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Sample records for putative phosphatase lsf1

  1. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    Science.gov (United States)

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag

    Czech Academy of Sciences Publication Activity Database

    Ulrych, Aleš; Holečková, Nela; Goldová, Jana; Doubravová, Linda; Benada, Oldřich; Kofroňová, Olga; Halada, Petr; Branny, Pavel

    2016-01-01

    Roč. 16, OCT 24 (2016), s. 247 ISSN 1471-2180 R&D Projects: GA ČR GAP302/12/0256; GA ČR GAP207/12/1568; GA MŠk LH12055 Institutional support: RVO:61388971 Keywords : Signal transduction * Protein phosphatase * Protein kinase Subject RIV: EE - Microbiology, Virology Impact factor: 2.644, year: 2016

  3. Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis.

    Science.gov (United States)

    Martins, Erica Soares; Monnerat, Rose Gomes; Queiroz, Paulo Roberto; Dumas, Vinicius Fiuza; Braz, Shélida Vasconcelos; de Souza Aguiar, Raimundo Wagner; Gomes, Ana Cristina Menezes Mendes; Sánchez, Jorge; Bravo, Alejandra; Ribeiro, Bergmann Morais

    2010-02-01

    Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. They interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in midgut epithelial cells lysis. In this work we had cloned, sequenced and expressed a cry1Ba toxin gene from the B thuringiensis S601 strain which was previously shown to be toxic to Anthonomus grandis, a cotton pest. The Cry1Ba6 protein expressed in an acrystaliferous B. thuringiensis strain was toxic to A. grandis in bioassays. The binding of Cry1Ba6 toxin to proteins located in the midgut brush border membrane of A. grandis was analyzed and we found that Cry1Ba6 binds to two proteins (62 and 65kDa) that showed alkaline phosphatase (ALP) activity. This work is the first report that shows the localization of Cry toxin receptors in the midgut cells of A. grandis. 2009. Published by Elsevier Ltd.

  4. Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme.

    Science.gov (United States)

    Bertone, V; Tarantola, E; Ferrigno, A; Gringeri, E; Barni, S; Vairetti, M; Freitas, I

    2011-02-08

    Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT). Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS). However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20) that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1), frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but most hepatocytes

  5. Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

    Directory of Open Access Journals (Sweden)

    V. Bertone

    2011-02-01

    Full Text Available Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT. Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS. However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20 that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1, frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa, using as controls lean Zucker (fa/+ and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but

  6. Virus-induced Gene Silencing-based Functional Analyses Revealed the Involvement of Several Putative Trehalose-6-Phosphate Synthase/Phosphatase Genes in Disease Resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in Tomato

    Directory of Open Access Journals (Sweden)

    Huijuan Zhang

    2016-08-01

    Full Text Available Trehalose and its metabolism have been demonstrated to play important roles in control of plant growth, development and stress responses. However, direct genetic evidence supporting the functions of trehalose and its metabolism in defense response against pathogens is lacking. In the present study, genome-wide characterization of putative trehalose-related genes identified 11 SlTPSs for trehalose-6-phosphate synthase, 8 SlTPPs for trehalose-6-phosphate phosphatase and one SlTRE1 for trehalase in tomato genome. Nine SlTPSs, 4 SlTPPs and SlTRE1 were selected for functional analyses to explore their involvement in tomato disease resistance. Some selected SlTPSs, SlTPPs and SlTRE1 responded with distinct expression induction patterns to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst DC3000 as well as to defense signaling hormones (e.g. salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-mediated silencing of SlTPS3, SlTPS4 or SlTPS7 led to deregulation of ROS accumulation and attenuated the expression of defense-related genes upon pathogen infection and thus deteriorated the resistance against B. cinerea or Pst DC3000. By contrast, silencing of SlTPS5 or SlTPP2 led to an increased expression of the defense-related genes upon pathogen infection and conferred an increased resistance against Pst DC3000. Silencing of SlTPS3, SlTPS4, SlTPS5, SlTPS7 or SlTPP2 affected trehalose level in tomato plants with or without infection of B. cinerea or Pst DC3000. These results demonstrate that SlTPS3, SlTPS4, SlTPS5, SlTPS7 and SlTPP2 play roles in resistance against B. cinerea and Pst DC3000, implying the importance of trehalose and tis metabolism in regulation of defense response against pathogens in tomato.

  7. Voltage-sensing phosphatase: its molecular relationship with PTEN.

    Science.gov (United States)

    Okamura, Yasushi; Dixon, Jack E

    2011-02-01

    Voltage-sensing phosphoinositide phosphatase (VSP) contains voltage sensor and cytoplasmic phosphatase domains. A unique feature of this protein is that depolarization-induced motions of the voltage sensor activate PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) phosphatase activities. VSP exhibits remarkable structural similarities with PTEN, the phosphatase and tensin homolog deleted on chromosome 10. These similarities include the cytoplasmic phosphatase region, the phosphoinositide binding region, and the putative membrane interacting C2 domain.

  8. Cdc14 phosphatase

    DEFF Research Database (Denmark)

    Machín, Félix; Quevedo Rodriguez, Oliver; Ramos-Pérez, Cristina

    2016-01-01

    and cancer cells uncontrollably divide, much attention has been put into knocking down CDK activity. However, much less is known on the consequences of interfering with the phosphatases that put an end to the cell cycle. We have addressed in recent years the consequences of transiently inactivating the only...

  9. The protein histidine phosphatase LHPP is a tumour suppressor.

    Science.gov (United States)

    Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

    2018-03-29

    Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

  10. A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis

    Directory of Open Access Journals (Sweden)

    Bennett Hayley J

    2010-08-01

    Full Text Available Abstract Background Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. Results We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. Conclusion This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases

  11. A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis.

    Science.gov (United States)

    Beresford, Nicola J; Saville, Charis; Bennett, Hayley J; Roberts, Ian S; Tabernero, Lydia

    2010-08-02

    Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR) are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. This study provides

  12. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  13. Protein tyrosine phosphatases: regulatory mechanisms.

    NARCIS (Netherlands)

    den Hertog, J.; Ostman, A.; Bohmer, F.D.

    2008-01-01

    Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and

  14. Glucose-6-phosphatase deficiency

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    Labrune Philippe

    2011-05-01

    Full Text Available Abstract Glucose-6-phosphatase deficiency (G6P deficiency, or glycogen storage disease type I (GSDI, is a group of inherited metabolic diseases, including types Ia and Ib, characterized by poor tolerance to fasting, growth retardation and hepatomegaly resulting from accumulation of glycogen and fat in the liver. Prevalence is unknown and annual incidence is around 1/100,000 births. GSDIa is the more frequent type, representing about 80% of GSDI patients. The disease commonly manifests, between the ages of 3 to 4 months by symptoms of hypoglycemia (tremors, seizures, cyanosis, apnea. Patients have poor tolerance to fasting, marked hepatomegaly, growth retardation (small stature and delayed puberty, generally improved by an appropriate diet, osteopenia and sometimes osteoporosis, full-cheeked round face, enlarged kydneys and platelet dysfunctions leading to frequent epistaxis. In addition, in GSDIb, neutropenia and neutrophil dysfunction are responsible for tendency towards infections, relapsing aphtous gingivostomatitis, and inflammatory bowel disease. Late complications are hepatic (adenomas with rare but possible transformation into hepatocarcinoma and renal (glomerular hyperfiltration leading to proteinuria and sometimes to renal insufficiency. GSDI is caused by a dysfunction in the G6P system, a key step in the regulation of glycemia. The deficit concerns the catalytic subunit G6P-alpha (type Ia which is restricted to expression in the liver, kidney and intestine, or the ubiquitously expressed G6P transporter (type Ib. Mutations in the genes G6PC (17q21 and SLC37A4 (11q23 respectively cause GSDIa and Ib. Many mutations have been identified in both genes,. Transmission is autosomal recessive. Diagnosis is based on clinical presentation, on abnormal basal values and absence of hyperglycemic response to glucagon. It can be confirmed by demonstrating a deficient activity of a G6P system component in a liver biopsy. To date, the diagnosis is most

  15. Identification and characterization of a pyridoxal 5'-phosphate phosphatase in the silkworm (Bombyx mori).

    Science.gov (United States)

    Huang, ShuoHao; Han, CaiYun; Ma, ZhenQiao; Zhou, Jie; Zhang, JianYun; Huang, LongQuan

    2017-03-01

    Vitamin B 6 comprises six interconvertible pyridine compounds, among which pyridoxal 5'-phosphate (PLP) is a coenzyme for over 140 enzymes. PLP is also a very reactive aldehyde. The most well established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. A human PLP-specific phosphatase has been identified and characterized. However, very little is known about the phosphatase in other living organisms. In this study, a cDNA clone of putative PLP phosphatase was identified from B. mori and characterized. The cDNA encodes a polypeptide of 343 amino acid residues, and the recombinant enzyme purified from E. coli exhibited properties similar to that of human PLP phosphatase. B. mori has a single copy of the PLPP gene, which is located on 11th chromosome, spans a 5.7kb region and contains five exons and four introns. PLP phosphatase transcript was detected in every larva tissue except hemolymph, and was most highly represented in Malpighian tube. We further down-regulated the gene expression of the PLP phosphatase in 5th instar larvae with the RNA interference. However, no significant changes in the gene expression of PLP biosynthetic enzymes and composition of B 6 vitamers were detected as compared with the control. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Phosphatases in Cancer : Shifting the balance

    NARCIS (Netherlands)

    E. Hoekstra (Elmer)

    2015-01-01

    markdownabstractAbstract The role of phosphatases in cancer is an ignored research field, mostly based on the dogma that phosphatases function as tumor suppressor genes. However, in our opinion dephosphorylation events by phosphatases can also enhance signaling in cancer. The current research

  17. Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

    DEFF Research Database (Denmark)

    Olsen, Line; Bressendorff, Simon; Troelsen, Jesper T

    2005-01-01

    The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the en...

  18. Increased liver alkaline phosphatase and aminotransferase ...

    African Journals Online (AJOL)

    The effect of daily, oral administration of ethanolic extract of Khaya senegalensis stem bark (2mg/kg body weight) for 18days on the alkaline phosphatase, aspartate and alanine aminotransferase activities of rat liver and serum were investigated. Compared with the control, the activities of liver alkaline phosphatase (ALP), ...

  19. Sensing charges of the Ciona intestinalis voltage-sensing phosphatase.

    Science.gov (United States)

    Villalba-Galea, Carlos A; Frezza, Ludivine; Sandtner, Walter; Bezanilla, Francisco

    2013-11-01

    Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.

  20. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Robert J.; Beazley, Melanie J.; Wilson, Jarad J.; Taillefert, Martial; Sobecky, Patricia A.

    2005-04-05

    The overall goal of this project is to examine the role of nonspecific phosphohydrolases present in naturally occurring subsurface microorganisms for the purpose of promoting the immobilization of radionuclides through the production of uranium [U(VI)] phosphate precipitates. Specifically, we hypothesize that the precipitation of U(VI) phosphate minerals may be promoted through the microbial release and/or accumulation of PO{sub 4}{sup 3-}. During this phase of the project we have been conducting assays to determine the effects of pH, inorganic anions and organic ligands on U(VI) mineral formation and precipitation when FRC bacterial isolates were grown in simulated groundwater medium. The molecular characterization of FRC isolates has also been undertaken during this phase of the project. Analysis of a subset of gram-positive FRC isolates cultured from FRC soils (Areas 1, 2 and 3) and background sediments have indicated a higher percentage of isolates exhibiting phosphatase phenotypes (i.e., in particular those surmised to be PO{sub 4}{sup 3-}-irrepressible) relative to isolates from the reference site. A high percentage of strains that exhibited such putatively PO{sub 4}{sup 3-}-irrepressible phosphatase phenotypes were also resistant to the heavy metals lead and cadmium. Previous work on FRC strains, including Arthrobacter, Bacillus and Rahnella spp., has demonstrated differences in tolerance to U(VI) toxicity (200 {micro}M) in the absence of organophosphate substrates. For example, Arthrobacter spp. exhibited the greatest tolerance to U(VI) while the Rahnella spp. have been shown to facilitate the precipitation of U(VI) from solution and the Bacillus spp. demonstrate the greatest sensitivity to acidic conditions and high concentrations of U(VI). PCR-based detection of FRC strains are being conducted to determine if non-specific acid phosphatases of the known molecular classes [i.e., classes A, B and C] are present in these FRC isolates. Additionally, these

  1. A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

    Science.gov (United States)

    Perego, M

    1997-08-05

    The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

  2. Structural and biochemical analysis of atypically low dephosphorylating activity of human dual-specificity phosphatase 28.

    Directory of Open Access Journals (Sweden)

    Bonsu Ku

    Full Text Available Dual-specificity phosphatases (DUSPs constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central β-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.

  3. Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.

    Science.gov (United States)

    Matsuda, Makoto; Takeshita, Kohei; Kurokawa, Tatsuki; Sakata, Souhei; Suzuki, Mamoru; Yamashita, Eiki; Okamura, Yasushi; Nakagawa, Atsushi

    2011-07-01

    Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

  4. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

  5. Posttranslational heterogeneity of bone alkaline phosphatase in metabolic bone disease.

    Science.gov (United States)

    Langlois, M R; Delanghe, J R; Kaufman, J M; De Buyzere, M L; Van Hoecke, M J; Leroux-Roels, G G

    1994-09-01

    Bone alkaline phosphatase is a marker of osteoblast activity. In order to study the posttranscriptional modification (glycosylation) of bone alkaline phosphatase in bone disease, we investigated the relationship between mass and catalytic activity of bone alkaline phosphatase in patients with osteoporosis and hyperthyroidism. Serum bone alkaline phosphatase activity was measured after lectin precipitation using the Iso-ALP test kit. Mass concentration of bone alkaline phosphatase was determined with an immunoradiometric assay (Tandem-R Ostase). In general, serum bone alkaline phosphatase mass and activity concentration correlated well. The activity : mass ratio of bone alkaline phosphatase was low in hyperthyroidism. Activation energy of the reaction catalysed by bone alkaline phosphatase was high in osteoporosis and in hyperthyroidism. Experiments with neuraminidase digestion further demonstrated that the thermodynamic heterogeneity of bone alkaline phosphatase can be explained by a different glycosylation of the enzyme.

  6. Defining Starch Binding by Glucan Phosphatases

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2015-01-01

    Starch is a vital energy molecule in plants that has a wide variety of uses in industry, such as feedstock for biomaterial processing and biofuel production. Plants employ a three enzyme cyclic process utilizing kinases, amylases, and phosphatases to degrade starch in a diurnal manner. Starch...... is comprised of the branched glucan amylopectin and the more linear glucan amylose. Our lab has determined the first structures of these glucan phosphatases and we have defined their enzymatic action. Despite this progress, we lacked a means to quickly and efficiently quantify starch binding to glucan...

  7. Evidence for phosphoprotein phosphatase in Streptomyces granaticolor

    Czech Academy of Sciences Publication Activity Database

    Bobek, J.; Hercík, K.; Dobrová, Zuzana; Branny, Pavel; Nádvorník, Richard; Janeček, Jiří

    2000-01-01

    Roč. 45, č. 4 (2000), s. 310-312 ISSN 0015-5632 R&D Projects: GA ČR GA204/99/1534 Institutional research plan: CEZ:AV0Z5020903 Keywords : streptomycetes * phosphoprotein phosphatase Subject RIV: EE - Microbiology, Virology Impact factor: 0.752, year: 2000

  8. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  9. Osteocalcin and bone-specific alkaline phosphatase in Sickle cell ...

    African Journals Online (AJOL)

    specific alkaline phosphatase (b-AP) total protein levels were evaluated as indicators of bone turnover in twenty patients with sickle cell haemoglobinopathies and in twenty normal healthy individuals. The serum bonespecific alkaline phosphatase ...

  10. Expression, purification and crystallization of an atypical class C acid phosphatase from Mycoplasma bovis

    International Nuclear Information System (INIS)

    Singh, Harkewal; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2011-01-01

    Methods for the expression, purification and crystallization of the class C acid phosphatase from M. bovis are reported. This enzyme is atypical in that it is nearly 20 kDa larger than other known class C acid phosphatases. Class C acid phosphatases (CCAPs) are 25–30 kDa bacterial surface proteins that are thought to function as broad-specificity 5′,3′-nucleotidases. Analysis of the newly published complete genome sequence of Mycoplasma bovis PG45 revealed a putative CCAP with a molecular weight of 49.9 kDa. The expression, purification and crystallization of this new family member are described here. Standard purification procedures involving immobilized metal-ion affinity chromatography and ion-exchange chromatography yielded highly pure and crystallizable protein. Crystals were grown in sitting drops at room temperature in the presence of PEG 3350 and HEPES buffer pH 7.5 and diffracted to 2.3 Å resolution. Analysis of diffraction data suggested a primitive monoclinic space group, with unit-cell parameters a = 78, b = 101, c = 180 Å, β = 92°. The asymmetric unit is predicted to contain six molecules, which are likely to be arranged as three dimers

  11. Purification of acidic phosphatase from mustard seedlings

    OpenAIRE

    sprotocols

    2014-01-01

    ### INTRODUCTION Phosphate esters are widely distributed in any organism. Nucleic acids, metabolic intermediates like glucose-6-phosphate, energy-rich substrates (AMP, creatine phosphate) are some obvious examples. While many metabolic intermediates are activated through the transfer of phosphate groups (e.g., by kinases) it is equally important that phosphate esters can also be rapidly broken down. The hydrolytic removal of phosphate groups from phosphoesters is catalyzed by phosphatases...

  12. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    Science.gov (United States)

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

  13. Phosphatase activity of Poa pratensis seeds. I. Preliminary studies on acid phosphatase II

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

    Full Text Available Acid phosphatase (EC 3.1.3.2 was extracted with 0.1 M sodium acetate buffer pH 5.1 from Poa pratensis seeds, and separated into three fractions by chromatography on DEAE cellulose. The highest activity was found in fraction Il-b (acid phosphatase II. The activity of the enzyme was optimal at pH 4.9. It hydrolyzed p-nitrophenyl phosphate most readily among the various phosphomonoesters examined. Acid phosphatase II showed also a high activity toward β-naphtyl phosphate and phenyl phosphate, very low activity towards β-glycero phosphate, 5'-GMP and no activity with glucose-1 phosphate. The enzyme was inhibited by Ca2+ and fluoride, but activated by Mg2+. EDTA had no influence on the activity of the enzyme.

  14. Phosphatase activity of Poa pratensis seeds. l. Preliminary studies on acid phosphatase II

    Energy Technology Data Exchange (ETDEWEB)

    Lorenc-Kubis, I.; Morawiecka, B.

    1973-01-01

    Acid phosphatase (EC 3.1.3.2) was extracted from 0.1 M sodium acetate buffer, pH 5.1 from Poa pratensis seeds, and separated into three fractions by chromatography on DEAE cellulose. The highest activity was found in fraction II-b (acid phosphatase II). The activity of the enzyme was optimal at pH 4.9. It hydrolyzed p-nitrophenyl phosphate most readily among the various phosphomonoesters examined. Acid phosphatase II showed also a high activity toward ..beta..-naphtyl phosphate and phenyl phosphate, very low activity towards ..beta..-glycero phosphate, 5'-GMP and no activity with glucose-1 phosphate. The enzyme was inhibited by Ca/sup 2 +/ and fluoride, but activated by Mg/sup 2 +/. EDTA had no influence on the activity of the enzyme. 12 references, 3 figures, 4 tables.

  15. Characterization of a double deletion mutant of Fusarium verticillioides lacking two putative trehalose-6-phosphate phosphatase genes

    Science.gov (United States)

    Fusarium verticillioides is a fungal pathogen that commonly infects the stalk, ear, and kernels of corn and can produce fumonisins, a family of mycotoxins linked to disease in livestock and humans. Our goal is to characterize the role of the disaccharide trehalose in growth and stress response in F....

  16. Construction and characterization of a double deletion mutant of Fusarium verticillioides lacking two putative trehalose-6-phosphate phosphatase genes

    Science.gov (United States)

    Fusarium verticillioides is a fungal pathogen that commonly infects the stalk, ear, and kernels of corn and can produce fumonisins, a family of mycotoxins linked to disease in livestock and humans. Our goal is to characterize the role of the disaccharide trehalose in growth and stress response in F....

  17. Identification and characterization of putative conserved IAM ...

    African Journals Online (AJOL)

    Available putative AMI sequences from a wide array of monocot and dicot plants were identified and the phylogenetic tree was constructed and analyzed. We identified in this tree, a clade that contained sequences from species across the plant kingdom suggesting that AMI is conserved and may have a primary role in plant ...

  18. Toddlers' Duration of Attention toward Putative Threat

    Science.gov (United States)

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  19. Persistently increased intestinal fraction of alkaline phosphatase

    DEFF Research Database (Denmark)

    Nathan, E; Baatrup, G; Berg, H

    1984-01-01

    Persistent elevation of the intestinal fraction of the alkaline phosphatase (API) as an isolated finding has to our knowledge not been reported previously. It was found in a boy followed during a period of 5.5 years. The only symptom was transient periodic fatigue observed at home, but not apparent...... during hospitalization. His blood type was O, RH+, Le (a-, b+) and he was a secretor of H-substance, which may be associated with rising API activity after fat-loading. In this case API was unchanged after fat-loading. Neither intestinal nor liver diseases were found, and no other cause for the elevated...

  20. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

    Science.gov (United States)

    Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi; Gasser, Robin B; Kim, Jeong-Sun; Coster, Mark J; Hofmann, Andreas

    2018-04-24

    The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

  1. Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach.

    Directory of Open Access Journals (Sweden)

    Joanne L Dunster

    2015-11-01

    Full Text Available We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2, provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in

  2. A generally applicable sequential alkaline phosphatase immunohistochemical double staining

    NARCIS (Netherlands)

    van der Loos, Chris M.; Teeling, Peter

    2008-01-01

    A universal type of sequential double alkaline phosphatase immunohistochemical staining is described that can be used for formalin-fixed, paraffin-embedded and cryostat tissue sections from human and mouse origin. It consists of two alkaline phosphatase detection systems including enzymatic

  3. Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research

    Directory of Open Access Journals (Sweden)

    Kirstin eHobiger

    2015-02-01

    Full Text Available The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs. Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs. In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs. Although PTPs have already been well characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.

  4. Direct determination of phosphatase activity from physiological substrates in cells.

    Directory of Open Access Journals (Sweden)

    Zhongyuan Ren

    Full Text Available A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1 mg(-1 for PPi, to 56 ± 11 nmol min(-1 mg(-1 for AMP, to 79 ± 23 nmol min(-1 mg(-1 for beta-glycerophosphate and to 73 ± 15 nmol min(-1 mg(-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  5. Hematopoietic cell phosphatase is recruited to CD22 following B cell antigen receptor ligation

    NARCIS (Netherlands)

    Lankester, A. C.; van Schijndel, G. M.; van Lier, R. A.

    1995-01-01

    Hematopoietic cell phosphatase is a nonreceptor protein tyrosine phosphatase that is preferentially expressed in hematopoietic cell lineages. Motheaten mice, which are devoid of (functional) hematopoietic cell phosphatase, have severe disturbances in the regulation of B cell activation and

  6. Phosphatase activity of Poa pratensis seeds. II. Purification and characterization of acid phosphatase Ia2 and Ia3

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

    Full Text Available Two acid phosphatases (Ia2, Ia3 have been isolated from Poa pratensis seeds and partially purified. Both enzymes showed maximal activity at pH 4,9. They exhibited high activity towards p-nitrophenyl phosphate, inorganic pyrophosphate and phenyl phosphate, much less activity towards glucose-6 phosphate, and mononucleotides. Phosphatases a2 and a3 differed in their activity towards ADP. Orthophosphate, fluoride and Zn2+ were effective inhibitors. EDTA, β-mercaptoethanol and Mg2+ activated phophatase a2 but had no effect on phosphatase a3. Zn2+ inhibited the activity of phosphatase a2 noncompetitively, whereas phosphatase a3 showed inhibition of mixed type. Trypsin, chymotrypsin and pronase had no effect on the enzyme activities of both molecular forms.

  7. Ten Putative Contributors to the Obesity Epidemic

    Science.gov (United States)

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  8. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  9. Alkaline phosphatase activity of rumen bacteria.

    Science.gov (United States)

    Cheng, K J; Costerton, J W

    1977-11-01

    Of the 54 strains of rumen bacteria examined for alkaline phosphatase (APase) production, 9 of 33 gram-negative strains and none of 21 gram-positive strains produced the enzyme. The APase of the cells of the three strains of Bacteroides ruminicola that produced significant amounts of the enzyme was located in the periplasmic area of the cell envelope, whereas the enzyme was located in the strains of Selenomonas ruminantium and Succinivibrio dextrinosolvens was associated with the outer membrane. The localization of APase production in the cells of natural populations of rumen bacteria from hay-fed sheep was accomplished by reaction product deposition, and both the proportion of APase-producing bacteria and the location of the enzyme in the cell envelope of the producing cells could be determined. We suggest that this procedure is useful in detecting shifts in the bacterial population and the release of cell-bound APase that accompany feedlot bloat and other sequelae of dietary manipulation in ruminants.

  10. Identification of human pulmonary alkaline phosphatase isoenzymes.

    Science.gov (United States)

    Capelli, A; Cerutti, C G; Lusuardi, M; Donner, C F

    1997-04-01

    An increase of alkaline phosphatase (ALP) activity has been observed in the bronchoalveolar lavage fluid (BALF) of patients affected by pulmonary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ agglutinin (WGA) precipitation, and an immunoassay specific for the bone-isoform of ALP. Only one anodic band representing a high-molecular-weight isoform of ALP (Mr approximately 2,000 kDa) was observed on electrophoresis of BALF. The inhibition assay results were consistent for a tissue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and resistant to L-phenylalanine and L-leucine. Less than 13% of ALP activity was heat-stable. After incubation of BALF specimens with glycosyl-phosphatidylinositol-phospholipase D plus Nonidet P-40, or with phosphatidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr approximately 220 kDa) appeared near the bone band of a standard serum. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less than 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.

  11. Alkaline Phosphatase, an Unconventional Immune Protein

    Directory of Open Access Journals (Sweden)

    Bethany A. Rader

    2017-08-01

    Full Text Available Recent years have seen an increase in the number of studies focusing on alkaline phosphatases (APs, revealing an expanding complexity of function of these enzymes. Of the four human AP (hAP proteins, most is known about tissue non-specific AP (TNAP and intestinal AP (IAP. This review highlights current understanding of TNAP and IAP in relation to human health and disease. TNAP plays a role in multiple processes, including bone mineralization, vitamin B6 metabolism, and neurogenesis, is the genetic cause of hypophosphatasia, influences inflammation through regulation of purinergic signaling, and has been implicated in Alzheimer’s disease. IAP regulates fatty acid absorption and has been implicated in the regulation of diet-induced obesity and metabolic syndrome. IAP and TNAP can dephosphorylate bacterial-derived lipopolysaccharide, and IAP has been identified as a potential regulator of the composition of the intestinal microbiome, an evolutionarily conserved function. Endogenous and recombinant bovine APs and recombinant hAPs are currently being explored for their potential as pharmacological agents to treat AP-associated diseases and mitigate multiple sources of inflammation. Continued research on these versatile proteins will undoubtedly provide insight into human pathophysiology, biochemistry, and the human holobiont.

  12. HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain.

    Directory of Open Access Journals (Sweden)

    Marie-Claude Gingras

    Full Text Available The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP. To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported.Here we report a rigorous enzymatic analysis demonstrating that the HD-PTP protein does not harbor tyrosine phosphatase or lipid phosphatase activity using the highly sensitive DiFMUP substrate and a panel of different phosphatidylinositol phosphates. We found that HD-PTP tyrosine phosphatase inactivity is caused by an evolutionary conserved amino acid divergence of a key residue located in the HD-PTP phosphatase domain since its back mutation is sufficient to restore the HD-PTP tyrosine phosphatase activity. Moreover, in agreement with a tumor suppressor activity, HD-PTP expression leads to colony growth reduction in human cancer cell lines, independently of its catalytic PTP activity status.In summary, we demonstrate that HD-PTP is a catalytically inactive protein tyrosine phosphatase. As such, we identify one residue involved in its inactivation and show that its colony growth reduction activity is independent of its PTP activity status in human cancer cell lines.

