPG was injected in water. An amphiphilic long-chained lipid, cholesteryl hemisuccinate polyethylene glycol 1500 (CHS-PEG) was mixed in the OLGPG Langmuir monolayers and nanoassemblies with the optimal proportion. The OLGPG and OLGPG/CHS-PEG nanoassemblies were spherical vesicles due to the hydrophobic interaction of lipid moieties with the small sizes of 50.33nm and 64.76nm, respectively. Phospholipase A2 (PLA2) is highly expressed in tumor tissues to specifically degrade the 2-acyl of phospholipid to lysophospholipid. OLGPG showed PLA2-sensitive degradation. The nanoassemblies showed higher in vitro anticancer effect on HepG2 cells than the parent drug gemcitabine. In the in vivo studies on the hepatocellular tumor-bearing mouse model, the OLGPG/CHS-PEG nanoassemblies group (eq. to 1/5 dose of the Gem group) showed the highest antitumor and tumor targeting effects compared to the other groups. The long-circulating phospholipid-like prodrug nanoassemblies are the promising anticancer nanomedicines based on the biomimetic strategy and specific tumor microenvironment.

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

    PubMed

    Chung, Hsiu-Ying; Li, Cheng Che

    2013-03-01

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

  8. A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar, Premolis semirufa, Is a Potent Complement System Activator

    PubMed Central

    Villas Boas, Isadora Maria; Pidde-Queiroz, Giselle; Magnoli, Fabio Carlos; Gonçalves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.

    2015-01-01

    Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly

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

    PubMed

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

    2011-07-01

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

  10. Endothelin-1 stimulates catalase activity through the PKCδ-mediated phosphorylation of serine 167.

    PubMed

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

    2014-02-01

    Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in 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 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 δV1.1, this phosphorylation was shown to be protein kinase Cδ (PKCδ) dependent. Mass spectrometry identified serine 167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from Escherichia coli or transiently transfected COS-7 cells demonstrated that S167D catalase had an increased ability to degrade H2O2 compared to the wild-type 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 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 wild-type and S167D catalase. We found that recombinant wild-type catalase was present as a mixture of monomers and dimers, whereas S167D catalase was primarily tetrameric. Further, the incubation of wild-type catalase with PKCδ was sufficient to convert wild-type catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity.

  11. 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

  12. Atorvastatin ameliorates cognitive impairment, Aβ1-42 production and Tau hyperphosphorylation in APP/PS1 transgenic mice.

    PubMed

    Zhou, Dongsheng; Liu, Huaxia; Li, Chenli; Wang, Fangyan; Shi, Yaosheng; Liu, Lingjiang; Zhao, Xin; Liu, Aiming; Zhang, Junfang; Wang, Chuang; Chen, Zhongming

    2016-06-01

    Amyloid-beta (Aβ) interacts with the serine/threonine protein kinase AKT (also known as protein kinase B)/glycogen synthase kinase 3β (GSK3β) pathway and deactivates GSK3β signaling, which result in microtubule protein tau phosphorylation. Atorvastatin, a HMG-CoA reductase inhibitor, has been proven to improve learning and memory performance, reduce Aβ and phosphorylated tau levels in mouse model of Alzheimer's disease (AD). However, it still remains unclear whether atorvastatin is responsible for regulation of AKT/GSK3β signaling and contributes to subsequent down-regulation of Aβ1-42 and phosphorylated tau in APP/PS1 transgenic (Tg APP/PS1) mice. Herein, we aimed to investigate the possible impacts of atorvastatin (10 mg/kg, p.o.) on the memory deficit by behavioral tests and changes of AKT/GSK3β signaling in hippocampus and prefrontal cortex by western blot test in Tg APP/PS1 mice. The results showed that treatment with atorvastatin significantly reversed the memory deficit in the Tg APP/PS1 mice in a novel object recognition and the Morris water maze tests. Moreover, atorvastatin significantly attenuated Aβ1-42 accumulation and phosphorylation of tau (Ser396) in the hippocampus and prefrontal cortex of Tg APP/PS1 mice. In addition, atorvastatin treatment also increased phosphorylation of AKT, inhibited GSK3β activity by increasing phosphorylation of GSK3β (Ser9) and decreasing the beta-site APP cleaving enzyme 1 (BACE1) expression. These results indicated that the memory ameliorating effect of atorvastatin may be, in part, by regulation the AKT/GSK3β signaling which may contribute to down-regulation of Aβ1-42 and tau hyperphosphorylation.

  13. Participation of phosphatidyl inositol 3-kinase/protein kinase B and ERK1/2 pathways in interleukin-1beta stimulation of lactate production in Sertoli cells.

    PubMed

    Riera, María Fernanda; Galardo, María Noel; Pellizzari, Eliana Herminia; Meroni, Silvina Beatriz; Cigorraga, Selva Beatriz

    2007-04-01

    Interleukin-1beta (IL1beta ) belongs to a set of intratesticular regulators that provide the fine-tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to analyze the signaling pathways that may participate in IL1beta regulation of Sertoli cell function. Sertoli cell cultures from 20-day-old rat were used. Stimulation of the cultures with IL1beta showed increments in phosphorylated protein kinase B (PKB), P70S6K, and ERK1/2 levels. A phosphatidyl inositol 3-kinase (PI3K) inhibitor (wortmannin (W)), a mammalian target of rapamycin inhibitor (rapamycin (R)), and a MEK inhibitor (PD98059 (PD)) were utilized to evaluate the participation of PI3K/PKB, P70S6K, and ERK1/2 pathways in the regulation of lactate production by IL1beta . PD and W, but not R, decreased IL1beta-stimulated lactate production. The participation of these pathways in the regulation of glucose uptake and lactate dehydrogenase (LDH) A mRNA levels by IL1beta was also analyzed. It was observed that W decreased IL1beta-stimulated glucose uptake, whereas PD and R did not modify it. On the other hand, PD decreased the stimulation of LDH A mRNA levels by IL1beta , whereas W and R did not modify it. In summary, results presented herein demonstrate that IL1beta stimulates PI3K/PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1beta utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH A mRNA levels. This study reveals that IL1beta utilizes different signal transduction pathways to modify the biochemical steps that are important to regulate lactate production in rat Sertoli cells.

  14. Glycosyl-Phosphatidyl-Inositol (GPI)-Anchors and Metalloproteases: Their Roles in the Regulation of Exosome Composition and NKG2D-Mediated Immune Recognition.

    PubMed

    López-Cobo, Sheila; Campos-Silva, Carmen; Valés-Gómez, Mar

    2016-01-01

    Communication within the immune system depends on the release of factors that can travel and transmit information at points distant from the cell that produced them. In general, immune cells use two key strategies that can occur either at the plasma membrane or in intracellular compartments to produce such factors, vesicle release and proteolytic cleavage. Release of soluble factors in exosomes, a subset of vesicles that originate from intracellular compartments, depends generally on biochemical and lipid environment features. This physical environment allows proteins to be recruited to membrane microdomains that will be later endocytosed and further released to the extracellular milieu. Cholesterol and sphingolipid rich domains (also known as lipid rafts or detergent-resistant membranes, DRMs) often contribute to exosomes and these membrane regions are rich in proteins modified with Glycosyl-Phosphatidyl-Inositol (GPI) and lipids. For this reason, many palmitoylated and GPI-anchored proteins are preferentially recruited to exosomes. In this review, we analyse the biochemical features involved in the release of NKG2D-ligands as an example of functionally related gene families encoding both transmembrane and GPI-anchored proteins that can be released either by proteolysis or in exosomes, and modulate the intensity of the immune response. The immune receptor NKG2D is present in all human Natural Killer and T cells and plays an important role in the first barrier of defense against tumor and infection. However, tumor cells can evade the immune system by releasing NKG2D-ligands to induce down-regulation of the receptor. Some NKG2D-ligands can be recruited to exosomes and potently modulate receptor expression and immune function, while others are more susceptible to metalloprotease cleavage and are shed as soluble molecules. Strikingly, metalloprotease inhibition is sufficient to drive the accumulation in exosomes of ligands otherwise released by metalloprotease

  15. Glycosyl-Phosphatidyl-Inositol (GPI)-Anchors and Metalloproteases: Their Roles in the Regulation of Exosome Composition and NKG2D-Mediated Immune Recognition

    PubMed Central

    López-Cobo, Sheila; Campos-Silva, Carmen; Valés-Gómez, Mar

    2016-01-01

    Communication within the immune system depends on the release of factors that can travel and transmit information at points distant from the cell that produced them. In general, immune cells use two key strategies that can occur either at the plasma membrane or in intracellular compartments to produce such factors, vesicle release and proteolytic cleavage. Release of soluble factors in exosomes, a subset of vesicles that originate from intracellular compartments, depends generally on biochemical and lipid environment features. This physical environment allows proteins to be recruited to membrane microdomains that will be later endocytosed and further released to the extracellular milieu. Cholesterol and sphingolipid rich domains (also known as lipid rafts or detergent-resistant membranes, DRMs) often contribute to exosomes and these membrane regions are rich in proteins modified with Glycosyl-Phosphatidyl-Inositol (GPI) and lipids. For this reason, many palmitoylated and GPI-anchored proteins are preferentially recruited to exosomes. In this review, we analyse the biochemical features involved in the release of NKG2D-ligands as an example of functionally related gene families encoding both transmembrane and GPI-anchored proteins that can be released either by proteolysis or in exosomes, and modulate the intensity of the immune response. The immune receptor NKG2D is present in all human Natural Killer and T cells and plays an important role in the first barrier of defense against tumor and infection. However, tumor cells can evade the immune system by releasing NKG2D-ligands to induce down-regulation of the receptor. Some NKG2D-ligands can be recruited to exosomes and potently modulate receptor expression and immune function, while others are more susceptible to metalloprotease cleavage and are shed as soluble molecules. Strikingly, metalloprotease inhibition is sufficient to drive the accumulation in exosomes of ligands otherwise released by metalloprotease

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

    PubMed

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

    2017-03-08

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

  17. Nimbolide, a neem limonoid inhibits Phosphatidyl Inositol-3 Kinase to activate Glycogen Synthase Kinase-3β in a hamster model of oral oncogenesis

    PubMed Central

    Sophia, Josephraj; Kiran Kishore T., Kranthi; Kowshik, Jaganathan; Mishra, Rajakishore; Nagini, Siddavaram

    2016-01-01

    Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase is frequently inactivated by the oncogenic signalling kinases PI3K/Akt and MAPK/ERK in diverse malignancies. The present study was designed to investigate GSK-3β signalling circuits in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis model and the therapeutic potential of the neem limonoid nimbolide. Inactivation of GSK-3β by phosphorylation at serine 9 and activation of PI3K/Akt, MAPK/ERK and β-catenin was associated with increased cell proliferation and apoptosis evasion during stepwise evolution of HBP carcinomas. Administration of nimbolide inhibited PI3K/Akt signalling with consequent activation of GSK-3β thereby inducing trafficking of β-catenin away from the nucleus and enhancing the expression of miR-126 and let-7. Molecular docking studies confirmed interaction of nimbolide with PI3K, Akt, ERK and GSK-3β. Furthermore, nimbolide attenuated cell proliferation and induced apoptosis as evidenced by increased p-cyclin D1Thr286 and pro-apoptotic proteins. The present study has unravelled aberrant phosphorylation as a key determinant for oncogenic signalling and acquisition of cancer hallmarks in the HBP model. The study has also provided mechanistic insights into the chemotherapeutic potential of nimbolide that may be a useful addition to the armamentarium of natural compounds targeting PI3K for oral cancer treatment. PMID:26902162

  18. Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

    PubMed Central

    Wright, David P; Ulijasz, Andrew T

    2014-01-01

    Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential. PMID:25603430

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

    PubMed Central

    Uversky, Vladimir N

    2015-01-01

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

  20. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. 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

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

    PubMed

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

    2007-04-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. PS foams at high pressure drop rates

    NASA Astrophysics Data System (ADS)

    Tammaro, Daniele; De Maio, Attilio; Carbone, Maria Giovanna Pastore; Di Maio, Ernesto; Iannace, Salvatore

    2014-05-01

    In this paper, we report data on PS foamed at 100 °C after CO2 saturation at 10 MPa in a new physical foaming batch that achieves pressure drop rates up to 120 MPa/s. Results show how average cell size of the foam nicely fit a linear behavior with the pressure drop rate in a double logarithmic plot. Furthermore, foam density initially decreases with the pressure drop rate, attaining a constant value at pressure drop rates higher than 40 MPa/s. Interestingly, furthermore, we observed that the shape of the pressure release curve has a large effect on the final foam morphology, as observed in tests in which the maximum pressure release rate was kept constant but the shape of the curve changed. These results allow for a fine tuning of the foam density and morphology for specific applications.

  5. PS2004 Light-harvesting Systems Workshop

    SciTech Connect

    Robert E. Blankenship

    2005-11-01

    This special issue of the international scientific research journal Photosynthesis Research consists of 25 original peer-reviewed contributions from participants in the PS 2004 Lisht-Harvesting Systems Workshop. This workshop was held from 26-29, 2004 at Hotel Le Chantecler, Sainte-Adele, Quebec, Canada. The workshop was a satellite meeting of the XIII International Congress on Photosynthesis held August 29-September 3, 2004 in Montreal, Canada. The workshope dealt with all types of photosynthetic antenna systems and types of organisms, including anoxygenic photosynthetic bacteria, cyanobacteria, algae and higher plants, as well as in vitro studies of isolated pigments. This collection of papers is a good representation of the highly interdisciplinary nature of modern research on photosynthetic antenna complexes, utilizing techniques of advanced spectroscopy, biochemistry, molecular biology, synthetic chemistry and structural determination to understand these diverse and elegant molecular complexes.

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

    PubMed

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

    2006-06-01

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

  7. 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.

  8. Energy and expectation values of the PsH system

    SciTech Connect

    Mitroy, J.

    2006-05-15

    Close to converged energies and expectation values for PsH are computed using a ground state wave function consisting of 1800 explicitly correlated gaussians. The best estimate of the Ps{sup {infinity}}H energy was -0.789 196 740 hartree which is the lowest variational energy to date. The 2{gamma} annihilation rate for Ps{sup {infinity}}H was 2.471 78x10{sup 9} s{sup -1}.

  9. Phosphorylation of human estrogen receptor-beta at serine 105 inhibits breast cancer cell migration and invasion.

    PubMed

    Lam, Hung-Ming; Suresh Babu, C V; Wang, Jiang; Yuan, Yong; Lam, Ying-Wai; Ho, Shuk-Mei; Leung, Yuet-Kin

    2012-07-06

    Multiple phosphorylation sites on the human estrogen receptor (hER)α were identified and shown to influence mammary carcinogenesis. In contrast, functional phosphorylation sites of hERβ have yet to be experimentally identified and validated. Here, using mass spectrometry, we uncovered three serines (S75, S87, and S105) in the N-terminus of hERβ as targets of ERK1/2 and p38 kinases. We raised a specific antibody against phosphorylated S105 (pS105) and demonstrated that this site was endogenously phosphorylated in MDA-MB-231 and BT-474 cells. A phospho-mimetic mutant generated from hERβ1 was found to exhibit higher transactivation activity than hERβ1. Ectopic expression of this mutant inhibited cell migration and invasion, but did not affect cell growth and cell-cycle progression in these cell models. In breast cancer specimens, pS105-hERβ immunoreactivity was detected with a higher prevalence and intensity than that of hERβ1. These results underscore the functional importance of the first experimentally identified hERβ-phosphorylation site in breast cancer.

  10. PS: A nonprocedural language with data types and modules

    NASA Technical Reports Server (NTRS)

    Gokhale, M. B.

    1986-01-01

    The Problem Specification (PS) nonprocedural language is a very high level language for algorithm specification. PS is suitable for nonprogrammers, who can specify a problem using mathematically-oriented equations; for expert programmers, who can prototype different versions of a software system for evaluation; and for those who wish to use specifications for portions (if not all) of a program. PS has data types and modules similar to Modula-2. The compiler generates C code. PS is first shown by example, and then efficiency issues in scheduling and code generation are discussed.

  11. 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.

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

    PubMed Central

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

    2012-01-01

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

  13. Membrane-anchored serine proteases in health and disease

    PubMed Central

    Bugge, Thomas; Wu, Qingyu

    2013-01-01

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

  14. Site-specific DNA Inversion by Serine Recombinases

    PubMed Central

    2015-01-01

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

  15. Dynamics simulation of the interaction between serine and water

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Zhang, Peng; Lu, Ying-Bo; Han, Sheng-Hao; Yu, Hui

    2013-05-01

    Using the first principles density functional theory (DFT), we simulated the neutron scattering spectra of the hydration dynamics of serine. Experimental data analyses have shown that dissociative H2O molecules were more likely to form hydrogen bonds (H-bonds) with an -OH group in monohydrated serine and easily shift to a -NH_3 ^ + group at a higher hydration level [P. Zhang, Y. Zhang, S. H. Han, Q. W. Yan, R. C. Ford, and J. C. Li, J. Phys. Chem. A 110, 5000 (2006), 10.1021/jp0569741]. We set the 1:1 ratio hydrated compounds at the two positions and found that the H2O could be optimized to form H-bonds with -OH and -NH3+ separately. When the simulated phonon signals of the -OH…H2O and -NH3+…H2O combinations were summed on a 3:1 scale, the calculating spectra were in good agreement with the experimental results, especially for the peak at 423 cm-1 of the -OH…H2O combination and the peak at 367 cm-1 of the -NH3+…H2O combination, which mutually complemented the real spectrum. We confirm that H2O may break the intermolecular H-bonds of the interlaced binding -OH to form a new structure, and that with the skeleton deformation of serine, H2O forms stronger H-bonds more often with the -NH3+ side indicating the flexible dynamic mechanism of the serine hydration process.

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

    PubMed

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

    2008-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  18. Structural Basis for Catalytic Activation of a Serine Recombinase

    SciTech Connect

    Keenholtz, Ross A.; Rowland, Sally-J.; Boocock, Martin R.; Stark, W. Marshall; Rice, Phoebe A.

    2014-10-02

    Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 {angstrom} crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.

  19. Spectroscopic Classification of PS16chs with SOAR/Goodman

    NASA Astrophysics Data System (ADS)

    Miller, J. A.; Hounsell, R. A.; Pan, Y.-C.; Foley, R. J.; Jha, S. W.; Rest, A.; Scolnic, D.

    2016-05-01

    We report the classification of PS16chs from spectroscopic observation with the Goodman spectrograph on the SOAR telescope. The observation was made on 2016 May 08 UT. We classify PS16chs as a SN Ia near maximum light at z = 0.19.

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

    PubMed

    Iijima, Jun; Naruke, Haruo; Takiyama, Hiroshi

    2013-10-19

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

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

    PubMed

    Tanti, Jean-François; Jager, Jennifer

    2009-12-01

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

  2. Final results for π± 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 π± production in proton nucleus or π± 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.

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

    PubMed

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

    2017-01-29

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

  4. Phosphorylated Histone 3 at Serine 10 Identifies Activated Spinal Neurons and Contributes to the Development of Tissue Injury-Associated Pain

    PubMed Central

    Torres-Pérez, Jose Vicente; Sántha, Péter; Varga, Angelika; Szucs, Peter; Sousa-Valente, Joao; Gaal, Botond; Sivadó, Miklós; Andreou, Anna P; Beattie, Sara; Nagy, Bence; Matesz, Klara; C. Arthur, J. Simon; Jancsó, Gábor; Nagy, Istvan

    2017-01-01

    Transcriptional changes in superficial spinal dorsal horn neurons (SSDHN) are essential in the development and maintenance of prolonged pain. Epigenetic mechanisms including post-translational modifications in histones are pivotal in regulating transcription. Here, we report that phosphorylation of serine 10 (S10) in histone 3 (H3) specifically occurs in a group of rat SSDHN following the activation of nociceptive primary sensory neurons by burn injury, capsaicin application or sustained electrical activation of nociceptive primary sensory nerve fibres. In contrast, brief thermal or mechanical nociceptive stimuli, which fail to induce tissue injury or inflammation, do not produce the same effect. Blocking N-methyl-D-aspartate receptors or activation of extracellular signal-regulated kinases 1 and 2, or blocking or deleting the mitogen- and stress-activated kinases 1 and 2 (MSK1/2), which phosphorylate S10 in H3, inhibit up-regulation in phosphorylated S10 in H3 (p-S10H3) as well as fos transcription, a down-stream effect of p-S10H3. Deleting MSK1/2 also inhibits the development of carrageenan-induced inflammatory heat hyperalgesia in mice. We propose that p-S10H3 is a novel marker for nociceptive processing in SSDHN with high relevance to transcriptional changes and the development of prolonged pain. PMID:28120884

  5. 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".

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

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

    2010-05-15

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

  9. PP and PS interferometric images of near-seafloor sediments

    USGS Publications Warehouse

    Haines, S.S.

    2011-01-01

    I present interferometric processing examples from an ocean-bottom cable OBC dataset collected at a water depth of 800 m in the Gulf of Mexico. Virtual source and receiver gathers created through cross-correlation of full wavefields show clear PP reflections and PS conversions from near-seafloor layers of interest. Virtual gathers from wavefield-separated data show improved PP and PS arrivals. PP and PS brute stacks from the wavefield-separated data compare favorably with images from a non-interferometric processing flow. ?? 2011 Society of Exploration Geophysicists.

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

    PubMed

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

    2017-03-02

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

  11. 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

  12. Inhibition of serine proteases by peptidyl fluoromethyl ketones

    SciTech Connect

    Imperiali, B.; Abeles, R.H.

    1986-07-01

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

  13. A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts.

    PubMed

    Picaud, A; Acker, S; Duranton, J

    1982-01-01

    After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.

  14. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

    Delepine, D.; Lucio M, J. L.; Mendoza S, J. A.; Ramirez, Carlos A.

    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.

  17. 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.

  18. S-wave Ps-Li and Ps-Na scattering

    SciTech Connect

    Chakraborty, Sumana; Ghosh, A.S.; Sinha, Prabal K.

    2004-05-01

    Projectile-elastic, target-elastic, and full close-coupling approximation models have been employed to investigate scattering of ortho-positronium off Li and Na atoms at low energies. Nine different basis sets for each system have been used to find the relative importance of atomic (target and projectile) states on the elastic scattering parameters and the convergence of the predicted results with added eigen- and pseudostates. The s-wave elastic phase shifts, scattering lengths and cross sections of both the systems are reported upto the incident energy k=0.5 a.u. and compared with the corresponding existing theoretical predictions. It has been found that the van der Waals and higher order long-range interactions play a crucial role in predicting s-wave elastic parameters for Ps-alkali-metal-atom systems at low energies.

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

    DTIC Science & Technology

    1999-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Listening to PS II: enthalpy, entropy, and volume changes.

    PubMed

    Hou, Harvey J M; Mauzerall, David

    2011-01-01

    Photosystem II, located in the thylakoid membranes of green plants, algae, and cyanobacteria, uses sunlight to split water into protons, electrons, and a dioxygen molecule. The mechanism of its electron transfers and oxygen evolution including the structure of the protein and rates of the S-state cycle has been extensively investigated. Substantial progress has been made; however, the thermodynamics of PS II electron transfer and of the oxygen cycle are poorly understood. Recent progress in thermodynamic measurements in photosynthesis provides novel insights on the enthalpic and entropic contribution to electron transfer in proteins. In this review the thermodynamic parameters including quantum yield, enthalpy, entropy, and volume changes of PS II photochemistry determined by photoacoustics and other laser techniques are summarized and evaluated. Light-driven volume changes via electrostriction are directly related to the photoreaction in PS II and thus can be a useful measurement of PS II activity and function. The enthalpy changes of the reactions observed can be directly measured by photoacoustics. The apparent reaction entropy can also be estimated when the free energy is known. Dissecting the free energy of a photoreaction into enthalpic and entropic components provides critical information about mechanisms of PS II function. Potential limitations and future direction of the study of the thermodynamics of PS II electron transfer and oxygen evolution are presented.

  2. Antibacterial activity of silver nanoparticles synthesized from serine.

    PubMed

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

    2015-04-01

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

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

    PubMed

    Dounay, A B; Forsyth, C J

    2002-11-01

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

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

    PubMed

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

    2008-03-01

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

  5. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

    PubMed

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

    2014-10-01

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

  6. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  7. Profiling the microRNA Expression in Human iPS and iPS-derived Retinal Pigment Epithelium

    PubMed Central

    Wang, Heuy-Ching; Greene, Whitney A; Kaini, Ramesh R; Shen-Gunther, Jane; Chen, Hung-I H; Cai, Hong; Wang, Yufeng

    2014-01-01

    The purpose of this study is to characterize the microRNA (miRNA) expression profiles of induced pluripotent stem (iPS) cells and retinal pigment epithelium (RPE) derived from induced pluripotent stem cells (iPS-RPE). MiRNAs have been demonstrated to play critical roles in both maintaining pluripotency and facilitating differentiation. Gene expression networks accountable for maintenance and induction of pluripotency are linked and share components with those networks implicated in oncogenesis. Therefore, we hypothesize that miRNA expression profiling will distinguish iPS cells from their iPS-RPE progeny. To identify and analyze differentially expressed miRNAs, RPE was derived from iPS using a spontaneous differentiation method. MiRNA microarray analysis identified 155 probes that were statistically differentially expressed between iPS and iPS-RPE cells. Up-regulated miRNAs including miR-181c and miR-129–5p may play a role in promoting differentiation, while down-regulated miRNAs such as miR-367, miR-18b, and miR-20b are implicated in cell proliferation. Subsequent miRNA–target and network analysis revealed that these miRNAs are involved in cellular development, cell cycle progression, cell death, and survival. A systematic interrogation of temporal and spatial expression of iPS-RPE miRNAs and their associated target mRNAs will provide new insights into the molecular mechanisms of carcinogenesis, eye differentiation and development. PMID:25392691

  8. Enantioselective inhibition of D-serine transport by (S)-ketamine

    PubMed Central

    Singh, Nagendra S; Bernier, Michel; Camandola, Simonetta; Khadeer, Mohammed A; Moaddel, Ruin; Mattson, Mark P; Wainer, Irving W

    2015-01-01

    Background and Purpose Patients with major depressive disorder receiving racemic ketamine, (R,S)-ketamine, experience transient increases in Clinician-Administered Dissociative States Scale scores and a coincident drop in plasma d-serine levels. The results suggest that (R,S)-ketamine produces an immediate, concentration-dependent pharmacological effect on d-serine plasma concentrations. One potential source of this effect is (R,S)-ketamine-induced inhibition of the transporter ASCT2, which regulates intracellular d-serine concentrations. In this study, we tested this hypothesis by examining the effect of (S)- and (R)-ketamine on ASCT2-mediated transport of d-serine in PC-12 and 1321N1 cells and primary neuronal cells in culture. Experimental Approach Intracellular and extracellular d-serine levels were determined using capillary electrophoresis–laser-induced fluorescence and liquid chromatography–mass spectrometry respectively. Expression of ASCT2, Asc-1 and serine racemase was determined utilizing Western blotting. Key Results (S)-Ketamine produced a concentration-dependent increase in intracellular d-serine and reduced extracellular d-serine accumulation. In contrast, (R)-ketamine decreased both intracellular and extracellular d-serine levels. The ASCT2 inhibitor, benzyl-d-serine (BDS), and ASCT2 gene knockdown mimicked the action of (S)-ketamine on d-serine in PC-12 cells, while the Asc-1 agonist d-isoleucine reduced intracellular d-serine and increased extracellular d-serine accumulation. This response to d-isoleucine was not affected by BDS or (S)-ketamine. Primary cultures of rat neuronal cells expressed ASCT2 and were responsive to (S)-ketamine and BDS. (S)- and (R)-ketamine increased the expression of monomeric serine racemase in all the cells studied, with (S)-ketamine having the greatest effect. Conclusions and Implications (S)-Ketamine decreased cellular export of d-serine via selective inhibition of ASCT2, and this could represent a possible source

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

    PubMed

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

    2012-04-01

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

  10. Anticancer Activity of VDR-Coregulator Inhibitor PS121912

    PubMed Central

    Sidhu, Preetpal S.; Teske, Kelly; Feleke, Belaynesh; Yuan, Nina Y.; Guthrie, Margaret L.; Fernstrum, Grant B.; Vyas, Nishita D.; Han, Lanlan; Preston, Joshua; Bogart, Jonathan W.; Silvaggi, Nicholas R.; Cook, James M.; Singh, Rakesh K.; Bikle, Daniel D.; Arnold, Leggy A.

