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Sample records for phosphatidyl serine ps

  1. Alteration by phosphatidyl serine of tension responses and 45Ca distribution in aortic smooth muscle.

    PubMed

    Goodman, F R; Weiss, G B; Goth, A

    1976-07-01

    The effects of phosphatidyl serine (PS) on 45Ca distribution, 45Ca movements and contractions were examined in rabbit aortic smooth muscle. Contractile responses to submaximal concentrations of norepinephrine and histamine were potentiated by prior exposure to PS, but equivalent responses to potassium were unaffected. Addition of PS to the incubation solution decreased 45Ca uptake; exposure of aortic strips to PS during washout of either 45Ca or promethium (147Pm) resulted in maintained increases in efflux. These PS-induced alterations in net loss of 45Ca or 147Pm can be attributed to a decreased membrane reuptake and/or rebinding. However, the presence of PS during the washout significantly reduced the increases in 45Ca efflux rate elicited with either 0.05 mM concentrations of Ca++ or ethylenediamine tetraacetic acid. Thus, in rabbit aortic smooth muscle, exogenous PS can alter the availability and/or exchangeability of a membrane-bound Ca++ fraction. By specifically increasing the affinity for Ca++ at relevant membrane sites or stores. PS may enhance the ability of vascular smooth muscle to respond to stimulatory agents that mobilize Ca++ from these sites and, in this manner, potentiate contractile responses. PMID:933004

  2. The interaction of calcium and strontium with phosphatidyl serine in the anaphylactic secretion of histamine

    PubMed Central

    Foreman, J. C.; Mongar, J. L.

    1973-01-01

    1. Spontaneous histamine release from isolated mast cells was found to be independent of calcium in the concentration range up to 1 m-mole/l. Phosphatidyl serine did not change the effect of calcium on spontaneous release. 2. Spontaneous histamine release was found to vary with the strontium ion concentration. Graded increase in the release occurred as the concentration of strontium was raised from 1 to 10 m-mole/l. Phosphatidyl serine potentiated this action of strontium; the potentiation showed a graded increase as the phosphatidyl serine concentration was raised from 1 to 100 μg/ml. 3. The activation of anaphylactic histamine release by calcium was potentiated by phosphatidyl serine; the degree of potentiation showed a graded increase as the calcium concentration was raised from 0·1 to 1·0 m-mole/l. 4. The activation of anaphylactic histamine release by strontium showed little, if any, potentiation by phosphatidyl serine. 5. The response of the mast cells, in terms of anaphylactic histamine release, to calcium, in the presence of optimal concentrations of phosphatidyl serine, was found to be similar to that observed in the presence of strontium alone. 6. These observations are discussed in terms of the concepts of affinity and efficacy of the ions at their receptor sites. PMID:4122664

  3. Structural Basis for Calcium and Phosphatidyl Serine Regulation of PLC δ1

    PubMed Central

    Lomasney, Jon W.; Cheng, Hwei-Fang; Kobayashi, Minae; King, Klim

    2012-01-01

    Many membrane associated enzymes including those of the phospholipase C (PLC) superfamily are regulated by specific interactions with lipids. Previously we have shown that the C2 domain of PLC δ1 is required for phosphatidyl serine (PS) dependent enzyme activation, and that activation requires the presence of Ca2+. To identify the site of interaction and the role of Ca2+ in the activation mechanism, we mutagenized three highly conserved Ca2+ binding residues (Asp 653, Asp-706 and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca2+ binding affinities of the mutant enzymes D653G, D706G and D708G were reduced by an order of magnitude and the maximal Ca2+ binding was reduced to half of that of the native enzyme. The Ca2+ dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca2+ dependence and maximal hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) was not altered in the mutants. However, the Ca2+ dependent PS stimulation was severely defective. PS reduces the Km of the native enzyme almost 20 fold, but far less for the mutants. Replacing Asp-653, Asp-706 and Asp-708 simultaneously to glycine in the C2 domain of PLC δ1, leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, 706 and 708 are required for Ca2+ binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands. PMID:22385159

  4. Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature.

    PubMed

    Shi, Jialan; Heegaard, Christian W; Rasmussen, Jan T; Gilbert, Gary E

    2004-11-17

    Lactadherin, a milk protein, contains discoidin-type lectin domains with homology to the phosphatidylserine-binding domains of blood coagulation factor VIII and factor V. We have found that lactadherin functions, in vitro, as a potent anticoagulant by competing with blood coagulation proteins for phospholipid binding sites [J. Shi and G.E. Gilbert, Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid binding sites, Blood 101 (2003) 2628-2636]. We wished to characterize the membrane-binding properties that correlate to the anticoagulant capacity. We labeled bovine lactadherin with fluorescein and evaluated binding to membranes of composition phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine, 4:20:76 supported by 2 mum diameter glass microspheres. Lactadherin bound saturably with an apparent KD of 3.3+/-0.4 nM in a Ca++ -independent manner. The number of lactadherin binding sites increased proportionally to the phosphatidylserine content over a range 0-2% and less rapidly for higher phosphatidylserine content. Inclusion of phosphatidylethanolamine in phospholipid vesicles did not enhance the apparent affinity or number of lactadherin binding sites. The number of sites was at least 4-fold higher on small unilamellar vesicles than on large unilamellar vesicles, indicating that lactadherin binding is enhanced by membrane curvature. Lactadherin bound to membranes with synthetic dioleoyl phosphatidyl-L-serine but not dioleoyl phosphatidyl-D-serine indicating stereoselective recognition of phosphatidyl-L-serine. We conclude that lactadherin resembles factor VIII and V with stereoselective preference for phosphatidyl-L-serine and preference for highly curved membranes. PMID:15533308

  5. Apoptosis in esophagus and pancreas carcinoma cells induced by circulating microparticles is related to phosphatidyl serine and microparticle-associated caspases.

    PubMed

    Schneider, Jan; Chromik, Ansgar M; Uhl, Waldemar; Mügge, Andreas; Bulut, Daniel

    2012-06-01

    Circulating microparticles (MPs) are recently discussed as "biologically active", participating in the pathology of diseases rather than being a marker of damaging processes. It was the purpose of the present study to investigate the effects of MPs, as isolated from the blood of healthy volunteers, on the induction of apoptosis and necrosis in cultured KYSE-270 esophageal and ASPC1 pancreas carcinoma cells. MPs were obtained from the blood of 20 healthy volunteers (11 women; mean age 33.3 years). Viability, apoptosis, and necrosis were determined by flow cytometry using Annexin V/propidium iodide and tetramethylrhodamine ethyl ester perchlorate (TMRE)/propidium iodide for staining. Incubation of KYSE and ASPC1 carcinoma cells with MPs (1-20.000/μl) for 48 h reduced significantly viability of the cells, and induced apoptosis, but not necrosis. This apoptotic effect was significant at a concentration of ≥1.000 MPs/μl in both cell types. Pre-treatment of MPs with either the global caspase inhibitor ZVAD-FMK or Annexin V which blocks phosphatidyl serine in the outer membrane of MPs with high affinity, almost abolished MP-induced apoptosis. A specific enzyme assay as well Western blot analysis confirmed the presence (activity, protein) of the apoptotic enzyme caspase-3 in MPs. Incubation of carcinoma cells with MPs (20.000/μl) resulted in an increase in caspase-3 protein in carcinoma cells; this increase could be prevented by pre-treatment of MPs with Annexin V. It is suggested that MPs induce concentration-dependent apoptosis in KSYE esophageal and ASPC1 pancreas carcinoma cells in vitro by transferring caspases into target cells. This process probably requires a target cell-MP interaction, and membrane-bound anionic phosphatidyl serine may be involved. PMID:21452043

  6. Interactions between titanium dioxide and phosphatidyl serine-containing liposomes: formation and patterning of supported phospholipid bilayers on the surface of a medically relevant material.

    PubMed

    Rossetti, Fernanda F; Bally, Marta; Michel, Roger; Textor, Marcus; Reviakine, Ilya

    2005-07-01

    Titanium is widely used in biomedical applications. Its mechanical properties and biocompatibility, conferred by a layer of oxide present on its surface, make titanium the material of choice for various implants (artificial hip and knee joints, dental prosthetics, vascular stents, heart valves). Furthermore, the high refractive index of titanium oxide is advantageous in biosensor applications based on optical detection methods. In both of the above fields of application, novel surface modification strategies leading to biointeractive interfaces (that trigger specific responses in biological systems) are continuously sought. In this report, we investigate the interactions between TiO2 and phosphatidyl serine-containing liposomes, present a novel approach for preparing supported phospholipid bilayers (SPBs) of various compositions on TiO2, and use the unique ability of liposomes to distinguish between different surfaces to create SPB corrals on SiO2/TiO2 structured substrates. These results represent an important first step toward the design of biointeractive interfaces on titanium oxide surfaces that are based on a cell membrane-like environment. PMID:15982052

  7. A serine carboxypeptidase gene (PsCP), expressed in early steps of reproductive and vegetative development in Pisum sativum, is induced by gibberellins.

    PubMed

    Cercós, Manuel; Urbez, Cristina; Carbonell, Juan

    2003-01-01

    A cDNA clone encoding a serine carboxypeptidase (PsCP), isolated from young fruits of Pisum sativum L., was used to study the temporal and spatial expression and hormonal regulation of serine carboxypeptidase during reproductive and vegetative development. In unpollinated pea ovaries PsCP transcript levels decreased during senescence. However, during early fruit development, PsCP transcript were accumulated in both pericarp and seeds, preferentially in the nucellus, with a polar distribution at the chalazal region of the embryo sac, suggesting a role in seed development. PsCP transcript levels increased also when fruit set was induced in unpollinated ovaries by gibberellins, although the distribution was uniform. PsCP expression was also induced by auxins but not cytokinins, indicating a selective hormonal regulation of PsCP transcription. Localization of PsCP transcript after pollination parallel reported changes in gibberellin distribution, suggesting that PsCP transcription in developing fruits and seeds is induced by gibberellins. PsCP is also expressed in developing seedlings but not in cotyledons, suggesting that it is not involved in the mobilization of storage materials. PsCP transcripts were suppressed by treatment of seedlings with paclobutrazol and restored by gibberellic acid (GA3) treatment. In addition, PsCP transcript levels decreased in etiolated pea seedlings when they were exposed to continuous light but not when exposed to light in the presence of GA3. These results indicate that PsCP transcript accumulation is induced by gibberellins in developing seedlings. This is the first report of a serine carboxypeptidase-like gene induced by gibberellins in reproductive and vegetative developing tissues in dicotyledoneous plants. PMID:12602875

  8. 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…

  9. 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

  10. PS Corner.

    ERIC Educational Resources Information Center

    Woods, D. R.

    1984-01-01

    Four papers are reviewed. They include a description of how instructional modules and small group problem-solving (PS) in a second-year plant anatomy course produced excellent PS skills and various ideas on PS in the context of chemistry. Also reviews guided-design as a strategy to help develop PS skills. (JN)

  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. PMID:26614404

  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. 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

    2014-10-01

    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. PMID:25255083

  14. Spectrophotometric determination of Sc in eriochrome cyanine R(chrome azurol S) - phosphatidyl choline system

    SciTech Connect

    Xu, Y.; Chen, X.; Hu, Z.

    1987-07-01

    Eriochrome cyanine R(chrome azurol S) is used as a color reagent to determine Sc in the presence of phosphatidyl choline, eta = 3.7 * 10/sup 4/ (4.5 * 10/sup 4/). This method has been connected to extraction separation to determine Sc in the presence of rare earth elements, and good results have been obtained. Phosphatidyl choline(PC) is a biochemical reagent, which can be used as a surfactant. It has been reported that chrome azurol S(CAS) can be used to determine Be in the presence of PC but it has not been reported that eriochrome cyanine R(ECR) and CAS can been used to determine Sc in the presence of PC. This paper has put forward a method by which Sc can be determined. ECR (CAS) has been used as a color reagent and PC as a surfactant. Conditional experiments have been made and this method has been connected to extraction separation. Tributyl phosphate (TBP) extracts Sc from rare earth elements to make a determination and good results have been obtained.

  15. Cloning and characterization of a gene encoding phosphatidyl inositol-specific phospholipase C from Trypanosoma cruzi.

    PubMed

    Nozaki, T; Toh-e, A; Fujii, M; Yagisawa, H; Nakazawa, M; Takeuchi, T

    1999-08-20

    A gene encoding phosphatidyl inositol-4,5-bisphosphate phospholipase C (PLC) was cloned from the protozoan parasite Trypanosoma cruzi. A partial cDNA encoding putative PLC was obtained by a polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved regions of PLCs. A 2178-bp protein coding region of the T. cruzi PLC gene, composed from cDNA and genomic clones, encodes a putative PLC with a calculated molecular mass of 82,032 Da and an isoelectric point of 5.93. The deduced amino acid sequence of T. cruzi PLC exhibited 23-42% overall identities with the PLCs from other organisms. Among them, PLC from Ictalurus punctatus revealed the highest identity to T. cruzi PLC. The percentage identities of the entire proteins and the catalytic X/Y domains suggested that T. cruzi PLC is more evolutionarily related to the PLCs of higher eukaryotes than to those of lower unicellular eukaryotes. The tetrad analysis of the segregants of the Saccharomyces cerevisiae PLC1/plc1::HIS3 diploid strain transformed with the T. cruzi PLC-expressing plasmid showed that expression of T. cruzi PLC suppressed the growth defect caused by the plc1 disruption in yeasts. Temperature-sensitive phenotype of the S. cerevisiae plc1-mutant haploid strain was also suppressed by the expression of T. cruzi PLC. The phosphatidyl inositol-4,5-biphosphate (PtdIns(4,5)P2) hydrolyzing activity of T. cruzi PLC was demonstrated in the lysate from the plc1-temperature sensitive yeast mutant strain transformed with the T. cruzi PLC-expressing plasmid. The yeast-expressed T. cruzi PLC showed an absolute Ca2+ dependence which was similar to mammalian PLC isoforms: the half-maximal activity at 0.5-1 x 10(-5) M Ca2+ and the maximal activity at 1-2 x 10(-4) M Ca2+. PMID:10498184

  16. 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

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

    PubMed

    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

  18. Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

    PubMed Central

    Groves, J T; Wlfing, C; Boxer, S G

    1996-01-01

    Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8913608

  19. 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

  20. 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.

  1. Orchestrating Serine Resolvases

    PubMed Central

    Rice, Phoebe A.; Mouw, Kent. W.; P.Montaño, Sherwin; Boocock, Martin R.; Rowland, Sally-J.; Stark, W. Marshall

    2013-01-01

    A remarkable feature of the Serine resolvases is their regulation: the WT enzymes will catalyze intra- but not intermolecular recombination, can sense the relative orientation of their sites, and can exchange strands directionally despite the fact that there is no net release of chemical bond energy. This key to this regulation is that they are only active within a large, intertwined complex called the “synaptosome.” Because substrate topology greatly facilitates (or, in other cases, inhibits) formation of the synaptosome, it acts as a “topological filter.” Within the defined topology of the synaptosome, strand exchange releases supercoiling tension, providing an energy source to bias the reaction direction. The regulatory portion of this complex contains additional copies of the recombinase and sometimes other DNA bending proteins. We are using a combination of x-ray crystallography, biochemistry and genetics to model the full synaptic complex and to understand how the regulatory portion activates the crossover-site bound recombinases. PMID:20298188

  2. 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

  3. Serine proteases of parasitic helminths.

    PubMed

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

    2015-02-01

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

  4. Photoregulated expression of the PsPK3 and PsPK5 genes in pea seedlings.

    PubMed

    Khanna, R; Lin, X; Watson, J C

    1999-01-01

    The PsPK3 and PsPK5 genes of the garden pea encode protein-serine/threonine kinases whose catalytic domains are closely related to known signal transducing kinases from animals and fungi. The PsPK3 polypeptide is predicted to be located in the nucleus, whereas PsPK5 is a homologue of NPH1, the probable blue light receptor for phototropism from Arabidopsis. We found previously that when etiolated pea seedlings are illuminated with continuous white light, PsPK3 and PsPK5 transcript levels within apical buds decline substantially, reaching their minimum levels within one day of exposure to light. The role of light in regulating the expression of the PsPK3 and PsPK5 genes was investigated further. To gain insight into the rapidity with which expression changes, 6-day old, dark-grown pea seedlings were transferred to continuous white light, and PsPK3 and PsPK5 RNA levels monitored over the ensuing 24 h. While transcripts from the RbcS gene family increase, the PsPK3 and PsPK5 mRNAs decline rapidly to their minimum levels. PsPK5 mRNA declines 10-fold in ca. 2 h, whereas PsPK3 mRNA declines 4-fold in ca. 8 h. We used single pulses of light to elucidate which photoreceptor triggers the negative regulation of PsPK3 and PsPK5 gene expression. To assess phytochrome involvement, etiolated seedlings were treated with single pulses of red light, red followed by far-red light, or far-red light alone. RbcS induction by a red light pulse is reversible with a subsequent far-red light pulse, clearly showing that phytochrome mediates its induction. Likewise, RbcS expression is induced with a single pulse of blue light or a dichromatic pulse of red+blue light. However, none of these pulses trigger the PsPK3 and PsPK5 mRNA levels to decline. Given the lack of effectiveness of light pulses, etiolated seedlings were transferred to continuous light of three different qualities to determine the spectral sensitivity of PsPK3 and PsPK5 gene expression. Exposure to continuous red, continuous far-red, or continuous blue light causes the PsPK3 and PsPK5 mRNAs to decline and transcripts from the RbcS and Cab gene families to increase. One likely explanation is that phytochrome A mediates the responses of these genes to continuous far-red light. The effectiveness of continuous red light and blue light in triggering the reduction in PsPK3 and PsPK5 mRNA levels and the increase in RbcS and Cab mRNAs may imply the participation of additional phytochromes and/or cryptochromes. Thus, the PsPK3 and PsPK5 genes exhibit responsiveness to continuous light, but a lack of responsiveness to single light pulses that is unusual, and perhaps unique, among light-regulated genes. PMID:10080691

  5. 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. PMID:23050313

  6. The PS1 Observatory

    NASA Astrophysics Data System (ADS)

    Kaiser, Nick; Morgan, J.; Pier, E.; Chambers, K.

    2007-12-01

    The Panoramic Survey Telescope and Rapid Response System (Pan-STARRS) will use gigapixel cameras on multi-aperture telescopes to survey the sky in the visible and near-infrared bands. The first surveys will begin in 2008 using a single telescope system (PS1) has been deployed on Haleakala, Maui. This facility is currently undergoing commissioning tests. The PS1 telescope is a 1.8-m f/4 Richey-Chretien design that employs three 50 cm diameter correcting lens. The optical system produces a 3 degree diameter field of view at the focal plane. Images will be recorded on a 1.4 gigapixel CCD camera (described in an accompanying poster presentation). The survey programs will be conducted using g, r, i, and z filters which closely approximate the band-pass and response of those used in the Sloan Digital Sky Survey. These filters will be supplemented with a y band filter further to the infrared of z and a wide w filter for solar system observations. The images from the PS1 camera are supplemented by an Imaging Sky Probe that will provide co-pointed photometric calibration images of each target field. An all-sky camera at the observatory monitors sky conditions and transparency. The operation of the PS1 telescope is supported by the Observatory, Telescope, and Instrument Software (OTIS) system. The OTIS software interfaces the telescope control software provided by the vendor and the CCD camera computer systems. OTIS also records and archives environmental metadata from the dome and the observatory weather station.

  7. 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.

  8. Association of anti phosphatidyl ethanolamine antibodies and low complement levels in systemic sclerosis patients--results of a cross-sectional study.

    PubMed

    Bălănescu, Paul; Lădaru, Anca; Bălănescu, Eugenia; Băicuş, Cristian; Dan, Gheorghe Andrei

    2015-10-01

    Some systemic sclerosis (Ssc) patients express antiphospholipid antibodies and their percentage varies within studies in the literature. The particular role of these antibodies in clinical manifestations of Ssc is still unknown. The aim of the study was to examine an extended panel of antiphospholipid antibodies in Ssc patients who did not have any clinical features of antiphospholipid antibody syndrome. A cross-sectional study was designed and 36 consecutive patients with Ssc were recruited. A relatively high proportion of patients (14 patients - 38.9%) had antiphospholipid antibody presence. Most Ssc patients (11 patients - 30.6%) had IgM anti phosphatidyl ethanolamine antibodies. Serum IgM anti phosphatidyl ethanolamine antibodies, IgM anti prothrombin and IgG anti β2 glycoprotein 1 antibodies were associated with low complement levels in Ssc patients. In multivariate analysis, only serum IgM anti phosphatidyl ethanolamine antibodies concentration and serum IgG anti β2 glycoprotein 1 antibodies concentration were independently associated with hypocomplementemia after adjusting for age and gender. No other correlations with Ssc clinical characteristics were found. In conclusion, antiphospholipid antibodies are present in a large proportion of Ssc patients who do not have clinical features or a history of antiphospholipid antibodies. IgM anti phosphatidyl ethanolamine antibodies seem to be more frequent and the dominant antiphospholipid antibody type in the group recruited from the Romanian Ssc population. PMID:26067612

  9. 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

  10. BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma

    PubMed Central

    Speranza, Maria-Carmela; Nowicki, Michal O.; Behera, Prajna; Cho, Choi-Fong; Chiocca, E. Antonio; Lawler, Sean E.

    2016-01-01

    Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma. PMID:26846842

  11. 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.

  12. Serine and glycine metabolism in cancer☆

    PubMed Central

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

    2014-01-01

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

  13. 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. PMID:24924182

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

    PubMed

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

    2016-01-01

    D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating that D-serine effects are mediated through postsynaptic NMDARs. Conversely, exogenous application of glycine has no such effects, suggesting D-serine, rather than glycine, modulates postsynaptic events. Taken together, our findings demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development, implicating D-serine/SR as regulators of cortical synaptic and circuit development. PMID:26941605

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

    PubMed Central

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

    2016-01-01

    D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating that D-serine effects are mediated through postsynaptic NMDARs. Conversely, exogenous application of glycine has no such effects, suggesting D-serine, rather than glycine, modulates postsynaptic events. Taken together, our findings demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development, implicating D-serine/SR as regulators of cortical synaptic and circuit development. PMID:26941605

  16. Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation.

    PubMed Central

    Elabbadi, N; Ancelin, M L; Vial, H J

    1997-01-01

    Erythrocytes infected with Plasmodium falciparum or Plasmodium knowlesi efficiently incorporated radioactive serine into phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Serine was also metabolized into ethanolamine (Etn) and phosphorylethanolamine (P-Etn) via direct serine decarboxylation; this is a major phenomenon since together these metabolites represent 60% of total radioactive water-soluble metabolites. They were identified by reverse-phase HPLC and two TLC-type analyses and confirmed by alkaline phosphatase treatment, which depleted the radioactive P-Etn peak completely with a concomitant increase in that of Etn. In the presence of 5 microM labelled serine, radioactivity appeared in Etn and P-Etn after a 25 min lag period, and isotopic equilibrium was reached at 40 and 95 min respectively. There was a similar lag period for PtdEtn formation, which accumulated steadily for at least 180 min. Incorporation of serine into phospholipids and water-soluble metabolites increased in the presence of up to 500 microM external serine. An apparent plateau was then reached for all metabolites except intracellular serine and Etn. Exogenous Etn (at 20 microM) induced a concomitant dramatic decrease in serine incorporation into P-Etn and all phospholipids, but not into Etn. Increasing exogenous serine to 100 microM decreased the incorporation of radioactive Etn into PtdEtn by only 30%, and the PtdCho level was not affected. 2-Hydroxyethylhydrazine significantly decreased serine incorporation into P-Etn and PtdEtn, whereas Etn was accumulated. No concomitant inhibition of PtdSer or PtdCho labelling from serine occurred, even when PtdEtn formation was decreased by 95%. This indicates that the PtdEtn pool derived from direct serine decarboxylation differed from that derived from PtdSer decarboxylation, and the latter appeared to be preferentially used for PtdCho biosynthesis. Hydroxylamine also inhibited phosphorylation of serine-derived Etn but not that of exogenous Etn. The rate of PtdSer synthesis from 10 microM L-serine was 3.1+/-0.5 and 2.95+/-1.3 nmol/5 h per 10(10) infected cells, whereas L-serine decarboxylation accounted for 7.1+/-1.5 and 9.9+/-3 nmol/5 h per 10(10) infected cells for P. falciparum and P. knowlesi respectively (means+/-S.E.M.). The serine decarboxylating reaction was not detected in other higher eukaryotic cells such as mouse fibroblasts and human lymphocytes. Finally, these results also indicate compartmentalization of phospholipid metabolism in Plasmodium-infected erythrocytes. PMID:9182701

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

    PubMed Central

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

    2015-01-01

    Neuronal insulin signaling abnormalities have been associated with Alzheimer's disease (AD). However, the specificity of this association and its underlying mechanisms have been unclear. This study investigated the expression of abnormal serine phosphorylation of insulin receptor substrate 1 (IRS1) in 157 human brain autopsy cases that included AD, tauopathies, α-synucleinopathies, TDP-43 proteinopathies, and normal aging. IRS1-pS616, IRS1-pS312 and downstream target Akt-pS473 measures were most elevated in AD but were also significantly increased in the tauopathies: Pick's disease, corticobasal degeneration and progressive supranuclear palsy. Double immunofluorescence labeling showed frequent co-expression of IRS1-pS616 with pathologic tau in neurons and dystrophic neurites. To further investigate an association between tau and abnormal serine phosphorylation of IRS1, we examined the presence of abnormal IRS1-pS616 expression in pathological tau-expressing transgenic mice and demonstrated that abnormal IRS1-pS616 frequently co-localizes in tangle-bearing neurons. Conversely, we observed increased levels of hyperphosphorylated tau in the high-fat diet-fed mouse, a model of insulin resistance. These results provide confirmation and specificity that abnormal phosphorylation of IRS1 is a pathological feature of AD and other tauopathies, and provide support for an association between insulin resistance and abnormal tau as well as amyloid-β. PMID:25107476

  18. Pharmacological Activities and Hydrolysis by Peptidases of [Phospho-Ser(6)]-Bradykinin (pS(6)-BK).

    PubMed

    Assis, Diego M; Juliano, Luiz; Paschoalin, Thaysa; Kouyoumdjian, Maria; Calixto, Joao B; Santos, Robson A S; Pertinhez, Thelma A; Gauthier, Francis; Moreau, Thierry; Blaber, Michael; Juliano, Maria A

    2015-09-15

    Phosphorylated kininogen and some of its fragments containing serine phosphorylated bradykinin ([pS(6)]-Bk) were identified in human serum and plasma by a phosphoproteomic approach. We report the kininogenase ability of human tissue and plasma kallikreins and tryptase to generate [pS(6)]-Bk or Lys-[pS(6)]-Bk having as substrate the synthetic human kininogen fluorescent fragment Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2. The pharmacological assays of [pS(6)]-Bk showed it as a full B2 bradykinin receptor agonist in smooth muscle, it produces a portal liver hypertensive response in rat and mouse paw edema that lasts longer than Bk. The rat hypotensive response to infusions of Bk is greater than that of [pS(6)]Bk, both if injected through femoral vein or aorta. [pS(6)]-Bk was more resistant than Bk to kininase digestion performed with angiotensin converting enzyme, neprilysin, thimet oligopeptidase, aminopeptidase P and carboxypeptidase M. (1)H-NMR experiments indicated that [pS(6)]-Bk has lower flexibility, with the pS(6)-P(7) bond restricted to the trans conformation, and can explain [pS(6)]-Bk resistance to hydrolysis. In conclusion, [pS(6)]-Bk presenting lower activity than Bk, with longer lasting effects and being slowly released by kininogenases from synthetic Abz-MISLMKRPPGF[pS(386)]PFRSSRI-NH2, suggests that phosphorylation of the kininogens can be an efficient kallikrein-kinin system regulator. PMID:26235942

  19. Serine:glyoxylate aminotransferase mutant of barley

    SciTech Connect

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

    1987-08-01

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

  20. Serine hydrolase targets of organophosphorus toxicants.

    PubMed

    Casida, John E; Quistad, Gary B

    2005-12-15

    Acetylcholinesterase (AChE) is one of several hundred serine hydrolases in people potentially exposed to about 80 organophosphorus (OP) compounds important as insecticides or chemical warfare agents. The toxicology of OPs was interpreted until recently almost solely on the basis of AChE inhibition. It is assumed that each serine hydrolase has a specific function and proposed that every OP compound has a unique inhibitory profile. This review considers the progress in sifting the expanding list of potential serine hydrolase toxicological targets. About 50 serine hydrolase targets have been recognized but only a few studied thoroughly. The toxicological relevance of known secondary OP targets is established mainly from observations with humans (butyrylcholinesterase and neuropathy target esterase-lysophospholipase) and studies with mice (cannabinoid CB1 receptor, carboxylesterase, lysophospholipase and platelet activating factor acetylhydrolase) and hen eggs (arylformamidase or kynurenine formamidase). Pesticides most commonly shown to inhibit these targets in experimental vertebrates are chlorpyrifos and tribufos. Generally the levels of environmental and occupational OP pesticide exposure are well below those causing in vivo inhibition of secondary serine hydrolase targets. Although exposure to OP insecticides is decreasing from stricter regulations and the development of resistant pest strains, it will continue to some degree for decades in the future. Only two OPs are used as pharmaceuticals, i.e. echothiophate as an ophthalmic for treatment of glaucoma and metrifonate as an anthelmintic for Schistosoma (and formerly as a candidate drug for improved cognitive function in Alzheimer's disease). In safety evaluations, knowledge on known OP targets must be balanced against major gaps in current understanding since more than 75% of the serine hydrolases are essentially unknown as to OP targeting and relevance, i.e. it is not clear if they play a role in OP toxicology. PMID:16243304

  1. 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.

  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. PMID:24082071

  3. Revealing the multiple structures of serine.

    PubMed

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

    2007-12-18

    We explored the conformational landscape of the proteinogenic amino acid serine [CH(2)OH CH(NH(2)) 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

  4. 21 CFR 582.5701 - Serine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  5. Cross-talk between diverse serine integrases

    PubMed Central

    Singh, Shweta; Rockenbach, Kate; Dedrick, Rebekah M.; VanDemark, Andrew

    2013-01-01

    Phage-encoded serine-integrases are large serine-recombinases that mediate integrative and excisive site-specific recombination of temperate phage genomes. They are well suited for use in heterologous systems and for synthetic genetic circuits as the attP and attB attachment sites are small (<50 bp), there are no host factor or DNA supercoiling requirements, and they are strongly directional, doing only excisive recombination in the presence of a recombination directionality factor. Combining different recombinases that function independently and without cross-talk to construct complex synthetic circuits is desirable, and several different serine-integrases are available. However, we show here that these functions are not reliably predictable, and we describe a pair of serine-integrases encoded by mycobacteriophages Bxz2 and Peaches with unusual and unpredictable specificities. The Integrases share only 59% amino acid sequence identity and the attP sites have fewer than 50% shared bases, but they use the same attB site and there is non-reciprocal cross-talk between the two systems. The DNA binding specificities do not result from differences in specific DNA contacts, but from the constraints imposed by the configuration of the component half-sites within each of the attachment site DNAs. PMID:24161951

  6. 21 CFR 582.5701 - Serine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Serine. 582.5701 Section 582.5701 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  7. 21 CFR 582.5701 - Serine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Serine. 582.5701 Section 582.5701 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  8. 21 CFR 582.5701 - Serine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  9. Topoisomerase-I PS506 as a Dual Function Cancer Biomarker

    PubMed Central

    Zhao, Ming; Gjerset, Ruth A.

    2015-01-01

    Novel biomarkers for cancer diagnosis and therapy selection are urgently needed to facilitate early detection and improve therapy outcomes. We have previously identified a novel phosphorylation site at serine 506 (PS506) on topoisomerase-I (topo-I) and have shown that it is widely expressed in cell lines derived from several cancers, including lung cancer, but is low in cell lines derived from non-cancerous tissues. Here we have investigated how PS506 expression in lung tissue specimens correlates with their malignant status. We find that PS506 expression is significantly elevated in malignant tumors of non-small cell lung cancer (NSCLC) compared to adjacent, non-cancerous lung tissue and benign lung tumors. PS506 expression was up to 6-fold higher in malignant specimens than in paired non-malignant tissue. Using the well-characterized NIH/NCI 60-cell line panel, we correlate the most elevated expression levels of PS506 in lung, ovarian, and colon cancer cells lines with increased sensitivity to camptothecin, a plant alkaloid that targets topo-I. This is consistent with our earlier studies in a smaller sampling of cell lines and with our finding that PS506 increases topo-I DNA binding. Two widely used chemotherapeutic drugs for ovarian and colon cancer, topotecan and irinotecan, respectively, are derived from camptothecin. Irinotecan has also displayed efficacy in clinical trials of NSCLC. Our results suggest that elevated PS506 expression may correlate with clinical chemosensitivity to these agents in ovarian, colon, and NSCLC. PS506 may therefore serve as a biomarker for diagnosis or therapy selection. PMID:26248194

  10. 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).

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

    PubMed Central

    2013-01-01

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

  12. HSAN1 mutations in serine palmitoyltransferase reveal a close structure-function-phenotype relationship.

    PubMed

    Bode, Heiko; Bourquin, Florence; Suriyanarayanan, Saranya; Wei, Yu; Alecu, Irina; Othman, Alaa; Von Eckardstein, Arnold; Hornemann, Thorsten

    2016-03-01

    Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from l-serine to l-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mildand severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile. PMID:26681808

  13. Polymerization of serine guanylate in the presence of montmorillonite

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1981-01-01

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

  14. Role of the serine-rich surface glycoprotein GspB of Streptococcus gordonii in the pathogenesis of infective endocarditis.

    PubMed

    Xiong, Yan Q; Bensing, Barbara A; Bayer, Arnold S; Chambers, Henry F; Sullam, Paul M

    2008-10-01

    The direct binding of bacteria to platelets is a central interaction in the pathogenesis of infective endocarditis. GspB is a serine-rich, cell wall glycoprotein of Streptococcus gordonii that mediates the binding of this organism to human platelets in vitro. To assess the contribution of this adhesin to the pathogenesis of endocarditis, we compared the virulence of S. gordonii M99 (which expresses GspB) with an isogenic, gspB mutant (PS846) in two rat models of endovascular infection. In the first group of experiments, animals were infected intravenously with M99 or PS846, and sacrificed 72 h later, to assess levels of bacteria within cardiac vegetations, kidneys, and spleens. When inoculated with 10(5)CFU, rats infected with PS846 had significantly lower densities of organisms within vegetations (mean: 3.84 log(10)CFU/g) as compared with M99-infected rats (6.67 log(10)CFU/g; P<0.001). Marked differences were also seen in rats co-infected with M99 and PS846, at a 1:1 ratio. While M99 was found at high levels within vegetations, kidneys and spleens (mean log(10)CFU/g: 6.62, 5.07 and 4.18, respectively) PS846 was not detected within these tissues. Thus, platelet binding by GspB appears to be a major interaction in the pathogenesis of endocarditis due to S. gordonii. PMID:18656529

  15. PP/PS anisotropic stereotomography

    NASA Astrophysics Data System (ADS)

    Nag, Steinar; Alerini, Mathias; Ursin, Bjørn

    2010-04-01

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

  16. Parallel software simulation using PS-nets

    SciTech Connect

    Markov, N.G.; Miroshnichenko, E.A.; Saraikin, A.V.

    1995-09-01

    The requirements of techniques for parallel software simulation are discussed. According to these requirements, techniques on the basis of PS-nets are suggested. The fundamentals of program system modeling by PS-nets are given. The tools developed for modeling are described.