  13. Redox Regulation of Receptor Protein-Tyrosine Phosphatases

    NARCIS (Netherlands)

    Groen, A.J.

    2006-01-01

    Phosphorylation is of major importance in cell signalling processes like cell migration, cell proliferation and cell differentiation within higher eukaryotic organisms. Therefore, the balance between phosphorylation, mediated by kinases, and dephosphorylation, mediated by phosphatases, must be

  14. Synthesis and phosphatase activity of a Cobalt(II) phenanthroline ...

    Indian Academy of Sciences (India)

    MAMONI GARAI

    2017-09-19

    Sep 19, 2017 ... Synthesis and phosphatase activity of a Cobalt(II) phenanthroline complex. MAMONI GARAIa ... tion, cobalt complexes have gained importance because of their application as ... 2.3 Physical measurements. Infrared spectrum ...

  15. Novel Combinatorial Chemistry-Derived Inhibitors of Oncogenic Phosphatases

    National Research Council Canada - National Science Library

    Lazo, John

    1999-01-01

    Our overall goal of this US Army Breast Cancer Grant entitled "Novel Combinatorial Chemistry-Derived Inhibitors of Oncogenic Phosphatases" is to identity and develop novel therapeutic agents for human breast cancer...

  16. Research on Phosphatases of Belladona Leaves and Their Purification

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1957-01-01

    Full Text Available Through experimentation with several leaves it has been possible for us to point out the existance of two different acid phosphatases. We have studied in more detail the phosphatases of belldon a leaves (Atropa Belladona L. Solanacees. The great part of the phosphatase activity is water extractable. We have compared the activity of the soluble fraction with that not directly extractable by means of water. The insoluble fraction could not be solubilized in a satisfaetC'fY m.anner.The digestion by papaine produced a slight solubilizing effect; on the other hand salt solutions, neutral or alkaline, or water glycerol mixtures had no solubilizing effect on the enzyme, It has been possible to demonstrate the existence of two different phosphatases in the insoluble fraction: the first of the type II,

  17. Putative neuroprotective agents in neuropsychiatric disorders.

    Science.gov (United States)

    Dodd, Seetal; Maes, Michael; Anderson, George; Dean, Olivia M; Moylan, Steven; Berk, Michael

    2013-04-05

    In many individuals with major neuropsychiatric disorders including depression, bipolar disorder and schizophrenia, their disease characteristics are consistent with a neuroprogressive illness. This includes progressive structural brain changes, cognitive and functional decline, poorer treatment response and an increasing vulnerability to relapse with chronicity. The underlying molecular mechanisms of neuroprogression are thought to include neurotrophins and regulation of neurogenesis and apoptosis, neurotransmitters, inflammatory, oxidative and nitrosative stress, mitochondrial dysfunction, cortisol and the hypothalamic-pituitary-adrenal axis, and epigenetic influences. Knowledge of the involvement of each of these pathways implies that specific agents that act on some or multiple of these pathways may thus block this cascade and have neuroprotective properties. This paper reviews the potential of the most promising of these agents, including lithium and other known psychotropics, aspirin, minocycline, statins, N-acetylcysteine, leptin and melatonin. These agents are putative neuroprotective agents for schizophrenia and mood disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Detection of phosphatase activity in aquatic and terrestrial cyanobacterial strains

    Directory of Open Access Journals (Sweden)

    Babić Olivera B.

    2013-01-01

    Full Text Available Cyanobacteria, as highly adaptable microorganisms, are characterized by an ability to survive in different environmental conditions, in which a significant role belongs to their enzymes. Phosphatases are enzymes produced by algae in relatively large quantities in response to a low orthophosphate concentration and their activity is significantly correlated with their primary production. The activity of these enzymes was investigated in 11 cyanobacterial strains in order to determine enzyme synthesis depending on taxonomic and ecological group of cyanobacteria. The study was conducted with 4 terrestrial cyanobacterial strains, which belong to Nostoc and Anabaena genera, and 7 filamentous water cyanobacteria of Nostoc, Oscillatoria, Phormidium and Microcystis genera. The obtained results showed that the activity of acid and alkaline phosphatases strongly depended on cyanobacterial strain and the environment from which the strain originated. Higher activity of alkaline phosphatases, ranging from 3.64 to 85.14 μmolpNP/s/dm3, was recorded in terrestrial strains compared to the studied water strains (1.11-5.96 μmolpNP/s/dm3. The activity of acid phosphatases was higher in most tested water strains (1.67-6.28 μmolpNP/s/dm3 compared to the activity of alkaline phosphatases (1.11-5.96 μmolpNP/s/dm3. Comparing enzyme activity of nitrogen fixing and non-nitrogen fixing cyanobacteria, it was found that most nitrogen fixing strains had a higher activity of alkaline phosphatases. The data obtained in this work indicate that activity of phosphatases is a strain specific property. The results further suggest that synthesis and activity of phosphatases depended on eco-physiological characteristics of the examined cyanobacterial strains. This can be of great importance for the further study of enzymes and mechanisms of their activity as a part of cyanobacterial survival strategy in environments with extreme conditions. [Projekat Ministarstva nauke Republike

  19. Effect of vanadium compounds on acid phosphatase activity

    OpenAIRE

    Vescina, Cecilia M.; Sálice, Viviana C.; Cortizo, Ana María; Etcheverry, Susana B.

    1996-01-01

    The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activi...

  20. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  1. Role of tyrosine phosphatase inhibitors in cancer treatment with emphasis on SH2 domain-containing tyrosine phosphatases (SHPs)

    NARCIS (Netherlands)

    Irandoust, Mahban; van den Berg, Timo K.; Kaspers, Gertjan J. L.; Cloos, Jacqueline

    2009-01-01

    Protein tyrosine phosphorylation is one of the key mechanisms involved in signal transduction pathways. This modification is regulated by concerted action of protein tyrosine phosphatases and protein tyrosine kinases. Deregulation of either of these key regulators lead to abnormal cellular

  2. Analysis of Hydraulic Flood Control Structure at Putat Boro River

    OpenAIRE

    Ruzziyatno, Ruhban

    2015-01-01

    Putat Boro River is one of the main drainage systems of Surakarta city which drains into Bengawan Solo river. The primary problem when flood occur is the higher water level of Bengawan Solo than Boro River and then backwater occur and inundates Putat Boro River. The objective of the study is to obtain operational method of Putat Boro River floodgate to control both inflows and outflows not only during flood but also normal condition. It also aims to know the Putat Boro rivers floodgate op...

  3. Serum alkaline phosphatase screening for vitamin D deficiency states

    International Nuclear Information System (INIS)

    Shaheen, S.; Barrakzai, Q.

    2012-01-01

    Objective: To determine whether serum vitamin D levels are correlated with serum levels of alkaline phosphatase or not. Study Design: Cross-sectional, observational study. Place and Duration of Study: Multi-centre study, conducted at Liaquat National Hospital and Medical College, National Medical Centre and Medicare Hospital, Karachi, from January to October 2009. Methodology: Patients attending the Orthopaedic OPDs with complaints of pain in different body regions and serum vitamin D/sub 3/ levels of greater or equal to 30 ng/ml were included in the study. Patients with vitamin D deficiency were further categorized into mild deficiency or insufficiency (vit. D/sub 3/ = 20-29 ng/ml), moderate deficiency (vit. D/sub 3/ = 5 - 19 ng/ml) and severe deficiency forms (vit. D/sub 3/ < 5 ng/ml). Pearson correlation was applied to test the correlation of serum alkaline phosphatase levels with serum vitamin D/sub 3/ levels. P-value < 0.05 was considered to be significant. Results: Out of 110 samples, 26 had mild (23%), 61 had moderate (55%) and 21 had severe (19.1%) vitamin D deficiencies. All of the patients in the three groups had alkaline phosphatase with in normal limits and the total mean value of the enzyme was 135.97 +- 68.14I U/L. The inter group comparison showed highest values of alkaline phosphatase in the moderate vitamin D deficiency group. The correlation coefficient of alkaline phosphatase and serum vitamin D/sub 3/ levels was r =0.05 (p =0.593). Conclusion: Serum vitamin D/sub 3/ levels may not be correlated with increased serum alkaline phosphatase levels. Therefore, alkaline phosphatase may not be used as a screening test to rule out vitamin D deficiency. (author)

  4. Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

    Science.gov (United States)

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

  5. Toddlers’ Duration of Attention towards Putative Threat

    Science.gov (United States)

    Kiel, Elizabeth J.; Buss, Kristin A.

    2010-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk for developing anxious behavior, toddlers’ attention towards a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined how attention towards an angry-looking gorilla mask in a room with alternative opportunities for play in 24-month-old toddlers predicted social inhibition when children entered kindergarten. Analyses examined attention to threat above and beyond and in interaction with both proximity to the mask and fear of novelty observed in other situations. Attention to threat interacted with proximity to the mask to predict social inhibition, such that attention to threat most strongly predicted social inhibition when toddlers stayed furthest from the mask. This relation occurred above and beyond the predictive relation between fear of novelty and social inhibition. Results are discussed within the broader literature of anxiety development and attentional processes in young children. PMID:21373365

  6. Acid phosphatase and lipid peroxidation in human cataractous lens epithelium

    Directory of Open Access Journals (Sweden)

    Vasavada Abhay

    1993-01-01

    Full Text Available The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium. In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium. From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

  7. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

    1999-01-01

    A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  8. Elevated Serum Level of Human Alkaline Phosphatase in Obesity

    International Nuclear Information System (INIS)

    Khan, A. R.; Awan, F. R.; Najam, S. S.; Islam, M.; Siddique, T.; Zain, M.

    2015-01-01

    Objective: To investigate a correlation between serum alkaline phosphatase level and body mass index in human subjects. Methods: The comparative cross-sectional study was carried out at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, from April 2012 to June 2013. Blood serum alkaline phosphatase levels were estimated and the subjects were divided into three sub-groups on the basis of their body mass index: normal weight (<25kg/m2), overweight (25-27kg/m2) and obese (>27kg/m2) subjects. The serum samples were used for the estimation of clinically important biochemical parameters, using commercial kits on clinical chemistry analyser. Results: Of the 197 subjects, 97(49 percent) were obese and 100(51 percent) were non-obese. The serum alkaline phosphatase level increased in obese (214±6.4 IU/L) compared to the non-obese subjects (184.5±5 IU/L). Furthermore, a significant linear relationship (r=0.3;p-0.0001) was found between serum alkaline phosphatase and body mass index. Other biochemical variables were not correlated to the body mass index. Conclusion: Over activity and higher amounts of alkaline phosphatase were linked to the development of obesity. (author)

  9. Domain-to-domain coupling in voltage-sensing phosphatase.

    Science.gov (United States)

    Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

    2017-01-01

    Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

  10. Twenty putative palmitoyl-acyl transferase genes with distinct ...

    African Journals Online (AJOL)

    There are 20 genes containing DHHC domain predicted to encode putative palmitoyltransferase in Arabidopsis thaliana genome. However, little is known about their characteristics such as genetic relationship and expression profile. Here, we present an overview of the putative PAT genes in A. thaliana focusing on their ...

  11. Putative radioresistant bacterial isolate from sewage water

    International Nuclear Information System (INIS)

    Ang, April; Chua, Patricia; Perez, Kristine; Rey, April; Rivor Kristel; San Pablo, Czarina; Santos, Ernestine

    2001-01-01

    Sewage water was collected from a stagnant body of water in Balara, Quezon City. approximately 150 ml was aseptically transferred into eight Erlenmeyer flasks. Seven flasks were then subjected to different doses of radiation at the 60 Co irradiation facility, PNRI (Philippine Nuclear Research Institute) which are as follows: 0.01 kGy, 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, and 15 kGy. The remaining flask was used as the control. After irradiation, all the different treatments were subjected to colony count at the culture collection laboratory, NSRI. Results showed that the colonies from sewage water treatments irradiated at 0.01 kGy (treatment A), 0.10 kGy (treatment B), and 0.50 kGy (treatment C) exhibited a decreasing trend with colony counts 4.60 x 10 3 CFU/ml, and 1.30 x 10 3 CFU/ml, and 26 CFU/ml, respectively. Contrastingly, at 1 kGy (treatment D), high colony count of 2.95 x 10 3 CFU/ml was observed which is even higher compared to the control (1.02 x 10 3 CFU/ml). Treatment E that was irradiated at 5 kGy manifested low survival rate (25 CFU/ml) indicating the presence of few putative intermediate radioresistant bacteria. Radiation dose treatments higher than 5 kGy (i.e., 10 kGy and 15 kGy) exhibited no bacterial survival. (Author)

  12. Putative radioresistant bacterial isolate from sewage water

    Energy Technology Data Exchange (ETDEWEB)

    Ang, April; Chua, Patricia; Perez, Kristine; Rey, April; Kristel, Rivor; San Pablo, Czarina; Santos, Ernestine

    2001-01-29

    Sewage water was collected from a stagnant body of water in Balara, Quezon City. approximately 150 ml was aseptically transferred into eight Erlenmeyer flasks. Seven flasks were then subjected to different doses of radiation at the {sup 60}Co irradiation facility, PNRI (Philippine Nuclear Research Institute) which are as follows: 0.01 kGy, 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, and 15 kGy. The remaining flask was used as the control. After irradiation, all the different treatments were subjected to colony count at the culture collection laboratory, NSRI. Results showed that the colonies from sewage water treatments irradiated at 0.01 kGy (treatment A), 0.10 kGy (treatment B), and 0.50 kGy (treatment C) exhibited a decreasing trend with colony counts 4.60 x 10{sup 3} CFU/ml, and 1.30 x 10{sup 3} CFU/ml, and 26 CFU/ml, respectively. Contrastingly, at 1 kGy (treatment D), high colony count of 2.95 x 10{sup 3} CFU/ml was observed which is even higher compared to the control (1.02 x 10{sup 3} CFU/ml). Treatment E that was irradiated at 5 kGy manifested low survival rate (25 CFU/ml) indicating the presence of few putative intermediate radioresistant bacteria. Radiation dose treatments higher than 5 kGy (i.e., 10 kGy and 15 kGy) exhibited no bacterial survival. (Author)

  13. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  14. Effect of vanadium compounds on acid phosphatase activity.

    Science.gov (United States)

    Vescina, C M; Sálice, V C; Cortizo, A M; Etcheverry, S B

    1996-01-01

    The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.

  15. Phosphotyrosine as a substrate of acid and alkaline phosphatases.

    Science.gov (United States)

    Apostoł, I; Kuciel, R; Wasylewska, E; Ostrowski, W S

    1985-01-01

    A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.

  16. Identification of genes required for secretion of the Francisella oxidative burst-inhibiting acid phosphatase AcpA

    Directory of Open Access Journals (Sweden)

    John S Gunn

    2016-04-01

    Full Text Available Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses.

  17. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    Science.gov (United States)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  18. Phosphatase and tensin homologue deleted on chromosome 10 ...

    African Journals Online (AJOL)

    Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene deleted or mutated in many human cancers such as glioblastoma, spinal tumors, prostate, bladder, adrenals, thyroid, breast, endometrium, and colon cancers. They result from loss of heterozygosity (LOH) for the PTEN ...

  19. Phosphatases as an index of biotic contamination of dust

    NARCIS (Netherlands)

    Kniest, F.M.; Bronswijk, van J.E.M.H.; Schober, G.; Bouma, C.

    1990-01-01

    Enzymatic (phosphatase) activity (naphthol-release made visible with diazonium salt) of 10 mattress dust samples was correlated with number of counted arthropods, fungal spores and bacteria. This method can be helpful in the evaluation of large number of dust samples e.g. from riskful areas or from

  20. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    Science.gov (United States)

    Kulkarni, Suhasini; Tran, Vu; Ho, Maggie K.-M.; Phan, Chieu; Chin, Elizabeth; Wemmer, Zeke; Sommerhalter, Monika

    2010-01-01

    This experiment was performed in an upper-level undergraduate biochemistry laboratory course. Students learned how to immobilize an enzyme in a sol-gel matrix and how to perform and evaluate enzyme-activity measurements. The enzyme acid phosphatase (APase) from wheat germ was encapsulated in sol-gel beads that were prepared from the precursor…

  1. Anxious moments for the protein tyrosine phosphatase PTP1B

    OpenAIRE

    Krishnan, Navasona; Tonks, Nicholas K.

    2015-01-01

    Chronic stress can lead to the development of anxiety and mood disorders. Thus, novel therapies for preventing adverse effects of stress are vitally important. Recently, the protein tyrosine phosphatase PTP1B was identified as a novel regulator of stress-induced anxiety. This opens up exciting opportunities to exploit PTP1B inhibitors as anxiolytics.

  2. WIP1 phosphatase as pharmacological target in cancer therapy

    Czech Academy of Sciences Publication Activity Database

    Pecháčková, Soňa; Burdová, Kamila; Macůrek, Libor

    2017-01-01

    Roč. 95, č. 6 (2017), s. 589-599 ISSN 0946-2716 R&D Projects: GA MŠk 7F14061; GA MŠk LO1220 Institutional support: RVO:68378050 Keywords : Cancer * Phosphatase * Checkpoint * DNA damage response * Inhibitor * p53 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Medicinal chemistry Impact factor: 4.686, year: 2016

  3. Defining carbohydrate binding of glucan phosphatases via Affinity gel electrophoresis

    DEFF Research Database (Denmark)

    Auger, Kyle; Raththagala, Madushi; Wilkens, Casper

    2016-01-01

    was to determine a technique to measure carbohydrate binding quickly and efficiently. We established a protocol to reproducibly and quantitatively measure the binding of the enzymes to glucans utilizing Affinity Gel Electrophoresis (AGE). The results show that the various glucan phosphatases possess differing...

  4. Dephosphorylation of endotoxin by alkaline phosphatase in vivo

    NARCIS (Netherlands)

    Poelstra, Klaas; Bakker, W.W; Klok, P.A; Kamps, J.AAM; Hardonk, M.J; Meijer, D.K F

    1997-01-01

    Natural substrates for alkaline phosphatase (AP) are at present not identified despite extensive investigations. Difficulties in imagining a possible physiological function involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. As endotoxin

  5. Targeted deletion of kidney glucose-6 phosphatase leads to nephropathy

    NARCIS (Netherlands)

    Clar, Julie; Gri, Blandine; Calderaro, Julien; Birling, Marie-Christine; Herault, Yann; Smit, G. Peter A.; Mithieux, Gilles; Rajas, Fabienne

    2014-01-01

    Renal failure is a major complication that arises with aging in glycogen storage disease type 1a and type 1b patients. In the kidneys, glucose-6 phosphatase catalytic subunit (encoded by G6pc) deficiency leads to the accumulation of glycogen, an effect resulting in marked nephromegaly and

  6. Impact of ionic aluminium on extracellular phosphatases in acidified lakes

    Czech Academy of Sciences Publication Activity Database

    Bittl, T.; Vrba, Jaroslav; Nedoma, Jiří; Kopáček, Jiří

    2001-01-01

    Roč. 3, č. 9 (2001), s. 578-587 ISSN 1462-2912 R&D Projects: GA ČR GA206/97/0072; GA ČR GA206/00/0063 Keywords : acid phosphatases * pH effect * inhibition Subject RIV: DA - Hydrology ; Limnology Impact factor: 3.276, year: 2001

  7. 21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte alkaline phosphatase test. 864.7660 Section 864.7660 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7660...

  8. In vitro production of growth regulators and phosphatase activity by ...

    African Journals Online (AJOL)

    The result showed that the population levels of phosphobacteria were higher in the rhizosphere soil of groundnut plant. Further, all the strains of phosphobacteria were able to produce phytohormones and phosphatase enzyme under in vitro conditions. Keywords: In vitro, phosphobacteria, growth regulators ...

  9. Alkaline phosphatase activity in gingival crevicular fluid during canine retraction.

    Science.gov (United States)

    Batra, P; Kharbanda, Op; Duggal, R; Singh, N; Parkash, H

    2006-02-01

    The aim of the study was to investigate alkaline phosphatase activity in the gingival crevicular fluid (GCF) during orthodontic tooth movement in humans. Postgraduate orthodontic clinic. Ten female patients requiring all first premolar extractions were selected and treated with standard edgewise mechanotherapy. Canine retraction was done using 100 g sentalloy springs. Maxillary canine on one side acted as experimental site while the contralateral canine acted as control. Gingival crevicular fluid was collected from mesial and distal of canines before initiation of canine retraction (baseline), immediately after initiation of retraction, and on 1st, 7th, 14th and 21st day and the alkaline phosphatase activity was estimated. The results show significant (p < 0.05) changes in alkaline phosphatase activity on the 7th, 14th and 21st day on both mesial and distal aspects of the compared experimental and control sides. The peak in enzyme activity occurred on the 14th day of initiation of retraction followed by a significant fall in activity especially on the mesial aspect. The study showed that alkaline phosphatase activity could be successfully estimated in the GCF using calorimetric estimation assay kits. The enzyme activity showed variation according to the amount of tooth movement.

  10. Yeast Acid Phosphatases and Phytases: Production, Characterization and Commercial Prospects

    Science.gov (United States)

    Kaur, Parvinder; Satyanarayana, T.

    The element phosphorus is critical to all life forms as it forms the basic component of nucleic acids and ATP and has a number of indispensable biochemical roles. Unlike C or N, the biogeochemical cycling of phosphorus is very slow, and thus making it the growth-limiting element in most soils and aquatic systems. Phosphohydrolases (e.g. acid phosphatases and phytases) are enzymes that break the C-O-P ester bonds and provide available inorganic phosphorus from various inassimilable organic forms of phosphorus like phytates. These enzymes are of significant value in effectively combating phosphorus pollution. Although phytases and acid phosphatases are produced by various plants, animals and micro organisms, microbial sources are more promising for the production on a commercial scale. Yeasts being the simplest eukaryotes are ideal candidates for phytase and phos-phatase research due to their mostly non-pathogenic and GRAS status. They have not, however, been utilized to their full potential. This chapter focuses attention on the present state of knowledge on the production, characterization and potential commercial prospects of yeast phytases and acid phosphatases.

  11. Decryptification of Acid Phosphatase in Arthrospores of Geotrichum Species Treated with Dimethyl Sulfoxide and Acetone

    Science.gov (United States)

    Cotter, David A.; Martel, Anita J.; MacDonald, Paul

    1975-01-01

    Decryptification of acid phosphatase in Geotrichum sp. arthrospores was accomplished using acetone or dimethyl sulfoxide treatment. Both dimethyl sulfoxide and acetone irreversibly destroyed the integrity of the spore membranes without solubilizing acid phosphatase. PMID:1167386

  12. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng

    2003-01-01

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

  13. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: Implications for immunoassays

    DEFF Research Database (Denmark)

    Jørgensen, Kaj Anker; Karamperis, Nikolaos; Koefoed-Nielsen, Pernille Bundgaard

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  14. Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays

    DEFF Research Database (Denmark)

    Karamperis, N; Koefoed-Nielsen, PB; Brahe, P

    2006-01-01

    by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites...... by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition...

  15. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    OpenAIRE

    Leis, J F; Kaplan, N O

    1982-01-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid pho...

  16. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

    Science.gov (United States)

    Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

    1999-01-01

    BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

  17. Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

    Science.gov (United States)

    Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

    1996-06-01

    We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

  18. Phosphatase activity of Poa pratensis seeds. III. Effect of fluoride, citrate, urea and other substances on the activity of acid phosphatase Ia2 and Ia3

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2015-01-01

    Full Text Available Effects of fluoride, citrate, urea and other substances on the activity of acid phosphatase a2 and a3 toward p-nitrophenylphosphate and phenylphosphate were investigated. Both enzymes were inhibited by fluoride, p-chloromercuribenzoate and oxalate. Fluoride inhibited acid phosphatase a2 noncorapetitively with p-mitrophenylphosphate, whereas acid phosphatase a3 showed inhibition of mixed type. Hydrolysis of phenylphosphate by both acid phosphatases was activated by citrate. Cytosine and uridine inhibited the activity of phosphatase a2 toward p-nitrophenylphosphate and phenylphosphate, but no effect was observed in case of acid phosphatase a3. After 30 min. incubation with 4 M urea both enzymes lost about 30% of activity.

  19. Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

    Science.gov (United States)

    Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

    2009-04-01

    Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

  20. Structure determination of T-cell protein-tyrosine phosphatase

    DEFF Research Database (Denmark)

    Iversen, L.F.; Møller, K. B.; Pedersen, A.K.

    2002-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly...... homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co...... the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme....

  1. Myosin phosphatase Fine-tunes Zebrafish Motoneuron Position during Axonogenesis.

    Directory of Open Access Journals (Sweden)

    Juliane Bremer

    2016-11-01

    Full Text Available During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, we show that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. We find that this alignment requires myosin phosphatase activity within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, we demonstrate that spinal motoneurons fine-tune their position during axonogenesis and we identify the myosin II regulatory network as a key regulator.

  2. Phosphatase activity tunes two-component system sensor detection threshold.

    Science.gov (United States)

    Landry, Brian P; Palanki, Rohan; Dyulgyarov, Nikola; Hartsough, Lucas A; Tabor, Jeffrey J

    2018-04-12

    Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the phosphatase activity of the sensor histidine kinase. We experimentally validate this result in engineered Bacillus subtilis nitrate and E. coli aspartate TCS sensors by tuning their detection threshold up to two orders of magnitude. We go on to apply our TCS tuning method to recently described tetrathionate and thiosulfate sensors by mutating a widely conserved residue previously shown to impact phosphatase activity. Finally, we apply TCS tuning to engineer B. subtilis to sense and report a wide range of fertilizer concentrations in soil. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.

  3. Central regulation of metabolism by protein tyrosine phosphatases

    Directory of Open Access Journals (Sweden)

    Ryan eTsou

    2013-01-01

    Full Text Available Protein tyrosine phosphatases (PTPs are important regulators of intracellular signaling pathways via the dephosphorylation of phosphotyrosyl residues on various receptor and non-receptor substrates. The phosphorylation state of central nervous system (CNS signaling components underlies the molecular mechanisms of a variety of physiological functions including the control of energy balance and glucose homeostasis. In this review, we summarize the current evidence implicating PTPs as central regulators of metabolism, specifically highlighting their interactions with the neuronal leptin and insulin signaling pathways. We discuss the role of a number of PTPs (PTP1B, SHP2, TCPTP, RPTPe, and PTEN, reviewing the findings from genetic mouse models and in vitro studies which highlight these phosphatases as key central regulators of energy homeostasis.

  4. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

    OpenAIRE

    Bharadwaj, Rajnish; Cunningham, Kathleen M.; Zhang, Ke; Lloyd, Thomas E.

    2015-01-01

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 mi...

  5. Light availability may control extracellular phosphatase production in turbid environments

    Czech Academy of Sciences Publication Activity Database

    Rychtecký, Pavel; Řeháková, Klára; Kozlíková, Eliška; Vrba, Jaroslav

    2015-01-01

    Roč. 69, č. 1 (2015), s. 37-44 ISSN 0095-3628 R&D Projects: GA ČR(CZ) GA206/09/0309; GA ČR(CZ) GAP504/11/2177; GA ČR(CZ) GAP504/11/2182 Institutional support: RVO:60077344 Keywords : phytoplankton * phosphatase activity * ELF97 phosphate Subject RIV: DA - Hydrology ; Limnology Impact factor: 3.232, year: 2015

  6. Membrane interaction and functional plasticity of inositol polyphosphate 5-phosphatases.

    Science.gov (United States)

    Braun, Werner; Schein, Catherine H

    2014-05-06

    In this issue of Structure, Trésaugues and colleagues determined the interaction of membrane-bound phosphoinositides with three clinically significant human inositol polyphosphate 5-phosphatases (I5Ps). A comparison to the structures determined with soluble substrates revealed differences in the binding mode and suggested how the I5Ps and apurinic endonuclease (APE1) activities evolved from the same metal-binding active center. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Acid phosphatases in seeds and developing of squash (Cucurbita ficifolia

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2014-01-01

    Full Text Available Changes in protein content and acid phosphatase activity were followed during germination (imbition through seedlings development in extracts from cotyledons of squash (Cucurbita ficifolia. It has been shown that the activity of acid phosphatase was initially low and than increased to a maximum after 6 days of imbition. Acid phosphates were isolated from cotyledons of seeds and from 6-, 10- and 22-days old seedlings by extraction the proteins with 0.1 M acetate buffer pH 5.1, precipitation with ethanol and by affinity chromatography on con A-Sepharose. Two glycoprotein enzymes AcPase Ba and AcPase Bb which differ in their affinity to immobilized con A were obtained. Both acid phosphatates retained the enzyme activity after binding to free con A. Rocket affinity electrophoresis of AcPase Ba and AcPase Bb, isolated from cotyledons of seeds and seedlings, revealed differences in their ability to bind to con A during seeds germination and seedling develop-ment indicating changes in their sugar component. Con A was found to activate both enzymes. The enzymes cross-reacted with monospecific antibodies raised against grass seed acid phosphatate Ba indicating an antigenic relationship between squash and grass acid phosphatases.