    2014-01-01

    Purpose PS121912 has been developed as selective vitamin D receptor (VDR)–coregulator inhibitor starting from a high throughput screening campaign to identify new agents that modulate VDR without causing hypercalcemia. Initial antiproliferative effects of PS121912 were observed that are characterized herein to enable future in vivo investigation with this molecule. Methods Antiproliferation and apoptosis was determined using four different cancer cell lines (DU145, Caco2, HL-60, and SKOV3) in the presence of PS121912, 1,25-(OH)2D3, or a combination of 1,25-(OH)2D3 and PS121912. VDR si-RNA was used to identify the role of VDR during this process. The application of ChIP enabled us to determine the involvement of coregulator recruitment during transcription, which was investigated by rt-PCR with VDR target genes and those affiliated with cell cycle progression. Translational changes of apoptotic proteins were determined with an antibody array. The preclinical characterization of PS121912 include the determination of metabolic stability and CYP3A4 inhibition. Results PS121912 induced apoptosis in all four cancer cells, with HL-60 cells being the most sensitive. At sub-micromolar concentrations, PS121912 amplified the growth inhibition of cancer cells caused by 1,25-(OH)2D3 without being antiproliferative by itself. A knockout study with VDR si-RNA confirmed the mediating role of VDR. VDR target genes induced by 1,25-(OH)2D3 were down-regulated with the co-treatment of PS121912. This process was highly dependent on the recruitment of coregulators that in case of CYP24A1 was SRC2. The combination of PS121912 and 1,25-(OH)2D3 reduced the presence of SRC2 and enriched the occupancy of corepressor NCoR at the promoter site. E2F transcription factor 1 and 4 were down-regulated in the presence of PS121912 and 1,25-(OH)2D3 that in turn reduced the transcription levels of cyclin A and D thus arresting HL-60 cells in the S or G2/M phase. In addition, proteins with

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    1979-04-01

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

  13. Pressure Monitoring Using Hybrid fs/ps Rotational CARS

    NASA Technical Reports Server (NTRS)

    Kearney, Sean P.; Danehy, Paul M.

    2015-01-01

    We investigate the feasibility of gas-phase pressure measurements at kHz-rates using fs/ps rotational CARS. Femtosecond pump and Stokes pulses impulsively prepare a rotational Raman coherence, which is then probed by a high-energy 6-ps pulse introduced at a time delay from the Raman preparation. Rotational CARS spectra were recorded in N2 contained in a room-temperature gas cell for pressures from 0.1 to 3 atm and probe delays ranging from 10-330 ps. Using published self-broadened collisional linewidth data for N2, both the spectrally integrated coherence decay rate and the spectrally resolved decay were investigated as means for detecting pressure. Shot-averaged and single-laser-shot spectra were interrogated for pressure and the accuracy and precision as a function of probe delay and cell pressure are discussed. Single-shot measurement accuracies were within 0.1 to 6.5% when compared to a transducer values, while the precision was generally between 1% and 6% of measured pressure for probe delays of 200 ps or more, and better than 2% as the delay approached 300 ps. A byproduct of the pressure measurement is an independent but simultaneous measurement of the gas temperature.

  14. SEA and strategy formation theories: From three Ps to five Ps

    SciTech Connect

    Cherp, Aleh Watt, Alan Vinichenko, Vadim

    2007-10-15

    A transition to environmentally sustainable societies should involve a significant and comprehensive - strategic - change. Much of the promise of SEA is associated precisely with its perceived capacity to facilitate such a strategic transformation by influencing selected 'strategic decisions'. This paper examines the potential effectiveness and limitations of such an approach in light of contemporary organizational strategy theories. Most of these theories separate 'strategies' from 'decisions' and also transcend the notion of strategies as formal plans, policies and programs (PPPs). Instead, they consider strategies as 'five Ps', adding 'Position', 'Perspective', 'Pattern' and 'Ploy' to the 'Plan'. Lessons from organizational strategy formation give rise to the following challenges for SEA theory and practice: 1.How to assess and influence informal as well as formal aspects of strategic initiatives? 2.How to extend SEA 'beyond decisions' to address 'emergent strategies' where strategic action is not necessarily preceded by a decision? 3.How to ensure that knowledge provided as a result of SEA is strategically relevant and communicated to key players in strategy formation? 4.How to deal with an uncontrollable and unpredictable environment in which strategic initiatives unfold? 5.How to recognize those situations when SEA can have most strategic influence? This paper takes a step towards examining these challenges by exploring the intellectual history of SEA in light of the main strategy formation theories and by identifying directions in which the SEA discourse may be further enhanced to meet these five challenges.

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

    PubMed

    Haley, Sheila A; Wessel, Gary M

    2004-05-01

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

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

    SciTech Connect

    Roskoski, Robert

    2010-08-27

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2001-12-05

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

  19. 75 FR 66734 - Proposed Voluntary Product Standard PS 2-10, Structural Plywood

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-29

    ... National Institute of Standards and Technology Proposed Voluntary Product Standard PS 2-10, Structural... Structural-Use Panels. This revised standard, PS 2-10, was prepared by the Standing Committee for PS 2 and... Acrobat File) of the proposed standard, PS 2-10, can be obtained at the following Web site:...

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

    PubMed

    Ozawa, Kazuo

    2008-06-01

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

  1. Normal mouse peritoneum contains a large population of Ly-1+ (CD5) B cells that recognize phosphatidyl choline. Relationship to cells that secrete hemolytic antibody specific for autologous erythrocytes

    PubMed Central

    1988-01-01

    We have found that, in the peritoneums of normal adult mice, 5-15% of lymphocytes bind a fluorescent liposome probe. In ontogeny, cells with this specificity were shown to appear by 8 d after birth, and increase to the adult frequency by 2-3 wk. Some older mice contain an expanded population of these cells. We have shown that liposome binding occurs by cell surface IgM recognizing the common membrane phospholipid, phosphatidyl choline (PtC). Virtually all of these PtC-specific cells bear the cell surface marker Ly-1. Our results indicate that roughly 1 in 10 peritoneal Ly-1+ B cells has this single specificity. We have found that the precursors to all the cells that form plaques on protease-treated autologous erythrocytes (BrMRBC) are included in the PtC-specific population and can be isolated by FACS. We believe this is the first report of sorting large numbers of B cells with a single antigen specificity from normal, unimmunized animals. This method will allow for in vitro and in vivo studies of differentiative and proliferative properties of Ly-1+ B cells, which may help define their role in development and disease. PMID:3045250

  2. Isolation of PsPIN2 and PsAUX1 from etiolated pea epicotyls and their expression on a three-dimensional clinostat

    NASA Astrophysics Data System (ADS)

    Hoshino, Tomoki; Hitotsubashi, Reiko; Miyamoto, Kensuke; Tanimoto, Eiichi; Ueda, Junichi

    We isolated novel cDNAs containing the complete open reading frames of a putative auxin influx carrier, PsAUX1, and a putative auxin efflux carrier, PsPIN2, from etiolated pea epicotyls. High levels of homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (Accession No. AY222857) and AtPINs. Phylogenetic analyses based on deduced amino acid sequences revealed that PsPIN2 belonged to a subclade including AtPIN3, AtPIN4 and AtPIN7, while PsPIN1 belonged to the same clade as AtPIN1. The results were similar for PsAUX1 and AtAUX1, where PsAUX1 belongs to the same subclade as AtAUX1 and CS-AUX1. Expression of PsPIN1, PsPIN2 and PsAUX1 in pea epicotyl segments was promoted upon incubation of the segments with auxin (indole-3-acetic acid). In 3.5-d-old etiolated pea seedlings, relatively high expression of PsPIN1 and PsAUX1 was observed in the hook region, growing epicotyls and root tips as compared with those in mature regions of epicotyls and roots. Expression of PsPIN2 in roots was less than that in shoots. Simulated microgravity conditions on a three-dimensional clinostat remarkably increased gene expression of PsPIN1 and PsAUX1 in the hook and the internodes of pea epicotyls, but the increase in PsPIN2 was less. In contrast, polar auxin transport of pea epicotyls was substantially suppressed under simulated microgravity conditions on a 3D clinostat, similar to data from a space experiment on STS-95. These results suggest that PsPINs and PsAUX1 are auxin-inducible genes, and that the expression of PsPINs and PsAUX1 genes is sensitive to gravistimulation.

  3. Spectroscopic Classification of AT2016axb (=PS16axi)

    NASA Astrophysics Data System (ADS)

    Blanchard, Peter; Berger, Edo; Bieryla, Allyson; Brzycki, Bryan; Cohn, Amy; Dillett, Sean; Feder-Staehle, Richard; Frostig, Danielle; Grell, Gabe; James, Nicholas; Johnson, April; Mahmood, Zahra; Merryman, Hunter; Peak, Barra; Sanders, Cecilia; Taylor, Anthony

    2016-03-01

    We obtained a low-resolution optical spectrum of AT2016axb (=PS16axi) reported by the Pan-STARRS Survey for Transients (PSST; Huber et al., ATel #7153). Observations were carried out on 2016 March 16.3 UT using the FAST spectrograph mounted on the F.L. Whipple Observatory 1.5-m telescope.

  4. Boiling treatment of ABS and PS plastics for flotation separation.

    PubMed

    Wang, Chong-qing; Wang, Hui; Wu, Bao-xin; Liu, Qun

    2014-07-01

    A new physical method, namely boiling treatment, was developed to aid flotation separation of acrylonitrile-butadiene-styrene (ABS) and polystyrene (PS) plastics. Boiling treatment was shown to be effective in producing a hydrophilic surface on ABS plastic. Fourier Transform Infrared analysis was conducted to investigate the mechanism of boiling treatment of ABS. Surface rearrangement of polymer may be responsible for surface change of boiling treated ABS, and the selective influence of boiling treatment on the floatability of boiling treated plastics may be attributed to the difference in the molecular mobility of polymer chains. The effects of flotation time, frother concentration and particle size on flotation behavior of simple plastic were investigated. Based on flotation behavior of simple plastic, flotation separation of boiling treatment ABS and PS with different particle sizes was achieved efficiently. The purity of ABS and PS was up to 99.78% and 95.80%, respectively; the recovery of ABS and PS was up to 95.81% and 99.82%, respectively. Boiling treatment promotes the industrial application of plastics flotation and facilitates plastic recycling.

  5. Seismic receiver function interpretation: Ps splitting or anisotropic underplating?

    NASA Astrophysics Data System (ADS)

    Liu, Zhen; Park, Jeffrey

    2017-03-01

    Crustal anisotropy is crucial to understanding the evolutionary history of Earth's lithosphere. Shear wave splitting of Moho P-to-S converted phases in receiver functions (RFs) have been often used to study crustal anisotropy. Harmonic variation of Moho Ps phases in delay times are used to infer splitting parameters of averaged anisotropy in the crust. However, crustal anisotropy may distribute at various levels within the crust due to complex deformational processes. Layered anisotropy requires careful investigation of the distribution of anisotropy before interpreting Moho Ps splitting. In this study, we show results from stations ARU in Russia, KIP in the Hawaiian Islands and LSA in Tibetan Plateau, where layered anisotropy is constrained well by intracrust Ps conversions at high frequencies using a harmonic-decomposition technique. Anisotropic velocity models are inferred by forward-modeling decomposed RF waveforms. We suggest that the harmonic variation of Moho Ps phases should always be investigated to check for anisotropic layering using RFs with frequency content above 1 Hz, rather than simply reporting averaged anisotropy of the whole crust.

  6. Data-Acquisition Board For IBM PS/2 Computer

    NASA Technical Reports Server (NTRS)

    Hoang, Phuong-Dung T.

    1990-01-01

    Circuit board containing microprocessors designed to control acquisition of data by IBM PS/2 computer. Plugged into one of 16-bit slots on mother board of computer. Controls transfer of data from as many as 48 discrete channels to Micro Channel Interface. With expansion of software, board recognizes and filters specified kinds of signal patterns, possibly to detect errors.

  7. Consistent scenario for B{yields}PS decays

    SciTech Connect

    Delepine, D.; Lucio M, J. L.; Mendoza S, J. A.; Ramirez, Carlos A.

    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.

  8. Seismic receiver function interpretation: Ps splitting or anisotropic underplating?

    NASA Astrophysics Data System (ADS)

    Liu, Zhen; Park, Jeffrey

    2016-12-01

    Crustal anisotropy is crucial to understanding the evolutionary history of Earth's lithosphere. Shear-wave splitting of Moho P-to-S converted phases in receiver functions have been often used to study crustal anisotropy. Harmonic variation of Moho Ps phases in delay times are used to infer splitting parameters of averaged anisotropy in the crust. However, crustal anisotropy may distribute at various levels within the crust due to complex deformational processes. Layered anisotropy requires careful investigation of the distribution of anisotropy before interpreting Moho Ps splitting. In this study, we show results from stations ARU in Russia, KIP in the Hawaiian Islands and LSA in Tibetan Plateau, where layered anisotropy is constrained well by intra-crust Ps conversions at high frequencies using a harmonic-decomposition technique. Anisotropic velocity models are inferred by forward-modeling decomposed RF waveforms. We suggest that the harmonic variation of Moho Ps phases should always be investigated to check for anisotropic layering using RFs with frequency content above 1 Hz, rather than simply reporting averaged anisotropy of the whole crust.

  9. 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…

  10. Solvent annealing of Micropatterned PS-b-PEO copolymer films

    NASA Astrophysics Data System (ADS)

    Kim, Tae Hee; Acharya, Himadri; Joeng, Hee June; Park, Cheolmin

    2009-03-01

    Solvent annealing of block copolymer thin films have been known as an effective way to control both orientation of microdomains with respect to the surface and their registration into a well ordered periodic lattice structure. We have recently demonstrated hierarchically ordered microdomains in a thin poly(styrene-b-ethylene oxide)(PS-b-PEO) film combined with microcontact printing. The solvent annealing gave rise to well ordered spherical PEO microdomains in large area by the confined dewetting of thin PS-b-PEO films which had been micropatterned on chemically modified surface during solvent annealing. In this presentation, we intentionally prepare a micropatterned dewet film of PS-b-PEO by spincoating a block copolymer solution on a topographic PDMS pre-pattern. Convex lens shaped spherical caps of PS-b-PEO individually located on each PDMS mesa were successfully transferred to a Si substrate by a conventional transfer printing technique. We investigate the effect of solvent on not only film wettability but also formation of hierarchical nanostructures.

  11. Framing Retention for Institutional Improvement: A 4 Ps Framework

    ERIC Educational Resources Information Center

    Kalsbeek, David H.

    2013-01-01

    A 4 Ps framework for student retention strategy is a construct for reframing the retention discussion in a way that enables institutional improvement by challenging some conventional wisdom and prevailing perspectives that have characterized retention strategy for years. It opens new possibilities for action and improvement by suggesting that…

  12. Microproteins (miPs) - the next big thing.

    PubMed

    Feller, Stephan

    2012-12-18

    With iPS cells, sncRNAs, chromatin modification regulation and cancer stem cells already cooling off again, i.e. not being guaranteed publications in the 'ultimate' journals anymore, what will be very soon the new red-hot (or super-cool, i.e. anything but lukewarm) 'kid on the block'? We would vote for microproteins.

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

    PubMed Central

    Sullivan, Steve J.; Miller, Robert F.

    2010-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

    Abbas, Haider

    2017-03-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

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

    PubMed

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

    2015-01-01

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

  19. Metabolic flux analyses for serine alkaline protease production.

    PubMed

    Çalik; Çalik; Takaç; Özdamar

    2000-12-01

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

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

    PubMed

    Basso, Manuela; Pennuto, Maria

    2015-09-01

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

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

    PubMed

    Minamizaki, Tomoko; Yoshiko, Yuji

    2015-01-01

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

  2. Conformation effects on the molecular orbitals of serine

    NASA Astrophysics Data System (ADS)

    Wang, Ke-Dong; Ma, Peng-Fei; Shan, Xu

    2011-03-01

    This paper calculates the five most stable conformers of serine with Hartree—Fock theory, density functional theory (B3LYP), Møller—Plesset perturbation theory (MP4(SDQ)) and electron propagation theory with the 6-311++G(2d,2p) basis set. The calculated vertical ionization energies for the valence molecular orbitals of each conformer are in agreement with the experimental data, indicating that a range of molecular conformations would coexist in an equilibrium sample. Information of the five outer valence molecular orbitals for each conformer is explored in coordinate and momentum spaces using dual space analysis to investigate the conformational processes, which are generated from the global minimum conformer Ser1 by rotation of C2-C3 (Ser4), C1-C2 (Ser5) and C1-O2 (Ser2 and Ser3). Orbitals 28a, 27a and 26a are identified as the fingerprint orbitals for all the conformational processes. Project supported by the Doctoral Research Fund of Henan Normal University, China (Grant No. 525449).

  3. Exploring a new serine protease from Cucumis sativus L.

    PubMed

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process.

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

    PubMed Central

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

    2011-01-01

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

  5. The Nature of D-Serine-Induced Nephrotoxicity

    PubMed Central

    Ganote, Charles E.; Peterson, Darryl R.; Carone, Frank A.

    1974-01-01

    Renal structural changes were studied sequentially between 1 hour and 6 days in rats treated with D-serine. Extensive necrosis of proximal straight tubules was rapid in onset and was followed by complete tubular regeneration 6 days post-treatment. The apparent progression of cellular changes was initial shrinkage, followed either by swelling and loss of apical cytoplasm or immediate lysis of cytoplasmic and nuclear contents. Tubular damage left only the basement membrane as a barrier between interstitial and luminal fluids. In similarly treated rats, proteinuria and glucosuria developed at the onset of tubular necrosis and disappeared when the tubules were completely relined by epithelium suggesting that they are due to diffusion of protein and glucose from interstitium into tubular fluid across the denuded basement membranes and that epithelial cells, under normal conditions, act as a barrier to diffusion of certain substances between the interstitium and tubular fluid. ImagesFig 10Fig 11Fig 1Fig 2Fig 3Fig 12Fig 13Fig 14Fig 15Fig 4Fig 5Fig 6Fig 7Fig 8Fig 9 PMID:4447130

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2010-06-01

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

  8. The role of a conserved membrane proximal cysteine in altering αPS2CβPS integrin diffusion

    NASA Astrophysics Data System (ADS)

    Syed, Aleem; Arora, Neha; Bunch, Thomas A.; Smith, Emily A.

    2016-12-01

    Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys1368 on the diffusion properties of αPS2CβPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys1368 in wild-type integrins with valine (Val1368) putatively blocks a PTM site and alters integrins’ ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys1368 contains a redox or palmitoylation PTM in αPS2CβPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.

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

    PubMed

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

    2017-01-01

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

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. The angiotensin receptor type 1-Gq protein-phosphatidyl inositol phospholipase Cbeta-protein kinase C pathway is involved in activation of proximal tubule Na+-ATPase activity by angiotensin(1-7) in pig kidneys.

    PubMed

    Lara, Lucienne S; Correa, Juliana S; Lavelle, Anouchka B; Lopes, Anibal G; Caruso-Neves, Celso

    2008-05-01

    In a previous study, we observed that angiotensin(1-7) (Ang(1-7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1-7) on Na+-ATPase activity through AT1R involves a Gq protein-phosphatidyl inositol-phospholipase Cbeta (PI-PLCbeta) pathway because: (1) the effect was reversed by GDPbetaS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPgammaS, a non-hydrolysable GTP analogue; (3) Ang(1-7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCbeta substrate; and (4) U73122, a specific inhibitor of PI-PLCbeta, abolished Ang(1-7)-induced stimulation of Na+-ATPase activity. Angiotensin(1-7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1-7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1-7) stimulates Na+-ATPase activity through the AT1R-Gq protein-PI-PLCbeta-PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Shiraiwa, Yuka; Ikushiro, Hiroko; Hayashi, Hideyuki

    2009-06-05

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

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

    PubMed Central

    Shiraiwa, Yuka; Ikushiro, Hiroko; Hayashi, Hideyuki

    2009-01-01

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

  16. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    SciTech Connect

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  17. The CERN PS/SL Controls Java Application Programming Interface

    SciTech Connect

    I. Deloose; J. Cuperus; P. Charrue; F. DiMaio; K. Kostro; M. Vanden Eynden; W. Watson

    1999-10-01

    The PS/SL Convergence Project was launched in March 1998. Its objective is to deliver a common control as infrastructure for the CERN accelerators by year 2001. In the framework of this convergence activity, a project was launched to develop a Java Application Programming Interface (API) between programs written in the Java language and the PS and SL accelerator equipment. This Java API was specified and developed in collaboration with TJNAF. It is based on the Java CDEV [1] package that has been extended in order to end up with a CERN/TJNAF common product. It implements a detailed model composed of devices organized in named classes that provide a property-based interface. It supports data subscription and introspection facilities. The device model is presented and the capabilities of the API are described with syntax examples. The software architecture is also described.

  18. PS3 CELL Development for Scientific Computation and Research

    NASA Astrophysics Data System (ADS)

    Christiansen, M.; Sevre, E.; Wang, S. M.; Yuen, D. A.; Liu, S.; Lyness, M. D.; Broten, M.

    2007-12-01

    The Cell processor is one of the most powerful processors on the market, and researchers in the earth sciences may find its parallel architecture to be very useful. A cell processor, with 7 cores, can easily be obtained for experimentation by purchasing a PlayStation 3 (PS3) and installing linux and the IBM SDK. Each core of the PS3 is capable of 25 GFLOPS giving a potential limit of 150 GFLOPS when using all 6 SPUs (synergistic processing units) by using vectorized algorithms. We have used the Cell's computational power to create a program which takes simulated tsunami datasets, parses them, and returns a colorized height field image using ray casting techniques. As expected, the time required to create an image is inversely proportional to the number of SPUs used. We believe that this trend will continue when multiple PS3s are chained using OpenMP functionality and are in the process of researching this. By using the Cell to visualize tsunami data, we have found that its greatest feature is its power. This fact entwines well with the needs of the scientific community where the limiting factor is time. Any algorithm, such as the heat equation, that can be subdivided into multiple parts can take advantage of the PS3 Cell's ability to split the computations across the 6 SPUs reducing required run time by one sixth. Further vectorization of the code can allow for 4 simultanious floating point operations by using the SIMD (single instruction multiple data) capabilities of the SPU increasing efficiency 24 times.

  19. The four "P"s of marketing are dead.

    PubMed

    English, J

    2000-01-01

    For several decades marketing planning in the United States has relied upon the "four Ps" model. Product, price, place, and promotion were considered the foundation of the marketing mix. This model, however, has never been a comfortable fit for health care and, as the new century dawns, we find that a new marketing model--emphasizing the "four Rs"--is emerging. The foundations of the new model are relevance, response, relationships, and results.

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2013-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-05-01

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

  4. Antitumor Activity of 3-Indolylmethanamines 31B and PS121912

    PubMed Central

    Guthrie, Margaret L; Sidhu, Preetpal S.; Hill, Emily K.; Horan, Timothy C.; Nandhikonda, Premchendar; Teske, Kelly A.; Yuan, Nina Y.; Sidorko, Marina; Kodali, Revathi; Cook, James M.; Han, Lanlan; Silvaggi, Nicholas R.; Bikle, Daniel D.; Moore, Richard G.; Singh, Rakesh K.; Arnold, Leggy A.

    2015-01-01

    Aim To investigate the in vivo effects of 3-indolylmethanamines 31B and PS121912 in treating ovarian cancer and leukemia, respectively. Materials and Methods Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and western blotting were applied to demonstrate the induction of apoptosis. Xenografted mice were investigated to show the antitumor effects of 3-indolylmethanamines. 13C-Nuclear magnetic resource (NMR) and western blotting were used to demonstrate inhibition of glucose metabolism. Results 31B inhibited ovarian cancer cell proliferation and activated caspase-3, cleaved poly [ADP-ribose] polymerase 1 (PARP-1), and phosphorylated mitogen-activated protein kinases (MAPK), jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. 31B reduced ovarian cancer xenograft tumor growth and PS121912 inhibited the growth of HL-60 derived xenografts without any sign of toxicity. Compound 31B inhibited de novo glycolysis and lipogenesis mediated by the reduction of fatty acid synthase and lactate dehydrogenase-A expression. Conclusion 3-Indolylmethanamines represent a new class of antitumor agents. We have shown for the first time the in vivo anticancer effects of 3-indolylmethanamines 31B and PS121912. PMID:26504023

  5. Stabilization of PS/PLA cocontinuous blends by interfacial graphene

    NASA Astrophysics Data System (ADS)

    Bai, Lian; He, Siyao; Fruehwirth, John; Stein, Andreas; Cheng, Xiang; Macosko, Christopher

    Reduced graphene oxide (r-GO) is known to be effective in increasing the conductivity of cocontinuous polymer blends with a lower electrical percolation threshold. However, little is known regarding the localization and dynamics of r-GO along with morphology change during annealing. In this study, we develop a facile method to stabilize the polystyrene (PS)/polylactic acid (PLA) cocontinuous blends with r-GO jammed at interface. In this method, the non-functionalized GO is premixed with PLA via solvent method, and then reduced in-situ at 210oC to obtain a PLA/r-GO polymer composite. This composite is further mixed with PS via batch melt compounding. We observe the migration of r-GO from the PLA phase to the interface during annealing. The interfacial r-GO suppresses the coarsening of cocontinuous morphology and increases the conductivity of the filled polymer blend. Moreover, we systematically investigate the relationship between r-GO localization, rheological and conductivity change during annealing of r-GO filled PLA/PS blends. University of Minnesota Industrial Partnership for Research in Interfacial and Materials Engineering (IPRIME).