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

    NASA Technical Reports Server (NTRS)

    1938-01-01

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

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

    NASA Technical Reports Server (NTRS)

    1938-01-01

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

  19. Inhibitors and inactivators of serine proteases

    SciTech Connect

    Abeles, R.H.

    1986-05-01

    Inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone results in the formation of a covalent adduct in which the ..gamma..-oxygen of serine-195 is covalently attached to C-1 of (Z)-2-benzyl-pentenedioic acid. The carboxylate group of the inhibitor forms a salt bridge with histidine-57 and this prevents access of water to the active site. The structure was established through NMR and x-ray diffraction analysis at 1.9 A resolution. Fluoromethyl-ketones are inhibitors of chymotrypsin and pancreatic elastase. /sup 19/F NMR shows that the carbonyl-carbon of the enzyme bound inhibitor is tetrahedral, most probably, due to adduct formation with the active site serine. The efficacy of these inhibitors increases with the number of fluorine substituents and with increasing length of the peptide. For the elastase inhibitor AcProAlaCF/sub 3/ K/sub i/ = 3 x 10/sup -3/ M and for AcAlaAlaProAlaCF/sub 3/ K/sub i/ = 0.34 x 10/sup -6/ M. For the tetrapeptide k/sub on/ = 290 s/sup -1/ M/sup -1/. The lowering of K/sub i/ concomitant with structural change correlates well with the variation in V/K for the corresponding substrates. AcLeuPheCFH/sub 2/ is a weak inhibitor of chymotrypsin, K/sub i/ = 0.28 x 10/sup -3/ M, which irreversibly inactivates the enzyme by alkylating a histidine.

  20. 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. PMID:25986490

  1. 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.

  2. Neonatal Disruption of Serine Racemase Causes Schizophrenia-Like Behavioral Abnormalities in Adulthood: Clinical Rescue by D-Serine

    PubMed Central

    Hagiwara, Hiroko; Iyo, Masaomi; Hashimoto, Kenji

    2013-01-01

    Background D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA) receptor, is synthesized from L-serine by serine racemase (SRR). Given the role of D-serine in both neurodevelopment and the pathophysiology of schizophrenia, we examined whether neonatal disruption of D-serine synthesis by SRR inhibition could induce behavioral abnormalities relevant to schizophrenia, in later life. Methodology/Principal Findings Neonatal mice (7–9 days) were injected with vehicle or phenazine methosulfate (Met-Phen: 3 mg/kg/day), an SRR inhibitor. Behavioral evaluations, such as spontaneous locomotion, novel object recognition test (NORT), and prepulse inhibition (PPI) were performed at juvenile (5–6 weeks old) and adult (10–12 weeks old) stages. In addition, we tested the effects of D-serine on PPI deficits in adult mice after neonatal Met-Phen exposure. Finally, we assessed whether D-serine could prevent the onset of schizophrenia-like behavior in these mice. Neonatal Met-Phen treatment reduced D-serine levels in the brain, 24 hours after the final dose. Additionally, this treatment caused behavioral abnormalities relevant to prodromal symptoms in juveniles and to schizophrenia in adults. A single dose of D-serine improved PPI deficits in adult mice. Interestingly, chronic administration of D-serine (900 mg/kg/day from P35 to P70) significantly prevented the onset of PPI deficits after neonatal Met-Phen exposure. Conclusions/Significance This study shows that disruption of D-serine synthesis during developmental stages leads to behavioral abnormalities relevant to prodromal symptoms and schizophrenia, in later life. Furthermore, early pharmacological intervention with D-serine may prevent the onset of psychosis in adult. PMID:23630632

  3. Interactions of Streptomyces serine-protease inhibitors with Streptomyces griseus metalloendopeptidase II.

    PubMed

    Kajiwara, K; Fujita, A; Tsuyuki, H; Kumazaki, T; Ishii, S

    1991-09-01

    Streptomyces griseus metalloendopeptidase II (SGMPII) was shown to form tight complexes with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites. PMID:1769961

  4. Effect of Serine Hydroxamate on Phospholipid Synthesis in Escherichia coli

    PubMed Central

    Pizer, Lewis I.; Merlie, John P.

    1973-01-01

    Serine hydroxamate, which inhibits the charging of seryl-transfer ribonucleic acid, reduced the synthesis of phospholipid and nucleic acids in Escherichia coli. This effect was analogous to depriving amino acid auxotrophs of their nutritional requirement and appears to be a manifestation of the stringent response shown by rel+ strains of E. coli. Amino acid starvation (serine or methionine) alone or serine hydroxamate treatment alone results in 60 to 80% inhibition of lipid accumulation, 90% inhibition of ribonucleic acid accumulation, and an increase in guanosine tetraphosphate (ppGpp). These three effects were reversed by addition of chloramphenicol (CM). A combination of serine starvation and serine hydroxamate treatment resulted in inhibition of lipid and RNA accumulation as well as an increase in ppGpp, but the consequences of the double block were not reversed by CM. We conclude that a strong interrelationship exists among these processes and that CM acts to relax a stringent response by mechanisms other than interference with ppGpp formation. All species of phospholipid were affected by a stringent response evoked by amino acid starvation or addition of serine hydroxamate, but in all cases the synthesis of phosphatidylethanolamine was most severely inhibited. Serine hydroxamate was not incorporated into lipid but specifically caused phosphatidylserine accumulation. Serine starvation produced a dramatic alteration of the distribution of isotope incorporated into phospholipid, which resulted from the stringent response compounded with the limitation of a substrate for phosphatidylserine synthesis. Images PMID:4576413

  5. On the phenotypic spectrum of serine biosynthesis defects.

    PubMed

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

    2016-05-01

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

  6. Serine catabolism regulates mitochondrial redox control during hypoxia

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  8. 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.

  9. 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

  10. Asiago spectroscopic classification of PS15cwx and PS15cwo

    NASA Astrophysics Data System (ADS)

    Tartaglia, L.; Pastorello, A.; Benetti, S.; Cappellaro, E.; Elias-Rosa, N.; Ochner, P.; Terreran, G.; Tomasella, L.; Turatto, M.

    2015-11-01

    The Asiago Transient Classification Program (Tomasella et al. 2014, AN, 335, 841) reports the spectroscopic classifications of PS15cwx in 2MFGC 04279 and PS15cwo in SDSS J031116.60+011124.3 The targets were supplied by the Pan-STARRS Survey for Transients (see Huber et al., ATel #7153).

  11. 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.

  12. Astrocytes release D-serine by a large vesicle

    PubMed Central

    Kang, Ning; Peng, Hong; Yu, Yufei; Stanton, Patric K.; Guilarte, Tomás R.; Kang, Jian

    2015-01-01

    Long-term potentiation (LTP) of synaptic transmission in the CA1 region of the hippocampus depends on activation of N-methyl-D-aspartate receptors (NMDARs), which can be regulated by Ca2+-dependent release of D-serine from astrocytes. The detailed mechanism underlying astrocytic D-serine release is still unknown. In this study, we found that clamping astrocytic [Ca2+] at 100-150 nM or puffing artificial cerebrospinal fluid (ACSF) into the extracellular space (weak mechanical stimulation) enhanced synaptic activation of NMDARs. The enhancement was blocked by the NMDAR glycine site antagonist DCKA, glycine saturation, and infusion of astrocytes with D-Amino Acid Oxidase (DAAO) and the serine racemase inhibitor L-erythro-3-hydroxyaspartate (HoAsp), suggesting the involvement of astrocytic D-serine release. Intracellular 100-150 nM [Ca2+] or puffing ACSF stimulated astrocytes to generate D-serine-containing large vesicles (1-3 μm), exocytotic fusion of which released D-serine. The formation of astrocytic large vesicles involved intracellular fusion of small vesicles and/or other organelles. Spontaneous fusion of large vesicles occurred occasionally in astrocytes at rest, contributing to baseline D-serine levels, which increased the rising slope of NMDAR post-burst potentiation (PBP) without altering the PBP peak amplitude. Thus, under physiological conditions, astrocytic D-serine release by large vesicles facilitated weak theta-burst (TBS consisting of 5 bursts), but not strong TBS (TBS consisting of 10 bursts) stimulation-induced LTP. PMID:23485803

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

    PubMed

    Montesinos Guevara, Camila; Mani, Ali R

    2016-06-01

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

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

    PubMed

    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 D1(Thr286) 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

  15. 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

  16. Phospholipid Composition and Metabolism of Micrococcus denitrificans

    PubMed Central

    Wilkinson, Brian J.; Morman, Manuel R.; White, David C.

    1972-01-01

    The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of 32P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of 32P or acetate-1-14C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found. Images PMID:4640503

  17. 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...

  18. 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

  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. Resource Letter PS-2: Physics of Sports

    NASA Astrophysics Data System (ADS)

    Frohlich, Cliff

    2011-06-01

    This Resource Letter provides a guide to the literature on the physics of sports, updating Resource Letter PS-1, published 25 years ago (Ref. 17). The intent is to suggest literature for anyone curious about the basic physics of particular sports, for physics teachers searching for sports examples to augment their teaching, and for physicists contemplating research on unsolved sports-related questions.

  1. 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…

  2. 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.

  3. 10th Anniversary P.S.

    ScienceCinema

    None

    2011-04-25

    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.....)

  4. Asiago spectroscopic classification of PS15cyk

    NASA Astrophysics Data System (ADS)

    Tartaglia, L.; Pastorello, A.; Benetti, S.; Cappellaro, E.; Elias-Rosa, N.; Ochner, P.; Terreran, G.; Tomasella, L.; Turatto, M.

    2015-12-01

    The Asiago Transient Classification Program (Tomasella et al. 2014, AN, 335, 841) reports the spectroscopic classification of PS15cyk in UGC 12687. The target is supplied by the Pan-STARRS Survey for Transients (see Huber et al., ATel #7153).

  5. Beyond metric gravity: Progress on PS-200

    SciTech Connect

    Goldman, T.; Brown, R.E.; Camp, J.B.; Darling, T.; Dyer, P.; Holzscheiter, M.H.; Hughes, R.J.; Jarmie, N.; King, N.S.P.; Lizon, D.C.; Nieto, M.M.; Schauer, M.M.M.; Schecker, J.A.; Cornford, S.; Hosea, K.; Kenefick, R.A.; Hoibraaten, S.; Midzor, M.M.; Parry, S.P.; Ristenen, R.A.; Witteborn, F.C.; Rochet, J.

    1993-03-01

    The reconciliation of quantum mechanics and gravity on varying distance scales requires changes to General Relativity that may be testable implications. We briefly review the status of tests with matter of the inverse square law and the principle of equivalence, then report on progress on the drift-tube measurement section of PS- 200, the experiment to measure the gravitational acceleration of antiprotons.

  6. Beyond metric gravity: Progress on PS-200

    SciTech Connect

    Goldman, T.; Brown, R.E.; Camp, J.B.; Darling, T.; Dyer, P.; Holzscheiter, M.H.; Hughes, R.J.; Jarmie, N.; King, N.S.P.; Lizon, D.C.; Nieto, M.M.; Schauer, M.M.M.; Schecker, J.A. ); Cornford, S.; Hosea, K.; Kenefick, R.A. ); Hoibraaten, S.; Midzor, M.M.; Parry, S.P.; Ristenen, R.A. (Colorado Univ., Boulder, CO (U

    1993-01-01

    The reconciliation of quantum mechanics and gravity on varying distance scales requires changes to General Relativity that may be testable implications. We briefly review the status of tests with matter of the inverse square law and the principle of equivalence, then report on progress on the drift-tube measurement section of PS- 200, the experiment to measure the gravitational acceleration of antiprotons.

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

    PubMed Central

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

    1980-01-01

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

  8. The Study of Interpenetration Length between dPS Films and PS-grafted Layers

    NASA Astrophysics Data System (ADS)

    Lee, Hoyeon; Jo, Seongjun; Hirata, Toyoaki; Yamada, Norifumi L.; Tanaka, Keiji; Ryu, Du Yeol

    In polymer thin film system, the type of interfacial interaction is a critical parameter to determining the thermal and physical properties of polymer films. Interestingly, the interfacial energy of grafted substrates with polymer chains is remarkably altered by simply controlling grafting density, which has been referred to as autophobicity. In this study, we investigated the interpenetrating interfaces between deuterated polystyrene (dPS) and grafted substrates with the same chemical identity. PS-grafted substrates were prepared using a grafting-to approach with hydroxyl end-functionalized polystyrene (PSOH) in a dry brush regime, where the brush thickness and grafting density were determined based on the chain length (or molecular weight, Mn) of PSOHs. The interpenetration lengths (ξ) at interfaces between dPS and PS-grafted layers were characterized using neutron reflectivity (NR) measurements (performed at the SOFIA beam-line at J-PARC, Japan). Academic adviser.

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

    PubMed Central

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

    2013-01-01

    Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells (PAEC) to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H2O2) levels. These changes correlated with an increase in serine phosphorylated catalase. Using the inhibitory peptide δV1.1, this phosphorylation was shown to be PKCδ dependent. Mass spectrometry identified serine167 as the phosphorylation site. Site-directed mutagenesis was used to generate a phospho-mimic (S167D) catalase. Activity assays using recombinant protein purified from E.coli or transiently transfected COS-7 cells, demonstrated that S167D-catalase had an increased ability to degrade H2O2 compared to the wildtype enzyme. Using a phospho-specific antibody, we were able to verify that pS167 catalase levels are modulated in lambs with acute increases in PBF in the presence and absence of the ET receptor antagonist, tezosentan. S167 is being located on the dimeric interface suggesting it could be involved in regulating the formation of catalase tetramers. To evaluate this possibility we utilized analytical gel-filtration to examine the multimeric structure of recombinant wildtype- and S167D-catalase. We found that recombinant wildtype catalase was present as a mixture of monomers and dimers while S167D catalase was primarily tetrameric. Further, the incubation of wildtype catalase with PKCδ was sufficient to convert wildtype catalase into a tetrameric structure. In conclusion, this is the first report indicating that the phosphorylation of catalase regulates its multimeric structure and activity. PMID:24211614

  10. 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 interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. Conclusion These data show that a serine protease present in the Premolis semirufa’s bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation. PMID:25760458

  11. Participation of d-serine in the development and reproduction of the silkworm Bombyx mori.

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2014-05-26

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

  13. An application of diagonal electrophoresis to the selective purification of serine phosphate peptides. Serine phosphate peptides from ovalbumin

    PubMed Central

    Milstein, Celia P.

    1968-01-01

    A diagonal-electrophoresis method for the selective purification of serine phosphate peptides was applied to tryptic, chymotryptic and peptic digests of oxidized ovalbumin. This method is based on the release of the phosphate group bound to serine by treatment with alkaline phosphatase on paper. The identified serine phosphate peptides were purified by paper electrophoresis at pH65 and 20, dephosphorylation with bacterial alkaline phosphatase, and paper electrophoresis at pH20 again, in that order. The presence of two groups of serine phosphate peptides was apparent from the amino acid composition. One group contained no lysine, cysteic acid, proline, leucine or isoleucine (sequence 1) and the other had all those amino acids (sequence 2). Further degradation with subtilisin of those peptides and `dansyl'Edman sequence analysis established their partial sequences. The proposed sequences are as follows (with `SerP' representing serine phosphate): sequence 1, -Ala-Gly-Arg-Glu-Val-Val-Gly-SerP-Ala-Glu-Ala-Gly-Asp-Val-Ala-Ala-Ser-(Val,Glx2,Ser,Phe)-Arg-; sequence 2, -Asp-Lys-Leu-Pro-Gly-Phe-Gly-Asp-SerP-Ile-Glx-Ala-Glx-CySO3H-Gly-(Thr,Ser,Val)-(Asp,His,Val)-. The partial sequence of one of the phosphopeptides, Asp-(Glu,Ile,SerP), reported by Flavin (1954) was used to establish the proposed sequence 2. PMID:4881141

  14. Spectroscopic Classification of PS16ccj with Mayall/KOSMOS

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

    We report the classification of PS16ccj from spectroscopic observation with KOSMOS on the Mayall telescope. The observation was made on 2016 May 05 UT. We classify PS16ccj as a SN Ia near maximum light.

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

    PubMed Central

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

    2012-01-01

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

  16. 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

  17. 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. PMID:26883430

  18. 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...

  19. A conserved extraordinarily long serine homopolymer in Dictyostelid amoebae

    PubMed Central

    Tian, X; Strassmann, J E; Queller, D C

    2014-01-01

    Eukaryotic protein sequences often contain amino-acid homopolymers that consist of a single amino acid repeated from several to dozens of times. Some of these are functional but others may persist largely because of high expansion rates due to DNA slippage. However, very long homopolymers with over a hundred repeats are very rare. We report an extraordinarily long homopolymer consisting of 306 tandem serine repeats from the single-celled eukaryote Dictyostelium discoideum, which also has a multicellular stage. The gene has a paralog with 132 repeats and orthologs, also with high serine repeat numbers, in various other Dictyostelid species. The conserved gene structure and protein sequences suggest that the homopolymer is functional. The high codon diversity and very poor alignment of serine codons in this gene between species similarly indicate functionality. This is because the serine homopolymer is conserved despite much DNA sequence change. A survey of other very long amino-acid homopolymers in eukaryotes shows that high codon diversity is the rule, suggesting that these too may be functional. PMID:24084645

  20. Protein targets of inflammatory serine proteases and cardiovascular disease

    PubMed Central

    2010-01-01

    Serine proteases are a key component of the inflammatory response as they are discharged from activated leukocytes and mast cells or generated through the coagulation cascade. Their enzymatic activity plays a major role in the body's defense mechanisms but it has also an impact on vascular homeostasis and tissue remodeling. Here we focus on the biological role of serine proteases in the context of cardiovascular disease and their mechanism(s) of action in determining specific vascular and tissue phenotypes. Protease-activated receptors (PARs) mediate serine protease effects; however, these proteases also exert a number of biological activities independent of PARs as they target specific protein substrates implicated in vascular remodeling and the development of cardiovascular disease thus controlling their activities. In this review both PAR-dependent and -independent mechanisms of action of serine proteases are discussed for their relevance to vascular homeostasis and structural/functional alterations of the cardiovascular system. The elucidation of these mechanisms will lead to a better understanding of the molecular forces that control vascular and tissue homeostasis and to effective preventative and therapeutic approaches. PMID:20804552

  1. A serine sensor for multicellularity in a bacterium

    PubMed Central

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

    2013-01-01

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

  2. Purification and properties of serine hydroxymethyltransferase from Sulfolobus solfataricus.

    PubMed Central

    Delle Fratte, S; White, R H; Maras, B; Bossa, F; Schirch, V

    1997-01-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the reversible cleavage of serine to glycine with the transfer of the one-carbon group to tetrahydrofolate to form 5,10-methylenetetrahydrofolate. No SHMT has been purified from a nonmethanogenic Archaea strain, in part because this group of organisms uses modified folates as the one-carbon acceptor. These modified folates are not readily available for use in assays for SHMT activity. This report describes the purification and characterization of SHMT from the thermophilic organism Sulfolobus solfataricus. The exchange of the alpha-proton of glycine with solvent protons in the absence of the modified folate was used as the activity assay. The purified protein catalyzes the synthesis of serine from glycine and a synthetic derivative of a fragment of the natural modified folate found in S. solfataricus. Replacement of the modified folate with tetrahydrofolate did not support serine synthesis. In addition, this SHMT also catalyzed the cleavage of both allo-threonine and beta-phenylserine in the absence of the modified folate. The cleavage of these two amino acids in the absence of tetrahydrofolate is a property of other characterized SHMTs. The enzyme contains covalently bound pyridoxal phosphate. Sequences of three peptides showed significant similarity with those of peptides of SHMTs from two methanogens. PMID:9393711

  3. Serine Transhydroxymethylase in Methionine Biosynthesis in Saccharomyces cerevisiae

    PubMed Central

    Botsford, J. L.; Parks, L. W.

    1969-01-01

    Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the ?-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme. PMID:5776524

  4. Rapid identification of metallo- and serine beta-lactamases.

    PubMed

    Payne, D J; Cramp, R; Bateson, J H; Neale, J; Knowles, D

    1994-05-01

    Simple methods to detect, identify, and differentiate metallo- and serine beta-lactamases were developed and used to differentiate enzymes produced by 17 clinical isolates of Xanthomonas maltophilia. All isolates exhibited beta-lactamase activity, and in 16 strains this was induced by imipenem. All but one isolate hydrolyzed imipenem (and meropenem), and in all cases this activity was inhibited by 1 mM EDTA. The metallo- and serine beta-lactamases in the cell extracts were distinguished on isoelectric focusing (IEF) gels by using the following procedures. (i) Cell lysates were preincubated with 83 mM EDTA prior to IEF and subsequent visualization with nitrocefin, and (ii) after IEF, the gels were overlaid with either 1 mM zinc sulfate or 100 microM BRL 42715 before staining with nitrocefin. Bands of beta-lactamase activity which were removed by BRL 42715 but unaffected by EDTA or zinc sulfate were categorized as serine beta-lactamases. Bands which were unaffected by BRL 42715 but inhibited by EDTA or enhanced by zinc sulfate were classified as metallo-beta-lactamases. By using this approach, seven metallo-beta-lactamases were differentiated with pI values of 4.8 (two strains), 5.5 (four strains), 5.7 (one strain), 6.0 (one strain), 6.4 (four strains), 6.6 (one strain), and 6.8 (three strains). The metallo-beta-lactamase band with a pI of 6.4 aligned with the recently characterized metallo-beta-lactamase from X. maltophilia 511. Heterogeneity was also observed for the serine beta-lactamases: 14 isolates elaborated serine beta-lactamase activity which focused with major bands with at least eight different pIs. The remaining three strains produced serine beta-lactamases which focused with five distinct bands with pIs of 6.4, 6.2, 5.7, 5.5, and 5.2. We conclude that X. maltophilia produces many types of metallo- and serine beta-lactamases distinguishable by these new methods and that the previously reported L-1 and L-2 enzymes are not solely representative of the beta-lactamases produced by this species. PMID:8067782

  5. PAN/PS elctrospun fibers for oil spill cleanup

    NASA Astrophysics Data System (ADS)

    Ying, Qiao; Lili, Zhao; Haixiang, Sun; Peng, Li

    2014-08-01

    A high-capacity oil sorbent was fabricated by electrospinning using PS/PAN blend. Morphology, contact angle and oil adsorption of PAN/PS fiber and PP nonwoven fabric were studied. It was found that the PAN/PS fiber had a smaller diameter than PP, and the maximum sorption capacities of the PAN/PS sorbent for pump oil, peanut oil, diesel, and gasoline were 194.85, 131.7, 66.75, and 43.38 g/g, which were far higher than those of PP. The sorbent PS/PAN fiber showed a contact angle of water144.32° and diesel oil 0°. The sorption kinetics of PAN/PS and PP sorbent were also investigated. Compared with the commercial PP fabric, the PAN/PS fiber seems to have the ability to be used in oil-spill cleanup application.

  6. 10 ps x-ray streak camera

    NASA Astrophysics Data System (ADS)

    Belolipetski, V. S.; Gornostaev, P. B.; Lozovoi, V. I.; Schelev, M. Ya.

    2008-11-01

    10ps, X-ray streak camera developed at GPI, Photoelectronics Department is intended for photographic recording of high-speed events in visible and soft X-ray spectral regions. The camera contains a picosecond streak tube of PV-003-X type with a photocathode being simultaneously sensitive in visible (250-700 nm) and soft X-ray regions (1-10KeV). Due to this unique feature the camera may be adjusted in the visible light range and afterward, without any further readjustment, be used for high-speed recording in X-ray spectral range. Both single-streak and single-frame modes are available. The streak duration range over the output phosphor screen of 25mm length is 2.5-250ns. Single frame exposure time is between 100 and 500 ns. Dynamic spatial resolution in X-ray spectral range is 7 line pairs/mm. Maximum dynamic recording range is close to 100. The camera triggering delay at maximum streak-speed is less than 50ns with the triggering jitter within +/-50ps.

  7. Drosophila PS2 and PS3 integrins play distinct roles in retinal photoreceptors-glia interactions.

    PubMed

    Tavares, Lígia; Pereira, Emiliana; Correia, Andreia; Santos, Marília A; Amaral, Nuno; Martins, Torcato; Relvas, João B; Pereira, Paulo S

    2015-07-01

    Cellular migration and differentiation are important developmental processes that require dynamic cellular adhesion. Integrins are heterodimeric transmembrane receptors that play key roles in adhesion plasticity. Here, we explore the developing visual system of Drosophila to study the roles of integrin heterodimers in glia development. Our data show that αPS2 is essential for retinal glia migration from the brain into the eye disc and that glial cells have a role in the maintenance of the fenestrated membrane (Laminin-rich ECM layer) in the disc. Interestingly, the absence of glial cells in the eye disc did not affect the targeting of retinal axons to the optic stalk. In contrast, αPS3 is not required for retinal glia migration, but together with Talin, it functions in glial cells to allow photoreceptor axons to target the optic stalk. Thus, we present evidence that αPS2 and αPS3 integrin have different and specific functions in the development of retinal glia. PMID:25731761

  8. Capillary electrophoresis method for determination of D-serine and its application for monitoring of serine racemase activity.

    PubMed

    Koval, Dusan; Jirásková, Jana; Strísovský, Kvido; Konvalinka, Jan; Kasicka, Václav

    2006-07-01

    Serine racemase (SR) is an enzyme responsible for the biosynthesis of D-serine, the coagonist of the N-methyl-D-aspartate receptor, in the brain. Therefore, it has been suggested as a possible therapeutic target for the treatment of various neurodegenerative diseases. To develop a potent inhibitor of SR, a simple, sensitive, fast, and robust assay is needed. In this paper, a new CE method for the determination of D-serine is described. Serine enantiomers are resolved in the form of o-phthaldialdehyde (OPA)/2-mercaptoethanol (2-ME) derivatives in an alkaline BGE composed of 50 mM sodium tetraborate, pH 9.7, and containing 40 mM 2-hydroxypropyl-gamma-CD as a chiral selector. The problem of time-limited stability of OPA/2-ME derivatives has been overcome by employing in-capillary derivatization of the sample, i.e., the derivatization reaction was carried out directly in the separation capillary in the first phase of the CE run. UV-absorption detection at 230 nm allowed concentration detection limit of 3 microM. Baseline resolution of D- and L-serine derivatives was achieved in less than 10 min. This fact, together with the simple sample pretreatment, allowed application of the method to medium-throughput monitoring of SR activity, such as the screening of potential SR inhibitors. A good agreement was achieved between the developed CE method and the previously established HPLC method for determination of the inhibition constant, K(i), of a new SR inhibitor, L-erythro-3-hydroxyaspartate. PMID:16721908

  9. Inhibition of Growth of Mycobacterium smegmatis and of Cell Wall Synthesis by d-Serine

    PubMed Central

    Yabu, Kunihiko; Huempfner, Herman R.

    1974-01-01

    d-Serine inhibited the growth of Mycobacterium smegmatis and induced the morphological alteration of the bacilli. The growth inhibitory action of d-serine was partially reduced by an equimolecular concentration of d-alanine. The combination of glycine with d-alanine reversed the growth inhibition produced by d-serine more than did d-alanine alone. In cells cultured in the presence of d-serine, the amounts of alanine, diaminopimelic acid, and glycine inserted into the cell wall mucopeptide were reduced, and serine was increased. The intracellular accumulation of a precursor of cell wall mucopeptide was increased by d-serine, and this accumulation was reduced by d-alanine. d-Serine competed with glycine for incorporation into the cell wall mucopeptide. The incorporation of l-aspartic acid into diaminopimelic acid residues in the cell wall mucopeptide was markedly inhibited by d-serine. Three mutants resistant to d-serine were isolated by nitrosoguanidine treatment. In these mutants the effects of d-serine on the sites of cell wall mucopeptide synthesis were all reduced. Thus, d-serine inhibition of the growth is due to replacement of glycine residues of the cell wall mucopeptide with d-serine and inhibition of the cell wall synthesis by blocking the formation of d-alanine and diaminopimelic acid. PMID:15828163

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

    PubMed Central

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

    2012-01-01

    Multiple phosphorylation sites on the human estrogen receptor (hER)? were identified and shown to influence mammary carcinogenesis. In contrast, functional phosphorylation sites of hER? 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 hER? 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 hER?1 was found to exhibit higher transactivation activity than hER?1. 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-hER? immunoreactivity was detected with a higher prevalence and intensity than that of hER?1. These results underscore the functional importance of the first experimentally identified hER?-phosphorylation site in breast cancer. PMID:22370157

  11. Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

    PubMed

    Beuvink, I; Hess, D; Flotow, H; Hofsteenge, J; Groner, B; Hynes, N E

    2000-04-01

    The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration. PMID:10744710

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

    PubMed Central

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

    2014-01-01

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

  13. Phospholipid Metabolism in Ferrobacillus ferrooxidans

    PubMed Central

    Short, Steven A.; White, David C.; Aleem, M. I. H.

    1969-01-01

    The lipid composition of the chemoautotroph Ferrobacillus ferrooxidans has been examined. Fatty acids represent 2% of the dry weight of the cells and 86% of the total are extractable with organic solvents. About 25% of the total fatty acids are associated with diacyl phospholipids. Polar carotenoids, the benzoquinone coenzyme Q-8, and most of the fatty acids are present in the neutral lipids. The phospholipids have been identified as phosphatidyl monomethylethanolamine (42%), phosphatidyl glycerol (23%), phosphatidyl ethanolamine (20%), cardiolipin (13%), phosphatidyl choline (1.5%), and phosphatidyl dimethylethanolamine (1%) by chromatography of the diacyl lipids, by chromatography in four systems of the glycerol phosphate esters derived from the lipids by mild alkaline methanolysis, and by chromatographic identification of the products of acid hydrolysis of the esters. No trace of phosphatidylserine (PS), glycerolphosphorylserine, or serine could be detected in the lipid extract or in derivatives of that extract. This casts some doubt on the postulated involvement of PS in iron metabolism. After growth in the presence of 14C and 32P, there was essentially no difference in the turnover of either isotope in the glycerolphosphate ester derived from each lipid in cells grown at pH 1.5 or 3.5. Images PMID:5802599

  14. Amino acid changes in Drosophila alphaPS2betaPS integrins that affect ligand affinity.

    PubMed

    Bunch, Thomas A; Helsten, Teresa L; Kendall, Timmy L; Shirahatti, Nikhil; Mahadevan, Daruka; Shattil, Sanford J; Brower, Danny L

    2006-02-24

    We developed a ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2betaPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavbeta3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2betaPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the betaPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the betaPS I domain (betaPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2betaPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin beta subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the betaPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because betaPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system. PMID:16371365

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

    PubMed

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

    2014-01-01

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

  16. 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

  17. Crystal structure of maize serine racemase with pyridoxal 5'-phosphate.

    PubMed

    Zou, Lingling; Song, Yang; Wang, Chengliang; Sun, Jiaqi; Wang, Leilei; Cheng, Beijiu; Fan, Jun

    2016-03-01

    Serine racemase (SR) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that is responsible for D-serine biosynthesis in vivo. The first X-ray crystal structure of maize SR was determined to 2.1 Å resolution and PLP binding was confirmed in solution by UV-Vis absorption spectrometry. Maize SR belongs to the type II PLP-dependent enzymes and differs from the SR of a vancomycin-resistant bacterium. The PLP is bound to each monomer by forming a Schiff base with Lys67. Structural comparison with rat and fission yeast SRs reveals a similar arrangement of active-site residues but a different orientation of the C-terminal helix. PMID:26919519

  18. Architecture of a Serine Recombinase-DNA Regulatory Complex

    PubMed Central

    Mouw, Kent W.; Rowland, Sally-J.; Gajjar, Mark M.; Boocock, Martin R.; Stark, W. Marshall; Rice, Phoebe A.

    2008-01-01

    Summary An essential feature of many site-specific recombination systems is their ability to regulate the direction and topology of recombination. Resolvases from the serine recombinase family assemble an interwound synaptic complex that harnesses negative supercoiling to drive the forward reaction and promote recombination between properly oriented sites. To better understand the interplay of catalytic and regulatory functions within these synaptic complexes, we have solved the structure of the regulatory site synapse in the Sin resolvase system. It reveals an unexpected synaptic interface between helix-turn-helix DNA-binding domains that is also highlighted in a screen for synapsis mutants. The tetramer defined by this interface provides the foundation for a robust model of the synaptic complex, assembled entirely from available crystal structures, that gives insight into how the catalytic activity of Sin and other serine recombinases may be regulated. PMID:18439894

  19. Photoinactivation of PS2 secondary donors by PS2 cation radicals and superoxide radicals

    SciTech Connect

    Chen, G.X.; Cheniae, G.M.; Blubaugh, D.J.; Golbeck, J.H.

    1991-12-31

    Illumination of Mn- and Cl-depleted PS2 causes rapid irreversible inactivation of specific redox-active components on the donor side of the PS2 Reaction Center (RC). Under aerobic conditions, weak light preillumination of NH{sub 2}OH-PS2 causes rapid loss of Y{sub Z}{sup {plus_minus}} formation, Y{sub Z} {yields} P{sub 680}{sup +}, the A{sub T}-band thermoluminescence emission, the Y{sub Z}{sup +}-dependent (Site 1) photooxidation of exogenous e{sup {minus}} donors, and the capability to photoligate Mn{sup 2+} into the water oxidizing enzyme (photoactivation), all without significantly affecting P{sub 680}{sup +}/Q{sub A}{sup {minus}} charge separation. In contrast, aerobic high light preillumination of Mn-depleted PS2 promotes very rapid and parallel loss of photoactivation and A{sub T}-band emission capabilities significantly than loss of either Y{sub Z}{sup +}-formation or P{sub 680}{sup +}/Q{sub A}{sup {minus}} charge separation capabilities. These photodamages and those to Cl-depleted thylakoids (4,5) generally are believed to be caused by reactions between the highly oxidizing cation radicals (P{sub 680}{sup +}/Chl{sup +}) and nearby amino acid residues of D{sub 1}>D{sub 2}. The reported promotion of the photodamages by e{sup {minus}} acceptors of Q{sub A}{sup {minus}}/Q{sub B}{sup {minus}} their inhibition by e{sup {minus}} donors to Y{sub Z}{sup +} and their occurrence under strict anaerobic conditions all tend to support the idea of direct damage by P{sub 680}{sup +}/Chl{sup +}. Our studies lead us to conclude that the photodamages to the donor side components are caused minimally by a rapid mechanism requiring both superoxide and PS2 cation radicals; and by a slower mechanism driven by the PS2 cation radicals only.

  20. 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

  1. 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.

  2. 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}.

  3. 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.