  8. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

    Directory of Open Access Journals (Sweden)

    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  9. A histochemical study of rat salivary gland acid phosphatase.

    Science.gov (United States)

    Isacsson, G

    1986-01-01

    Male Sprague-Dawley rats received 4 mg pilocarpine/100 g body wt intraperitoneally or physiological saline as control and were killed at various intervals. Acid phosphatase was reacted on frozen sections from soft palate, parotid and submandibular glands using sodium-alpha-naphthyl acid phosphate as substrate. Various inhibitors were added to the incubation medium. The strongest acid phosphatase activity was in the parotid gland acinar and proximal secretory duct cells; the mucous minor glands of the palate were completely negative. Activity was found in the acinar cells, proximal secretory duct cells, granular and striated duct and excretory duct cells. Pilocarpine injection slightly reduced the activity up to 6 h after injection. Cupric chloride added to the incubation medium lowered the overall activity. Fluoride and molybdate inhibited the acid phosphatase reaction in all structures. Tartrate inhibited the reaction in all structures except the submandibular striated duct cells. The tartrate-resistant activity may be a Na+K+-dependent ATPase involved in re-absorbing water and electrolytes from the primary saliva.

  10. A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

    Science.gov (United States)

    Hughes, J.F.; Brown, G.K.

    2004-01-01

    Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

  11. Influence of triethyl phosphate on phosphatase activity in shooting range soil: Isolation of a zinc-resistant bacterium with an acid phosphatase.

    Science.gov (United States)

    Story, Sandra; Brigmon, Robin L

    2017-03-01

    Phosphatase-mediated hydrolysis of organic phosphate may be a viable means of stabilizing heavy metals via precipitation as a metal phosphate in bioremediation applications. We investigated the effect of triethyl phosphate (TEP) on soil microbial-phosphatase activity in a heavy-metal contaminated soil. Gaseous TEP has been used at subsurface sites for bioremediation of organic contaminants but not applied in heavy-metal contaminated areas. Little is known about how TEP affects microbial activity in soils and it is postulated that TEP can serve as a phosphate source in nutrient-poor groundwater and soil/sediments. Over a 3-week period, TEP amendment to microcosms containing heavy-metal contaminated soil resulted in increased activity of soil acid-phosphatase and repression of alkaline phosphatase, indicating a stimulatory effect on the microbial population. A soil-free enrichment of microorganisms adapted to heavy-metal and acidic conditions was derived from the TEP-amended soil microcosms using TEP as the sole phosphate source and the selected microbial consortium maintained a high acid-phosphatase activity with repression of alkaline phosphatase. Addition of 5mM zinc to soil-free microcosms had little effect on acid phosphatase but inhibited alkaline phosphatase. One bacterial member from the consortium, identified as Burkholderia cepacia sp., expressed an acid-phosphatase activity uninhibited by high concentrations of zinc and produced a soluble, indigo pigment under phosphate limitation. The pigment was produced in a phosphate-free medium and was not produced in the presence of TEP or phosphate ion, indicative of purple acid-phosphatase types that are pressed by bioavailable phosphate. These results demonstrate that TEP amendment was bioavailable and increased overall phosphatase activity in both soil and soil-free microcosms supporting the possibility of positive outcomes in bioremediation applications. Copyright © 2016. Published by Elsevier Inc.

  12. Proteomic analysis of protein phosphatase Z1 from Candida albicans.

    Directory of Open Access Journals (Sweden)

    Bernadett Márkus

    Full Text Available Protein phosphatase Z is a "novel type" fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0 that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly

  13. Proteomic analysis of protein phosphatase Z1 from Candida albicans

    Science.gov (United States)

    Pfliegler, Walter P.; Petrényi, Katalin; Boros, Enikő; Pócsi, István; Tőzsér, József; Dombrádi, Viktor

    2017-01-01

    Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for

  14. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    International Nuclear Information System (INIS)

    Sakisaka, Yukihiko; Tsuchiya, Masahiro; Nakamura, Takashi; Tamura, Masato; Shimauchi, Hidetoshi; Nemoto, Eiji

    2015-01-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression

  15. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    Energy Technology Data Exchange (ETDEWEB)

    Sakisaka, Yukihiko [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tsuchiya, Masahiro [Department of Oral Diagnosis, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tohoku Fukushi University, Sendai 989-3201 (Japan); Nakamura, Takashi [Department of Pediatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Liason Center for Innovative Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Tamura, Masato [Department of Biochemistry and Molecular Biology, Hokkaido University Graduate School of Dentistry, Sapporo 060-8586 (Japan); Shimauchi, Hidetoshi [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan); Nemoto, Eiji, E-mail: e-nemoto@dent.tohoku.ac.jp [Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai 980-8575 (Japan)

    2015-08-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.

  16. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    International Nuclear Information System (INIS)

    Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

    1987-01-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

  17. Putative golden proportions as predictors of facial esthetics in adolescents.

    NARCIS (Netherlands)

    Kiekens, R.M.A.; Kuijpers-Jagtman, A.M.; Hof, M.A. van 't; Hof, B.E. van 't; Maltha, J.C.

    2008-01-01

    INTRODUCTION: In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. METHODS: Seventy-six adult laypeople

  18. Exploring universal partnerships and putative marriages as tools for ...

    African Journals Online (AJOL)

    Following upon the Supreme Court of Appeal's judgment in Butters v Mncora 2012 4 SA 1 (SCA), which broadened the criteria and consequences of universal partnerships in cohabitation relationships, this article investigates the potential of universal partnerships and putative marriages to allocate rights to share in ...

  19. Putative Lineage of Novel African Usutu Virus, Central Europe

    Centers for Disease Control (CDC) Podcasts

    2015-10-15

    Sarah Gregory reads an abridged version of "Putative Lineage of Novel African Usutu Virus, Central Europe.".  Created: 10/15/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 10/15/2015.

  20. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    In this work, a computational study of expressed sequence tags (ESTs) of soybean was performed by data mining methods and bio-informatics tools and as a result 78 putative P450 genes were identified, including 57 new ones. These genes were classified into five clans and 20 families by sequence similarities and among ...

  1. Differential expressions of putative genes in various floral organs of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-03

    Jun 3, 2009 ... Full Length Research Paper. Differential expressions of putative genes in various floral organs of the Pigeon orchid (Dendrobium crumenatum) using GeneFishing. Faridah, Q. Z.1, 2, Ng, B. Z.3, Raha, A. R.4, Umi, K. A. B.5 and Khosravi, A. R.2*. 1Department of Biology, Faculty Science, University Putra ...

  2. Inhibitory Synaptic Plasticity - Spike timing dependence and putative network function.

    Directory of Open Access Journals (Sweden)

    Tim P Vogels

    2013-07-01

    Full Text Available While the plasticity of excitatory synaptic connections in the brain has been widely studied, the plasticity of inhibitory connections is much less understood. Here, we present recent experimental and theoretical □ndings concerning the rules of spike timing-dependent inhibitory plasticity and their putative network function. This is a summary of a workshop at the COSYNE conference 2012.

  3. Alkenenitrile Transmissive Olefination: Synthesis of the Putative Lignan "Morinol I"

    Science.gov (United States)

    Fleming, Fraser F.; Liu, Wang; Yao, Lihua; Pitta, Bhaskar; Purzycki, Matthew; Ravikumar, P. C.

    2012-01-01

    Grignard reagents trigger an addition-elimination with α'-hydroxy acrylonitriles to selectively generate Z-alkenenitriles. The modular assembly of Z-alkenenitriles from a Grignard reagent, acrylonitrile, and an aldehyde is ideal for stereoselectively synthesizing alkenes as illustrated in the synthesis of the putative lignan "morinol I." PMID:22545004

  4. Phosphoprotein phosphatase of bovine spleen cell nuclei: physicochemical properties

    International Nuclear Information System (INIS)

    Rezyapkin, V.I.; Leonova, L.E.; Komkova, A.I.

    1986-01-01

    The physicochemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were studied. The enzyme possesses broad substrate specificity and catalyzes the dephosphorylation of phosphocasein, ATP, ADP, and p-nitrophenyl phosphate (pNPP). K/sub m/ for ATP, ADP, and pNPP are equal to 0.44, 0.43, and 1.25 mM, respectively. M/sub r/ of the enzyme, according to the data of gel filtraction of Sephadex G-75 and electrophoresis in polyacrylamide gel of various concentrations is ∼ 33,000. In electrophoresis in the presence of SDS, two protein bands with M/sub r/ 12,000 and 18,000 are detected. In the enzyme molecule, acid amino acid residues predominate; two free SH groups and two disulfide bridges are detected. Phosphoprotein phosphatase is a glycoprotein, containing ∼ 22% carbonhydrates. The protein possesses a supplementary absorption maximum at 560 nm. Ammonium molybdate is a competitive inhibitor with K/sub i/ 0.37 μM, while sodium fluoride is a noncompetitive inhibitor with K/sub i/ 1.3 mM. Incubation in the presence of 2 mM phenylmethylsulfonyl fluoride for 25 h leads to a loss of ∼ 46% of the enzymatic activity. Ammonium molybdate, sodium fluoride, and PMSF are reversible inhibitors. Modifications of the SH groups, NH 2 groups, and histidine leads to a decrease in the enzymatic activity. Incubation of phosphoprotein phosphatase with [γ- 32 P]ATP leads to the incorporation of 0.33 mole 33 P per mole of the enzyme. The mechanism of hydrolysis of the phosphodiester bond, catalyzed by the enzyme, is discussed

  5. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

    Science.gov (United States)

    Bharadwaj, Rajnish; Cunningham, Kathleen M; Zhang, Ke; Lloyd, Thomas E

    2016-02-15

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Tea Contains Potent Inhibitors of Tyrosine Phosphatase PTP1B

    OpenAIRE

    Ma, Junfeng; Li, Zhe; Xing, Shu; Ho, Wanting Tina; Fu, Xueqi; Zhao, Zhizhuang Joe

    2011-01-01

    Tea is widely consumed all over the world. Studies have demonstrated the role of tea in prevention and treatment of various chronic diseases including diabetes and obesity, but the underlying mechanism is unclear. PTP1B is a widely expressed tyrosine phosphatase which has been defined as a target for therapeutic drug development to treat diabetes and obesity. In screening for inhibitors of PTP1B, we found that aqueous extracts of teas exhibited potent PTP1B inhibitory effects with an IC50 val...

  7. A double antibody radioimmunoassay specific for placental alkaline phosphatase

    International Nuclear Information System (INIS)

    Dass, S.; Bagshawe, K.D.

    1984-01-01

    Placental alkaline phosphatase (PLAP) is normally found in enzymically measurable amounts in second and third trimester pregnancy serum. Its occurrence in sera and tumours from patients with malignant disease has led to the development of methods to specifically identify and quantitate the enzyme. Recently immunological techniques have been used, employing antibodies raised to purified PLAP; these include solid phase radioimmunoassays and enzyme-immunoassay. The development of a sensitive, specific, automated double-antibody radioimmunoassay for the measurement of PLAP in serum is reported. (Auth.)

  8. Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects

    Energy Technology Data Exchange (ETDEWEB)

    Pradalier, N; Canal, P; Pujol, A; Fregevu, Y [Groupe de Recherches du Centre Claudius-Regaud, Toulouse (France); Soula, G [Faculte des Sciences Pharmaceutiques, Toulouse (France)

    1982-01-01

    We propose a double antibody radioimmunoassay for human prostatic acid phosphatase (PAP) in serum for diagnosis and management of prostatic adenocarcinoma under treatment. The antigen is purified from human prostatic fluid by a gel-filtration on Sephadex G 100 followed by affinity chromatography on Con A Sepharose. A specific antibody is raised in rabbits and purified by immunoadsorption with a female serum. The described technique offers both radioisotopic sensibility and immunologic specificity. Physiological values determined in the serum of 125 healthy males are below 2 ng/ml. No significative differences are observed with age. The proposed technique also shows significant differences between values evaluated for benign prostatic hyperplasia and prostatic adenocarcinoma.

  9. Autophagy Signaling in Prostate Cancer: Identification of a Novel Phosphatase

    Science.gov (United States)

    2013-01-01

    chloroquine treatment] and LC3 accumulation used as a measure of autophagic flux (Tanida et al., 2005). We found that both knockdown of PTPs and amino acid...6 Jeffrey P. MacKeigan,3 Kyle A. Furge,2 H. Eric Xu1,7†D ow nloaded The antimalaria drug chloroquine has been used as an anti-inflammatory agent for...verified and characterized for phosphatase activity in vitro (task 2). In addition to inserting these constructs into mammalian expression vectors, used

  10. Chromatographic separation of alkaline phosphatase from dental enamel

    DEFF Research Database (Denmark)

    Moe, D; Kirkeby, S; Salling, E

    1989-01-01

    Alkaline phosphatase (AP) was prepared from partly mineralized bovine enamel by extraction in phosphate buffer, centrifugation and various chromatographic techniques. Chromatofocusing showed that the enamel enzyme possessed five isoelectric points at the acid pH level ranging from pH 5.7 to pH 4.......4. Three enzyme peaks were eluted using low pressure chromatography with a Bio-gel column. With a HPLC gel filtration column the separation of the enamel extract resulted in only one peak with AP activity. The fractions of this peak were used to produce an antibody against bovine AP....

  11. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y...... not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation....

  12. Phosphoglycolate phosphatase and 2,3-diphosphoglycerate in red cells of normal and anemic subjects.

    Science.gov (United States)

    Somoza, R; Beutler, E

    1983-10-01

    Red cell phosphoglycolate phosphatase (PGP) and 2,3-diphosphoglycerate (2,3-DPG) were investigated in normal and anemic patients and rabbits. In hemolytic anemia and blood-loss anemia, characterized by a young red cell population, there was an increase in both phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. In aplastic anemia, the phosphoglycolate phosphatase activity was normal, but the 2,3-diphosphoglycerate values were nonetheless increased. Thus, no relationship was found between phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. The lack of correlation between the activity of phosphoglycolate phosphatase and 2,3-DPG levels suggests that modulation of phosphoglycolate phosphatase activity does not control the level of 2,3-DPG in erythrocytes.

  13. Simplified preparation of a phosphatase inhibitor and further studies of its action.

    Science.gov (United States)

    Coburn, S P; Schaltenbrand, W E

    1978-05-01

    1-Pyrrolidinecarbothioic acid (2-pyridylmethylene) hydrazide chelates Zn2+ but not Mg2+. This compound is about twice as effective as EDTA for inhibiting alkaline phosphatase from calf mucosa, and approx. 1000-fold more effective than EDTA for inhibiting acid phosphatase from wheat germ. The compound did not inhibit pyridoxine kinase activity in human leucocytes at the highest concentration tested (33 micron). Therefore it may be a useful tool for either examining or eliminating the effects of phosphatases in complex enzyme systems.

  14. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    OpenAIRE

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

  15. Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

    Science.gov (United States)

    Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

    1996-09-01

    We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

  16. Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

    Directory of Open Access Journals (Sweden)

    Gilbert Christophe

    2008-04-01

    Full Text Available Abstract Background Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS biosynthesis loci of numerous lactic acid bacteria genomes. Results The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP, with an optimal activity in presence of bovine serum albumin (BSA 1% at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S showed close to 20% increase in phosphatase activity. Conclusion These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.

  17. Growth and extracellular phosphatase activity of arbuscular mycorrhizal hyphae as influenced by soil organic matter

    DEFF Research Database (Denmark)

    Joner, E.J.; Jakobsen, I.

    1995-01-01

    Two experiments were set up to investigate the influence of soil organic matter on growth of arbuscular mycorrhizal (AM) hyphae and concurrent changes in soil inorganic P, organic P and phosphatase activity. A sandy loam soil was kept for 14 months under two regimes (outdoor where surplus...... additions. In soil with added clover alkaline phosphatase activity increased due to the presence of mycorrhizal hyphae. We suggest that mycorrhizas may influence the exudation of acid phosphatase by roots. Hyphae of G. invermaium did apparently not excrete extracellular phosphatases, but their presence may...

  18. Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

    Science.gov (United States)

    Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

    2005-11-01

    Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in

  19. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  20. Semi-Automatic Rating Method for Neutrophil Alkaline Phosphatase Activity.

    Science.gov (United States)

    Sugano, Kanae; Hashi, Kotomi; Goto, Misaki; Nishi, Kiyotaka; Maeda, Rie; Kono, Keigo; Yamamoto, Mai; Okada, Kazunori; Kaga, Sanae; Miwa, Keiko; Mikami, Taisei; Masauzi, Nobuo

    2017-01-01

    The neutrophil alkaline phosphatase (NAP) score is a valuable test for the diagnosis of myeloproliferative neoplasms, but it has still manually rated. Therefore, we developed a semi-automatic rating method using Photoshop ® and Image-J, called NAP-PS-IJ. Neutrophil alkaline phosphatase staining was conducted with Tomonaga's method to films of peripheral blood taken from three healthy volunteers. At least 30 neutrophils with NAP scores from 0 to 5+ were observed and taken their images. From which the outer part of neutrophil was removed away with Image-J. These were binarized with two different procedures (P1 and P2) using Photoshop ® . NAP-positive area (NAP-PA) and granule (NAP-PGC) were measured and counted with Image-J. The NAP-PA in images binarized with P1 significantly (P < 0.05) differed between images with NAP scores from 0 to 3+ (group 1) and those from 4+ to 5+ (group 2). The original images in group 1 were binarized with P2. NAP-PGC of them significantly (P < 0.05) differed among all four NAP score groups. The mean NAP-PGC with NAP-PS-IJ indicated a good correlation (r = 0.92, P < 0.001) to results by human examiners. The sensitivity and specificity of NAP-PS-IJ were 60% and 92%, which might be considered as a prototypic method for the full-automatic rating NAP score. © 2016 Wiley Periodicals, Inc.

  1. Alkaline phosphatase as a screening test for osteomalacia.

    Science.gov (United States)

    Chinoy, Muhammad Amin; Javed, Muhammad Imran; Khan, Alamzeb; Sadruddin, Nooruddin

    2011-01-01

    Vitamin D deficiency remains common in children and adults in Pakistan despite adequate sunlight exposure. Diagnosis in adults is usually delayed and is made following pathological fractures that result in significant morbidity. The objective of this study was to see whether Serum Alkaline Phosphatase levels could be used as a screening test for osteomalacia. The Study was conducted at Fatima Hospital, Baqai Medical University, Gadap, Karachi, between July 2002 and June 2005. Serum calcium levels are commonly used to screen patients suspected of osteomalacia, and raised serum alkaline phosphatase (SALP) is considered a diagnostic finding. We used SALP to screen patients who presented with back or non-specific aches and pain of more than six months duration. Three hundred thirty-four (334) patients were screened of which 116 (35%) had raised SALP. Osteomalacia was diagnosed in 92 (79.3%) of these 116 either by plain radiographs, bone biopsy or isotope bone scan. Fifty-four (53.4%) of the 101 cases had a normal level of serum calcium. Osteomalacia is likely to be missed if only serum calcium is used to screen patients. Serum Alkaline Phosphate should be used as the preferred method for screening these patients.

  2. Spatial control of protein phosphatase 2A (de)methylation

    International Nuclear Information System (INIS)

    Longin, Sari; Zwaenepoel, Karen; Martens, Ellen; Louis, Justin V.; Rondelez, Evelien; Goris, Jozef; Janssens, Veerle

    2008-01-01

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A C ) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A C antibodies revealed a good correlation with the methylation status of PP2A C , demethylated PP2A C being substantially nuclear. Throughout mitosis, demethylated PP2A C is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A C in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1

  3. SH2-inositol phosphatase 1 negatively influences early megakaryocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Lia E Perez

    Full Text Available The SH2-containing-5'inositol phosphatase-1 (SHIP influences signals downstream of cytokine/chemokine receptors that play a role in megakaryocytopoiesis, including thrombopoietin, stromal-cell-derived-Factor-1/CXCL-12 and interleukin-3. We hypothesize that SHIP might control megakaryocytopoiesis through effects on proliferation of megakaryocyte progenitors (MKP and megakaryocytes (MK.Herein, we report the megakaryocytic phenotype and MK functional assays of hematopoietic organs of two strains of SHIP deficient mice with deletion of the SHIP promoter/first exon or the inositol phosphatase domain. Both SHIP deficient strains exhibit a profound increase in MKP numbers in bone marrow (BM, spleen and blood as analyzed by flow cytometry (Lin(-c-Kit+CD41+ and functional assays (CFU-MK. SHIP deficient MKP display increased phosphorylation of Signal Transducers and Activators of Transcription 3 (STAT-3, protein kinase B (PKB/AKT and extracellular signal-regulated kinases (ERKs. Despite increased MKP content, total body number of mature MK (Lin(-c-kit(-CD41+ are not significantly changed as SHIP deficient BM contains reduced MK while spleen MK numbers are increased. Reduction of CXCR-4 expression in SHIP deficient MK may influence MK localization to the spleen instead of the BM. Endomitosis, process involved in MK maturation, was preserved in SHIP deficient MK. Circulating platelets and red blood cells are also reduced in SHIP deficient mice.SHIP may play an important role in regulation of essential signaling pathways that control early megakaryocytopoiesis in vivo.

  4. PTPN13, a Fas-associated protein tyrosine phosphatase, is located on the long arm of chromosome 4 at band q21.3

    Energy Technology Data Exchange (ETDEWEB)

    Inazawa, Johji; Ariyama, Takeshi; Abe, Tatsuo [Kyoto Prefectural Univ. of Medicine (Japan)] [and others

    1996-01-15

    PTPN13 is a protein tyrosine phosphatase that associates with the C-terminal negative regulatory domain in the Fas (APO-1/CD95) receptor. The PTPN13 protein contains six GLGF repeats that have been found in the rat postsynaptic density protein (PSD-95) and the Drosophila tumor suppressor protein, lethal-(1)-disclarge-1 (dlg-1). The localization of the PTPN13 gene to human chromosome 4q21.3 was determined by both FISH and PCR analysis of somatic cell hybrids. This 4q21.3 chromosomal region contains a gene for autosomal dominant polycystic kidney disease as well as the region frequently deleted in liver and ovarian cancers, suggesting that PTPN13 is a candidate for one of the putative tumor suppressor genes on the long arm of chromosome 4. 21 refs., 1 fig.

  5. Zinc and glutamate dehydrogenase in putative glutamatergic brain structures.

    Science.gov (United States)

    Wolf, G; Schmidt, W

    1983-01-01

    A certain topographic parallelism between the distribution of histochemically (TIMM staining) identified zinc and putative glutamatergic structures in the rat brain was demonstrated. Glutamate dehydrogenase as a zinc containing protein is in consideration to be an enzyme synthesizing transmitter glutamate. In a low concentration range externally added zinc ions (10(-9) to 10(-7) M) induced an increase in the activity of glutamate dehydrogenase (GDH) originating from rat hippocampal formation, neocortex, and cerebellum up to 142.4%. With rising molarity of Zn(II) in the incubation medium, the enzyme of hippocampal formation and cerebellum showed a biphasic course of activation. Zinc ions of a concentration higher than 10(-6) M caused a strong inhibition of GDH. The effect of Zn(II) on GDH originating from spinal ganglia and liver led only to a decrease of enzyme activity. These results are discussed in connection with a functional correlation between zinc and putatively glutamatergic system.

  6. TORC1 regulates Pah1 phosphatidate phosphatase activity via the Nem1/Spo7 protein phosphatase complex.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Dubots

    Full Text Available The evolutionarily conserved target of rapamycin complex 1 (TORC1 controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.

  7. Dynamic Changes in Yeast Phosphatase Families Allow for Specialization in Phosphate and Thiamine Starvation.

    Science.gov (United States)

    Nahas, John V; Iosue, Christine L; Shaik, Noor F; Selhorst, Kathleen; He, Bin Z; Wykoff, Dennis D

    2018-05-10

    Convergent evolution is often due to selective pressures generating a similar phenotype. We observe relatively recent duplications in a spectrum of Saccharomycetaceae yeast species resulting in multiple phosphatases that are regulated by different nutrient conditions - thiamine and phosphate starvation. This specialization is both transcriptional and at the level of phosphatase substrate specificity. In Candida glabrata , loss of the ancestral phosphatase family was compensated by the co-option of a different histidine phosphatase family with three paralogs. Using RNA-seq and functional assays, we identify one of these paralogs, CgPMU3 , as a thiamine phosphatase. We further determine that the 81% identical paralog CgPMU2 does not encode thiamine phosphatase activity; however, both are capable of cleaving the phosphatase substrate, 1-napthyl-phosphate. We functionally demonstrate that members of this family evolved novel enzymatic functions for phosphate and thiamine starvation, and are regulated transcriptionally by either nutrient condition, and observe similar trends in other yeast species. This independent, parallel evolution involving two different families of histidine phosphatases suggests that there were likely similar selective pressures on multiple yeast species to recycle thiamine and phosphate. In this work, we focused on duplication and specialization, but there is also repeated loss of phosphatases, indicating that the expansion and contraction of the phosphatase family is dynamic in many Ascomycetes. The dynamic evolution of the phosphatase gene families is perhaps just one example of how gene duplication, co-option, and transcriptional and functional specialization together allow species to adapt to their environment with existing genetic resources. Copyright © 2018, G3: Genes, Genomes, Genetics.

  8. The TriTryp Phosphatome: analysis of the protein phosphatase catalytic domains

    Directory of Open Access Journals (Sweden)

    Huxley-Jones Julie

    2007-11-01

    Full Text Available Abstract Background The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome of the three parasites. Results An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. Conclusion The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent

  9. Supplementary data: Variation in the PTEN-induced putative kinase ...

    Indian Academy of Sciences (India)

    Variation in the PTEN-induced putative kinase 1 gene associated with the increase risk of type 2 diabetes in northern Chinese. Yanchun Qu, Liang Sun, Ze Yang and Ruifa Han. J. Genet. 90, 125–128. Table 1. Clinical characteristics of cases and controls. Phenotype. T2DM. Controls. P value. Age (years). 49.5 ± 11.1. 50.4 ± ...

  10. Protein phosphatase 2A dysfunction in Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Jean-Marie eSontag

    2014-03-01

    Full Text Available Protein Phosphatase 2A (PP2A is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer’s disease (AD field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. PP2A dysfunction has been linked to Tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/B holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/B isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/B-Tau protein interactions likely contribute to Tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/B, enhanced phosphorylation of Tau and amyloid precursor protein, Tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie PP2A dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes.

  11. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

    Science.gov (United States)

    Frey, Stefan; Reschka, Eva J; Pöggeler, Stefanie

    2015-01-01

    The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

  12. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK Complex.

    Directory of Open Access Journals (Sweden)

    Stefan Frey

    Full Text Available The striatin-interacting phosphatase and kinase (STRIPAK complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP. In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

  13. 3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP).

    Science.gov (United States)

    Kurokawa, Tatsuki; Takasuga, Shunsuke; Sakata, Souhei; Yamaguchi, Shinji; Horie, Shigeo; Homma, Koichi J; Sasaki, Takehiko; Okamura, Yasushi

    2012-06-19

    Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.