  6. The proteasome inhibitor, PS-341, causes cytokeratin aggresome formation.

    PubMed

    Bardag-Gorce, Fawzia; Riley, Nora E; Nan, Li; Montgomery, Rosalyn O; Li, Jun; French, Barbara A; Lue, Yan H; French, Samuel W

    2004-02-01

    Mallory body (MB) experimental induction takes 10 weeks of drug ingestion. Therefore, it is difficult to study the dynamics and mechanisms involved in vivo. Consequently, an in vitro study was done using primary tissue culture of hepatocytes from drug-primed mice livers in which MBs had already formed. The hypothesis to be tested was that MBs are cytokeratin aggresomes, which form when hepatocytes have a defective ubiquitin-proteasome pathway by which turnover of cytokeratin proteins is prevented. To test this hypothesis, primary tissue cultures of the hepatocytes from normal and MB-forming livers were incubated with the proteasome inhibitor PS-341 and then the cytokeratin filaments and the filament connecting proteins, that is, beta-actin, and ZO1, were visualized by immunofluorescence microscopy. PS-341 caused detachment of the cytokeratins from the cell surface plasma membrane. The cytokeratin filaments retracted toward the nucleus and cytokeratin aggresomes formed. In human livers, MBs showed colocalization of cytokeratin-8 (CK-8) with ubiquitin but not with beta-actin or ZO1. Mouse hepatoma cell lines were studied using PS-341 to induce cytokeratin aggresome formation. In these cell lines, the cytokeratin filaments first retracted toward the nucleus then formed cytokeratin-ubiquitin aggresomes polarized at one side of the nucleus. At the same time, the cells became dissociated from each other, however. The results simulated MB formation. MBs differ from cytokeratin aggresomes both morphologically and in ultrastructure.

  7. iPS cell transplantation for traumatic spinal cord injury

    PubMed Central

    Goulão, Miguel; Lepore, Angelo C.

    2016-01-01

    A large body of work has been published on transplantation of a wide range of neural stem and progenitor cell types derived from the developing and adult CNS, as well as from pluripotent embryonic stem cells, in models of traumatic spinal cord injury (SCI). However, many of these cell-based approaches present practical issues for clinical translation such as ethical cell derivation, generation of potentially large numbers of homogenously prepared cells, and immune rejection. With the advent of induced Pluripotent Stem (iPS) cell technology, many of these issues may potentially be overcome. To date, a number of studies have demonstrated integration, differentiation into mature CNS lineages, migration and long-term safety of iPS cell transplants in a variety of SCI models, as well as therapeutic benefits in some cases. Given the clinical potential of this advance in stem cell biology, we present a concise review of studies published to date involving iPS cell transplantation in animal models of SCI. PMID:26201863

  8. Region-specific metabolic alterations in the brain of the APP/PS1 transgenic mice of Alzheimer's disease.

    PubMed

    González-Domínguez, Raúl; García-Barrera, Tamara; Vitorica, Javier; Gómez-Ariza, José Luis

    2014-12-01

    Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide, but its etiology is still not completely understood. The identification of underlying pathological mechanisms is becoming increasingly important for the discovery of biomarkers and therapies, for which metabolomics presents a great potential. In this work, we studied metabolic alterations in different brain regions of the APP/PS1 mice by using a high-throughput metabolomic approach based on the combination of gas chromatography-mass spectrometry and ultra-high performance liquid chromatography-mass spectrometry. Multivariate statistics showed that metabolomic perturbations are widespread, affecting mainly the hippocampus and the cortex, but are also present in regions not primarily associated with AD such as the striatum, cerebellum and olfactory bulbs. Multiple metabolic pathways could be linked to the development of AD-type disorders in this mouse model, including abnormal purine metabolism, bioenergetic failures, dyshomeostasis of amino acids and disturbances in membrane lipids, among others. Interestingly, region-specific alterations were observed for some of the potential markers identified, associated with abnormal fatty acid composition of phospholipids and sphingomyelins, or differential regulation of neurotransmitter amino acids (e.g. glutamate, glycine, serine, N-acetyl-aspartate), not previously described to our knowledge. Therefore, these findings could provide a new insight into brain pathology in Alzheimer's disease.

  9. Expectation values of the e{sup +}PsH system

    SciTech Connect

    Zhang, J.-Y.; Mitroy, J.

    2007-07-15

    Close to converged energies and expectation values for e{sup +}PsH are computed using a ground-state wave function consisting of 1500 explicitly correlated Gaussians. The best estimate of the e{sup +}PsH{sup {infinity}} energy was -0.810 254 hartrees, which has a binding energy of 0.021 057 hartrees against dissociation into e{sup +}+PsH. The 2{gamma} annihilation rate was 2.7508x10{sup 9} s{sup -1}. Binding energies and annihilation rates are also given for the different finite-mass variants of e{sup +}PsH. Comparisons between expectation values for e{sup +}PsH and PsH provide compelling evidence that the e{sup +}PsH ground state can be regarded as consisting of a weakly bound positron orbiting the PsH ground state.

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

    PubMed Central

    Cao, Song; Sun, Mengjie; Li, Youyan

    2016-01-01

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

  11. UV-solvent annealing of PDMS-majority and PS-majority PS-b-PDMS block copolymer films

    NASA Astrophysics Data System (ADS)

    Lee, Keehong; Kreider, Melissa; Bai, Wubin; Cheng, Li-Chen; Safari Dinachali, Saman; Tu, Kun-Hua; Huang, Tao; Ntetsikas, K.; Liontos, G.; Avgeropoulos, A.; Ross, C. A.

    2016-11-01

    The response of polystyrene-block-poly(dimethylsiloxane) (PS-b-PDMS) thin films to UV exposure during solvent vapor annealing is described, in order to improve their applicability in nanolithography and nanofabrication. Two BCPs were examined, one with the PS block as majority (f PS = 68%, M n = 53 kg mol-1), the other with PDMS block as majority (f PDMS = 67%, M n = 44 kg mol-1). A 5 min UV irradiation was applied during solvent vapor annealing which led to both partial crosslinking of the polymer and a small increase in the temperature of the annealing chamber. This approach was effective for improving the correlation length of the self-assembled microdomain arrays and in limiting subsequent flow of the PDMS in the PDMS-majority BCP to preserve the post-anneal morphology. Ordering and orientation of microdomains were controlled by directed self-assembly of the BCPs in trench substrates. Highly-ordered perpendicular nanochannel arrays were obtained in the PDMS-majority BCP.

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

    PubMed

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

    2016-03-30

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

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

    PubMed

    Mino, Koshiki; Ishikawa, Kazuhiko

    2003-09-11

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

  14. PS integrins and laminins: key regulators of cell migration during Drosophila embryogenesis.

    PubMed

    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 αPSPS (PS1) and/or αPSPS (PS3) integrins are required in migrating cells, whereas αPSPS (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 α1,2 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.

  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 αPSPS (PS1) and/or αPSPS (PS3) integrins are required in migrating cells, whereas αPSPS (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 α1,2 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. 7 CFR 1753.37 - Plans and specifications (P&S).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Plans and specifications (P&S). 1753.37 Section 1753... Installation of Central Office Equipment § 1753.37 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S, the borrower shall review with the GFR the current and future requirements...

  17. 7 CFR 1753.26 - Plans and specifications (P&S).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 11 2013-01-01 2013-01-01 false Plans and specifications (P&S). 1753.26 Section 1753... Buildings § 1753.26 Plans and specifications (P&S). (a) For headquarters and commercial office buildings only, the borrower shall prepare preliminary P&S showing the floor plan and general...

  18. 7 CFR 1753.26 - Plans and specifications (P&S).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 11 2014-01-01 2014-01-01 false Plans and specifications (P&S). 1753.26 Section 1753... Buildings § 1753.26 Plans and specifications (P&S). (a) For headquarters and commercial office buildings only, the borrower shall prepare preliminary P&S showing the floor plan and general...

  19. 7 CFR 1753.37 - Plans and specifications (P&S).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 11 2014-01-01 2014-01-01 false Plans and specifications (P&S). 1753.37 Section 1753... Installation of Central Office Equipment § 1753.37 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S, the borrower shall review with the GFR the current and future requirements...

  20. 7 CFR 1753.47 - Plans and specifications (P&S).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 11 2013-01-01 2013-01-01 false Plans and specifications (P&S). 1753.47 Section 1753... Construction by Contract § 1753.47 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S for the construction project: (i) A review shall be made of the outside plant requirements,...

  1. 7 CFR 1753.37 - Plans and specifications (P&S).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 11 2012-01-01 2012-01-01 false Plans and specifications (P&S). 1753.37 Section 1753... Installation of Central Office Equipment § 1753.37 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S, the borrower shall review with the GFR the current and future requirements...

  2. 7 CFR 1753.26 - Plans and specifications (P&S).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 11 2012-01-01 2012-01-01 false Plans and specifications (P&S). 1753.26 Section 1753... Buildings § 1753.26 Plans and specifications (P&S). (a) For headquarters and commercial office buildings only, the borrower shall prepare preliminary P&S showing the floor plan and general...

  3. 7 CFR 1753.47 - Plans and specifications (P&S).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 11 2012-01-01 2012-01-01 false Plans and specifications (P&S). 1753.47 Section 1753... Construction by Contract § 1753.47 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S for the construction project: (i) A review shall be made of the outside plant requirements,...

  4. 7 CFR 1753.26 - Plans and specifications (P&S).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 11 2011-01-01 2011-01-01 false Plans and specifications (P&S). 1753.26 Section 1753... Buildings § 1753.26 Plans and specifications (P&S). (a) For headquarters and commercial office buildings only, the borrower shall prepare preliminary P&S showing the floor plan and general...

  5. 7 CFR 1753.37 - Plans and specifications (P&S).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 11 2011-01-01 2011-01-01 false Plans and specifications (P&S). 1753.37 Section 1753... Installation of Central Office Equipment § 1753.37 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S, the borrower shall review with the GFR the current and future requirements...

  6. 7 CFR 1753.37 - Plans and specifications (P&S).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 11 2013-01-01 2013-01-01 false Plans and specifications (P&S). 1753.37 Section 1753... Installation of Central Office Equipment § 1753.37 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S, the borrower shall review with the GFR the current and future requirements...

  7. 7 CFR 1753.47 - Plans and specifications (P&S).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 11 2014-01-01 2014-01-01 false Plans and specifications (P&S). 1753.47 Section 1753... Construction by Contract § 1753.47 Plans and specifications (P&S). (a) General. (1) Prior to the preparation of P&S for the construction project: (i) A review shall be made of the outside plant requirements,...

  8. Recurrent trisomy and Robertsonian translocation of chromosome 14 in murine iPS cell lines.

    PubMed

    Chen, Qian; Shi, Xiaoyun; Rudolph, Cornelia; Yu, Yong; Zhang, Ding; Zhao, Xiaoyu; Mai, Sabine; Wang, Gang; Schlegelberger, Brigitte; Shi, Qinghua

    2011-10-01

    Induced pluripotent stem (iPS) cells have greatly provoked people's interest due to their enormous potential of clinical applications. Increasing care is taken with the genetic safety of iPS cells. However, up to now, the chromosomal integrity of murine iPS (miPS) cells has been largely unknown. We have observed recurrent trisomy and/or Robertsonian translocation (Rb) of chromosome 14 in six out of nine independent miPS cell lines from three laboratories by G-banding, fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) analyses, while all the miPS cell lines were derived from mouse embryonic fibroblasts (MEFs) or neural precursor cells (NPCs) with a normal karyotype. The miPS cells with trisomy and/or Rb of chromosome 14 showed growth advantage over the miPS cells with a normal karyotype. We found a significantly higher frequency of Rbs in the miPS cell lines induced with c-Myc than those without c-Myc. Our findings demonstrate that miPS cell lines have the propensity for chromosomal aberrations and there is an obvious correlation between the extent of chromosomal aberrations in miPS cells and the transcriptional factors used for their reprogramming. Therefore, our study raises awareness of the need for improvements of the induction conditions of miPS cells in order to avoid the chromosomal aberrations and ensure future safe applications.

  9. 7 CFR 1753.26 - Plans and specifications (P&S).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Buildings § 1753.26 Plans and specifications (P&S). (a) For headquarters and commercial office buildings only, the borrower shall prepare preliminary P&S showing the floor plan and general architectural... 7 Agriculture 11 2010-01-01 2010-01-01 false Plans and specifications (P&S). 1753.26 Section...

  10. Communicating Knowing through Communities of Practice: Exploring Internal Communicative Processes and Differences among CoPs

    ERIC Educational Resources Information Center

    Iverson, Joel O.; McPhee, Robert D.

    2008-01-01

    Knowing is an enacted, communicated process that is difficult to observe, let alone manage, in organizations. Communities of practice (CoPs) offer a productive solution for improving knowledge and knowledge management, but the communicative processes that enact CoPs have not been explored, leaving CoPs as an organizational black box. This research…

  11. PsHint1, associated with the G-protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae.

    PubMed

    Zhang, Xin; Zhai, Chunhua; Hua, Chenlei; Qiu, Min; Hao, Yujuan; Nie, Pingping; Ye, Wenwu; Wang, Yuanchao

    2016-02-01

    Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G-protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1-interacting proteins, including PsHint1, a histidine triad (HIT) domain-containing protein orthologous to human HIT nucleotide-binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms of PsGPA1. An analysis of the gene-silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1-silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα-independent pathway involved in the pathogenicity of P. sojae.

  12. Combustion of PMMA, PE, and PS in a ramjet

    SciTech Connect

    van der Geld, C.W.M. ); Korting, P.A.O.G. ); Wijchers, T. )

    1990-03-01

    This paper reports the combustion behavior of polymethylmetharcrylate (PMMA), polyethylene (PE), and polystyrene (PS) with air investigated in a connected pipe test facility; spectroscopy showed the presence of OH, C{sub 2}, and CH and temperatures between 1300 and 3000 K during combustion. Particular attention was focused on regression rate and combustion efficiency and the role of temperature and soot production. The present investigation gives an understanding of the most important phenomena that control (or emanate from) the combustion of a cylindrical solid fuel with a rearward facing step, and this has application for solid fuel ramjets, the safe burning of toxic waste, and hot gas generators. The results are summarized.

  13. BioMaPS: A Roadmap for Success

    PubMed Central

    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 biology faculty and has produced research insights and curriculum developments at the intersection of these two disciplines. The goals, structure, achievements, and curriculum initiatives are described in relation to the effects they have had to enhance the study of biomathematics. PMID:20810948

  14. Antiprotonic potentials from global fits to the PS209 data

    NASA Astrophysics Data System (ADS)

    Friedman, E.; Gal, A.

    2004-01-01

    The experimental results for strong interaction effects in antiprotonic atoms by the PS209 collaboration consist of high quality data for several sequences of isotopes along the periodic table. Global analysis of these data in terms of a p¯-nucleus optical potential achieves good description of the data using a s-wave finite-range p¯N interaction. Equally good fits are also obtained with a poorly-defined zero-range potential containing a p-wave term.

  15. Swift/XRT detection of PS17bgn/2017bcc

    NASA Astrophysics Data System (ADS)

    Sokolovsky, K.; Wyrzykowski, L.; Hamanowicz, A.; Gromadzki, M.

    2017-03-01

    PS17bgn was discovered on 2017-02-18 0.3 arcsec. from the center of the galaxy SDSS J113152.97+295944.8. Spectroscopic observations with the NTT (PESSTO, ATel #10105) on 2017-02-20 and NOT 2017-02-28 suggest this may be a superluminous Type II supernova (SLSN-II) or an AGN flare at z=0.148, however, the re-examination of the spectrum shows it is at redshift z=0.133, lower than the one reported in ATel #10105.

  16. Genetic transformation in the methanogen Methanococcus voltae PS

    NASA Technical Reports Server (NTRS)

    Bertani, G.; Baresi, L.

    1987-01-01

    Mutations causing requirements for histidine, purine, and vitamin B12 were obtained in strain PS of Methanococcus voltae (archaebacteria) upon irradiation with UV or gamma rays. The first two mutations were shown to revert at low frequencies and were used to demonstrate the occurrence of transformation with homologous, wild-type DNA. The transformation rates obtained for these presumably chromosomal markers were in the range of 2 to 100 transformants per microgram of DNA. Mutants resistant to 2-bromoethanesulfonate and to 5-methyl-DL-tryptophan were also isolated.

  17. Quality control of the tribological coating PS212

    NASA Technical Reports Server (NTRS)

    Sliney, Harold E.; Dellacorte, Christopher; Deadmore, Daniel L.

    1989-01-01

    PS212 is a self-lubricating, composite coating that is applied by the plasma spray process. It is a functional lubricating coating from 25 C (or lower) to 900 C. The coating is prepared from a blend of three different powders with very dissimilar properties. Therefore, the final chemical composition and lubricating effectiveness of the coatings are very sensitive to the process variables used in their preparation. Defined here are the relevant variables. The process and analytical procedures that will result in satisfactory tribological coatings are discussed.

  18. 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.

  19. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    PubMed

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.

  20. A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins.

    PubMed Central

    Stepanov, V M; Rudenskaya, G N; Revina, L P; Gryaznova, Y B; Lysogorskaya, E N; Filippova IYu; Ivanova, I I

    1992-01-01

    A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins). Images Fig. 3. PMID:1637313

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

    PubMed Central

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

    2016-01-01

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

  2. D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period

    PubMed Central

    Meunier, Claire N. J.; Dallérac, Glenn; Le Roux, Nicolas; Sacchi, Silvia; Levasseur, Grégoire; Amar, Muriel; Pollegioni, Loredano; Mothet, Jean-Pierre; Fossier, Philippe

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors. PMID:27003418

  3. D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

    PubMed

    Meunier, Claire N J; Dallérac, Glenn; Le Roux, Nicolas; Sacchi, Silvia; Levasseur, Grégoire; Amar, Muriel; Pollegioni, Loredano; Mothet, Jean-Pierre; Fossier, Philippe

    2016-01-01

    N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

  4. Functional roles of endogenous D-serine in pain-induced ultrasonic vocalization.

    PubMed

    Tsuzuki, Hitomi; Maekawa, Masao; Konno, Ryuichi; Hori, Yuuichi

    2012-11-14

    The N-methyl-D-aspartate receptor (NMDAR) is crucial for pain-related behaviors. D-Serine is synthesized from L-serine by serine racemase (SR) and modulates NMDAR functions by acting as an agonist at the glycine-binding site. We analyzed noxious stimulus-induced ultrasonic vocalization and locomotor activity in the open-field test using SR knockout (SR-KO) mice to examine the role of endogenous D-serine in mammalian behaviors. SR-KO mice emitted less ultrasonic vocalization after noxious stimulation (VAS) than wild-type (WT) mice. The locomotor activity of WT mice decreased with repeated daily exposures to the open field, whereas that of SR-KO mice remained unchanged. VAS was significantly enhanced during arthritis in WT mice, whereas it was not enhanced during arthritis in SR-KO mice. These results indicate that mice lacking the ability to produce D-serine endogenously in the brain differ from normal mice with respect to the chronic pain-induced behavioral changes.

  5. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.

    PubMed

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

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

    PubMed

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

    2009-10-01

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

  7. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei.

    PubMed

    Qin, Zhen; Yan, Qiaojuan; Ma, Qingjun; Jiang, Zhengqiang

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5'-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0-9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering.

  8. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase

    PubMed Central

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-01-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE. PMID:26790714

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

    PubMed

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

    2009-12-10

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

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

    PubMed

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  12. 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

  13. [Features of Expression of the PsSst] and PsIgn1 Genes in Nodules of Pea (Pisum sativum L.) Symbiotic Mutants].

    PubMed

    Zhukova, V A; Rychagova, T S; Fedorina, Ya V; Pinaeva, A G; Andronova, E E; Borisova, A Yu; Tikhonovich, I A

    2016-04-01

    The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotusjaponicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix⁻ (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.

  14. 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.

  15. iPS cell technology: Future impact on renal care.

    PubMed

    Freedman, Benjamin S; Steinman, Theodore I

    2015-08-01

    iPS cells from patients with kidney disease are a new tool with the potential to impact the future of renal care. They can be used in the laboratory to model the pathophysiology of human kidney disease, and have the potential to establish a new area of immunocompatible, on-demand renal transplantation. Critical challenges remain before the full potential of these cells can be accurately assessed. We need to understand whether the derived cell types are mature and can replace kidney function(s). To what extent can iPS cells model kidney disease in the simplified environment of cell culture? Ultimately, successful integration of these cells as autograft therapies will require demonstration of safety and efficacy equal or superior to the existing gold standards of kidney allograft transplantation and dialysis. Specific educational and infrastructural changes will be necessary if these specialized technologies are to be adopted as an accepted modalities in clinical medicine. Given these barriers, the first fruit of these labors is likely to be improved understanding of pathophysiological pathways in human IPS cell disease models, followed by drug discovery and testing. These experiments will lead naturally to improvements in differentiation and experiments in animal models testing function. The time course to achieve the desired goals remains unknown, but the ultimate hope is that new, more effective and less expensive modalities for renal replacement therapy will occur in the foreseeable future. A new standard of care for patients is anticipated that addresses limitations of currently available treatments.

  16. Simple sub-50-ps rise-time high voltage generator

    NASA Astrophysics Data System (ADS)

    Kekez, M. M.

    1991-12-01

    This article relates to the development of an ultrafast (nanoseconds-picoseconds time scale) compact system(s) readily applicable to the field of EMP/radiation, x-ray-induced nondestructive testing, plasma fusion (energy) experiments, bioelectromagnetic (food-drug) sterilization, drivers for x-ray preionized XeCl laser and similar applications. The present work shows that the Marx and the Pulse forming section can be integrated into a single unit. The stray capacitance present in each stage acts as a peaking capacitor. For a charging voltage per stage of <40 kV, the rise time of the output pulse is below 50 ps at 200 kV into a 100-Ω load. Work is in progress to extend the voltage amplitude to 1.6 MV while maintaining the relative pulse waveform. With a contemporary optical diagnostic technique it is believed that the present concept may achieve 1-10 ps rise-time pulses at a megavolt level in ``smart gas mixtures.'' In addition a solution for the classical peaking circuit has been obtained and presented in the Appendix.

  17. 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.

  18. On the roles of the alanine and serine in the β-sheet structure of fibroin.

    PubMed

    Carrascoza Mayen, Juan Francisco; Lupan, Alexandru; Cosar, Ciprian; Kun, Attila-Zsolt; Silaghi-Dumitrescu, Radu

    2015-02-01

    In its silk II form, fibroin is almost exclusively formed from layers of β-sheets, rich in glycine, alanine and serine. Reported here are computational results on fibroin models at semi-empirical, DFT levels of theory and molecular dynamics (MD) for (Gly)10, (Gly-Ala)5 and (Gly-Ser)5 decapeptides. While alanine and serine introduce steric repulsions, the alanine side-chain adds to the rigidity of the sheet, allowing it to maintain a properly pleated structure even in a single β-sheet, and thus avoiding two alternative conformations which would interfere with the formation of the multi-layer pleated-sheet structure. The role of the serine is proposed to involve modulation of the hydrophobicity in order to construct the supramolecular assembly as opposed to random precipitation due to hydrophobicity.

  19. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms.

    PubMed

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M

    2014-08-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding.

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

    PubMed

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

    2013-07-01

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

  1. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms

    PubMed Central

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M.

    2014-01-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding. PMID:24878526

  2. Adaptation of caddisfly larval silks to aquatic habitats by phosphorylation of h-fibroin serines.

    PubMed

    Stewart, Russell J; Wang, Ching Shuen

    2010-04-12

    Aquatic caddisflies diverged from a silk-spinning ancestor shared with terrestrial moths and butterflies. Caddisfly larva spin adhesive silk underwater to construct protective shelters with adventitiously gathered materials. A repeating (SX)(n) motif conserved in the H-fibroin of several caddisfly species is densely phosphorylated. In total, more than half of the serines in caddisfly silk may be phosphorylated. Major molecular adaptations allowing underwater spinning of an ancestral dry silk appear to have been phosphorylation of serines and the accumulation of basic residues in the silk proteins. The amphoteric nature of the silk proteins could contribute to silk fiber assembly through electrostatic association of phosphorylated blocks with arginine-rich blocks. The presence of Ca(2+) in the caddisfly larval silk proteins suggest phosphorylated serines could contribute to silk fiber periodic substructure through Ca(2+) crossbridging.

  3. TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases.

    PubMed

    Busch, Florian; Rajendran, Chitra; Mayans, Olga; Löffler, Patrick; Merkl, Rainer; Sterner, Reinhard

    2014-09-30

    The rapid increase of the number of sequenced genomes asks for the functional annotation of the encoded enzymes. We used a combined computational-structural approach to determine the function of the TrpB2 subgroup of the tryptophan synthase β chain/β chain-like TrpB1-TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are O-phospho-l-serine dependent tryptophan synthases, whereas TrpB1 enzymes catalyze the l-serine dependent synthesis of tryptophan. We found a single residue being responsible for the different substrate specificities of TrpB1 and TrpB2 and confirmed this finding by mutagenesis studies and crystallographic analysis of a TrpB2 enzyme with bound O-phospho-l-serine.

  4. Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: an advantageous alternative to ES and iPS cells.

    PubMed

    Amoh, Yasuyuki; Kanoh, Maho; Niiyama, Shiro; Hamada, Yuko; Kawahara, Katsumasa; Sato, Yuichi; Hoffman, Robert M; Katsuoka, Kensei

    2009-08-01

    The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K-15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary-acidic-protein (GFAP)-positive Schwann cells and promoted the recovery of pre-existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells.

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

    PubMed

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

    1987-07-01

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

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

    PubMed

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

    2016-01-15

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

  7. Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β-Lactone

    PubMed Central

    2015-01-01

    Flavaglines are a class of natural products with potent insecticidal and anticancer activities. β-Lactones are a privileged structural motif found in both therapeutic agents and chemical probes. Herein, we report the synthesis, unexpected light-driven di-epimerization, and activity-based protein profiling of a novel rocaglate-derived β-lactone. In addition to in vitro inhibition of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases CTSA and SCPEP1, in PC3 cells. PMID:24447064

  8. Comparison of implosion core metrics: A 10 ps dilation X-ray imager vs a 100 ps gated microchannel plate

    NASA Astrophysics Data System (ADS)

    Nagel, S. R.; Benedetti, L. R.; Bradley, D. K.; Hilsabeck, T. J.; Izumi, N.; Khan, S.; Kyrala, G. A.; Ma, T.; Pak, A.

    2016-11-01

    The dilation x-ray imager (DIXI) [T. J. Hilsabeck et al., Rev. Sci. Instrum. 81, 10E317 (2010); S. R. Nagel et al., ibid. 83, 10E116 (2012); S. R. Nagel et al., ibid. 85, 11E504 (2014)] is a high-speed x-ray framing camera that uses the pulse-dilation technique to achieve a temporal resolution of less than 10 ps. This is a 10 × improvement over conventional framing cameras currently employed on the National Ignition Facility (NIF) (100 ps resolution), and otherwise only achievable with 1D streaked imaging. A side effect of the dramatically reduced gate width is the comparatively lower detected signal level. Therefore we implement a Poisson noise reduction with non-local principal component analysis method [J. Salmon et al., J. Math. Imaging Vision 48, 279294 (2014)] to improve the robustness of the DIXI data analysis. Here we present results on ignition-relevant experiments at the NIF using DIXI. In particular we focus on establishing that/when DIXI gives reliable shape metrics (P0, P2, and P4 Legendre modes, and their temporal evolution/swings).