  4. Serine racemase: a key player in apoptosis and necrosis

    PubMed Central

    Canu, Nadia; Ciotti, Maria Teresa; Pollegioni, Loredano

    2014-01-01

    A fine balance between cell survival and cell death is required to sculpt the nervous system during development. However, an excess of cell death can occur following trauma, exposure to neurotoxins or alcohol, and some developmental and neurodegenerative diseases, such as Alzheimer's disease (AD). N-Methyl-D-aspartate receptors (NMDARs) support synaptic plasticity and survival of many neuronal populations whereas inappropriate activation may promote various forms of cell death, apoptosis, and necrosis representing the two extremes of a continuum of cell death processes both “in vitro” and “in vivo.” Hence, by identifying the switches controlling pro-survival vs. apoptosis and apoptosis vs. pro-excitotoxic outcome of NMDAR stimulation, NMDAR modulators could be developed that selectively block the cell death enhancing pro-survival signaling or synaptic plasticity mediated by NMDAR. Among these modulators, a role is emerging for the enzyme serine racemase (SR) that synthesizes D-serine, a key co-agonist with glutamate at NMDAR. This review summarizes the experimental evidence from “in vitro” neuronal cultures—with special emphasis on cerebellar granule neurons (CGNs)—and “in vivo” models of neurodegeneration, where the dual role of the SR/D-serine pathway as a master regulator of apoptosis and the apoptosis-necrosis shift will be discussed. PMID:24795622

  5. Structural basis for catalytic activation of a serine recombinase

    PubMed Central

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

    2011-01-01

    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 1.86Å 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. PMID:21645851

  6. A new serine protease from the leaves of Thespesia populnea.

    PubMed

    Ishwarya, S; Sangeetha, R

    2013-01-01

    A serine protease was purified 6.9-fold from the leaves of Thespesia populnea using ammonium sulfate fractionation followed by CM-cellulose and Sephadex G-100 chromatography. The purified enzyme was named populnein and was characterized. It was made up of a single polypeptide, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis showed that the enzyme had a molecular mass of 14,518 Da. Inhibition of enzyme activity by phenyl methane sulfonyl fluoride indicates that populnein belongs to the class of serine proteases. The enzyme had appreciable pH and temperature stability. The activity of the enzyme was optimal at pH 8.0 and temperature 40°C. The enzyme was thermostable and retained 85% of its activity at 70°C after 1 hr. The enzyme was also resistant to autodigestion. The stabilization of the membrane of red blood cells exhibited by the protease populnein was found to be higher than for diclofenac. More studies are necessary to investigate the biological activity and applications of serine protease of T. populnea. PMID:23215657

  7. 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.

  8. Resonances in PsH below the p+Ps^- threshold

    NASA Astrophysics Data System (ADS)

    Yan, Zong-Chao; Ho, Y. K.

    2008-05-01

    Recently, Di Rienzi and Drachman carried out an investigation of high-lying resonances in the Ps-H system lying below the Ps^- + H^+ threshold [1]. Using an effective potential formalism, they calculated the resonance energies for some S, P and D states. In the present work, we present a calculation of S-wave resonances lying below the p + Ps^- threshold by using the method of complex-coordinate rotation [2]. We use elaborate Hylleraas wave functions [3] in which all the six inter-particle coordinates are included [4]. Using up to 6412 terms in the basis functions, resonance energies and widths for several lower members of a Rydberg series are calculated. At the meeting, we will compare our preliminary results with those of the earlier calculation [1]. [1] J. Di Rienzi and R. J. Drachman, Phys. Rev. A 76 (2007) 032705. [2] Y. K. Ho, Phys. Rept. 99 (1983) 1, and references therein. [3] Z.-C. Yan and Y. K. Ho, Phys. Rev. A 59 (1999) 2697. [4] Z.-C. Yan and G. W. F. Drake, J. Phys. B. 30 (1997) 4723.

  9. Serine protease inhibitors: novel therapeutic targets for stroke?

    PubMed

    Vivien, D; Buisson, A

    2000-05-01

    Although the thrombolytic activity of tissue-type plasminogen activator (t-PA) may be beneficial in the acute treatment of stroke, recent studies have suggested that this serine protease could also play a critical role in determining the extent of neuronal death after injury to the central nervous system (CNS). This hypothesis is based on several experimental results: t-PA-deficient mice are resistant to excitotoxic neuronal death induced by the intrahippocampal injection of kainate; the infarct volume induced by occlusion of the middle cerebral artery is reduced in t-PA knockout mice; and the intravenous injection of t-PA can under certain circumstances potentiate the infarct volume in animals subjected to middle cerebral artery occlusion. In the CNS, the serine proteases have been identified to occur both in neurons and glial cells. Their enzymatic activity regulates the balance between the accumulation and the degradation of the extracellular matrix. They are involved in many physiologic functions, ranging from synaptic outgrowth during perinatal development to plasticity in adults. For instance, thrombin and t-PA are known to modulate neurite outgrowth and tissue remodeling in the early stages of development. In the adult brain, t-PA may contribute to the late phase of long-term potentiation and to the subsequent synaptic growth in the hippocampal mossy fiber pathway. This balance between the degradation and accumulation of the extracellular matrix may also be integral to various pathologic processes involved in acute brain injury. For example, compounds that modulate the activity of serine proteases exhibit neuroprotective activity. Based on the above, numerous studies have focused on the production and modulation of the endogenously produced serine protease inhibitors, termed serpins, such as type 1 plasminogen activator inhibitor, neuroserpin, and protease nexin-1. In the present review, we will discuss the need to distinguish between the potentially neurotoxic effects of t-PA and its beneficial effect on reperfusion. We will present data supporting the idea that the modulation of serine protease activity may represent a novel and efficient strategy for the treatment of acute cerebral injury in humans. PMID:10826525

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

    PubMed

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

    2007-01-01

    It has been suggested that oxidative stress is involved in d-serine-induced nephrotoxicity. The purpose of this study was to assess if oxidative stress is involved in this experimental model using several approaches including (a) the determination of several markers of oxidative stress and the activity of some antioxidant enzymes in kidney and (b) the use of compounds with antioxidant or prooxidant effects. Rats were sacrificed at several periods of time (from 3 to 24h) after a single i.p. injection of d-serine (400mg/kg). Control rats were injected with l-serine (400mg/kg) and sacrificed 24h after. The following markers were used to assess the temporal aspects of renal damage: (a) urea nitrogen (BUN) and creatinine in blood serum, (b) kidney injury molecule (KIM-1) mRNA levels, and (c) tubular necrotic damage. In addition, creatinine clearance, proteinuria, and urinary excretion of N-acetyl-beta-d-glucosaminidase (NAG) were measured 24h after d-serine injection. Protein carbonyl content, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), fluorescent products of lipid peroxidation, reactive oxygen species (ROS), glutathione (GSH) content, and heme oxygenase-1 (HO-1) expression were measured as markers of oxidative stress in the kidney. Additional experiments were performed using the following compounds with antioxidant or pro-oxidant effects before d-serine injection: (a) alpha-phenyl-tert-butyl-nitrone (PBN), a spin trapping agent; (b) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) (FeTPPS), a soluble complex able to metabolize peroxynitrite; (c) aminotriazole (ATZ), a catalase (CAT) inhibitor; (d) stannous chloride (SnCl(2)), an HO-1 inductor; (e) tin mesoporphyrin (SnMP), an HO inhibitor. In the time-course study, serum creatinine and BUN increased significantly on 15-24 and 20-24h, respectively, and KIM-1 mRNA levels increased significantly on 6-24h. Histological analyses revealed tubular necrosis at 12h. The activity of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase remained unchanged at all times studied. Protein carbonyl content, MDA, 4-HNE, and ROS remained unchanged at all time-points studied. GSH content decreased transiently on 9 and 12h. Interestingly, fluorescent products of lipid peroxidation decreased significantly on 3-24h. HO-1 expression was undetectable by Western blot and the immunohistochemistry studies revealed that the intensity of HO-1 staining was weak. The administration of PBN, FeTPPS, ATZ, SnCl(2), and SnMP did not prevent or enhance renal damage induced by d-serine. Our data taken as a whole suggest that oxidative stress is not involved in the early phase of the nephrotoxicity induced by d-serine. PMID:17110013

  11. A wavelength tunable 2-ps pulse VECSEL

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

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

    PubMed Central

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

    2012-01-01

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

  13. pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model

    PubMed Central

    Neuberger, Georg; Schneider, Georg; Eisenhaber, Frank

    2007-01-01

    Background Protein kinase A (cAMP-dependent kinase, PKA) is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal. Results Approximately 20 sequence positions flanking the phosphorylated residue on both sides have been found to be restricted in their sequence variability (region -18...+23 with the site at position 0). The conserved physical pattern can be rationalized in terms of a qualitative binding model with the catalytic cleft of the protein kinase A. Positions -6...+4 surrounding the phosphorylation site are influenced by direct interaction with the kinase in a varying degree. This sequence stretch is embedded in an intrinsically disordered region composed preferentially of hydrophilic residues with flexible backbone and small side chain. This knowledge has been incorporated into a simplified analytical model of productive binding of substrate proteins with PKA. Conclusion The scoring function of the pkaPS predictor can confidently discriminate PKA phosphorylation sites from serines/threonines with non-permissive sequence environments (sensitivity of ~96% at a specificity of ~94%). The tool "pkaPS" has been applied on the whole human proteome. Among new predicted PKA targets, there are entirely uncharacterized protein groups as well as apparently well-known families such as those of the ribosomal proteins L21e, L22 and L6. Availability The supplementary data as well as the prediction tool as WWW server are available at . Reviewers Erik van Nimwegen (Biozentrum, University of Basel, Switzerland), Sandor Pongor (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy), Igor Zhulin (University of Tennessee, Oak Ridge National Laboratory, USA). PMID:17222345

  14. Safeguarding Nonhuman Primate iPS Cells With Suicide Genes

    PubMed Central

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

    2011-01-01

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

  15. 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.

  16. Annihilation of electron-positron pairs in the positronium ion Ps{sup -} and bipositronium Ps{sub 2}

    SciTech Connect

    Frolov, Alexei M.

    2009-07-15

    Rates of the two-, three-, four-, and five-photon annihilations of the electron-positron pairs are determined numerically for the three-body positronium ion Ps{sup -}(e{sup -}e{sup +}e{sup -}) and four-body bipositronium ''molecule''Ps{sub 2}(e{sup -}e{sup +}e{sup -}e{sup +}). The values obtained in our computations are {gamma}{sub 2{gamma}}(Ps{sup -}){approx_equal}2.080 485 305 25x10{sup 9} s{sup -1}, {gamma}{sub 3{gamma}}(Ps{sup -}){approx_equal}5.636 415 155 0x10{sup 6} s{sup -1}, {gamma}{sub 4{gamma}}(Ps{sup -}){approx_equal}3.075x10{sup 3} s{sup -1}, {gamma}{sub 5{gamma}}(Ps{sup -}){approx_equal}5.383 s{sup -1}, and {gamma}{sub 2{gamma}}(Ps{sub 2}){approx_equal}4.438 595 2x10{sup 9} s{sup -1}, {gamma}{sub 3{gamma}}(Ps{sub 2}){approx_equal}1.202 497x10{sup 7} s{sup -1}, {gamma}{sub 4{gamma}}(Ps{sub 2}){approx_equal}6.562x10{sup 3} s{sup -1}, {gamma}{sub 5{gamma}}(Ps{sub 2}){approx_equal}11.484 s{sup -1}. The four- and five-photon annihilation rates are significantly smaller than the corresponding two- and three-photon annihilation rates known for these systems. We also determine the rates of one- and zero-photon annihilation for the Ps{sup -} ion and Ps{sub 2} system. The corresponding numerical values are {gamma}{sub 1{gamma}}(Ps{sup -}){approx_equal}3.824 91x10{sup -2} s{sup -1}, {gamma}{sub 1{gamma}}(Ps{sub 2}){approx_equal}1.941 88x10{sup -1} s{sup -1}, and {gamma}{sub 0{gamma}}(Ps{sub 2}){approx_equal}2.321 97x10{sup -9} s{sup -1}.

  17. Nitrate respiration of Klebsiella pneumoniae on amino acids, especially on serine.

    PubMed

    Kikuchi, S; Ishimoto, M

    1980-01-01

    Anaerobic growth on an amino acids mixture in the presence of nitrate of a strain of K. pneumoniae isolated from soil depended on the presence of serine in the mixture. This organism grew on serine as sole carbon source under anaerobic conditions in the presence of nitrate but not in its absence. Glycylglycine could replace serine. Serine was degraded to acetate, formate, pyruvate, acetone and acetoin with formation of nitrite under these conditions. These products were practically not different from those formed from glucose. Resting cells decomposed serine also in the absence of nitrate forming butanediol and a large amount of formate in place of decreased amounts of acetate and pyruvate. These cells also anaerobically decomposed alanine to products similar to those from serine and phenylalanine to phenylacetate and formate only in the presence of nitrate. Valine was not catabolized under the same conditions. Serine was decomposed to pyruvate and ammonia in the crude extract in the presence and absence of nitrate, indicating participation of serine dehydratase in its breakdown. Phenylalanine appears to be decomposed to phenylpyruvate by transamination coupled to glutamate oxidation and then to phenylacetate and formate. Little growth on serine under anaerobic conditions in the absence of nitrate may be accounted for by the small gain of ATP in the anaerobic breakdown of serine by this organism. PMID:6775428

  18. Sphingoid Bases and the Serine Catabolic Enzyme CHA1 Define a Novel Feedforward/Feedback Mechanism in the Response to Serine Availability*

    PubMed Central

    Montefusco, David J.; Newcomb, Benjamin; Gandy, Jason L.; Brice, Sarah E.; Matmati, Nabil; Cowart, L. Ashley; Hannun, Yusuf A.

    2012-01-01

    Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation. PMID:22277656

  19. Exfoliated CdPS 3 Single Layers and Restacked Films

    NASA Astrophysics Data System (ADS)

    Yang, D.; Westreich, P.; Frindt, R. F.

    2002-07-01

    The layered compound CdPS3 has been exfoliated to form a suspension by ion exchange and washing in distilled water. The X-ray diffraction patterns of the suspension show that the material is two-dimensional, indicating the formation of single molecular layers of Cd0.8PS3. The 3.5 eV optical band-gap absorption and the refractive index of bulk CdPS3 have been measured. The measured absorption of single layers of Cd0.8PS3 in water suspension indicates that there is no significant change in electronic structure for the single layers. Single layers of Cd0.8PS3 have been restacked with different included molecules, opening up another approach for the synthesis of new layered nanocomposites.

  20. Telomere Reprogramming and Maintenance in Porcine iPS Cells

    PubMed Central

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

    2013-01-01

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

  1. 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.

  2. 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

  3. 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.

  4. LLNL PuPS Weld Qualification Plan

    SciTech Connect

    Dodson, K E; Riley, D

    2001-08-24

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

  5. 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

  6. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease.

    PubMed

    Majeed, Tanveer; Tabassum, Romana; Orts, William J; Lee, Charles C

    2013-01-01

    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 that the enzyme had optimal activity under alkaline conditions (pH 8-10). In addition, the enzyme had an elevated optimum temperature (60°C). The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market. PMID:24453843

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

    PubMed

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

    2010-10-01

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

  8. An Intergenic Stem-Loop Mutation in the Bacillus subtilis ccpA-motPS Operon Increases motPS Transcription and the MotPS Contribution to Motility†

    PubMed Central

    Terahara, Naoya; Fujisawa, Makoto; Powers, Benjamin; Henkin, Tina M.; Krulwich, Terry A.; Ito, Masahiro

    2006-01-01

    A stem-loop mutation between ccpA and motP in the Bacillus subtilis ccpA-motPS operon increased motPS transcription and membrane-associated MotPS levels, motility, and number of flagella/cell when MotPS is the sole stator and the MotPS contribution to motility at high pH, Na+, and viscosity when MotAB is also present. PMID:16547058

  9. An intergenic stem-loop mutation in the Bacillus subtilis ccpA-motPS operon increases motPS transcription and the MotPS contribution to motility.

    PubMed

    Terahara, Naoya; Fujisawa, Makoto; Powers, Benjamin; Henkin, Tina M; Krulwich, Terry A; Ito, Masahiro

    2006-04-01

    A stem-loop mutation between ccpA and motP in the Bacillus subtilis ccpA-motPS operon increased motPS transcription and membrane-associated MotPS levels, motility, and number of flagella/cell when MotPS is the sole stator and the MotPS contribution to motility at high pH, Na+, and viscosity when MotAB is also present. PMID:16547058

  10. 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.

  11. Regulation of scleraxis transcriptional activity by serine phosphorylation.

    PubMed

    Bagchi, Rushita A; Wang, Ryan; Jahan, Fahmida; Wigle, Jeffrey T; Czubryt, Michael P

    2016-03-01

    Cardiac fibroblasts are the major extracellular matrix producing cells in the heart. Our laboratory was the first to demonstrate that the transcription factor scleraxis induces collagen 1?2 expression in both cardiac fibroblasts and myofibroblasts. Here we identify a novel post-translational mechanism by which scleraxis activity is regulated and determine its effect on transcription of genes targeted by scleraxis. Putative serine phosphorylation sites on scleraxis were revealed by in silico analysis using motif prediction software. Mutation of key serine residues to alanine, which cannot be phosphorylated, significantly attenuated the expression of fibrillar type I collagen and myofibroblast marker genes that are normally induced by scleraxis. Down-regulation of collagen 1?2 expression was due to reduced binding of the non-phosphorylated scleraxis mutant to specific E-box DNA-binding sites within the promoter as determined by chromatin immunoprecipitation in human cardiac myofibroblast cells and by electrophoretic mobility shift assay. This is the first evidence suggesting that scleraxis is phosphorylated under basal conditions. The phosphorylation sequence matched that targeted by Casein Kinase 2, and inhibition of this kinase activity disrupted the ability of scleraxis to modulate the expression of its target genes while also attenuating TGF?-induced expression of type I collagen and myofibroblast phenotype conversion marker genes. These results demonstrate a novel mechanism for regulation of scleraxis activity, which may prove to be tractable for pharmacologic manipulation. PMID:26883788

  12. Neutrophil serine proteases exert proteolytic activity on endothelial cells.

    PubMed

    Jerke, Uwe; Hernandez, Daniel Perez; Beaudette, Patrick; Korkmaz, Brice; Dittmar, Gunnar; Kettritz, Ralph

    2015-10-01

    Neutrophil serine proteases (NSPs) are released from activated neutrophils during inflammation. Here we studied the transfer of the three major NSPs, namely proteinase 3, human neutrophil elastase, and cathepsin G, from neutrophils to endothelial cells and used an unbiased approach to identify novel endothelial NSP substrates. Enzymatically active NSPs were released from stimulated neutrophils and internalized by endothelial cells in a dose- and time-dependent manner as shown by immunoblotting, flow cytometry, and the Boc-Ala substrate assay. Using terminal-amine isotopic labeling of substrates in endothelial cells, we identified 121 peptides from 82 different proteins consisting of 36 substrates for proteinase 3, 30 for neutrophil elastase, and 28 for cathepsin G, respectively. We characterized the extended cleavage pattern and provide corresponding IceLogos. Gene ontology analysis showed significant cytoskeletal substrate enrichment and confirmed several cytoskeletal protein substrates by immunoblotting. Finally, ANCA-stimulated neutrophils released all three active NSPs into the supernatant. Supernatants increased endothelial albumin flux and disturbed the endothelial cell cytoskeletal architecture. Serine protease inhibition abrogated this effect. Longer exposure to NSPs reduced endothelial cell viability and increased apoptosis. Thus, we identified novel NSP substrates and suggest NSP inhibition as a therapeutic measure to inhibit neutrophil-mediated inflammatory vascular diseases. PMID:26061547

  13. Activity-based protein profiling: The serine hydrolases

    PubMed Central

    Liu, Yongsheng; Patricelli, Matthew P.; Cravatt, Benjamin F.

    1999-01-01

    With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression. PMID:10611275

  14. Rhodamine-based compounds as fluorogenic substrates for serine proteinases.

    PubMed Central

    Leytus, S P; Melhado, L L; Mangel, W F

    1983-01-01

    A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50--300-fold. The physical basis for this increase in sensitivity is discussed. PMID:6342611

  15. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation.

    PubMed

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

    2016-01-01

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

  16. 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. PMID:18164783

  17. Dynamic properties of extremophilic subtilisin-like serine-proteases.

    PubMed

    Tiberti, Matteo; Papaleo, Elena

    2011-04-01

    The investigation of the structural determinants of enzymatic temperature adaptation is a crucial pre-requisite both in terms of fundamental research and industrial applications to develop new biocatalysts active at different temperature ranges. In several cases, the differences related to cold- or warm-adaptation are related to subtle structural and aminoacidic differences at the molecular level, often hard to detect. In this context, we present a comparative study of psychrophilic, mesophilic and thermophilic subtilisin-like serine proteases by all-atom molecular dynamics (MD) simulations in explicit solvent using a multiple-replica approach. Our results strongly enforce the current view on localized flexibility in crucial functional regions for cold-adapted serine proteases and point out a different optimization and usage of salt-bridge interactions and networks in cold- and warm-adapted enzymes. The analyses allow to identify a subset of structural and dynamic features strictly associated to cold adaptation and which change from cold- to heat-active subtilisins. In particular, the thermophilic subtilisin presents a high affinity calcium binding site which is not structurally conserved in the mesophilic and psychrophilic counterparts, which, as it turns out from the MD analyses, at the same position show a stable salt bridge network and no stabilizing intra-molecular interactions, respectively. These aspects, along with differential flexibility in regions close to the active site or substrate binding pocket, can be an indication of evolution at this protein site toward a lower stability moving from high to low temperature conditions. PMID:21276854

  18. Antibacterial activity of silver nanoparticles synthesized from serine.

    PubMed

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

    2015-04-01

    Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV-Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443nm. The emission spectrum of Ag NPs showed an emission band at 484nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO3 against Gram-positive and Gram-negative bacteria. PMID:25686955

  19. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  1. Pharmacokinetics of oral D-serine in D-amino acid oxidase knockout mice.

    PubMed

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

    2012-11-01

    D-Amino acid oxidase (DAAO) catalyzes the oxidative deamination of D-amino acids including D-serine, a full agonist at the glycine modulatory site of the N-methyl-d-aspartate (NMDA) receptor. To evaluate the significance of DAAO-mediated metabolism in the pharmacokinetics of oral D-serine, plasma D-serine levels were measured in both wild-type mice and transgenic mice lacking DAAO. Although D-serine levels were rapidly diminished in wild-type mice (t(½) = 1.2 h), sustained drug levels over the course of 4 h (t(½) > 10 h) were observed in mice lacking DAAO. Coadministration of D-serine with 6-chlorobenzo[d]isoxazol-3-ol (CBIO), a small-molecule DAAO inhibitor, in wild-type mice resulted in the enhancement of plasma D-serine levels, although CBIO seems to have only temporary effects on the plasma D-serine levels due to glucuronidation of the key hydroxyl group. These findings highlight the predominant role of DAAO in the clearance of D-serine from the systemic circulation. Thus, a potent DAAO inhibitor with a longer half-life should be capable of maintaining high plasma D-serine levels over a sustained period of time and might have therapeutic implications for the treatment of schizophrenia. PMID:22837388

  2. Regional organisation of brain activity during paradoxical sleep (PS).

    PubMed

    Maquet, P; Ruby, P; Schwartz, S; Laureys, S; Albouy, G; Dang-Vu, T; Desseilles, M; Boly, M; Melchior, G; Peigneux, P

    2004-07-01

    Human brain function is regionally organised during paradoxical sleep (PS) in a very different way than during wakefulness or slow wave sleep. The important activity in the pons and in the limbic/paralimbic areas constitutes the key feature of the functional neuroanatomy of PS, together with a relative quiescence of prefrontal and parietal associative cortices. Two questions are still outstanding. What neurocognitive and neurophysiological mechanisms may explain this original organization of brain function during PS? How the pattern of regional brain function may relate to dream content? Although some clues are already available, the experimental answer to both questions is still pending. PMID:15493545

  3. 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.

  4. Synthesis and Structures of New One-Dimensional KTiPS 5and RbTiPS 5

    NASA Astrophysics Data System (ADS)

    Do, Junghwan; Lee, Kunsoo; Yun, Hoseop

    1996-08-01

    Two new compounds KTiPS5and RbTIPS5have been synthesized through reactions of the elements with alkali metal halide fluxes. KTiPS5crystallizes in the space groupC32h-C2/mof the monoclinic system with four formula units in a cell of dimensionsa= 17.678(8),b= 7.080(4),c= 6.325(3) Å, β = 97.99(4)°. RbTiPS5crystallizes in the space groupC32h-C2/mof the monoclinic system with eight formula units in a cell of dimensionsa= 18.159(7),b= 7.081(3),c= 15.352(6) Å, β = 124.07(2)°. The structures of both compounds have been determined by single crystal X-ray techniques. Both structures consist of one-dimensional1∞[TiPS-5] chains separated from one another by K+or Rb+cations. The linear chains are built up from the basic repeating unit, [Ti2(PS4)2S2-2], which is composed of two edge-shared TiS6octahedra and two PS4tetrahedra. There is no interchain bonding except the van der Waals interactions. The classical charge of the two compounds can be represented by [A+][Ti4+][PS3-4][S2-] (A= K or Rb).

  5. Spectral characteristics of PS II reaction centres: as isolated preparations and when integral to PS II core complexes.

    PubMed

    Krausz, Elmars; Cox, Nicholas; Arsköld, Sindra Peterson

    2008-01-01

    The discovery that the native PS II enzyme undergoes charge separation via an absorption extending to 730 nm has led us to re-examine the low-temperature absorption spectra of Nanba-Satoh PS II reaction centre preparations with particular focus on the long wavelength region. It is shown that these preparations do not exhibit absorption in the 700-730 nm region at 1.7 K. Absorption in the Nanba-Satoh type preparations analogous to the 'red tail' as observed in functional PS II core complexes is likely shifted to higher energy by >20 nm. Spectral changes associated with the stable reduction of pheo(a) in chemically treated reaction centre preparations are also revisited. Dithionite treatment of PS II preparations in the dark leads to changes of pigment-pigment and/or pigment-protein interactions, as evidenced by changes in absorption and CD spectra. Absorption and CD changes associated with stable Pheo(D1) photo-reduction in PS II core complexes and Nanba-Satoh preparations are compared. For Nanba-Satoh preparations, Q(y) bleaches are approximately 3x broader than in PS II core complexes and are blue-shifted by approximately 4 nm. These data are discussed in terms of current models of PS II, and suggest a need to consider protein-induced changes of some electronic properties of reaction centre pigments. PMID:18663598

  6. Phosphatidylserine (PS) Is Exposed in Choroidal Neovascular Endothelium: PS-Targeting Antibodies Inhibit Choroidal Angiogenesis In Vivo and Ex Vivo

    PubMed Central

    Li, Tao; Aredo, Bogale; Zhang, Kaiyan; Zhong, Xin; Pulido, Jose S.; Wang, Shusheng; He, Yu-Guang; Huang, Xianming; Brekken, Rolf A.; Ufret-Vincenty, Rafael L.

    2015-01-01

    Purpose Choroidal neovascularization (CNV) accounts for 90% of cases of severe vision loss in patients with advanced age-related macular degeneration. Identifying new therapeutic targets for CNV may lead to novel combination therapies to improve outcomes and reduce treatment burden. Our goal was to test whether phosphatidylserine (PS) becomes exposed in the outer membrane of choroidal neovascular endothelium, and whether this could provide a new therapeutic target for CNV. Methods Choroidal neovascularization was induced in C57BL/6J mice using laser photocoagulation. Choroidal neovascularization lesions costained for exposed PS and for intercellular adhesion molecule 2 (or isolectin B4) were imaged in flat mounts and in cross sections. The laser CNV model and a choroidal sprouting assay were used to test the effect of PS-targeting antibodies on choroidal angiogenesis. Choroidal neovascularization lesion size was determined by intercellular adhesion molecule 2 (ICAM-2) staining of flat mounts. Results We found that PS was exposed in CNV lesions and colocalized with vascular endothelial staining. Treatment with PS-targeting antibodies led to a 40% to 80% reduction in CNV lesion area when compared to treatment with a control antibody. The effect was the same as that seen using an equal dose of an anti-VEGF antibody. Results were confirmed using the choroid sprouting assay, an ex vivo model of choroidal angiogenesis. Conclusions We demonstrated that PS is exposed in choroidal neovascular endothelium. Furthermore, targeting this exposed PS with antibodies may be of therapeutic value in CNV. PMID:26529048

  7. 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.

  8. Assessment of slope stability using PS-InSAR technique

    NASA Astrophysics Data System (ADS)

    Dwivedi, R.; Varshney, P.; Tiwari, A.; Singh, A. K.; Dikshit, O.

    2014-11-01

    In this research work, PS-InSAR approach is envisaged to monitor slope stability of landslides prone areas in Nainital and Tehri region of Uttarakhand, India. For the proposed work, Stanford Method for Persistent Scatterers (StaMPS) based PS-InSAR is used for processing ENVISAT ASAR C-Band data stacks of study area which resulted in a time series 1D-Line of Sight (LOS) map of surface displacement. StaMPS efficiently extracted the PS pixels on the unstable slopes in both areas and the time series 1D-LOS displacement map of PS pixels indicates that those areas in Nainital and Tehri region have measurement pixels with maximum displacement away from the satellite of the order of 22 mm/year and 17.6 mm/year respectively

  9. Doubly excited 3P(e) resonant states in Ps(-)

    NASA Technical Reports Server (NTRS)

    Ho, Y. K.; Bhatia, A. K.

    1992-01-01

    Doubly excited 3P(e) resonant states in Ps(-) are calculated using a method of complex-coordinate rotation. Resonance parameters (both resonance positions and widths) for doubly excited states associated with the n = 2, 3, 4, 5, and 6 thresholds of positronium atoms are evaluated using elaborate Hylleraas-type functions. In addition to ten Feshbach-type resonances lying below various Ps thresholds, three shape resonances were identified, one each lying above the n = 2, 4, and 6 Ps thresholds. It is further noted that the energy levels for the present 3P(e) states are nearly degenerate with respect to the previously calculated 1P(0) states. Such a symmetric character suggests that the highly and doubly excited Ps(-), similar to its counterpart in H(-), would exhibit rovibrational behaviors analogous to those of XYX triatomic molecules.

  10. Serine uptake by luminal and basolateral membrane vesicles from rabbit kidney.

    PubMed Central

    Kragh-Hansen, U; Sheikh, M I

    1984-01-01

    The mechanism of renal transport of L- and D-serine by membrane vesicles prepared from either whole cortex, pars convoluta or pars recta of rabbit proximal tubule was studied by a rapid filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Transport studies carried out with different salt gradients and by employing various ionophores showed that uptake of both L- and D-serine by luminal membrane vesicles from whole cortex was mediated by an Na+-dependent and electrogenic transport process. Eadie-Hofstee analysis of experimental data, obtained under extravesicular greater than intravesicular NaCl gradients, revealed the existence of multiple transport systems for L-serine but only one system for the D-isomer. The value of KA (the concentration producing a half-maximal optical response) for the D-serine transport system was calculated to be approximately 30 mM. Luminal membrane vesicles from pars convoluta take up both L- and D-serine by a single and common transport system. KA values for L- and D-serine transport were calculated to be 3.7 and 30 mM, respectively. Luminal membrane vesicles from pars recta take up L-serine by means of two transport systems, one of high affinity (KA = 0.37 mM) and the other of low affinity (KA = 10 mM). By contrast, no D-serine transport by these membrane vesicles could be detected. Uptake of L-serine by basolateral membrane vesicles is Na+ independent and electroneutral. Filtration studies showed that the transport is saturable (Km = 25-30 mM) and is inhibited by the presence of L-phenylalanine (but not by D-serine), indicating carrier-mediated uptake of L-serine. PMID:6434730

  11. Engineering of high yield production of L-serine in Escherichia coli.

    PubMed

    Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre; Nielsen, Alex Toftgaard

    2016-04-01

    L-serine is a widely used amino acid that has been proposed as a potential building block biochemical. The high theoretical yield from glucose makes a fermentation based production attractive. In order to achieve this goal, serine degradation to pyruvate and glycine in E. coli MG1655 was prevented by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very high serine production yield (0.45 g/g glucose) during small-scale batch fermentation in minimal medium. Serine, however, was found to be highly toxic even at low concentrations to this strain, which lead to slow growth and production during fed batch fermentation, resulting in a serine production of 8.3 g/L. The production strain was therefore evolved by random mutagenesis to achieve increased tolerance towards serine. Additionally, overexpression of eamA, a cysteine/homoserine transporter was demonstrated to increase serine tolerance from 1.6 g/L to 25 g/L. During fed batch fermentation, the resulting strain lead to the serine production titer of 11.7 g/L with yield of 0.43 g/g glucose, which is the highest yield reported so far for any organism. Biotechnol. Bioeng. 2016;113: 807-816. © 2015 Wiley Periodicals, Inc. PMID:26416585

  12. Thermal Behaviors of PS Thin Films on Grafted PS Layer with Varied Grafting Density

    NASA Astrophysics Data System (ADS)

    Lee, Hoyeon; Park, Sungmin; Lee, Yonghoon; Choi, Eunyoung; Ryu, Du Yeol; Trombly, David M.; Ganesan, Venkat

    2012-02-01

    The thermal behavior properties (especially glass transition) are the key parameters for determining the mechanical properties of polymer system. In thin film system, the interfacial interactions at the substrate/polymer and polymer/air influence the transition behavior. In this study, we investigated the inter-relationship between the interfacial interactions arising from grafted polymer layers and the glass transition behavior (Tg) of thin polymer films with same chemical identity. We controlled grafting density of hydroxyl-terminated polystyrene (HO-PS). For polymer chains on the brushes of the same chemical identity with high density, the autophobic dewetting behavior can be attributed to the entropic effects of the polymer and the grafted polymer chains.

  13. [Application for Lifestyle disease by iPS cells technologies].

    PubMed

    Takashima, Yasuhiro

    2016-03-01

    Currently it is less advanced to understand the pathology of lifestyle disease by using iPS cells because there is partly less direct connection between life style disease and iPS cells. So much more scientists focus on regenerative medicine such as beta cells therapy using iPS cells technologies. It will be indeed a powerful tool to generate beta cells from iPS cells as even in type2 diabetes patients, hyposecretion of insulin from beta cells in pancreas is one of causes. Another reason is complexity of the pathology of life style disease. There are a lot of reasons to cause lifestyle disease. Lifestyle diseases include cancer, chronic liver disease, Type 2 diabetes, heart disease, metabolic syndrome, chronic renal failure, stroke, and obesity. Since obesity is one of major causes of lifestyle diseases, we want to focus on adipogenesis from iPS cells in this review. We analysed and established the differentiation protocol into adipocytes from mouse ES cells and human iPS cells. The other point in this review is the starting pluripotent cells for differentiation. Quality of pluripotent stem cells are one of most critical factors to succeed in getting well-differentiated cells. Recently, we have developed new naive human pluripotent stem cells (PSC),"Reset cells". Naive PSC have more similar to human epibast cells than conventional human PSC. They will be more ideal cells for differentiation because of their hypomethylated status and earlier stage of development. PMID:26923982

  14. Gene-delivery systems for iPS cell generation

    PubMed Central

    Shao, Lijian; Wu, Wen-Shu

    2009-01-01

    Importance of the field Induced pluripotent stem (iPS) cells offer extraordinary promise for regenerative medicine applications, and provide new opportunities for use in disease modeling, drug screening and drug toxicology. Areas coved in this review iPS cell technology is still in its infancy. In this review article, we present a comprehensive survey of reprogramming approaches focusing on gene-delivery systems used for generation of iPS cells from somatic cells, categorize gene-delivery vectors, and discuss their advantages and limitations for somatic cell reprogramming. We include pertinent literature published between 2006 and the present. What the reader will gain Although iPS cell technology has been improved via the use of various gene-delivery vectors, it still suffers from either low reprogramming efficiency or too many genomic modification steps. Extensive work is still required to improve current vectors or explore new vectors for effectively reprogramming human somatic cells into iPS cells, with or without minimal genomic modification steps. Take home message A single non-integrating reprogramming vector system with high reprogramming efficiency is probably essential for generation of clinically translatable human iPS cells. PMID:20088717

  15. 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. PMID:21530300

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

    PubMed Central

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

    2013-01-01

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

  17. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Astrocytes are involved in trigeminal dynamic mechanical allodynia: potential role of D-serine.