  14. Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

    Directory of Open Access Journals (Sweden)

    Kellie Burnside

    2010-06-01

    Full Text Available Exotoxins, including the hemolysins known as the alpha (alpha and beta (beta toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1 were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1 increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU, serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE and a hypothetical protein (NWMN_1123 were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

  15. Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics

    NARCIS (Netherlands)

    Groves, M R; Yao, Z J; Roller, P P; Burke, T R; Barford, D

    1998-01-01

    Protein tyrosine phosphatases regulate diverse cellular processes and represent important targets for therapeutic intervention in a number of diseases. The crystal structures of protein tyrosine phosphatase 1B (PTP1B) in complex with small molecule inhibitors based upon two classes of

  16. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol

    NARCIS (Netherlands)

    Cerneus, D. P.; Ueffing, E.; Posthuma, G.; Strous, G. J.; van der Ende, A.

    1993-01-01

    Alkaline phosphatase is anchored to the outer leaflet of the plasma membrane by a covalently attached glycosyl-phosphatidylinositol anchor. We have studied the biosynthetic transport and endocytosis of alkaline phosphatase in the choriocarcinoma cell line BeWo, which endogenously expresses this

  17. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signal...

  18. Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

    Science.gov (United States)

    Scollar, M P; Sigal, G; Klibanov, A M

    1985-03-01

    Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

  19. Coupling between the voltage-sensing and phosphatase domains of Ci-VSP.

    Science.gov (United States)

    Villalba-Galea, Carlos A; Miceli, Francesco; Taglialatela, Maurizio; Bezanilla, Francisco

    2009-07-01

    The Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

  20. The tillage effect on the soil acid and alkaline phosphatase activity

    Directory of Open Access Journals (Sweden)

    Lacramioara Oprica

    2011-12-01

    Full Text Available Phosphatases (acid and alkaline are important in soils because these extracellular enzymes catalyze the hydrolysis of organic phosphate esters to orthophosphate; thus they form an important link between biologically unavailable and mineral phosphorous. Phosphatase activity is sensitive to environmental perturbations such as organic amendments, tillage, waterlogging, compaction, fertilizer additions and thus it is often used as an environmental indicator of soil quality in riparian ecosystems. The aim of the study was to assess the effect of tillage systems on phosphatases activity in a field experiment carried out in Ezăreni farm. The phosphatase activitiy were determined at two depths (7-10 cm and 15-25cm layers of a chernozem soil submitted to conventional tillage (CT in a fertilised and unfertilised experiment. Monitoring soil alkaline phosphatase activity showed, generally, the same in fertilized soil profiles collected from both depths; the values being extremely close. In unfertilized soils, alkaline phosphatase activity is different only in soils that were exposed to unconventional work using disc harrows and 30cm tillage. Both works type (no tillage and conventional tillage cause an intense alkaline phosphatase activity in 7-10 cm soil profile. Acid phosphatase activity is highly fluctuating in both fertilized as well unfertilized soil, this enzyme being influenced by the performed works.

  1. Phylogenetic characterization of phosphatase-expressing bacterial communities in Baltic Sea sediments

    NARCIS (Netherlands)

    Steenbergh, Anne; Bodelier, Paul; Hoogveld, H.L.; Slomp, C.P; Laanbroek, H.J.

    2015-01-01

    Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is

  2. Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk.

    Science.gov (United States)

    Kim, Dong-Hyeon; Chon, Jung-Whan; Lim, Jong-Soo; Kim, Hong-Seok; Kang, Il-Byeong; Jeong, Dana; Song, Kwang-Young; Kim, Hyunsook; Kim, Kwang-Yup; Seo, Kun-Ho

    2016-01-01

    The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 μg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk.

  3. Radioimmunoassay for prostatic acid phosphatase in human serum. Methodologic aspects

    International Nuclear Information System (INIS)

    Pradalier, N.; Canal, P.; Pujol, A.; Fregevu, Y.; Soula, G.

    1982-01-01

    We propose a double antibody radioimmunoassay for human prostatic acid phosphatase (PAP) in serum for diagnosis and management of prostatic adenocarcinoma under treatment. The antigen is purified from human prostatic fluid by a gel-filtration on Sephadex G 100 followed by affinity chromatography on Con A Sepharose. A specific antibody is raised in rabbits and purified by immunoadsorption with a female serum. The described technique offers both radioisotopic sensibility and immunologic specificity. Physiological values determined in the serum of 125 healthy males are below 2 ng/ml. No significative differences are observed with age. The proposed technique also shows significant differences between values evaluated for benign prostatic hyperplasia and prostatic adenocarcinoma [fr

  4. The influence of complexing pharmaceutical compositions on alkaline phosphatase

    Science.gov (United States)

    Atyaksheva, L. F.; Chukhrai, E. S.; Stepina, N. D.; Novikova, N. N.; Yur'eva, E. A.

    2011-06-01

    It is established that the pharmaceutical compositions xydiphon, medifon, succimer, and EDTA, which are used as complexing agents for accelerating the excretion of heavy metals from human organism, at certain concentrations inhibit enzyme alkaline phosphatase (AP). It is concluded that xydiphon and EDTA have a noticeable effect on AP activity at concentrations over 0.01 mM; medifon and succimer, at concentrations of over 0.3-0.5 mM. The enzyme's inhibition constants and type of inhibition are determined. Xydiphon is found to manifest the highest affinity to AP ( K I = 0.35 mM). It is shown by kinetic analysis that dissociative chemoinactivation of the enzyme takes place under the action of complexing agents. The corresponding kinetic parameters are calculated.

  5. Serum creatinine and alkaline phosphatase levels are associated with severe chronic periodontitis.

    Science.gov (United States)

    Caúla, A L; Lira-Junior, R; Tinoco, E M B; Fischer, R G

    2015-12-01

    Periodontitis may alter systemic homeostasis and influence creatinine and alkaline phosphatase levels. Therefore, the aim of this study was to evaluate the relationship between severe chronic periodontitis and serum creatinine and alkaline phosphatase levels. One hundred patients were evaluated, 66 with severe chronic periodontitis (test group) and 34 periodontally healthy controls (control group). Medical, demographic and periodontal parameters were registered. Blood sample was collected after an overnight fast and serum creatinine and alkaline phosphatase levels were determined. There were significant differences between test and control groups in ethnicity, gender and educational level (p creatinine level (p creatinine and alkaline phosphatase levels. Severe chronic periodontitis was associated to lower creatinine and higher alkaline phosphatase levels. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. The involvement of glucose-6-phosphatase in mucilage secretion by root cap cells of Zea mays

    Science.gov (United States)

    Moore, R.; McClelen, C. E.

    1985-01-01

    In order to determine the involvement of glucose-6-phosphatase in mucilage secretion by root cap cells, we have cytochemically localized the enzyme in columella and peripheral cells of root caps of Zea mays. Glucose-6-phosphatase is associated with the plasmalemma and cell wall of columella cells. As columella cells differentiate into peripheral cells and begin to produce and secrete mucilage, glucose-6-phosphatase staining intensifies and becomes associated with the mucilage and, to a lesser extent, the cell wall. Cells being sloughed from the cap are characterized by glucose-6-phosphatase staining being associated with the vacuole and plasmalemma. These changes in enzyme localization during cellular differentiation in root caps suggest that glucose-6-phosphatase is involved in the production and/or secretion of mucilage by peripheral cells of Z. mays.

  7. Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

    Science.gov (United States)

    Crans, D C; Simone, C M; Saha, A K; Glew, R H

    1989-11-30

    A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

  8. Alkaline phosphatase levels in patients with coronary heart disease saliva and its relation with periodontal status

    Science.gov (United States)

    Yunita, Dina Suci; Masulili, Sri Lelyati C.; Tadjoedin, Fatimah M.; Radi, Basuni

    2017-02-01

    Coronary heart disease (CHD) is a disease that causes narrowing of the coronary arteries. Currently, there is a hypothesis regarding periodontal infection that increases risk for heart disease. Alkaline phosphatase (ALP) as a marker of inflammation will increase in atherosclerosis and periodontal disease. The objective of this research is analyzing the relationship between the levels of alkaline phosphatase in saliva with periodontal status in patients with CHD and non CHD. Here, saliva of 104 subjects were taken, each 1 ml, and levels of Alkaline Phosphatase was analyzed using Abbott ci4100 architect. We found that no significant difference of Alkaline Phosphatase levels in saliva between CHD patients and non CHD. Therefore, it can be concluded that Alkaline Phosphatase levels in patients with CHD saliva was higher than non CHD and no association between ALP levels with periodontal status.

  9. Immunocytochemical detection of the microsomal glucose-6-phosphatase in human brain astrocytes.

    Science.gov (United States)

    Bell, J E; Hume, R; Busuttil, A; Burchell, A

    1993-10-01

    Using an antibody raised against the catalytic subunit of glucose-6-phosphatase, this enzyme was immunolocalized in many astrocytes in 20 normal human brains. Double immunofluorescence studies showed co-localization of glial fibrillary acidic protein (GFAP) with glucose-6-phosphatase in astrocytes. However, not all GFAP-positive cells were also glucose-6-phosphatase positive, indicating that some astrocytes do not contain demonstrable expression of this enzyme. Reactive astrocytes in a variety of abnormal brains were strongly glucose-6-phosphatase positive, but neoplastic astrocytes were often only weakly positive. Expression of the enzyme could not be demonstrated in radial glia, neurons or oligodendroglia. Astrocytes normally contain glycogen and the demonstration that some astrocytes also contain glucose-6-phosphatase indicates that they are competent for both glycogenolysis and gluconeogenesis, which may be critical for neuronal welfare.

  10. Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways.

    Directory of Open Access Journals (Sweden)

    Francesca eSacco

    2014-05-01

    Full Text Available Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI#K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26 respectively.

  11. Regulation of Akt/Protein Kinase B Signaling by a Novel Protein Phosphatase in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Brognard, John; Newton, Alexandra

    2008-01-01

    ...: cell proliferation, growth, and apoptosis. Finally, since this phosphatase resides in a location of frequent loss of heterozygosity in breast cancer, we sought to determine if this phosphatase played a role in breast tumorigenesis...

  12. Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study

    OpenAIRE

    Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

    2016-01-01

    Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis ...

  13. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    Science.gov (United States)

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  14. COMPARISON OF METHODS FOR ALKALINE PHOSPHATASE AND PEROXIDASE DETECTION IN MILK

    Directory of Open Access Journals (Sweden)

    felipe Nael Seixas

    2014-02-01

    Full Text Available This study evaluated the performance of strips for colorimetric detection of alkaline phosphatase and peroxidase in milk, comparing them with a kit of reagents for alkaline phosphatase and the official methodology for peroxidase. The samples were analyzed at the Laboratory Inspection of Products of Animal Origin, State University of Londrina. For the comparison tests for the detection of alkaline phosphatase four treatments were made by adding different percentages of raw milk (1%, 2%, 5% and 10% in the pasteurized milk, plus two control treatments. Thirty-eight samples triplicate for each treatment were analyzed. To compare the performance of tests for peroxidase 80 pasteurized milk samples were evaluated simultaneously by official methodology and by colorimetric strips. The performance of the alkaline phosphatase were different for the treatments with 1% and 2% of raw milk which had all the strips change color as the reagent kit showed the presence of phosphatase in just 2.63% and 5.26% the cases, respectively for each treatment. The colorimetric strips for alkaline phosphatase are more sensitive for the identification of small quantities compared to the reagent kit. The performance of tests for peroxidase showed no difference. The strips for the detection of peroxidase or alkaline phosphatase were effective and can replace traditional methods.

  15. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

  16. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-03-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

  17. Characterization of four plasma membrane aquaporins in tulip petals: a putative homolog is regulated by phosphorylation.

    Science.gov (United States)

    Azad, Abul Kalam; Katsuhara, Maki; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2008-08-01

    We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

  18. Putative golden proportions as predictors of facial esthetics in adolescents.

    Science.gov (United States)

    Kiekens, Rosemie M A; Kuijpers-Jagtman, Anne Marie; van 't Hof, Martin A; van 't Hof, Bep E; Maltha, Jaap C

    2008-10-01

    In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. Seventy-six adult laypeople evaluated sets of photographs of 64 adolescents on a visual analog scale (VAS) from 0 to 100. The facial esthetic value of each subject was calculated as a mean VAS score. Three observers recorded the position of 13 facial landmarks included in 19 putative golden proportions, based on the golden proportions as defined by Ricketts. The proportions and each proportion's deviation from the golden target (1.618) were calculated. This deviation was then related to the VAS scores. Only 4 of the 19 proportions had a significant negative correlation with the VAS scores, indicating that beautiful faces showed less deviation from the golden standard than less beautiful faces. Together, these variables explained only 16% of the variance. Few golden proportions have a significant relationship with facial esthetics in adolescents. The explained variance of these variables is too small to be of clinical importance.

  19. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    Energy Technology Data Exchange (ETDEWEB)

    Baig, M.; Brown, A.; Eswaramoorthy, S.; Swaminathan, S.

    2009-01-01

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

  20. A new putative deltapartitivirus recovered from Dianthus amurensis.

    Science.gov (United States)

    An, Hongliu; Tan, Guanlin; Xiong, Guihong; Li, Meirong; Fang, Shouguo; Islam, Saif Ul; Zhang, Songbai; Li, Fan

    2017-09-01

    Two double stranded RNAs (dsRNA), likely representing the genome of a novel deltapartitivirus, provisionally named carnation cryptic virus 3 (CCV3), were recovered from Dianthus amurensis. The two dsRNAs were 1,573 (dsRNA1) and 1,561 (dsRNA2) bp in size, each containing a single open reading frame (ORF) encoding a 475- and 411-aa protein, respectively. The 475-aa protein contains a conserved RNA dependent RNA polymerase (RdRp) domain which shows significant homology to RdRps of established or putative partitiviruses, particularly those belonging to the genus Deltapartitivirus. However, it shares an amino acid identity of 75% with its closest relative, the RdRp of the deltapartitivirus beet cryptic virus 2 (BCV2), and is <62% identical to the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRps of selected partitiviruses, CCV3 clustered with BCV2 and formed a well-supported monophyletic clade with known or putative deltapartitiviruses.

  1. The Putative Chemosignal Androstadienone Makes Women More Generous.

    Science.gov (United States)

    Perrotta, Valentina; Graffeo, Michele; Bonini, Nicolao; Gottfried, Jay A

    2016-06-01

    Putative human chemosignals have been shown to influence mood states and emotional processing, but the connection between these effects and higher-order cognitive processing is not well established. This study utilized an economic game (Dictator Game) to test whether androstadienone (AND), an odorous compound derived from testosterone, impacts on altruistic behavior. We predicted that the female participants would act more generously in the AND condition, exhibiting a significant interaction effect between gender and AND on Dictator Game contributions. We also expected that the presence of AND should increase the positive mood of the female participants, compared to a control odor condition and also compared to the mood of the male participants. The results confirm our hypotheses: for women the subliminal perception of AND led to larger monetary donations, compared to a control odor, and also increased positive mood. These effects were absent or significantly weaker in men. Our findings highlight the capacity of human putative chemosignals to influence emotions and higher cognitive processes - in particular the processes used in the context of economic decisions - in a gender-specific way.

  2. Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings.

    Science.gov (United States)

    Cheng, H F; Tao, M

    1989-10-19

    A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.

  3. Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

    Directory of Open Access Journals (Sweden)

    Janetti R Signorelli

    Full Text Available There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate. The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1 NCM; 2 NCM plus inhibitors; 3 RCM; and 4 RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important

  4. Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

    Directory of Open Access Journals (Sweden)

    Norris-Mullins Brianna

    2014-01-01

    Full Text Available Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5 in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention.

  5. Research on Phosphatases of Belladona Leaves and Their Purification (Part 1

    Directory of Open Access Journals (Sweden)

    M. Khorsand

    1956-12-01

    Full Text Available Belladona leaves as well as all other studied leaves contains two distinct phosphatase fractions belonging respectively to types II and IIIi the major parts of these enzymes is extraetible by water. It was not possible to extract the non soluble fraction which is solidly retained by the cellular constituents. Phosphatase II does not differ from other phosphatnses of the same type. Whereas phosphatase III is distinetely different from enzymes of the same type of vegetal or animal origins. It is activated by bivalent metallic ions which are specific activators of the alkaline phcspbatnses: Mg-Zn-Ni and Co.

  6. Regulation of tyrosine phosphatases in the adventitia during vascular remodelling

    International Nuclear Information System (INIS)

    Micke, Patrick; Hackbusch, Daniel; Mercan, Sibel; Stawowy, Philipp; Tsuprykov, Oleg; Unger, Thomas; Ostman, Arne; Kappert, Kai

    2009-01-01

    Protein tyrosine phosphatases (PTPs) are regulators of growth factor signalling in vascular remodelling. The aim of this study was to evaluate PTP expression in the context of PDGF-signalling in the adventitia after angioplasty. Utilising a rat carotid artery model, the adventitial layers of injured and non-injured vessels were laser microdissected. The mRNA expression of the PDGF β-receptor, the ligands PDGF-A/B/C/D and the receptor-antagonising PTPs (DEP-1, TC-PTP, SHP-2, PTP1B) were determined and correlated to vascular morphometrics, proliferation markers and PDGF β-receptor phosphorylation. The levels of the PDGF β-receptor, PDGF-C and PDGF-D were upregulated concurrently with the antagonising PTPs DEP-1 and TC-PTP at day 8, and normalised at day 14 after vessel injury. Although the proliferation parameters were time-dependently altered in the adventitial layer, the phosphorylation of the PDGF β-receptor remained unchanged. The expression dynamics of specific PTPs indicate a regulatory role of PDGF-signalling also in the adventitia during vascular remodelling.

  7. Monomeric tartrate resistant acid phosphatase induces insulin sensitive obesity.

    Directory of Open Access Journals (Sweden)

    Pernilla Lång

    2008-03-01

    Full Text Available Obesity is associated with macrophage infiltration of adipose tissue, which may link adipose inflammation to insulin resistance. However, the impact of inflammatory cells in the pathophysiology of obesity remains unclear. Tartrate resistant acid phosphatase (TRAP is an enzyme expressed by subsets of macrophages and osteoclasts that exists either as an enzymatically inactive monomer or as an active, proteolytically processed dimer.Using mice over expressing TRAP, we show that over-expression of monomeric, but not the dimeric form in adipose tissue leads to early onset spontaneous hyperplastic obesity i.e. many small fat cells. In vitro, recombinant monomeric, but not proteolytically processed TRAP induced proliferation and differentiation of mouse and human adipocyte precursor cells. In humans, monomeric TRAP was highly expressed in the adipose tissue of obese individuals. In both the mouse model and in the obese humans the source of TRAP in adipose tissue was macrophages. In addition, the obese TRAP over expressing mice exhibited signs of a low-grade inflammatory reaction in adipose tissue without evidence of abnormal adipocyte lipolysis, lipogenesis or insulin sensitivity.Monomeric TRAP, most likely secreted from adipose tissue macrophages, induces hyperplastic obesity with normal adipocyte lipid metabolism and insulin sensitivity.

  8. Uranium Biomineralization By Natural Microbial Phosphatase Activities in the Subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Taillefert, Martial [Georgia Tech Research Corporation, Atlanta, GA (United States)

    2015-04-01

    This project investigated the geochemical and microbial processes associated with the biomineralization of radionuclides in subsurface soils. During this study, it was determined that microbial communities from the Oak Ridge Field Research subsurface are able to express phosphatase activities that hydrolyze exogenous organophosphate compounds and result in the non-reductive bioimmobilization of U(VI) phosphate minerals in both aerobic and anaerobic conditions. The changes of the microbial community structure associated with the biomineralization of U(VI) was determined to identify the main organisms involved in the biomineralization process, and the complete genome of two isolates was sequenced. In addition, it was determined that both phytate, the main source of natural organophosphate compounds in natural environments, and polyphosphate accumulated in cells could also be hydrolyzed by native microbial population to liberate enough orthophosphate and precipitate uranium phosphate minerals. Finally, the minerals produced during this process are stable in low pH conditions or environments where the production of dissolved inorganic carbon is moderate. These findings suggest that the biomineralization of U(VI) phosphate minerals is an attractive bioremediation strategy to uranium bioreduction in low pH uranium-contaminated environments. These efforts support the goals of the SBR long-term performance measure by providing key information on "biological processes influencing the form and mobility of DOE contaminants in the subsurface".

  9. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    Energy Technology Data Exchange (ETDEWEB)

    Sobecky, Patricia A. [Univ. of Alabama, Tuscaloosa, AL (United States)

    2015-04-06

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  10. Allosteric substrate switching in a voltage-sensing lipid phosphatase.

    Science.gov (United States)

    Grimm, Sasha S; Isacoff, Ehud Y

    2016-04-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We found that the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), has not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage-sensing domain (VSD). Using fast fluorescence resonance energy transfer (FRET) reporters of PIPs to monitor enzyme activity and voltage-clamp fluorometry to monitor conformational changes in the VSD, we found that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage-sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This two-step allosteric control over a dual-specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility, endocytosis and exocytosis.

  11. Allosteric substrate switching in a voltage sensing lipid phosphatase

    Science.gov (United States)

    Grimm, Sasha S.; Isacoff, Ehud Y.

    2016-01-01

    Allostery provides a critical control over enzyme activity, biasing the catalytic site between inactive and active states. We find the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP), which modifies phosphoinositide signaling lipids (PIPs), to have not one but two sequential active states with distinct substrate specificities, whose occupancy is allosterically controlled by sequential conformations of the voltage sensing domain (VSD). Using fast FRET reporters of PIPs to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, we find that Ci-VSP switches from inactive to a PIP3-preferring active state when the VSD undergoes an initial voltage sensing motion and then into a second PIP2-preferring active state when the VSD activates fully. This novel 2-step allosteric control over a dual specificity enzyme enables voltage to shape PIP concentrations in time, and provides a mechanism for the complex modulation of PIP-regulated ion channels, transporters, cell motility and endo/exocytosis. PMID:26878552

  12. Identification of a variant form of tyrosine phosphatase LYP

    Directory of Open Access Journals (Sweden)

    Ho Wanting T

    2010-11-01

    Full Text Available Abstract Background Protein tyrosine phosphatases (PTPs are important cell signaling regulators with major pathological implications. LYP (also known as PTPN22 is an intracellular enzyme initially found to be predominately expressed in lymphocytes. Importantly, an allelic R620W variant of LYP is strongly associated with multiple autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and autoimmune thyroid disease. Results In this study, we isolated a novel isoform of LYP designated LYP3. LYP3 differs from LYP1, the known isoform of LYP, in that it lacks a 28 amino acid segment right after the R620W site embedded in a proline-rich protein-protein interaction motif. Genomic sequence analysis revealed that LYP3 resulted from alternative splicing of the LYP gene located on chromosome 1p 13.3-13.1. Reverse transcription PCR analyses of 48 human tissues demonstrated that both LYP1 and LYP3 are predominantly expressed in primary and secondary lymphoid tissues but the relative expression levels of the two isoforms varies in different human tissues and individuals. Conclusions We thus identified a new variant form of LYP and conducted a comprehensive analysis of LYP tissue expressions. Considering the pathogenesis of LYP R620W, we believe that the expression of LYP3 may have an important role in regulating activity and function of LYP and may be implicated in autoimmune diseases.

  13. Changes of serum alkaline phosphatase isoenzymes in fasted rats.

    Science.gov (United States)

    Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

    1996-10-01

    Changes of serum alkaline phosphatase (sALP) isoenzymes under fasting conditions were examined using polyacrylamide gel electrophoresis (PAGE), amino-acids (L-phenylalanine (L-Phe), L-homoarginine (L-HArg)) inhibition and wheat germ agglutinin (WGA) treatment. The sALP of non-fasted rats was separated into three bands (S1, S2, S3) by PAGE. The molecular weight (M.W.) of S1 corresponded to that of an isoenzyme found in the ileum. By the addition of L-Phe, the staining intensity of S1 was weakened, S2 and S3 remained unchanged and the total activity of the isoenzymes extracted from intestine decreased. On the other hand, the activity of isoenzymes extracted from kidney and bone decreased by the addition of L-HArg. Therefore, S1 was judged to be derived from intestine. The activities of total sALP and S1 decreased from 16 h of fasting. Total sALP activity and sALP activity of the supernatant prepared by WGA treatment decreased, whereas the ALP activity of the precipitate (difference between total sALP activity and supernatant sALP activity) did not change. The activity band of the precipitate corresponded to that of S3 by PAGE. Therefore, S3 was judged to be derived from bone. In conclusion, under fasting conditions, the activity of S1 decreased while the activities of S2 and S3 remained unchanged.

  14. Uranium Biomineralization by Natural Microbial Phosphatase Activities in the Subsurface

    International Nuclear Information System (INIS)

    Sobecky, Patricia A.

    2015-01-01

    In this project, inter-disciplinary research activities were conducted in collaboration among investigators at The University of Alabama (UA), Georgia Institute of Technology (GT), Lawrence Berkeley National Laboratory (LBNL), Brookhaven National Laboratory (BNL), the DOE Joint Genome Institute (JGI), and the Stanford Synchrotron Radiation Light source (SSRL) to: (i) confirm that phosphatase activities of subsurface bacteria in Area 2 and 3 from the Oak Ridge Field Research Center result in solid U-phosphate precipitation in aerobic and anaerobic conditions; (ii) investigate the eventual competition between uranium biomineralization via U-phosphate precipitation and uranium bioreduction; (iii) determine subsurface microbial community structure changes of Area 2 soils following organophosphate amendments; (iv) obtain the complete genome sequences of the Rahnella sp. Y9-602 and the type-strain Rahnella aquatilis ATCC 33071 isolated from these soils; (v) determine if polyphosphate accumulation and phytate hydrolysis can be used to promote U(VI) biomineralization in subsurface sediments; (vi) characterize the effect of uranium on phytate hydrolysis by a new microorganism isolated from uranium-contaminated sediments; (vii) utilize positron-emission tomography to label and track metabolically-active bacteria in soil columns, and (viii) study the stability of the uranium phosphate mineral product. Microarray analyses and mineral precipitation characterizations were conducted in collaboration with DOE SBR-funded investigators at LBNL. Thus, microbial phosphorus metabolism has been shown to have a contributing role to uranium immobilization in the subsurface.

  15. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

    Science.gov (United States)

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

  16. Influence of acid phosphatase activity on the saccharification of potato maltodextrins by Aspergillus niger glucoamylase

    Energy Technology Data Exchange (ETDEWEB)

    Zyla, K. (Akademia Rolnicza, Cracow (Poland). Dept. of Biotechnology)

    1990-01-01

    A preparation of Aspergillus niger acid phosphatase, which had the temperature optimum 60deg C, pH optimum 1.8-3.0; good stability at pH 4-5, the ability to hydrolyze glucose-6-phosphate at a high rate, and substantial lack of glucogenic activities, was used simultaneously with a glucoamylase in order to learn its influence on the saccharification of potato maltodextrins. The addition of the acid phosphatase activity in amounts that gave the 50 fold increase, as compared to phosphatase activity which naturally occurs in the gluocoamylase (GA) preparation 'AMG-200', was found to influence on the DE level, mainly at the high substrate concentration (40% d.s.) and low glucoamylase dosage (60-100 GAU/kg d.s.). It may also be possible, when using the acid phosphatase addition, to shorten the saccharification time. (orig.).

  17. Acceleration of gelation and promotion of mineralization of chitosan hydrogels by alkaline phosphatase

    NARCIS (Netherlands)

    Douglas, T.E.L.; Skwarczynska, A.; Modrzejewska, Z.; Balcaen, L.; Schaubroeck, D.; Lycke, S.; Vanhaecke, F.; Vandenabeele, P.; Dubruel, P.; Jansen, J.A.; Leeuwenburgh, S.C.G.

    2013-01-01

    Thermosensitive chitosan hydrogels containing sodium beta-glycerophosphate (beta-GP), whose gelation is induced by increasing temperature to body temperature, were functionalized by incorporation of alkaline phosphatase (ALP), an enzyme involved in mineralization of bone. ALP incorporation led to

  18. Diagnosis of prostate cancer using a radioimmunoassay for prostatic acid phosphatase in serum

    International Nuclear Information System (INIS)

    Lea, O.A.; Hoeisaeter, P.Aa.