  9. Laser-free RF-gun as a combined source of THz and ps-sub-ps X-rays

    DOE PAGES

    Agustsson, R.; Boucher, S.; Finn, O.; ...

    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

  10. Laser-free RF-gun as a combined source of THz and ps-sub-ps X-rays

    SciTech Connect

    Agustsson, R.; Boucher, S.; Finn, O.; Hartzell, J.; Ruelas, M.; Smirnov, A. V.; Storms, S.; Ning, Z.; Murokh, A.; Campese, T.; Faillace, L.; Verma, A.; Kim, Y.; Buaphad, P.; Andrews, A.; Berls, B.; Eckman, C.; Folkman, K.; Knowles-Swingle, A.; O’Neill, C.; Smith, M.; Grandsaert, T.; van der Geer, B.; de Loos, M.; Berg, W. J.; Sereno, N. S.; Sun, Y.; Zholents, A. A.

    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.

  11. Comparison of implosion core metrics: A 10 ps dilation X-ray imager vs a 100 ps gated microchannel plate.

    PubMed

    Nagel, S R; Benedetti, L R; Bradley, D K; Hilsabeck, T J; Izumi, N; Khan, S; Kyrala, G A; Ma, T; Pak, A

    2016-11-01

    The dilation x-ray imager (DIXI) [T. J. Hilsabeck et al., Rev. Sci. Instrum. 81, 10E317 (2010); S. R. Nagel et al., ibid. 83, 10E116 (2012); S. R. Nagel et al., ibid. 85, 11E504 (2014)] is a high-speed x-ray framing camera that uses the pulse-dilation technique to achieve a temporal resolution of less than 10 ps. This is a 10 × improvement over conventional framing cameras currently employed on the National Ignition Facility (NIF) (100 ps resolution), and otherwise only achievable with 1D streaked imaging. A side effect of the dramatically reduced gate width is the comparatively lower detected signal level. Therefore we implement a Poisson noise reduction with non-local principal component analysis method [J. Salmon et al., J. Math. Imaging Vision 48, 279294 (2014)] to improve the robustness of the DIXI data analysis. Here we present results on ignition-relevant experiments at the NIF using DIXI. In particular we focus on establishing that/when DIXI gives reliable shape metrics (P0, P2, and P4 Legendre modes, and their temporal evolution/swings).

  12. Transient development of SRS and SBS in ps-time scale by using sub-ps Thomson diagnostic

    NASA Astrophysics Data System (ADS)

    Rousseaux, C.; Casanova, M.; Gremillet, L.; Loiseau, P.; Rabec Le Gloahec, M.; Baton, S. D.; Amiranoff, F.; Audebert, P.; Popescu, H.; Adam, J. C.; Héron, A.; Hüller, S.; Mora, P.; Pesme, D.

    2006-06-01

    The control of parametric instabilities in large plasmas remains a challenge for the ICF program. Clearly, kinetic effects play an important role in the saturation mechanisms. Sub-picosecond Thomson analysis associated with short pulse interaction permits to explore these topics. A set of experiments have been performed in preformed, He plasmas using the 100-TW laser facility at LULI. The spectra of the electrostatic waves driven by stimulated Raman and Brillouin backscatterings generated in the 1.5 ps, ω laser interaction have been measured with 0.3 ps time-resolution by using a short Thomson probe. Additionally, space-resolved and k-resolved spectra have been obtained. The experiments show that the fastest instability -B-SRS- first develops in the rising part of the pump. The B-SBS-driven IAW grows more slowly. B-SRS then abruptly vanishes around the maximum of the pump, while the IAW can be detected tens of picoseconds after the pump, allowing direct measurement of the IAW damping. The EPW k-spectra show that the EPW dispersion relation significantly deviates from the standard one. They exhibit a k-feature which could be related to the presence of a hot electron population produced in the B-SRS saturation process.

  13. 150-ps broadband low dispersion mirror thin film damage competition

    NASA Astrophysics Data System (ADS)

    Stolz, Christopher J.; Negres, Raluca A.; Kafka, Kyle; Chowdhury, Enam; Kirchner, Matt; Shea, Kevin; Daly, Meaghan

    2015-11-01

    Broadband low dispersion mirrors are fluence-limiting and pulse-shape-limiting components in short pulse lasers. To better understand the current technology state of broadband low dispersion mirrors, a laser damage competition was held at the 2015 Laser Damage Conference. Participants were asked to submit mirrors that met a minimum reflection of 99.5% at 45 degrees incidence angle at "P" polarization with a Group Dispersion Delay (GDD) of <± 100 fs2 over a spectral range of 773 nm ± 50 nm. The participants selected the coating materials, design, and deposition method. Laser damage testing was performed using the raster scan method with a 150 ps pulse length on a single testing facility to enable a direct comparison among the participants. GDD measurements were performed to validate specification compliance. Unfortunately nearly half of the submitted samples were found to not meet the GDD specifications. Details of the deposition processes, cleaning method, coating materials, and layer count are also shared.

  14. Development and characterization of sub-100 ps photomultiplier tubes

    SciTech Connect

    Horsfield, C. J.; Rubery, M. S.; Mack, J. M.; Young, C. S.; Herrmann, H. W.; Caldwell, S. E.; Evans, S. C.; Sedilleo, T. J.; Kim, Y. H.; McEvoy, A.; Milnes, J. S.; Howorth, J.; Davis, B.; O'Gara, P. M.; Garza, I.; Miller, E. K.; Stoeffl, W.; Ali, Z.

    2010-10-15

    We describe the evaluation of a microchannel plate (MCP) photomultiplier tube (PMT), incorporating a 3 {mu}m pore MCP and constant voltage anode and cathode gaps. The use of the small pore size results in PMTs with response functions of the order of 85 ps full-width-half-maximum, while the constant electric field across the anode and cathode gaps produces a uniform response function over the entire operating range of the device. The PMT was characterized on a number of facilities and employed on gas Cherenkov detectors fielded on various deuterium tritium fuel (DT) implosions on the Omega Laser Facility at the University of Rochester. The Cherenkov detectors are part of diagnostic development to measure Gamma ray reaction history for DT implosions on the National Ignition Facility.

  15. 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.

  16. 75 FR 16748 - Final Voluntary Product Standard; DOC PS 20-10 “American Softwood Lumber Standard”

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-02

    ... National Institute of Standards and Technology Final Voluntary Product Standard; DOC PS 20-10 ``American... DOC PS 20-10 ``American Softwood Lumber Standard'' which will supersede DOC PS 20-05. The Standard..., the Board of Review, and the National Grading Rule Committee. DATES: DOC PS 20-10 ``American...

  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. Coexistence of WiFi and WiMAX systems based on PS-request protocols.

    PubMed

    Kim, Jongwoo; Park, Suwon; Rhee, Seung Hyong; Choi, Yong-Hoon; Chung, Young-uk; Hwang, Ho Young

    2011-01-01

    We introduce both the coexistence zone within the WiMAX frame structure and a PS-Request protocol for the coexistence of WiFi and WiMAX systems sharing a frequency band. Because we know that the PS-Request protocol has drawbacks, we propose a revised PS-Request protocol to improve the performance. Two PS-Request protocols are based on the time division operation (TDO) of WiFi system and WiMAX system to avoid the mutual interference, and use the vestigial power management (PwrMgt) bit within the Frame Control field of the frames transmitted by a WiFi AP. The performance of the revised PS-Request protocol is evaluated by computer simulation, and compared to those of the cases without a coexistence protocol and to the original PS-Request protocol.

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

    PubMed

    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.

  20. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  1. 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.

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

    PubMed Central

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-01-01

    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 α-chymotrypsin to 9.4% residual activity and also inhibited α-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

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

    PubMed

    Jürgensen, V Würtz; Jalkanen, K

    2006-02-22

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

    PubMed

    Rudenskaya, G N; Bogacheva, A M; Preusser, A; Kuznetsova, A V; Dunaevsky YaE; Golovkin, B N; Stepanov, V M

    1998-10-23

    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 and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6-9 in the presence of 2 mM Ca2+, temperature optimum is at 40 degrees C; Km=0.37+/-0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases, cucumisin and macluralisin. The taraxalisin N-terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.

  5. Identification and characterization of a chymotrypsin-like serine protease from periodontal pathogen, Tannerella forsythia.

    PubMed

    Hockensmith, K; Dillard, K; Sanders, B; Harville, B A

    2016-11-01

    Tannerella forsythia is a bacteria associated with severe periodontal disease. This study reports identification and characterization of a membrane-associated serine protease from T. forsythia. The protease was isolated from T. forsythia membrane fractions and shown to cleave both gelatin and type I collagen. The protease was able to cleave both substrates over a wide range of pH values, however optimal cleavage occurred at pH 7.5 for gelatin and 8.0 for type I collagen. The protease was also shown to cleave both gelatin and type I collagen at the average reported temperature for the gingival sulcus however it showed a lack of thermal stability with a complete loss of activity by 60 °C. When treated with protease inhibitors the enzyme's activity could only be completely inhibited by serine protease inhibitors antipain and phenylmethanesulfonyl fluoride (PMSF). Further characterization of the protease utilized serine protease synthetic peptides. The protease cleaved N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide but not Nα-benzoyl-dl-arginine p-nitroanilide (BAPNA) or N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide indicating that the protease is a chymotrypsin-like serine protease. Since type I collagen is a major component in the gingival tissues and periodontal ligament, identification and characterization of this enzyme provides important information regarding the role of T. forsythia in periodontal disease.

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

    PubMed

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

    1983-10-01

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

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

    PubMed

    Ogawara, Hiroshi

    2016-09-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  10. Characterization and expression analysis of a trypsin-like serine protease from planarian Dugesia japonica.

    PubMed

    Zhou, Luming; Wu, Suge; Liu, Dianchen; Xu, Bo; Zhang, Xiufang; Zhao, Bosheng

    2012-06-01

    Trypsin-like serine proteases are involved in large number of processes, especially in digestive degradation and immune responses. Here, we identify the characterization of a trypsin-like serine protease in planarian, Djtry, which interestingly has the incompletely conserved catalytic triad (K, D, and S). Phylogenetic analysis suggests that Djtry is an ancient type of trypsin-like serine proteases. The spatial and temporal expression patterns of Djtry are shown during regenerating and embryonic development by whole-mount in situ hybridization. Djtry is found to display a tissue specific expression pattern, with a predominant expression detected in whole gut region of intact and regenerating planarian. While the tissue- and stage-specific expression patterns during the embryonic development imply the roles of Djtry involve in yolk degradation and gut formation. Quantitative real-time PCR was carried out to analyze the function of this protease in vivo after planarians were stimulated to a bacterial challenge and food. The results showed that Djtry increased after a bacterial challenge and was basically stable for food. Therefore, the trypsin-like serine protease might be involved in the innate defense reactions against bacterial infection.

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

    PubMed Central

    Santos, M A; Tuite, M F

    1995-01-01

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

  12. Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein

    PubMed Central

    Tyshchuk, Oksana; Völger, Hans Rainer; Bulau, Patrick; Koll, Hans; Mølhøj, Michael

    2017-01-01

    ABSTRACT Molecular mass determination by electrospray ionization mass spectrometry of a recombinant IgG-based fusion protein (mAb1-F) produced in human embryonic kidney (HEK) cells demonstrated the presence of a dominant +79 Da product variant. Using LC-MS tryptic peptide mapping analysis and collision-induced dissociation (CID) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the C-terminal serine residue of a glycine-serine linker [(G4S)2] of a fused heavy chain containing in total 2 (G4S)2-linkers. The modification was identified as a phosphorylation (+79.97 Da) by the presence of a 98 Da neutral loss reaction with CID, by spiking a synthetic phosphoserine peptide, and by dephosphorylation with alkaline phosphatase. A thermolysin digest combined with higher-energy collision dissociation (HCD) positioned the phosphoserine to one specific glycine-serine linker of the fused heavy chain, and the relative level of phosphorylated linker was determined to be 11.3% and 0.4% by LC-MS when the fusion protein was transiently expressed in HEK or in stably transformed Chinese hamster ovary cells, respectively. This observation demonstrates that fusions with glycine-serine linker sequences should be carefully evaluated during drug development to prevent the introduction of a phosphorylation site in therapeutic fusion proteins. PMID:27661266

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

    PubMed

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

    2016-01-01

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

  14. Serine/threonine protein phosphatases: multi-purpose enzymes in control of defense mechanisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine/threonine protein phosphatases are a group of enzymes involved in the regulation of defense mechanisms in plants. This paper describes the effects of an inhibitor of these enzymes on the expression of all of the genes associated with these defense mechanisms. The results suggest that inhibi...

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

    PubMed

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

    2015-01-01

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

  16. Phase I clinical trial of safety of L-serine for ALS patients.

    PubMed

    Levine, Todd D; Miller, Robert G; Bradley, Walter G; Moore, Dan H; Saperstein, David S; Flynn, Lynne E; Katz, Jonathan S; Forshew, Dallas A; Metcalf, James S; Banack, Sandra A; Cox, Paul A

    2017-02-01

    We performed a randomized, double-blind phase I clinical trial for six months on the effects of oral L-serine in patients with ALS. The protocol called for enrollment of patients with a diagnosis of probable or definite ALS, age 18-85 years, disease duration of less than three years and forced vital capacity (FVC) ≥ 60%. Patients were randomly assigned to four different oral twice-daily dose regimens (0.5, 2.5, 7.5, or 15 g/dose). Blood, urine and CSF samples, ALS Functional Rating Scale-Revised (ALSFRS-R) scores and forced vital capacity (FVC) were obtained throughout the trial. Disease progression was compared with matched historical placebo controls from five previous ALS therapeutic trials. Of 20 patients enrolled, one withdrew before receiving study drug and two withdrew with gastro-intestinal problems. Three patients died during the trial. L-serine was generally well tolerated by the patients and L-serine did not appear to accelerate functional decline of patients as measured by slope of their ALSFRS-R scores. Based on this small study, L-serine appears to be generally safe for patients with ALS.

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

    PubMed

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

    2016-08-04

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

  18. The natural killer cell serine protease gene Lmet1 maps to mouse chromosome 10

    SciTech Connect

    Thia, K.Y.T.; Smyth, M.J.; Jenkins, N.A.; Gilbert, D.J.; Copeland, N.G.

    1995-01-01

    Cytotoxic lymphocytes play a key role in immune responses against viruses and tumors. Lymphocyte-mediated cytolysis by both cytotoxic T lymphocytes (CTL) and natural killer (NK) cells is often associated with the formation of membrane lesions on target cells caused by exocytosis of cytoplasmic granule serine proteases and a pore-forming protein, perforin. A variety of granzymes have been found to reside within the cytoplasmic granules of cytotoxic lymphocytes, but unlike perforin, isolated serine proteases are not intrinsically lytic. However, a role for serine proteases in cellular cytotoxicity has been supported by the ability of protease inhibitors to completely abrogate lymphocyte cytotoxicity, and the demonstration that serine proteases can initiate DNA fragmentation in target cells transfected or pretreated with a sublytic concentration of perforin. Granzymes cloned in human, mouse, and rat encode four granzyme activities and all are expressed in either T cells, their thymic precursors, and/or NK cells. In particular, a rat granzyme that cleaves after methionine residues, but not phenylalanine residues and its human equivalent, human Met-ase 1, are unique granzymes with restricted expression in CD3-NK cells. 24 refs., 2 figs.

  19. 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...

  20. 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...

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

    PubMed Central

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

    2016-01-01

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

  2. A PC-Based Time Interval Counter With 200 PS Resolution

    DTIC Science & Technology

    2004-09-01

    35th Annual Precise Time and Time Interval (PTTI) Meeting 359 A PC-BASED TIME INTERVAL COUNTER WITH 200 PS RESOLUTION Józef...CMOS FPGA technology, the 200 ps resolution (1 LSB - Least Significant Bit) was achieved in single-shot measurements of time intervals within the...time counter with 200 ps resolution in a single CMOS FPGA (Complementary-Metal-Oxide- Semiconductor Field-Programmable-Gate-Array) device [1,2], as a

  3. MATRIX: a 15 ps resistive interpolation TDC ASIC based on a novel regular structure

    NASA Astrophysics Data System (ADS)

    Mauricio, J.; Gascón, D.; Ciaglia, D.; Gómez, S.; Fernández, G.; Sanuy, A.

    2016-12-01

    This paper presents a 4-channel TDC ASIC with the following features: 15-ps LSB (9.34 ps after calibration), 10-ps jitter, < 4-ps time resolution, up to 10 MHz of sustained input rate per channel, 45 mW of power consumption and very low area (910×215 μm2) in a commercial 180 nm technology. The main contribution of this work is the novel design of the clock interpolation circuitry based on a resistive interpolation mesh circuit (patented), a two-dimensional regular structure with very good properties in terms of power consumption, area and low process variability.

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

    PubMed Central

    Southorn, B G; Thompson, J R

    1987-01-01

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

  5. 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

    2014-09-01

    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.

  6. 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.

  7. Neural stem cells differentiated from iPS cells spontaneously regain pluripotency.

    PubMed

    Choi, Hyun Woo; Kim, Jong Soo; Choi, Sol; Hong, Yean Ju; Kim, Min Jung; Seo, Han Geuk; Do, Jeong Tae

    2014-10-01

    Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cell-derived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP(+) ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP(+) cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.

  8. Detergent-protein interactions in aqueous buffer suspensions of Photosystem I (PS I).

    PubMed

    Mukherjee, Dibyendu; May, Mark; Khomami, Bamin

    2011-06-15

    Systematic and uniform monolayer formation of Photosystem I (PS I) onto self-assembled monolayer (SAM) substrates to enable unidirectional electron transfer is crucial for its successful use in the fabrication of bio-hybrid solid-state electronic or photovoltaic devices. Yet, our recent studies (Mukherjee et al., 2010) indicate that surface self-assembly of PS I from aqueous buffer suspensions onto alkanethiolate SAM/Au substrates frequently leads to complex columnar structures due to solution phase protein aggregations. We investigate the effect of two prototypical non-ionic detergents, n-Dodecyl-β-D-Maltoside (DM) and Triton X-100 (TX-100), on protein-protein interactions via the protein-detergent interfacial chemistry. Dynamic light scattering (DLS) experiments are used to demonstrate the impact of relative protein/detergent concentrations on aggregation dynamics of PS I suspensions. In turn, the surface attachment characteristics of PS I adsorbed from the aforementioned suspensions onto SAM/Au substrate is examined by atomic force (AFM) microscopy. Our results indicate that relative concentration of PS I and detergents (DM or, TX-100) with respect to their critical micelle concentrations (CMC) determines the extent of self-association between PS I complexes driven by the screening induced by detergent micelles and/or, inter-protein distances. Such interfacial phenomena during the PS I-detergent complexation process drives the colloidal system through various regimes of phase separations, suspension and/or, de-aggregation, wherein individual PS I complexes can exist in a frustrated state that prevents favorable orientations for PS I-PS I interactions. The present study presents a novel strategy, heretofore not considered, for tailoring inter-protein distances and protein-protein interactions in solution phase, thereby allowing a superior control on the surface attachment of PS I onto SAM/Au substrates.

  9. Generation of Endoderm derived Human iPS cells from Primary Hepatocytes

    PubMed Central

    Liu, Hua; Ye, Zhaohui; Kim, Yong-Hak; Sharkis, Saul; Jang, Yoon-Young

    2010-01-01

    Recent advances in induced pluripotent stem (iPS) cell research significantly changed our perspective on regenerative medicine. Patient specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to hES cells or safer than hES cells. There are several important issues which need to be addressed and foremost are the safety and efficacy of human iPS cells from different origins. Human iPS cells have been derived mostly from cells originated from mesoderm, with a few cases from ectoderm. So far there has been no report of endoderm derived human iPS cells, preventing comprehensive comparative investigations on the quality of human iPS cells from different origins. Here we show for the first time reprogramming of human endoderm derived cells (i.e. primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from human embryonic stem cells in colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells were able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. The technology to develop endoderm derived human iPS cell lines, together with other established cell lines, will provide a foundation to elucidate the mechanisms of cellular reprogramming and to study the safety and efficacy of differentially originated human iPS cells for cell therapy. For studying liver disease pathogenesis, this technology also provides a potentially more amenable system to generate liver disease specific iPS cells. PMID:20432258

  10. Stereodefined phosphorothioate analogues of DNA: relative thermodynamic stability of the model PS-DNA/DNA and PS-DNA/RNA complexes.

    PubMed

    Boczkowska, Małgorzata; Guga, Piotr; Stec, Wojciech J

    2002-10-15

    Thermodynamic data regarding the influence of P-chirality on stability of duplexes formed between phosphorothioate DNA oligonucleotides (of either stereo-defined all-R(P) or all-S(P) or random configuration at the P atoms) and complementary DNA or RNA strands are presented. Measured melting temperatures and calculated DeltaG(37)(o) values showed that duplexes formed by PS-DNA oligomers with DNA strands are less stable than their unmodified counterparts. However, relative stability of the duplexes ([all-R(P)]-PS-DNA/DNA vs [all-S(P)]-PS-DNA/DNA) depends on their sequential composition rather than on the absolute configuration of PS-oligos, contrary to the results of theoretical considerations and molecular modeling reported in the literature. On the other hand, for all six analyzed pairs of diastereomers, the [all-R(P)]-PS isomers form more stable duplexes with RNA templates, but the origin of stereodifferentiation depends on the sequence with more favorable entropy and enthalpy factors which correlated with dT-rich and dA/dG-rich PS-oligomers, respectively.

  11. Chromosome 14 familial Alzheimer's disease: the clinical and neuropathological characteristics of a family with a leucine→serine (L250S) substitution at codon 250 of the presenilin 1 gene

    PubMed Central

    Harvey, R.; Ellison, D.; Hardy, J.; Hutton, M.; Roques, P.; Collinge, J.; Fox, N.; Rossor, M.

    1998-01-01

    BACKGROUND—Seven affected members are described from a kindred with autosomal dominant familial Alzheimer's disease associated with a novel mutation in the presenilin 1 (PS1) gene on chromosome 14that results in a leucine to serine substitution at codon 250(L250S).
METHOD—Clinical information on the pedigree was collected directly from family members including affected members and their carers and also from hospital records.
RESULTS—Detailed clinical information was available on five members. All had an early age at onset with a median age of 52 (95% confidence interval (95% CI) 49.4-54.9). Age at onset varied between 49 and 56 years, with duration of illness varying between six years and 15 years. Myoclonus, depression, and psychosis were features of this pedigree; seizures were not reported.
CONCLUSIONS—PS1 L250S familial Alzheimer's disease is an early onset form of Alzheimer's disease with clinical features similar to other reported familial Alzheimer's disease pedigrees, except that seizures were absent.

 PMID:9436726

  12. Anisotropic Polymer Conformations in Aligned SWCNT/PS Nanocomposites

    NASA Astrophysics Data System (ADS)

    Tung, Wei-Shao; Composto, Russell; Clarke, Nigel; Winey, Karen

    2015-03-01

    In our previous study of isotropic SWCNT/polystyrene (PS) nanocomposites, we found that the polymer radius of gyration (Rg) increases and the SWCNT mesh size decreases with increasing SWCNT concentration. Here, we investigate the effect of aligning SWCNTs on Rg and the SWNCT mesh. SWCNT alignment was accomplished by melt fiber spinning or extrusion, and small angle neutron scattering was used to probe the structure. As expected, SWCNT alignment produces anisotropic meshes. Below 2 wt% SWCNT, no significant change in Rg is observed and Rg parallel and perpendicular to the direction of SWCNT alignment are comparable. More interestingly, at higher wt% SWCNT the polymer conformations are anisotropic with a larger Rg perpendicular to the SWCNT than along the SWCNT. For example, with 6 wt% SWCNT, the Rg perpendicular to the SWCNT is ~ 15% larger than parallel to the SWNCT. This anisotropy in the polymer conformation becomes more prominent at higher SWCNT concentrations, perhaps because at higher SWCNT concentrations the distribution of mesh shifts to smaller meshes (<2Rg) so that a single polymer chain interacts with multiple SWCNTs. Implications of this finding will be discussed. National Science Foundation DMR-12-10379.

  13. A Critical Appraisal of NLO+PS Matching Methods

    SciTech Connect

    Hoeche, Stefan; Krauss, Frank; Schonherr, Marek; Siegert, Frank; /Freiburg U.

    2012-03-19

    In this publication, uncertainties in and differences between the MC{at}NLO and POWHEG methods for matching next-to-leading order QCD calculations with parton showers are discussed. Implementations of both algorithms within the event generator SHERPA are employed to assess the impact on a representative selection of observables. In the MC{at}NLO approach a phase space restriction has been added to subtraction and parton shower, which allows to vary in a transparent way the amount of non-singular radiative corrections that are exponentiated. Effects on various observables are investigated, using the production of a Higgs boson in gluon fusion, with or without an associated jet, as a benchmark process. The case of H+jet production is presented for the first time in an NLO+PS matched simulation. Uncertainties due to scale choices and non-perturbative effects are explored in the production of W{sup {+-}} and Z bosons in association with a jet. Corresponding results are compared to data from the Tevatron and LHC experiments.

  14. Optimisation of freeze drying conditions for purified serine protease from mango (Mangifera indicaCv. Chokanan) peel.

    PubMed

    Mehrnoush, Amid; Tan, Chin Ping; Hamed, Mirhosseini; Aziz, Norashikin Ab; Ling, Tau Chuan

    2011-09-01

    This study investigated the possible relationship between the encapsulation variables, namely serine protease content (9-50mg/ml, X1), Arabic gum (0.2-10%(w/w), X2), maltodextrin (2-5%(w/w), X3) and calcium chloride (1.3-5.5%(w/w), X4) on the enzymatic properties of encapsulated serine protease. The study demonstrated that Arabic gum, maltodextrin and calcium chloride, as coating agents, protected serine protease from activity loss during freeze-drying. The overall optimum region resulted in a suitable freeze drying condition with a yield of 92% for the encapsulated serine protease, were obtained using 29.5mg/ml serine protease content, 5.1%(w/w) Arabic gum, 3.5%(w/w) maltodextrin and 3.4%(w/w) calcium chloride. It was found that the interaction effect of Arabic gum and calcium chloride improved the serine protease activity, and Arabic gum was the most effective amongst the examined coating agents. Thus, Arabic gum should be considered as potential protection in freeze drying of serine protease.

  15. Identification of potential transmembrane protease serine 4 inhibitors as anti-cancer agents by integrated computational approach.