    PubMed

    Dieb, W; Hafidi, A

    2013-09-01

    Trigeminal neuropathic pain affects millions of people worldwide. Despite decades of study on the neuronal processing of pain, mechanisms underlying enhanced pain states after injury remain unclear. N-methyl-D-aspartate (NMDA) receptor-dependent changes play a critical role in triggering central sensitization in neuropathic pain. These receptors are regulated at the glycine site through a mandatory endogenous co-agonist D-serine, which is synthesized by astrocytes. Therefore, the present study was carried out to determine whether astrocytes are involved, through D-serine secretion, in dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN) in rats. Two weeks after CCI-IoN, an important reaction of astrocytes was present in the medullary dorsal horn (MDH), as revealed by an up-regulation of glial fibrillary acidic protein (GFAP) in allodynic rats. In parallel, an increase in D-serine synthesis, which co-localized with its synthesis enzyme serine racemase, was strictly observed in astrocytes. Blocking astrocyte metabolism by intracisternal delivery of fluorocitrate alleviated DMA. Furthermore, the administration of D-amino-acid oxidase (DAAO), a D-serine-degrading enzyme, or that of L-serine O-sulfate (LSOS), a serine racemase inhibitor, significantly decreased pain behavior in allodynic rats. These results demonstrate that astrocytes are involved in the modulation of orofacial post-traumatic neuropathic pain via the release of the gliotransmitter D-serine. PMID:23881719

  20. COMPARATIVE ANALYSIS OF SERINE PROTEASE-RELATED GENES IN THE HONEY BEE GENOME.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera. Most of these genes encode putative secreted proteins, but 4 SPs and 3 SPHs may associate with the plasma membrane via a transmembrane region. Clip domains represent the most abunda...

  1. 2.8-A structure of yeast serine carboxypeptidase.

    PubMed

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

    1994-09-20

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

  2. Serine O-sulfation probed by IRMPD spectroscopy.

    PubMed

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

    2015-10-21

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

  3. Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase.

    PubMed

    di Salvo, M L; Delle Fratte, S; Maras, B; Bossa, F; Wright, H T; Schirch, V

    1999-12-15

    Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme. PMID:10600164

  4. 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.

  5. Activity based protein profiling to detect serine hydrolase alterations in virus infected cells

    PubMed Central

    Shahiduzzaman, Md.; Coombs, Kevin M.

    2012-01-01

    Activity-based protein profiling (ABPP) is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified, but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation, and reproduction. ABPP has been used recently to investigate hostvirus interactions and to understand the molecular pathogenesis of virus infections. Monitoring the altered serine hydrolases during viral infection gives insight into the catalytic activity of these enzymes that will help to identify novel targets for diagnostic and therapeutic application. This review presents the usefulness of ABPP in detecting and analyzing functional annotation of host cell serine hydrolases as a result of hostvirus interaction. PMID:23024641

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

    PubMed

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

    2015-07-01

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

  7. Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest, Eurygaster integriceps.

    PubMed

    Saadati, Fatemeh; Bandani, Ali R

    2011-01-01

    In the current study the effects of serine proteinase inhibitors (TLCK, TPCK, SBTI, and a combination of SBTI and TPCK) with concentrations of 1% and 4% of dietary protein in artificial diets were tested against growth of the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), development, and its gut serine proteinase targets. Analysis of variance indicated that protease inhibitors affected nymphal development time, adult weight, and survival. Mean development time of third instar nymphs in control, SBTI (1%), TLCK (1%), and TPCK was 7.18, 9.74, 9.97, and 8.52 days, respectively. The highest mortality (100 % mortality) was observed when a combination of TPCK and SBTI, both at 4% of dietary protein, was used followed by TPCK (4%) that produced 95% mortality. There were significant differences in proteinase activity between treatments and controls when BApNA and SAAPFpNA were used as substrates for trypsin and chymotrypsin, respectively. Reduction of trypsin activity in insects fed with low doses of SBTI (1%), TLCK (1%), and both doses of TPCK (1% and 4%) was 40, 26, 23, and 17%, respectively. Inhibition of chymotrypsin activity was seen in the insects fed on SBTI (1%), TLCK (1%), and TPCK (4%) where inhibition was 14, 9, and 36%, respectively. Maximum inhibition of chymotrypsin activity was observed in the insects fed on diets containing high doses of TPCK (4%). In gel assays, the greatest effects were observed when E. integriceps were fed on high doses of SBTI and TPCK. Therefore, TPCK followed by SBTI proved to be the most effective proteinase inhibitors of E. integriceps. PMID:21867440

  8. Effects of Serine Protease Inhibitors on Growth and Development and Digestive Serine Proteinases of the Sunn Pest, Eurygaster integriceps

    PubMed Central

    Saadati, Fatemeh; Bandani, Ali R.

    2011-01-01

    In the current study the effects of serine proteinase inhibitors (TLCK, TPCK, SBTI, and a combination of SBTI and TPCK) with concentrations of 1% and 4% of dietary protein in artificial diets were tested against growth of the Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), development, and its gut serine proteinase targets. Analysis of variance indicated that protease inhibitors affected nymphal development time, adult weight, and survival. Mean development time of third instar nymphs in control, SBTI (1%), TLCK (1%), and TPCK was 7.18, 9.74, 9.97, and 8.52 days, respectively. The highest mortality (100 % mortality) was observed when a combination of TPCK and SBTI, both at 4% of dietary protein, was used followed by TPCK (4%) that produced 95% mortality. There were significant differences in proteinase activity between treatments and controls when BApNA and SAAPFpNA were used as substrates for trypsin and chymotrypsin, respectively. Reduction of trypsin activity in insects fed with low doses of SBTI (1%), TLCK (1%), and both doses of TPCK (1% and 4%) was 40, 26, 23, and 17%, respectively. Inhibition of chymotrypsin activity was seen in the insects fed on SBTI (1%), TLCK (1%), and TPCK (4%) where inhibition was 14, 9, and 36%, respectively. Maximum inhibition of chymotrypsin activity was observed in the insects fed on diets containing high doses of TPCK (4%). In gel assays, the greatest effects were observed when E. integriceps were fed on high doses of SBTI and TPCK. Therefore, TPCK followed by SBTI proved to be the most effective proteinase inhibitors of E. integriceps. PMID:21867440

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

    SciTech Connect

    Cha, Yoon-Jong; Choe, Soonja

    1995-12-01

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

  10. High-resolution PS-OCT of Enamel Remineralization.

    PubMed

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

    2008-03-01

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

  11. Assessment of dentin remineralization with PS-OCT

    NASA Astrophysics Data System (ADS)

    Manesh, Saman K.; Darling, Cynthia L.; Fried, Daniel

    2009-02-01

    Previous studies have demonstrated that polarization sensitive optical coherence tomography (PS-OCT) can be used to image natural root caries lesions, measure non-destructively the severity of dentin demineralization and determine the efficacy of intervention with anti-caries agents including fluoride and lasers. The objective of this study was to determine if PS-OCT could be used to nondestructively measure the formation of a layer of remineralized dentin on the surface of dentin lesions after exposure to a remineralization solution. In this study images of artificial dentin lesions on extracted human teeth were acquired using PS-OCT after exposure to an artificial demineralizing solution at pH 4.9 for six days and after subsequent exposure to a remineralizing solution at pH 7.0 for 20 days. Polarized light microscopy and microradiography were used to examine histological thin sections from the samples for comparison. PS-OCT successfully measured the formation of a layer of increased mineral content near the lesion surface. PLM and TMR corroborated those results. This study demonstrates the potential use of PS-OCT for the nondestructive measurement of the remineralization of dentin surfaces.

  12. High-resolution PS-OCT of Enamel Remineralization

    PubMed Central

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

    2011-01-01

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

  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. Development of a high-throughput method for the determination of pharmacological levels of plasma D-serine.

    PubMed

    Alt, Jesse; Rojas, Camilo; Wozniak, Krystyna; Wu, Ying; Ferraris, Dana; Tsukamoto, Takashi; Slusher, Barbara

    2011-12-15

    D-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, D-Serine must be administered at high doses to observe clinical effects. This is due in large part to D-Serine undergoing oxidation by D-Serine acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of D-Serine with a DAAO inhibitor has been suggested as a way to lower the dose of D-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma D-Serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure D-Serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor D-Serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate D-Serine from plasma followed by quantitation of D-Serine using the DAAO-catalyzed reaction. Plasma D-Serine determination using this assay could also be used as pharmacodynamic marker and as biomarker. PMID:21889486

  15. Pan-STARRS PS1 Observatory, Telescope and Instrument Control

    NASA Astrophysics Data System (ADS)

    Pier, E.; Chambers, K.

    An ultimate goal for the final Pan-STARRS array (PS4) is fully robotic facility operations. To facilitate that development, PS1 is a remotely operable observatory that includes many features of the future robotic Pan-STARRS. Both remote and robotic operational concepts require sufficient knowledge of environmental conditions to support survey scheduling, and this requires an auxiliary instrumentation suite to measure meteorological and atmospheric conditions in addition to the control systems for the observatory and telescope. For the Pan-STARRS PS1 prototype, the monitoring, control, and summit subsystem coordination are handled by the HW/SW within the Observatory, Telescope, and Instrumentation Subsystem (OTIS). In this talk, we present the functional capabilities of OTIS, and the HW and SW architectures designed to meet the subsystem operational requirements. OTIS performance through early commissioning is described as well as the future plans to incorporate the Pan-STARRS scheduler into system operations.

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

    PubMed

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

    2016-06-01

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

  17. Serine-borate complex as a transition-state inhibitor of gamma-glutamyl transpeptidase.

    PubMed Central

    Tate, S S; Meister, A

    1978-01-01

    gamma-Glutamyl transpeptidase, a membrane-bound enzyme, functions in the gamma-glutamyl cycle to catalyze utilization of glutathione. It has been postulated that the amino-acid-stimulated utilization of glutathione by gamma-glutamyl transpeptidase reflects an aspect of amino acid translocation. As one approach to the effective in vivo inhibition of this enzyme, the inhibition of the enzyme by L-serine in the presence of borate buffers [Revel, J.P. & Ball, E.G. (1959) J. Biol. Chem. 234, 577-582] was reinvestigated. Inhibition by L-serine, D-serine, and alpha-methyl-DL-serine in the presence of borate is competitive with respect to gamma-glutamyl substrate and such inhibition is parallel to the activity of transpeptidase toward L-gamma-glutamyl, D-gamma-glutamyl, and L-gamma-(alpha-methyl)glutamyl derivatives. L-Serine and borate effectively protect against inactivation of the enzyme by the gamma-glutamyl analogs, 6-diazo-5-oxonorleucine and azaserine, which bind to the gamma-glutamyl site of the enzyme. These studies, kinetic investigations, equilibrium dialysis experiments, and other data support the view that inhibition is produced by formation of serine-borate complex which binds at the gamma-glutamyl binding site of the light subunit of gamma-glutamyl transpeptidase. The data indicate that serine-borate complex is a transition state inhibitor of gamma-glutamyl transpeptidase. PMID:33382

  18. 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. PMID:26853817

  19. Serine racemase is associated with schizophrenia susceptibility in humans and in a mouse model

    PubMed Central

    Labrie, Viviane; Fukumura, Ryutaro; Rastogi, Anjali; Fick, Laura J.; Wang, Wei; Boutros, Paul C.; Kennedy, James L.; Semeralul, Mawahib O.; Lee, Frankie H.; Baker, Glen B.; Belsham, Denise D.; Barger, Steven W.; Gondo, Yoichi; Wong, Albert H.C.; Roder, John C.

    2009-01-01

    Abnormal N-methyl-d-aspartate receptor (NMDAR) function has been implicated in the pathophysiology of schizophrenia. d-serine is an important NMDAR modulator, and to elucidate the role of the d-serine synthesis enzyme serine racemase (Srr) in schizophrenia, we identified and characterized mice with an ENU-induced mutation that results in a complete loss of Srr activity and dramatically reduced d-serine levels. Mutant mice displayed behaviors relevant to schizophrenia, including impairments in prepulse inhibition, sociability and spatial discrimination. Behavioral deficits were exacerbated by an NMDAR antagonist and ameliorated by d-serine or the atypical antipsychotic clozapine. Expression profiling revealed that the Srr mutation influenced several genes that have been linked to schizophrenia and cognitive ability. Transcript levels altered by the Srr mutation were also normalized by d-serine or clozapine treatment. Furthermore, analysis of SRR genetic variants in humans identified a robust association with schizophrenia. This study demonstrates that aberrant Srr function and diminished d-serine may contribute to schizophrenia pathogenesis. PMID:19483194

  20. D-serine as a gliotransmitter and its roles in brain development and disease

    PubMed Central

    Horn, Marion R. Van; Sild, Mari; Ruthazer, Edward S.

    2013-01-01

    The development of new techniques to study glial cells has revealed that they are active participants in the development of functional neuronal circuits. Calcium imaging studies demonstrate that glial cells actively sense and respond to neuronal activity. Glial cells can produce and release neurotransmitter-like molecules, referred to as gliotransmitters, that can in turn influence the activity of neurons and other glia. One putative gliotransmitter, D-serine is believed to be an endogenous co-agonist for synaptic N-methyl-D-aspartate receptors (NMDARs), modulating synaptic transmission and plasticity mediated by this receptor. The observation that D-serine levels in the mammalian brain increase during early development, suggests a possible role for this gliotransmitter in normal brain development and circuit refinement. In this review we will examine the data that D-serine and its associated enzyme serine racemase are developmentally regulated. We will consider the evidence that D-serine is actively released by glial cells and examine the studies that have implicated D-serine as a critical player involved in regulating NMDAR-mediated synaptic transmission and neuronal migration during development. Furthermore, we will consider how dysregulation of D-serine may play an important role in the etiology of neurological and psychiatric diseases. PMID:23630460

  1. The nonconserved hydrophilic loop domain of presenilin (PS) is not required for PS endoproteolysis or enhanced abeta 42 production mediated by familial early onset Alzheimer's disease-linked PS variants.

    PubMed

    Saura, C A; Tomita, T; Soriano, S; Takahashi, M; Leem, J Y; Honda, T; Koo, E H; Iwatsubo, T; Thinakaran, G

    2000-06-01

    Presenilin 1 (PS1) and presenilin 2 (PS2) are polytopic membrane proteins that are mutated in the majority of early onset familial Alzheimer's disease (FAD) cases. Two lines of evidence establish a critical role for PS in the production of beta-amyloid peptides (Abeta). FAD-linked PS mutations elevate the levels of highly amyloidogenic Abeta ending at residue 42 (Abeta42), and cells with ablated PS1 alleles secrete low levels of Abeta. Several recent reports have shown that the hydrophilic loop (HL) domain, located between transmembrane domains 6 and 7, contains sites for phosphorylation, caspase cleavage, and sequences that bind several PS-interacting proteins. In the present report, we examined the metabolism of PS polypeptides lacking the HL domain and the influence of these molecules on Abeta production. We report that the deletion of the HL domain does not have a deleterious effect on the regulated endoproteolysis of PS, saturable accumulation of PS fragments, or the self-association of PS fragments. Abeta production was not significantly altered in cells expressing HL-deleted PS polypeptides compared with cells expressing full-length PS. Importantly, deletion of the HL domain did not affect FAD mutation-mediated elevation in the production of Abeta42. Furthermore, the deletion of the HL domain did not impair the role of PS1 or PS2 in facilitating Notch processing. Thus, our results argue against a biologically or pathologically relevant role for the HL domain phosphorylation and caspase cleavage and the association of PS HL domain-interacting proteins, in amyloid precursor protein metabolism and Abeta production or Notch cleavage. PMID:10748144

  2. D-Serine and D-Cycloserine Reduce Compulsive Alcohol Intake in Rats.

    PubMed

    Seif, Taban; Simms, Jeffrey A; Lei, Kelly; Wegner, Scott; Bonci, Antonello; Messing, Robert O; Hopf, F Woodward

    2015-09-01

    There is considerable interest in NMDAR modulators to enhance memory and treat neuropsychiatric disorders such as addiction, depression, and schizophrenia. D-serine and D-cycloserine, the NMDAR activators at the glycine site, are of particular interest because they have been used in humans without serious adverse effects. Interestingly, D-serine also inhibits some NMDARs active at hyperpolarized potentials (HA-NMDARs), and we previously found that HA-NMDARs within the nucleus accumbens core (NAcore) are critical for promoting compulsion-like alcohol drinking, where rats consume alcohol despite pairing with an aversive stimulus such as quinine, a paradigm considered to model compulsive aspects of human alcohol use disorders (AUDs). Here, we examined the impact of D-serine and D-cycloserine on this aversion-resistant alcohol intake (that persists despite adulteration with quinine) and consumption of quinine-free alcohol. Systemic D-serine reduced aversion-resistant alcohol drinking, without altering consumption of quinine-free alcohol or saccharin with or without quinine. Importantly, D-serine within the NAcore but not the dorsolateral striatum also selectively reduced aversion-resistant alcohol drinking. In addition, D-serine inhibited EPSCs evoked at -70 mV in vitro by optogenetic stimulation of mPFC-NAcore terminals in alcohol-drinking rats, similar to reported effects of the NMDAR blocker AP5. Further, D-serine preexposure occluded AP5 inhibition of mPFC-evoked EPSCs, suggesting that D-serine reduced EPSCs by inhibiting HA-NMDARs. Systemic D-cycloserine also selectively reduced intake of quinine-adulterated alcohol, and D-cycloserine inhibited NAcore HA-NMDARs in vitro. Our results indicate that HA-NMDAR modulators can reduce aversion-resistant alcohol drinking, and support testing of D-serine and D-cycloserine as immediately accessible, FDA-approved drugs to treat AUDs. PMID:25801502

  3. Cell Selective Conditional Null Mutations of Serine Racemase Demonstrate a Predominate Localization in Cortical Glutamatergic Neurons

    PubMed Central

    Benneyworth, Michael A.; Li, Yan; Basu, Alo C.; Bolshakov, Vadim Y.

    2016-01-01

    D-Serine, which is synthesized by the enzyme serine racemase (SR), is a co-agonist at the N-methyl-D-aspartate receptor (NMDAR). Crucial to an understanding of the signaling functions of D-serine is defining the sites responsible for its synthesis and release. In order to quantify the contributions of astrocytes and neurons to SR and D-serine localization, we used recombinant DNA techniques to effect cell type selective suppression of SR expression in astrocytes (aSRCKO) and in forebrain glutamatergic neurons (nSRCKO). The majority of SR is expressed in neurons: SR expression was reduced by ~65% in nSRCKO cerebral cortex and hippocampus, but only ~15% in aSRCKO as quantified by western blots. In contrast, nSRCKO is associated with only modest decreases in D-serine levels as quantified by HPLC, whereas D-serine levels were unaffected in aSRCKO mice. Liver expression of SR was increased by 35% in the nSRCKO, suggesting a role for peripheral SR in the maintenance of brain D-serine. Electrophysiologic studies of long-term potentiation (LTP) at the Schaffer collateral–CA1 pyramidal neuron synapse revealed no alterations in the aSRCKO mice versus wild-type. LTP induced by a single tetanic stimulus was reduced by nearly 70% in the nSRCKO mice. Furthermore, the mini-excitatory post-synaptic currents mediated by NMDA receptors but not by AMPA receptors were significantly reduced in nSRCKO mice. Our findings indicate that in forebrain, where D-serine appears to be the endogenous co-agonist at NMDA receptors, SR is predominantly expressed in glutamatergic neurons, and co-release of glutamate and D-serine is required for optimal activation of post-synaptic NMDA receptors. PMID:22362148

  4. Crystal structure of a homolog of mammalian serine racemase from Schizosaccharomyces pombe.

    PubMed

    Goto, Masaru; Yamauchi, Takae; Kamiya, Nobuo; Miyahara, Ikuko; Yoshimura, Tohru; Mihara, Hisaaki; Kurihara, Tatsuo; Hirotsu, Ken; Esaki, Nobuyoshi

    2009-09-18

    D-serine is an endogenous coagonist for the N-methyl-D-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5'-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of L-serine to yield D-serine and vice versa. The enzyme also catalyzes the dehydration of D- and L-serine. Both reactions are enhanced by Mg.ATP in vivo. We have determined the structures of the following three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe: the wild-type enzyme, the wild-type enzyme in the complex with an ATP analog, and the modified enzyme in the complex with serine at 1.7, 1.9, and 2.2 A resolution, respectively. On binding of the substrate, the small domain rotates toward the large domain to close the active site. The ATP binding site was identified at the domain and the subunit interface. Computer graphics models of the wild-type enzyme complexed with L-serine and D-serine provided an insight into the catalytic mechanisms of both reactions. Lys-57 and Ser-82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique "lysino-D-alanyl" residue at the active site, also exhibits catalytic activities. The crystal-soaking experiment showed that the substrate serine was actually trapped in the active site of the modified enzyme, suggesting that the lysino-D-alanyl residue acts as a catalytic base in the same manner as inherent Lys-57 of the wild-type enzyme. PMID:19640845

  5. The collision between positronium (Ps) and muonium (Mu)

    NASA Astrophysics Data System (ADS)

    Ray, Hasi; De, Rina

    2015-06-01

    The collision between a positronium(Ps) and a muonium(Mu) is studied for the first time using the static-exchange model and considering the system as a four-centerCoulomb problem in the centerof mass frame. An exact analysis is made to find the s-wave elastic phase-shifts, the scattering-lengths for both singlet and triplet channels, the integrated/total elastic cross section and the quenching cross section due to orthoto para conversion of Ps and the conversion ratio.

  6. iPS cell modeling of cardiometabolic diseases.

    PubMed

    Nakamura, Kenta; Hirano, Ken-ichi; Wu, Sean M

    2013-02-01

    Cardiometabolic diseases encompass simple monogenic enzyme deficiencies with well-established pathogenesis and clinical outcomes to complex polygenic diseases such as the cardiometabolic syndrome. The limited availability of relevant human cell types such as cardiomyocytes has hampered our ability to adequately model and study pathways or drugs relevant to these diseases in the heart. The recent discovery of induced pluripotent stem (iPS) cell technology now offers a powerful opportunity to establish translational platforms for cardiac disease modeling, drug discovery, and pre-clinical testing. In this article, we discuss the excitement and challenges of modeling cardiometabolic diseases using iPS cell and their potential to revolutionize translational research. PMID:23070616

  7. Physiologically generated presenilin 1 lacking exon 8 fails to rescue brain PS1−/− phenotype and forms complexes with wildtype PS1 and nicastrin

    PubMed Central

    Brautigam, Hannah; Moreno, Cesar L.; Steele, John W.; Bogush, Alexey; Dickstein, Dara L.; Kwok, John B.J.; Schofield, Peter R.; Thinakaran, Gopal; Mathews, Paul M.; Hof, Patrick R.; Gandy, Sam; Ehrlich, Michelle E.

    2015-01-01

    The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer’s disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1∆exon8). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1∆exon8 reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1∆exon8 did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1∆exon8 is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1∆exon8 in vivo by crossing PS1∆exon8 transgenics with either PS1-null or Dutch APPE693Q mice. As a control, we crossed APPE693Q with mice expressing a deletion in an adjacent exon (PS1∆exon9). PS1∆exon8 did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1∆exon8 interacts with nicastrin, participating in the γ–secretase complex formation. These data support that catalytically inactive PS1∆exon8 is generated physiologically and participates in protein-protein interactions. PMID:26608390

  8. Physiologically generated presenilin 1 lacking exon 8 fails to rescue brain PS1-/- phenotype and forms complexes with wildtype PS1 and nicastrin.

    PubMed

    Brautigam, Hannah; Moreno, Cesar L; Steele, John W; Bogush, Alexey; Dickstein, Dara L; Kwok, John B J; Schofield, Peter R; Thinakaran, Gopal; Mathews, Paul M; Hof, Patrick R; Gandy, Sam; Ehrlich, Michelle E

    2015-01-01

    The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1(∆exon8)). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1(∆exon8) reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1(∆exon8) is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1(∆exon8) in vivo by crossing PS1(∆exon8) transgenics with either PS1-null or Dutch APP(E693Q) mice. As a control, we crossed APP(E693Q) with mice expressing a deletion in an adjacent exon (PS1(∆exon9)). PS1(∆exon8) did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation. These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions. PMID:26608390

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

    NASA Astrophysics Data System (ADS)

    Kishimoto, Naoki; Harayama, Manami; Ohno, Koichi

    2016-05-01

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

  10. Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus.

    PubMed

    Prokesová, L; Potuzníková, B; Potempa, J; Zikán, J; Radl, J; Hachová, L; Baran, K; Porwit-Bobr, Z; John, C

    1992-02-15

    The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses--IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of Mr (approx. 41,000 to less than 12,400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus that may protect bacteria against the defence mechanisms of the host. PMID:1372285

  11. Regulating the regulators: serine/arginine-rich proteins under scrutiny.

    PubMed

    Risso, Guillermo; Pelisch, Federico; Quaglino, Ana; Pozzi, Berta; Srebrow, Anabella

    2012-10-01

    Serine/arginine-rich (SR) proteins are among the most studied splicing regulators. They constitute a family of evolutionarily conserved proteins that, apart from their initially identified and deeply studied role in splicing regulation, have been implicated in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export and mRNA translation. Remarkably, this list of SR protein activities seems far from complete, as unexpected functions keep being unraveled. An intriguing aspect that awaits further investigation is how the multiple tasks of SR proteins are concertedly regulated within mammalian cells. In this article, we first discuss recent findings regarding the regulation of SR protein expression, activity and accessibility. We dive into recent studies describing SR protein auto-regulatory feedback loops involving different molecular mechanisms such asunproductive splicing, microRNA-mediated regulation and translational repression. In addition, we take into account another step of regulation of SR proteins, presenting new findings about a variety of post-translational modifications by proteomics approaches and how some of these modifications can regulate SR protein sub-cellular localization or stability. Towards the end, we focus in two recently revealed functions of SR proteins beyond mRNA biogenesis and metabolism, the regulation of micro-RNA processing and the regulation of small ubiquitin-like modifier (SUMO) conjugation. PMID:22941908

  12. MBL-associated serine proteases (MASPs) and infectious diseases.

    PubMed

    Beltrame, Marcia H; Boldt, Angelica B W; Catarino, Sandra J; Mendes, Hellen C; Boschmann, Stefanie E; Goeldner, Isabela; Messias-Reason, Iara

    2015-09-01

    The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases. PMID:25862418

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process. PMID:25577345

  15. 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.

  16. 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

  17. Effects of Chronic D-Serine Elevation on Animal Models of Depression and Anxiety-Related Behavior.

    PubMed

    Otte, David-Marian; Barcena de Arellano, Maria Luisa; Bilkei-Gorzo, Andras; Albayram, Onder; Imbeault, Sophie; Jeung, Haang; Alferink, Judith; Zimmer, Andreas

    2013-01-01

    NMDA receptors are activated after binding of the agonist glutamate to the NR2 subunit along with a co-agonist, either L-glycine or D-serine, to the NR1 subunit. There is substantial evidence to suggest that D-serine is the most relevant co-agonist in forebrain regions and that alterations in D-serine levels contribute to psychiatric disorders. D-serine is produced through isomerization of L-serine by serine racemase (Srr), either in neurons or in astrocytes. It is released by astrocytes by an activity-dependent mechanism involving secretory vesicles. In the present study we generated transgenic mice (SrrTg) expressing serine racemase under a human GFAP promoter. These mice were biochemically and behaviorally analyzed using paradigms of anxiety, depression and cognition. Furthermore, we investigated the behavioral effects of long-term administration of D-serine added to the drinking water. Elevated brain D-serine levels in SrrTg mice resulted in specific behavioral phenotypes in the forced swim, novelty suppression of feeding and olfactory bulbectomy paradigms that are indicative of a reduced proneness towards depression-related behavior. Chronic dietary D-serine supplement mimics the depression-related behavioral phenotype observed in SrrTg mice. Our results suggest that D-serine supplementation may improve mood disorders. PMID:23805296

  18. DEVELOPMENT OF A HIGH THROUGHPUT METHOD FOR THE DETERMINATION OF PHARMACOLOGICAL LEVELS OF PLASMA D-SERINE

    PubMed Central

    Alt, Jesse; Rojas, Camilo; Wozniak, Krystyna; Wu, Ying; Ferraris, Dana; Tsukamoto, Takashi; Slusher, Barbara

    2011-01-01

    D-serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, D-serine has to be administered at high doses in order to observe clinical effects. This is in large part due to D-serine undergoing oxidation by D-amino acid oxidase (DAAO) before it reaches the brain. Consequently, co-administration of D-serine with a DAAO inhibitor has been suggested as a way to lower the dose of D-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma D-serine levels after oral co-administration. Current HPLC-based methodologies to measure D-serine in plasma are time consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well-format assay to monitor D-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate D-serine from plasma followed by quantitation of D-serine using the DAAO catalyzed reaction. Plasma D-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker. PMID:21889486

  19. 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

  20. 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

  1. 3Ps, Task-Based Learning, and the Japanese Learner.

    ERIC Educational Resources Information Center

    Tanasarnsanee, Mika

    2002-01-01

    Summarizes the findings of a work in progress that attempted to investigate to what extent task-based learning was more effective than the 3Ps approach in the teaching of Japanese as a foreign language in Thailand. (Author/VWL)

  2. 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.

  3. Spectroscopic Classification of PS15cwx as a Type Ia

    NASA Astrophysics Data System (ADS)

    Kilpatrick, Charles; Milne, Peter; Andrews, Jennifer; Smith, Nathan

    2015-11-01

    The AZTEC (Arizona Transient Exploration and Characterization) collaboration reports an optical spectrum of PS15cwx (ATEL #8299) obtained on UT 2015 Nov. 19.33 with the 2.3m Bok telescope (+ Boller & Chivens spectrograph) on Kitt Peak, Arizona.

  4. A 100 ps gated x-ray spectrometer

    SciTech Connect

    Walsh, P.J.; Blake, R.L.; Caldwell, S.; Hockaday, M.; Chrien, R.; Smith, R.C.

    1995-09-01

    Material opacities are of interest in many fields. We have developed a Bragg reflection spectrometer that is gated for imaging samples in a laser heated environment for opacity measurement. A micro-channel plate is coated with a photocathode material and a fast pulse is launched across it. Electrons are converted to photons in a phosphor and recorded on film. Optical gate pulse widths of 100 ps are achieved. Some optical pulse width and sensitivity enhancements are noted at launch and termination. Events of interest are 200 ps long. The framing window is approximately 250 ps in length. Timing jitter is a problem. The instrument timing networks have been examined, and the source of jitter is still unknown. Timing to 50 ps resolution is desired. Close in proximity to the laser-driven event leads to complications in shielding from hard x-rays, hot electrons and shock-driven damage. High Z materials provide shielding from hard x-rays. Magnets screen out hot electrons produced by laser-matter interactions Filters provide energy fiducials. PCD`s provide high resolution timing measurements. Data is recorded on film in a specially designed film pack. The instrument is designed to be used in the NOVA Laser Facility at Lawrence Livermore National Laboratory.

  5. 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.

  6. 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…

  7. 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.

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

    PubMed

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

    2015-07-01

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

  9. 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…

  10. The Pan-STARRS PS4 telescope suite

    NASA Astrophysics Data System (ADS)

    Morgan, Jeffrey S.; Burgett, William; Teran, Jose U.

    2008-07-01

    The Pan-STARRS project is planning to build a suite of four telescopes (PS4) on the summit of Mauna Kea at the site of the current University of Hawaii 2.2-m telescope. These telescopes will have the goal of surveying the entire sky visible from a single site in 5 colors (g, r, i, z, and y) on the time scale of approximately 1 week at a spatial resolution limited primarily by the quality of the site. To accomplish this task each of these four telescopes will be equipped with a Giga-Pixel camera, a camera shutter, and a 6 filter mechanism. A prototype telescope for this project (PS1) that includes all of these subsystems is already under going commissioning. The project is currently involved in developing the Environmental Impact Statement that is required to build the PS4 array of telescopes. We give an overview here of the scientific goals, the instrumentation package, the telescope design, and the enclosure design for the PS4 system.

  11. Small Molecule Activation of PKM2 in Cancer Cells Induces Serine Auxotrophy

    PubMed Central

    Kung, Charles; Hixon, Jeff; Choe, Sung; Marks, Kevin; Gross, Stefan; Murphy, Erin; DeLaBarre, Byron; Cianchetta, Giovanni; Sethumadhavan, Shalini; Wang, Xiling; Yan, Shunqi; Gao, Yi; Fang, Cheng; Wei, Wentao; Jiang, Fan; Wang, Shaohui; Qian, Kevin; Saunders, Jeff; Driggers, Ed; Woo, Hin Koon; Kunii, Kaiko; Murray, Stuart; Yang, Hua; Yen, Katharine; Liu, Wei; Cantley, Lewis C.; Vander Heiden, Matthew G.; Su, Shinsan M.; Jin, Shengfang; Salituro, Francesco G.; Dang, Lenny

    2013-01-01

    SUMMARY Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress. PMID:22999886

  12. Small molecule activation of PKM2 in cancer cells induces serine auxotrophy.

    PubMed

    Kung, Charles; Hixon, Jeff; Choe, Sung; Marks, Kevin; Gross, Stefan; Murphy, Erin; DeLaBarre, Byron; Cianchetta, Giovanni; Sethumadhavan, Shalini; Wang, Xiling; Yan, Shunqi; Gao, Yi; Fang, Cheng; Wei, Wentao; Jiang, Fan; Wang, Shaohui; Qian, Kevin; Saunders, Jeff; Driggers, Ed; Woo, Hin Koon; Kunii, Kaiko; Murray, Stuart; Yang, Hua; Yen, Katharine; Liu, Wei; Cantley, Lewis C; Vander Heiden, Matthew G; Su, Shinsan M; Jin, Shengfang; Salituro, Francesco G; Dang, Lenny

    2012-09-21

    Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress. PMID:22999886

  13. Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity and role in virulence

    PubMed Central

    Ruiz-Perez, Fernando; Nataro, James P.