    1981-01-01

    The paper describes the development and evaluation of a specific radioimmunoassay for the determination of prostatic acid phosphatase in serum as a useful aid in the detection of prostatic cancer. (Auth.)

  19. Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B

    International Nuclear Information System (INIS)

    Brown-Shimer, S.; Johnson, K.A.; Bruskin, A.; Green, N.R.; Hill, D.E.; Lawrence, J.B.; Johnson, C.

    1990-01-01

    The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases function as growth suppressors, the authors have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1,305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2

  20. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    International Nuclear Information System (INIS)

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-01-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table

  1. Acid phosphatase from stored Poa pratensis caryopses and its ability for binding to lectins

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2014-01-01

    Full Text Available The effect of the storage period of Poa pratensis caryopses on acid phosphatase activity and on the ability of this enzyme to interact with lectins has been studied. It has been shown that after ten years of caryopses storage, the activity of acid phosphatase decreased about 50 per cent, while the content of proteins and carbohydrates did not change. The decrease of enzyme activity during the long period of storage was found only in seeds, but not in chaffs. Acid phosphatase was isolated from caryopses stored one, two, three, five and ten years. The enzyme showed the ability to bind to immoblized as well as to free conA during the whole period of storage, hut did not react with Wheat Germen Agglutinin (WGA. The activation of acid phosphatase by binding to conA decreased with the length of storage period.

  2. Bone mineralisation in premature infants cannot be predicted from serum alkaline phosphatase or serum phosphate

    DEFF Research Database (Denmark)

    Faerk, J; Peitersen, Birgit; Petersen, S

    2002-01-01

    BACKGROUND: The bone mineral content of premature infants at term is lower than in mature infants at the same postconceptional age. Serum alkaline phosphatase and serum phosphate are often used as indicators of bone mineralisation. OBJECTIVE: To analyse the association between bone mineral content...... content was measured at term (mean gestational age 41 weeks) by dual energy x ray absorptiometry and corrected for body size. RESULTS: Serum alkaline phosphatase was significantly negatively associated with serum phosphate (p mineral content was not associated with mean serum alkaline...... and serum alkaline phosphatase and serum phosphate. METHODS: Serum alkaline phosphatase and phosphate were measured at weekly intervals during admission in 108 premature infants of gestational age below 32 weeks (mean (SD) gestational age 29 (2) weeks; mean (SD) birth weight 1129 (279) g). Bone mineral...

  3. Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

    Science.gov (United States)

    Schröter, W; Neuvians, M

    1970-12-01

    Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

  4. Disparate subcellular location of putative sortase substrates in Clostridium difficile.

    Science.gov (United States)

    Peltier, Johann; Shaw, Helen A; Wren, Brendan W; Fairweather, Neil F

    2017-08-23

    Clostridium difficile is a gastrointestinal pathogen but how the bacterium colonises this niche is still little understood. Sortase enzymes covalently attach specific bacterial proteins to the peptidoglycan cell wall and are often involved in colonisation by pathogens. Here we show C. difficile proteins CD2537 and CD3392 are functional substrates of sortase SrtB. Through manipulation of the C-terminal regions of these proteins we show the SPKTG motif is essential for covalent attachment to the cell wall. Two additional putative substrates, CD0183 which contains an SPSTG motif, and CD2768 which contains an SPQTG motif, are not cleaved or anchored to the cell wall by sortase. Finally, using an in vivo asymmetric cleavage assay, we show that despite containing a conserved SPKTG motif, in the absence of SrtB these proteins are localised to disparate cellular compartments.

  5. Putative benefits of microalgal astaxanthin on exercise and human health

    Directory of Open Access Journals (Sweden)

    Marcelo P. Barros

    2011-04-01

    Full Text Available Astaxanthin (ASTA is a pinkish-orange carotenoid produced by microalgae, but also commonly found in shrimp, lobster and salmon, which accumulate ASTA from the aquatic food chain. Numerous studies have addressed the benefits of ASTA for human health, including the inhibition of LDL oxidation, UV-photoprotection and prophylaxis of bacterial stomach ulcers. ASTA is recognized as a powerful scavenger of reactive oxygen species (ROS, especially those involved in lipid peroxidation. Both aerobic and anaerobic exercise are closely related to overproduction of ROS in muscle tissue. Post-exercise inflammatory processes can even exacerbate the oxidative stress imposed by exercise. Thus, ASTA is suggested here as a putative nutritional alternative/coadjutant for antioxidant therapy to afford additional protection to muscle tissues against oxidative damage induced by exercise, as well as for an (overall integrative redox re-balance and general human health.

  6. Hepatology may have problems with putative surrogate outcome measures

    DEFF Research Database (Denmark)

    Gluud, Christian; Brok, Jesper; Gong, Yan

    2007-01-01

    A surrogate outcome measure is a laboratory measurement, a physical sign, or another intermediate substitute that is able to predict an intervention's effect on a clinically meaningful outcome. A clinical outcome detects how a patient feels, functions, or survives. Surrogate outcome measures occur...... faster or more often, are cheaper, and/or are less invasively achieved than the clinical outcome. In practice, validation is surprisingly often overlooked, especially if a biologic plausible rationale is proposed. Surrogate outcomes must be validated before use. The first step in validation...... predicts the intervention's effect on the clinical outcome. In hepatology a number of putative surrogate outcomes are used both in clinical research and in clinical practice without having been properly validated. Sustained virological response to interferons and ribavirin in patients with chronic...

  7. Basal ganglia calcification as a putative cause for cognitive decline

    Directory of Open Access Journals (Sweden)

    João Ricardo Mendes de Oliveira

    Full Text Available ABSTRACT Basal ganglia calcifications (BGC may be present in various medical conditions, such as infections, metabolic, psychiatric and neurological diseases, associated with different etiologies and clinical outcomes, including parkinsonism, psychosis, mood swings and dementia. A literature review was performed highlighting the main neuropsychological findings of BGC, with particular attention to clinical reports of cognitive decline. Neuroimaging studies combined with neuropsychological analysis show that some patients have shown progressive disturbances of selective attention, declarative memory and verbal perseveration. Therefore, the calcification process might represent a putative cause for dementia syndromes, suggesting a probable link among calcinosis, the aging process and eventually with neuronal death. The increasing number of reports available will foster a necessary discussion about cerebral calcinosis and its role in determining symptomatology in dementia patients

  8. Basal ganglia calcification as a putative cause for cognitive decline.

    Science.gov (United States)

    de Oliveira, João Ricardo Mendes; de Oliveira, Matheus Fernandes

    2013-01-01

    Basal ganglia calcifications (BGC) may be present in various medical conditions, such as infections, metabolic, psychiatric and neurological diseases, associated with different etiologies and clinical outcomes, including parkinsonism, psychosis, mood swings and dementia. A literature review was performed highlighting the main neuropsychological findings of BGC, with particular attention to clinical reports of cognitive decline. Neuroimaging studies combined with neuropsychological analysis show that some patients have shown progressive disturbances of selective attention, declarative memory and verbal perseveration. Therefore, the calcification process might represent a putative cause for dementia syndromes, suggesting a probable link among calcinosis, the aging process and eventually with neuronal death. The increasing number of reports available will foster a necessary discussion about cerebral calcinosis and its role in determining symptomatology in dementia patients.

  9. Purification and properties of acid phosphatase from Avena elatior L. seeds

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    E. Wieczorek

    2015-01-01

    Full Text Available Acid phosphatase F1 from Avena elatior seeds was isolated and partially purified by means of alcohol precepitation, DEAE-, CM-column chromatography, Sephadex G-150, Sephadex G-200 and Sepharose 4B - gel filtration. The enzyme was stable at 50°C, pH 5.1. The pH optimum for phosphatase activity was 4.2. Fluoride, Zn2+, molybdate were effective inhibitors. EDTA and l, 10-phenanthroline activated the enzyme.

  10. Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

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    Consuelo Giménez-Pardo

    2004-03-01

    Full Text Available Cholinesterase and acid phosphatase (AP, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.

  11. Tetranucleotide repeat polymorphism at the human prostatic acid phosphatase (ACPP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Polymeropoulos, M H; Xiao, Hong; Rath, D S; Merril, C R [National Inst. of Mental Health Neuroscience Center, Washington, DC (United States)

    1991-09-11

    The polymorphic (AAAT){sub n} repeat begins at base pair 2342 of the human prostatic acid phosphatase gene on chromosome 3q21-qter. The polymorphism can be typed using the polymerase chain reaction (PCR) as described previously. The predicted length of the amplified sequence was 275 bp. Co-dominant segregation was observed in two informative families. The human prostatic acid phosphatase gene has been assigned to chromosome 3q21-qter.

  12. Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

    OpenAIRE

    Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

    1990-01-01

    Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

  13. Phosphorylcholine Phosphatase: A Peculiar Enzyme of Pseudomonas aeruginosa

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    Carlos Eduardo Domenech

    2011-01-01

    Full Text Available Pseudomonas aeruginosa synthesizes phosphorylcholine phosphatase (PchP when grown on choline, betaine, dimethylglycine or carnitine. In the presence of Mg2+ or Zn2+, PchP catalyzes the hydrolysis of p-nitrophenylphosphate (p-NPP or phosphorylcholine (Pcho. The regulation of pchP gene expression is under the control of GbdR and NtrC; dimethylglycine is likely the metabolite directly involved in the induction of PchP. Therefore, the regulation of choline metabolism and consequently PchP synthesis may reflect an adaptive response of P. aeruginosa to environmental conditions. Bioinformatic and biochemistry studies shown that PchP contains two sites for alkylammonium compounds (AACs: one in the catalytic site near the metal ion-phosphoester pocket, and another in an inhibitory site responsible for the binding of the alkylammonium moiety. Both sites could be close to each other and interact through the residues 42E, 43E and 82YYY84. Zn2+ is better activator than Mg2+ at pH 5.0 and it is more effective at alleviating the inhibition produced by the entry of Pcho or different AACs in the inhibitory site. We postulate that Zn2+ induces at pH 5.0 a conformational change in the active center that is communicated to the inhibitory site, producing a compact or closed structure. However, at pH 7.4, this effect is not observed because to the hydrolysis of the [Zn2+L2−1L20(H2O2] complex, which causes a change from octahedral to tetrahedral in the metal coordination geometry. This enzyme is also present in P. fluorescens, P. putida, P. syringae, and other organisms. We have recently crystallized PchP and solved its structure.

  14. Imaging of alkaline phosphatase activity in bone tissue.

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    Terence P Gade

    Full Text Available The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP using a small imaging molecule in combination with (19Flourine magnetic resonance spectroscopic imaging ((19FMRSI. 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19Fluorine magnetic resonance spectroscopy ((19FMRS and (19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19FMRS and (19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.

  15. [Glucose-6-phosphatase from nuclear envelope in rat liver].

    Science.gov (United States)

    González-Mujica, Freddy

    2008-06-01

    Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform.

  16. Expression of Prostatic Acid Phosphatase in Rat Circumvallate Papillae.

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    Kentaro Nishida

    Full Text Available ATP and its metabolites are important for taste signaling in taste buds, and thus a clearance system for them would play critical roles in maintenance of gustatory function. A previous report revealed that mRNAs for ecto-5'-nucleotidase (NT5E and prostatic acid phosphatase (PAP were expressed by taste cells of taste buds, and NT5E-immunoreactivity was detected in taste cells. However, there was no information on PAP-immunoreactivity in taste buds. In this study, we examined the expression profile of PAP in rat taste buds. In the isolated rat taste buds, we detected expression of mRNA for PAP, but NT5E was not detected differing from the case of mouse ones (Dando et al., 2012, J Neuroscience. On immunohistochemical analysis, PAP-immunoreactivity was found predominantly in NTPDase2-positive type I and SNAP25-positive type III taste cells, while there were no apparent signals of it in PLC-β2-positive type II, α-gustducin-positive type II, AADC-positive type III and 5HT-positive type III ones. As for NT5E, we could not detect its immunoreactivity in rat taste buds, and co-localization of it with any taste cell markers, although mouse taste buds expressed NT5E as reported previously. These findings suggest that PAP expressed by type I and one of type III taste cells of rats may contribute to metabolic regulation of the extracellular levels of adenine nucleotides in the taste buds of circumvallate papillae, and the regulating mechanisms for adenine nucleotides in taste buds might be different between rats and mice.

  17. Response to DNA damage: why do we need to focus on protein phosphatases?

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    Midori eShimada

    2013-01-01

    Full Text Available Eukaryotic cells are continuously threatened by unavoidable errors during normal DNA replication or various sources of genotoxic stresses that cause DNA damage or stalled replication. To maintain genomic integrity, cells have developed a coordinated signaling network, known as the DNA damage response (DDR. Following DNA damage, sensor molecules detect the presence of DNA damage and transmit signals to downstream transducer molecules. This in turn conveys the signals to numerous effectors, which initiate a large number of specific biological responses, including transient cell cycle arrest mediated by checkpoints, DNA repair, and apoptosis. It is recently becoming clear that dephosphorylation events are involved in keeping DDR factors inactive during normal cell growth. Moreover, dephosphorylation is required to shut off checkpoint arrest following DNA damage and has been implicated in the activation of the DDR. Spatial and temporal regulation of phosphorylation events is essential for the DDR, and fine-tuning of phosphorylation is partly mediated by protein phosphatases. While the role of kinases in the DDR has been well documented, the complex roles of protein dephosphorylation have only recently begun to be investigated. Therefore, it is important to focus on the role of phosphatases and to determine how their activity is regulated upon DNA damage. In this work, we summarize current knowledge on the involvement of serine/threonine phosphatases, especially the protein phosphatase 1, protein phosphatase 2A, and protein phosphatase Mg2+/Mn2+-dependent families, in the DDR.

  18. A study of the alkaline and acid phosphatase activities in acute uranium intoxication

    International Nuclear Information System (INIS)

    Bokova, N.; Pavlova, V.; Stancheva, Yu.; Khadzhirusev, S.; Kiradzhiev, G.

    1975-01-01

    Comparative study of the ability of the sodium salt of diethylbarbituric acid and acetazolamide to protect the kidneys is conducted under conditions of acute uranium intoxication in rats. The parameters studied are alkaline and acid phosphatase activities in the serum and urine and phosphatase activity in the kidneys (histochemically as described by Gomori) followed up until the 30th day after the total uranyl acetate dose was reached (2 or 7 mg per kg bodyweight). Either compound exerted only minor effect on serum alkaline phosphatase activity. Sodium diethylbarbiturate induced distinct fluctuations in urinary alkaline phosphatase activity throughout the entire study period, but the differences never reached statistical significance. Acetazolamide caused essential decrease in urinary alkaline phosphatase activity. In either case renal tissue protection from the action of the uranyl ion may be suggested. This assumption is supported by the histochemical analysis. The compounds appeared to have no effect on serum acid phosphatase activity which showed high variability both in control and in treated rats. (Ch.K.)

  19. The impact of phosphatases on proliferative and survival signaling in cancer.

    Science.gov (United States)

    Narla, Goutham; Sangodkar, Jaya; Ryder, Christopher B

    2018-05-03

    The dynamic and stringent coordination of kinase and phosphatase activity controls a myriad of physiologic processes. Aberrations that disrupt the balance of this interplay represent the basis of numerous diseases. For a variety of reasons, early work in this area portrayed kinases as the dominant actors in these signaling events with phosphatases playing a secondary role. In oncology, these efforts led to breakthroughs that have dramatically altered the course of certain diseases and directed vast resources toward the development of additional kinase-targeted therapies. Yet, more recent scientific efforts have demonstrated a prominent and sometimes driving role for phosphatases across numerous malignancies. This maturation of the phosphatase field has brought with it the promise of further therapeutic advances in the field of oncology. In this review, we discuss the role of phosphatases in the regulation of cellular proliferation and survival signaling using the examples of the MAPK and PI3K/AKT pathways, c-Myc and the apoptosis machinery. Emphasis is placed on instances where these signaling networks are perturbed by dysregulation of specific phosphatases to favor growth and persistence of human cancer.

  20. Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

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    Xi'en Chen

    Full Text Available Protein phosphatase 5 (PP5, a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa, 490 (55.82 kDa and 491 (56.07 kDa amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests.

  1. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    Science.gov (United States)

    Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

    2013-01-01

    Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

  2. Transcriptional profiling of putative human epithelial stem cells

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    Koçer Salih S

    2008-07-01

    Full Text Available Abstract Background Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells. Results Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells. Conclusion This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells

  3. Associations between renal hyperfiltration and serum alkaline phosphatase.

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    Se Won Oh

    Full Text Available Renal hyperfiltration, which is associated with renal injury, occurs in diabetic or obese individuals. Serum alkaline phosphatase (ALP level is also elevated in patients with diabetes (DM or metabolic syndrome (MS, and increased urinary excretion of ALP has been demonstrated in patients who have hyperfiltration and tubular damage. However, little was investigated about the association between hyperfiltration and serum ALP level. A retrospective observational study of the 21,308 adults in the Korea National Health and Nutrition Examination Survey IV-V databases (2008-2011 was performed. Renal hyperfiltration was defined as exceeding the age- and sex-specific 97.5th percentile. We divided participants into 4 groups according to their estimated glomerular filtration rate (eGFR: >120, 90-119, 60-89, and 120 mL/min/1.73 m2 showed the highest risk for MS, in the highest ALP quartiles (3.848, 95% CI, 1.876-7.892, compared to the lowest quartile. Similarly, the highest risk for DM, in the highest ALP quartiles, was observed in participants with eGFR >120 ml/min/1.73 m2 (2.166, 95% CI, 1.084-4.329. ALP quartiles were significantly associated with albuminuria in participants with eGFR ≥ 60 ml/min/1.73m2. The highest ALP quartile had a 1.631-fold risk elevation for albuminuria with adjustment of age and sex. (95% CI, 1.158-2.297, P = 0.005. After adjustment, the highest ALP quartile had a 1.624-fold risk elevation, for renal hyperfiltration (95% CI, 1.204-2.192, P = 0.002. In addition, hyperfiltration was significantly associated with hemoglobin, triglyceride, white blood cell count, DM, smoking, and alcohol consumption (P<0.05. The relationship between serum ALP and metabolic disorders is stronger in participants with an upper-normal range of eGFR. Higher ALP levels are significantly associated with renal hyperfiltration in Korean general population.

  4. Voltage-sensing phosphatase modulation by a C2 domain

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    Paul M. Castle

    2015-04-01

    Full Text Available The voltage-sensing phosphatase (VSP is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD, the inter-domain linker, the cytosolic catalytic domain and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5P2 (Kalli et al., 2014. Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  5. Voltage-sensing phosphatase modulation by a C2 domain.

    Science.gov (United States)

    Castle, Paul M; Zolman, Kevin D; Kohout, Susy C

    2015-01-01

    The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  6.  Alkaline phosphatase normalization is a biomarker of improved survival in primary sclerosing cholangitis.

    Science.gov (United States)

    Hilscher, Moira; Enders, Felicity B; Carey, Elizabeth J; Lindor, Keith D; Tabibian, James H

    2016-01-01

     Introduction. Recent studies suggest that serum alkaline phosphatase may represent a prognostic biomarker in patients with primary sclerosing cholangitis. However, this association remains poorly understood. Therefore, the aim of this study was to investigate the prognostic significance and clinical correlates of alkaline phosphatase normalization in primary sclerosing cholangitis. This was a retrospective cohort study of patients with a new diagnosis of primary sclerosing cholangitis made at an academic medical center. The primary endpoint was time to hepatobiliaryneoplasia, liver transplantation, or liver-related death. Secondary endpoints included occurrence of and time to alkaline phosphatase normalization. Patients who did and did not achieve normalization were compared with respect to clinical characteristics and endpoint-free survival, and the association between normalization and the primary endpoint was assessed with univariate and multivariate Cox proportional-hazards analyses. Eighty six patients were included in the study, with a total of 755 patient-years of follow-up. Thirty-eight patients (44%) experienced alkaline phosphatase normalization within 12 months of diagnosis. Alkaline phosphatase normalization was associated with longer primary endpoint-free survival (p = 0.0032) and decreased risk of requiring liver transplantation (p = 0.033). Persistent normalization was associated with even fewer adverse endpoints as well as longer survival. In multivariate analyses, alkaline phosphatase normalization (adjusted hazard ratio 0.21, p = 0.012) and baseline bilirubin (adjusted hazard ratio 4.87, p = 0.029) were the only significant predictors of primary endpoint-free survival. Alkaline phosphatase normalization, particularly if persistent, represents a robust biomarker of improved long-term survival and decreased risk of requiring liver transplantation in patients with primary sclerosing cholangitis.

  7. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    International Nuclear Information System (INIS)

    Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

    2012-01-01

    Highlights: ► AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. ► AtPP2CG1 up-regulates the expression of marker genes in different pathways. ► AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2–3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter–GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

  8. Rapid Discrimination Among Putative Mechanistic Models of Biochemical Systems.

    Science.gov (United States)

    Lomnitz, Jason G; Savageau, Michael A

    2016-08-31

    An overarching goal in molecular biology is to gain an understanding of the mechanistic basis underlying biochemical systems. Success is critical if we are to predict effectively the outcome of drug treatments and the development of abnormal phenotypes. However, data from most experimental studies is typically noisy and sparse. This allows multiple potential mechanisms to account for experimental observations, and often devising experiments to test each is not feasible. Here, we introduce a novel strategy that discriminates among putative models based on their repertoire of qualitatively distinct phenotypes, without relying on knowledge of specific values for rate constants and binding constants. As an illustration, we apply this strategy to two synthetic gene circuits exhibiting anomalous behaviors. Our results show that the conventional models, based on their well-characterized components, cannot account for the experimental observations. We examine a total of 40 alternative hypotheses and show that only 5 have the potential to reproduce the experimental data, and one can do so with biologically relevant parameter values.

  9. The inducible CAM plants in putative lunar lander experiments

    Science.gov (United States)

    Burlak, Olexii; Zaetz, Iryna; Soldatkin, Olexii; Rogutskyy, Ivan; Danilchenko, Boris; Mikheev, Olexander; de Vera, Jean-Pierre; Vidmachenko, Anatolii; Foing, Bernard H.; Kozyrovska, Natalia

    Precursory lunar lander experiments on growing plants in locker-based chambers will increase our understanding of effect of lunar conditions on plant physiology. The inducible CAM (Cras-sulacean Acid Metabolism)-plants are reasonable model for a study of relationships between environmental challenges and changes in plant/bacteria gene expression. In inducible CAM-plants the enzymatic machinery for the environmentally activated CAM switches on from a C3-to a full-CAM mode of photosynthesis in response to any stresses (Winter et al., 2008). In our study, Kalanchoe spp. are shown to be promising candidates for putative lunar experiments as resistant to irradiation and desiccation, especially after inoculation with a bacterial consortium (Boorlak et al., 2010). Within frames of the experiment we expect to get information about the functional activity of CAM-plants, in particular, its organogenesis, photosystem, the circadian regulation of plant metabolism on the base of data gaining with instrumental indications from expression of the reporter genes fused to any genes involved in vital functions of the plant (Kozyrovska et al., 2009). References 1. Winter K., Garcia M., Holtum J. (2008) J. Exp. Bot. 59(7):1829-1840 2. Bourlak O., Lar O., Rogutskyy I., Mikheev A., Zaets I., Chervatyuk N., de Vera J.-P., Danilchenko A.B. Foing B.H., zyrovska N. (2010) Space Sci. Technol. 3. Kozyrovska N.O., Vidmachenko A.P., Foing B.H. et al. Exploration/call/estec/ESA. 2009.

  10. Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

    International Nuclear Information System (INIS)

    Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

    1986-01-01

    In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with [ 14 C]-5-aminolevulinic acid [ 14 C]-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H 2 O 2 stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [ 14 C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed

  11. Alkaline phosphatase expression during relapse after orthodontic tooth movement

    Directory of Open Access Journals (Sweden)

    Pinandi Sri Pudyani

    2014-03-01

    Full Text Available Background: The increasing of osteoblast activities during bone formation will be accompanied with the increasing expression of alkaline phosphatase enzyme (ALP. ALP can be obtained from clear fluid excreted by gingival crevicular fluid (GCF. Bone turnover, especially bone formation process, can be monitored through the expression of ALP secreted by GCF during orthodontic treatment. Thus, retention period is an important period that can be monitored through the level of bone metabolism around teeth. Purpose: This research were aimed to determine the relation of distance change caused by tooth relapse and ALP activities in gingival crevicular fluid after orthodontic; and to determine ALP as a potential biomarker of bone formation during retention period. Methods: Lower incisors of 25 guinea pigs were moved 3 mm to the distally by using open coil spring. Those relapse distance were measured and the gingival crevicular fluid was taken by using paper points to evaluate ALP levels on days 0, 3, 7, 14 and 21 respectivelly by using a spectrophotometer (405 nm. t-test and ANOVA test were conducted to determine the difference of ALP activities among the time intervals. The correlation regression analysis was conducted to determine the relation of distance change caused by the relapse tooth movement and ALP activities. Results: The greatest relapse movement was occurred on day 3 after open coil spring was removed. There was significant difference of the average of distance decrease among groups A1-A5 (p<0.05. It was also known that ALP level was increased on day 3, but there was no significant difference of the average level of ALP among groups A1-A5 (p>0.05. Finally, based on the results of correlation analysis between the ALP level decreasing and the relapse distance on both right and left of mesial and distal sides, it is known that there was no relation between those two variables (p>0.05. Conclusion: It can be concluded that relapse after orthodontic

  12. Loss of a putative tumor suppressor locus after gamma-ray-induced neoplastic transformation of HeLa x Skin fibroblast human cell hybrids

    International Nuclear Information System (INIS)

    Mendonca, M.S.; Redpath, J.L.; Fasching, C.L.

    1995-01-01

    The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to γ radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several γ-ray-induced IAP-expression mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice. Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. 49 refs., 3 figs., 2 tabs

  13. Acid phosphatase turnover during repressed and derepressed cultivation of Aspergillus niger

    International Nuclear Information System (INIS)

    Komano, Teruya

    1975-01-01

    Enhancement of the activity of acid phosphatase (EC 3.1.3.2) by phosphate starvation in growing Aspergillus niger mycelia was prevented by cycloheximide. This indicates that the enhancement was due to de novo protein synthesis caused by derepression. Radioactive acid phosphatase extracted from mycelia labeled with 14 C-amino acid was separated into at least four fractions. Experiments on pulse labeling and the chasing of the four acid phosphatases revealed the synthesis and degradation of each fraction occurred at different rates; showing a different rate of turnover of the enzyme molecules. The results of similar experiments performed during culture in the presence of phosphate (partially repressed condition) suggested that the marked change in the activity ratios of the four acid phosphatases during cultivation was the result of the active turnover of enzyme molecules. In contrast, the slight changes in the ratios observed during derepressed cultivation seemed to be the result of similar of synthesis and degradation of each phosphatase fraction. (auth.)

  14. Elevated serum tartrate-resistant acid phosphatase isoform 5a levels in metabolic syndrome.

    Science.gov (United States)

    Huang, Yi-Jhih; Huang, Tsai-Wang; Chao, Tsu-Yi; Sun, Yu-Shan; Chen, Shyi-Jou; Chu, Der-Ming; Chen, Wei-Liang; Wu, Li-Wei

    2017-09-29

    Tartrate-resistant phosphatase isoform 5a is expressed in tumor-associated macrophages and is a biomarker of chronic inflammation. Herein, we correlated serum tartrate-resistant phosphatase isoform 5a levels with metabolic syndrome status and made comparisons with traditional markers of inflammation, including c-reactive protein and interleukin-6. One hundred healthy volunteers were randomly selected, and cut-off points for metabolic syndrome related inflammatory biomarkers were determined using receiver operating characteristic curves. Linear and logistic regression models were subsequently used to correlate inflammatory markers with the risk of metabolic syndrome. Twenty-two participants met the criteria for metabolic syndrome, and serum tartrate-resistant phosphatase isoform 5a levels of >5.8 μg/L were associated with metabolic syndrome (c-statistics, 0.730; p = 0.001; 95% confidence interval, 0.618-0.842). In addition, 1 μg/L increases in tartrate-resistant phosphatase isoform 5a levels were indicative of a 1.860 fold increase in the risk of metabolic syndrome (p = 0.012). Elevated serum tartrate-resistant phosphatase isoform 5a levels are associated with the risk of metabolic syndrome, with a cut-off level of 5.8 μg/L.