    PubMed

    Ilamathi, M; Hemanth, R; Nishanth, S; Sivaramakrishnan, V

    2016-01-21

    Transmembrane protease serine 4 is a well known cell surface protease facilitating the extracellular matrix degradation and epithelial mesenchymal transition in hepatocellular carcinoma. Henceforth targeting transmembrane protease serine 4 is strongly believed to provide therapeutic intervention against hepatocellular carcinoma. Owing to lack of crystal structure for human transmembrane protease serine 4, we predicted its three dimensional structure for the first time in this study. Experimentally proven inhibitor-Tyroserleutide (TSL) against hepatocellular carcinoma via transmembrane protease serine 4 was used as a benchmark to identify structurally similar candidates from PubChem database to create the TSL library. Virtual screening of TSL library against modeled transmembrane protease serine 4 revealed the top four potential inhibitors. Further binding free energy (ΔGbind) analysis of the potential inhibitors revealed the best potential lead compound against transmembrane protease serine 4. Drug likeliness nature of the top four potential hits were additionally analyzed in comparison to TSL to confirm on the best potential lead compound with the highest % of human oral absorption. Consequently, e-pharmacophore mapping of the best potential lead compound yielded a six point feature. It was observed to contain four hydrogen bond donor sites (D), one positively ionizable site (P) and one aromatic ring (R). Such e-pharmacophore insight obtained from structural determinants by integrated computational analysis could serve as a framework for further advancement of drug discovery process of new anti-cancer agents with less toxicity and high specificity targeting transmembrane protease serine 4 and hepatocellular carcinoma.

  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. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    PubMed

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  18. NASA PS400: A New Temperature Solid Lubricant Coating for High Temperature Wear Applications

    NASA Technical Reports Server (NTRS)

    DellaCorte, C.; Edmonds, B. J.

    2009-01-01

    A new solid lubricant coating, NASA PS400, has been developed for high temperature tribological applications. This plasma sprayed coating is a variant of the patented PS304 coating and has been formulated to provide higher density, smoother surface finish and better dimensional stability than PS304. PS400 is comprised of a nickel-molybdenum binder that provides strength, creep resistance and extreme oxidative and dimensional stability. Chromium oxide, silver and barium-calcium fluoride eutectic are added to the binder to form PS400.Tribological properties were evaluated with a pin-on-disk test rig in sliding contact to 650 C. Coating material samples were exposed to air, argon and vacuum at 760 C followed by cross section microscopic analysis to assess microstructure stability. Oil-Free microturbine engine hot section foil bearing tests were undertaken to assess PS400 s suitability for hot foil gas bearing applications. The preliminary results indicate that PS400 exhibits tribological characteristics comparable to the PS304 coating but with enhanced creep resistance and dimensional stability suitable for demanding, dynamic applications.

  19. SALT spectroscopic classification of PS15bzz as a type-Ia supernova at maximum light

    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.; Miszalski, B.

    2015-09-01

    We obtained SALT (+RSS) spectroscopy of PS15bzz on 2015 Aug 16.9 UT, covering the wavelength range 360-820 nm. Cross-correlation of the spectrum with a template library using SNID (Blondin & Tonry 2007, ApJ, 666, 1024) shows PS15bzz is a type-Ia supernova within a few days of maximum light.

  20. 76 FR 35683 - Medicare Program; Conditions of Participation (CoPs) for Community Mental Health Centers

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-17

    ... 42 CFR Part 485 Medicare Program; Conditions of Participation (CoPs) for Community Mental Health... 485 RIN 0938-AP51 Medicare Program; Conditions of Participation (CoPs) for Community Mental Health... (no password required). I. Background A. Introduction In 2007, 224 certified Community Mental...

  1. The directed differentiation of human iPS cells into kidney podocytes.

    PubMed

    Song, Bi; Smink, Alexandra M; Jones, Christina V; Callaghan, Judy M; Firth, Stephen D; Bernard, Claude A; Laslett, Andrew L; Kerr, Peter G; Ricardo, Sharon D

    2012-01-01

    The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

  2. Beginning Reading and Math in PS 133, New York City. A Report.

    ERIC Educational Resources Information Center

    Schwartz, Richard; Saxi, Ellen

    This booklet describes the program in Harlem (PS 133) which is based upon Dr. Caleb Gattegno's reading and mathematics in color. The program was primarily intended to test the teaching of Gattegno's methods and materials. PS 133 was considered to be a good site for the program because it was stable, pedagogically traditional, and staffed by a…

  3. 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

  4. 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.

  5. 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. ECμPs 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 ECμPs 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 ECμPs were functionalized to bind prostate specific antigen (PSA) or IgG-phycoerythrin (PE) in aqueous media (buffer, plasma, blood); whereas monodisperse ECμPs were functionalized to bind a high density lipoprotein in the aqueous media. Polydisperse ECμPs functionalized to bind PSA in a physiological buffer (PBS pH 7.4) demonstrated nanomolar detection using flow cytometry analysis; whereas ECμPs functionalized to bind IgG-PE demonstrated picomolar detection in 10% porcine plasma. ECμPs have a specific density of ~1.03 and are more compressible than their surrounding aqueous media; which allowed the ECμPs to exhibit negative acoustic contrast properties under an ultrasonic acoustic standing wave field. The negative acoustic contrast property of ECμPs was advantageously utilized in an IgG-PE assay conducted in 0.1% whole porcine blood. The ligand-bound ECμPs suspended in the diluted blood sample were flowed through an acoustofluidic device where the application of an ultrasonic acoustic standing wave

  6. iPS cell technologies: significance and applications to CNS regeneration and disease.

    PubMed

    Okano, Hideyuki; Yamanaka, Shinya

    2014-03-31

    In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinson's disease and Alzheimer's disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine.

  7. TG/FTIR analysis on co-pyrolysis behavior of PE, PVC and PS.

    PubMed

    Wu, Jingli; Chen, Tianju; Luo, Xitao; Han, Dezhi; Wang, Zhiqi; Wu, Jinhu

    2014-03-01

    The pyrolysis and co-pyrolysis behaviors of polyethylene (PE), polystyrene (PS) and polyvinyl chloride (PVC) under N2 atmosphere were analyzed by Thermal gravimetric/Fourier transform infrared (TG/FTIR). The volatile products were analyzed to investigate the interaction of the plastic blends during the thermal decomposition process. The TGA results showed that the thermal stability increased followed by PVC, PS and PE. The pyrolysis process of PE was enhanced when mixed with PS. However, PS was postponed when mixed with PVC. As for PE and PVC, mutual block was happened when mixed together. The FTIR results showed that the free radical of the decomposition could combine into a stable compound. When PE mixed with PVC or PS, large amount of unsaturated hydrocarbon groups existed in products while the content of alkynes was decreased. The methyl (-CH3) and methylene (-CH2-) bonds were disappeared while PVC mixed with PE.

  8. Study of mechanical properties of polyvinyl chloride (PVC) and polystyrene (PS) polymers and their blends

    NASA Astrophysics Data System (ADS)

    Agarwal, Shalini; Saxena, N. S.; Agrawal, R.; Saraswat, Vibhav K.

    2013-06-01

    Presented work is an effort to observe the variation in mechanical properties of two thermoplastic materials PVC, PS and their blends. PVC and PS are taken in the ratio of 100:0, 70:30, 50:50, and 0:100. Mixing of PVC and PS is carried out by solution casting method using tetra hydro furan as solvent. Dynamical mechanical analyzer (DMA) is used to study mechanical properties. The storage modulus, loss modulus and mechanical loss factor (tan δ) are determined with temperature. The pallets of pure PS, PVC and their blends are scanned over a temperature range from room to 140 °C. The variation of modulus, tan δ of pure PVC & pure PS and their blends with temperature were studied. The observed variation in modulus and tan δ could be accounted for their thermal behavior and compositions.

  9. iPS cell technologies: significance and applications to CNS regeneration and disease

    PubMed Central

    2014-01-01

    In 2006, we demonstrated that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating induced pluripotent stem (iPS) cells. Since that time, there has been an enormous increase in interest regarding the application of iPS cell technologies to medical science, in particular for regenerative medicine and human disease modeling. In this review article, we outline the current status of applications of iPS technology to cell therapies (particularly for spinal cord injury), as well as neurological disease-specific iPS cell research (particularly for Parkinson’s disease and Alzheimer’s disease). Finally, future directions of iPS cell research are discussed including a) development of an accurate assay system for disease-associated phenotypes, b) demonstration of causative relationships between genotypes and phenotypes by genome editing, c) application to sporadic and common diseases, and d) application to preemptive medicine. PMID:24685317

  10. Tribology and Microstructure of PS212 with a Cr2O3 Seal Coat

    NASA Technical Reports Server (NTRS)

    Sliney, Harold E.; Benoy, Patricia A.; Korenyi-Both, Andras; Dellacorte, Christopher

    1994-01-01

    PS212 is a plasma sprayed metal bonding chrome carbide coating with solid lubricant additives which has lubricating properties at temperatures up to about 900 deg C. The coating is diamond ground to achieve an acceptable tribological surface. But, as with many plasma spray coatings, PS212 is not fully-dense. In this study, a chromium oxide base seal coating is used in an attempt to seal any porosity that is open to the surface of the PS212 coating, and to study the effect of the sealant on the tribological properties of PS212. The results indicate that the seal coating reduces friction and wear when it is applied and then diamond ground leaving a thin layer of seal coating which fills in the surface pits of the PS212 coating.

  11. [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.

  12. Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis

    PubMed Central

    Xu, Chang; Wang, Yan; Wang, Lu; Wang, Qin; Du, Li-Qing; Fan, Saijun; Liu, Qiang; Li, Lei

    2015-01-01

    Chromosome instability usually leads to tumorigenesis. Bloom syndrome (BS) is a genetic disease associated with chromosome instability. The BS gene product, BLM, has been reported to function in the spindle assembly checkpoint (SAC) to prevent chromosome instability. BTR complex, composed of BLM, topoisomerase IIIα (Topo IIIα), RMI1 (RecQ-mediated genome instability protein 1, BLAP75) and RMI2 (RecQ-mediated genome instability protein 2, BLAP18), is crucial for maintaining genome stability. Recent work has demonstrated that RMI2 also plays critical role in SAC. However, little is know about RMI1 regulation during the cell cycle. Here we present that RMI1 protein level does not change through G1, S and G2 phases, but significantly increases in M phase. Moreover, phosphorylation of RMI1 occurs in mitosis. Upon microtubule-disturbing agent, RMI1 is phosphorylated primarily at the sites of Serine 284 and Serine 292, which does not interfere with the formation of BTR complex. Additionally, this phosphorylation is partially reversed by roscovitine treatment, implying cycling-dependent kinase 1 (CDK1) might be one of the upstream kinases. PMID:26556339

  13. Rtr1 is a CTD Phosphatase that Regulates the Transition from Serine 5 to Serine 2 Phosphorylation during the RNA Polymerase II Transcription Cycle

    PubMed Central

    Mosley, Amber L.; Pattenden, Samantha G.; Carey, Michael; Venkatesh, Swaminathan; Gilmore, Joshua M.; Florens, Laurence; Workman, Jerry L.; Washburn, Michael P.

    2010-01-01

    SUMMARY Messenger RNA processing is coupled to RNA Polymerase II (RNAPII) transcription through coordinated recruitment of accessory proteins to the Rpb1 C-terminal domain (CTD). Dynamic changes in CTD phosphorylation during transcription elongation are responsible for their recruitment, with serine 5 phosphorylation (S5-P) occurring towards the 5’ end of genes and serine 2 phosphorylation (S2-P) occurring towards the 3’ end. The proteins responsible for regulation of the transition state between S5-P and S2-P CTD remain elusive. We show that a conserved protein of unknown function, Rtr1, localizes within coding regions, with maximum levels of enrichment occurring between the peaks of S5-P and S2-P RNAPII. Upon deletion of Rtr1, the S5-P form of RNAPII accumulates in both whole cell extracts and throughout coding regions; additionally, RNAPII transcription is decreased and termination defects are observed. Functional characterization of Rtr1 reveals its role as a CTD phosphatase essential for the S5- to S2- P transition. PMID:19394294

  14. On the theory of ps and sub-ps laser pulse interaction with metals. II. Spatial temperature distribution

    NASA Astrophysics Data System (ADS)

    Hüttner, Bernd; Rohr, Gernot C.

    1998-04-01

    The interaction of short laser pulses with metals is described in terms of a new model involving a generalised nonlocal heat flow in time. The resulting system of coupled differential equations contains additional terms that originate in the thermal inertia of the electron subsystem. They are connected to the time derivative of both the laser intensity and the coupling of the electrons to the phonons. After briefly discussing the importance of the coefficient of heat exchange and of the different contributions to its main parts, the electron-phonon coupling and the average of the squared phonon frequencies, we present snap-shot-like calculations of the spatial temperature distribution along the z-axis for Al, Au, Pb and Nb for different intensities but fixed pulse duration τL=250 fs. In addition, the electron and phonon temperatures inside a thin Au film are studied in detail. Our results are compared to those arising from a standard two temperature model (TTM) and those of the conventional theory. While the traditional theory becomes invalid in most cases at least in the ps-range the differences between our approach and the TTM become the more pronounced the smaller the coefficient of heat exchange.

  15. Intron-exon organization of the active human protein S gene PS. alpha. and its pseudogene PS. beta. : Duplication and silencing during primate evolution

    SciTech Connect

    Ploos van Amstel, H.; Reitsma, P.H.; van der Logt, C.P.; Bertina, R.M. )

    1990-08-28

    The human protein S locus on chromosome 3 consists of two protein S genes, PS{alpha} and PS{beta}. Here the authors report the cloning and characterization of both genes. Fifteen exons of the PS{alpha} gene were identified that together code for protein S mRNA as derived from the reported protein S cDNAs. Analysis by primer extension of liver protein S mRNA, however, reveals the presence of two mRNA forms that differ in the length of their 5{prime}-noncoding region. Both transcripts contain a 5{prime}-noncoding region longer than found in the protein S cDNAs. The two products may arise from alternative splicing of an additional intron in this region or from the usage of two start sites for transcription. The intron-exon organization of the PS{alpha} gene fully supports the hypothesis that the protein S gene is the product of an evolutional assembling process in which gene modules coding for structural/functional protein units also found in other coagulation proteins have been put upstream of the ancestral gene of a steroid hormone binding protein. The PS{beta} gene is identified as a pseudogene. It contains a large variety of detrimental aberrations, viz., the absence of exon I, a splice site mutation, three stop codons, and a frame shift mutation. Overall the two genes PS{alpha} and PS{beta} show between their exonic sequences 96.5% homology. Southern analysis of primate DNA showed that the duplication of the ancestral protein S gene has occurred after the branching of the orangutan from the African apes. A nonsense mutation that is present in the pseudogene of man also could be identified in one of the two protein S genes of both chimpanzee and gorilla. This implicates that silencing of one of the two protein S genes must have taken place before the divergence of the three African apes.

  16. The Ames dwarf mutation attenuates Alzheimer's disease phenotype of APP/PS1 mice.

    PubMed

    Puig, Kendra L; Kulas, Joshua A; Franklin, Whitney; Rakoczy, Sharlene G; Taglialatela, Giulio; Brown-Borg, Holly M; Combs, Colin K

    2016-04-01

    APP/PS1 double transgenic mice expressing human mutant amyloid precursor protein (APP) and presenilin-1 (PS1) demonstrate robust brain amyloid beta (Aβ) peptide containing plaque deposition, increased markers of oxidative stress, behavioral dysfunction, and proinflammatory gliosis. On the other hand, lack of growth hormone, prolactin, and thyroid-stimulating hormone due to a recessive mutation in the Prop 1 gene (Prop1df) in Ames dwarf mice results in a phenotype characterized by potentiated antioxidant mechanisms, improved learning and memory, and significantly increased longevity in homozygous mice. Based on this, we hypothesized that a similar hormone deficiency might attenuate disease changes in the brains of APP/PS1 mice. To test this idea, APP/PS1 mice were crossed to the Ames dwarf mouse line. APP/PS1, wild-type, df/+, df/df, df/+/APP/PS1, and df/df/APP/PS1 mice were compared at 6 months of age through behavioral testing and assessing amyloid burden, reactive gliosis, and brain cytokine levels. df/df mice demonstrated lower brain growth hormone and insulin-like growth factor 1 concentrations. This correlated with decreased astrogliosis and microgliosis in the df/df/APP/PS1 mice and, surprisingly, reduced Aβ plaque deposition and Aβ 1-40 and Aβ 1-42 concentrations. The df/df/APP/PS1 mice also demonstrated significantly elevated brain levels of multiple cytokines in spite of the attenuated gliosis. These data indicate that the df/df/APP/PS1 line is a unique resource in which to study aging and resistance to disease and suggest that the affected pituitary hormones may have a role in regulating disease progression.

  17. Time-Dependent Increase of Chitinase1 in APP/PS1 Double Transgenic Mice.

    PubMed

    Xiao, Qian; Shi, Rui; Yang, Wenxiu; Zou, Yan; Du, Yinshi; Zhang, Man; Yu, Weihua; Lü, Yang

    2016-07-01

    It is reported that chitinase1 increases in Alzheimer's disease (AD). However, the alteration of chitinase1 in the progress of AD is still unclear. Thus, we designed the present study to detect chitinase1 level in different stages of APP/PS1 double transgenic mice. Experimental models were APP/PS1 double transgenic mice with 4, 12 and 22 months. Cognitive function was detected by Morris water maze test in APP/PS1 mice as well as controls. ELISA and the quantitative RT-PCR were used to detect chitinase1 level in different groups. The study displayed that expression of chitinase1 gradually increased in a time-dependent manner in APP/PS1 mice, while there were no statistical differences among the wild-type mice in varies ages. Moreover, chitnase1 increased significantly in APP/PS1 mice aged 12 and 22 months compared with the age matched wild-type group, respectively. However, no difference of chitnase1 was found between 4 months-old APP/PS1 mice and wild-type mice. Comparing with the age matched wild type group, the consequences of mRNA on the increase in chitnase1 is in accordance with protein in APP/PS1 mice. Furthermore, Morris water maze showed that 4 months-old APP/PS1 mice have normal spatial learning and impaired spatial memory; both spatial learning and spatial memory in 12 and 22 months-old APP/PS1 mice were declined. Time-dependent increase of chitnase1 in APP/PS1 double transgenic mice indicates that the level of chitinase1 is associated with decline of cognition. Therefore, chitinase1 might be a biomarker of disease progression in AD.

  18. 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.

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

    PubMed

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

    2009-08-13

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

  20. Purification and Characterization of a Thermostable Caseinolytic Serine Protease from the Latex of Euphorbia heterophylla L.

    PubMed

    Singh, Sorokhaibam J; Singh, Laishram R; Devi, Sanjenbam K; Singh, Senjam S; Devi, Chingsubam B; Rully, Huidrom

    2015-01-01

    A new thermostable caseinolytic serine protease was purified from the latex of Euphorbia heterophylla L. to electrophoretic homogeneity by a procedure involving successive steps of pretreatment of the latex, PEG fractionation, CM-cellulose chromatography and DEAE-cellulose chromatography. The purified protease was found to be a monomeric protein of molecular weight 77.2 kDa. It exhibited caseinolytic activity with hyperbolic azocasein saturation with Vmax and Km values of 0.11 units.mL(-1) and 0.55 mg.mL(-1) respectively. Specific inhibitory studies revealed the enzyme to be a serine protease. The protease was characterized by pH optimum of 8.0 and high thermostability with T1/2 of 75°C. Based on the results of peptide mass fingerprinting analysis, the protease was shown to be a new protein not characterized earlier.

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

    PubMed

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

    2016-01-18

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and -beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function.

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

    PubMed Central

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

    2004-01-01

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

  3. The importance of SERINE DECARBOXYLASE1 (SDC1) and ethanolamine biosynthesis during embryogenesis of Arabidopsis thaliana.

    PubMed

    Yunus, Ian Sofian; Liu, Yu-Chi; Nakamura, Yuki

    2016-11-01

    In plants, ethanolamine is considered a precursor for the synthesis of choline, which is an essential dietary nutrient for animals. An enzyme serine decarboxylase (SDC) has been identified and characterized in Arabidopsis, which directly converts serine to ethanolamine, a precursor to phosphorylethanolamine and its subsequent metabolites in plants. However, the importance of SDC and ethanolamine production in plant growth and development remains unclear. Here, we show that SDC is required for ethanolamine biosynthesis in vivo and essential in plant embryogenesis in Arabidopsis. The knockout of SDC1 caused an embryonic lethal defect due to the developmental arrest of the embryos at the heart stage. During embryo development, the expression was observed at the later stages, at which developmental defect occurred in the knockout mutant. Overexpression of SDC1 in planta increased levels of ethanolamine, phosphatidylethanolamine, and phosphatidylcholine both in leaves and siliques. These results suggest that SDC1 plays an essential role in ethanolamine biosynthesis during the embryogenesis in Arabidopsis.

  4. Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice.

    PubMed

    Wang, Dekai; Liu, Heqin; Li, Sujuan; Zhai, Guowei; Shao, Jianfeng; Tao, Yuezhi

    2015-09-01

    Serine hydroxymethyltransferase (SHMT) is important for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development, and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, OsSHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller, lethal phenotype under natural ambient CO2 concentrations, but could be restored to wild type with normal growth under elevated CO2 levels (0.5% CO2 ), showing a typical photorespiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to significant oxidative stress. Also, OsSHM1 was expressed in all organs tested (root, culm, leaf, and young panicle) but predominantly in leaves. OsSHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the OsSHM1 gene is conserved in rice and Arabidopsis.

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

    PubMed Central

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

    2005-01-01

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

  6. PS2013 Satellite Workshop on Photosynthetic Light-Harvesting Systems

    SciTech Connect

    Niederman, Robert A.; Blankenship, Robert E.; Frank, Harry A.

    2015-02-07

    These funds were used for partial support of the PS2013 Satellite Workshop on Photosynthetic Light-Harvesting Systems, that was held on 8-11 August, 2013, at Washington University, St. Louis, MO. This conference, held in conjunction with the 16th International Congress on Photosynthesis/St. Louis, continued a long tradition of light-harvesting satellite conferences that have been held prior to the previous six international photosynthesis congresses. In this Workshop, the basis was explored for the current interest in replacing fossil fuels with energy sources derived form direct solar radiation, coupled with light-driven electron transport in natural photosynthetic systems and how they offer a valuable blueprint for conversion of sunlight to useful energy forms. This was accomplished through sessions on the initial light-harvesting events in the biological conversion of solar energy to chemically stored energy forms, and how these natural photosynthetic processes serve as a guide to the development of robust bio-hybrid and artificial systems for solar energy conversion into both electricity or chemical fuels. Organized similar to a Gordon Research Conference, a lively, informal and collegial setting was established, highlighting the exchange of exciting new data and unpublished results from ongoing studies. A significant amount of time was set aside for open discussion and interactive poster sessions, with a special session devoted to oral presentations by talented students and postdoctoral fellows judged to have the best posters. This area of research has seen exceptionally rapid progress in recent years, with the availability of a number of antenna protein structures at atomic resolution, elucidation of the molecular surface architecture of native photosynthetic membranes by atomic force microscopy and the maturing of ultrafast spectroscopic and molecular biological techniques for the investigation and manipulation of photosynthetic systems. The conferees

  7. iPS cells to model CDKL5-related disorders

    PubMed Central

    Amenduni, Mariangela; De Filippis, Roberta; Cheung, Aaron Y L; Disciglio, Vittoria; Epistolato, Maria Carmela; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hayek, Youssef; Renieri, Alessandra; Ellis, James; Meloni, Ilaria

    2011-01-01

    Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene, whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. PMID:21750574

  8. Terminally coordinated AsS and PS ligands.

    PubMed

    Balázs, Gábor; Green, Jennifer C; Scheer, Manfred

    2006-11-15

    The terminal AsS and PS complexes [(N(3)N)W(ES)] (N(3)N=N(CH(2)CH(2)NSiMe(3))(3); E=P (3), As (4)) were synthesised by reaction of [(N(3)N)W[triple chemical bond]As] and [(N(3)N)W[triple chemical bond]P], respectively, with cyclohexene sulfide. Both complexes present very short W--E and E--S bond lengths. The bonding was investigated by density functional theory (DFT) calculations using the fragment calculation method and natural bond orbital (NBO) analysis. According to the fragment analysis, in which the complexes were separated in an ES and a (N(3)N)W fragment, the bonding in complexes 3, 4 and [(N(3)N)W(SbS)] (5) is realised over a set of two sigma (1 sigma and 2 sigma) and two degenerate pi molecular orbitals (MOs) (1 pi and 2 pi). The 1 sigma MO is a bonding MO extended over the N(ax)-W-E-S core, whereas the 2 sigma MO is localised mainly on the E-S fragment. The 1 pi set is a E-S localised bonding molecular orbital, whereas the 2 pi set is in phase with respect to W-E but in antiphase with respect to E-S. Both methods indicate bond orders around two for both the E--S and the W--E bonds. The polarity of the complexes was examined by Hirshfeld charge analysis. This shows that complexes 3 and 4 are only slightly polarised, whereas 5 is moderately polarised toward the sulphur. As suggested by the computational results, the pi system in complexes 3-5 is best described by two three-centre four-electron bonds.

  9. Evidence for an oxyanion hole in serine beta-lactamases and DD-peptidases.

    PubMed Central

    Murphy, B P; Pratt, R F

    1988-01-01

    A thionocephalosporin is shown to be a much poorer substrate of representative serine beta-lactamases of class A (RTEM-2) and class C (Enterobacter cloacae P99) and a much poorer inhibitor of the Streptomyces R61 DD-peptidase than is the analogous oxo beta-lactam. These results provide kinetic evidence for the existence of a catalytic oxyanion hole in these enzymes. PMID:3066349

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

    PubMed

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

    2005-10-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-06-01

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

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

    PubMed

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

    2007-09-14

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

  14. Alkali metal-cationized serine clusters studied by sonic spray ionization tandem mass spectrometry.

    PubMed

    Nanita, Sergio C; Sokol, Ewa; Cooks, R Graham

    2007-05-01

    Serine solutions containing salts of alkali metals yield magic number clusters of the type (Ser(4)+C)(+), (Ser(8)+C)(+), (Ser(12)+C)(+), and (Ser(17)+2C)(+2) (where C = Li(+), Na(+), K(+), Rb(+), or Cs(+)), in relative abundances which are strongly dependent on the cation size. Strong selectivity for homochirality is involved in the formation of serine tetramers cationized by K(+), Rb(+), and Cs(+). This is also the case for the octamers cationized by the smaller alkalis but there is a strong preference for heterochirality in the octamers cationized by the larger alkali cations. Tandem mass spectrometry shows that the octamers and dodecamers cationized by K(+), Rb(+), and Cs(+) dissociate mainly by the loss of Ser(4) units, suggesting that the neutral tetramers are the stable building blocks of the observed larger aggregates, (Ser(8)+C)(+) and (Ser(12)+C)(+). Remarkably, although the Ser(4) units are formed with a strong preference for homochirality, they aggregate further regardless of their handedness and, therefore, with a preference for the nominally racemic 4D:4L structure and an overall strong heterochiral preference. The octamers cationized by K(+), Rb(+), or Cs(+) therefore represent a new type of cluster ion that is homochiral in its internal subunits, which then assemble in a random fashion to form octamers. We tentatively interpret the homochirality of these tetramers as a consequence of assembly of the serine molecules around a central metal ion. The data provide additional evidence that the neutral serine octamer is homochiral and is readily cationized by smaller ions.