    2013-01-01

    Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now been also identified in Citrobacter rodentium, Salmonella, and Edwarsiella species. Here, we focus on proteins belonging to the Serine Protease Autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs’ contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis. PMID:23689588

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

    PubMed

    Sugimoto, Hiroshi; Kakehi, Masaaki; Jinno, Fumihiro

    2015-10-15

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

  15. D-Amino-Acid Oxidase Inhibition Increases D-Serine Plasma Levels in Mouse But not in Monkey or Dog.

    PubMed

    Rojas, Camilo; Alt, Jesse; Ator, Nancy A; Thomas, Ajit G; Wu, Ying; Hin, Niyada; Wozniak, Krystyna; Ferraris, Dana; Rais, Rana; Tsukamoto, Takashi; Slusher, Barbara S

    2016-05-01

    D-serine has been shown to improve positive, negative, and cognitive symptoms when used as add-on therapy for the treatment of schizophrenia. However, D-serine has to be administered at high doses to observe clinical effects. This is thought to be due to D-serine undergoing oxidation by D-amino-acid oxidase (DAAO) before it reaches the brain. Consequently, co-administration of D-serine with a DAAO inhibitor could be a way to lower the D-serine dose required to treat schizophrenia. Early studies in rodents to evaluate this hypothesis showed that concomitant administration of structurally distinct DAAO inhibitors with D-serine enhanced plasma and brain D-serine levels in rodents compared with administration of D-serine alone. In the present work we used three potent DAAO inhibitors and confirmed previous results in mice. In a follow-up effort, we evaluated plasma D-serine levels in monkeys after oral administration of D-serine in the presence or absence of these DAAO inhibitors. Even though the compounds reached steady state plasma concentrations exceeding their Ki values by >60-fold, plasma D-serine levels remained the same as those in the absence of DAAO inhibitors. Similar results were obtained with dogs. In summary, in contrast to rodents, DAAO inhibition in monkeys and dogs did not influence the exposure to exogenously administered D-serine. Results could be due to differences in D-serine metabolism and/or clearance mechanisms and suggest that the role of DAAO in the metabolism of D-serine is different across species. These data provide caution regarding the utility of DAAO inhibition for patients with schizophrenia. PMID:26471255

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

    PubMed

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

    2015-02-10

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

  17. Nuclear targeting of the serine protease granzyme A (fragmentin-1).

    PubMed

    Jans, D A; Briggs, L J; Jans, P; Froelich, C J; Parasivam, G; Kumar, S; Sutton, V R; Trapani, J A

    1998-09-01

    Cytolytic granule-mediated target cell killing is effected in part through synergistic action of the membrane-acting protein perforin and serine proteases such as granzymes A (GrA) or B (GrB). In the present study we examine GrA cellular entry and nuclear uptake in intact mouse myeloid FDC-P1 cells exposed to perforin using confocal laser scanning microscopy, as well as reconstitute GrA nuclear uptake in vitro. GrA alone was found to be able to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of perforin, it specifically accumulated in the cell nuclei, with maximal levels about 2.5 times those in the cytoplasm after 2. 5 hours. In vitro, GrA accumulated in the nucleus and nucleolus maximally to levels that were four- and sixfold, respectively, those in the cytoplasm. In contrast, the active form of the apoptotic cysteine protease CPP32 did not accumulate in nuclei in vitro. Nuclear/nucleolar import of GrA in vitro was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTPgammaS, but was dependent on exogenously added cytosol. Importantly, GrA was found to be able to accumulate in the nucleus of semi-intact cells in the presence of the nuclear envelope-permeabilizing detergent CHAPS, implying that the mechanism of nuclear accumulation was through binding to insoluble factors in the nucleus. GrB was found for the first time to be similar in this regard. The results support the contention that GrA and GrB accumulate in the nucleus through a novel nuclear import pathway, and that this is integral to induction of the nuclear changes associated with cytolytic granule-mediated apoptosis. PMID:9701563

  18. Physicochemical properties of alkaline serine proteases in alcohol.

    PubMed

    Chen, S T; Chen, S Y; Tu, C C; Chiou, S H; Wang, K T

    1995-05-01

    The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1 micrograms/ml and that of alcalase was 48.1 micrograms/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters, L-Ala-L-Ala-(D- or L-)Pro-L-Phe-OMe and L-Ala-L-Ala-(D- or L-)Ala-L-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents. PMID:7662108

  19. Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization

    PubMed Central

    2012-01-01

    Background Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT) and putative mitochondria SHMT (mSHMT), but their roles have not been fully investigated. Methods The presence of Plasmodium SHMT isoforms in the intra-erythrocytic stage was assessed based on their gene expression using reverse transcription PCR (RT-PCR). Localization studies of Plasmodium SHMT isoforms were performed by transfection of fluorescent-tagged gene constructs into P. falciparum and expressions of fluorescent fusion proteins in parasites were observed using a laser scanning confocal microscope. Genetic targeting through homologous recombination was used to study the essentiality of SHMT in Plasmodium spp. Results Semi-quantitative RT-PCR revealed the expression of these two genes throughout intra-erythrocytic development. Localization studies using P. falciparum expressing fluorescent-tagged SHMT showed that PfcSHMT-red fluorescent fusion protein (PfcSHMT-DsRed) is localized in the cytoplasm, while PfmSHMT-green fluorescent fusion protein (PfmSHMT-GFP) co-localized with Mitotracker™-labelled mitochondria as predicted. The essentiality of plasmodial cSHMT was inferred from transfection experiments where recovery of viable knock-out parasites was not achieved, unless complemented with a functional equivalent copy of shmt. Conclusions Distinct compartment localizations of PfSHMT were observed between cytoplasmic and mitochondrial isoforms, and evidence was provided for the indispensable role of plasmodial cSHMT indicating it as a valid target for development of novel anti-malarials. PMID:23173711

  20. 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.

  1. 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.

  2. Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells

    PubMed Central

    Singh, Nagendra S.; Rutkowska, Ewelina; Plazinska, Anita; Khadeer, Mohammed; Moaddel, Ruin; Jozwiak, Krzysztof; Bernier, Michel; Wainer, Irving W.

    2016-01-01

    D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer’s and Parkinsons’ diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac)-dehydronorketamine and (2S,6S)-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine. PMID:27096720

  3. 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 34.5kDa. 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. PMID:23537874

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

    PubMed Central

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

    2015-01-01

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

  5. CSF d-serine concentrations are similar in Alzheimer's disease, other dementias, and elderly controls.

    PubMed

    Biemans, Elisanne A L M; Verhoeven-Duif, Nanda M; Gerrits, Johan; Claassen, Jurgen A H R; Kuiperij, H Bea; Verbeek, Marcel M

    2016-06-01

    Cerebrospinal fluid (CSF) levels of d-serine were recently reported as a potential new biomarker for Alzheimer's disease (AD), showing a perfect distinction between AD patients and healthy controls. In this study, we aimed to confirm these results and extend these previous findings to dementia with Lewy bodies and frontotemporal dementia. d-Serine levels in CSF of 29 AD patients, 8 dementia with Lewy bodies patients, 14 frontotemporal dementia patients, and 28 nondemented controls were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. In contrast to previous findings, in our study CSF d-serine levels were only slightly increased in AD patients compared with controls. CSF d-serine in AD did not differ from other dementias and was also not correlated to mini-mental state examination-scores. Owing to the large overlap of d-serine levels, we conclude that CSF d-serine is neither a suitable biomarker for AD nor for cognitive decline. PMID:27143438

  6. IGF-1 mediated phosphorylation of specific IRS-1 serines in Ames dwarf fibroblasts is associated with longevity

    PubMed Central

    Papaconstantinou, John; Hsieh, Ching-Chyuan

    2015-01-01

    Insulin/IGF-1 signaling involves phosphorylation/dephosphorylation of serine/threonine or tyrosine residues of the insulin receptor substrate (IRS) proteins and is associated with hormonal control of longevity determination of certain long-lived mice. The stimulation of serine phosphorylations by IGF-1 suggests there is insulin/IGF-1 crosstalk that involves the phosphorylation of the same serine residues. By this mechanism, insulin and IGF-1 mediated phosphorylation of specific IRS-1 serines could play a role in longevity determination. We used fibroblasts from WT and Ames dwarf mice to examine whether: (a) IGF-1 stimulates phosphorylation of IRS-1 serines targeted by insulin; (b) the levels of serine phosphorylation differ in WT vs. Ames fibroblasts; and (c) aging affects the levels of these serine phosphorylations which are altered in the Ames dwarf mutant. We have shown that IRS-1 is a substrate for IGF-1 induced phosphorylation of Ser307, Ser612, Ser636/639, and Ser1101; that the levels of phosphorylation of these serines are significantly lower in Ames vs. WT cells; that IGF-1 mediated phosphorylation of these serines increases with age in WT cells. We propose that insulin/IGF-1 cross talk and level of phosphorylation of specific IRS-1 serines may promote the Ames dwarf longevity phenotype. PMID:26474286

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

    PubMed

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

    2016-03-30

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

  8. N-methyl-d-aspartate receptor coagonist d-serine suppresses intake of high-preference food.

    PubMed

    Sasaki, Tsutomu; Kinoshita, Yoshihiro; Matsui, Sho; Kakuta, Shigeru; Yokota-Hashimoto, Hiromi; Kinoshita, Kuni; Iwasaki, Yusaku; Kinoshita, Toshio; Yada, Toshihiko; Amano, Naoji; Kitamura, Tadahiro

    2015-09-01

    d-Serine is abundant in the forebrain and physiologically important for modulating excitatory glutamatergic neurotransmission as a coagonist of synaptic N-methyl-d-aspartate (NMDA) receptor. NMDA signaling has been implicated in the control of food intake. However, the role of d-serine on appetite regulation is unknown. To clarify the effects of d-serine on appetite, we investigated the effect of oral d-serine ingestion on food intake in three different feeding paradigms (one-food access, two-food choice, and refeeding after 24-h fasting) using three different strains of male mice (C57Bl/6J, BKS, and ICR). The effect of d-serine was also tested in leptin signaling-deficient db/db mice and sensory-deafferented (capsaicin-treated) mice. The expression of orexigenic neuropeptides [neuropeptide Y (Npy) and agouti-related protein (Agrp)] in the hypothalamus was compared in fast/refed experiments. Conditioned taste aversion for high-fat diet (HFD) was tested in the d-serine-treated mice. Under the one-food-access paradigm, some of the d-serine-treated mice showed starvation, but not when fed normal chow. HFD feeding with d-serine ingestion did not cause aversion. Under the two-food-choice paradigm, d-serine suppressed the intake of high-preference food but not normal chow. d-Serine also effectively suppressed HFD intake but not normal chow in db/db mice and sensory-deafferented mice. In addition, d-serine suppressed normal chow intake after 24-h fasting despite higher orexigenic gene expression in the hypothalamus. d-Serine failed to suppress HFD intake in the presence of L-701,324, the selective and full antagonist at the glycine-binding site of the NMDA receptor. Therefore, d-serine suppresses the intake of high-preference food through coagonism toward NMDA receptors. PMID:26157056

  9. Expression of fibroblast growth factor receptor 1, fibroblast growth factor 2, phosphatidyl inositol 3 phosphate kinase and their clinical and prognostic significance in early and advanced stage of squamous cell carcinoma of the lung

    PubMed Central

    Usul Afsar, Cigdem; Sahin, Berksoy; Gunaldi, Meral; Kılıc Bagir, Emine; Gumurdulu, Derya; Burgut, Refik; Erkisi, Melek; Kara, Ismail Oguz; Paydas, Semra; Karaca, Feryal; Ercolak, Vehbi

    2015-01-01

    Aim: Non-small cell lung carcinoma is the leading cause of cancer related to death in the world. Squamous cell lung carcinoma (SqCLC) is the second most frequent histological subtype of lung carcinomas. Recently, growth factors, growth factor receptors, and signal transduction system-related gene amplifications and mutations are extensively under investigation to estimate the prognosis and to develop individualized therapies in SqCLC. In this study, besides the signal transduction molecule phosphatidyl inositol-3-phosphate kinase (IP3K) p110α, we explored the expressions of fibroblast growth factor 2 (FGF2) and receptor-1 (FGFR1) in tumor tissue and also their clinical and prognostic significance in patients with early/advanced SqCLC. Materials and methods: From 2005 to 2013, 129 patients (23 early, 106 advanced disease) with a histopathological SqCLC diagnosis were selected from the hospital files of Cukurova University Medical Faculty for this study. Two independent pathologists evaluated FGFR1, FGF2, and PI3K (p110α) expressions in both tumor and stromal tissues from 99 of the patients with sufficient tissue samples, using immunohistochemistry. Considering survival analysis separately for patients with both early and advanced stage diseases, the relationship between the clinical features of the patients and expressions were evaluated by univariate and multivariate analyses. Results: FGFR1 expression was found to be low in 59 (60%) patients and high in 40 (40%) patients. For FGF2; 12 (12%) patients had high, 87 (88%) patients had low expression and for IP3K; 31 (32%) patients had high and 66 (68%) patients had low expressions. In univariate analysis, overall survival (OS) was significantly associated with stage of the disease and the performance status of the patient (P<0.0001 and P<0.001). There was no significant difference in OS of the patients with either low or high expressions of FGFR1, FGF2, and IP3K. When the patients with early or advanced stage disease were separately taken into consideration, the relationship did not differ, either. Any of FGFR1, FGF2 or IP3K expressions was not found predictive for the treatment of early or advanced staged patients. On the other hand, the expressions of both FGFR1 and FGF2 were significantly different with respect to smoking, scar of tuberculosis and scar of radiotherapy (P=0.002; P=0.06 and P=0.05, respectively). Discussion: There has not been identified an effective individualized treatment for SqCLC yet. Therefore, in order to be able to develop such a treatment in the future, it is essential to identify the genetic abnormalities that are responsible for the biological behaviors and carcinogenesis of SqCLC. Although we could not show the prognostic and predictive significance of FGFR1, FGF2 and IP3K expressions in SqCLC, we determined the expression rates of FGFR1, FGF2 and IP3K as a reference for Turkish patients. In conclusion, we want to put some emphasis on the fact that, pulmonary fibrosis which is a late complication of radiotherapy at stage III disease, and the scar of tuberculosis could be associated with FGFR1 and FGF2 expressions. PMID:26617686

  10. Spectra and relaxation dynamics of the pseudohalide (PS) vibrational bands for Ru(bpy)2(PS)2 complexes, PS = CN, NCS and N3

    NASA Astrophysics Data System (ADS)

    Compton, Ryan; Gerardi, Helen K.; Weidinger, Daniel; Brown, Douglas J.; Dressick, Walter J.; Heilweil, Edwin J.; Owrutsky, Jeffrey C.

    2013-08-01

    Static and transient infrared spectroscopy were used to investigate cis-Ru(bpy)2(N3)2 (bpy = 2,2‧-bipyridine), cis-Ru(bpy)2(NCS)2, and cis-Ru(bpy)2(CN)2 in solution. The NC stretching IR band for cis-Ru(bpy)2(NCS)2 appears at higher frequency (∼2106 cm-1 in DMSO) than for the free NCS- anion while the IR bands for the azide and cyanide complexes are closer to those of the respective free anions. The vibrational energy relaxation (VER) lifetime for the azide complex is found to be much shorter (∼5 ps) than for either the NCS or CN species (both ∼70 ps in DMSO) and the lifetimes resemble those for each corresponding free anion in solution. However, for cis-Ru(bpy)2(N3)2, it is determined that the transition frequency depends more on the solvent than the VER lifetime implying that intramolecular vibrational relaxation is predominant over solvent energy-extracting interactions. These results are compared to the behavior of other related metal complexes in solution.

  11. Trapping kinetics in isolated cyanobacterial PS I complexes

    NASA Astrophysics Data System (ADS)

    Slavov, Chavdar; El-Mohsnawy, Eithar; Rögner, Matthias; Holzwarth, Alfred R.

    2009-02-01

    The excitation energy trapping and the role of the 'red' chlorophyll (Chl) states in the photosystem (PS) I monomers and trimers from the cyanobacterium Thermosynechococcus elongatus were studied. We demonstrate the adequacy of the "charge recombination" model for the trapping kinetics. On the basis of this model the reaction center excited state can be resolved. The overall kinetics is shown to be trap-limited even though the presence of the 'red' Chls induces a substantial slowing down (˜60%) of the trapping. Two kinetically different 'red' Chl pools were resolved. Both of these 'red' pools originate from the same groups of pigments in either of the two aggregation states. This indicates that careful isolation does not disturb substantially the 'red' Chls and we can exclude their location at the monomer-monomer interface. Acceleration of the secondary electron transfer step in the studied complexes as compared to PS I from mesophilic organisms is observed.

  12. Four-frame gated optical imager with 120-ps resolution

    SciTech Connect

    Young, P.E.; Hares, J.D.; Kilkenny, J.D.; Phillion, D.W.; Campbell, E.M.

    1988-04-01

    In this paper we describe the operation and applications of a framing camera capable of four separate two-dimensional images with each frame having a 120-ps gate width. Fast gating of a single frame is accomplished by using a wafer image intensifier tube in which the cathode is capacitively coupled to an external electrode placed outside of the photocathode of the tube. This electrode is then pulsed relative to the microchannel plate by a narrow (120 ps), high-voltage pulse. Multiple frames are obtained by using multiple gated tubes which share a single bias supply and pulser with relative gate times selected by the cable lengths between the tubes and the pulser. A beamsplitter system has been constructed which produces a separate image for each tube from a single scene. Applications of the framing camera to inertial confinement fusion experiments are discussed.

  13. Functional Characterization of Calcineurin Homologs PsCNA1/PsCNB1 in Puccinia striiformis f. sp. tritici Using a Host-Induced RNAi System

    PubMed Central

    Zhang, Hong; Guo, Jun; Voegele, Ralf T.; Zhang, Jinshan; Duan, Yinghui; Luo, Huaiyong; Kang, Zhensheng

    2012-01-01

    Calcineurin plays a key role in morphogenesis, pathogenesis and drug resistance in most fungi. However, the function of calcineurin genes in Puccinia striiformis f. sp. tritici (Pst) is unclear. We identified and characterized the calcineurin genes PsCNA1 and PsCNB1 in Pst. Phylogenetic analyses indicate that PsCNA1 and PsCNB1 form a calcium/calmodulin regulated protein phosphatase belonging to the calcineurin heterodimers composed of subunits A and B. Quantitative RT-PCR analyses revealed that both PsCNA1 and PsCNB1 expression reached their maximum in the stage of haustorium formation, which is one day after inoculation. Using barely stripe mosaic virus (BSMV) as a transient expression vector in wheat, the expression of PsCNA1 and PsCNB1 in Pst was suppressed, leading to slower extension of fungal hyphae and reduced production of urediospores. The immune-suppressive drugs cyclosporin A and FK506 markedly reduced the germination rates of urediospores, and when germination did occur, more than two germtubes were produced. These results suggest that the calcineurin signaling pathway participates in stripe rust morphogenetic differentiation, especially the formation of haustoria during the early stage of infection and during the production of urediospores. Therefore PsCNA1 and PsCNB1 can be considered important pathogenicity genes involved in the wheat-Pst interaction. PMID:23139840

  14. 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.

  15. An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei

    PubMed Central

    Moss, Catherine X.; Brown, Elaine; Hamilton, Alana; Van der Veken, Pieter; Augustyns, Koen; Mottram, Jeremy C.

    2015-01-01

    The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival. PMID:25816352

  16. Antimicrobial activity of a honeybee (Apis cerana) venom Kazal-type serine protease inhibitor.

    PubMed

    Kim, Bo Yeon; Lee, Kwang Sik; Zou, Feng Ming; Wan, Hu; Choi, Yong Soo; Yoon, Hyung Joo; Kwon, Hyung Wook; Je, Yeon Ho; Jin, Byung Rae

    2013-12-15

    Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or trypsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor. PMID:24076031

  17. Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease

    PubMed Central

    Ibrahim, Yara; Eini, Amir; Naor, Ronit; Rosen, Graciela; Bachrach, Gilad

    2014-01-01

    Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61–65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55–101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96–113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55–65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections. PMID:25357190

  18. EphrinBs regulate D-serine synthesis and release in astrocytes

    PubMed Central

    Zhuang, Zhiye; Yang, Bing; Theus, Michelle H; Sick, Justin T; Bethea, John R; Sick, Thomas J; Liebl, Daniel J

    2010-01-01

    There is growing evidence that astrocytes play critical roles in neuron-glial interactions at the synapse. Astrocytes are believed to regulate pre- and post-synaptic structures and functions, in part, by the release of gliotransmitters such as glutamate, ATP and D-serine; however, little is known of how neurons and astrocytes communicate to regulate these processes. Here, we investigated a family of transmembrane proteins called ephrins and Eph receptors that are expressed in the synapse and are known to regulate synaptic transmission and plasticity. In addition to their presence on CA1 hippocampal neurons, we determined that ephrins and Eph receptors are also expressed on hippocampal astrocytes. Stimulation of hippocampal astrocytes with soluble ephrinB3, known to be expressed on CA1 post-synaptic dendrites, enhanced D-serine synthesis and release in culture. Conversely, ephrinB3 had no effect on D-serine release from astrocytes deficient in EphB3 and EphA4, which are the primary receptors for ephrinB3. Eph receptors mediate this response through interactions with PICK1 and by dephosphorylating PKCα to activate the conversion of L-serine to D-serine by serine racemase. These findings are supported in vivo, where reduced D-serine levels and synaptic transmissions are observed in the absence of EphB3 and EphA4. These data support a role for ephrins and Eph receptors in regulating astrocyte gliotransmitters, which may have important implications on synaptic transmission and plasticity. PMID:21106840

  19. 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).

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  1. A new shape resonance in the Ps^- system

    NASA Astrophysics Data System (ADS)

    Ho, Yew Kam

    2012-06-01

    There have been continues experimental and theoretical investigations on the positronium negative ion (Ps^-), one of the simplest three-lepton systems interacting through Coulomb forces. In the present work, we use highly correlated Hylleraas wave functions up to N=1078 terms together with employing the complex-coordinate rotation method [1] to investigate resonances in the Ps^- system. We have located a new S-wave shape resonance lying above the Ps (n=2) threshold. Our preliminary results for the resonance parameters are Er = - 0.0498788 a.u. and γ / 2 = 0.0139470 a.u., where Er and γ denote the resonance energy and width, respectively. This stabilized complex eigenvalue has never been reported in the literature, to the best of our knowledge. Here, by changing the mass of the positively charged particle from one unit of the electron mass to infinitely heavy, we have traced this resonance pole from the positronium negative ion to the hydrogen negative ion [2]. Detailed calculations will be presented at the meeting. [4pt] [1]. Y. K. Ho, Phys. Reports 99, 1 (1983) and references therein. [0pt] [2]. A. Burgers and E. Lindroth, Euro. Phys. J. D 10, 327 (2000).

  2. 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

  3. 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

  4. Anomalous high ionic conductivity of nanoporous -Li3PS4

    SciTech Connect

    Liu, Zengcai; Fu, Wujun; Payzant, E Andrew; Yu, Xiang; Wu, Zili; Dudney, Nancy J; Kiggans, Jim; Hong, Kunlun; Rondinone, Adam Justin; Liang, Chengdu

    2013-01-01

    Lithium-ion conducting solid electrolytes hold the promise for enabling high-energy battery chemistries and circumventing safety issues of conventional lithium batteries1-3. Achieving the combination of high ionic conductivity and broad electrochemical window in solid electrolytes is a grand challenge for the synthesis of battery materials. Herein we show an enhancement of room-temperature lithium-ion conductivity of 3 orders of magnitude by creating nanostructured Li3PS4. This material has a wide (5V) electrochemical window and superior chemical stability against lithium metal. The nanoporous structure of Li3PS4 reconciles two vital effects that enhance ionic conductivity: (1) The reduced dimension to nanometer-sized framework stabilizes the high conduction beta phase that occurs at elevated temperatures1,4; and (2) The high surface-to-bulk ratio of nanoporous -Li3PS4 promotes surface conduction5,6. Manipulating the ionic conductivity of solid electrolytes has far-reaching implications for materials design and synthesis in a broad range of applications such as batteries, fuel-cells, sensors, photovoltaic systems, and so forth3,7.

  5. X-ray photoelectron spectroscopy of CdPS3

    NASA Astrophysics Data System (ADS)

    Curró, G. M.; Grasso, V.; Silipigni, L.

    1998-12-01

    Single CdPS3 crystals have been investigated at room temperature by means of x-ray photoemission spectroscopy (XPS) and x-ray induced Auger spectroscopy. Special attention has been paid to the Cd 3d and 4d and S and P 2p core levels and to the Cd Auger M4,5N4,5N4,5 transitions. As far as the XPS spectra are concerned, a single-peak structure has been observed for all the core levels investigated. Moreover, the Cd 4d levels are discrete and well localized, in good agreement with the transition-metal weakly interacting model according to which the transition metal-sulfur bond is weak. Further information about the nature of the cadmium-sulfur bond in CdPS3 was obtained from the calculated modified Auger parameter for Cd. A correlation has been observed between the change in the Cd modified Auger parameter in CdPS3 and the energy gap of this compound.

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    1988-08-01

    The human pS2 gene is specifically expressed under estrogen transcriptional control in a subclass of estrogen receptor-containing human breast cancer cells. The pS2 gene encodes an 84-amino acid protein that is secreted after signal peptide cleavage. The distribution of pS2 protein in normal human tissues was studied with antibodies to pS2; pS2 was specifically expressed and secreted by mucosa cells of the normal stomach antrum and body of both female and male individuals. Moreover, no estrogen receptor could be detected in these cells, indicating that pS2 gene expression is estrogen-independent in the stomach. The function of the pS2 protein in the gastrointestinal tract is unknown. However, the pS2 protein is similar in sequence to a porcine pancreatic protein that has been shown to inhibit gastrointestinal motility and gastric secretion.

  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. Identification of Serine/Threonine Kinase Substrates in the Human Pathogen Group B Streptococcus

    PubMed Central

    Silvestroni, Aurelio; Jewell, Kelsea A.; Lin, Wan-Jung; Connelly, James E.; Ivancic, Melanie M.; Tao, W. Andy; Rajagopal, Lakshmi

    2010-01-01

    All living organisms respond to changes in their internal and external environment for their survival and existence. Signaling is primarily achieved through reversible phosphorylation of proteins in both prokaryotes and eukaryotes. A change in the phosphorylation state of a protein alters its function to enable the control of cellular responses. A number of serine/threonine kinases regulate the cellular responses of eukaryotes. Although common in eukaryotes, serine/threonine kinases have only recently been identified in prokaryotes. We have described that the human pathogen Group B Streptococcus (GBS, Streptococcus agalactiae) encodes a single membrane-associated, serine/threonine kinase (Stk1) that is important for virulence of this bacterium. In this study, we used a combination of phosphopeptide enrichment and mass spectrometry to enrich and identify serine (S) and threonine (T) phosphopeptides of GBS. A comparison of S/T phosphopeptides identified from the Stk1 expressing strains to the isogenic stk1 mutant indicates that 10 proteins are potential substrates of the GBS Stk1 enzyme. Some of these proteins are phosphorylated by Stk1 in vitro and a site-directed substitution of the phosphorylated threonine to an alanine abolished phosphorylation of an Stk1 substrate. Collectively, these studies provide a novel approach to identify serine/threonine kinase substrates for insight into their signaling in human pathogens like GBS. PMID:19309132

  10. Identification of serine/threonine kinase substrates in the human pathogen group B streptococcus.

    PubMed

    Silvestroni, Aurelio; Jewell, Kelsea A; Lin, Wan-Jung; Connelly, James E; Ivancic, Melanie M; Tao, W Andy; Rajagopal, Lakshmi

    2009-05-01

    All living organisms respond to changes in their internal and external environment for their survival and existence. Signaling is primarily achieved through reversible phosphorylation of proteins in both prokaryotes and eukaryotes. A change in the phosphorylation state of a protein alters its function to enable the control of cellular responses. A number of serine/threonine kinases regulate the cellular responses of eukaryotes. Although common in eukaryotes, serine/threonine kinases have only recently been identified in prokaryotes. We have described that the human pathogen Group B Streptococcus (GBS, Streptococcus agalactiae) encodes a single membrane-associated, serine/threonine kinase (Stk1) that is important for virulence of this bacterium. In this study, we used a combination of phosphopeptide enrichment and mass spectrometry to enrich and identify serine (S) and threonine (T) phosphopeptides of GBS. A comparison of S/T phosphopeptides identified from the Stk1 expressing strains to the isogenic stk1 mutant indicates that 10 proteins are potential substrates of the GBS Stk1 enzyme. Some of these proteins are phosphorylated by Stk1 in vitro and a site-directed substitution of the phosphorylated threonine to an alanine abolished phosphorylation of an Stk1 substrate. Collectively, these studies provide a novel approach to identify serine/threonine kinase substrates for insight into their signaling in human pathogens like GBS. PMID:19309132

  11. Approaches to the simultaneous inactivation of metallo-and serine-β-lactamases

    PubMed Central

    Ganta, Sudhakar Reddy; Perumal, Senthil; Pagadala, Sundar Ram Reddy; Samuelsen, Ørjan; Spencer, James; Pratt, R. F.; Buynak, John D.

    2010-01-01

    A series of cephalosporin-derived reverse hydroxamates and oximes were prepared and evaluated as inhibitors of representative metallo- and serine-β-lactamases. The reverse hydroxamates showed submicromolar inhibition of the GIM-1 metallo-β-lactamase. With respect to interactions with the classes A, C, and D serine β-lactamases, as judged by their correspondingly low Km values, the reverse hydroxamates were recognized in a manner similar to the non-hydroxylated N-H amide side chains of the natural substrates of these enzymes. This indicates that, with respect to recognition in the active site of the serine β-lactamases, the O=C-NR-OH functionality can function as a structural isostere of the O=C-NR-H group, with the NO-H group presumably replacing the amide N-H group as a hydrogen bond donor to the appropriate backbone carbonyl oxygen of the protein. The reverse hydroxamates, however, displayed kcat values up to three orders of magnitude lower than the natural substrates, thus indicating substantial slowing of the hydrolytic action of these serine β-lactamases. Although the degree of inactivation is not yet enough to be clinically useful, these initial results are promising. The substitution of the amide N-H bond by N-OH may represent a useful strategy for the inhibition of other serine hydrolases. PMID:19243936

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-01

    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. PMID:25386732

  14. The Cutting Edge: Membrane Anchored Serine Protease Activities in the Pericellular Microenvironment

    PubMed Central

    Antalis, Toni M.; Buzza, Marguerite S.; Hodge, Kathryn M.; Hooper, John D.; Netzel-Arnett, Sarah

    2013-01-01

    Synopsis The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Recent studies have identified members of this family which contain amino- or carboxy-terminal domains that serve to tether the serine protease catalytic domain directly at the plasma membrane. These membrane anchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homeostasis. Substrates activated by membrane anchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies. This review discusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease. PMID:20507279

  15. 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. PMID:26790714

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

    PubMed

    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; Warren, J David; Fendt, Sarah-Maria; Asara, John M; DeNicola, Gina M; Lyssiotis, Costas A; Lairson, Luke L; Cantley, Lewis C

    2016-02-16

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

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

    PubMed Central

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

    2013-01-01

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

  18. 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

  19. 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. PMID:21949686

  20. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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…

  5. 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

  6. 7 CFR 1753.26 - Plans and specifications (P&S).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... details of the building to be constructed using loan funds. In particular, the preliminary P&S shall... subject to RUS approval. (b) The borrower shall prepare P&S for construction of all buildings. Each set of P&S shall include: (1) RUS Contract Form 257, Contract to Construct Buildings, completed to...

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

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... details of the building to be constructed using loan funds. In particular, the preliminary P&S shall... subject to RUS approval. (b) The borrower shall prepare P&S for construction of all buildings. Each set of P&S shall include: (1) RUS Contract Form 257, Contract to Construct Buildings, completed to...

  8. 7 CFR 1753.26 - Plans and specifications (P&S).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... details of the building to be constructed using loan funds. In particular, the preliminary P&S shall... subject to RUS approval. (b) The borrower shall prepare P&S for construction of all buildings. Each set of P&S shall include: (1) RUS Contract Form 257, Contract to Construct Buildings, completed to...

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

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... details of the building to be constructed using loan funds. In particular, the preliminary P&S shall... subject to RUS approval. (b) The borrower shall prepare P&S for construction of all buildings. Each set of P&S shall include: (1) RUS Contract Form 257, Contract to Construct Buildings, completed to...

  10. 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. PMID:25976113

  11. 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. PMID:24878526

  12. Mosquito Saliva Serine Protease Enhances Dissemination of Dengue Virus into the Mammalian Host

    PubMed Central

    Conway, Michael J.; Watson, Alan M.; Colpitts, Tonya M.; Dragovic, Srdjan M.; Li, Zhiyong; Wang, Penghua; Feitosa, Fabiana; Shepherd, Denueve T.; Ryman, Kate D.; Klimstra, William B.; Anderson, John F.

    2014-01-01

    Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo, marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease. PMID:24131723

  13. Mitogen inactivation of glycogen synthase kinase-3 beta in intact cells via serine 9 phosphorylation.

    PubMed Central

    Stambolic, V; Woodgett, J R

    1994-01-01

    Glycogen synthase kinase-3 (GSK-3), a protein-serine kinase implicated in cell-fate determination and differentiation, phosphorylates several regulatory proteins that are activated by dephosphorylation in response to hormones or growth factors. GSK-3 beta is phosphorylated in vitro at serine 9 by p70 S6 kinase and p90rsk-1, resulting in its inhibition [Sutherland, Leighton, and Cohen (1993) Biochem. J. 296, 15-19]. Using HeLa cells expressing GSK-3 beta or a mutant containing alanine at residue 9, we demonstrate that serine 9 is modified in intact cells and is targeted specifically by p90rsk-1, and that phosphorylation leads to loss of activity. Since p90rsk-1 is directly activated by mitogen-activated protein kinases, agonists of this pathway, such as insulin, repress GSK-3 function. Images Figure 1 Figure 2 PMID:7980435

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

    PubMed

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

    2016-03-01

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

  15. 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. PMID:20196534

  16. Microfluidic analysis of serine levels using seryl-tRNA synthetase coupled with spectrophotometric detection.

    PubMed

    Kugimiya, Akimitsu; Matsuzaki, Emi

    2014-12-01

    The measurement of amino acid content is useful for the diagnosis of several types of diseases, including cancer and diabetes. In this study, a microfluidic method for the analysis of serine using enzymatic reactions coupled with spectrophotometric detection was developed. The assay system has some advantages in the analytical field, such as the ability to detect small amounts of analyte and reaction solution and a rapid and efficient reaction. For the specific detection of serine, seryl-tRNA synthetase was coupled with the generation of hydrogen peroxide, which was then detected by the Trinder reagent spectrophotometric method. Seryl- and other aminoacyl-tRNA synthetases are involved in the biosynthesis of peptides and proteins in the human body and should allow precise recognition of the corresponding amino acids. This approach provided selective quantitation of up to 250 ?M serine in 100 mM Tris-HCl buffer (pH 8.0) in a semiautomatic system. PMID:25190303

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-07-15

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

  19. 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.

  20. 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.

  1. 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

  2. Genetic transformation in the methanogen Methanococcus voltae PS.

    PubMed Central

    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. Images PMID:3034867

  3. Science spin: iPS cell research in the news.

    PubMed

    Caulfield, T; Rachul, C

    2011-05-01

    Big scientific developments have always been spun to meet particular social agendas. We have seen it in the context of global warming, nuclear power, and genetically modified organisms. But few stories illustrate the phenomenon of spin as well as the reaction, and concomitant media coverage, that surrounded the November 2007 announcement regarding the reprogramming of skin cells to produce cells with qualities comparable to those of human embryonic stem cells (hESCs) known as induced pluripotent stem (iPS) cells. PMID:21512523

  4. 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. PMID:26704265

  5. Tau Phosphorylation at Serine 396 Residue Is Required for Hippocampal LTD

    PubMed Central

    Regan, Philip; Piers, Thomas; Yi, Jee-Hyun; Kim, Dong-Hyun; Huh, Seonghoo; Park, Se Jin; Ryu, Jong Hoon; Whitcomb, Daniel J.