  15. Sensitive detection of alkaline phosphatase by switching on gold nanoclusters fluorescence quenched by pyridoxal phosphate.

    Science.gov (United States)

    Halawa, Mohamed Ibrahim; Gao, Wenyue; Saqib, Muhammad; Kitte, Shimeles Addisu; Wu, Fengxia; Xu, Guobao

    2017-09-15

    In this work, we designed highly sensitive and selective luminescent detection method for alkaline phosphatase using bovine serum albumin functionalized gold nanoclusters (BSA-AuNCs) as the nanosensor probe and pyridoxal phosphate as the substrate of alkaline phosphatase. We found that pyridoxal phosphate can quench the fluorescence of BSA-AuNCs and pyridoxal has little effect on the fluorescence of BSA-AuNCs. The proposed mechanism of fluorescence quenching by PLP was explored on the basis of data obtained from high-resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS), UV-vis spectrophotometry, fluorescence spectroscopy, fluorescence decay time measurements and circular dichroism (CD) spectroscopy. Alkaline phosphatase catalyzes the hydrolysis of pyridoxal phosphate to generate pyridoxal, restoring the fluorescence of BSA-AuNCs. Therefore, a recovery type approach has been developed for the sensitive detection of alkaline phosphatase in the range of 1.0-200.0U/L (R 2 =0.995) with a detection limit of 0.05U/L. The proposed sensor exhibit excellent selectivity among various enzymes, such as glucose oxidase, lysozyme, trypsin, papain, and pepsin. The present switch-on fluorescence sensing strategy for alkaline phosphatase was successfully applied in human serum plasma with good recoveries (100.60-104.46%), revealing that this nanosensor probe is a promising tool for ALP detection. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. [Spectroscopic analysis of the interaction of ethanol and acid phosphatase from wheat germ].

    Science.gov (United States)

    Xu, Dong-mei; Liu, Guang-shen; Wang, Li-ming; Liu, Wei-ping

    2004-11-01

    Conformational and activity changes of acid phosphatase from wheat germ in ethanol solutions of different concentrations were measured by fluorescence spectra and differential UV-absorption spectra. The effect of ethanol on kinetics of acid phosphatase was determined by using the double reciprocal plot. The results indicate the ethanol has a significant effect on the activity and conformation of acid phosphatase. The activity of acid phosphatase decreased linearly with increasing the concentration of ethanol. Differential UV-absorption spectra of the enzyme denatured in ethanol solutions showed two positive peaks at 213 and 234 nm, respectively. The peaks on the differential UV-absorption spectra suggested that the conformation of enzyme molecule changed from orderly structure to out-of-order crispation. The fluorescence emission peak intensity of the enzyme gradually strengthened with increasing ethanol concentration, which is in concordance with the conformational change of the microenvironments of tyrosine and tryptophan residues. The results indicate that the expression of the enzyme activity correlates with the stability and integrity of the enzyme conformation to a great degree. Ethanol is uncompetitive inhibitor of acid phosphatase.

  17. Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

    Science.gov (United States)

    Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

    2017-07-14

    The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Relationship of serum and saliva calcium, phosphorus and alkaline phosphatase with dry mouth feeling in menopause.

    Science.gov (United States)

    Agha-Hosseini, Farzaneh; Mirzaii-Dizgah, Iraj; Moosavi, Mahdieh-Sadat

    2012-06-01

    The aim of this study was to compare serum and saliva calcium, phosphorus and alkaline phosphatase of menopausal women with/without dry mouth (DM) feeling. The composition of saliva in menopause women with/without DM feeling is different. Some of these differences are in hormones that are related to bone turnover. A case-control study was carried out on 60 selected menopausal women aged 45-79 years with or without DM feeling (30 as case, 30 as control), conducted at the Clinic of Oral Medicine, Tehran University of Medical Sciences. The phosphorus concentration was measured by photometrical measurement of the blue colour formed after the addition of ammonium molybdate and stannous chloride; calcium was measured by Arsenazo reaction; and alkaline phosphatase by the pNPP-AMP method. Statistical analysis of Student's t-test was used. The mean serum phosphorus and alkaline phosphatase, stimulated and unstimulated saliva calcium and alkaline phosphatase levels were significantly higher in the menopausal women suffering from DM. There were no significant differences between groups regarding saliva phosphorus and serum calcium concentration. Calcium, phosphorus and alkaline phosphatase appear associated with DM feeling in menopause. © 2012 The Gerodontology Society and John Wiley & Sons A/S.

  19. Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

    Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

  20. Phosphate-solubility and phosphatase activity in Gangetic alluvial soil as influenced by organophosphate insecticide residues.

    Science.gov (United States)

    Majumder, Shyam Prasad; Das, Amal Chandra

    2016-04-01

    An experiment was conducted under laboratory conditions to investigate the effect of four organophosphate insecticides, viz. monocrotophos, profenophos, quinalphos and triazophos at their field application rates (0.75, 1.0, 0.5 and 0.6 kg a.i.ha(-1), respectively), on the growth and activities of phosphate solubilizing microorganisms in relation to availability of insoluble phosphates in the Gangetic alluvial soil of West Bengal, India. The proliferation of phosphate solubilizing microorganisms was highly induced with profenophos (38.3%), while monocrotophos exerted maximum stimulation (20.8%) towards the solubility of insoluble phosphates in soil. The phosphatase activities of the soil (both acid phosphatase and alkaline phosphatase) were significantly increased due to the incorporation of the insecticides in general, and the augmentation was more pronounced with quinalphos (43.1%) followed by profenophos (27.6%) for acid phosphatase, and with monocrotophos (25.2%) followed by profenophos (16.1%) for alkaline phosphatase activity in soil. The total phosphorus was highly retained by triazophos (19.9%) followed by monocrotophos (16.5%), while incorporation of triazophos and quinalphos manifested greater availability of water soluble phosphorus in soil. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

    Science.gov (United States)

    Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

    2015-01-01

    Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

  2. A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

    DEFF Research Database (Denmark)

    Johannesson, Martina; Semb, Tor Henrik; Serup, Palle

    2012-01-01

    . Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed. Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos...... to determine any occurrence of integration. Putative DE was analyzed by gene and protein expression prior to transplantation and 48 h post transplantation. Results: Putative DE, derived from mouse and human ESCs, was successfully integrated within the chick endoderm. Endoderm-specific genes were expressed...... result show that putative DE integrates with the chick endoderm and participate in the development of the chicken gut, indicating the generation of functional DE from ESCs. This functional assay can be used to assess the generation of functional DE derived from both human and mouse ESCs and provides...

  3. Evidence That Speciation of Oxovanadium Complexes Does Not Solely Account for Inhibition of Leishmania Acid Phosphatases

    Science.gov (United States)

    Dorsey, Benjamin M.; McLauchlan, Craig C.; Jones, Marjorie A.

    2018-01-01

    Leishmaniasis is an endemic disease affecting a diverse spectra of populations, with 1.6 million new cases reported each year. Current treatment options are costly and have harsh side effects. New therapeutic options that have been previously identified, but still underappreciated as potential pharmaceutical targets, are Leishmania secreted acid phosphatases (SAP). These acid phosphatases, which are reported to play a role in the survival of the parasite in the sand fly vector, and in homing to the host macrophage, are inhibited by orthovanadate and decavanadate. Here, we use L. tarentolae to further evaluate these inhibitors. Using enzyme assays, and UV-visible spectroscopy, we investigate which oxovanadium starting material (orthovanadate or decavanadate) is a better inhibitor of L. tarentolae secreted acid phosphatase activity in vitro at the same total moles of vanadium. Considering speciation and total vanadium concentration, decavanadate is a consistently better inhibitor of SAP in our conditions, especially at low substrate:inhibitor ratios. PMID:29707535

  4. Evidence that Speciation of Oxovanadium Complexes does not Solely Account for Inhibition of Leishmania Acid Phosphatases

    Science.gov (United States)

    Dorsey, Benjamin M.; McLauchlan, Craig C.; Jones, Marjorie A.

    2018-04-01

    Leishmaniasis is an endemic disease affecting a diverse spectra of populations, with 1.6 million new cases reported each year. Current treatment options are costly and have harsh side effects. New therapeutic options that have been previously identified, but still underappreciated as potential pharmaceutical targets, are Leishmania secreted acid phosphatases (SAP). These acid phosphatases, which are reported to play a role in the survival of the parasite in the sand fly vector, and in homing to the host macrophage, are inhibited by orthovanadate and decavanadate. Here, we use L. tarentolae to further evaluate these inhibitors. Using enzyme assays, and UV-visible spectroscopy, we investigate which oxovanadium starting material (orthovanadate or decavanadate) is a better inhibitor of L. tarentolae secreted acid phosphatase activity in vitro at the same total moles of vanadium. Considering speciation and total vanadium concentration, decavanadate is a consistently better inhibitor of SAP in our conditions, especially at low substrate:inhibitor ratios.

  5. The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

    Science.gov (United States)

    Srivastava, Pramod Kumar; Anand, Asha

    2015-01-01

    Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

  6. Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

    Science.gov (United States)

    Catarsi, S; Drapeau, P

    1997-08-01

    Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

  7. Osteomalacia with low alkaline phosphatase: a not so rare condition with important consequences.

    Science.gov (United States)

    Belkhouribchia, Jamal; Bravenboer, Bert; Meuwissen, Marije; Velkeniers, Brigitte

    2016-01-28

    Hypophosphatasia is a genetic disorder, characterised by a dysfunctional tissue-non-specific isoenzyme of alkaline phosphatase that impacts bone metabolism and predisposes to osteomalacia or rickets. The clinical presentation is very diverse, depending on the age of onset and the severity of the disease. Several forms of hypophosphatasia are recognised. We present a case of a 50-year-old woman with low impact fractures and loss of teeth at a young age. She also had a low alkaline phosphatase and was diagnosed with adult hypophosphatasia. Although the severe forms of hypophosphatasia are rather rare, the adult form is thought to occur quite frequently. As this condition is not well known by healthcare professionals, the time to diagnosis and initiation of adequate treatment is often postponed. When encountering a patient with low alkaline phosphatase, low bone density or a history of bone fractures, the possibility of hypophosphatasia should be considered. 2016 BMJ Publishing Group Ltd.

  8. Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli

    DEFF Research Database (Denmark)

    Brøndsted, Lone; Atlung, Tove

    1996-01-01

    The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene has been investigated during different environmental conditions using single copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase...... of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high expression of the operon was obtained in glucose medium supplemented with formate, where E.coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented...... in this paper indicate a clear difference in the regulation of the cyx-appA operon compared to the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function at even more oxygen limiting conditions than cytochrome d oxidase. The expression of the app...

  9. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    Energy Technology Data Exchange (ETDEWEB)

    Felts, Richard L. [Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States); Reilly, Thomas J. [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Calcutt, Michael J. [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Tanner, John J., E-mail: tannerjj@missouri.edu [Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States); Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States)

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  10. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    International Nuclear Information System (INIS)

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2005-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4 1 2 1 2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4 1 2 1 2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

  11. Functional diversity of voltage-sensing phosphatases in two urodele amphibians.

    Science.gov (United States)

    Mutua, Joshua; Jinno, Yuka; Sakata, Souhei; Okochi, Yoshifumi; Ueno, Shuichi; Tsutsui, Hidekazu; Kawai, Takafumi; Iwao, Yasuhiro; Okamura, Yasushi

    2014-07-16

    Voltage-sensing phosphatases (VSPs) share the molecular architecture of the voltage sensor domain (VSD) with voltage-gated ion channels and the phosphoinositide phosphatase region with the phosphatase and tensin homolog (PTEN), respectively. VSPs enzymatic activities are regulated by the motions of VSD upon depolarization. The physiological role of these proteins has remained elusive, and insights may be gained by investigating biological variations in different animal species. Urodele amphibians are vertebrates with potent activities of regeneration and also show diverse mechanisms of polyspermy prevention. We cloned cDNAs of VSPs from the testes of two urodeles; Hynobius nebulosus and Cynops pyrrhogaster, and compared their expression and voltage-dependent activation. Their molecular architecture is highly conserved in both Hynobius VSP (Hn-VSP) and Cynops VSP (Cp-VSP), including the positively-charged arginine residues in the S4 segment of the VSD and the enzymatic active site for substrate binding, yet the C-terminal C2 domain of Hn-VSP is significantly shorter than that of Cp-VSP and other VSP orthologs. RT-PCR analysis showed that gene expression pattern was distinct between two VSPs. The voltage sensor motions and voltage-dependent phosphatase activities were investigated electrophysiologically by expression in Xenopus oocytes. Both VSPs showed "sensing" currents, indicating that their voltage sensor domains are functional. The phosphatase activity of Cp-VSP was found to be voltage dependent, as shown by its ability to regulate the conductance of coexpressed GIRK2 channels, but Hn-VSP lacked such phosphatase activity due to the truncation of its C2 domain. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  12. 2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate

    International Nuclear Information System (INIS)

    Stankiewicz, P.J.; Gresser, M.J.; Tracey, A.S.; Hass, L.F.

    1987-01-01

    The binding of inorganic vanadate (V/sub i/) to rabbit muscle phosphoglycerate mutase (PGM), studied by using 51 V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating [V/sub i/]. The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 x 10 -6 M. The relevance of this result to other studies which have shown that the V/sub i/-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on [V/sub i/] with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 μM V/sub i/ has little effect on the phosphatase activity. However, in the presence of 25 μM V/sub i/, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed

  13. Subcellular localisation and properties of histone phosphate phosphatase in human polymorphonuclear leukocytes: alterations in pregnancy and chronic granulocytic leukaemia and relationship to alkaline phosphatase

    International Nuclear Information System (INIS)

    Smith, G.P.; Peters, T.J.

    1981-01-01

    Using [ 32 P]histone as substrate, an assay for histone phosphate phosphatase was optimised for human polymorphonuclear leukocytes. Kinetic studies showed that the activity was optimal at pH 6.8, was stimulated by Mn 2+ and Co 2+ , and inhibited by sodium sulphite and zinc chloride. The apparent Ksub(m) of the enzyme for histone phosphate was 0.89 μmol/l. (Auth.)

  14. [Effect of elevated atmospheric CO2 on soil urease and phosphatase activities].

    Science.gov (United States)

    Chen, Lijun; Wu, Zhijie; Huang, Guohong; Zhou, Likai

    2002-10-01

    The response of soil urease and phosphatase activities at different rice growth stages to free air CO2 enrichment (FACE) was studied. The results showed that comparing with the ambient atmospheric CO2 concentration (370 mumol.mol-1), FACE (570 mumol.mol-1) significantly increased the urease activity of 0-5 cm soil layer at the vigorous growth stage of rice, whole that of 5-10 cm layer had no significant change during the whole growing season. Phosphatase activity of 0-5 cm and 5-10 cm soil layers significantly increased, and the peak increment was at the vigorous growth stage of rice.

  15. Water Quality Interaction with Alkaline Phosphatase in the Ganga River: Implications for River Health.

    Science.gov (United States)

    Yadav, Amita; Pandey, Jitendra

    2017-07-01

    Carbon, nitrogen and phosphorus inputs through atmospheric deposition, surface runoff and point sources were measured in the Ganga River along a gradient of increasing human pressure. Productivity variables (chlorophyll a, gross primary productivity, biogenic silica and autotrophic index) and heterotrophy (respiration, substrate induced respiration, biological oxygen demand and fluorescein diacetate hydrolysis) showed positive relationships with these inputs. Alkaline phosphatase (AP), however, showed an opposite trend. Because AP is negatively influenced by available P, and eutrophy generates a feedback on P fertilization, the study implies that the alkaline phosphatase can be used as a high quality criterion for assessing river health.

  16. Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol.

    Science.gov (United States)

    Tomaszewski, M; Buchowicz, J

    1971-08-01

    The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.

  17. Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase.

    Science.gov (United States)

    Rosasco, Mario G; Gordon, Sharona E; Bajjalieh, Sandra M

    2015-12-15

    Voltage-sensitive phosphatases (VSPs) are proteins that directly couple changes in membrane electrical potential to inositol lipid phosphatase activity. VSPs thus couple two signaling pathways that are critical for cellular functioning. Although a number of nonmammalian VSPs have been characterized biophysically, mammalian VSPs are less well understood at both the physiological and biophysical levels. In this study, we aimed to address this gap in knowledge by determining whether the VSP from mouse, Mm-VSP, is expressed in the brain and contains a functional voltage-sensing domain (VSD) and a phosphatase domain. We report that Mm-VSP is expressed in neurons and is developmentally regulated. To address whether the functions of the VSD and phosphatase domain are retained in Mm-VSP, we took advantage of the modular nature of these domains and expressed each independently as a chimeric protein in a heterologous expression system. We found that the Mm-VSP VSD, fused to a viral potassium channel, was able to drive voltage-dependent gating of the channel pore. The Mm-VSP phosphatase domain, fused to the VSD of a nonmammalian VSP, was also functional: activation resulted in PI(4,5)P2 depletion that was sufficient to inhibit the PI(4,5)P2-regulated KCNQ2/3 channels. While testing the functionality of the VSD and phosphatase domain, we observed slight differences between the activities of Mm-VSP-based chimeras and those of nonmammalian VSPs. Although the properties of VSP chimeras may not completely reflect the properties of native VSPs, the differences we observed in voltage-sensing and phosphatase activity provide a starting point for future experiments to investigate the function of Mm-VSP and other mammalian VSPs. In conclusion, our data reveal that both the VSD and the lipid phosphatase domain of Mm-VSP are functional, indicating that Mm-VSP likely plays an important role in mouse neurophysiology. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All

  18. A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

    Directory of Open Access Journals (Sweden)

    Bruno L Bozaquel-Morais

    Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

  19. Putative cryomagma interaction with aerosols deposit at Titan's surface

    Science.gov (United States)

    Coll, Patrice; Navarro-Gonzalez, Rafael; Raulin, Francois; Coscia, David; Ramirez, Sandra I.; Buch, Arnaud; Szopa, Cyril; Poch, Olivier; Cabane, Michel; Brassé, Coralie

    The largest moon of Saturn, Titan, is known for its dense, nitrogen-rich atmosphere. The organic aerosols which are produced in Titan’s atmosphere are of great astrobiological interest, particularly because of their potential evolution when they reach the surface and may interact with putative ammonia-water cryomagma [1]. In this context we have followed the evolution of alkaline pH hydrolysis (25wt% ammonia-water) of Titan aerosol analogues, that have been qualified as representative of Titan’s aerosols [2]. Indeed the first results obtained by the ACP experiment onboard Huygens probe revealed that the main products obtained after thermolysis of Titan’s collected aerosols, were ammonia (NH3) and hydrogen cyanide (HCN). Then performing a direct comparison of the volatiles produced after a thermal treatment done in conditions similar to the ones used by the ACP experiment, we may estimate that the tholins we used are relevant to chemical analogues of Titan’s aerosols, and to note free of oxygen. Taking into account recent studies proposing that the subsurface ocean may contain a lower fraction of ammonia (about 5wt% or less [3]), and assuming the presence of specific gas species [4, 5], in particular CO2 and H2S, trapped in likely internal ocean, we determine a new probable composition of the cryomagma which could potentially interact with deposited Titan’s aerosols. We then carried out different hydrolyses, taking into account this composition, and we established the influence of the hydrolysis temperature on the organic molecules production. References: [1] Mitri et al., 2008. Resurfacing of Titan by ammonia-water cryomagma. Icarus. 196, 216-224. [2] Coll et al. 2013, Can laboratory tholins mimic the chemistry producing Titan's aerosols? A review in light of ACP experimental results, Planetary and Space Science 77, 91-103. [3] Tobie et al. 2012. Titan’s Bulk Composition Constrained by Cassini-Huygens: implication for internal outgassing. The

  20. In vitro silencing of Brugia malayi trehalose-6-phosphate phosphatase impairs embryogenesis and in vivo development of infective larvae in jirds.

    Directory of Open Access Journals (Sweden)

    Susheela Kushwaha

    Full Text Available The trehalose metabolic enzymes have been considered as potential targets for drug or vaccine in several organisms such as Mycobacterium, plant nematodes, insects and fungi due to crucial role of sugar trehalose in embryogenesis, glucose uptake and protection from stress. Trehalose-6-phosphate phosphatase (TPP is one of the enzymes of trehalose biosynthesis that has not been reported in mammals. Silencing of tpp gene in Caenorhabditis elegans revealed an indispensable functional role of TPP in nematodes.In the present study, functional role of B. malayi tpp gene was investigated by siRNA mediated silencing which further validated this enzyme to be a putative antifilarial drug target. The silencing of tpp gene in adult female B. malayi brought about severe phenotypic deformities in the intrauterine stages such as distortion and embryonic development arrest. The motility of the parasites was significantly reduced and the microfilarial production as well as their in vitro release from the female worms was also drastically abridged. A majority of the microfilariae released in to the culture medium were found dead. B. malayi infective larvae which underwent tpp gene silencing showed 84.9% reduced adult worm establishment after inoculation into the peritoneal cavity of naïve jirds.The present findings suggest that B. malayi TPP plays an important role in the female worm embryogenesis, infectivity of the larvae and parasite viability. TPP enzyme of B. malayi therefore has the potential to be exploited as an antifilarial drug target.

  1. Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

    Science.gov (United States)

    Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

    1993-06-01

    The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

  2. DMPD: DUSP meet immunology: dual specificity MAPK phosphatases in control of theinflammatory response. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17114416 DUSP meet immunology: dual specificity MAPK phosphatases in control of the...ml) (.csml) Show DUSP meet immunology: dual specificity MAPK phosphatases in control of theinflammatory resp...onse. PubmedID 17114416 Title DUSP meet immunology: dual specificity MAPK phospha

  3. Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases.

    Science.gov (United States)

    Hossain, Md Israil; Iwasaki, Hirohide; Okochi, Yoshifumi; Chahine, Mohamed; Higashijima, Shinichi; Nagayama, Kuniaki; Okamura, Yasushi

    2008-06-27

    The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

  4. Gamma-glutamyltransferase, aspartate aminotransferase and alkaline phosphatase as markers of alcohol consumption in out-patient alcoholics

    DEFF Research Database (Denmark)

    Gluud, C; Andersen, I; Dietrichson, O

    1981-01-01

    and alkaline phosphatase in 18% and 7%. Neither the activity of gamma-glutamyltransferase, aspartate aminotransferase nor alkaline phosphatase showed any significant (P greater than 0.05) correlation with the history of alcohol consumption. The activities of gamma-glutamyltransferase and aspartate...

  5. Identification of protein phosphatase involvement in the AT-receptor induced activation of endothelial nitric oxide synthase

    DEFF Research Database (Denmark)

    Peluso, A Augusto; Bertelsen, Jesper Bork; Andersen, Kenneth

    2018-01-01

    -antagonist), L-NAME (10µM; eNOS inhibitor), MK-2206 (100nM; Akt-inhibitor) sodium fluoride (1nM; serine/threonine-phosphatase inhibitor) or sodium orthovanadate (10nM; tyrosine-phosphatase inhibitor). NO release was estimated by quantifying DAF-FM fluorescence. The phosphorylation status of activating (e...

  6. Effects of sub-lethal dose of gamma-irradiation on levels of acid phosphatase in cerebellum of pigeons

    International Nuclear Information System (INIS)

    Shah, V.C.; Gadhia, P.K.

    1980-01-01

    The changes in the activities of acid phosphatase in the sham-irradiated and γ-irradiated cerebellum of pigeons have been studied both biochemically as well as histochemically after 400 rads. The specific activity of acid phosphatase decreased significantly after 48h and 72h of irradiation. The histochemical observations following total body irradiation confirmed the results obtained by quantitative biochemical studies. (author)

  7. Alkaline phosphatase immobilization onto Bio-Gide(R) and Bio-Oss(R) for periodontal and bone regeneration.

    NARCIS (Netherlands)

    Oortgiesen, D.A.W.; Plachokova, A.S.; Geenen, C.; Meijer, G.J.; Walboomers, X.F.; Beucken, J.J.J.P van den; Jansen, J.B.M.J.

    2012-01-01

    AIM: To evaluate the effect of alkaline phosphatase (ALP) immobilization onto Bio-Gide((R)) in vitro, and to study the in vivo performance of ALP-enriched Bio-Gide((R)) and/or Bio-Oss((R)) with the purpose to enhance periodontal regeneration. MATERIALS AND METHODS: Alkaline phosphatase ALP was

  8. HAB1–SWI3B Interaction Reveals a Link between Abscisic Acid Signaling and Putative SWI/SNF Chromatin-Remodeling Complexes in Arabidopsis[C][W

    Science.gov (United States)

    Saez, Angela; Rodrigues, Americo; Santiago, Julia; Rubio, Silvia; Rodriguez, Pedro L.

    2008-01-01

    Abscisic acid (ABA) has an important role for plant growth, development, and stress adaptation. HYPERSENSITIVE TO ABA1 (HAB1) is a protein phosphatase type 2C that plays a key role as a negative regulator of ABA signaling; however, the molecular details of HAB1 action in this process are not known. A two-hybrid screen revealed that SWI3B, an Arabidopsis thaliana homolog of the yeast SWI3 subunit of SWI/SNF chromatin-remodeling complexes, is a prevalent interacting partner of HAB1. The interaction mapped to the N-terminal half of SWI3B and required an intact protein phosphatase catalytic domain. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the interaction of HAB1 and SWI3B in the nucleus of plant cells. swi3b mutants showed a reduced sensitivity to ABA-mediated inhibition of seed germination and growth and reduced expression of the ABA-responsive genes RAB18 and RD29B. Chromatin immunoprecipitation experiments showed that the presence of HAB1 in the vicinity of RD29B and RAB18 promoters was abolished by ABA, which suggests a direct involvement of HAB1 in the regulation of ABA-induced transcription. Additionally, our results uncover SWI3B as a novel positive regulator of ABA signaling and suggest that HAB1 modulates ABA response through the regulation of a putative SWI/SNF chromatin-remodeling complex. PMID:19033529

  9. Purification and characterization of an alkaline phosphatase induced by phosphorus starvation in common bean (Phaseolus vulgaris L.) roots

    Energy Technology Data Exchange (ETDEWEB)

    Morales, L.; Gutierrez, N.; Maya, V.; Parra, C.; Martinez B, E.; Coello, P., E-mail: pcoello@servidor.unam.mx [UNAM, Facultad de Quimica, Departamento de Bioquimica, Ciudad Universitaria, 04510 Mexico D. F. (Mexico)

    2012-07-01

    Two phosphatase isoforms from roots of the common bean (Phaseolus vulgaris L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-Page and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of ph 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phospho enol-pyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyro phosphatase activity of this enzyme, the hydrolysis of pyro phosphatase increased substantially in the presence of Mg{sup 2+}.

  10. Purification and characterization of an alkaline phosphatase induced by phosphorus starvation in common bean (Phaseolus vulgaris L.) roots

    International Nuclear Information System (INIS)

    Morales, L.; Gutierrez, N.; Maya, V.; Parra, C.; Martinez B, E.; Coello, P.

    2012-01-01

    Two phosphatase isoforms from roots of the common bean (Phaseolus vulgaris L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-Page and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of ph 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phospho enol-pyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyro phosphatase activity of this enzyme, the hydrolysis of pyro phosphatase increased substantially in the presence of Mg 2+ .

  11. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a repre...

  12. Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

    International Nuclear Information System (INIS)

    Yang, S.D.; Fong, Y.L.

    1985-01-01

    Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32 P-labeled myelin basic protein (MBP) and [ 32 P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues

  13. The manometric determination of thiamine pyrophosphate and the inhibition of the acid yeast phosphatase

    NARCIS (Netherlands)

    Steyn-Parvé, Elizabeth P.