  15. Evidence for an oxyanion hole in serine beta-lactamases and DD-peptidases.

    PubMed

    Murphy, B P; Pratt, R F

    1988-12-01

    A thionocephalosporin is shown to be a much poorer substrate of representative serine beta-lactamases of class A (RTEM-2) and class C (Enterobacter cloacae P99) and a much poorer inhibitor of the Streptomyces R61 DD-peptidase than is the analogous oxo beta-lactam. These results provide kinetic evidence for the existence of a catalytic oxyanion hole in these enzymes.

  16. New cyanopeptide-derived low molecular weight inhibitors of trypsin-like serine proteases.

    PubMed

    Radau, Gregor; Schermuly, Sonja; Fritsche, Alexandra

    2003-08-01

    This paper deals with the design, syntheses, and inhibition tests of new low molecular weight thrombin inhibitors utilizing cyanopeptides, the secondary metabolites of cyanobacteria with interesting biological activities, as new lead structures. Starting with aeruginosin 98-B (1) as a lead structure, we have developed and synthesised new, selective acting inhibitors of serine proteases (RA-1005 and RA-1009, which are suitable targets for further structure-activity studies.

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2015-01-07

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

  19. Corynebactin and a Serine Trilactone Based Analogue-Chirality and Molecular Modeling of ferric Complexes

    SciTech Connect

    Bluhm, Martin E.; Hay, Benjamin P.; Kim, Sangoo S.; Dertz, Emily A.; Raymond, Kenneth N.

    2002-09-14

    The chirality of ferric siderophore complexes is a determinant for their cellular recognition and transport. Corynebactin (first isolated from a Gram-positive bacterium) contains L-threonine, unlike the closely related enterobactin, which contains L-serine. Also unlike enterobactin, ferric corynebactin is preferentially L at the iron center. Experimental (circular dichroism spectra and synthesis of a corynebactin/enterobactin hybrid) and theoretical (MM3 and density functional theory calculations) results explain ferric corynebactin's properties.

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

    PubMed Central

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

    2012-01-01

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

  1. A novel family of serine/threonine kinases participating in spermiogenesis.

    PubMed

    Kueng, P; Nikolova, Z; Djonov, V; Hemphill, A; Rohrbach, V; Boehlen, D; Zuercher, G; Andres, A C; Ziemiecki, A

    1997-12-29

    The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of approximately 65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms.

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

    PubMed

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

    2016-12-01

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

  3. Structures of Plasmodium vivax serine hydroxymethyltransferase: implications for ligand-binding specificity and functional control

    PubMed Central

    Chitnumsub, Penchit; Jaruwat, Aritsara; Riangrungroj, Pinpunya; Ittarat, Wanwipa; Noytanom, Krittikar; Oonanant, Worrapoj; Vanichthanankul, Jarunee; Chuankhayan, Phimonphan; Maenpuen, Somchart; Chen, Chun-Jung; Chaiyen, Pimchai; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree

    2014-01-01

    Plasmodium parasites, the causative agent of malaria, rely heavily on de novo folate biosynthesis, and the enzymes in this pathway have therefore been explored extensively for antimalarial development. Serine hydroxymethyltransferase (SHMT) from Plasmodium spp., an enzyme involved in folate recycling and dTMP synthesis, has been shown to catalyze the conversion of l- and d-serine to glycine (Gly) in a THF-dependent reaction, the mechanism of which is not yet fully understood. Here, the crystal structures of P. vivax SHMT (PvSHMT) in a binary complex with l-serine and in a ternary complex with d-serine (d-Ser) and (6R)-5-formyl­tetra­hydro­folate (5FTHF) provide clues to the mechanism underlying the control of enzyme activity. 5FTHF in the ternary-complex structure was found in the 6R form, thus differing from the previously reported structures of SHMT–Gly–(6S)-5FTHF from other organisms. This suggested that the presence of d-Ser in the active site can alter the folate-binding specificity. Investigation of binding in the presence of d-Ser and the (6R)- or (6S)-5FTHF enantiomers indicated that both forms of 5FTHF can bind to the enzyme but that only (6S)-5FTHF gives rise to a quinonoid intermediate. Likewise, a large surface area with a highly positively charged electrostatic potential surrounding the PvSHMT folate pocket suggested a preference for a polyglutamated folate substrate similar to the mammalian SHMTs. Furthermore, as in P. falciparum SHMT, a redox switch created from a cysteine pair (Cys125–Cys364) was observed. Overall, these results assert the importance of features such as stereoselectivity and redox status for control of the activity and specificity of PvSHMT. PMID:25478836

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

    PubMed Central

    Connolly, James PR; Goldstone, Robert J; Burgess, Karl; Cogdell, Richard J; Beatson, Scott A; Vollmer, Waldemar; Smith, David GE; Roe, Andrew J

    2015-01-01

    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

  5. Studies on alkaline serine protease produced by Bacillus clausii GMBE 22.

    PubMed

    Kazan, Dilek; Bal, Hulya; Denizci, Aziz Akin; Ozturk, Nurcin Celik; Ozturk, Hasan Umit; Dilgimen, Aydan Salman; Ozturk, Dilek Coskuner; Erarslan, Altan

    2009-01-01

    An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60 degrees C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The K(m) and k(cat) values for hydrolysis of this substrate are 0.347 mM and 1141 min(-1) respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30 degrees C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus clausii reported in the literature.

  6. Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway

    PubMed Central

    Deng, Yihong; Ran, Wei; Man, Suqin; Li, Xueping; Gao, Hongjian; Tang, Wei

    2015-01-01

    Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections. PMID:26014935

  7. Controlled rotation mechanism of DNA strand exchange by the Hin serine recombinase

    PubMed Central

    Xiao, Botao; McLean, Meghan M.; Lei, Xianbin; Marko, John F.; Johnson, Reid C.

    2016-01-01

    DNA strand exchange by serine recombinases has been proposed to occur by a large-scale rotation of halves of the recombinase tetramer. Here we provide the first direct physical evidence for the subunit rotation mechanism for the Hin serine invertase. Single-DNA looping assays using an activated mutant (Hin-H107Y) reveal specific synapses between two hix sites. Two-DNA “braiding” experiments, where separate DNA molecules carrying a single hix are interwound, show that Hin-H107Y cleaves both hix sites and mediates multi-step rotational relaxation of the interwinding. The variable numbers of rotations in the DNA braid experiments are in accord with data from bulk experiments that follow DNA topological changes accompanying recombination by the hyperactive enzyme. The relatively slow Hin rotation rates, combined with pauses, indicate considerable rotary friction between synapsed subunit pairs. A rotational pausing mechanism intrinsic to serine recombinases is likely to be crucial for DNA ligation and for preventing deleterious DNA rearrangements. PMID:27032966

  8. Characterization of a Mn sup 2+ -dependent membrane serine kinase that is activated by tyrosine phosphorylation

    SciTech Connect

    Singh, T.J. )

    1991-03-11

    It is hypothesized that the insulin receptor (IR) tyrosine kinase may directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases as well as their modes of activation are unclear. The authors have described a serine kinase from rat liver membranes that copurifies with the IR on wheat germ agglutinin (WGA)-sepharose. The kinase is activated after phosphorylation of the WGA-sepharose-purified fraction by casein kinase-1, casein kinase-2, or casein kinase-3. A tyrosine kinase, possibly IR tyrosine kinase, also participates in the activation process since a phosphotyrosine phosphatase inhibitor such as vanadate, p-nitrophenyl phosphate, or phosphotyrosine is required in reaction mixtures for activation to be observed. By contrast, phosphoserine and phosphothreonine do not support activation. The activated kinase can use IR {beta}-subunit, myelin basic protein (MBP), and histones as substrates. IR {beta}-subunit phosphorylation was stimulated by MBP, histones, and polylysine, and inhibited by heparin and poly(glu, tyr). The kinase prefers Mn{sup 2+} over Mg{sup 2+} as a metal cofactor.

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

    PubMed Central

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

    2015-01-01

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

  10. D-Serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis

    PubMed Central

    Sasabe, Jumpei; Chiba, Tomohiro; Yamada, Marina; Okamoto, Koichi; Nishimoto, Ikuo; Matsuoka, Masaaki; Aiso, Sadakazu

    2007-01-01

    Excitotoxicity has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). More recently, glial involvement has been shown to be essential for ALS-related motoneuronal death. Here, we identified an N-methyl-D-aspartate (NMDA) receptor co-agonist, D-serine (D-Ser), as a glia-derived enhancer of glutamate (Glu) toxicity to ALS motoneurons. Cell death assay indicated that primary spinal cord neurons from ALS mice were more vulnerable to NMDA toxicity than those from control mice, in a D-Ser-dependent manner. Levels of D-Ser and its producing enzyme, serine racemase, in spinal cords of ALS mice were progressively elevated, dominantly in glia, with disease progression. In vitro, expression of serine racemase was induced not only by an extracellular pro-inflammatory factor, but also by transiently expressed G93A-superoxide dismutase1 in microglial cells. Furthermore, increases of D-Ser levels were also observed in spinal cords of both familial and sporadic ALS patients. Collectively, Glu toxicity enhanced by D-Ser overproduced in glia is proposed as a novel mechanism underlying ALS motoneuronal death, and this mechanism may be regarded as a potential therapeutic target for ALS. PMID:17762863

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

    PubMed

    Ksiazek, Miroslaw; Karim, Abdulkarim Y; Bryzek, Danuta; Enghild, Jan J; Thøgersen, 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.

  12. Adjustments of serine proteases of Daphnia pulex in response to temperature changes.

    PubMed

    Dölling, Ramona; Becker, Dörthe; Hawat, Susan; Koch, Marita; Schwarzenberger, Anke; Zeis, Bettina

    2016-01-01

    Elevated temperatures considerably challenge aquatic invertebrates, and enhanced energy metabolism and protein turnover require adjustments of digestion. In Daphnia, the serine proteases chymotrypsin and trypsin represent the major proteolytic enzymes. Daphnia pulex acclimated to different temperature conditions or subjected to acute heat stress showed increased expression level of serine proteases with rising temperatures. Transcripts of trypsin isoforms were always present in higher amounts than observed for chymotrypsin. Additionally, trypsin isoform transcripts were induced by elevated temperatures to a larger extent. Correspondingly, trypsin activity dominated in cold-acclimated animals. However, the enzymatic activity of chymotrypsin increased at elevated temperatures, whereas trypsin activity slightly decreased, resulting in a shift to dominating chymotrypsin activity in warm-acclimated animals. Zymograms revealed eight bands with proteolytic activity in the range of 20 to 86 kDa. The single bands were assigned to trypsin or chymotrypsin activity applying specific inhibitors or from casein cleavage products identified by mass spectrometric analysis. The total amount of proteolytic activity was elevated with acclimation temperature increase and showed a transient decrease under acute heat stress. The contribution of the different isoforms to protein digestion indicated induction of chymotrypsin with increasing acclimation temperature. For trypsin, the share of one isoform decreased with elevated temperature, while another isoform was enhanced. Thus differential expression of serine proteases was observed in response to chronic and acute temperature changes. The observed phenotypic plasticity adjusts the set of active proteases to the altered needs of protein metabolism optimizing protein digestion for the temperature conditions experienced in the habitat.

  13. Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway.

    PubMed

    Deng, Yihong; Ran, Wei; Man, Suqin; Li, Xueping; Gao, Hongjian; Tang, Wei; Tachibana, Hiroshi; Cheng, Xunjia

    2015-08-01

    Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections.

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

    PubMed

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

    2004-04-01

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

  15. Serine and SAM Responsive Complex SESAME Regulates Histone Modification Crosstalk by Sensing Cellular Metabolism.

    PubMed

    Li, Shanshan; Swanson, Selene K; Gogol, Madelaine; Florens, Laurence; Washburn, Michael P; Workman, Jerry L; Suganuma, Tamaki

    2015-11-05

    Pyruvate kinase M2 (PKM2) is a key enzyme for glycolysis and catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, which supplies cellular energy. PKM2 also phosphorylates histone H3 threonine 11 (H3T11); however, it is largely unknown how PKM2 links cellular metabolism to chromatin regulation. Here, we show that the yeast PKM2 homolog, Pyk1, is a part of a novel protein complex named SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex), which contains serine metabolic enzymes, SAM (S-adenosylmethionine) synthetases, and an acetyl-CoA synthetase. SESAME interacts with the Set1 H3K4 methyltransferase complex, which requires SAM synthesized from SESAME, and recruits SESAME to target genes, resulting in phosphorylation of H3T11. SESAME regulates the crosstalk between H3K4 methylation and H3T11 phosphorylation by sensing glycolysis and glucose-derived serine metabolism. This leads to auto-regulation of PYK1 expression. Thus, our study provides insights into the mechanism of regulating gene expression, responding to cellular metabolism via chromatin modifications.

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

    PubMed

    Pułaski, 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.

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

    The genome of Tannerella forsythia, an etiologic 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) 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

  20. [Interaction between gabapentin and D-serin in the formalin orofacial test].

    PubMed

    Quiñónez, Belkis; Silva, Elizabeth; González, Luis E; Hernández, Luis

    2009-12-01

    Gabapentin is a useful agent for the relief of trigeminal neuralgia and orofacial phantom pain. However, there is scarce information on the gabapentin analgesic effect in orofacial pain models. We tested the analgesic action of gabapentin on the formalin-induced face grooming in the rat, an orofacial pain paradigm. IP Gabapentin (10 mg/kg), induced a drastic reduction in face grooming during phase I and II, indicating a clear-cut antinociceptive effect. However, at 1 mg/kg, gabapentin had an analgesic effect only on phase I. D-serine (100 microg, ICV) was silent when given alone and did not antagonize the antinociceptive effect of gabapentin. On the contrary, gabapentin 1 mg/kg plus D-serine significantly reduced face grooming in phase II. These results show a difference between gabapentin induced orofacial analgesia and previous studies showing gabapentin-induced hind paw analgesia in the formalin test, only during phase II, as well as D-serine antagonism of gabapentin. The results are discussed in terms of different pain processing of hind paw, versus orofacial nociceptive stimulation.

  1. Purification and partial characterization of serine-metallokeratinase from a newly isolated Bacillus pumilus NRC21.

    PubMed

    Tork, Sanaa E; Shahein, Yasser E; El-Hakim, Amr E; Abdel-Aty, Azza M; Aly, Magda M

    2016-05-01

    A serine metallokeratinase enzyme (30 kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000 units/mg protein against 170 units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na(+), K(+), Mg(2+)), Hg(+2) had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzyme. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45 °C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60 °C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5 mM Mg(+2). These results suggest that the purified keratinase may be used in several industrial applications.

  2. Discrimination of chiral solids: a terahertz spectroscopic investigation of L- and DL-serine.

    PubMed

    King, Matthew D; Hakey, Patrick M; Korter, Timothy M

    2010-03-04

    The terahertz (THz, far-infrared) spectra of enantiomerically pure and racemic crystalline serine were investigated using time-domain THz spectroscopy and solid-state density functional theory (DFT) in the spectral range of 10-90 cm(-1). The experimental THz spectra of L- and DL-serine at 78 K appear quite similar despite the significant differences in arrangement of the molecules in their crystal structures. Structural analyses of the two systems and calculation of the vibrational modes and intensities were performed using DFT with periodic boundary conditions employing the B3LYP and PW91 density functionals with the 6-31G(d,p) and 6-311G(d,p) basis sets. The applied computational methods produced simulations of the THz spectra in good agreement with experiment, with accurate predictions of the subtle differences in the THz spectra of the two chiral solid-state mixtures of serine. The observed spectral features are assigned as primarily external lattice translations and rotations with lesser contributions due to intramolecular torsions of the -NH(3)(+) and -COO(-) groups modified by intermolecular hydrogen bonding.

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

    PubMed Central

    Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

    2016-01-01

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

  4. Change in activity of serine palmitoyltransferase affects sensitivity to syringomycin E in yeast Saccharomyces cerevisiae.

    PubMed

    Toume, Moeko; Tani, Motohiro

    2014-09-01

    Syringomycin E is a cyclic lipodepsipeptide produced by strains of the plant bacterium Pseudomonas syringae pv. syringae. Genetic studies involving the yeast Saccharomyces cerevisiae have revealed that complex sphingolipids play important roles in the action of syringomycin E. Here, we found a novel mutation that confers resistance to syringomycin E on yeast; that is, a deletion mutant of ORM1 and ORM2, which encode negative regulators of serine palmitoyltransferase catalyzing the initial step of sphingolipid biosynthesis, exhibited resistance to syringomycin E. On the contrary, overexpression of Orm2 resulted in high sensitivity to the toxin. Moreover, overexpression of Lcb1 and Lcb2, catalytic subunits of serine palmitoyltransferase, causes resistance to the toxin, whereas partial repression of expression of Lcb1 had the opposite effect. Partial reduction of complex sphingolipids by repression of expression of Aur1, an inositol phosphorylceramide synthase, also resulted in high sensitivity to the toxin. These results suggested that an increase in sphingolipid biosynthesis caused by a change in the activity of serine palmitoyltransferase causes resistance to syringomycin E.

  5. High-power passively mode-locked Nd:YVO4 oscillator with adjustable pulse duration between 46 ps and 12 ps

    NASA Astrophysics Data System (ADS)

    Nadeau, Marie-Christine; Petit, Stéphane; Balcou, Philippe; Czarny, Romain; Montant, Sébastien; Simon-Boisson, Christophe

    2010-05-01

    We report on a high-power, passively mode-locked, TEM00 Nd:YVO4 oscillator with adjustable pulse duration between 46 and 12ps. The laser is end-pumped by an 888nm laser diode and mode-locking is achieved with a semiconductor saturable absorber mirror (SESAM). The laser has a repetition rate of 91MHz and the M2 beam quality factor is better than 1.2 at 15ps. At the optimum output coupler, it provides a maximum average output power of 45W with 32ps pulse duration. In literature, the presence of spatial hole burning (SHB) often helps to shorten the pulse length down to few picoseconds. However, SHB might be an issue for some specific application requiring e.g. low noise picosecond oscillators. In this contribution, we demonstrate that it is possible to shorten the pulse duration by lowering the intracavity losses without SHB. Pulse tunability from 46 to 12ps is achieved by changing the output coupler of the cavity while staying in the continuous-wave mode-locked regime. Pulse duration is almost linear with the output coupler transmission and increases from 12 to 32ps with average output power ranging from 15 to 45W. In this range of output power, we demonstrate the shortest pulses directly from a Nd:YVO4 oscillator.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  8. 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.

  9. 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.

  10. Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes.

    PubMed

    Bergersen, L H; Morland, C; Ormel, L; Rinholm, J E; Larsson, M; Wold, J F H; Røe, A T; Stranna, A; Santello, M; Bouvier, D; Ottersen, O P; Volterra, A; Gundersen, V

    2012-07-01

    Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

  11. Reprogramming in vivo produces teratomas and iPS cells with totipotency features.

    PubMed

    Abad, María; Mosteiro, Lluc; Pantoja, Cristina; Cañamero, Marta; Rayon, Teresa; Ors, Inmaculada; Graña, Osvaldo; Megías, Diego; Domínguez, Orlando; Martínez, Dolores; Manzanares, Miguel; Ortega, Sagrario; Serrano, Manuel

    2013-10-17

    Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

  12. An efficient compressive sensing based PS-DInSAR method for surface deformation estimation

    NASA Astrophysics Data System (ADS)

    Li, J. T.; Xu, H. P.; Shan, L.; Liu, W.; Chen, G. Z.

    2016-11-01

    Permanent scatterers differential interferometric synthetic aperture radar (PS-DInSAR) is a technique for detecting surface micro-deformation, with an accuracy at the centimeter to millimeter level. However, its performance is limited by the number of SAR images available (normally more than 20 are needed). Compressive sensing (CS) has been proven to be an effective signal recovery method with only a very limited number of measurements. Applying CS to PS-DInSAR, a novel CS-PS-DInSAR method is proposed to estimate the deformation with fewer SAR images. By analyzing the PS-DInSAR process in detail, first the sparsity representation of deformation velocity difference is obtained; then, the mathematical model of CS-PS-DInSAR is derived and the restricted isometry property (RIP) of the measurement matrix is discussed to validate the proposed CS-PS-DInSAR in theory. The implementation of CS-PS-DInSAR is achieved by employing basis pursuit algorithms to estimate the deformation velocity. With the proposed method, DInSAR deformation estimation can be achieved by a much smaller number of SAR images, as demonstrated by simulation results.

  13. Phase Transition and Mechanical Properties of PS/PVC/CdS Polymeric Nanocomposites

    NASA Astrophysics Data System (ADS)

    Mathur, Vishal; Dixit, Manasvi; Saxena, N. S.; Sharma, Kananbala

    2010-06-01

    The present study reports the phase transition temperature and mechanical properties of CdS dispersed PS-PVC nanocomposite through Dynamic Mechanical Analyzer (DMA). Thick films of polymeric nanocomposites have been synthesized by dispersing nano-filler particles of CdS in PS/PVC binary blend matrix. The surface morphology of PS/PVC blend samples has been characterized by Scanning Electron Microscopy (SEM) while the nanostructure of the CdS filler in PS/PVC/CdS composite has been ascertained through small angle X-ray Diffraction (XRD) technique. The phase transition temperature study of PS/PVC polymeric blends reveals that glass transition temperature, Tg, of the PS phase shifts towards lower temperature with the increase in PVC content in the blend whereas for CdS embedded polymeric phases of blends i.e. for PS/PVC/CdS samples, an increase in respective Tg values have been observed. This is suggestive to the fact that phase transition temperature and mechanical properties have been significantly influenced through the dispersion of CdS nano-filler particles in the studied polymeric blend series.

  14. Evaluation of ECHO PS Positioning System in a Porcine Model of Simulated Laparoscopic Ventral Hernia Repair

    PubMed Central

    Hanna, Erin M.; Voeller, Guy R.; Roth, J. Scott; Scott, Jeffrey R.; Gagne, Darcy H.; Iannitti, David A.

    2013-01-01

    Purpose. Operative efficiency improvements for laparoscopic ventral hernia repair (LVHR) have focused on reducing operative time while maintaining overall repair efficacy. Our objective was to evaluate procedure time and positioning accuracy of an inflatable mesh positioning device (Echo PS Positioning System), as compared to a standard transfascial suture technique, using a porcine model of simulated LVHR. Methods. The study population consisted of seventeen general surgeons (n = 17) that performed simulated LVHR on seventeen (n = 17) female Yorkshire pigs using two implantation techniques: (1) Ventralight ST Mesh + Echo PS Positioning System (Echo PS) and (2) Ventralight ST Mesh + transfascial sutures (TSs). Procedure time and mesh centering accuracy overtop of a simulated surgical defect were evaluated. Results. Echo PS demonstrated a 38.9% reduction in the overall procedure time, as compared to TS. During mesh preparation and positioning, Echo PS demonstrated a 60.5% reduction in procedure time (P < 0.0001). Although a trend toward improved centering accuracy was observed for Echo PS (16.2%), this was not significantly different than TS. Conclusions. Echo PS demonstrated a significant reduction in overall simulated LVHR procedure time, particularly during mesh preparation/positioning. These operative time savings may translate into reduced operating room costs and improved surgeon/operating room efficiency. PMID:23762628

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

    PubMed

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

    2014-09-07

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

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

    PubMed Central

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

    1982-01-01

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

  17. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

    PubMed

    Peng, Hu; Zhuang, Yugang; Harbeck, Mark C; He, Donghong; Xie, Lishi; Chen, Weiguo

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

  18. Real-life experience of using conventional disease-modifying anti-rheumatic drugs (DMARDs) in psoriatic arthritis (PsA). Retrospective analysis of the efficacy of methotrexate, sulfasalazine, and leflunomide in PsA in comparison to spondyloarthritides other than PsA and literature review of the use of conventional DMARDs in PsA

    PubMed Central

    Roussou, Euthalia; Bouraoui, Aicha

    2017-01-01

    Objective With the aim of assessing the response to treatment with conventional disease-modifying anti-rheumatic drugs (DMARDs) used in patients with psoriatic arthritis (PsA), data on methotrexate, sulfasalazine (SSZ), and leflunomide were analyzed from baseline and subsequent follow-up (FU) questionnaires completed by patients with either PsA or other spondyloarthritides (SpAs). Material and Methods A single-center real-life retrospective analysis was performed by obtaining clinical data via questionnaires administered before and after treatment. The indices used were erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Function Index (BASFI), wellbeing (WB), and treatment effect (TxE). The indices measured at baseline were compared with those measured on one occasion in a FU visit at least 1 year later. Results A total of 73 patients, 51 with PsA (mean age 49.8±12.8 years; male-to-female ratio [M:F]=18:33) and 22 with other SpAs (mean age 50.6±16 years; M:F=2:20), were studied. BASDAI, BASFI, and WB displayed consistent improvements during FU assessments in both PsA patients and controls in comparison to baseline values. SSZ exhibited better efficacy as confirmed by TxE in both PsA patients and controls. ESR and CRP displayed no differences in either the PsA or the SpA group between the cases before and after treatment. Conclusion Real-life retrospective analysis of three DMARDs used in PsA (and SpAs other than PsA) demonstrated that all three DMARDs that were used brought about improvements in BASDAI, BASFI, TxE, and WB. However, the greatest improvements at FU were seen with SSZ use in both PsA and control cohorts. PMID:28293446

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

    PubMed

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

    2012-09-01

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

  20. Unlike pregnant adult women, pregnant adolescent girls cannot maintain glycine flux during late pregnancy because of decreased synthesis from serine.

    PubMed

    Hsu, Jean W; Thame, Minerva M; Gibson, Raquel; Baker, Tameka M; Tang, Grace J; Chacko, Shaji K; Jackson, Alan A; Jahoor, Farook

    2016-03-14

    During pregnancy, glycine and serine become more important because they are the primary suppliers of methyl groups for the synthesis of fetal DNA, and more glycine is required for fetal collagen synthesis as pregnancy progresses. In an earlier study, we reported that glycine flux decreased by 39% from the first to the third trimester in pregnant adolescent girls. As serine is a primary precursor for glycine synthesis, the objective of this study was to measure and compare glycine and serine fluxes and inter-conversions in pregnant adolescent girls and adult women in the first and third trimesters. Measurements were made after an overnight fast by continuous intravenous infusions of 2H2-glycine and 15N-serine in eleven adolescent girls (17·4 (se 0·1) years of age) and in ten adult women (25·8 (se 0·5) years of age) for 4 h. Adolescent girls had significantly slower glycine flux and they made less glycine from serine in the third (P<0·05) than in the first trimester. Baby birth length was significantly shorter of adolescent girls (P=0·04) and was significantly associated with third trimester glycine flux. These findings suggest that the pregnant adolescent cannot maintain glycine flux in late pregnancy compared with early pregnancy because of decreased synthesis from serine. It is possible that the inability to maintain glycine synthesis makes her fetus vulnerable to impaired cartilage synthesis, and thus linear growth.

  1. 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.

  2. Role for neonatal D-serine signaling: prevention of physiological and behavioral deficits in adult Pick1 knockout mice.