    2015-01-01

    Tau is required for the induction of long-term depression (LTD) of synaptic transmission in the hippocampus. Here we probe the role of tau in LTD, finding that an AMPA receptor internalization mechanism is impaired in tau KO mice, and that LTD causes specific phosphorylation at the serine 396 and 404 residues of tau. Surprisingly, we find that phosphorylation at serine 396, specifically, is critical for LTD but has no role in LTP. Finally, we show that tau KO mice exhibit deficits in spatial reversal learning. These findings underscore the physiological role for tau at the synapse and identify a behavioral correlate of its role in LTD. PMID:25810511

  6. 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

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

    PubMed

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

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

  8. TRPV-1-mediated elimination of residual iPS cells in bioengineered cardiac cell sheet tissues.

    PubMed

    Matsuura, Katsuhisa; Seta, Hiroyoshi; Haraguchi, Yuji; Alsayegh, Khaled; Sekine, Hidekazu; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Yamazaki, Kenji; Okano, Teruo

    2016-01-01

    The development of a suitable strategy for eliminating remaining undifferentiated cells is indispensable for the use of human-induced pluripotent stem (iPS) cell-derived cells in regenerative medicine. Here, we show for the first time that TRPV-1 activation through transient culture at 42 °C in combination with agonists is a simple and useful strategy to eliminate iPS cells from bioengineered cardiac cell sheet tissues. When human iPS cells were cultured at 42 °C, almost all cells disappeared by 48 hours through apoptosis. However, iPS cell-derived cardiomyocytes and fibroblasts maintained transcriptional and protein expression levels, and cardiac cell sheets were fabricated after reducing the temperature. TRPV-1 expression in iPS cells was upregulated at 42 °C, and iPS cell death at 42 °C was TRPV-1-dependent. Furthermore, TRPV-1 activation through thermal or agonist treatment eliminated iPS cells in cardiac tissues for a final concentration of 0.4% iPS cell contamination. These findings suggest that the difference in tolerance to TRPV-1 activation between iPS cells and iPS cell-derived cardiac cells could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues, which will further reduce the risk of tumour formation. PMID:26888607

  9. TRPV-1-mediated elimination of residual iPS cells in bioengineered cardiac cell sheet tissues

    PubMed Central

    Matsuura, Katsuhisa; Seta, Hiroyoshi; Haraguchi, Yuji; Alsayegh, Khaled; Sekine, Hidekazu; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Yamazaki, Kenji; Okano, Teruo

    2016-01-01

    The development of a suitable strategy for eliminating remaining undifferentiated cells is indispensable for the use of human-induced pluripotent stem (iPS) cell-derived cells in regenerative medicine. Here, we show for the first time that TRPV-1 activation through transient culture at 42 °C in combination with agonists is a simple and useful strategy to eliminate iPS cells from bioengineered cardiac cell sheet tissues. When human iPS cells were cultured at 42 °C, almost all cells disappeared by 48 hours through apoptosis. However, iPS cell-derived cardiomyocytes and fibroblasts maintained transcriptional and protein expression levels, and cardiac cell sheets were fabricated after reducing the temperature. TRPV-1 expression in iPS cells was upregulated at 42 °C, and iPS cell death at 42 °C was TRPV-1-dependent. Furthermore, TRPV-1 activation through thermal or agonist treatment eliminated iPS cells in cardiac tissues for a final concentration of 0.4% iPS cell contamination. These findings suggest that the difference in tolerance to TRPV-1 activation between iPS cells and iPS cell-derived cardiac cells could be exploited to eliminate remaining iPS cells in bioengineered cell sheet tissues, which will further reduce the risk of tumour formation. PMID:26888607

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

    PubMed Central

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

    2013-01-01

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

  11. 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. PMID:26454909

  12. 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.

  13. 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.

  14. The elite and stochastic model for iPS cell generation: multilineage-differentiating stress enduring (Muse) cells are readily reprogrammable into iPS cells.

    PubMed

    Wakao, Shohei; Kitada, Masaaki; Dezawa, Mari

    2013-01-01

    Induced pluripotent stem (iPS) cells have attracted a great deal of attention, although the mechanism by which they are generated is still not fully understood. Currently, two theories, the stochastic and elite models, have been proposed. Some reports provide theoretical support for the stochastic model. Other reports, however, support the elite model. For example, some human fibroblasts, such as Multilineage-differentiating stress enduring (Muse) cells, are reported to be pluripotent and a primary source of iPS cells. Thus, the mechanism of iPS cell generation continues to be debated. In this review, we discuss the properties of the original cell source, such as the components of the original populations and the potential of each population to become iPS cells, and further discuss the implications of the two theories for iPS cell research. PMID:22693162

  15. Cloning and expression studies during vegetative growth and sexual development of Psp2, a serine protease gene from Pyrenopeziza brassicae.

    PubMed

    Keniry, Catherine A; Li, Donghui; Ashby, Alison M

    2002-08-19

    A serine protease gene designated Pyrenopeziza brassicae serine protease 2 (Psp2) was cloned from the plant pathogenic fungus Pyrenopeziza brassicae and its expression analysed. Psp2 was transcribed in equal levels during both vegetative growth and sexual development and showed no differential expression in the presence of exogenous protein or during starvation. PMID:12151110

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

    PubMed

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

    2016-01-21

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

  17. Tryptophanase-catalyzed L-tryptophan synthesis from D-serine in the presence of diammonium hydrogen phosphate.

    PubMed

    Shimada, Akihiko; Ozaki, Haruka; Saito, Takeshi; Noriko, Fujii

    2009-06-01

    Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of l-tryptophan from l-serine and indole through a beta-substitution mechanism of the ping-pong type, and has no activity on d-serine. We previously reported that tryptophanase changed its stereospecificity to degrade d-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH(4))(2)HPO(4) solution. The present study provided the same stereospecific change seen in the d-tryptophan degradation reaction also occurs in tryptophan synthesis from d-serine. Tryptophanase became active to d-serine to synthesize l-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. d-serine seems to undergo beta-replacement via an enzyme-bonded alpha-aminoacylate intermediate to yield l-tryptophan. PMID:19582219

  18. 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

  19. 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

  20. Investigations on recyclisation and hydrolysis in avibactam mediated serine β-lactamase inhibition.

    PubMed

    Choi, Hwanho; Paton, Robert S; Park, Hwangseo; Schofield, Christopher J

    2016-04-26

    β-Lactams inhibit penicillin-binding proteins (PBPs) and serine β-lactamases by acylation of a nucleophilic active site serine. Avibactam is approved for clinical use in combination with ceftazidime, and is a breakthrough non β-lactam β-lactamase inhibitor also reacting via serine acylation. Molecular dynamics (MD) and quantum chemical calculations on avibactam-mediated inhibition of a clinically relevant cephalosporinase reveal that recyclisation of the avibactam derived carbamoyl complex is favoured over hydrolysis. In contrast, we show that analogous recyclisation in β-lactam mediated inhibition is disfavoured. Avibactam recyclisation is promoted by a proton shuttle, a 'structural' water protonating the nucleophilic serine, and stabilization of the negative charge developed on aminocarbonyl oxygen. The results imply the potential of calculations for distinguishing between bifurcating pathways during inhibition and in generating hypotheses for predicting resistance. The inability of β-lactams to undergo recyclisation may be an Achilles heel, but one that can be addressed by suitably functionalized reversibly binding inhibitors. PMID:27072755

  1. 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...

  2. 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...

  3. 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.

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

    PubMed

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

    2015-05-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

    PubMed Central

    Ramesh, K S; Appaji Rao, N

    1980-01-01

    The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes. Images Fig. 1. Fig. 2. Fig. 3. Fig. 6. PMID:6821365

  7. 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.

  8. 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

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

    PubMed

    Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

    2013-09-01

    Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited 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

  10. Specific inhibition of mature fungal serine proteinases and metalloproteinases by their propeptides.

    PubMed Central

    Markaryan, A; Lee, J D; Sirakova, T D; Kolattukudy, P E

    1996-01-01

    The function of the long propeptides of fungal proteinases is not known. Aspergillus fumigatus produces a 33-kDa serine proteinase of the subtilisin family and a 42-kDa metalloproteinase of the thermolysin family. These extracellular enzymes are synthesized as preproenzymes containing large amino-terminal propeptides. Recombinant propeptides were produced in Escherichia coli as soluble fusion proteins with glutathione S-transferase or thioredoxin and purified by affinity chromatography. A. fumigatus serine proteinase propeptide competitively inhibited serine proteinase, with a Ki of 5.3 x 10(-6) M, whereas a homologous serine proteinase from A. flavus was less strongly inhibited and subtilisin was not inhibited. Binding of metalloproteinase propeptide from A. fumigatus to the mature metalloenzyme was demonstrated. This propeptide strongly inhibited its mature enzyme, with a Ki of 3 x 10(-9) M, whereas thermolysin and a metalloproteinase from A. flavus were not inhibited by this propeptide. Enzymatically inactive metalloproteinase propeptide complex could be completely activated by trypsin treatment. These results demonstrate that the propeptides of the fungal proteinases bind specifically and inhibit the respective mature enzymes, probably reflecting a biological role of keeping these extracellular enzymes inactive until secretion. PMID:8636020

  11. POLYMORPHISMS IN CYTOPLASMIC SERINE HYDROXYMETHYLTRANSFERASE AND METHYLENETETRAHYDROFOLATE REDUCTASE AFFECT THE RISK OF CARDIOVASCULAR DISEASE IN MEN

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic variation in folate-regulating enzymes contributes to the risk of cardiovascular disease (CVD). The cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme is proposed to regulate a key metabolic intersection in folate metabolism. We hypothesized that a variant in cSHMT (cSHMT 1420CT) aff...

  12. Regulation of the EBNA1 Epstein-Barr Virus Protein by Serine Phosphorylation and Arginine Methylation

    PubMed Central

    Shire, Kathy; Kapoor, Priya; Jiang, Ke; Hing, Margaret Ng Thow; Sivachandran, Nirojini; Nguyen, Tin; Frappier, Lori

    2006-01-01

    The Epstein-Barr virus (EBV) EBNA1 protein is important for the replication and mitotic segregation of EBV genomes in latently infected cells and also activates the transcription of some of the viral latency genes. A Gly-Arg-rich region between amino acids 325 and 376 is required for both the segregation and transcriptional activation functions of EBNA1. Here we show that this region is modified by both arginine methylation and serine phosphorylation. Mutagenesis of the four potentially phosphorylated serines in this region indicated that phosphorylation of multiple serines contributes to the efficient segregation of EBV-based plasmids by EBNA1, at least in part by increasing EBNA1 binding to hEBP2. EBNA1 was also found to bind the arginine methyltransferases PRMT1 and PRMT5. Multiple arginines in the 325-376 region were methylated in vitro by PRMT1 and PRMT5, as was an N-terminal Gly-Arg-rich region between amino acids 41 and 50. EBNA1 was also shown to be methylated in vivo, predominantly in the 325-376 region. Treatment of cells with a methylation inhibitor or down-regulation of PRMT1 altered EBNA1 localization, resulting in the formation of EBNA1 rings around the nucleoli. The results indicate that EBNA1 function is influenced by both serine phosphorylation and arginine methylation. PMID:16699006

  13. In-silico comparative study of inhibitory mechanism of Plant Serine Proteinase Inhibitors

    PubMed Central

    Siva Prasad, Chekkara Venkata Sathya; Gupta, Saurabh; Gaponenko, Alex; Dhar, Murli

    2012-01-01

    The nematodes like root-knot and cyst are plant-parasitic pest found in horticultural and agricultural crops. They do damages in the roots of plants as a result losses million tons of production. High cost of nematicides and environment safety concern has necessitated finding of some alternative methods. Under Integrated Pest Management (IPM) such problems are solving significantly by means of target gene inhibition, agrobacterium mediated transformation etc. One of this strategy use Plant Proteinase Inhibitors (PIs) gene which are used to control the proteolysis mechanism of Pest by inhibiting gut Serine Proteinase (SP). Present work investigates the utility of computer aided methods to study the mechanism of Protein-Protein interactions and thereby inhibition of Serine Proteinase by PIs. Hence 3D models of Serine Proteinase as well as Serine Proteinase Inhibitors (SPIs) generated using homology modeling. Validations of constructed models have been done by PROCHECK, VERIFY3D, ERRAT and PROSA. Prediction of Protein interacting surface patches and site specific protein docking was performed by using ZDOCK Server. Backbone refinement of output protein complexes was executed in Fiber Dock server. Interaction study between SP and SPIs complexes shows their comparative inhibition efficacy, measured in terms of number of hydrogen bonds, Van dar wall attraction and docking energy. This work reported that Vigna marina and Phaseolus oligospermus are having better inhibition efficiency in comparison to other inhibitors. PMID:23055608

  14. 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...

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

    PubMed

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

    2012-08-01

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

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

    PubMed

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

    2016-01-01

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

  17. 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...

  18. Related Arabidopsis serine carboxypeptidase-like sinapoylglucose acyltransferases display distinct but overlapping substrate specificities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis genome encodes fifty-one proteins annotated as serine carboxypeptidase-like (SCPL) enzymes. Nineteen of these SCPL proteins are highly similar to one another, and represent a clade that appears to be unique to plants. Two of these proteins have been characterized to date: sinapoylgl...

  19. Cross-Linking of Serine Racemase Dimer by Reactive Oxygen Species and Reactive Nitrogen Species

    PubMed Central

    Wang, Wei; Barger, Steven W.

    2011-01-01

    Serine racemase (SR) is the only identified enzyme in mammals responsible for isomerization of L-serine to D-serine, a co-agonist at NMDA receptors in the forebrain. Our previous data reported that an apparent SR dimer resistant to SDS and ?-mercaptoethanol was elevated in microglial cells after proinflammatory activation. Because the activation of microglia is typically associated with an oxidative burst, oxidative cross-linking between SR subunits was speculated. In this study, an siRNA technique was employed to confirm the identity of this SR dimer band. The oxidative species potentially responsible for the cross-linking was investigated with recombinant SR protein. The data indicate that nitric oxide, peroxynitrite, and hydroxyl radical were the likely candidates, while superoxide and hydrogen peroxide per se failed to contribute. Furthermore, the mechanism of formation of SR dimer by peroxynitrite oxidation was studied by mass spectrometry. A disulfide bond between Cys6 and Cys113 was identified in both SIN-1 treated SR monomer and dimer. Activity assays indicated that SIN-1 treatment decreased SR activity, confirming our previous conclusion that noncovalent dimer is the most active form of SR. These findings suggest a compensatory feedback whereby the consequences of neuroinflammation might dampen D-serine production to limit excitotoxic stimulation of NMDA receptors. PMID:22354542

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  2. Laser-Free RF-Gun as a Combined Source of Thz and Ps-Sub-Ps X-Rays

    NASA Astrophysics Data System (ADS)

    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.

    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.

  3. 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.

  4. 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.

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

    DOE PAGESBeta

    Agustsson, R.; Boucher, S.; Finn, O.; Hartzell, J.; Ruelas, M.; Smirnov, A. V.; Storms, S.; Ning, Z.; Murokh, A.; Campese, T.; et al

    2015-01-01

    A coherent, mm-sub-mm-wave source driven by a RF electron gun is proposed for wide research applications as well as auxiliary inspection and screening, safe imaging, cancer diagnostics, surface defectoscopy, and enhanced time-domain spectroscopy. It allows generation of high peak and average THz-sub-THz radiation power provided by beam pre-bunching and chirping in the RF gun followed by microbunching in magnetic compressor, and resonant Cherenkov radiation of an essentially flat beam in a robust, ~inch-long, planar, mm-sub-mm gap structure. The proof-of-principle has been successfully demonstrated in Phase I on a 5 MeV beam of L-band thermionic injector of Idaho Accelerator Center. Themore » system can also deliver an intense, ps-sub-ps bursts of low-to-moderate dose of relativistic electrons and X-ray radiation produced by the same beam required for pulsed radiolysis as well as to enhance screening efficiency, throughput and safety.« less

  6. Absolute Magnitudes of Pan-STARRS PS1 Asteroids

    NASA Astrophysics Data System (ADS)

    Veres, Peter; Jedicke, R.; Fitzsimmons, A.; Denneau, L.; Wainscoat, R.; Bolin, B.; PS1SC Collaboration

    2013-10-01

    Absolute magnitude (H) of an asteroid is a fundamental parameter describing the size and the apparent brightness of the body. Because of its surface shape, properties and changing illumination, the brightness changes with the geometry and is described by the phase function governed by the slope parameter (G). Although many years have been spent on detailed observations of individual asteroids to provide H and G, vast majority of minor planets have H based on assumed G and due to the input photometry from multiple sources the errors of these values are unknown. We compute H of ~ 180 000 and G of few thousands asteroids observed with the Pan-STARRS PS1 telescope in well defined photometric systems. The mean photometric error is 0.04 mag. Because on average there are only 7 detections per asteroid in our sample, we employed a Monte Carlo (MC) technique to generate clones simulating all possible rotation periods, amplitudes and colors of detected asteroids. Known asteroid colors were taken from the SDSS database. We used debiased spin and amplitude distributions dependent on size, spectral class distributions of asteroids dependent on semi-major axis and starting values of G from previous works. H and G (G12 respectively) were derived by phase functions by Bowell et al. (1989) and Muinonen et al. (2010). We confirmed that there is a positive systematic offset between H based on PS1 asteroids and Minor Planet Center database up to -0.3 mag peaking at 14. Similar offset was first mentioned in the analysis of SDSS asteroids and was believed to be solved by weighting and normalizing magnitudes by observatory codes. MC shows that there is only a negligible difference between Bowell's and Muinonen's solution of H. However, Muinonen's phase function provides smaller errors on H. We also derived G and G12 for thousands of asteroids. For known spectral classes, slope parameters agree with the previous work in general, however, the standard deviation of G in our sample is twice as larger, most likely due to sparse phase curve sampling. In the near future we plan to complete the H and G determination for all PS1 asteroids (500,000) and publish H and G values online. This work was supported by NASA grant No. NNX12AR65G.

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

    PubMed Central

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

    2014-01-01

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

  8. Serine 216 Phosphorylation ofEstrogen Receptor ? in Neutrophils: Migration and Infiltration into the Mouse Uterus

    PubMed Central

    Shindo, Sawako; Moore, Rick; Flake, Gordon; Negishi, Masahiko

    2013-01-01

    Background Whereas estrogen receptors are present in immune cells, it is not known if they are phosphorylated to regulate immune cell functions. Here we determined the phosphorylation status of estrogen receptor ? (ER?) at residue serine 216 in mouse neutrophils and examined its?role in migration and infiltration. Serine 216 is the conserved phosphorylation site within the DNA binding domains found in the majority of nuclear receptors. Methodology/Principal Findings A phospho-peptide antibody specific to phosphorylated serine 216 and ER? KO mice were utilized in immunohistochemistry, double immuno-staining or Western blot to detect phosphorylation of ER? in peripheral blood as well as infiltrating neutrophils in the mouse uterus. Transwell assays were performed to examine migration of neutrophils. An anti-Ly6G antibody identified neutrophils. About 20% of neutrophils expressed phosphorylated ER? at serine 216 in peripheral white blood cells (WBC) from C3H/HeNCrIBR females. Phosphorylation was additively segregated between C3H/HeNCrIBR and C57BL/6 females. Only neutrophils that expressed phosphorylated ER? migrated in Transwell assays as well as infiltrated the mouse uterus during normal estrous cycles. Conclusions/Significance ER? was phosphorylated at serine 216 in about 20% of mouse peripheral blood neutrophils. Only those that express phosphorylated ER? migrate and infiltrate the mouse uterus. This phosphorylation was the first to be characterized in endogenous ER? found in normal tissues and cells. Phosphorylated ER? may have opened a novel research direction for biological roles of phosphorylation in ER? actions and can be developed as a drug target for treatment of immune-related diseases. PMID:24386386

  9. An Unorthodox Sensory Adaptation Site in the Escherichia coli Serine Chemoreceptor

    PubMed Central

    Han, Xue-Sheng

    2014-01-01

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

  10. Small-size meshless 50 ps streak tube

    NASA Astrophysics Data System (ADS)

    Ageeva, N. V.; Andreev, S. V.; Belolipetski, V. S.; Bryukhnevich, G. I.; Greenfield, D. E.; Ivanova, S. R.; Kaverin, A. M.; Khohlova, A. N.; Kuz'menko, E. A.; Levina, G. P.; Makushina, V. A.; Monastyrskiy, M. A.; Schelev, M. Ya.; Semichastnova, Z. M.; Serdyuchenko, Yu. N.; Skaballanovich, T. A.; Sokolov, V. E.

    2008-11-01

    In contrast to the conventional image intensifier with large work area, a streak image tube should possess additional important feature - the comparatively small temporal distortion at the entire work area of the photocathode. With this additional engineering restriction taken into account, a novel small-size meshless streak image tube has been developed by means of numerical optimization. The tube with 25-mm wide work area contains a pair of deflection plates to sweep the electron image along the 25 mm output phosphor screen that is separated by 100 mm from the photocathode. The electron image can be shuttered with a 300 V blanking electric pulse. Electron-optical magnification of the tube is unit; spatial resolution reaches 30 lp/mm over the entire photocathode work area; temporal resolution lies in the 20 - 50 ps range, depending on the accelerating voltage (6 - 15 kV).

  11. Sub-10ps monolithic and low-power photodetector readout

    SciTech Connect

    Varner, Gary S.; Ruckman, Larry L.

    2009-02-20

    Recent advances in photon detectors have resulted in high-density imaging arrays that offer many performance and cost advantages. In particular, the excellent transit time spread of certain devices show promise to provide tangible benefits in applications such as Positron Emission Tomography (PET). Meanwhile, high-density, high-performance readout techniques have not kept on pace for exploiting these developments. Photodetector readout for next generation high event rate particle identification and time-resolved PET requires a highly-integrated, low-power, and cost-effective readout technique. We propose fast waveform sampling as a method that meets these criteria and demonstrate that sub-10ps resolution can be obtained for an existing device.

  12. Midinfrared Nonlinear Optical Thiophosphates from LiZnPS4 to AgZnPS4: A Combined Experimental and Theoretical Study.

    PubMed

    Zhou, Molin; Kang, Lei; Yao, Jiyong; Lin, Zheshuai; Wu, Yicheng; Chen, Chuangtian

    2016-04-18

    Our earlier theoretical calculation and preliminary experiment highlighted LiZnPS4 as a good mid-infrared (mid-IR) nonlinear optical (NLO) material. However, this compound suffers from problems including corrosion of the silica tubes, a pungent smell, deliquescence, and incongruent-melting behavior in the further single crystal growth and applications. In order to overcome these problems, herein, we investigate the analogues of LiZnPS4 and propose that AgZnPS4 would be a good candidate. The combination of experimental and theoretical study demonstrates that AgZnPS4 exhibits a much stronger NLO effect than that of LiZnPS4 despite the relatively smaller energy band gap. More importantly, AgZnPS4 melts congruently with a melting point as low as 534 °C, much lower than those of traditional IR NLO crystals, and is nondeliquescent with enough stability in the air. Such a good crystal growth habit and chemical stability enable AgZnPS4 to possess much better overall performance for the practical mid-IR NLO applications. PMID:27015097

  13. Performance Analysis of the ertPS Algorithm and Enhanced ertPS Algorithm for VoIP Services in IEEE 802.16e Systems

    NASA Astrophysics Data System (ADS)

    Kim, Bong Joo; Hwang, Gang Uk

    In this paper, we analyze the extended real-time Polling Service (ertPS) algorithm in IEEE 802.16e systems, which is designed to support Voice-over-Internet-Protocol (VoIP) services with data packets of various sizes and silence suppression. The analysis uses a two-dimensional Markov Chain, where the grant size and the voice packet state are considered, and an approximation formula for the total throughput in the ertPS algorithm is derived. Next, to improve the performance of the ertPS algorithm, we propose an enhanced uplink resource allocation algorithm, called the e2rtPS algorithm, for VoIP services in IEEE 802.16e systems. The e2rtPS algorithm considers the queue status information and tries to alleviate the queue congestion as soon as possible by using remaining network resources. Numerical results are provided to show the accuracy of the approximation analysis for the ertPS algorithm and to verify the effectiveness of the e2rtPS algorithm.

  14. Hormone interactions and regulation of Unifoliata, PsPK2, PsPIN1 and LE gene expression in pea (Pisum sativum) shoot tips.

    PubMed

    Bai, Fang; DeMason, Darleen A

    2006-07-01

    The Unifoliata (Uni) gene plays a major role in development of the compound leaf in pea, but its regulation is unknown. In this study, we examined the effects of plant hormones on the expression of Uni, PsPK2 (the gene for a pea homolog of Arabidopsis PID, a regulator of PIN1 targeting), PsPIN1 (the major gene for a putative auxin efflux carrier) and LE (a gibberellin biosynthesis gene, GA3ox), and also examined mutual hormonal regulation of these genes, in pea shoot tips, including a number of mutants. The Uni promoter possessed putative auxin and gibberellin response elements. The PsPIN1 mRNA levels were increased in afila, which replaces leaflets with branched tendrils; and reduced in tendrilless, which replaces tendrils with leaflets, compared with the wild type (WT). In contrast, mRNA levels of LE were increased in uni and tendrilless and decreased in afila compared with the WT. Uni, PsPK2 and PsPIN1 are positively regulated by gibberellin and auxin, and were induced to higher levels by simultaneous application of auxin and gibberellin. Auxin induction of Uni, PsPK2 and PsPIN1 did not require de novo protein synthesis. LE was positively regulated by auxin and cytokinin. In conclusion, these results support the hypothesis that auxin and gibberellin positively regulate Uni, which controls pea compound leaf development. Also, Uni, PsPIN1, PsPK2 and LE are expressed differentially in the leaf mutants, suggesting that mutual regulation by auxin and gibberellin promotes compound leaf development. PMID:16760220

  15. Study of the crystalline structures of the syndiotactic polystyrene (sPS) under mechanical deformation

    NASA Astrophysics Data System (ADS)

    Nagasaka, Suguru; Hotta, Atsushi

    2008-03-01

    Polystyrene (PS) has become one of the important, yet complicated semi-crystalline materials since the successful synthesis of the syndiotactic PS (sPS) by Ziegler-Natta catalyst. Since then, sPS has been actively investigated and four different crystalline forms (α, β, γ and δ), two mesomorphic forms and various clathrate forms have been found, indicating complex feature of its crystalline structures. Among the four crystalline forms, α and β-crystals can be obtained by different annealing processes and both crystalline structures comprise the same all-trans planar conformation. In this work, β-crystal structure was made by high temperature annealing and the sPS with β-crystal structure was mechanically stretched above the glass transition temperature of sPS, followed by the crystalline transition analysis studied by FT-IR.

  16. Coexistence of WiFi and WiMAX Systems Based on PS-Request Protocols†

    PubMed Central

    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. PMID:22163721

  17. 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. PMID:22163721

  18. 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.

  19. Kristallstruktur und Schwingungsspektrum des Praseodym-ortho-Thiophosphates PrPS4 / Crystal Structure and Vibrational Spectrum of PrPS4

    NASA Astrophysics Data System (ADS)

    Wibbelmann, Claus; Brockner, Wolfgang; Eisenmann, Brigitte; Schäfer, Herbert

    1984-02-01

    PrPS4 crystallizes in the tetragonal system, space group I41/acd with the lattice constants a= 1091.4(5) pm. c = 1936.1(8) pm. In the structure there are slightly distorted PS3-4 tetrahedra as discrete structural units, placed along the b-axis. Far infrared, infrared and Raman spectra of this compound have been recorded. The observed frequencies are assigned rangewise by factor group analysis and the Td-D4h-correlation. The Raman data of rare-earth ortho thiophosphates LnPS4 with Ln = La, Ce, Pr, Nd, Sm, Tb and Tm are given in a line diagram.

  20. DRiPs Solidify: Progress in Understanding Endogenous MHC Class I Antigen Processing

    PubMed Central

    Yewdell, Jonathan W.

    2011-01-01

    Defective Ribosomal Products (DRiPs) are a subset of rapidly degraded polypeptides that provide peptide ligands for MHC class I molecules. Here, I review recent progress in understanding DRiP biogenesis. These findings place DRiPs at the center of the MHC class I antigen processing pathway, linking immunosurveillance of viruses and tumors to mechanisms of specialized translation and cellular compartmentalization. DRiPs enable the immune system to rapidly and sensitively detect alterations in cellular gene expression. PMID:21962745

  1. Kristallstruktur und Schwingungsspektrum des Thallium(I)-Zinn(II)-ortho-Thiophosphates TlSnPS4

    NASA Astrophysics Data System (ADS)

    Becker, Robert; Brockner, Wolfgang; Eisenmann, Brigitte

    1987-11-01

    TlSnPS4 crystallizes in the orthorhombic system, space group Pna21 (Nr. 33), Z = 4 with the lattice constants a = 1175.8 (5) pm, b = 890.1 (4) pm, c = 663.3 (4) pm. In the structure are sligthly distorted discrete PS3-4 anions. The far infrared, infrared and Raman spectrum is assigned on the basis of PS3-4 -units with C3v symmetry. According to the DTA data the melting point for TlSnPS4 is 575 ± 5 °C. The title compound is not moisture sensitive and semi-conducting.

  2. Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system.

    PubMed

    Pernot, Pierre; Mothet, Jean-Pierre; Schuvailo, Oleg; Soldatkin, Alexey; Pollegioni, Loredano; Pilone, Mirella; Adeline, Marie-Thrse; Cespuglio, Raymond; Marinesco, Stphane

    2008-03-01

    d-Serine is an endogenous ligand for N-methyl-d-aspartate (NMDA) receptors, and alterations in its concentration have been related to several brain disorders, especially schizophrenia. It is therefore an important target neuromodulator for the pharmaceutical industry. To monitor d-serine levels in vivo, we have developed a microbiosensor based on cylindrical platinum microelectrodes, covered with a membrane of poly-m-phenylenediamine (PPD) and a layer of immobilized d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). By detecting the hydrogen peroxide produced by enzymatic degradation of d-serine, this microbiosensor shows a detection limit of 16 nM and a mean response time of 2 s. Interferences by ascorbic acid, uric acid, l-cysteine, and by biogenic amines and their metabolites are rejected at more than 97% by the PPD layer. Although several d-amino acids are potential substrates for RgDAAO, d-serine was the only endogenous substrate present in sufficient concentration to be detected by our microbiosensor in the central nervous system. When implanted in the cortex of anesthetized rats, this microbiosensor detected the increase in concentration of d-serine resulting from its diffusion across the blood-brain barrier after an intraperitoneal injection. This new device will make it possible to investigate in vivo the variations in d-serine concentrations occurring under normal and pathological conditions and to assess the pharmacological potency of new drugs designed to impact d-serine metabolism. PMID:18229946

  3. Pyruvate kinase M2 promotes de novo serine synthesis to sustain mTORC1 activity and cell proliferation

    PubMed Central

    Ye, Jiangbin; Mancuso, Anthony; Tong, Xuemei; Ward, Patrick S.; Fan, Jing; Rabinowitz, Joshua D.; Thompson, Craig B.

    2012-01-01

    Despite the fact that most cancer cells display high glycolytic activity, cancer cells selectively express the less active M2 isoform of pyruvate kinase (PKM2). Here we demonstrate that PKM2 expression makes a critical regulatory contribution to the serine synthetic pathway. In the absence of serine, an allosteric activator of PKM2, glycolytic efflux to lactate is significantly reduced in PKM2-expressing cells. This inhibition of PKM2 results in the accumulation of glycolytic intermediates that feed into serine synthesis. As a consequence, PKM2-expressing cells can maintain mammalian target of rapamycin complex 1 activity and proliferate in serine-depleted medium, but PKM1-expressing cells cannot. Cellular detection of serine depletion depends on general control nonderepressible 2 kinase-activating transcription factor 4 (GCN2-ATF4) pathway activation and results in increased expression of enzymes required for serine synthesis from the accumulating glycolytic precursors. These findings suggest that tumor cells use serine-dependent regulation of PKM2 and GCN2 to modulate the flux of glycolytic intermediates in support of cell proliferation. PMID:22509023

  4. 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 to promote TLR2-dependent bone loss as is reported in experimental periodontitis following oral infection with P. gingivalis. These results also support the conclusion that serine dipeptide lipids are involved in alveolar bone loss in chronic periodontitis. PMID:26409254

  5. The midgut of Aedes albopictus females expresses active trypsin-like serine peptidases

    PubMed Central

    2014-01-01

    Background Aedes albopictus is widely distributed across tropical and sub-tropical regions and is associated with the transmission of several arboviruses. Although this species is increasingly relevant to public health due its ability to successfully colonize both urban and rural habitats, favoring the dispersion of viral infections, little is known about its biochemical traits, with all assumptions made based on studies of A. aegypti. In previous studies we characterized the peptidase profile of pre-imaginal stages of A. albopictus and we reported the first proteomic analysis of the midgut from sugar-fed females of this insect species. Methods In the present work, we further analyzed the peptidase expression in the midgut of sugar-fed females using 1DE-substrate gel zymography, two-dimensional electrophoresis (2DE), mass spectrometry (MS), and protein identification based on similarity. Results The combination of zymography, in solution assays using fluorescent substrates and 2DE-MS/MS allowed us to identify the active serine peptidase “fingerprint” in the midgut of A. albopictus females. Zymographic analysis revealed a proteolytic profile composed of at least 13 bands ranging from ~25 to 250 kDa, which were identified as trypsin-like serine peptidases by using specific inhibitors of this class of enzymes. Concomitant use of the fluorogenic substrate Z-Phe-Arg-AMC and trypsin-like serine protease inhibitors corroborated the zymographic findings. Our proteomic approach allowed the identification of two different trypsin-like serine peptidases and one chymotrypsin in protein spots of the alkaline region in 2DE map of the A. albopictus female midgut. Identification of these protein coding genes was achieved by similarity to the A. aegypti genome sequences using Mascot and OMSSA search engines. Conclusion These results allowed us to detect, identify and characterize the expression of active trypsin-like serine peptidases in the midgut of sugar-fed A. albopictus females. In addition, proteomic analysis allowed us to confidently assign the expression of two trypsin genes and one chymotrypsin gene to the midgut of this mosquito. These results contribute to the gene annotation in this species of unknown genome and represent a small but important step toward the protein-level functional and localization assignment of trypsin-like serine peptidase genes in the Aedes genus. PMID:24886160

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

    PubMed

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

    2015-08-01

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

  7. 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.

  8. Screening of serine protease inhibitors with antimicrobial activity using iron oxide nanoparticles functionalized with dextran conjugated trypsin and in silico analyses of bacterial serine protease inhibition.

    PubMed

    Mandal, Santi M; Porto, William F; De, Debasis; Phule, Ajit; Korpole, Suresh; Ghosh, Ananta K; Roy, Sanat K; Franco, Octavio L

    2014-01-21

    Plants produce a variety of proteins and peptides which are involved in their defense against pathogens. Serine protease inhibitors are a well-established class of inhibitors correlated with plant defense. Increased levels of protease inhibitors delay cell damage and expand the cell's life-span. Recently, the rapid emergence of antibiotic-resistant microbial pathogens has prompted immense interest in purifying novel antimicrobial proteins or peptides from plant sources. Usually, the purification of protease inhibitors is accomplished by salt-extraction, ultrafiltration and affinity chromatography. Here, we developed a novel approach based on iron oxide nanoparticles conjugated to dextran functionalized with trypsin beads that accelerate the quick screening and purification of antimicrobial peptides with serine protease inhibitor activity. The method described here also works for screening other inhibitors using particular protein kinases, and it is therefore a novel tool for use as the leading method in the development of novel antimicrobial agents with protease inhibitory activity. Finally, and no less important, molecular modelling and dynamics studies of a homologous inhibitor studied here with Escherichia coli trypsin and chymotrypsin are provided in order to shed some light on inhibitor-enzyme interactions. PMID:24294628

  9. 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

  10. 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. PMID:24898298

  11. 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

  12. Tobacco serine/threonine protein kinase gene NrSTK enhances black shank resistance.

    PubMed

    Gao, Y-L; Wang, B-W; Xu, Z-L; Li, M-Y; Song, Z-B; Li, W-Z; Li, Y-P

    2015-01-01

    A serine/threonine protein kinase gene (NrSTK) was cloned from Nicotiana repanda based on the sequence of a previously isolated resistance gene analog (RGA). Expression of RGA was induced by challenge with the pathogen black shank. The NrSTK gene was predicted to encode a protein kinase that contained an ATP binding site at residues 41-69 and a serine/threonine protein kinase activation sequence spanning the region 161-173. Overexpression of NrSTK in the susceptible tobacco variety Honghuadajinyuan significantly enhanced resistance to black shank, indicating that NrSTK plays a role in incompatibility reactions between tobacco and the pathogen. Characterization of NrSTK will help elucidate the molecular mechanisms involved in black shank resistance in N. repanda. PMID:26662438

  13. An anticoagulant serine protease from the wasp venom of Vespa magnifica.

    PubMed

    Han, Junyou; You, Dewen; Xu, Xueqing; Han, Wenyu; Lu, Yi; Lai, Ren; Meng, Qingxiong

    2008-04-01

    Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin, contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X. PMID:18308355

  14. 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.