    1962-01-01

    Sodium molybdate is a powerful inhibitor of the acid yeast phosphatase in both fresh baker's yeast and dried brewer's yeast, provided that the yeast is suspended in a suitable buffer. It displays no action in citrate or phosphate buffers, but is active in acetate or maleate buffers, both at the

  14. Enzymatic mineralization of hydrogels for bone tissue engineering by incorporation of alkaline phosphatase.

    NARCIS (Netherlands)

    Douglas, T.E.L.; Messersmith, P.B.; Chasan, S.; Mikos, A.G.; Mulder, E.L.W. de; Dickson, G.; Schaubroeck, D.; Balcaen, L.; Vanhaecke, F.; Dubruel, P.; Jansen, J.A.; Leeuwenburgh, S.C.G.

    2012-01-01

    Alkaline phosphatase (ALP), an enzyme involved in mineralization of bone, is incorporated into three hydrogel biomaterials to induce their mineralization with calcium phosphate (CaP). These are collagen type I, a mussel-protein-inspired adhesive consisting of PEG substituted with catechol groups,

  15. Advances in lanthanide-based luminescent peptide probes for monitoring the activity of kinase and phosphatase.

    Science.gov (United States)

    Pazos, Elena; Vázquez, M Eugenio

    2014-02-01

    Signaling pathways based on protein phosphorylation and dephosphorylation play critical roles in the orchestration of complex biochemical events and form the core of most signaling pathways in cells (i.e. cell cycle regulation, cell motility, apoptosis, etc.). The understanding of these complex signaling networks is based largely on the biochemical study of their components, i.e. kinases and phosphatases. The development of luminescent sensors for monitoring kinase and phosphatase activity is therefore an active field of research. Examples in the literature usually rely on the modulation of the fluorescence emission of organic fluorophores. However, given the exceptional photophysical properties of lanthanide ions, there is an increased interest in their application as emissive species for monitoring kinase and phosphatase activity. This review summarizes the advances in the development of lanthanide-based luminescent peptide sensors as tools for the study of kinases and phosphatases and provides a critical description of current examples and synthetic approaches to understand these lanthanide-based luminescent peptide sensors. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

    Science.gov (United States)

    Witherow, D. Scott

    2016-01-01

    This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…

  17. Soil phosphatase and urease activities impacted by cropping systems and water management

    Science.gov (United States)

    Soil enzymes can play an important role in nutrient availability to plants. Consequently, soil enzyme measurements can provide useful information on soil fertility for crop production. We examined the impact of cropping system and water management on phosphatase, urease, and microbial biomass C in s...

  18. Investigations into the stabilisation of drugs by sugar glasses : I. Tablets prepared from stabilised alkaline phosphatase

    NARCIS (Netherlands)

    Eriksson, H.J.C.; Hinrichs, W.L.J.; van Veen, B.; Somsen, G.W.; de Jong, G.J.; Frijlink, H.W.

    2002-01-01

    The aim of this study was to investigate the formulation of sugar glass stabilised alkaline phosphatase from bovine intestine (BIAP) into tablets. Two major subjects of tablet formulation were investigated. First, the compaction behaviour of the inulin sugar glass was investigated. Secondly, the

  19. The effect of potassium iodide on the production of acid phosphatase by Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    P. S. Grover

    2003-06-01

    Full Text Available The present study was undertaken to find out the in vitro effect of potassium iodide (KI on the production of acid phosphatase by fully characterized strain of S.schenckii isolated from a patient of Cutaneous Sporotrichosis. The enzyme acid phosphatase was estimated during the 3 phases of growth of S.schenckii, without and with three concentrations of KI incorporated in the culture medium. In the control and in the test proper, with various concentrations of KI, no adverse effect of KI was observed on the production of acid phosphatase in early and mid log phase of fungal growth. Whereas in the exponential phase in test proper, there was a statistical significant decrease in the enzyme production with 0.8% and 3.2% of KI. The low activity at 0.8% and 3.2% KI indicates that KI has inhibitory effect on the growth of S.schenckii and has led to decrease in the activity of the enzyme. (Med J Indones 2003; 12: 65-8 Keywords: S.schenckii, acid phosphatase, potassium iodide

  20. The effects of drought stress on the activity of acid phosphatase and ...

    African Journals Online (AJOL)

    A model of drought was created on pigweed and the effects of drought stress on the activity of acid phosphatase and its protective enzymes were examined. The pot-cultured pigweeds were divided into 4 groups (ten plants per group) when they reached 6 leaves. (1) In the control group, the culture media contained 70 ...

  1. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    Science.gov (United States)

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Dragmacidins: new protein phosphatase inhibitors from a southern australian deep-water marine sponge, spongosorites sp

    Science.gov (United States)

    Capon; Rooney; Murray; Collins; Sim; Rostas; Butler; Carroll

    1998-05-01

    A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

  3. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

    Directory of Open Access Journals (Sweden)

    Alessandra Pasquo

    Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

  4. Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

    Science.gov (United States)

    Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

    2017-08-15

    The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Benomyl inhibits phosphorus transport but not fungal alkaline phosphatase activity in a Glomus-cucumber symbiosis

    DEFF Research Database (Denmark)

    Larsen, J.; Thingstrup, I.; Jakobsen, I.

    1996-01-01

    when benomyl was applied to the HC at 10 µg g-1 soil, whereas the uptake of 32P from RHC I roots + hyphae) was reduced only at the highest dose of application to the RHC (100 µ g g-1 soil). In contrast to the marked reduction of benomyl on fungal P transport, the activity of fungal alkaline phosphatase...

  6. Phosphatase-regulated recruitment of the spindle- and kinetochore-associated (Ska complex to kinetochores

    Directory of Open Access Journals (Sweden)

    Sushama Sivakumar

    2017-11-01

    Full Text Available Kinetochores move chromosomes on dynamic spindle microtubules and regulate signaling of the spindle checkpoint. The spindle- and kinetochore-associated (Ska complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, has been implicated in both roles. Phosphorylation of kinetochore components by the well-studied mitotic kinases Cdk1, Aurora B, Plk1, Mps1, and Bub1 regulate chromosome movement and checkpoint signaling. Roles for the opposing phosphatases are more poorly defined. Recently, we showed that the C terminus of Ska1 recruits protein phosphatase 1 (PP1 to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes that are also seen after depletion of Ska. Artificial tethering of PP1 to the outer kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and it reduces but does not fully rescue chromosome alignment and metaphase arrest defects seen after Ska depletion. We propose that Ska has multiple functions in promoting mitotic progression and that kinetochore-associated phosphatases function in a positive feedback cycle to reinforce Ska complex accumulation at kinetochores.

  7. Intercropping Acacia mangium stimulates AMF colonization and soil phosphatase activity in Eucalyptus grandis

    Directory of Open Access Journals (Sweden)

    Daniel Bini

    Full Text Available ABSTRACT: Arbuscular mycorrhizal fungi (AMF are very important to plant nutrition, mostly in terms of acquisition of P and micronutrients. While Acacia mangium is closely associated with AMF throughout the whole cycle, Eucalyptus grandis presents this symbiosis primarily at the seedling stage. The aim of this study was to evaluate the dynamics of AMF in these two tree species in both pure and mixed plantations during the first 20 months after planting. We evaluated the abundance, richness and diversity of AMF spores, the rate of AMF mycorrhizal root colonization, enzymatic activity and soil and litter C, N and P. There was an increase in AMF root colonization of E. grandis when intercropped with A. mangium as well as an increase in the activity of acid and alkaline phosphatase in the presence of leguminous trees. AMF colonization and phosphatase activities were both involved in improvements in P cycling and P nutrition in soil. In addition, P cycling was favored in the intercropped plantation, which showed negative correlation with litter C/N and C/P ratios and positive correlation with soil acid phosphatase activity and soil N and P concentrations. Intercropping A. mangium and E. grandis maximized AMF root colonization of E. grandis and phosphatase activity in the soil, both of which accelerate P cycling and forest performance.

  8. [Serum calcium and phosphorus concentration and alkaline phosphatase activity in healthy children during growth and development].

    Science.gov (United States)

    Savić, Ljiljana; Savić, Dejan

    2008-01-01

    Many changes happen during growth and development in an organism as a result of important hormon changes, especially biohumoral ones. These changes make a problem when interpreting biochemical results in pediatric population. The most important changes are intensive calcium and phosphorus metabolic turnover in bone tissue with changes in alkaline phosphatase activity as a result of osteoblast activity. The aim of this study was to follow the serum calcium and phosphorus concentration and alkaline phosphatase activity in children 1-15 years old in different growth and development period and of different sexes and to fortify the influence of growth and development dynamics on biohumoral status in healthy male and female children. We evaluated 117 healthy children of both sexes from 1-15 years of age and divided them into three age groups: 1-5, 6-10 and 11-15 years. We followed the serum calcium and phosphorus concentration and alkaline phosphatase activity in different groups and in different sexes. Our investigation found significantly higher values of serum calcium in boys than in girls with no important changes between the age groups and significantly higher values of serum phosphorus in the youngest age group in all children and in different sexes with no important sex differences. Alkaline phosphatase activity followed the growth spurt and was the biggest in 6-10 years group in girls and in 11-15 years group in boys.

  9. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains...

  10. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki

    2004-01-01

    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

  11. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    Science.gov (United States)

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  12. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...

  13. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

    DEFF Research Database (Denmark)

    Lund, I K; Hansen, J A; Andersen, H S

    2005-01-01

    Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators...

  14. Pterocarpans with inhibitory effects on protein tyrosine phosphatase 1B from Erythrina lysistemon Hutch

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Nguyen, Phi Hung; Thuong, Phuong Thien

    2009-01-01

    ',5':3,4]-2'',2''-dimethyldihydropyrano[6'',5'':9,10]pterocarpan (1), furano[5',4':3,4]-9-hydroxy-10-prenylpterocarpan (2), and 8-formyl-3,9-dihydroxy-4,10-diprenylpterocarpan (3), based on spectroscopic analyses. All the isolates, with the exception of 3, 6, and 11, strongly inhibited protein tyrosine phosphatase 1B (PTP1B) activity...

  15. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J

    1993-01-01

    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

  16. Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation

    DEFF Research Database (Denmark)

    Daum, G; Regenass, S; Sap, J

    1994-01-01

    Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G...

  17. Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

    NARCIS (Netherlands)

    van Noorden, C. J.; Jonges, G. N.

    1987-01-01

    The indoxyl-tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections

  18. Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis

    Science.gov (United States)

    Background and Aims Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparat...

  19. In vitro antiprogestational/antiglucocorticoid activity and progestin and glucocorticoid receptor binding of the putative metabolites and synthetic derivatives of CDB-2914, CDB-4124, and mifepristone.

    Science.gov (United States)

    Attardi, Barbara J; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R

    2004-03-01

    In determining the biological profiles of various antiprogestins, it is important to assess the hormonal and antihormonal activity, selectivity, and potency of their proximal metabolites. The early metabolism of mifepristone is characterized by rapid demethylation and hydroxylation. Similar initial metabolic pathways have been proposed for CDB-2914 (CDB: Contraceptive Development Branch of NICHD) and CDB-4124, and their putative metabolites have been synthesized. We have examined the functional activities and potencies, in various cell-based assays, and relative binding affinities (RBAs) for progesterone receptors (PR) and glucocorticoid receptors (GR) of the putative mono- and didemethylated metabolites of CDB-2914, CDB-4124, and mifepristone and of the 17alpha-hydroxy and aromatic A-ring derivatives of CDB-2914 and CDB-4124. The binding affinities of the monodemethylated metabolites for rabbit uterine PR and human PR-A and PR-B were similar to those of the parent compounds. Monodemethylated mifepristone bound to rabbit thymic GR with higher affinity than monodemethylated CDB-2914 or CDB-4124. T47D-CO cells were used to assess inhibition of R5020-stimulated endogenous alkaline phosphatase activity and transactivation of the PRE(2)-thymidine kinase (tk)-luciferase (LUC) reporter plasmid in transient transfections. The antiprogestational potency was as follows: mifepristone/CDB-2914/CDB-4124/monodemethylated metabolites (IC(50)'s approximately 10(-9)M) > aromatic A-ring derivatives (IC(50)'s approximately 10(-8)M) > didemethylated/17alpha-hydroxy derivatives (IC(50)'s approximately 10(-7)M). Antiglucocorticoid activity was determined by inhibition of dexamethasone-stimulated transcriptional activity in HepG2 cells. The mono- and didemethylated metabolites of CDB-2914 and CDB-4124 had less antiglucocorticoid activity (IC(50)'s approximately 10(-6)M) than monodemethylated mifepristone (IC(50) approximately 10(-8)M) or the other test compounds. At 10(-6)M in

  20. Somatic cell count and alkaline phosphatase activity in milk for evaluation of mastitis in buffalo

    Science.gov (United States)

    Patil, M. P.; Nagvekar, A. S.; Ingole, S. D.; Bharucha, S. V.; Palve, V. T.

    2015-01-01

    Background and Aim: Mastitis is a serious disease of dairy animals causing great economic losses due to a reduction in milk yield as well as lowering its nutritive value. The application of somatic cell count (SCC) and alkaline phosphatase activity in the milk for diagnosis of mastitis in buffalo is not well documented. Therefore, the present study was conducted to observe the SCC and alkaline phosphatase activity for evaluation of mastitis in buffalo. Materials and Methods: Milk samples of forty apparently healthy lactating buffaloes were selected and categorized into five different groups viz. normal buffaloes, buffaloes with subclinical mastitis with CMT positive milk samples (+1 Grade), (+2 Grade), (+3 Grade), and buffaloes with clinical mastitis with 8 animals in each group. The milk samples were analyzed for SCC and alkaline phosphatase activity. Results: The levels of SCC (×105 cells/ml) and alkaline phosphatase (U/L) in different groups were viz. normal (3.21±0.179, 16.48±1.432), subclinical mastitis with CMT positive milk samples with +1 Grade (4.21±0.138, 28.11±1.013), with +2 Grade (6.34±0.183, 34.50±1.034), with +3 Grade (7.96±0.213, 37.73±0.737) and buffaloes with clinical mastitis (10.21±0.220, 42.37±0.907) respectively, indicating an increasing trend in the values and the difference observed among various group was statistically significant. Conclusion: In conclusion, the results of the present study indicate that the concentration of milk SCC and alkaline phosphatase activity was higher in the milk of buffaloes with mastitis than in the milk of normal buffaloes. PMID:27047098

  1. The phosphatase inhibitor menadione (vitamin K3) protects cells from EGFR inhibition by erlotinib and cetuximab.

    Science.gov (United States)

    Perez-Soler, Roman; Zou, Yiyu; Li, Tianhong; Ling, Yi He

    2011-11-01

    Skin toxicity is the main side effect of epidermal growth factor receptor (EGFR) inhibitors, often leading to dose reduction or discontinuation. We hypothesized that phosphatase inhibition in the skin keratinocytes may prevent receptor dephosphorylation caused by EGFR inhibitors and be used as a new potential strategy for the prevention or treatment of this side effect. Menadione (Vitamin K3) was used as the prototype compound to test our hypothesis. HaCat human skin keratinocyte cells and A431 human squamous carcinoma cells were used. EGFR inhibition was measured by Western blotting and immunofluorescence. Phosphatase inhibition and reactive oxygen species (ROS) generation were measured by standard ELISA and fluorescence assays. Menadione caused significant and reversible EGFR activation in a dose-dependent manner starting at nontoxic concentrations. EGFR activation by menadione was associated with reversible protein tyrosine phosphatase inhibition, which seemed to be mediated by ROS generation as exposure to antioxidants prevented both menadione-induced ROS generation and phosphatase inhibition. Short-term coincubation of cells with nontoxic concentrations of menadione and the EGFR inhibitors erlotinib or cetuximab prevented EGFR dephosphorylation. Seventy-two-hour coincubation of cells with the highest nontoxic concentration of menadione and erlotinib provided for a fourfold cell growth inhibitory protection in HaCat human keratinocyte cells. Menadione at nontoxic concentrations causes EGFR activation and prevents EGFR dephosphorylation by erlotinib and cetuximab. This effect seems to be mediated by ROS generation and secondary phosphatase inhibition. Mild oxidative stress in skin keratinocytes by topical menadione may protect the skin from the toxicity secondary to EGFR inhibitors without causing cytotoxicity. ©2011 AACR

  2. Somatic cell count and alkaline phosphatase activity in milk for evaluation of mastitis in buffalo

    Directory of Open Access Journals (Sweden)

    M. P. Patil

    2015-03-01

    Full Text Available Background and Aim: Mastitis is a serious disease of dairy animals causing great economic losses due to a reduction in milk yield as well as lowering its nutritive value. The application of somatic cell count (SCC and alkaline phosphatase activity in the milk for diagnosis of mastitis in buffalo is not well documented. Therefore, the present study was conducted to observe the SCC and alkaline phosphatase activity for evaluation of mastitis in buffalo. Materials and Methods: Milk samples of forty apparently healthy lactating buffaloes were selected and categorized into five different groups viz. normal buffaloes, buffaloes with subclinical mastitis with CMT positive milk samples (+1 Grade, (+2 Grade, (+3 Grade, and buffaloes with clinical mastitis with 8 animals in each group. The milk samples were analyzed for SCC and alkaline phosphatase activity. Results: The levels of SCC (×105 cells/ml and alkaline phosphatase (U/L in different groups were viz. normal (3.21±0.179, 16.48±1.432, subclinical mastitis with CMT positive milk samples with +1 Grade (4.21±0.138, 28.11±1.013, with +2 Grade (6.34±0.183, 34.50±1.034, with +3 Grade (7.96±0.213, 37.73±0.737 and buffaloes with clinical mastitis (10.21±0.220, 42.37±0.907 respectively, indicating an increasing trend in the values and the difference observed among various group was statistically significant. Conclusion: In conclusion, the results of the present study indicate that the concentration of milk SCC and alkaline phosphatase activity was higher in the milk of buffaloes with mastitis than in the milk of normal buffaloes.

  3. Phosphatase activity in sandy soil influenced by mycorrhizal and non-mycorrhizal cover crops

    Directory of Open Access Journals (Sweden)

    Alceu Kunze

    2011-06-01

    Full Text Available Cover crops may difffer in the way they affect rhizosphere microbiota nutrient dynamics. The purpose of this study was to evaluate the effect of mycorrhizal and non-mycorrhizal cover crops on soil phosphatase activity and its persistence in subsequent crops. A three-year experiment was carried out with a Typic Quartzipsamment. Treatments were winter species, either mycorrhizal black oat (Avena strigosa Schreb or the non-mycorrhizal species oilseed radish (Raphanus sativus L. var. oleiferus Metzg and corn spurry (Spergula arvensis L.. The control treatment consisted of resident vegetation (fallow in the winter season. In the summer, a mixture of pearl millet (Pennisetum americanum L. with sunnhemp (Crotalaria juncea L. or with soybean (Glycine max L. was sown in all plots. Soil cores (0-10 cm and root samples were collected in six growing seasons (winter and summer of each year. Microbial biomass P was determined by the fumigation-extraction method and phosphatase activity using p-nitrophenyl-phosphate as enzyme substrate. During the flowering stage of the winter cover crops, acid phosphatase activity was 30-35 % higher in soils with the non-mycorrhizal species oilseed radish, than in the control plots, regardless of the amount of P immobilized in microbial biomass. The values of enzyme activity were intermediate in the plots with corn spurry and black oat. Alkaline phosphatase activity was 10-fold lower and less sensitive to the treatments, despite the significant relationship between the two phosphatase activities. The effect of plant species on the soil enzyme profile continued in the subsequent periods, during the growth of mycorrhizal summer crops, after completion of the life cycle of the cover crops.

  4. Purification and characterization of a polyisoprenyl phosphate phosphatase from pig brain. Possible dual specificity.

    Science.gov (United States)

    Frank, D W; Waechter, C J

    1998-05-08

    Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-P) (Sumbilla, C. A., and Waechter, C. J. (1985) Methods Enzymol. 111, 471-482). The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P-40 and purified approximately 1,107-fold by a combination of anion exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2+ > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate Km for C95-Dol-P of 45 microM; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoyl-phosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F-, phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg2+-independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid

  5. Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

    Directory of Open Access Journals (Sweden)

    Andaleeb Sajid

    2011-03-01

    Full Text Available The integrated functions of 11 Ser/Thr protein kinases (STPKs and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP is regulated through phosphorylation by STPKs.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+-ions and inorganic phosphate (Pi. PstP is differentially phosphorylated on the cytosolic domain with Thr(137, Thr(141, Thr(174 and Thr(290 being the target residues of PknB while Thr(137 and Thr(174 are phosphorylated by PknA. The Mn(2+-ion binding residues Asp(38 and Asp(229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c (D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

  6. The function of Shp2 tyrosine phosphatase in the dispersal of acetylcholine receptor clusters

    Directory of Open Access Journals (Sweden)

    Madhavan Raghavan

    2008-07-01

    Full Text Available Abstract Background A crucial event in the development of the vertebrate neuromuscular junction (NMJ is the postsynaptic enrichment of muscle acetylcholine (ACh receptors (AChRs. This process involves two distinct steps: the local clustering of AChRs at synapses, which depends on the activation of the muscle-specific receptor tyrosine kinase MuSK by neural agrin, and the global dispersal of aneural or "pre-patterned" AChR aggregates, which is triggered by ACh or by synaptogenic stimuli. We and others have previously shown that tyrosine phosphatases, such as the SH2 domain-containing phosphatase Shp2, regulate AChR cluster formation in muscle cells, and that tyrosine phosphatases also mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli, although the specific phosphatases involved in this latter step remain unknown. Results Using an assay system that allows AChR cluster assembly and disassembly to be studied separately and quantitatively, we describe a previously unrecognized role of the tyrosine phosphatase Shp2 in AChR cluster disassembly. Shp2 was robustly expressed in embryonic Xenopus muscle in vivo and in cultured myotomal muscle cells, and treatment of the muscle cultures with an inhibitor of Shp2 (NSC-87877 blocked the dispersal of pre-patterned AChR clusters by synaptogenic stimuli. In contrast, over-expression in muscle cells of either wild-type or constitutively active Shp2 accelerated cluster dispersal. Significantly, forced expression in muscle of the Shp2-activator SIRPα1 (signal regulatory protein α1 also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPα1 mutant that suppresses Shp2 signaling inhibited cluster disassembly. Conclusion Our results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPα1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.

  7. Molecular cloning and characterization of a putative OGG_N domain ...

    African Journals Online (AJOL)

    Molecular cloning and characterization of a putative OGG_N domain from the camel, Camelus dromedarius. Farid Shokry Ataya, Mohammad Saud Alanazi, Dalia Fouad, Hehsam Mahmoud Saeed, Mohammad Bazzi ...

  8. Human 2'-phosphodiesterase localizes to the mitochondrial matrix with a putative function in mitochondrial RNA turnover

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave; Andersen, Kasper Røjkjær; Kjær, Karina Hansen

    2011-01-01

    . Interestingly, 2′-PDE shares both functionally and structurally characteristics with the CCR4-type exonuclease–endonuclease–phosphatase family of deadenylases. Here we show that 2′-PDE locates to the mitochondrial matrix of human cells, and comprise an active 3′–5′ exoribonuclease exhibiting a preference...

  9. Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

  10. [Kinetic study on inhibition effects of dansyl-L-phenylalanine and L-phenylalanine on calf intestinal alkaline phosphatase].

    Science.gov (United States)

    Li, Li-Na; Wu, Yu-Qing; Buchet, René

    2009-10-01

    To evaluate the inhibition effect of dansyl-L-phenylalanine on calf intestinal alkaline phosphatase (CIAP), UV-Vis spectrophotometric method was employed. It was found that dansyl-L-phenylalanine can selectively inhibit CIAP. The kinetic inhibition processes of dansyl-L-phenylalanine and L-phenylalanine were comparatively studied. The authors' finding elucidates that at the optimized alkaline pH of alkaline phosphatase (pH 10.4) and 37 degrees C, dansyl-L-phenylalanine can inhibit alkaline phosphatase activity of CIAP efficiently and specifically, similar as L-phenylalanine. Both inhibition types were uncompetitive inhibition resulting from the double reciprocal curve fitting of upsilon versus substrate concentrations, and the inhibition constants Ki of both inhibitors were determined to be 2.3 and 1.1 mmol L(-1) respectively, both of which were at millimolar level. The investigation of the inhibition effect of dansyl modified L-phenylalanine on calf intestinal alkaline phosphatase not only helped get insight into the detailed inhibition mechanism of L-phenylalanine on tissue specific alkaline phosphatase, such as in the case of intestinal alkaline phosphatase, but also provided the possibility to employ fluorescence spectroscopy by labeling the specific inhibitors of alkaline phosphatase with chromophoric groups.

  11. Structural and biochemical analysis of a unique phosphatase from Bdellovibrio bacteriovorus reveals its structural and functional relationship with the protein tyrosine phosphatase class of phytase.

    Directory of Open Access Journals (Sweden)

    Robert J Gruninger

    Full Text Available Bdellovibrio bacteriovorus is an unusual δ-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

  12. Beryllium and growth. III. The effect of beryllium on plant phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Hoagland, M B

    1952-01-01

    The purpose of the investigations was to correlate the apparent ability of beryllium to substitute for magnesium in plant growth with a specific biochemical effect of the metal. Through association with earlier work on beryllium inhibition of animal alkaline phosphatase, a study was made of the effect of beryllium and other metals upon the activity of a phosphatase derived from tomato leaves. Although only indirect evidence is available that this enzyme system was magnesium-activated, beryllium was found to inhibit reversibly the splitting of GP and ATP. Other metals were also found to be inhibitory but the ATP-ase inhibition - and especially the ratio of P split from GP to P split from ATP - was higher for beryllium than for any other metal studied. The significance of this finding in relation to energy metabolism, growth, and beryllium toxicity is discussed. 12 references, 5 figures, 2 tables.

  13. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Science.gov (United States)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2013-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites. PMID:22116026

  14. Low molecular weight protein tyrosine phosphatases control antibiotic production in Streptomyces coelicolor A3(2)

    DEFF Research Database (Denmark)

    Sohoni, Sujata Vijay; Lieder, Sarah; Bapat, Prashant Madhusudhan

    2014-01-01

    3700 was established usingpara-nitrophenyl phosphate and the tyrosine-phosphorylated protein PtkA from Bacillus subtilis as substrates. Theoptimum pH for the Sco3700 phosphatase activity was 6.8, and KM for pNPP was 14.3 mM compared to pH 6.0and KM0.75 mM for PtpA. The potential of Sco3700...... of ACT in the ptpA over expression strain. Furthermore, a significantly earlier onset of ACT productionwas observed when ptpA was over expressed. Sco3700 overexpression had a pleiotropic effect on the cell, and thestrain exhibited lower productivities and final concentrations of antibiotics. We conclude...... that Sco3700 is indeed atyrosine phosphatase, and it contributes to regulation of antibiotic production in S. coelicolor affecting the timing ofonset of the antibiotic production...

  15. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    International Nuclear Information System (INIS)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2012-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  16. Development of conductometric biosensors based on alkaline phosphatases for the water quality control

    Science.gov (United States)

    Berezhetskyy, A.

    2008-09-01

    Researches are focused on the elaboration of enzymatic microconductometric device for heavy metal ions detection in water solutions. The manuscript includes a general introduction, the first chapter contains bibliographic review, the second chapter described the fundamentals of conductometric transducers, the third chapter examining the possibility to create and to optimize conductometric biosensor based on bovine alkaline phosphatase for heavy metals ions detection, the fourth chapter devoted to creation and optimization of conductometric biosensor based on alkaline phosphatase active microalgae and sol gel technology, the last chapter described application of the proposed algal biosensor for measurements of heavy metal ions toxicity of waste water, general conclusions stating the progresses achieved in the field of environmental monitoring

  17. Study on alkaline and acid phosphatase activity in acute uranium intoxication

    International Nuclear Information System (INIS)

    Bokova, N.V.; Pavlova, V.B.; Stancheva, Yu.A.; Khadzhirusev, S.B.; Kiradzhiev, G.D.