    PubMed

    Nomura, J; Jaaro-Peled, H; Lewis, E; Nuñez-Abades, P; Huppe-Gourgues, F; Cash-Padgett, T; Emiliani, F; Kondo, M A; Furuya, A; Landek-Salgado, M A; Ayhan, Y; Kamiya, A; Takumi, T; Huganir, R; Pletnikov, M; O'Donnell, P; Sawa, A

    2016-03-01

    NMDA glutamate receptors have key roles in brain development, function and dysfunction. Regulatory roles of D-serine in NMDA receptor-mediated synaptic plasticity have been reported. Nonetheless, it is unclear whether and how neonatal deficits in NMDA-receptor-mediated neurotransmission affect adult brain functions and behavior. Likewise, the role of D-serine during development remains elusive. Here we report behavioral and electrophysiological deficits associated with the frontal cortex in Pick1 knockout mice, which show D-serine deficits in a neonatal- and forebrain-specific manner. The pathological manifestations observed in adult Pick1 mice are rescued by transient neonatal supplementation of D-serine, but not by a similar treatment in adulthood. These results indicate a role for D-serine in neurodevelopment and provide novel insights on how we interpret data of psychiatric genetics, indicating the involvement of genes associated with D-serine synthesis and degradation, as well as how we consider animal models with neonatal application of NMDA receptor antagonists.

  3. Resonant-photoemission identification of the valence states of NiPS 3

    NASA Astrophysics Data System (ADS)

    Kelly, M. K.; Daniels, R. R.; Margaritondo, G.; Lévy, F.

    1984-04-01

    We monitored the resonant behavior of the Ni d satellite peaks in the valence band photoemission spectra of NiPS 3 at photon energies immediately below and immediately above the Ni3p threshold. The observed resonance gives an unequivocal identification of the satellite peaks and of the corresponding main Ni d features. The study of the electronic structure of this material and of the related compounds FePS 3 and HgPS 3 was extended to unoccupied states by means of partial-yield synchrotron-radiation photoemission spectroscopy.

  4. Tunneling transport of mono- and few-layers magnetic van der Waals MnPS3

    NASA Astrophysics Data System (ADS)

    Lee, Sungmin; Choi, Ki-Young; Lee, Sangik; Park, Bae Ho; Park, Je-Geun

    2016-08-01

    We have investigated the tunneling transport of mono- and few-layers of MnPS3 by using conductive atomic force microscopy. Due to the band alignment of indium tin oxide/MnPS3/Pt-Ir tip junction, the key features of both Schottky junction and Fowler-Nordheim tunneling (FNT) were observed for all the samples with varying thickness. Using the FNT model and assuming the effective electron mass (0.5 me) of MnPS3, we estimate the tunneling barrier height to be 1.31 eV and the dielectric breakdown strength as 5.41 MV/cm.

  5. Virus-induced gene silencing of PEAM4 affects floral morphology by altering the expression pattern of PsSOC1a and PsPVP in pea.

    PubMed

    Chen, Zhe-Hao; Jia, Fei-Fei; Hu, Jiang-Qin; Pang, Ji-Liang; Xu, Lei; Wang, Li-Lin

    2014-01-15

    pea-MADS4 (PEAM4) regulates floral morphology in Pisum sativum L., however, its molecular mechanisms still remain unclear. Virus-induced gene silencing (VIGS) is a recently developed reverse genetic approach that facilities an easier and more rapid study of gene functions. In this study, the PEAM4 gene was effectively silenced by VIGS using a pea early browning virus (PEBV) in wild type pea JI992. The infected plants showed abnormal phenotypes, as the floral organs, especially the sepals and petals changed in both size and shape, which made the corolla less closed. The petals changed in morphology and internal symmetry with, the stamens reduced and carpel dehisced. Larger sepals and longer tendrils with small cauline leaves appeared, with some sepals turning into bracts, and secondary inflorescences with fused floral organs were formed, indicating a flower-to-inflorescence change. The infected plants also displayed a delayed and prolonged flowering time. The PEAM4-VIGS plants with altered floral morphology were similar to the pim (proliferating inflorescence meristem) mutant and also mimicked the phenotypes of ap1 mutants in Arabidopsis. The expression pattern of the homologous genes PsSOC1a and PsSVP, which were involved in flowering time and florescence morphological control downstream of PEAM4, were analyzed by real-time RT-PCR and mRNA in situ hybridization. PsSOC1a and PsSVP were ectopically expressed and enhanced in the floral meristems from PEAM4-silenced plants. Our data suggests that PEAM4 may have a similar molecular mechanism as AtAP1, which inhibits the expression of PsSOC1a and PsSVP in the floral meristem from the early stages of flower development. As such, in this way PEAM4 plays a crucial role in maintaining floral organ identity and flower development in pea.

  6. Room temperature light emission from the low-dimensional semiconductors AZrPS6 (A = K, Rb, Cs).

    PubMed

    Banerjee, Santanu; Szarko, Jodi M; Yuhas, Benjamin D; Malliakas, Christos D; Chen, Lin X; Kanatzidis, Mercouri G

    2010-04-21

    The new semiconducting thiophosphate compounds KZrPS(6), RbZrPS(6), and CsZrPS(6) exhibit red light emission at room temperature. The materials have longer photoluminescence lifetimes than most of the inorganic chalcogenide semiconductors. They can be solution processed into thin films for potential device fabrication.

  7. Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly.

    PubMed

    Devenport, Danelle; Bunch, Thomas A; Bloor, James W; Brower, Danny L; Brown, Nicholas H

    2007-08-15

    The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.

  8. Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds.

    PubMed

    Yamazoe, Taiji; Shiraki, Nobuaki; Kume, Shoen

    2016-01-01

    The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.

  9. Optimization of a multi-ring detector for Ps time of flight measurements

    NASA Astrophysics Data System (ADS)

    Di Noto, L.; Benetti, M.; Mariazzi, S.; Dalla Betta, G.-F.; Brusa, R. S.

    2013-06-01

    We have designed a multi-ring detector (MRD) based on Bismuth Germanate (BGO) crystals, coupled to Silicon PhotoMultipliers (SiPM) for measuring the Ps time of flight (TOF). The set-up geometry was optimized by Monte Carlo simulations to take into account at different Ps velocities: (i) the background noise due to backscattered positrons, (ii) the crosstalk between adjacent detectors, (iii) the lifetime of Ps decay. Three parameters were defined to evaluate the different configurations and a figure of merit was obtained. This allows the choice of the best set up configuration for measuring Ps emitted with a particular energy range, optimizing the signal to noise ratio and keeping the acquisition time acceptable.

  10. Studies on the controlled morphology and wettability of PS surfaces by electrospinning or electrospraying.

    NASA Astrophysics Data System (ADS)

    Zheng, Jianfen; He, Aihua; Han, Charles

    2007-03-01

    Electrospinning/electrospraying is a simple and effective way to fabricate various polymer surfaces such as beads, fibers and other shapes in the range of micro- to nanometer. Various surface morphologies have been produced by electrospinning or electrospraying: beads with different sizes and shapes, bead-on-string structure with different aspect ratios and fibers with different diameters and shapes. Physical properties of the PS solutions such as viscosity, surface tension and conductivity greatly influence the electrospun or electrosprayed PS morphology. The wettability of a solid surface is greatly influenced by its surface morphology: A spin-coated PS membrane has a water contact angle of 97^o, while electrospun PS membranes have water contact angles around 150^o. The most hydrophobic membrane has a water CA of 159.5^o.

  11. 2. P.S. Rittermann, Photographer February 1995 BUILDING 990, WEST SIDE. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. P.S. Rittermann, Photographer February 1995 BUILDING 990, WEST SIDE. - Presidio of San Francisco, Flammable Storage Building Submarine Mine Depot, Fort Point vicinity, Long Avenue, San Francisco, San Francisco County, CA

  12. The Photodetachment of Ps ion and Low-Energy e(+) -H Collisions

    NASA Technical Reports Server (NTRS)

    Ward, S.J.

    2007-01-01

    Two calculations in the area of positron collisions are presented. The first is the calculation of the photodetachment cross section of the positronium negative ion (Ps-) using accurate variational wave functions for both the initial bound-state and the final P continuum state. The second is the calculation of partial wave cross sections for Ps(1s)-formation in ef -H(ls) collisions using the hyperspherical hidden crossing method. Since the S-wave Stiickelberg phase is close to pi, the very small S-wave Ps(1s) formation cross section can be understood in terms of destructive interference. Other examples in positron collisions are given where it is either known or expected that destructive interference is the cause of the small S-wave Ps(1s) formation cross section. In addition, examples are presented of processes in atomic physics where the Stiickelberg phase is a multiple of pi/2.

  13. Necrotic Cells Actively Attract Phagocytes through the Collaborative Action of Two Distinct PS-Exposure Mechanisms

    PubMed Central

    Li, Zao; Venegas, Victor; Nagaoka, Yuji; Morino, Eri; Raghavan, Prashant; Audhya, Anjon; Nakanishi, Yoshinobu; Zhou, Zheng

    2015-01-01

    Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca2+-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common “eat me” signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively. PMID:26061275

  14. A 45 PS Time-Interval Counter Board with a PCI Interface

    DTIC Science & Technology

    2007-11-01

    39th Annual Precise Time and Time Interval (PTTI) Meeting A 45 PS TIME-INTERVAL COUNTER BOARD WITH A PCI INTERFACE Ryszard Szplet...precise time-interval and frequency counter board with PCI interface. The counter utilizes two two-stage interpolators that provide 45 ps resolution...synthesis and for generation of the four -phase clock used in the first interpolation stage. The fast arithmetic carry chains are used as high

  15. E-Cloud Drivent Single-Bunch Instabilities in PS2

    SciTech Connect

    Venturini, M.; Furman, M.; Penn, G.; Secondo, R.; Vay, J-L.; De Maria, R.; Papaphilippou, Y.; Rumolo, G.

    2010-05-23

    One of the proposals under consideration for future upgrades of the LHC injector complex entails the replacement of the PS with the PS2, a longer circumference and higher energy synchrotron, with electron cloud effects representing a potentially serious limitation to the achievement of the upgrade goals. We report on ongoing numerical studies aiming at estimating the e-cloud density threshold for the occurrence of single bunch instabilities.

  16. Perceptions of Community Health Workers (CHWs/PS) in the U.S.-Mexico border HEART CVD study.

    PubMed

    Balcazar, Hector G; Wise, Sherrie; Redelfs, Alisha; Rosenthal, E Lee; de Heer, Hendrik D; Burgos, Ximena; Duarte-Gardea, Maria

    2014-02-10

    Although prior research has shown that Community Health Workers/Promotores de Salud (CHW/PS) can facilitate access to care, little is known about how CHW/PS are perceived in their community. The current study reports the findings of a randomized telephone survey conducted in a high-risk urban community environment along the U.S.-Mexico border. In preparation for a community-based CHW/PS intervention called the HEART ecological study, the survey aimed to assess perceptions of CHW/PS, availability and utilization of community resources (recreational and nutrition related) and health behaviors and intentions. A total of 7,155 calls were placed to complete 444 surveys in three zip codes in El Paso, Texas. Results showed that participants felt that healthful community resources were available, but utilization was low and variable: 35% reported going to a park, 20% reported having taken a health class, few reported using a gym (12%), recreation center (8%), or YMCA/YWCA (0.9%). Awareness and utilization of CHW/PS services were low: 20% of respondents had heard of CHW/PS, with 8% reporting previous exposure to CHW/PS services. Upon review of a definition of CHW/PS, respondents expressed positive views of CHW/PS and their value in the healthcare system. Respondents who had previous contact with a CHW/PS reported a significantly more positive perception of the usefulness of CHW/PS (p = 0.006), were more likely to see CHW/PS as an important link between providers and patients (p = 0.008), and were more likely to ask a CHW/PS for help (p = 0.009). Participants who utilized CHW/PS services also had significantly healthier intentions to reduce fast food intake. Future research is needed to evaluate if CHW/PS can facilitate utilization of available community resources such as recreational facilities among Hispanic border residents at risk for CVD.

  17. New molecular-scale information on polystyrene dynamics in PS and PS-BaTiO3 composites from FTIR spectroscopy.

    PubMed

    Olmos, D; Martín, E V; González-Benito, J

    2014-11-28

    A new idea to understand the macromolecular motion occurring along the thermal relaxations of polystyrene (PS) and PS-barium titanate composites is proposed. Detailed analysis of PS infrared bands provides a better knowledge of the factors affecting polymer dynamics. Average spectral positions and integrated absorbance of bands in the region of C-H out-of-plane vibrations showed a continuous decrease with temperature, whereas those in the region of aliphatic and aromatic C-H stretching vibrations showed the sharpest changes with temperature. Relaxation temperatures were determined from the changes observed in the band wavenumber or area with temperature. These results were attributed to changes in the distribution of the phenyl π-electron cloud, causing important dipole moment variations in the different vibration modes when the thermal transitions are taking place. Finally, although the presence of BaTiO3 particles does not seem to exert any specific effect on the PS dynamics in the glassy state, the Curie transition of these particles might induce a kind of confinement effect observable by FTIR.

  18. PCaPs, possible regulators of PtdInsP signals on plasma membrane.

    PubMed

    Kato, Mariko; Nagasaki-Takeuchi, Nahoko; Ide, Yuki; Tomioka, Rie; Maeshima, Masayoshi

    2010-07-01

    In plants, Ca(2+), phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates are major components of intracellular signaling. Several kinds of proteins and enzymes, such as calmodulin (CaM), protein kinase, protein phosphatase, and the Ca(2+) channel, mediate the signaling. Two new Ca(2+)-binding proteins were identified from Arabidopsis thaliana and named PCaP1 and PCaP2 [plasma membrane (PM)-associated Ca(2+) (cation)-binding protein 1 and 2]. PCaP1 has an intrinsically disordered region in the central and C-terminal parts. The PCaP1 gene is expressed in most tissues and the PCaP2 gene is expressed predominantly in root hairs and pollen tubes. We recently demonstrated that these proteins are N-myristoylated, stably anchored in the PM, and are bound with phosphatidylinositol phosphates, especially PtdInsP2s. Here we propose a model for the switching mechanism of Ca (2+)-signaling mediated by PtdInsPs. Ca(2+) forms a complex with CaM (Ca(2+)-CaM) when there is an increase in the cytosol free Ca(2+). The binding of PCaPs with Ca(2+)-CaM causes PCaPs to release PtdInsPs. Until the release of PtdInsPs, the signaling is kept in the resting state.

  19. PCaPs, possible regulators of PtdInsP signals on plasma membrane

    PubMed Central

    Kato, Mariko; Nagasaki-Takeuchi, Nahoko; Ide, Yuki; Tomioka, Rie

    2010-01-01

    In plants, Ca2+, phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates are major components of intracellular signaling. Several kinds of proteins and enzymes, such as calmodulin (CaM), protein kinase, protein phosphatase, and the Ca2+ channel, mediate the signaling. Two new Ca2+-binding proteins were identified from Arabidopsis thaliana and named PCaP1 and PCaP2 [plasma membrane (PM)-associated Ca2+(cation)-binding protein 1 and 2]. PCaP1 has an intrinsically disordered region in the central and C-terminal parts. The PCaP1 gene is expressed in most tissues and the PCaP2 gene is expressed predominantly in root hairs and pollen tubes. We recently demonstrated that these proteins are N-myristoylated, stably anchored in the PM, and are bound with phosphatidylinositol phosphates, especially PtdInsP2s. Here we propose a model for the switching mechanism of Ca2+-signaling mediated by PtdInsPs. Ca2+ forms a complex with CaM (Ca2+-CaM) when there is an increase in the cytosol free Ca2+. The binding of PCaPs with Ca2+-CaM causes PCaPs to release PtdInsPs. Until the release of PtdInsPs, the signaling is kept in the resting state. PMID:20448467

  20. Protection of tree shrews by pVAX-PS DNA vaccine against HBV infection.

    PubMed

    Zhou, Feng-Juan; Hu, Zhen-Lin; Dai, Jian-Xin; Chen, Rui-Wen; Shi, Ke; Lin, Yi; Sun, Shu-Han

    2003-07-01

    The immunological protection of pVAX-PS, a DNA vaccine, was assessed in the tree shrews model. pVAX-PS was constructed by inserting the gene encoding the middle (pre-S2 plus S) envelope protein of HBV into a plasmid vector pVAX1. Tree shrews (Tupaia belangeri chinenesis) were experimentally infected with human HBV by inoculation with human serum positive for HBV markers. DNA vaccination-induced seroconversion and antibody to HBV surface antigen (anti-HBs) were analyzed by ELISA, and protective effects elicited by pVAX-PS vaccination against subsequent HBV challenge were evaluated by detection of HBV seromarkers and observation of hepatic lesions in HBV-infected tree shrews. The results shown that anti-HBs were detectable in serum at week 2 after pVAX-PS vaccination and peaked at week 4, and immunization with pVAX-PS decreased the positive conversion rate of HBV seromarkers and relieved hepatic lesions in tree shrews challenged with HBV. These results indicated that pVAX-PS immunization could induce remarkable humoral immune response and prevent the experimental tree shrews from infection of HBV.

  1. Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review

    PubMed Central

    Malhotra, Neeraj

    2016-01-01

    iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions. PMID:27572712

  2. Application of induced pluripotent stem (iPS) cells in periodontal tissue regeneration.

    PubMed

    Duan, Xuejing; Tu, Qisheng; Zhang, Jin; Ye, Jinhai; Sommer, Cesar; Mostoslavsky, Gustavo; Kaplan, David; Yang, Pishan; Chen, Jake

    2011-01-01

    Tissue engineering provides a new paradigm for periodontal tissue regeneration in which proper stem cells and effective cellular factors are very important. The objective of this study was, for the first time, to investigate the capabilities and advantages of periodontal tissue regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). In this study the effect of EMD gel on iPS cells in vitro was first determined, and then tissue engineering technique was performed to repair periodontal defects in three groups: silk scaffold only; silk scaffold + EMD; and silk scaffold + EMD + iPS cells. EMD greatly enhanced the mRNA expression of Runx2 but inhibited the mRNA expression of OC and mineralization nodule formation in vitro. Transplantation of iPS cells showed higher expression levels of OC, Osx, and Runx2 genes, both 12 and 24 days postsurgery. At 24 days postsurgery in the iPS cell group, histological analysis showed much more new alveolar bone and cementum formation with regenerated periodontal ligament between them. The results showed the commitment role that EMD contributes in mesenchymal progenitors to early cells in the osteogenic lineage. iPS cells combined with EMD provide a valuable tool for periodontal tissue engineering, by promoting the formation of new cementum, alveolar bone, and normal periodontal ligament.

  3. Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review.

    PubMed

    Malhotra, Neeraj

    2016-11-30

    iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions.

  4. Community Perspectives Associated With the African PsA-TT (MenAfriVac) Vaccine Trials

    PubMed Central

    Idoko, Olubukola T.; Diallo, Aldiouma; Sow, Samba O.; Hodgson, Abraham; Akinsola, Adebayo; Diarra, Bou; Haidara, Fadima Cheick; Ansah, Patrick Odum; Kampmann, Beate; Bouma, Enricke; Preziosi, Marie-Pierre; Enwere, Godwin C.

    2015-01-01

    Background. The Meningitis Vaccine Project (MVP) was established to address epidemic meningitis as a public health problem in sub-Saharan Africa and, to that end, worked to develop a group A meningococcal conjugate vaccine, PsA-TT. Methods. Experiences in 4 clinical trial sites are described. Culturally sensitive collaborative strategies were adopted to manage acceptable communication methods, peculiarities with the consent process, participant medical issues, community care, and death. Results. The clinical trials were completed successfully through community acceptance and active community collaboration. The trials also strengthened the capacities in the participating communities, and actively worked to resolve community problems. Conclusions. The understanding and integration of sociocultural realities of communities were major assets in the conduct and acceptance of these trials. MVP succeeded in these sites and provided a sound example for future clinical studies in Africa. Clinical Trials Registration. ISRTCN78147026 (PsA-TT 002); ISRCTN87739946 (PsA-TT 003); ISRCTN82484612 (PsA-TT 004); PACTR ATMR2010030001913177 (PsA-TT 006); and PACTR201110000328305 (PsA-TT 007). PMID:26553669

  5. Directed self-assembly materials for high resolution beyond PS-b-PMMA

    NASA Astrophysics Data System (ADS)

    Hirahara, Eri; Paunescu, Margareta; Polishchuk, Orest; Jeong, EunJeong; Ng, Edward; Shan, Jianhui; Yin, Jian; Kim, Jihoon; Cao, Yi; Li, Jin; Hong, SungEun; Baskaran, Durairaj; Lin, Guanyang

    2016-03-01

    To extend directed self-assembly (DSA) of poly(styrene-b-methyl methacrylate) (PS-b-PMMA) for higher resolution, placement accuracy and potentially improved pattern line edge roughness (LER), we have developed a next-generation material platform of organic high-χ block copolymers ("HC series", AZEMBLYTM EXP PME-3000 series). The new material platform has a built-in orientation control mechanism which enables block copolymer domains to vertically selforient without topcoat/additive or delicate solvent vapor annealing. Furthermore, sub-10 nm lines and spaces (L/S) patterning by two major chemoepitaxy DSA, LiNe and SMARTTM processes, was successfully implemented on 12" wafer substrates by using the PME-3000 lamellar series. The results revealed that the new material platform is compatible with the existing PS-b-PMMA-based chemical prepatterns and standard protocols. We also introduced the built-in orientation control strategy to the conventional PS-b-PMMA system, producing a new generation of PS-b-PMMA materials with facile orientation control. The modified PS-b-PMMA (m-PS-b-PMMA) performed LiNe flow DSA yielding a comparable CD process window with improved LER/LWR/SWR after the L/S patterns were transferred into a Si substrate.

  6. Relation of P-S4 interval to left ventricular end-diastolic pressure.

    PubMed Central

    Schapira, J N; Fowles, R E; Bowden, R E; Alderman, E L; Popp, R L

    1982-01-01

    Reports have suggested that the interval between P wave onset and the fourth heart sound (P-S4 interval) reflects changes in left ventricular myocardial stiffness. We made simultaneous measurements of the P-S4 or atrial electrogram to S4 (A-S4) interval and left ventricular pressure in 19 patients with coronary artery disease who were studied before and after atrial pacing. Thirteen patients developed angina accompanied by significant rises in their end-diastolic pressure and a consistent decrease in P-S4 or A-S4 interval; whereas the six patients who had atrial pacing without the development of angina had no change in end-diastolic pressure, P-S4, or A-S4 interval. The resting data showed in inverse correlation between left ventricular end-diastolic pressure and the P-S4 interval. In addition, the P-S4 interval let us discriminate between patients with normal and abnormal end-diastolic pressure (greater than 15 mmHg). Images PMID:7059403

  7. Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex.

    PubMed

    Purohit, Vivek S; Ramani, Karthik; Sarkar, Rita; Kazazian, Haig H; Balasubramanian, Sathyamangalam V

    2005-05-06

    Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.

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

    PubMed Central

    Lai, Run-Zhi; Parkinson, John S.

    2014-01-01

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

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

    PubMed

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

    2016-02-19

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

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

    PubMed

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

    1988-03-05

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

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

    SciTech Connect

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

    1990-05-01

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

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

    PubMed

    Caughey, G H

    1994-12-01

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

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

    PubMed

    Lai, Run-Zhi; Parkinson, John S

    2014-10-23

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.

  14. Synthesis and characterization of a new fluorogenic active-site titrant of serine proteases.

    PubMed

    Livingston, D C; Brocklehurst, J R; Cannon, J F; Leytus, S P; Wehrly, J A; Peltz, S W; Peltz, G A; Mangel, W F

    1981-07-21

    The molecule 3',6'-bis(4-guanidinobenzoyloxy)-5-[N'-(4-carboxyphenyl)thioureido[spirop]isobenzofuran-1-(3H),9'-[9H]xanthen]-3-one, abbreviated FDE, was designed and synthesized as a fluorogenic active-site titrant for serine proteases. It is an analogue of p-nitrophenyl p-guanidino-benzoate (NPGB) in which a fluorescein derivative is substituted for p-nitrophenol. FDE and NPGB exhibit similar kinetic characteristics in an active-site titration of trypsin in phosphate-buffered saline, pH 7.2. The rate of acylation with FDE is extremely fast (k2 = 1.05 s-1) and the rate of deacylation extremely slow (k3 = 1.66 X 10(-5) s-1). The Ks is 3.06 X 10(-6) M, and the Km(app) is 4.85 X 10(-11) M. With two of the serine proteases involved in fibrinolysis, the rate of acylation with FDE is also fast, K2 = 0.112 s-1 for urokinase and 0.799 s-1 for plasmin, and the rate of deacylation is slow, k3 = 3.64 X 10(-4) s-1 for urokinase and 6.27 X 10(-6) s-1 for plasmin. The solubility limit of FDE in phosphate-buffered saline is 1.3 X 10(-5) M, and the first-order rate constant for spontaneous hydrolysis is 5.1 X 10(-6) s-1. The major difference between FDE and NPGB is the detectability of the product in an active-site titration. p-Nitrophenol can be detected at concentrations no lower than 10(-6) M whereas fluorescein can be detected at concentrations as low as 10(-12) M. Thus, FDE should be useful in quantitatively assaying serine proteases as very low concentrations.

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

    PubMed Central

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

    1990-01-01

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the approximately 1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family. Images PMID:2187193

  16. Interaction of ortho-Phospho-l-serine with Hydroxyapatite: Formation of a Surface Complex

    PubMed

    Misra

    1997-10-01

    ortho-Phospho-l-serine (H2Psi, where Psi represents the serinephosphato ion), a constituent of salivary proteins, seems to play an important role in the mineralization of teeth. To understand the basic mechanism of this interaction, the uptake of o-phospho-l-serine from relatively concentrated aqueous solutions (up to 100 mmol/L) onto synthetic hydroxyapatite was studied. Previous studies have shown that in the dilute concentration range (<12.5 mmol/L) the uptake followed a regular Langmuirian adsorption plot. At higher concentrations the uptake curve increased steeply, but no formation of a separate phase in the reacted apatite was discernible, either by optical or by scanning electron microscopy. The dissolution of apatite released phosphate and calcium ions into the solution in amounts linearly related to the uptake of serine with P/Psi = 1 and Ca/Psi = 2. The charge and mass balance of the reaction can be reconciled with the formation of the surface complex (shown within brackets):Ca10(OH)2(PO4)6 + 6H2Psi --> [Ca6(HPsi)2(HPO4)2(PO4)2] + 4Ca2+ + 2HPsi1- + 2Psi2- + 2H2PO1-4 + 2H2O.The formation of two other surface complexes is possible; however, the complex shown above probably disrupts the apatite lattice the least. Traces of CaPsi·H2O precipitate out from the filtrates of highly concentrated solutions after 6 days. Copyright 1997 Academic Press. Copyright 1997Academic Press

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

    PubMed Central

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

    2016-01-01

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

  18. 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

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

    PubMed

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

    1995-09-01

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

  20. BbrzSP-32, the first serine protease isolated from Bothrops brazili venom: Purification and characterization.

    PubMed

    Zaqueo, Kayena D; Kayano, Anderson M; Domingos, Thaisa F S; Moura, Laura A; Fuly, André L; da Silva, Saulo L; Acosta, Gerardo; Oliveira, Eliandre; Albericio, Fernando; Zanchi, Fernando B; Zuliani, Juliana P; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M

    2016-05-01

    Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated.