  15. Molecular cloning and characterization of two digestive serine proteases from the Hessian fly, Mayetiola destructor.

    PubMed

    Mittapalli, O; Stuart, J J; Shukle, R H

    2005-06-01

    Full-length cDNA and genomic sequences for two genes (designated mdesprot-I and mdesprot-II) encoding digestive serine proteases in Hessian fly, Mayetiola destructor, have been cloned and characterized. The deduced amino acid sequences revealed similarity with trypsin-like digestive serine proteases from other Dipterans. Both mdesprot-I and mdesprot-II encoded proteins with secretion signal peptides at the N-terminals, indicating the proteins are secreted proteases that should function as midgut digestive proteases. A cytological analysis with fluorescent in situ hybridization revealed the cytological localization of mdesprot-I and mdesprot-II on the long arm of Autosome 2. Results are discussed in the context of the efficacy of potential protease inhibitors to develop Hessian fly resistant wheat through genetic engineering approaches. PMID:15926900

  16. A novel L-serine deaminase activity in Escherichia coli K-12.

    PubMed Central

    Su, H; Newman, E B

    1991-01-01

    We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely. Both mutations lie near 60.5 min on the E. coli genetic map. The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation. Images PMID:2013569

  17. Histochemical demonstration of chymotrypsin like serine esterases in mucosal mast cells in four species including man.

    PubMed Central

    Huntley, J F; Newlands, G F; Gibson, S; Ferguson, A; Miller, H R

    1985-01-01

    Serine esterases were detected in the granules of mucosal mast cells from rat, mouse, sheep, and man. Successful demonstration of enzyme activity required brief fixation (6 h) of tissues in 4% paraformaldehyde. Staining with naphthol AS-D chloroacetate produced an intense red reaction product in intraepithelial mucosal mast cells (globule leucocytes) and mucosal mast cells within the lamina propria of the gastrointestinal tract. The mast cell identity of cells stained for esterase was confirmed by sequential staining with toluidine blue (pH 0.5). Furthermore, the numbers of cells detected after staining for esterases or with toluidine blue were highly correlated. Esterase activity within mucosal mast cells/globule leucocytes from all species was inhibited with the serine enzyme inhibitor phenylmethylsulphonyl fluoride. Further histochemical studies with the substrate, N-acetyl-DL-phenylalanine B-naphthyl ester, indicated that mucosal mast cells and globule leucocytes contain esterases which are chymotrypsin like in substrate specificity. Images PMID:3988950

  18. Serine protease activity in excretory-secretory products of Oestrus ovis (Diptera: Oestridae) larvae.

    PubMed

    Tabouret, G; Bret-Bennis, L; Dorchies, Ph; Jacquiet, Ph

    2003-06-25

    The sheep bot fly, Oestrus ovis, is a very common myiasis of nasal and sinus cavities of sheep and goats causing severe welfare and production implications. As the viability of O. ovis adult flies strictly depends on larval abilities to assimilate and to stock nutrients from the host, it was necessary to investigate proteolytic activities in larval excretory/secretory products (ESP). ESP of O. ovis larvae degrade mucosal and plasmatic components such as mucin, albumin or immunoglobulin G. A preliminary biochemical characterization, using substrate gel analysis and inhibitor sensitivity, demonstrated the presence of at least six major serine proteases (molecular weights from 20 to 100 kDa), mainly trypsin-like, secreted in the digestive tube of larvae. Their involvement in larval trophic activity and evasion from the host immune response is further discussed as O. ovis excretory/secretory serine proteases could represent potential vaccinal targets. PMID:12809756

  19. Unique glycine-activated riboswitch linked to glycine-serine auxotrophy in SAR11

    PubMed Central

    Tripp, H. James; Schwalbach, Michael S.; Meyer, Michelle M.; Kitner, Joshua B.; Breaker, Ronald R.; Giovannoni, Stephen J.

    2008-01-01

    The genome sequence of the marine bacterium ‘Candidatus Pelagibacter ubique’ and subsequent analyses have shown that, while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that ‘Cand. P. ubique’ is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 μM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 ×106 cells ml−1 to 8.16 ×106 cells ml−1 (R2 = 0.992). Serine was capable of substituting for glycine at 1.5 μM. ‘Cand. P. ubique’ contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogs such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine, or threonine. We conclude that ‘Cand. P. ubique’ is therefore a glycine-serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade. PMID:19125817

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Vorlov, Barbora; Nachtigallov, Dana; Jirskov-Van?kov, Jana; Ajani, Haresh; Jansa, Petr; Rez?, Jan; Fanfrlk, Jind?ich; Otyepka, Michal; Hobza, Pavel; Konvalinka, Jan; Lepk, Martin

    2015-01-01

    Overactivation of NMDA receptors has been implicated in various neuropathological conditions, including brain ischaemia, neurodegenerative disorders and epilepsy. Production of d-serine, an NMDA receptor co-agonist, from l-serine is catalyzed invivo 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 4011mM for 2,2-bis(hydroxymethyl)malonate down to 571?M for 2,2-dichloromalonate, the most effective competitive mSR inhibitor known to date. The structure-activity relationship of the whole series in the human orthologue (hSR) was interpreted using Glide docking, WaterMap analysis of hydration and quantum mechanical calculations based on the X-ray structure of the hSR/malonate complex. Docking into the hSR active site with three thermodynamically favourable water molecules was able to discern qualitatively between good and weak inhibitors. Further improvement in ranking was obtained using advanced PM6-D3H4X/COSMO semiempirical quantum mechanics-based scoring which distinguished between the compounds with IC50 better/worse than 2mM. We have thus not only found a new potent hSR inhibitor but also worked out a computer-assisted protocol to rationalize the binding affinity which will thus aid in search for more effective SR inhibitors. Novel, potent hSR inhibitors may represent interesting research tools as well as drug candidates for treatment of diseases associated with NMDA receptor overactivation. PMID:25462239

  3. Inhibition of human serine racemase, an emerging target for medicinal chemistry.

    PubMed

    Jirásková-Vaníčková, Jana; Ettrich, Rudiger; Vorlová, Barbora; Hoffman, Hillary E; Lepšík, Martin; Jansa, Petr; Konvalinka, Jan

    2011-06-01

    Proteins of glutamatergic NMDA receptor signaling pathways have been studied as targets for intervention in a variety of neuropathological conditions, including neurodegenerations, epilepsy, neuropathic pain, drug addiction, and schizophrenia. High activity NMDA-blocking agents have been designed to treat some of these disorders; however, their effect is often compromised by undesirable side effects. Therefore, alternative ways of modulating NMDA receptor function need to be sought after. The opening of the NMDA receptor ion channel requires occupation of two distinct binding sites, the glutamate site and the glycine site. It has been shown that D-serine, rather than glycine, can trigger the physiological NMDA receptor function. D-serine is a product of the activity of a specific enzyme, serine racemase (SR), which was identified a decade ago. SR has therefore emerged as a new potential target for the NMDA-receptor-based diseases. There is evidence linking increased levels of D-Ser to amyotrophic lateral sclerosis and Alzheimer's disease and decreased concentrations of D-serine to schizophrenia. SR is a pyridoxal-5'-phosphate dependent enzyme found in the cytosol of glial and neuronal cells. It is activated by ATP, divalent cations like Mg(2+) or Ca(2+), and reducing agents. This paper reviews the present literature on the activity and inhibition of mammalian SRs. It summarizes approaches that have been applied to design SR inhibitors and lists the known active compounds. Based on biochemical and docking analyses, i) we delineate for the first time the ATP binding site of human SR, and ii) we suggest possible mechanisms of action for the active compounds. In the end, we discuss the SR features that make the discovery of its inhibitors a challenging, yet very important, task of medicinal chemistry. PMID:21291385

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. Sulfonate salts of amino acids: novel inhibitors of the serine proteinases.

    PubMed

    Groutas, W C; Brubaker, M J; Zandler, M E; Stanga, M A; Huang, T L; Castrisos, J C; Crowley, J P

    1985-04-16

    A series of amino acid-derived sulfonate salts have been synthesized. They were found to inactivate efficiently and selectively human leukocyte elastase. The sulfonate salts of the methyl esters of L-norleucine, L-norvaline and L-valine were the most potent. The enzyme is inactivated irreversibly with concomitant release of bisulfite ion. The results demonstrate for the first time that ionic compounds can indeed function as novel inhibitors for the serine proteinases. PMID:3885950

  7. Structure of Soybean Serine Acetyltransferase and Formation of the Cysteine Regulatory Complex as a Molecular Chaperone*

    PubMed Central

    Yi, Hankuil; Dey, Sanghamitra; Kumaran, Sangaralingam; Lee, Soon Goo; Krishnan, Hari B.; Jez, Joseph M.

    2013-01-01

    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. Formation of the cysteine regulatory complex (CRC) is a critical biochemical control feature in plant sulfur metabolism. Here we present the 1.75–3.0 Å resolution x-ray crystal structures of soybean (Glycine max) SAT (GmSAT) in apoenzyme, serine-bound, and CoA-bound forms. The GmSAT-serine and GmSAT-CoA structures provide new details on substrate interactions in the active site. The crystal structures and analysis of site-directed mutants suggest that His169 and Asp154 form a catalytic dyad for general base catalysis and that His189 may stabilize the oxyanion reaction intermediate. Glu177 helps to position Arg203 and His204 and the β1c-β2c loop for serine binding. A similar role for ionic interactions formed by Lys230 is required for CoA binding. The GmSAT structures also identify Arg253 as important for the enhanced catalytic efficiency of SAT in the CRC and suggest that movement of the residue may stabilize CoA binding in the macromolecular complex. Differences in the effect of cold on GmSAT activity in the isolated enzyme versus the enzyme in the CRC were also observed. A role for CRC formation as a molecular chaperone to maintain SAT activity in response to an environmental stress is proposed for this multienzyme complex in plants. PMID:24225955

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

    PubMed

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

    2016-02-01

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

  9. Three-Dimensional Molecular Modeling of a Diverse Range of SC Clan Serine Proteases

    PubMed Central

    Laskar, Aparna; Chatterjee, Aniruddha; Chatterjee, Somnath; Rodger, Euan J.

    2012-01-01

    Serine proteases are involved in a variety of biological processes and are classified into clans sharing structural homology. Although various three-dimensional structures of SC clan proteases have been experimentally determined, they are mostly bacterial and animal proteases, with some from archaea, plants, and fungi, and as yet no structures have been determined for protozoa. To bridge this gap, we have used molecular modeling techniques to investigate the structural properties of different SC clan serine proteases from a diverse range of taxa. Either SWISS-MODEL was used for homology-based structure prediction or the LOOPP server was used for threading-based structure prediction. The predicted models were refined using Insight II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core, differences in overall amino acid distribution, and unique surface electrostatic potential patterns between species. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on SC clan serine proteases. PMID:23213528

  10. The epidermal barrier function is dependent on the serine protease CAP1/Prss8

    PubMed Central

    Leyvraz, Céline; Charles, Roch-Philippe; Rubera, Isabelle; Guitard, Marjorie; Rotman, Samuel; Breiden, Bernadette; Sandhoff, Konrad; Hummler, Edith

    2005-01-01

    Serine proteases are proteolytic enzymes that are involved in the regulation of various physiological processes. We generated mice lacking the membrane-anchored channel-activating serine protease (CAP) 1 (also termed protease serine S1 family member 8 [Prss8] and prostasin) in skin, and these mice died within 60 h after birth. They presented a lower body weight and exhibited severe malformation of the stratum corneum (SC). This aberrant skin development was accompanied by an impaired skin barrier function, as evidenced by dehydration and skin permeability assay and transepidermal water loss measurements leading to rapid, fatal dehydration. Analysis of differentiation markers revealed no major alterations in CAP1/Prss8-deficient skin even though the epidermal deficiency of CAP1/Prss8 expression disturbs SC lipid composition, corneocyte morphogenesis, and the processing of profilaggrin. The examination of tight junction proteins revealed an absence of occludin, which did not prevent the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface. This study shows that CAP1/Prss8 expression in the epidermis is crucial for the epidermal permeability barrier and is, thereby, indispensable for postnatal survival. PMID:16061697

  11. Attachment site recognition and regulation of directionality by the serine integrases.

    PubMed

    Rutherford, Karen; Yuan, Peng; Perry, Kay; Sharp, Robert; Van Duyne, Gregory D

    2013-09-01

    Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between 'attachment sites' in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function. PMID:23821671

  12. Partial characterization of matrix-associated serine protease inhibitors from human skin cells.

    PubMed

    Rao, C N; Peavey, C L; Liu, Y Y; Lapiere, J C; Woodley, D T

    1995-03-01

    Serine protease inhibitors have important regulatory roles in angiogenesis, intravascular fibrinolysis, wound healing, and cell migration. In this study, the extracellular matrix secreted by cultured human keratinocytes, foreskin fibroblasts, and SV-40-transformed human skin fibroblasts was analyzed for serine protease inhibitors by substrate reverse zymography. We found that the extracellular matrix deposited by these cells contained three inhibitors (M(r) 33,000, 31,000, and 27,000). These inhibitors protected the degradation of gelatin by trypsin and elastase, and of casein by plasmin. In contrast, the gelatinolytic activities of thermolysin and papain were not inhibited. Compared to untreated cells, cells treated with phorbol 12-myristate 13-acetate showed a two- to 10-fold increase in the expression of these inhibitors. Cycloheximide and actinomycin D decreased the cellular expression of these inhibitors, suggesting the involvement of de novo protein and mRNA synthesis. Antitrypsin activity of these inhibitors was resistant to heat and sodium dodecylsulfate, but was lost after reduction of disulfide bonds. The inhibitors bound specifically to trypsin and could be eluted from a trypsin column in active form. Collectively, these data suggest that the extracellular matrix deposited by keratinocytes and dermal fibroblasts contains active serine protease inhibitors. PMID:7861006

  13. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases.

    PubMed

    Waldner, Birgit J; Fuchs, Julian E; Huber, Roland G; von Grafenstein, Susanne; Schauperl, Michael; Kramer, Christian; Liedl, Klaus R

    2016-01-21

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design. PMID:26709959

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

    SciTech Connect

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

    2006-01-01

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

  15. HATL5: A Cell Surface Serine Protease Differentially Expressed in Epithelial Cancers

    PubMed Central

    Miller, Gregory S.; Zoratti, Gina L.; Murray, Andrew S.; Bergum, Christopher; Tanabe, Lauren M.; List, Karin

    2014-01-01

    Over the last two decades, cell surface proteases belonging to the type II transmembrane serine protease (TTSP) family have emerged as important enzymes in the mammalian degradome, playing critical roles in epithelial biology, regulation of metabolic homeostasis, and cancer. Human airway trypsin-like protease 5 (HATL5) is one of the few family members that remains uncharacterized. Here we demonstrate that HATL5 is a catalytically active serine protease that is inhibited by the two Kunitz type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and 2, as well as by serpinA1. Full-length HATL5 is localized on the cell surface of cultured mammalian cells as demonstrated by confocal microscopy. HATL5 displays a relatively restricted tissue expression profile, with both transcript and protein present in the cervix, esophagus, and oral cavity. Immunohistochemical analysis revealed an expression pattern where HATL5 is localized on the cell surface of differentiated epithelial cells in the stratified squamous epithelia of all three of these tissues. Interestingly, HATL5 is significantly decreased in cervical, esophageal, and head and neck carcinomas as compared to normal tissue. Analysis of cervical and esophageal cancer tissue arrays demonstrated that the squamous epithelial cells lose their expression of HATL5 protein upon malignant transformation. PMID:24498351

  16. Evidence for the existence of granzyme-like serine proteases in teleost cytotoxic cells.

    PubMed

    Praveen, Kesavannair; Evans, Donald L; Jaso-Friedmann, Liliana

    2004-04-01

    Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with approximately 50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure-function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years. PMID:15114423

  17. Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

    PubMed Central

    Hasson, S. S.; Mothana, R. A.; Sallam, T. A.; Al-balushi, M. S.; Rahman, M. T.; Al-Jabri, A. A.

    2010-01-01

    Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs) from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization) the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent. PMID:20936075

  18. 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

  19. Phosphorylation on TRPV4 Serine 824 Regulates Interaction with STIM1.

    PubMed

    Shin, Sung H; Lee, Eun J; Chun, Jaesun; Hyun, Sunghee; Kang, Sang S

    2015-01-01

    The TRPV4 cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, we identified stromal interaction molecule 1 precursor (STIM1) as an auxiliary protein of this epithelial Ca2+channel using confocal microscopy analysis and GST pull-down assay. The STIM1 protein associates specifically with the C-terminal tail of TRPV4 to form a complex. In previous reports, we demonstrated that the serine824 residue of TRPV4 is one of the target phosphorylation sites of serum/glucocorticoid regulated kinase 1 (SGK1). In this report we further identified the role of serine 824 phosphorylation. The TRPV4 mutant S824D (not S824A) exhibited a diminished capacity to bind STIM1. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STIM1 is part of the TRPV4 protein complex. Our observations clearly suggest that the formation of a complex between TRPV4 and STIM1 and its plasma membrane localization are regulated through phosphorylation of serine824 of TRPV4, and that the STIM1-TRPV4 complex plays crucial roles in routing TRPV4 to the plasma membrane from the endoplasmic reticulum and in maintaining its function. PMID:25972993

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

    PubMed

    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

  1. A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence

    PubMed Central

    Connolly, James P. R.; Gabrielsen, Mads; Goldstone, Robert J.; Grinter, Rhys; Wang, Dai; Cogdell, Richard J.; Walker, Daniel; Smith, David G. E.; Roe, Andrew J.

    2016-01-01

    The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to “sense” levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host. PMID:26727373

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

    SciTech Connect

    Grer, J.

    1980-10-01

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

  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. 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. PMID:25391881

  5. 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

  6. 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

  7. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases

    PubMed Central

    2015-01-01

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design. PMID:26709959

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

    PubMed

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

    2012-08-01

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

  9. Phosphorylation of Serine 106 in Asef2 Regulates Cell Migration and Adhesion Turnover

    PubMed Central

    2015-01-01

    Asef2, a 652-amino acid protein, is a guanine nucleotide exchange factor (GEF) that regulates cell migration and other processes via activation of Rho family GTPases, including Rac. Binding of the tumor suppressor adenomatous polyposis coli (APC) to Asef2 is known to induce its GEF activity; however, little is currently known about other modes of Asef2 regulation. Here, we investigated the role of phosphorylation in regulating Asef2 activity and function. Using high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS), we obtained complete coverage of all phosphorylatable residues and identified six phosphorylation sites. One of these, serine 106 (S106), was particularly intriguing as a potential regulator of Asef2 activity because of its location within the APC-binding domain. Interestingly, mutation of this serine to alanine (S106A), a non-phosphorylatable analogue, greatly diminished the ability of Asef2 to activate Rac, while a phosphomimetic mutation (serine to aspartic acid, S106D) enhanced Rac activation. Furthermore, expression of these mutants in HT1080 cells demonstrated that phosphorylation of S106 is critical for Asef2-promoted migration and for cell-matrix adhesion assembly and disassembly (adhesion turnover), which is a process that facilitates efficient migration. Collectively, our results show that phosphorylation of S106 modulates Asef2 GEF activity and Asef2-mediated cell migration and adhesion turnover. PMID:24874604

  10. Expression and partial biochemical characterization of a recombinant serine protease from Bothrops pauloensis snake venom.

    PubMed

    Isabel, Thais F; Costa, Guilherme Nunes Moreira; Pacheco, Isabela B; Barbosa, Luana G; Santos-Junior, Célio D; Fonseca, Fernando P P; Boldrini França, Johara; Henrique-Silva, Flávio; Yoneyama, Kelly A G; Rodrigues, Renata S; Rodrigues, Veridiana de Melo

    2016-06-01

    Snake venom serine proteases (SVSPs) are enzymes capable of interfering at several points of hemostasis. Some serine proteases present thrombin-like activity, which makes them targets for the development of therapeutics agents in the treatment of many hemostatic disorders. In this study, a recombinant thrombin-like serine protease, denominated rBpSP-II, was obtained from cDNA of the Bothrops pauloensis venom gland and was characterized enzymatically and biochemically. The enzyme rBpSP-II showed clotting activity on bovine plasma and proteolytic activity on fibrinogen, cleaving exclusively the Aα chain. The evaluation of rBpSP-II activity on chromogenic substrates demonstrated thrombin-like activity of the enzyme due to its capacity to hydrolyze the thrombin substrate. These characteristics make rBpSP-II an attractive molecule for additional studies. Further research is needed to verify whether rBpSP-II can serve as a template for the synthesis of therapeutic agents to treat hemostatic disorders. PMID:26965926

  11. 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. PMID:26014935

  12. Screening and in vitro testing of antifolate inhibitors of human cytosolic serine hydroxymethyltransferase.

    PubMed

    Paiardini, Alessandro; Fiascarelli, Alessio; Rinaldo, Serena; Daidone, Frederick; Giardina, Giorgio; Koes, David R; Parroni, Alessia; Montini, Giulia; Marani, Marina; Paone, Alessio; McDermott, Lee A; Contestabile, Roberto; Cutruzzol, Francesca

    2015-03-01

    Metabolic reprogramming of tumor cells toward serine catabolism is now recognized as a hallmark of cancer. Serine hydroxymethyltransferase (SHMT), the enzyme providing one-carbon units by converting serine and tetrahydrofolate (H4 PteGlu) to glycine and 5,10-CH2 -H4 PteGlu, therefore represents a target of interest in developing new chemotherapeutic drugs. In this study, 13 folate analogues under clinical evaluation or in therapeutic use were in silico screened against SHMT, ultimately identifying four antifolate agents worthy of closer evaluation. The interaction mode of SHMT with these four antifolate drugs (lometrexol, nolatrexed, raltitrexed, and methotrexate) was assessed. The mechanism of SHMT inhibition by the selected antifolate agents was investigated in vitro using the human cytosolic isozyme. The results of this study showed that lometrexol competitively inhibits SHMT with inhibition constant (Ki ) values in the low micromolar. The binding mode of lometrexol to SHMT was further investigated by molecular docking. These results thus provide insights into the mechanism of action of antifolate drugs and constitute the basis for the rational design of novel and more potent inhibitors of SHMT. PMID:25677305

  13. 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 (30kDa) 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 2000units/mg protein against 170units/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 5mMMg(+2). These results suggest that the purified keratinase may be used in several industrial applications. PMID:26802243

  14. PAK-dependent STAT5 serine phosphorylation is required for BCR-ABL-induced leukemogenesis

    PubMed Central

    Berger, A; Hoelbl-Kovacic, A; Bourgeais, J; Hoefling, L; Warsch, W; Grundschober, E; Uras, I Z; Menzl, I; Putz, E M; Hoermann, G; Schuster, C; Fajmann, S; Leitner, E; Kubicek, S; Moriggl, R; Gouilleux, F; Sexl, V

    2014-01-01

    The transcription factor STAT5 (signal transducer and activator of transcription 5) is frequently activated in hematological malignancies and represents an essential signaling node downstream of the BCR-ABL oncogene. STAT5 can be phosphorylated at three positions, on a tyrosine and on the two serines S725 and S779. We have investigated the importance of STAT5 serine phosphorylation for BCR-ABL-induced leukemogenesis. In cultured bone marrow cells, expression of a STAT5 mutant lacking the S725 and S779 phosphorylation sites (STAT5SASA) prohibits transformation and induces apoptosis. Accordingly, STAT5SASA BCR-ABL+ cells display a strongly reduced leukemic potential in vivo, predominantly caused by loss of S779 phosphorylation that prevents the nuclear translocation of STAT5. Three distinct lines of evidence indicate that S779 is phosphorylated by group I p21-activated kinase (PAK). We show further that PAK-dependent serine phosphorylation of STAT5 is unaffected by BCR-ABL tyrosine kinase inhibitor treatment. Interfering with STAT5 phosphorylation could thus be a novel therapeutic approach to target BCR-ABL-induced malignancies. PMID:24263804

  15. 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

  16. A Bisbenzamidine Phosphonate as a Janus-faced Inhibitor for Trypsin-like Serine Proteases.

    PubMed

    Huler, Daniela; Scheidt, Tamara; Stirnberg, Marit; Steinmetzer, Torsten; Gtschow, Michael

    2015-10-01

    A hybrid approach was applied for the design of an inhibitor of trypsin-like serine proteases. Compound 16 [(R,R)- and (R,S)-diphenyl (4-(1-(4-amidinobenzylamino)-1-oxo-3-phenylpropan-2-ylcarbamoyl)phenylamino)(4-amidinophenyl)methylphosphonate hydrochloride], prepared in a convergent synthetic procedure, possesses a phosphonate warhead prone to react with the active site serine residue in a covalent, irreversible manner. Each of the two benzamidine moieties of 16 can potentially be accommodated in the S1 pocket of the target enzyme, but only the benzamidine close to the phosphonate group would then promote an irreversible interaction. The Janus-faced inhibitor 16 was evaluated against several serine proteases and caused a pronounced inactivation of human thrombin with a second-order rate constant (kinac /Ki) of 59?500?M(-1) ?s(-1). With human matriptase, 16 showed preference for a reversible mode of inhibition (IC50 =2.6??M) as indicated by linear progress curves and enzyme reactivation. PMID:26306030

  17. Cytoplasmic serine hydroxymethyltransferase regulates the metabolic partitioning of methylenetetrahydrofolate but is not essential in mice.

    PubMed

    MacFarlane, Amanda J; Liu, Xiaowen; Perry, Cheryll A; Flodby, Per; Allen, Robert H; Stabler, Sally P; Stover, Patrick J

    2008-09-19

    The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm. PMID:18644786

  18. Non-criteria anti-phospholipid antibodies and cognitive impairment in SLE.

    PubMed

    Luggen, Michael E; Gulati, Gaurav; Zhang, Bin; Willis, Rohan A; Gonzalez, Emilio B

    2016-01-01

    The pathogenesis of cognitive impairment (CI) in patients with systemic lupus erythematosus (SLE) is unknown. Anti-phospholipid antibodies (APL) have been implicated. The APL which have been evaluated have variably included anti-cardiolipin (ACL) antibodies, lupus anticoagulant (LAC), and antibodies to beta-2 glycoprotein I (β2GPI). Few studies have examined other APL (so-called non-criteria APL). We evaluated the association of CI with a broad spectrum of non-criteria APL. Subjects meeting SLE classification criteria were recruited from three different patient populations. Cognitive function was assessed with the Automated Neuropsychologic Assessment Metrics (ANAM), a validated computer-based assessment tool. The total throughput score (TTS = number of correct responses/time) was used as the primary outcome measure. The following APL of all three isotypes were assessed by ELISA using standardized techniques: anti-β2GPI, anti-phosphatidyl ethanolamine (aPE), anti-phosphatidyl choline (aPC), anti-phosphatidyl inositol (aPI), anti-phosphatidyl serine (aPS), anti-phosphatidyl glycerol (aPG), anti-phosphatidic acid (aPA). Fifty-seven (57) patients were evaluated. Of the 57, 12 had definite CI (>1.5 SD below the mean of an age-, sex-, and race-matched RA population). The two groups were significantly different with regard to age, ethnicity, and family income. There was no significant difference between groups with regard to the presence of any non-criteria APL. When titers of specific non-criteria APL were compared with TTS, no significant correlations were found. Using multiple linear regression and adjusting for relevant covariates including age, ethnicity, and family income, neither the presence nor the titer of any non-criteria APL significantly influenced TTS. In this cross-sectional study, non-criteria APL were not associated with CI. PMID:26563139

  19. 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.

  20. A critical appraisal of NLO+PS matching methods

    NASA Astrophysics Data System (ADS)

    Höche, Stefan; Krauss, Frank; Schönherr, Marek; Siegert, Frank

    2012-09-01

    In this publication, uncertainties in and differences between the M C@NLO and P OWHEG methods for matching next-to-leading order QCD calculations with parton showers are discussed. Implementations of both algorithms within the event generator S HERPA and based on Catani-Seymour subtraction are employed to assess the impact on a representative selection of observables. In the case of M C@NLO a substantial simplification is achieved by using dipole subtraction terms to generate the first emission. A phase space restriction is employed, 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 ± and Z bosons in association with a jet. Corresponding results are compared to data from the Tevatron and LHC experiments.

  1. 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.

  2. Phytophthora sojae effector PsCRN70 suppresses plant defenses in Nicotiana benthamiana.

    PubMed

    Rajput, Nasir Ahmed; Zhang, Meixiang; Ru, Yanyan; Liu, Tingli; Xu, Jing; Liu, Li; Mafurah, Joseph Juma; Dou, Daolong

    2014-01-01

    Phytophthora sojae, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in P. sojae. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulation in the PsCRN70-transgenic plants was reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection. PMID:24858571

  3. Genome Sequence of Non-O1 Vibrio cholerae PS15

    PubMed Central

    Kumar, Sanath; Lindquist, Ingrid E.; Sundararajan, Anitha; Rajanna, Chythanya; Floyd, Jared T.; Smith, Kenneth P.; Andersen, Jody L.; He, Guixin; Ayers, Ryan M.; Johnson, Judith A.; Werdann, James J.; Sandoval, Ava A.; Mojica, Nadia M.; Schilkey, Faye D.; Mudge, Joann

    2013-01-01

    The draft genome sequence of a non-O1 Vibrio cholerae strain, PS15, organized into 3,512 open reading frames within a 3.9-Mb genome, was determined. The PS15 genome sequence will allow for the study of the evolution of virulence and environmental adaptation in V. cholerae. PMID:23409261

  4. Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules.

    PubMed Central

    Dahiya, P; Kardailsky, I V; Brewin, N J

    1997-01-01

    The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development. PMID:9414555

  5. 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

  6. 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…

  7. 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.

  8. Spectral classification of PS15blq/MASTER OT J173142.37+435334.1

    NASA Astrophysics Data System (ADS)

    Huber, M.; Chambers, K. C.; Smith, K. W.; Wright, D.; Smartt, S. J.; Flewelling, H.; Willman, M.; Primak, N.; Schultz, A.; Gibson, B.; Magnier, E.; Waters, C.; Tonry, J.; Wainscoat, R. J.; Foley, R. J.; Jha, S. W.; Rest, A.; Scolnic, D.

    2015-10-01

    We report spectroscopic classification of PS15blq from the Pan-STARRS Survey for Transients (PSST; Huber et al., ATel #7153; http://star.pst.qub.ac.uk/ps1threepi/ ). This object was discovered independently by both PSST and the MASTER survey - see Shumkov et al ATel #8104, and Smith et al ATel.

  9. 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.

  10. 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 field focused the ligand-bound ECμPs to pressure antinodes and the positive acoustic contrast blood cells to the central pressure node of the microchannel. As a result of laminar flow, focused ligand-bound ECμPs and blood cells were flowed into properly aligned outlet channels at the downstream trifurcation, where they where collected separately off-chip. The cell-free fraction containing ligand-bound ECμPs was analyzed using flow cytometry; where the detection of IgG-PE was in the picomolar range. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.

  11. Retinoid Uptake, Processing, and Secretion in Human iPS-RPE Support the Visual Cycle

    PubMed Central

    Muñiz, Alberto; Greene, Whitney A.; Plamper, Mark L.; Choi, Jae Hyek; Johnson, Anthony J.; Tsin, Andrew T.; Wang, Heuy-Ching

    2014-01-01

    Purpose. Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE. Methods. iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium–derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids. Results. Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media. Conclusions. iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE. PMID:24255038

  12. [Current status and perspective on regenerative medicine for spinal cord injury using iPS cell].

    PubMed

    Nakamura, Masaya; Toyama, Yoshiaki; Okano, Hideyuki

    2013-01-01

    Stimulated by the 2012 Nobel Prize in Physiology or Medicine awarded for Shinya Yamanaka and Sir John Gurdon, there is an increasing interest in the iPS cells and reprogramming technologies in medical science. While iPS cells are expected to open new era providing enormous opportunities in the biomedical sciences in terms of cell therapies for regenerative medicine, safety-related concerns for iPS cell-based cell therapy should be resolved prior to the clinical application of iPS cells. In this symposium, the pre-clinical investigations of cell therapy for SCI using neural stem/progenitor cells derived from iPS cells, and their safety issues in vivo are outlined. PMID:24291863

  13. 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

  14. 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.

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

    PubMed Central

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

    2013-01-01

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

  16. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation

    PubMed Central

    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. PMID:26560496

  17. 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. PMID:26973101

  18. Retromer and Rab2-dependent trafficking mediate PS1 degradation by proteasomes in endocytic disturbance.

    PubMed

    Ueda, Naoya; Tomita, Taisuke; Yanagisawa, Katsuhiko; Kimura, Nobuyuki

    2016-05-01

    Accumulating evidence suggests that endocytic pathway deficits are involved in Alzheimer's disease pathogenesis. Several reports show that endocytic disturbance affects β-amyloid peptide (Aβ) cleavage from β-amyloid precursor protein (APP). Presenilin-1 (PS1) is the catalytic core of the γ-secretase complex required for Aβ generation. Previously, we showed that aging induces endocytic disturbance, resulting in the accumulation of Aβ and APP in enlarged endosomes. It remains unclear, however, whether PS1 localization and function are affected with endocytic disturbance. Here, we report that in endocytic disturbance, PS1 is transported from endosomes to ER/Golgi compartments via retromer trafficking, and that PS1 interacts with vacuolar protein sorting-associated protein 35 both in vitro and in vivo. Moreover, PS1 is degraded by proteasomes via a Rab2-dependent trafficking pathway, only during endocytic disturbance. These findings suggest that PS1 levels and localization in endosomes are regulated by retromer trafficking and ER-associated degradation system, even if endocytic disturbance significantly induces the endosomal accumulation of APP and β-site APP-cleaving enzyme 1. Results of this study also suggest that retromer deficiency can affect PS1 localization in endosomes, where Aβ cleavage mainly occurs, possibly leading to enhanced Aβ pathology. We proposed the following mechanism for intracellular transport of presenilin-1 (PS1). When endosome/lysosome trafficking is disturbed, PS1 is transported from endosome to endoplasmic reticulum (ER)/Golgi compartments via retromer and Rab2-mediated trafficking, and then degraded by endoplasmic reticulum-associated degradation (ERAD). Perturbations in this trafficking can cause abnormal endosomal accumulation of PS1, and then may lead to exacerbated Aβ pathology. Cover Image for this issue: doi: 10.1111/jnc.13318. PMID:26896628

  19. PsTRXh1 and PsTRXh2 Are Both Pea h-Type Thioredoxins with Antagonistic Behavior in Redox Imbalances12

    PubMed Central

    Traverso, José A.; Vignols, Florence; Cazalis, Roland; Pulido, Amada; Sahrawy, Mariam; Cejudo, Francisco Javier; Meyer, Yves; Chueca, Ana

    2007-01-01

    Thioredoxins (TRXs) are small ubiquitous oxidoreductases involved in disulfide bond reduction of a large panel of target proteins. The most complex cluster in the family of plant TRXs is formed by h-type TRXs. In Arabidopsis (Arabidopsis thaliana), nine members of this subgroup were described, which are less well known than their plastidial counterparts. The functional study of type-h TRXs is difficult because of the high number of isoforms and their similar biochemical characteristics, thus raising the question whether they have specific or redundant functions. Type-h TRXs are involved in seed germination and self incompatibility in pollen-pistil interaction. Their function as antioxidants has recently been proposed, but further work is needed to clarify this function in plants. In this study, we describe two new h-type TRXs from pea (Pisum sativum; stated PsTRXh1 and PsTRXh2). By functional complementation of a yeast (Saccharomyces cerevisiae) trx1Δ trx2Δ double mutant, we demonstrate that PsTRXh1 is involved in the redox-imbalance control, possibly through its interaction with peroxiredoxins. In contrast, PsTRXh2 provokes a phenotype of hypersensitivity to hydrogen peroxide in the yeast mutant. Furthermore, we show differential gene expression and protein accumulation of the two isoforms, PsTRXh1 protein being abundantly detected in vascular tissue and flowers, whereas PsTRXh2 gene expression was hardly detectable. By comparison with previous data of additional PsTRXh isoforms, our results indicate specific functions for the pea h-type TRXs so far described. PMID:17098852

  20. 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.