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

    PubMed Central

    Geissler, S; Götz, F; Kupke, T

    1996-01-01

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

  2. Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

    PubMed Central

    Karlyshev, A.V.; Thacker, G.; Jones, M.A.; Clements, M.O.; Wren, B.W.

    2014-01-01

    According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection. PMID:24918062

  3. A Serine-Substituted P450 Catalyzes Highly Efficient Carbene Transfer to Olefins In Vivo

    PubMed Central

    Coelho, Pedro S.; Wang, Z. Jane; Ener, Maraia E.; Baril, Stefanie A.; Kannan, Arvind A.; Arnold, Frances H.; Brustad, Eric M.

    2013-01-01

    Genetically encoded catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a unique serine-heme ligated cytochrome “P411” that catalyzes efficient and selective carbene transfers from diazoesters to olefins in intact Escherichia coli cells. The mutation C400S in cytochrome P450BM3 gives a signature ferrous-CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting state FeIII/II reduction potential, and significantly improves NAD(P)H-driven cyclopropanation activity. PMID:23792734

  4. A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo.

    PubMed

    Coelho, Pedro S; Wang, Z Jane; Ener, Maraia E; Baril, Stefanie A; Kannan, Arvind; Arnold, Frances H; Brustad, Eric M

    2013-08-01

    Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450(BM3) gives a signature ferrous CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting-state Fe(III)-to-Fe(II) reduction potential and substantially improves NAD(P)H-driven activity.

  5. Substituted isatoic anhydrides: selective inactivators of trypsin-like serine proteases.

    PubMed

    Gelb, M H; Abeles, R H

    1986-04-01

    Derivatives of isatoic anhydride were prepared and tested as inhibitors of serine proteases. A number of isatoic anhydrides with positively charged substituents irreversibly inactivated several trypsin-like enzymes and preferentially inactivated trypsin over chymotrypsin. Further selectivity was obtained by introduction of an aromatic group on the N-1 position of isatoic anhydride. 7-(Aminomethyl)-1-benzylisatoic anhydride was prepared and was a selective inactivator of thrombin; thus it is possible to prepare derivatives of isatoic anhydride that are highly enzyme selective without attaching peptide recognition structures.

  6. 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°, Δr G°, Δr H°, and Δr S°) of the studied equilibria are calculated. The final results are compared with the corresponding data on the related compounds.

  7. 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.

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

    PubMed

    Yakovlev, Ilya; Deming, Timothy J

    2015-04-01

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

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

    PubMed

    Sano, S

    2000-01-01

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

  10. 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.

  11. 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.

  12. Paper sludge (PS) to bioethanol: Evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentation.

    PubMed

    Boshoff, Sonja; Gottumukkala, Lalitha Devi; van Rensburg, Eugéne; Görgens, Johann

    2016-03-01

    Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20 FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11 FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2g/L at 66.9% and 45.5 g/L at 78.2%, respectively.

  13. 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

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

    PubMed

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

    1988-01-01

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

  15. Control of Appetite and Food Preference by NMDA Receptor and Its Co-Agonist d-Serine

    PubMed Central

    Sasaki, Tsutomu; Matsui, Sho; Kitamura, Tadahiro

    2016-01-01

    Obesity causes a significant negative impact on health of human beings world-wide. The main reason for weight gain, which eventually leads to obesity, is excessive ingestion of energy above the body’s homeostatic needs. Therefore, the elucidation of detailed mechanisms for appetite control is necessary to prevent and treat obesity. N-methyl-d-aspartate (NMDA) receptor is a post-synaptic glutamate receptor and is important for excitatory neurotransmission. It is expressed throughout the nervous system, and is important for long-term potentiation. It requires both ligand (glutamate) and co-agonist (d-serine or glycine) for efficient opening of the channel to allow calcium influx. d-serine is contained in fermented foods and marine invertebrates, and brain d-serine level is maintained by synthesis in vivo and supply from food and gut microbiota. Although the NMDA receptor has been reported to take part in the central regulation of appetite, the role of d-serine had not been addressed. We recently reported that exogenous d-serine administration can suppress appetite and alter food preference. In this review, we will discuss how NMDA receptor and its co-agonist d-seine participate in the control of appetite and food preference, and elaborate on how this system could possibly be manipulated to suppress obesity. PMID:27399680

  16. Supplementation with D-serine prevents the onset of cognitive deficits in adult offspring after maternal immune activation

    PubMed Central

    Fujita, Yuko; Ishima, Tamaki; Hashimoto, Kenji

    2016-01-01

    Prenatal maternal infection contributes to the etiology of schizophrenia, with D-serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA) receptor, playing a role in the pathophysiology of this disease. We examined whether supplementation with D-serine during juvenile and adolescent stages could prevent the onset of cognitive deficits, prodromal and the core symptoms of schizophrenia in adult offspring after maternal immune activation (MIA). Juvenile offspring exposed prenatally to poly(I:C) showed reduced expression of NMDA receptor subunits in the hippocampus. Supplementing drinking water with D-serine (600 mg/L from P28 to P56) prevented the onset of cognitive deficits in adult offspring after MIA, in a significant manner. This study shows that supplementing offspring with D-serine during juvenile and adolescent stages could prevent the onset of psychosis in adulthood, after MIA. Therefore, early intervention with D-serine may prevent the occurrence of psychosis in high-risk subjects. PMID:27853241

  17. Converted Ps amplitude variations on the dipping slab Moho beneath the Kii Peninsula, central Japan

    NASA Astrophysics Data System (ADS)

    Shiomi, K.; Park, J. J.

    2011-12-01

    Receiver function (RF) analysis is a very useful method to detect seismic velocity discontinuities beneath a seismic station. One can estimate the depth of interfaces using the delay time of Ps converted phases in RFs. One can also evaluate elastic properties at an interface from changes of Ps polarity and Ps amplitude. Ps amplitude depends primarily on the impedance contrast at an interface, but the variation of Ps amplitude on back azimuth (BAZ) of the incoming P wave is affected if the interface is dipping and/or anisotropic rock surrounds the interface. Moreover, variation in the incidence angle of incoming P waves also causes variation in Ps amplitude. Shiomi and Park (2009; AGU FM) defined 'standard amplitude' of a converted phase at a dipping interface beneath a station, based on back azimuth dependence of the Ps amplitude. They did not consider ray parameter dependence to the Ps amplitude evaluation, but it affects the accuracy of the standard amplitude estimate. In this study, we check ray parameter dependence, and apply the method to the stations in the Kii Peninsula, central Japan. We select earthquakes with high signal-to-noise ratio observed from October 2000 to August 2010 with magnitudes 6.0 or greater. Checking distribution of BAZs and incidence angles of teleseismic waveforms observed at each station, we confirm that 80% of the selected events are located to the south of stations. Ray parameters of 10% of the events are larger than 0.077. In the case of dipping interface, Ps amplitude variation with BAZ is larger for incoming P waves with larger ray parameters. Since events located in the west or northeast of stations are fewer than other directions, the contribution of events with large ray parameter is not small in these directions. These directions correspond to the dipping direction of the subducting Philippine Sea slab, thus the Ps amplitudes tend to become large. This means the Ps amplitude may be overestimated when we do not take ray

  18. Transmission of PM-QPSK and PS-QPSK with different fiber span lengths.

    PubMed

    Sjödin, Martin; Puttnam, Ben J; Johannisson, Pontus; Shinada, Satoshi; Wada, Naoya; Andrekson, Peter A; Karlsson, Magnus

    2012-03-26

    We perform experimental and numerical investigations of the transmission reach of polarization-switched QPSK (PS-QPSK) and polarization-multiplexed QPSK (PM-QPSK) for three different fiber span lengths: 83, 111 and 136 km. In the experimental comparison we investigate the performance of PS-QPSK at 20 Gbaud and PM-QPSK at the same bit rate (60 Gbit/s) and at the same symbol rate, both the single channel case and a WDM system with 9 channels on a 50 GHz grid. We show that PS-QPSK gives significant benefits in transmission reach for all span lengths. Compared to PM-QPSK, use of PS-QPSK increases the reach with more than 41% for the same symbol rate and 21% for the same bit rate. In the numerical simulations we use the same data rates as in the experiment. The simulation results agree well with the experimental findings, but the transmission reach is longer due to the absence of various non-ideal effects and higher back-to-back sensitivity. Apart from using data coded in the absolute phase in the simulations, we also investigate differentially coded PS-QPSK for the first time and compare with PM-QPSK with differential coding. The power efficiency advantage of PS-QPSK then increases with approximately 0.3 dB at a bit error rate of 10⁻³, resulting in a further relative transmission reach improvement over PM-QPSK. Both the experimental and the numerical results indicate that PS-QPSK has slightly higher tolerance to inter-channel nonlinear crosstalk than PM-QPSK.

  19. Assessment of coronary plaque collagen with polarization sensitive optical coherence tomography (PS-OCT)

    NASA Astrophysics Data System (ADS)

    Giattina, Susanne D.; Courtney, Brian K.; Herz, Paul R.; Harman, Michelle; Shortkroff, Sonya; Stamper, Debra L.; Liu, Bin; Fujimoto, James G.; Brezinski, Mark E.

    2006-02-01

    Current evidence indicates that most plaques classified as vulnerable or ruptured plaques do not lead to unstable angina or myocardial infarction. Improved methods are needed to risk stratify plaques to identify those which lead to most acute coronary syndromes. Collagen depletion in the intima overlying lipid collections appears to be a critical component of unstable plaques. In this study, we use polarization sensitive optical coherence tomography (PS-OCT) for the assessment of coronary plaque collagen. Collagen is birefringent, meaning that different polarization states travel through it at different velocities. Changes in PS-OCT images are a measure of tissue birefringence. Twenty-two coronary artery segments were imaged with PS-OCT and analyzed by picrosirius staining (a measure of collagen intensity and fiber size) and trichrome blue. The regression plot between PS-OCT changes and measured collagen yielded a correlation coefficient value of 0.475 (p<0.002). Good correlation was noted between two blinded investigators both with respect to PS-OCT measurements as well as luminosity as assessed by picrosirius. The predictive value of a PS-OCT measurement of negligible birefringence (less than 33% change) for minimal collagen was 93% while the predictive value of high birefringence (greater than 66% change) for high collagen concentrations was 89%. The effect of fiber type (chemical composition) was minimal relative to the effect due to fiber concentration. The capability of PS-OCT to assess plaque collagen content, in addition to its ability to generate high resolution structural assessments, make it a potentially powerful technology for identifying high risk plaques.

  20. Development and initial validation of a self-scored COPD Population Screener Questionnaire (COPD-PS).

    PubMed

    Martinez, Fernando J; Raczek, Anastasia E; Seifer, Frederic D; Conoscenti, Craig S; Curtice, Tammy G; D'Eletto, Thomas; Cote, Claudia; Hawkins, Clare; Phillips, Amy L

    2008-04-01

    COPD has a profound impact on daily life, yet remains underdiagnosed and undertreated. We set out to develop a brief, reliable, self-scored questionnaire to identify individuals likely to have COPD. COPD-PS development began with a list of concepts identified for inclusion using expert opinion from a clinician working group comprised of pulmonologists (n = 5) and primary care clinicians (n = 5). A national survey of 697 patients was conducted at 12 practitioner sites. Logistic regression identified items discriminating between patients with and without fixed airflow obstruction (AO, postbronchodilator FEV(1)/FVC < 70%). ROC analyses evaluated screening accuracy, compared scoring options, and assessed concurrent validity. Convergent and discriminant validity were assessed via COPD-PS and SF-12v2 score correlations. For known-groups validation, COPD-PS differences between clinical groups were tested. Test-retest reliability was evaluated in a 20% sample. Of 697 patients surveyed, 295 patients met expert review criteria for spirometry performance; 38% of these (n = 113) had results indicating AO. Five items positively predicted AO (p < 0.0001): breathlessness, productive cough, activity limitation, smoking history, and age. COPD-PS scores accurately classified AO status (area under ROC curve = 0.81) and reliable (r = 0.91). Patients with spirometry indicative of AO scored significantly higher (6.8, SD = 1.9; p< 0.0001) than patients without AO (4.0, SD = 2.3). Higher scores were associated with more severe AO, bronchodilator use, and overnight hospitalization for breathing problems. With the prevalence of COPD in the studied cohort, a score on the COPD-PS of greater than five was associated with a positive predictive value of 56.8% and negative predictive value of 86.4%. The COPD-PS accurately classified physician-reported COPD (AUC = 0.89). The COPD-PS is a brief, accurate questionnaire that can identify individuals likely to have COPD.

  1. Phenotypic correction of murine hemophilia A using an iPS cell-based therapy.

    PubMed

    Xu, Dan; Alipio, Zaida; Fink, Louis M; Adcock, Dorothy M; Yang, Jianchang; Ward, David C; Ma, Yupo

    2009-01-20

    Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons-including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.

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

    PubMed Central

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

    1990-01-01

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

  3. Determination of activable proacrosin/acrosin in bovine sperm using an irreversible isocoumarin serine protease inhibitor.

    PubMed

    Palencia, D D; Garner, D L; Hudig, D; Holcombe, D W; Burner, C A; Redelman, D; Fernandez, G C; Abuelyaman, A S; Kam, C M; Powers, J C

    1996-09-01

    The activable proacrosin/acrosin levels in bovine sperm were examined using fluorescent staining and flow cytometry. The proportion of sperm with active acrosin were determined using the biotinylated isocoumarin serine protease inhibitor, Bi-Aca-Aca-OMe-IC (BIC). The presence of bound inhibitor on sperm was then determined by secondary labeling with avidin fluorescein conjugate. The proportion of sperm with activable proacrosin/acrosin was assessed by using detergent treatment to expose the active acrosin in intact sperm. The difference between untreated and detergent-treated aliquots was used to estimate the proportion of sperm with activable proacrosin/acrosin. In the 24-h stored samples from six bulls, the mean proportion of sperm with activable proacrosin/acrosin was 78.8 +/- 2.8%, whereas the mean proportion with exposed acrosin after cryopreservation of these samples was 55.8 +/- 4.1%. Significant differences (p < 0.05) were found among bulls in the proportion of sperm with activable proacrosin/acrosin both before and after cryopreservation. Activable proacrosin/acrosin levels in samples of cryopreserved sperm from five bulls were not correlated with fertility. These results do indicate, however, that the irreversible isocoumarin serine protease inhibitor BIC can be used to determine the proportion of sperm cells that retain activable proacrosin/acrosin after cryopreservation and thawing.

  4. 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

  5. Benghalensin, a highly stable serine protease from the latex of medicinal plant Ficus benghalensis.

    PubMed

    Sharma, Anurag; Kumari, Moni; Jagannadham, M V

    2009-12-09

    A serine protease was purified to homogeneity from the latex of medicinal plant Ficus benghalensis by a single step procedure using anion exchange chromatography. The enzyme, named benghalensin, has a molecular mass of 47 kDa (MALDI-TOF and SDS-PAGE). The purified protein is a glycoprotein, and the enzymatic activity is solely inhibited by PMSF and chymostatin, indicating that the enzyme belongs to the serine protease class. The isoelectric point of the enzyme is pH 4.4 with optimum pH and temperature of pH 8.0 and 55 degrees C respectively. The extinction coefficient (epsilon(1%)(280)) of the enzyme is 29.25, and the molecular structure consists of 17 tryptophan, 31 tyrosine and 09 cysteine residues. Peptide mass fingerprinting and de novo sequencing of tryptic-digested fragments of the protein did not find any putative conserved domains in BLAST analysis. The enzyme is stable and retains full activity over a broad range of pH and temperature or prolonged storage at 4 degrees C. Simple purification, high yield and stability enable exploration of the protein for structure-function relationship studies as well as other applications.

  6. 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

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

    PubMed

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

    2005-05-25

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

  8. 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.

  9. Multiple interfaces between a serine recombinase and an enhancer control site-specific DNA inversion

    PubMed Central

    McLean, Meghan M; Chang, Yong; Dhar, Gautam; Heiss, John K; Johnson, Reid C

    2013-01-01

    Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism. DOI: http://dx.doi.org/10.7554/eLife.01211.001 PMID:24151546

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

    PubMed Central

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

    1993-01-01

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

  11. LKB1 loss links serine metabolism to DNA methylation and tumorigenesis.

    PubMed

    Kottakis, Filippos; Nicolay, Brandon N; Roumane, Ahlima; Karnik, Rahul; Gu, Hongcang; Nagle, Julia M; Boukhali, Myriam; Hayward, Michele C; Li, Yvonne Y; Chen, Ting; Liesa, Marc; Hammerman, Peter S; Wong, Kwok Kin; Hayes, D Neil; Shirihai, Orian S; Dyson, Nicholas J; Haas, Wilhelm; Meissner, Alexander; Bardeesy, Nabeel

    2016-11-17

    Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.

  12. Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement

    PubMed Central

    Ortmann, Weronika; Kolaczkowska, Elzbieta

    2016-01-01

    Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates. PMID:27416067

  13. Decrease of mitochondrial p53 during late apoptosis is linked to its dephosphorylation on serine 20

    PubMed Central

    Castrogiovanni, Cédric; Vandaudenard, Marie; Waterschoot, Béranger; De Backer, Olivier; Dumont, Patrick

    2015-01-01

    Following a genotoxic stress, the tumor suppressor p53 translocates to mitochondria to take part in direct induction of apoptosis, via interaction with BCL-2 family members such as BAK and BAX. We determined the kinetics of the mitochondrial translocation of p53 in HCT-116 and PA-1 cells exposed to different genotoxic stresses (doxorubicin, camptothecin, UVB). This analysis revealed an early escalation in the amount of mitochondrial p53, followed by a peak amount and a decrease of mitochondrial p53 at later time points. We show that the serine 20 phosphorylated form of p53 is present at the mitochondria and that the decrease of p53 mitochondrial level during late apoptosis correlates with a decrease of Ser-20 phosphorylation. Moreover, the S20A p53 mutant translocates well to mitochondria after a genotoxic stress but its mitochondrial localization is very low during late apoptosis when compared to wt p53. The S20A mutant also appears to be compromised for interaction with BAK. We propose here that the level of serine 20 phosphorylation is influential on p53 mitochondrial localization during late apoptosis. Additionally, we report the presence of a new ≃45 kDa caspase-cleaved fragment of p53 in the cytosolic and mitochondrial fractions of apoptotic cells. PMID:26252178

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

    PubMed

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

    2016-01-01

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

  15. Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1.

    PubMed

    Hennige, Anita M; Stefan, Norbert; Kapp, Katja; Lehmann, Rainer; Weigert, Cora; Beck, Alexander; Moeschel, Klaus; Mushack, Joanne; Schleicher, Erwin; Häring, Hans-Ulrich

    2006-06-01

    Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine-318 as a protein kinase C (PKC)-dependent phosphorylation site in Irs1 (Ser-318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity-related insulin resistance, promotes phosphorylation of Ser-318 in Irs1 by a janus kinase 2, Irs2, and PKC-dependent pathway. Mutation of Ser-318 to alanine abrogates the inhibitory effect of leptin on insulin-induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser-318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet-induced obesity Ser-318 phosphorylation levels were already up-regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser-318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser-318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser-318 in Irs1 mediates the inhibitory signal of leptin on the insulin-signaling cascade in obese subjects.

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

    Elsea, S.H.; Juyal, R.C.; Jiralerspong, S.

    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.

  18. Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

    PubMed

    Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

    2016-01-01

    Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways.

  19. 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.

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

    SciTech Connect

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

    2016-02-01

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

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

    PubMed

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

    2011-01-01

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

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

    PubMed Central

    Caughey, George H.

    2008-01-01

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

  3. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

    PubMed

    Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

    2012-07-01

    A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis.

  4. Effect of serine protease inhibitors on posttraumatic brain injury and neuronal apoptosis.

    PubMed

    Movsesyan, V A; Yakovlev, A G; Fan, L; Faden, A I

    2001-02-01

    N-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like serine protease (CSP), prevents DNA fragmentation and apoptotic cell death in certain blood cell lines and was reported to reduce hippocampal neuronal damage caused by cerebral ischemia. We examined the role of CSP on recovery after lateral fluid percussion-induced traumatic brain injury (TBI) in rats, as well as on cell survival in various in vitro models of neuronal cell death. TBI caused significant time-dependent upregulation of CSP activity, but not trypsin-like serine protease activity in injured cortex. Intracerebroventricular administration of TPCK to rats after TBI did not significantly affect deficits of spatial learning but exacerbated motor dysfunction after injury. Moreover, TPCK did not prevent apoptotic neuronal cell death caused by serum/K(+) deprivation or by application of staurosporine or etoposide in cultured rat cerebellar granule cells, rat cortical neurons, or in the human neuroblastoma SH-SY5Y cell line. Instead, at doses from 10 to 100 microM, TPCK was cytotoxic in all cultures tested. Similar results were obtained in cultures treated with another CSP inhibitor, 3,4-dichloroisocoumarin. Cell death caused by CSP inhibitors was neither caspase-dependent nor associated with oligonucleosomal DNA fragmentation. Taken together, these data do not support a neuroprotective role for CSP inhibitors. Rather, they suggest that CSPs may serve an endogenous neuroprotective role, possibly by modulating necrotic cell death.

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

    PubMed

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

    2007-12-01

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

  6. Suppression of collagen-induced arthritis with a serine proteinase inhibitor (serpin) derived from myxoma virus.

    PubMed

    Brahn, Ernest; Lee, Sarah; Lucas, Alexandra; McFadden, Grant; Macaulay, Colin

    2014-08-01

    Many viruses encode virulence factors to facilitate their own survival by modulating a host's inflammatory response. One of these factors, secreted from cells infected with myxoma virus, is the serine proteinase inhibitor (serpin) Serp-1. Because Serp-1 had demonstrated anti-inflammatory properties in arterial injury models and viral infections, it was cloned and evaluated for therapeutic efficacy in collagen-induced arthritis (CIA). Clinical severity was significantly lower in the Serp-1 protocols (p<0.0001) and blinded radiographs indicated that the Serp-1 group had significantly less erosions than the controls (p<0.01). Delayed-type hypersensitivity was lower in the Serp-1 group but antibody titers to type II collagen were not significantly altered. Recipients had minimal histopathologic synovial changes and did not develop neutralizing antibodies to Serp-1. These results indicate that Serp-1 impedes the pathogenesis of CIA and suggests that the therapeutic potential of serine proteinase inhibitors in inflammatory joint diseases, such as rheumatoid arthritis, should be investigated further.

  7. Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

    NASA Technical Reports Server (NTRS)

    Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

    2003-01-01

    Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

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

    PubMed Central

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

    1991-01-01

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

  9. Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47.

    PubMed

    Guerra-Castellano, Alejandra; Díaz-Moreno, Irene; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Quintana, Antonio

    2016-04-01

    Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c - namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death - is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.

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

    PubMed Central

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

    2016-01-01

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

  11. Stepwise Versus Concerted Mechanisms in General-Base Catalysis by Serine Proteases.

    PubMed

    Uritsky, Neta; Shokhen, Michael; Albeck, Amnon

    2016-01-26

    General-base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp-His-Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser-His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pKa  values of catalytic residues were QM calculated by the MD-QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pKa  values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand.

  12. Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection

    PubMed Central

    Lawrence, Amba; Fraser, Tamieka; Gillett, Amber; Tyndall, Joel D. A.; Timms, Peter; Polkinghorne, Adam; Huston, Wilhelmina M.

    2016-01-01

    The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas. PMID:27530689

  13. Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis.

    PubMed

    Bouacem, Khelifa; Bouanane-Darenfed, Amel; Laribi-Habchi, Hassiba; Elhoul, Mouna Ben; Hmida-Sayari, Aïda; Hacene, Hocine; Ollivier, Bernard; Fardeau, Marie-Laure; Jaouadi, Bassem; Bejar, Samir

    2015-11-01

    Caldicoprobacter guelmensis isolated from the hydrothermal hot spring of Guelma (Algeria) produced high amounts of extracellular thermostable serine alkaline protease (called SAPCG) (23,000U/mL). The latter was purified by ammonium sulphate precipitation, UNO Q-6 FPLC and Zorbex PSM 300 HPLC, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 55,824.19Da. The 19 N-terminal residue sequence of SAPCG showed high homology with those of microbial proteases. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine protease family. It showed optimum protease activity at pH 10 and 70°C with casein as a substrate. The thermoactivity and thermostability of SAPCG were enhanced in the presence of 2mM Ca(2+). Its half-life times at 80 and 90°C were 180 and 60min, respectively. Interestingly, the SAPCG protease exhibited significant compatibility with iSiS and Persil, and wash performance analysis revealed that it could remove blood-stains effectively. Overall, SAPCG displayed a number of attractive properties that make it a promising candidate for future applications as an additive in detergent formulations.

  14. Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55.

    PubMed

    Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Karutha Pandian, Shunmugiah

    2011-01-01

    The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.

  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.

  16. Inhibition of Aeromonas sobria serine protease (ASP) by α2-macroglobulin.

    PubMed

    Murakami, Yoji; Wada, Yoshihiro; Kobayashi, Hidetomo; Irie, Atsushi; Hasegawa, Makoto; Yamanaka, Hiroyasu; Okamoto, Keinosuke; Eto, Masatoshi; Imamura, Takahisa

    2012-10-01

    ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.

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

    PubMed

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

    1998-06-01

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

  18. Integrated Innate Mechanisms Involved in Airway Allergic Inflammation to the Serine Protease Subtilisin

    PubMed Central

    Florsheim, Esther; Yu, Shuang; Bragatto, Ivan; Faustino, Lucas; Gomes, Eliane; Ramos, Rodrigo N.; Barbuto, José Alexandre M.; Medzhitov, Ruslan; Russo, Momtchilo

    2015-01-01

    Proteases are recognized environmental allergens, but little is known about the mechanisms responsible for sensing enzyme activity and initiating the development of allergic inflammation. Because usage of the serine protease subtilisin in the detergent industry resulted in an outbreak of occupational asthma in workers, we sought to develop an experimental model of allergic lung inflammation to subtilisin and to determine the immunological mechanisms involved in type 2 responses. By using a mouse model of allergic airway disease, we have defined here that subcutaneous or intranasal sensitization followed by airway challenge to subtilisin induces prototypic allergic lung inflammation, characterized by airway eosinophilia, type 2 cytokines release, mucus production, high levels of serum IgE, and airway reactivity. These allergic responses were dependent on subtilisin protease activity, protease-activated receptor (PAR)-2, IL-33 receptor ST2, and MyD88 signaling. Also, subtilisin stimulated the expression of the pro-allergic cytokines IL-1α, IL-33, TSLP, and the growth factor amphiregulin in a human bronchial epithelial cell line. Notably, acute administration of subtilisin into the airways increased lung IL-5-producing type 2 innate lymphoid cells, which required PAR-2 expression. Finally, subtilisin activity acted as a Th2 adjuvant to an unrelated airborne antigen promoting allergic inflammation to inhaled OVA. Therefore, we established a murine model of occupational asthma to a serine protease and characterized the main molecular pathways involved in allergic sensitization to subtilisin that potentially contribute to initiate allergic airway disease. PMID:25876764

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

    PubMed

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

    2013-04-01

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

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

    PubMed

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

    2013-01-01

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