  1. Loss-of-function mutation of serine racemase attenuates excitotoxicity by intravitreal injection of N-methyl-D-aspartate.

    PubMed

    Jiang, Haiyan; Wang, Xianwei; Zhang, He; Chang, Yuhua; Feng, Meiling; Wu, Shengzhou

    2016-01-01

    Convincing data demonstrate that D-serine, a racemized product of serine racemase (SR), contributes to neurotoxicity. Furthermore, a line of evidence suggests that SR/D-serine contributes to retinal neurodegeneration in a diabetic retinopathy rat model and diabetic retinopathy patients. However, the connection between SR/D-serine and retinal neurodegeneration remains unclear. Herein, we report that intravitreal injection of N-methyl-D-aspartate (NMDA) induces excitotoxicity in rodent retina; this retinal neurodegeneration was attenuated in retina carrying a loss-of-function of mutation in Srr, the gene for SR, termed Srr(ochre269). Under the condition of NMDA injection, either posterior pole or middle - but not peripheral - retina from Srr(ochre269) mice was found to retain more retinal ganglion cells (RGC) than the counterpart from w/t (RGCs were identified with retrograde labeling). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining further demonstrated reduced RGC apoptosis from Srr(ochre269) compared to w/t mice under the condition of NMDA injection. Collectively, our studies demonstrate a pivotal role of SR/D-serine in retinal neurotoxicity. We demonstrated that loss-of-function mutation of the gene encoding serine racemase significantly attenuates excitotoxicity in retina; excitotoxicity accounts for retinal ganglion cell (RGC) demise in diabetic retinopathy (DR). We think that our findings deepen the current knowledge of the mechanisms of RGC degeneration. PMID:26485193

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

    PubMed Central

    Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura

    2015-01-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. PMID:25986610

  3. 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

    2016-03-01

    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. PMID:26785702

  4. 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 represented a diverse international and multidisciplinary group, with over 160 individuals attending from a total of 17 different countries. Attendees came from a wide range of fields assuring that the widest possible interdisciplinary exchanges. They included prominent biochemists, biophysicists, plant physiologists, chemical physicists, as well as theoretical and computational physical chemists, who presented their research findings or to hear the latest advances in this very dynamic field. In the choice of speakers, a balance was created between established scientists and young, emerging researchers, given this opportunity to showcase their results. Sessions were held on electronic and vibrational coherence including coherent sharing of excitations among donor and acceptor molecules during excitation energy transfer, nonphotochemical quenching, acclimation to light environments, evolution, adaptation and biodiversity of light-harvesting pigment-protein complexes, their structure and membrane organization, spectroscopy and dynamics, as well as artificial antenna systems. A joint session was also held with the participants from the Cyanobacterial Satellite Conference. A special issue of Photosynthesis Research devoted to light harvesting (Volume 121, Issue No. 1, July 2014) has recently appeared which contains peer-reviewed original research contributions arising from talks and posters presented at the PS2013 Satellite Workshop on Photosynthetic Light-Harvesting Systems. Edited by the Organizers of the Workshop, Robert E. Blankenship, Harry A. Frank and Robert A. Niederman, it includes topics ranging from the isolation of new bacteriochlorophyll species from green bacteria, temperature effects on the excited states of the newly discovered chlorophyll (Chl) ƒ, new architectures for enhancing energy capture by biohybrid light-harvesting complexes, forces governing the formation of light-harvesting rings, spectroscopy of carotenoids of algae and diatoms and the supramolecular organization of caroteno-Chl proteins in diatoms, the molecular basis for urea dissociation of phycocyanin trimers and the role of vibronic molecular excitation theory in describing the spectral dynamics of pigment-protein complexes.

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

    PubMed

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

    2016-05-01

    Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability. PMID:27073009

  6. Capillary electrophoresis-laser induced fluorescence (CE-LIF) assay for measurement of intra-cellular D-Serine and serine racemase activity

    PubMed Central

    Singh, Nagendra S.; Paul, Rajib K.; Sichler, Megan; Moaddel, Ruin; Bernier, Michel; Wainer, Irving W.

    2011-01-01

    An enantioselective capillary electrophoresis–laser induced fluorescence (CE-LIF) method for the analysis of D-Serine (D-Ser) in cellular matrices has been developed. The assay involves derivatization with FITC followed by CE-LIF using 0.5 mM hydroxyl propyl-β-cyclodextrin in borate buffer [80 mM, pH 9.3]. The method was able to resolve D-Ser and L-Ser with an enantioselectivity (α) of 1.03 and a Resolution (Rs) of 1.37. Linearity was established from 0.25 μM – 100.00 μM. The assay was also able to enantioselectively resolve 6 additional amino acid racemates. The method was applied to the determination of intra-cellular D-Ser concentrations in PC-12, C6, 1312N1 and HepG2 cell lines. This method was used to determine the concentration-dependent increases in D-Ser and associated EC50 values produced by L-Ser and the concentration-dependent decreases in D-Ser and associated IC50 values produced by glycine, a competitive inhibitor of serine racemase (SR). Western blot analysis determined that the PC-12 and C6 cell lines contained monomeric and dimeric forms of SR while the 1321N1 and HepG2 cells contained only the monomeric form. Although the SR dimer has been identified as the active form of the enzyme, all four of the tested cell lines expressed enzymatically active SR. PMID:22037294

  7. Genital warts in Burmeister's porpoises: characterization of Phocoena spinipinnis papillomavirus type 1 (PsPV-1) and evidence for a second, distantly related PsPV.

    PubMed

    Van Bressem, Marie-Françoise; Cassonnet, Patricia; Rector, Annabel; Desaintes, Christian; Van Waerebeek, Koen; Alfaro-Shigueto, Joanna; Van Ranst, Marc; Orth, Gérard

    2007-07-01

    We identified sequences from two distantly related papillomaviruses in genital warts from two Burmeister's porpoises, including a PV antigen-positive specimen, and characterized Phocoena spinipinnis papillomavirus type 1 (PsPV-1). The PsPV-1 genome comprises 7879 nt and presents unusual features. It lacks an E7, an E8 and a bona fide E5 open reading frame (ORF) and has a large E6 ORF. PsPV-1 L1 ORF showed the highest percentage of nucleotide identity (54-55 %) with human papillomavirus type 5, bovine papillomavirus type 3 (BPV-3) and Tursiops truncatus papillomavirus type 2 (TtPV-2). This warrants the classification of PsPV-1 as the prototype of the genus Omikronpapillomavirus. PsPV-1 clustered with TtPV-2 in the E6 and E1E2 phylogenetic trees and with TtPV-2 and BPV-3 in the L2L1 tree. This supports the hypothesis that PV evolution may not be monophyletic across all genes. PMID:17554024

  8. An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

    PubMed Central

    Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

    2015-01-01

    The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation. PMID:25934799

  9. 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. PMID:24025773

  10. Expression and localization in the fish retina of a homologue of the Alzheimer's related PS1 gene.

    PubMed

    Archer, S; Hirano, J; Diss, J K; Fraser, S P; Djamgoz, M B

    1998-06-22

    Early-onset familial Alzheimer's disease (early-onset FAD) has been linked with mutations in the presenilin gene, PS1. Mutations in PS1 may affect the processing/trafficking of b-amyloid precursor-protein (bAPP) and favour the production of toxic amyloid-b fragments that are associated with neural degeneration. This study reports the expression of a PS1-like cDNA in the carp (Cyprinus carpio) retina (the encoded protein shows 76% identity to the human PS1 protein). Carp PS1 mRNA was localized by in situ hybridization to the photoreceptor cell, inner nuclear and ganglion cell layers. Expression of the PS1 gene in the rat retina was also confirmed. The retinal expression of PS1 raises the possibility that PS1 mutations also lead to neural degeneration in the retina. PMID:9674592

  11. Expression and localization in the fish retina of a homologue of the Alzheimer's related PS1 gene.

    PubMed

    Archer, S; Hirano, J; Diss, J K; Fraser, S P; Djamgoz, M B

    1998-09-14

    Early-onset familial Alzheimer's disease (early-onset FAD) has been linked with mutations in the presenilin gene, PS1. Mutations in PS1 may affect the processing/ trafficking of beta-amyloid precursor-protein (betaAPP) and favour the production of toxic amyloid-beta fragments that are associated with neural degeneration. This study reports the expression of a PS1-like cDNA in the carp (Cyprinus carpio) retina (the encoded protein shows 76% identity to the human PS1 protein). Carp PS1 mRNA was localized by in situ hybridization to the photoreceptor cell, inner nuclear and ganglion cell layers. Expression of the PS1 gene in the rat retina was also confirmed. The retinal expression of PS1 raises the possibility that PS1 mutations also lead to neural degeneration in the retina. PMID:9804326

  12. Dynamic dipole polarizabilities of H- and Ps- in weakly coupled plasmas

    NASA Astrophysics Data System (ADS)

    Kar, Sabyasachi; Li, H. W.; Jiang, Pinghui

    2013-08-01

    The effects of weakly coupled plasmas on the dynamic dipole polarizabilities of the H- and Ps- ions are investigated using highly correlated exponential wave functions. The Debye-Hückel shielding approach of plasma modeling is used to represent weakly coupled plasma environments. In free-atomic cases, results obtained from the present study for H- are in agreement with the available calculations and results for Ps- are reported for the first time. Frequency-dependent polarizabilities of H- and Ps- as functions of screening parameter are also presented for the first time.

  13. The Characteristics of Murine iPS Cells and siRNA Transfection Under Hypoxia.

    PubMed

    Sugimoto, K; Hayashi, Yoshihiko

    2016-01-01

    iPS cells are attractive for the regenerative medicine. The creation of pluripotent cells from somatic cells has great potential for basic and clinical research and application. Retroviral transduction of four or three transfection factors has been shown to initiate a reprogramming process. Here, we describe the effect of transcription factors regarding the growth and differentiation of mouse iPS cells in normoxia or hypoxia. Furthermore, we introduce the function of hypoxia-inducible factors (HIFs) in mouse iPS cells in hypoxia using RT-PCR and western blotting together with HIFs knockdown techniques. PMID:25476444

  14. 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. PMID:24331430

  15. Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324

    PubMed Central

    Santhosh, K T; Sikarwar, A S; Hinton, M; Chelikani, P; Dakshinamurti, S

    2014-01-01

    Background and Purpose Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. Experimental Approach Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. Key Results The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. Conclusions and Implications In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity. PMID:24490858

  16. Irreversible inhibition of serine proteases by peptide derivatives of (. alpha. -aminoalkyl)phosphonate dephenyl esters

    SciTech Connect

    Oleksyszyn, J.; Powers, J.C. )

    1991-01-01

    Peptidyl derivatives of diphenyl ({alpha}-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentration. Z-Phe{sup P}(OPh){sub 2} irreversibly reacts with chymotrypsin and does not react with two elastases. The best inhibitor for most chymotryspin-like enzymes including bovine chymotrypsin, cathespin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-Phe{sup P}(OPh){sub 2} which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-Val{sup P}(OPh){sub 2} is specific for elastase and reacts with human leukocyte elastase but not with chymotrypsin. The tripeptide Boc-Val-Pro-Val{sup P}(OPh){sub 2}, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE and porcine pancreatic elastase. The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-Phe{sup P}(OPh){sub 2} and PPE and HLE by MeO-Suc-Ala-Ala-Prov-Val{sup P}(OPh){sub 2} were decreased 2-5 fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-Peh{sup P}(OPh){sub 2} reacts with chymotrypsin and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the {sup 31}P NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was {ge}48 h for the inhibited elastases and 7.5-26 h for chymotrypsin. Peptidyl derivatives of diphenyl ({alpha}-aminoalkyl)phosphonates are relatively easy to synthesize, are chemically stable in buffer and in human plasma, form very stable derivatives with serine proteases, do not react with acetylcholinesterase, and thus should have considerable potential utility as therapeutic agents.

  17. 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

  18. 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; Sll, Dieter

    2016-02-19

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

  19. 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 86kDa. 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. PMID:26773656

  20. Utilizing the activation mechanism of serine proteases to engineer hepatocyte growth factor into a Met antagonist

    PubMed Central

    Kirchhofer, Daniel; Lipari, Michael T.; Santell, Lydia; Billeci, Karen L.; Maun, Henry R.; Sandoval, Wendy N.; Moran, Paul; Ridgway, John; Eigenbrot, Charles; Lazarus, Robert A.

    2007-01-01

    Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase Met, is secreted as single chain pro-HGF that lacks signaling activity. Pro-HGF acquires functional competence upon cleavage between R494 and V495, generating a disulfide-linked ?/?-heterodimer, where the ?-chain of HGF (HGF ?) has a serine protease fold that lacks enzymatic activity. We show that, like serine proteases, insertion of the newly formed N terminus in the ?-chain is critical for activity, here by allosterically stabilizing interactions with Met. The HGF ? crystal structure shows that V495 inserts into the activation pocket near the Met binding site where the positively charged N terminus forms a salt bridge with the negatively charged D672, and the V495 side chain has hydrophobic interactions with main- and side-chain residues. Full-length two-chain HGF mutants designed to interrupt these interactions (D672N, V495G, V495A, G498I, and G498V) displayed <10% activity in Met receptor phosphorylation, cell migration, and proliferation assays. Impaired signaling of full-length mutants correlated with >50-fold decreases in Met binding of the low-affinity HGF ? domain alone bearing the same mutations and further correlated with impaired N-terminal insertion. Because high-affinity binding resides in the HGF ?-chain, full-length mutants maintained normal Met binding and efficiently inhibited HGF-mediated Met activation. Conversion of HGF from agonist to antagonist was achieved by as little as removal of two methyl groups (V495A) or a single charge (D672N). Thus, although serine proteases and HGF have quite distinct functions in proteolysis and Met signal transduction, respectively, they share a similar activation mechanism. PMID:17372204

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

    PubMed Central

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

    2013-01-01

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

  2. Attachment site recognition and regulation of directionality by the serine integrases

    PubMed Central

    Rutherford, Karen; Yuan, Peng; Perry, Kay; Sharp, Robert; Van Duyne, Gregory D.

    2013-01-01

    Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between ‘attachment sites’ in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function. PMID:23821671

  3. 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.

  4. Isolation and characterization of a serine protease, Ba III-4, from Peruvian Bothrops atrox venom.

    PubMed

    Ponce-Soto, L A; Bonfim, V L; Novello, J C; Navarro Oviedo, R; Yarlequé Chocas, A; Marangoni, S

    2007-09-01

    A serine protease from Bothrops atrox (Peruvian specimen's venom) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. This protein was denominated Ba III-4 (33,080.265 Da determinated by MALDI-TOF mass spectrometry) and showed pI of 5.06, Km 0.2 x 10(-1 ) M and the V (máx) 4.1 x 10(-1 )nmoles p-NA/lt/min on the synthetic substrate BapNA. Ba III-4 also showed ability to coagulate bovine fibrinogen. The serine protease was inhibited by soyben trypsin inhibitor and DA2II, which is an anti-hemorrhagic factor isolated from the opossum specie Didelphis albiventris. The primary structure of Ba III-4 showed the presence of His(44), Asp(94) and Ser(193) residues in the corresponding positions to the catalytic triad established in the serine proteases and Ser(193) are inhibited by phenylmethylsulfonylfluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro, as well as 12 half-cysteine residues. Ba III-4 contained 293 amino acid residues and the primary structure of VIGGDECDIN EHPFLAFMYY SPRYFCGMTL INQEWVLTAA HCRYFCGMTL IHLGVHRESE KANYDEVRRF PKEKYFIFCD NNFTDDEVDK DIMLIRLDKP VSNSEHIAPL SLPSNPPSVG SVCRIMGWGQ TTTSPIDVLS PDEPHCANIN LFDNTVCHTA HPQVANTRTS TDTLCAGDLQ GGRDTCNGDS GGPLICNEQL HGILSWGGDP CAQPNKPAFY TKVYYFDHPW IKSIIAGNKK TVNFTCPPLR SDAKDDSTTY INQEWDWVLT AEHCDRTHMR NSFYDYSSIN SDS. Titration experiments did not show the presence of free sulfhydryl groups after 4 h incubation, nor were differences found in relation to titration kinetics in the presence of nondenaturating buffer. The isolation of this protein, Ba III-4, is of potential interest for the understanding of the pathomechanism of the snake venom action and for the identification of new blood coagulation enzymes of natural sources. PMID:17522968

  5. 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 36kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520Da. 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. PMID:26827743

  6. 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

  7. Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

    PubMed Central

    Gelpi, C; Sontheimer, E J; Rodriguez-Sanchez, J L

    1992-01-01

    We describe an autoantibody specificity present in a subgroup of patients with a severe form of autoimmune chronic active hepatitis. These antibodies precipitate a 90-nucleotide RNA from human whole cell extracts and recognize a 48-kDa polypeptide in immunoblotting assays. The RNA is a UGA suppressor serine tRNA that carries selenocysteine (tRNA[Ser]Sec)), as shown by sequence analysis. The protein does not appear to be seryl-tRNA synthetase; rather, it is an excellent candidate for a factor involved in cotranslational selenocysteine incorporation in human cells. Images PMID:1409691

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

    PubMed Central

    Canova, Marc J.; Molle, Virginie

    2014-01-01

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

  9. Tick-Encoded serine proteinase inhibitors (serpins); potential target antigens for tick vaccine development.

    PubMed

    Muleng, A; Sugino, M; Nakajim, M; Sugimoto, C; Onuma, M

    2001-10-01

    Immunological protection of hosts against tick infestation is at present the most practically sustainable alternative tick control method to the current use of acaricides that is riddled with serious limitations. The current focus of tick vaccine research is the identification, cloning and in vitro production of recombinant tick vaccine candidate antigens. We have examined a selected number of reports on the roles of parasite-encoded members of the serine proteinase inhibitor (serpin) superfamily in modulation of mammalian anti-parasite defense and developed some food for thought commentaries on the possibility of targeting this class of proteins for anti-tick vaccine development. PMID:11714020

  10. A tri-serine tri-lactone scaffold for the quantification of citrate in urine.

    PubMed

    Akdeniz, Ali; Caglayan, Mehmet Gokhan; Anzenbacher, Pavel

    2016-01-21

    Tri-serine tri-lactone based C3 symmetry fluorescent sensors were synthesized. Citrate is shown to bind to sensors, while displaying an increase in fluorescence intensity for the sensor with thiourea and a quenching for the sensor with sulfonamide. Information-rich responses of the sensors enable us to discriminate structurally similar anions, including mono-, di- and tri-carboxylates with 100% correct classification. A simple two-sensor array enables the determination of the concentration of citrate in urine without any sample preparation with high accuracy (error < 2%). PMID:26669653

  11. 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.

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

    PubMed Central

    Locasale, Jason W

    2013-01-01

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

  13. An analysis of 3D solvation structure in biomolecules: application to coiled coil serine and bacteriorhodopsin.

    PubMed

    Hirano, Kenji; Yokogawa, Daisuke; Sato, Hirofumi; Sakaki, Shigeyoshi

    2010-06-17

    Three-dimensional (3D) solvation structure around coiled coil serine (Coil-Ser) and inner 3D hydration structure in bacteriorhodopsin (bR) were studied using a recently developed method named multicenter molecular Ornstein-Zernike equation (MC-MOZ) theory. In addition, a procedure for analyzing the 3D solvent distribution was proposed. The method enables us to calculate the coordination number of solvent water as well as the strength of hydrogen bonding between the water molecule and the protein. The results for Coil-Ser and bR showed very good agreement with the experimental observations. PMID:20469882

  14. 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

  15. 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.

  16. Room temperature light emission from the low-dimensional semiconductors AZrPS{sub 6} ( A = K, Rb, Cs).

    SciTech Connect

    Banerjee, S.; Szarko, J. M.; Yuhas, B. D.; Malliakas, C. D.; Chen, L. X.; Kanatzidis, M. G.

    2010-03-29

    The new semiconducting thiophosphate compounds KZrPS{sub 6}, RbZrPS{sub 6}, and CsZrPS{sub 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.

  17. 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

    ... the Federal Register on April 3, 2009 (74 FR 15255) announced NIST's circulation of the revision for... 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...

  18. Synthesis and Optical Properties of ZnO Nanoparticles in Submicron PS Hollow Reactors

    NASA Astrophysics Data System (ADS)

    Huang, Xue-feng; Ju, Fei-fei; Wang, Mo-zhen; Ge, Xue-wu

    2012-12-01

    ZnO nanoparticles were first encapsulated in submicron PS hollow microspheres through two-step swelling process of core-shell structured PMMA/PS (PMMA: polymethyl methaorylate) microspheres in acid-alkali solution, and the ZnO precursors, i.e. the ethanol solutions of (CH3COO)2Zn and LiOH. The transmission electron microscope, X-ray diffraction, and thermogravimetric analysis results show that the feeding order of ethanol solutions of (CH3COO)2Zn and LiOH in the second swelling step has great influence on the loading efficiency and the size of ZnO nanoparticles, but little on their crystal form. The photoluminescence and UV-Vis absorption behavior of ZnO/PS microspheres show that the PS shell can effectively avoid the fluorescence quenching effect.

  19. Chemically defined conditions for human iPS cell derivation and culture

    PubMed Central

    Chen, Guokai; Gulbranson, Daniel R.; Hou, Zhonggang; Bolin, Jennifer M.; Ruotti, Victor; Probasco, Mitchell D.; Smuga-Otto, Kimberly; Howden, Sara E.; Diol, Nicole R.; Propson, Nicholas E.; Wagner, Ryan; Lee, Garrett O.; Antosiewicz-Bourget, Jessica; Teng, Joyce M. C.; Thomson, James A.

    2011-01-01

    We reexamine the individual components for human ES and iPS cell culture, and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces, and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal or human sourced albumin batches have previously plagued human ES and iPS cell culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPS cells with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ES and iPS cells and their derivatives, and should be applicable to other reprogramming methods. PMID:21478862

  20. Hyperspherical hidden crossing calculation of Ps formation in low-energy e+-Na collisions

    NASA Astrophysics Data System (ADS)

    Ward, S. J.; Shertzer, J.

    2011-05-01

    The hyperspherical hidden crossing method (HHCM) can provide important insight into scattering processes. Previously, we have used the HHCM to calculate the Ps(1s)-formation cross section in low-energy e+-H and e+-Li collisions. Here we apply the HHCM to low-energy e+-Na collisions. We use the Peach model potential and treat e+e-Na+ as an effective three-body system. We calculate the Ps(1s)-formation cross sections for 0 <= L <= 3 and compare our results with a hyperspherical close-coupling calculation. The HHCM provides an explanation for the small S-wave Ps(1s)-formation cross section. The S-wave Stückelberg phase is close to π for the three collision systems due to destructive interference between the two amplitudes that correspond to different paths leading to Ps(1s) formation.

  1. Normal Amplitude of Electroretinography and Visual Evoked Potential Responses in AβPP/PS1 Mice.

    PubMed

    Leinonen, Henri; Lipponen, Arto; Gurevicius, Kestutis; Tanila, Heikki

    2016-01-19

    Alzheimer's disease has been shown to affect vision in human patients and animal models. This may pose the risk of bias in behavior studies and therefore requires comprehensive investigation. We recorded electroretinography (ERG) under isoflurane anesthesia and visual evoked potentials (VEP) in awake amyloid expressing AβPPswe/PS1dE9 (AβPP/PS1) and wild-type littermate mice at a symptomatic age. The VEPs in response to patterned stimuli were normal in AβPP/PS1 mice. They also showed normal ERG amplitude but slightly shortened ERG latency in dark-adapted conditions. Our results indicate subtle changes in visual processing in aged male AβPP/PS1 mice specifically at a retinal level. PMID:26836173

  2. VLA search for a radio counterpart to the superluminous supernova PS15ae

    NASA Astrophysics Data System (ADS)

    Alexander, K. D.; Nicholl, M.; Berger, E.; Margutti, R.

    2016-01-01

    We observed the position of the superluminous supernova PS15ae (=CSS141223-113342+004332 = MLS150211-113342+004333) with the Very Large Array on 2015 December 11.67 (UT) when the VLA was in D configuration.

  3. 7 CFR 1753.37 - Plans and specifications (P&S).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., DEPARTMENT OF AGRICULTURE TELECOMMUNICATIONS SYSTEM CONSTRUCTION POLICIES AND PROCEDURES Purchase and... “General Specification for Digital, Stored Program Controlled Central Office Equipment.” (4) P&S...

  4. 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

  5. PS microspheres coated by AuNPs via thermodynamic driving heterocoagulation and their high catalytic activity.

    PubMed

    Pan, Yunfei; Wang, Jianing; Wang, Yong; Wang, Zhaoqun

    2014-03-01

    A unique method of fabricating PS/AuNPs composite particles in ex situ mode is proposed on the basis of thermodynamically driving mechanism. It is facile and versatile as it eliminates the need for surface functionalizations and modifications of both PS microspheres and AuNPs. The PS/AuNPs composite particles take on a raspberry-like morphology with controllable coverage according to some thermodynamic factors, which have been extensively characterized by scanning electron microscopy, transmission electron microscopy, and thermogravimetric analysis. More importantly, the PS/AuNPs composite particles hold higher catalytic efficiency and better repeatability than the previously reported results, which are confirmed in two oxidation-reduction reactions of 2-nitroaniline/NaBH(4) and rhodamine B/NaBH(4). PMID:24474588

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

    PubMed Central

    Neuenschwander, Urs; Suter, Marianne; Brunold, Christian

    1991-01-01

    The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5′-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5′-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of 35SO42− into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5′-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from 35SO42− into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulfate assimilation. PMID:16668378

  7. Converted Ps amplitude variations on a dipping interface: Application to receiver functions in central Japan

    NASA Astrophysics Data System (ADS)

    Shiomi, K.; Park, J. J.

    2009-12-01

    Using the delay times of Ps converted phases in receiver functions (RFs), one can estimate the depth of interfaces underneath a seismic station. 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 φ and ray parameter of the incoming P wave is affected if the interface is dipping and/or anisotropic rock surrounds the interface. In this study, we estimate RFs with various back azimuths and define a 'standard amplitude' of a converted phase at a dipping interface beneath a station, based on back-azimuth dependence of the Ps amplitudes. We apply this analysis to the stations located within the Kii Peninsula, central Japan. First, we estimate the plunge azimuth of the dipping oceanic Moho beneath a station from the delay-time moveout of the Moho-converted Ps phase using the method by Park et al. (2007; AGU FM). Using the theoretical Ps arrival time evaluated from this information, we read the amplitude of RFs. Since this amplitude data shows strong scatter, we calculate an average and its standard deviation for each 5°bin, and fit a simple function constructed with sin(φ), sin(2φ) and constant harmonic terms with a least-squares algorithm. The component of sin(φ) corresponds mainly to the contribution from the dipping interface, and that of sin(2φ) indicates the strength of anisotropy near the velocity interface. At almost all stations, the sin(φ) component dominates, but 4-lobed back-azimuth dependence is clearly confirmed at several stations in the southern part of the peninsula, where strongly anisotropic rocks have been proposed by other researchers. We found that the standard amplitudes and bias components depend on oceanic-Moho depth. As the oceanic Moho deepens to ~40 km, the Ps amplitudes decrease from 13% to 6% of the primary P wave. Ps amplitudes flatten at 5-7% of the primary P wave for oceanic-Moho depths >40km. In the Kii Peninsula region, temperature at the bottom of the oceanic Moho is ~400°C. Thus, Ps amplitude decay likely reflects the lawsonite blueschist to epidote amphibolite transition as water is released to the overlying mantle wedge. These metamorphic fluids likely influence the occurrence of low-frequency nonvolcanic tremor and megathrust earthquakes along the subducting slab.

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

    PubMed

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

    2011-01-01

    A 'Heat treatment aqueous two phase system' was employed for the first time to purify serine protease from kesinai (Streblus asper) leaves. In this study, introduction of heat treatment procedure in serine protease purification was investigated. In addition, the effects of different molecular weights of polyethylene glycol (PEG 4000, 6000 and 8000) at concentrations of 8, 16 and 21% (w/w) as well as salts (Na-citrate, MgSO₄ and K₂HPO₄) at concentrations of 12, 15, 18% (w/w) on serine protease partition behavior were studied. Optimum conditions for serine protease purification were achieved in the PEG-rich phase with composition of 16% PEG6000-15% MgSO₄. Also, thermal treatment of kesinai leaves at 55 °C for 15 min resulted in higher purity and recovery yield compared to the non-heat treatment sample. Furthermore, this study investigated the effects of various concentrations of NaCl addition (2, 4, 6 and 8% w/w) and different pH (4, 7 and 9) on the optimization of the system to obtain high yields of the enzyme. The recovery of serine protease was significantly enhanced in the presence of 4% (w/w) of NaCl at pH 7.0. Based on this system, the purification factor was increased 14.4 fold and achieved a high yield of 96.7%. PMID:22158589

  9. Evidence against a role for serine 129 in determining murine cytochrome P450 Cyp2e-1 protein levels.

    PubMed

    Freeman, J E; Wolf, C R

    1994-11-29

    The cytochrome P450 CYP2E subfamily plays a central role in drug and carcinogen metabolism. The cellular content of this protein is regulated at both the transcriptional and posttranslational levels. CYP2E1 is degraded by both rapid and slow acting proteolytic systems. In the presence of a substrate, CYP2E1 becomes stabilized, and the contribution of the rapid actinig proteolytic pathway to its destruction decreases. It has been suggested that phosphorylation at serine 129 acts as a switch to initiate the fast acting degradative pathway. Phosphorylation at serine 129 has also been suggested to be the point at which hormones, such as insulin, exert actions on the stability of this protein. In order to investigate the role of phosphorylation in determining murine Cyp2e-1 levels, serine 129 was changed by site-directed mutagenesis to amino acids that could not be phosphorylated and the recombinant proteins expressed in COS 7 cells. Replacement of serine 129 with alanine and glycine does not lead to Cyp2e-1 accumulation. In the presence of insulin, although Cyp2e-1 levels increase slightly, specific stabilization of the wild-type protein relative to the two mutant forms is not observed. These observations provide evidence that insulin can act by stabilization of Cyp2e-1 protein but suggest that the phosphorylation of serine 129 is not the molecular basis of stabilization observed. PMID:7947804

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

    PubMed

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

    2015-05-14

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

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

    TOXLINE Toxicology Bibliographic Information

    Hecker SJ; Reddy KR; Totrov M; Hirst GC; Lomovskaya O; Griffith DC; King P; Tsivkovski R; Sun D; Sabet M; Tarazi Z; Clifton MC; Atkins K; Raymond A; Potts KT; Abendroth J; Boyer SH; Loutit JS; Morgan EE; Durso S; Dudley MN

    2015-05-14

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

  12. 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.

  13. SALT spectroscopic classification of PS15bqc as a type-IIb SN before 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.

    2015-08-01

    We obtained SALT (+RSS) spectroscopy of PS15bqc on 2015 Aug 10.8 UT, covering the wavelength range 340-920 nm. Cross-correlation of the spectrum with a template library using SNID (Blondin & Tonry 2007, ApJ, 666, 1024) shows PS15bqc is likely a type-IIb supernova a few days before maximum light, with good matches to spectra of SN 1993J at -3 or -2 days from maximum.

  14. 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

  15. Head-tail instability and microwave instability in the KEK-PS

    SciTech Connect

    Toyama, T.; Arakawa, D.; Igarashi, S.; Kishiro, J.; Koba, K.; Nakamura, E.; Takayama, K.; Yoshii, M.

    1999-12-03

    Head-tail instability and microwave instability has been studied and cured in the course of the intensity up-grade program of the KEK-PS. Experimental results, its interpretations and cures are given on the head-tail instabilities, emerging just after the acceleration start, and the microwave instability, arising after the transition energy in the 12 GeV PS main ring.

  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

    2014-02-01

    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. PMID:24518646

  17. Preparation of pancreatic ?-cells from human iPS cells with small molecules

    PubMed Central

    2012-01-01

    Human induced pluripotent stem (iPS) cells obtained from patients are expected to be a useful source for cell transplantation therapy, because many patients (including those with type 1 diabetes and severe type 2 diabetes) are on waiting lists for transplantation for a long time due to the shortage of donors. At present, many concerns related to clinical application of human iPS cells have been raised, but rapid development of methods for the establishment, culture, and standardization of iPS cells will lead autologous cell therapy to be realistic sooner or later. However, establishment of a method for preparing some of desired cell types is still challenging. Regarding pancreatic ?-cells, there have been many reports about differentiation of these cells from human embryonic stem (ES)/iPS cells, but a protocol for clinical application has still not been established. Since there is clear proof that cell transplantation therapy is effective for diabetes based on the results of clinical islet transplantation, pancreatic ?-cells prepared from human iPS cells are considered likely to be effective for reducing the burden on patients. In this article, the current status of procedures for preparing pancreatic ?-cells from human ES/iPS cells, including effective use of small molecules, is summarized, and some of the problems that still need to be overcome are discussed. PMID:22722666

  18. 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

  19. Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

    PubMed

    Li, Hongmei Lisa; Gee, Peter; Ishida, Kentaro; Hotta, Akitsu

    2016-05-15

    Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells. PMID:26525194

  20. Cloning and expression of the sucrose transporter gene PsSUT1 from tree peony leaf.

    PubMed

    Li, Y H; Guo, T; Cui, Y; Li, Y; He, D

    2015-01-01

    This study reports the cloning of a sucrose transporter gene, PsSUT1, from the leaf of tree peony (Paeonia suffruticosa Lind. cv 'Huhong'). Expression patterns were examined in different organs and at different developmental stages. The full-length cDNA of PsSUT1 consisted of a 2001-bp sequence containing a 1557-bp open reading frame, encoding 519 amino acids with a conserved domain typical of the glycoside-pentoside-hexuronide superfamily. The amino acid sequence of PsSUT1 in tree peony shared high homology with that of other plants. At different developmental stages, PsSUT1 was expressed in roots, stems, leaves, and petals. Its expression level in stems was 10.9-fold higher than in petals at the flowering stage. Expression of PsSUT1 at the flowering stage was highest during flower development. The significant differences in PsSUT1 expression observed among developmental stages and organs were closely related to changes in sucrose content during flower opening. These results form the basis for further research on the molecular mechanisms of carbohydrate metabolism and transport during flower development in tree peony. PMID:26505390