    1975-01-01

    The protective potential of diethyl barbituric acid sodium salt is studied, in comparison with that of acetazolamide, on kidneys under acute uranium intoxication. Experiments involved rats given intraperitoneal injections with uranyl acetate on 12 successive days up to a total dose of 0.5, 2.0 or 7.0 mg/kg. The resulting effects are measured by chemical assays of serum and urine for alkaline and acid phosphatase and histochemical assays for phosphatase activities in kidneys, kinetics being followed over a 30-day period after total dose administration. Protection of kidneys from toxic uranium effects was found to be of about the same degree with sodium diethyl barbiturate as with acetazolamide. (A.B.)

  18. Significance of bone specific alkaline phosphatase as a tumor marker in malignant bone tumor

    International Nuclear Information System (INIS)

    Kim, Sug Jun; Jeon, Dae Geun; Huh, Kwang

    1998-01-01

    The relationship between total alkaline phosphatase activity and bone forming lesion is a well known fact. But alkaline phosphatase consist mainly of two portion (liver, bone). To clarify the exact activity of bone forming tissue, quantitative measurement of BALP is essential. Two finds of tests were performed for their feasibility as a laboratory test (wheat germ lectin vs electrophoresis). We analyzed 40 bony lesion and got 58 samples. Lectin method was simple, economic, with reliable resproducability. Owing to the small number of test sample, we could not identify the relationship between the disease activity and measured BALP level. Further collection of clinical sample and analysis the pattern of BALP on each clinical settings. (author). 8 refs

  19. Phosphatase activity in Antarctica soil samples as a biosignature of extant life

    Science.gov (United States)

    Sato, Shuji; Itoh, Yuki; Takano, Yoshinori; Fukui, Manabu; Kaneko, Takeo; Kobayashi, Kensei

    Microbial activities have been detected in such extreme terrestrial environments as deep lithosphere, a submarine hydrothermal systems, stratosphere, and Antarctica. Microorganisms have adapted to such harsh environments by evolving their biomolecules. Some of these biomolecules such as enzymes might have different characteristics from those of organisms in ordinary environments. Many biosignatures (or biomarkers) have been proposed to detect microbial activities in such extreme environments. A number of techniques are proposed to evaluate biological activities in extreme environments including cultivation methods, assay of metabolism, and analysis of bioorganic compounds like amino acids and DNA. Enzyme activities are useful signature of extant life in extreme environments. Among many enzymes, phosphatase could be a good indicator of biological activities, since phosphate esters are essential for all the living terrestrial organisms. In addition, alkaline phosphatase is known as a typical zinc-containing metalloenzyme and quite stable in environments. We analyzed phosphatase activities in Antarctica soil samples to see whether they can be used as biosignatures for extant life. In addition, we characterized phosphatases extracted from the Antarctica soil samples, and compared with those obtained from other types of environments. Antarctica surface environments are quite severe environments for life since it is extremely cold and dry and exposed to strong UV and cosmic rays. We tried to evaluate biological activities in Antarctica by measuring phosphatase activities. Surface soil samples are obtained at the Sites 1-8 near Showa Base in Antarctica during the 47th Japan Antarctic exploration mission in 2005-6. Activities of acid phosphatase (ACP) and alkaline phosphatase (ALP) are measured spectrophotometrically after mixing the powdered sample and p-nitrophenyl phosphate solution (pH 6.5 for ACP, pH 8.0 for ALP). ALP was characterized after extraction from soils with

  20. Assessment of corticosteroid-induced alkaline phosphatase as a prognostic indicator in canine lymphoma.

    Science.gov (United States)

    Wiedemann, A L; Charney, S C; Barger, A M; Schaeffer, D J; Kitchell, B E

    2005-04-01

    To examine the incidence of elevated corticosteroid-induced alkaline phosphatase (sALP) in dogs with lymphoma and to determine if sALP is a reliable prognostic indicator in canine lymphoma. The medical records of 62 canine lymphoma patients treated with a combination chemotherapy protocol from 1994 to 2003 at the University of Illinois Veterinary Teaching Hospital were examined. Variables assessed with respect to response rate and remission duration included age, bodyweight, sex, breed, World Health Organization stage (I to V), substage (a or b), pretreatment administration of corticosteroid, and serum levels of alkaline phosphatase, sALP and alanine aminotransferase. sALP was not statistically significant with respect to response rate or duration of remission, nor was preinduction glucocorticoid administration. Stage was significant with respect to achieving remission. It was found that sALP is not a useful prognostic indicator for response rate and remission duration in dogs with lymphoma.

  1. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  2. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  3. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin, E-mail: fangfei6073@126.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn [Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150040 (China); Cai, Hua, E-mail: small-big@sohu.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Ji, Wei, E-mail: iwei_j@hotmail.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Luo, Xiao, E-mail: luoxiao2010@yahoo.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Li, Jing, E-mail: lijing@neau.edu.cn [Plant Secondary Metabolism Laboratory, Northeast Agricultural University, Harbin 150030 (China); Bai, Xi, E-mail: baixi@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

  4. Effect of Exogenous Phytase Addition on Soil Phosphatase Activities: a Fluorescence Spectroscopy Study.

    Science.gov (United States)

    Yang, Xiao-zhu; Chen, Zhen-hua; Zhang, Yu-lan; Chen, Li-jun

    2015-05-01

    The utilization of organic phosphorus (P) has directly or indirectly improved after exogenous phytase was added to soil. However, the mechanism by which exogenous phytase affected the soil phosphatases (phosphomonoesterase and phosphodiesterase) activities was not clear. The present work was aimed to study red soil, brown soil and cinnamon soil phosphomonoesterase (acid and alkaline) (AcP and AlP) and phosphodiesterase (PD) activities responding to the addition of exogenous phytase (1 g phytase/50 g air dry soil sample) based on the measurements performed via a fluorescence detection method combined with 96 microplates using a TECAN Infinite 200 Multi-Mode Microplate Reader. The results indicated that the acid phosphomonoesterase activity was significantly enhanced in red soil (p≤0. 01), while it was significantly reduced in cinnamon soil; alkaline phosphomonoesterase activity was significantly enhanced in cinnamon soil (p≤ 0. 01), while it was significantly reduced in red soil; phosphodiesterase activity was increased in three soils but it was significantly increased in brown soil (p≤0. 01) after the addition of exogenous phytase. The activities still remained strong after eight days in different soils, which indicated that exogenous phytase addition could be enhance soil phosphatases activities effectively. This effect was not only related to soil properties, such as pH and phosphorus forms, but might also be related to the excreted enzyme amount of the stimulating microorganism. Using fluorescence spectroscopy to study exogenous phytase addition influence on soil phosphatase activities was the first time at home and abroad. Compared with the conventional spectrophotometric method, the fluorescence microplate method is an accurate, fast and simple to use method to determine the relationships among the soil phosphatases activities.

  5. Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

    Science.gov (United States)

    Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas D Y; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan W M; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

    2015-02-17

    A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. © 2014 American Heart Association, Inc.

  6. Extracellular phosphatases in a Mediterranean reservoir: seasonal, spatial and kinetic heterogeneity

    Czech Academy of Sciences Publication Activity Database

    Nedoma, Jiří; García, J.C.; Comerma, M.; Šimek, Karel; Armengol, J.

    2006-01-01

    Roč. 51, č. 7 (2006), s. 1264-1276 ISSN 0046-5070 R&D Projects: GA ČR(CZ) GA206/99/0028 Grant - others:SICST(ES) HID96-1374-CO2; ICST(ES) 1997SGR-122 Institutional research plan: CEZ:AV0Z60170517 Keywords : alkaline phosphatase * eutrophication * P limitation Subject RIV: DJ - Water Pollution ; Quality Impact factor: 2.502, year: 2006

  7. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

    Directory of Open Access Journals (Sweden)

    Guillaume Manat

    Full Text Available Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2 catalyze the dephosphorylation of C55-PP molecules on the same (outer side of the plasma membrane.

  8. Dimerization of the Glucan Phosphatase Laforin Requires the Participation of Cysteine 329

    Science.gov (United States)

    Sánchez-Martín, Pablo; Raththagala, Madushi; Bridges, Travis M.; Husodo, Satrio; Gentry, Matthew S.; Sanz, Pascual; Romá-Mateo, Carlos

    2013-01-01

    Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin’s oligomerization. PMID:23922729

  9. Genetic interaction network of the Saccharomyces cerevisiae type 1 phosphatase Glc7

    Directory of Open Access Journals (Sweden)

    Neszt Michael

    2008-07-01

    Full Text Available Abstract Background Protein kinases and phosphatases regulate protein phosphorylation, a critical means of modulating protein function, stability and localization. The identification of functional networks for protein phosphatases has been slow due to their redundant nature and the lack of large-scale analyses. We hypothesized that a genome-scale analysis of genetic interactions using the Synthetic Genetic Array could reveal protein phosphatase functional networks. We apply this approach to the conserved type 1 protein phosphatase Glc7, which regulates numerous cellular processes in budding yeast. Results We created a novel glc7 catalytic mutant (glc7-E101Q. Phenotypic analysis indicates that this novel allele exhibits slow growth and defects in glucose metabolism but normal cell cycle progression and chromosome segregation. This suggests that glc7-E101Q is a hypomorphic glc7 mutant. Synthetic Genetic Array analysis of glc7-E101Q revealed a broad network of 245 synthetic sick/lethal interactions reflecting that many processes are required when Glc7 function is compromised such as histone modification, chromosome segregation and cytokinesis, nutrient sensing and DNA damage. In addition, mitochondrial activity and inheritance and lipid metabolism were identified as new processes involved in buffering Glc7 function. An interaction network among 95 genes genetically interacting with GLC7 was constructed by integration of genetic and physical interaction data. The obtained network has a modular architecture, and the interconnection among the modules reflects the cooperation of the processes buffering Glc7 function. Conclusion We found 245 genes required for the normal growth of the glc7-E101Q mutant. Functional grouping of these genes and analysis of their physical and genetic interaction patterns bring new information on Glc7-regulated processes.

  10. and alanine (EC. 2.6.1.2) transaminases, and alkaline phosphatase

    African Journals Online (AJOL)

    The activities of aspartate (E.C. 2.6.1.1) and alanine (E.C. 2.6.1.2) transaminases (AST and ALT, respectively), and alkaline phosphatase (ALP) (E.C. 3.1.3.1) were determined in erythrocytes obtained from 20 HbAA, 15 HbAS and 12 HbSS human subjects. The results showed that the three enzymes had different levels of ...

  11. Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

    Directory of Open Access Journals (Sweden)

    Lalima L Madan

    Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

  12. Phosphorylation-mediated regulation of the Staphylococcus aureus secreted tyrosine phosphatase PtpA.

    Science.gov (United States)

    Brelle, Solène; Baronian, Grégory; Huc-Brandt, Sylvaine; Zaki, Laila Gannoun; Cohen-Gonsaud, Martin; Bischoff, Markus; Molle, Virginie

    2016-01-15

    Due to the emergence of methicillin-resistant strains, Staphylococcus aureus has become as major public-health threat. Studies aimed at deciphering the molecular mechanism of virulence are thus required to identify new targets and develop efficient therapeutic agents. Protein phosphorylations are known to play key regulatory functions and their roles in pathogenesis are under intense scrutiny. Here we analyzed the protein tyrosine phosphatase PtpA of S. aureus, a member of the family of low molecular weight protein tyrosine phosphatases that are often secreted by pathogenic bacteria. We report for the first time that PtpA is phosphorylated in vitro by the S. aureus tyrosine kinase CapA1B2. A mass spectrometry approach allowed determining that Tyr122 and Tyr123 were the only two residues phosphorylated by this kinase. This result was confirmed by analysis of a double PtpA_Y122A/Y123A mutant that showed no phosphorylation by CapA1B2. Interestingly, PtpA phosphatase activity was abrogated in this mutant, suggesting a key regulatory function for these two tyrosine residues. This was further reinforced by the observation that CapA1B2-mediated phosphorylation significantly increased PtpA phosphatase activity. Moreover, we provide evidence that PtpA is secreted during growth of S. aureus. Together our results suggest that PtpA is an exported S. aureus signaling molecule controlled by tyrosine phosphorylation which may interfere with host cell signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    Science.gov (United States)

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  14. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Science.gov (United States)

    Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

    2012-02-03

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

  15. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    Energy Technology Data Exchange (ETDEWEB)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Iuliano, Rodolfo [Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, 88100 Catanzaro (Italy); Fusco, Alfredo [Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Instituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples (Italy); NOGEC (Naples Oncogenomocs Center)-CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples (Italy); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  16. YbiV from E. coli K12 is a HAD phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Anne; Lee, Seok-Yong; McCullagh, Emma; Silversmith, Ruth E.; Wemmer, David E.

    2004-03-16

    The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized ybiV as a HAD phosphatase. The crystal structure of YbiV reveals a two domain protein, one with the characteristic HAD hydrolase fold, the other an inserted a/b fold. In an effort to understand the mechanism we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3) which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate ybiV may be a sugar phosphatase, which is supported by biochemical assays which measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.

  17. Inhibition of AcpA phosphatase activity with ascorbate attenuates Francisella tularensis intramacrophage survival.

    Science.gov (United States)

    McRae, Steven; Pagliai, Fernando A; Mohapatra, Nrusingh P; Gener, Alejandro; Mahmou, Asma Sayed Abdelgeliel; Gunn, John S; Lorca, Graciela L; Gonzalez, Claudio F

    2010-02-19

    Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (K(i) = 380 +/- 160 microM) and 2-phosphoascorbate (K(i) = 3.2 +/- 0.85 microM) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia.

  18. Endocytosis of lysosomal acid phosphatase; involvement of mannose receptor and effect of lectins.

    Science.gov (United States)

    Imai, K; Yoshimura, T

    1994-08-01

    Acid phosphatase and beta-glucosidase are unique among lysosomal enzymes in that they have both high mannose and complex type sugasr chains, whereas oligosaccharide chains of lysosomal enzymes in matrix are of high mannose type. We have previously shown that beta-glucosidase was endocytosed into macrophages via an unidentified receptor different from a mannose/fucose receptor (K. Imai, Cell Struct. Funct. 13, 325-332, 1988). Here, we show that uptake of acid phosphatase purified from rat liver lysosomes into rat macrophages was inhibited by ligands for a mannose/fucose receptor and was mediated via an apparently single binding site with Kuptake of 24.7 nM. These results indicate that acid phosphatase and beta-glucosidase recognize different types of receptors even if they have similar sugar chains. Polyvalent concanavalin A which binds both to the enzyme and to macrophages specifically stimulated the uptake in a dose dependent manner, whereas wheat germ agglutinin and phytohaemagglutinin did not.

  19. The effects of Zinc supplementation on serum zinc, alkaline phosphatase activity and fracture healing of bones

    International Nuclear Information System (INIS)

    Sadighi, A.; Moradi, A.; Roshan, Marjan M.; Ostadrahimi, A.

    2009-01-01

    Objective was to determine the effect of zinc supplementation on callus information, serum zinc and alkaline phosphatase activity in humans. This randomized, double-blind, placebo controlled clinical trial was conducted on 60 patients with traumatic bone fracture referred to Shohada Hospital of Tabriz, Iran from August to December 2007. Subjects were randomly divided into 2 groups: cases (n=30), receiving one capsule of zinc sulfate consists of 50 mg zinc each day and the controls (n=30), receiving placebo for 60 days. Individual and clinical information was determined by a questionnaire: nutritional intake by 3 days food records at the beginning and the end of trial. Serum zinc and alkaline phosphatase was measured by atomic absorption spectroscopy and by enzymatic method. Callus information during fracture healing was evaluated by radiography of the bone. There was no significant difference in physical activity, gender, age, type of fractures and nutrient intake, between the 2 groups. The administration of zinc caused a significant elevation of serum zinc and alkaline phosphatase activity. Assessment of bone x-rays showed a significant progress in callus formation in cases compared to the controls. This study shows that zinc supplementation can stimulate fracture healing, however, it needs further study. (author)

  20. Alkaline and Acid Phosphatase Activity in Blood Plasma of Chickens Irradiated by Low dose Gamma Radiation

    International Nuclear Information System (INIS)

    Petar, K.; Marinko, V.; Saveta, M.; Miljenko, S.

    2004-01-01

    In our previous paper (Kraljevic et, al, 2000; Kraljevic et al 2002) we showed that the growth of the chickens hatched from eggs irradiated with 0.15 Gy gamma-rays before incubation was significantly higher than in controls during the fattening period (1-42 days). The concentration of total protein, glucose and cholesterol in the blood plasma of the same chickens was also significantly changed. In this paper an attempt was made to determine the effect of irradiation of eggs by low dose ionizing radiation before incubation upon activity of alkaline and acid phosphatase in the blood plasma of chickens hatched from irradiated eggs. The eggs of heavy breeding chickens were irradiated by dose of 0.15 Gy gamma radiation (60 Co) before incubation. Along with the chickens which were hatched from irradiated eggs, there was a control group of chickens hatched from nonirradiated eggs. All other conditions were the same for both groups. After hatching, blood samples were taken from the wing vein on days 1, 3, 5, 6, 10, 20, 30 and 42. The activity of both enzymes was determined spectrophotometrically by using Boehring Mannheim GmbH optimized kits. the activity of alkaline phosphatase in blood plasma was decreased on days 42, and the activity of acid phosphatase in the blood plasma of the same chickens was increased on day 42. Obtained results confirm our early obtained results that low dose of gamma radiation has effects upon metabolic processes in the chickens hatched from eggs irradiated before incubation. (Author)

  1. Prostatic acid phosphatase is required for the antinociceptive effects of thiamine and benfotiamine.

    Science.gov (United States)

    Hurt, Julie K; Coleman, Jennifer L; Fitzpatrick, Brendan J; Taylor-Blake, Bonnie; Bridges, Arlene S; Vihko, Pirkko; Zylka, Mark J

    2012-01-01

    Thiamine (Vitamin B1) is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT)-a phosphorylated derivative of thiamine-have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP) can dephosphorylate BT in vitro, in dorsal root ganglia (DRG) neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT) then decomposes to O-benzoylthiamine (O-BT) and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP) and thiamine-a compound that is not phosphorylated-were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap(-/-) mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP.

  2. Prostatic acid phosphatase is required for the antinociceptive effects of thiamine and benfotiamine.

    Directory of Open Access Journals (Sweden)

    Julie K Hurt

    Full Text Available Thiamine (Vitamin B1 is an essential vitamin that must be obtained from the diet for proper neurological function. At higher doses, thiamine and benfotiamine (S-benzoylthiamine O-monophosphate, BT-a phosphorylated derivative of thiamine-have antinociceptive effects in animals and humans, although how these compounds inhibit pain is unknown. Here, we found that Prostatic acid phosphatase (PAP, ACPP can dephosphorylate BT in vitro, in dorsal root ganglia (DRG neurons and in primary-afferent axon terminals in the dorsal spinal cord. The dephosphorylated product S-benzoylthiamine (S-BT then decomposes to O-benzoylthiamine (O-BT and to thiamine in a pH-dependent manner, independent of additional enzymes. This unique reaction mechanism reveals that BT only requires a phosphatase for conversion to thiamine. However, we found that the antinociceptive effects of BT, thiamine monophosphate (TMP and thiamine-a compound that is not phosphorylated-were entirely dependent on PAP at the spinal level. Moreover, pharmacokinetic studies with wild-type and Pap(-/- mice revealed that PAP is not required for the conversion of BT to thiamine in vivo. Taken together, our study highlights an obligatory role for PAP in the antinociceptive effects of thiamine and phosphorylated thiamine analogs, and suggests a novel phosphatase-independent function for PAP.

  3. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    International Nuclear Information System (INIS)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-01-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit

  4. Biomineralization of Uranium by PhoY Phosphatase Activity Aids Cell Survival in Caulobacter crescentus

    Energy Technology Data Exchange (ETDEWEB)

    Yung, M C [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jiao, Y [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2014-07-22

    Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-Pi precipitates via its native alkaline phosphatase activity. The U-Pi precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/Pi ratio than do chemically produced precipitates. The enzyme that is responsible for the phosphatase activity and thus the biomineralization process is identified as PhoY, a periplasmic alkaline phosphatase with broad substrate specificity. Furthermore, PhoY is shown to confer a survival advantage on C. crescentus toward U(VI) under both growth and nongrowth conditions. Results obtained in this study thus highlight U(VI) biomineralization as a resistance mechanism in microbes, which not only improves our understanding of bacterium-mineral interactions but also aids in defining potential ecological niches for metal-resistant bacteria.

  5. Identification and analysis of OsttaDSP, a phosphoglucan phosphatase from Ostreococcus tauri.

    Directory of Open Access Journals (Sweden)

    Julieta B Carrillo

    Full Text Available Ostreococcus tauri, the smallest free-living (non-symbiotic eukaryote yet described, is a unicellular green alga of the Prasinophyceae family. It has a very simple cellular organization and presents a unique starch granule and chloroplast. However, its starch metabolism exhibits a complexity comparable to higher plants, with multiple enzyme forms for each metabolic reaction. Glucan phosphatases, a family of enzymes functionally conserved in animals and plants, are essential for normal starch or glycogen degradation in plants and mammals, respectively. Despite the importance of O. tauri microalgae in evolution, there is no information available concerning the enzymes involved in reversible phosphorylation of starch. Here, we report the molecular cloning and heterologous expression of the gene coding for a dual specific phosphatase from O. tauri (OsttaDSP, homologous to Arabidopsis thaliana LSF2. The recombinant enzyme was purified to electrophoretic homogeneity to characterize its oligomeric and kinetic properties accurately. OsttaDSP is a homodimer of 54.5 kDa that binds and dephosphorylates amylopectin. Also, we also determined that residue C162 is involved in catalysis and possibly also in structural stability of the enzyme. Our results could contribute to better understand the role of glucan phosphatases in the metabolism of starch in green algae.

  6. Application of Scharer's quantitative method for the determination of residual alkaline phosphatase activity in standard Minas

    Directory of Open Access Journals (Sweden)

    C.F. Soares

    2013-08-01

    Full Text Available Milk pasteurization is a critical issue in the dairy industry, and failures in this process can affect final product safety. Scharer's enzymatic method is still traditionally used to verify pasteurization efficiency compliance, and it is based on screening for residual alkaline phosphatase in milk. Although several methods are used to quantify enzymatic activity to assess milk pasteurization efficiency, there is a small amount of published data regarding the use of these methods to quantify alkaline phosphatase in cheese. In this study, the Scharer's modified method was used to determine the levels of residual alkaline phosphatase in standard minas cheese, before and after 20 days of ripening. The cheeses were made using raw or pasteurized milk with the addition of different concentrations of raw milk (0; 0.05%; 0.10%; 0.20%; and 0.50%. In the fresh cheese samples, the method showed a sensitivity of only 0.50% with the addition of raw milk to the pasteurized milk used to make cheese. In addition, levels of up 0.20% of raw milk in pasteurized milk, the concentrations of phenol was inferior to 1μg phenol/g of dairy product which is the preconized indicator value for adequate pasteurization.

  7. Natural compounds as a source of protein tyrosine phosphatase inhibitors : Application to the rational design of small-molecule derivatives

    NARCIS (Netherlands)

    Ferreira, Carmen V.; Justo, Giselle Z.; Souza, Ana C. S.; Queiroz, Karla C. S.; Zambuzzi, William F.; Aoyama, Hiroshi; Peppelenbosch, Maikel P.

    2006-01-01

    Reversible phosphorylation of tyrosine residues is a key regulatory mechanism for numerous cellular events. Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) have a pivotal role in regulating both normal cell physiology and pathophysiology. Accordingly, deregulated activity of both

  8. Loss of Function Studies in Mice and Genetic Association Link Receptor Protein Tyrosine Phosphatase a to Schizophrenia

    DEFF Research Database (Denmark)

    Takahashi, Nagahide; Nielsen, Karin Sandager; Aleksic, Branko

    2011-01-01

    Solid evidence links schizophrenia (SZ) susceptibility to neurodevelopmental processes involving tyrosine phosphorylation-mediated signaling. Mouse studies implicate the Ptpra gene, encoding protein tyrosine phosphatase RPTPa, in the control of radial neuronal migration, cortical cytoarchitecture...

  9. Radioimmunoassay of human prostate-specific acid phosphatase in the diagnosis and follow-up of therapy of prostatic cancer

    International Nuclear Information System (INIS)

    Vihko, P.

    1981-01-01

    The author describes the development of a radioimmunoassay for the determination of serum prostate-specific acid phosphatase and studies its application to the diagnosis and follow-up of therapy of prostatic carcinoma. (Auth./C.F.)

  10. Alkaline phosphatase activity at the southwest coast of India: A comparison of locations differently affected by upwelling..

    Digital Repository Service at National Institute of Oceanography (India)

    Mamatha, S.S.; Malik, A.; Varik, S.; Parvathi, V.; Jineesh, V.K.; Gauns, M.; LokaBharathi, P.A.

    coastal waters. As alkaline phosphatase activity (APA) indicates the status of P for primary production in aquatic environments, we asked the following question: is the level of APA indicative of P sufficiency or deficiency in coastal waters, especially...

  11. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

    International Nuclear Information System (INIS)

    Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L.

    1988-01-01

    Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity

  12. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    Science.gov (United States)

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  13. A quantitative method for measurement of lysosomal acid phosphatase latency in cultured rat heart cells with 210Pb

    International Nuclear Information System (INIS)

    Hale, T.W.; Wenzel, D.G.

    1978-01-01

    A method is described for measuring the latency of lysomal acid phosphatase in cultured rat heart endotheloid cells. 210 Pb was added to a medium used to demonstrate acid phosphatase activity by the Gomori lead method, and the amount of lead deposited was measured with a liquid scintillation counter. Deposition rates were measured after enzyme activation pretreatments with acetate buffer (pH 5.0) at various osmolalities, and after formaldehyde fixation. Formaldehyde, alloxan, or fluoride in the Gomori medium were evaluated for their differential effects on lysosomal and non-lysosomal acid phosphatase The method was found to provide a sensitive, rapid and quantitative evaluation of acid phosphatase latency and should be useful for studying the integrity of lysosomes within cells. (author)

  14. Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

    Energy Technology Data Exchange (ETDEWEB)

    Kumar M.; Sharma M.K.; Saxena P.S.; Kumar A. [Rajasthan Univ., Jaipur (India)

    2003-03-01

    The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

  15. Loss of SYNJ1 dual phosphatase activity leads to early onset refractory seizures and progressive neurological decline

    DEFF Research Database (Denmark)

    Hardies, Katia; Cai, Yiying; Jardel, Claude

    2016-01-01

    SYNJ1 encodes a polyphosphoinositide phosphatase, synaptojanin 1, which contains two consecutive phosphatase domains and plays a prominent role in synaptic vesicle dynamics. Autosomal recessive inherited variants in SYNJ1 have previously been associated with two different neurological diseases...... with intractable epilepsy and tau pathology. We performed whole exome or genome sequencing in three independent sib pairs with early onset refractory seizures and progressive neurological decline, and identified novel segregating recessive SYNJ1 defects. A homozygous missense variant resulting in an amino acid...

  16. Radioprotective effect of Panax ginseng on the phosphatases and lipid peroxidation level in testes of Swiss albino mice

    International Nuclear Information System (INIS)

    Kumar, M.; Sharma, M.K.; Saxena, P.S.; Kumar, A.

    2003-01-01

    The Panax ginseng has been used as traditional medicine for past several years among oriental people. The present investigation has been made to assess the radioprotective efficacy of ginseng root extract in the testicular enzymes of Swiss albino mice. The Swiss albino mice were divided into different groups. Ginseng treated group: The animals were administered 10 mg/kg body weight ginseng root extract intraperitoneal (i.p.). Radiation treated group: The animals were exposed to 8 Gy gamma radiation at the dose rate of 1.69 Gy/min at the distance of 80 cm. Combination group: Animals were administered ginseng extract continuously for 4 d and on 4th day they were irradiated to 8 Gy gamma radiation after 30 min of extract administration. The animals from above groups were autopsied on day 1, 3, 7, 14 and 30. Biochemical estimations of acid and alkaline phosphatases and Lipid peroxidation (LPO) in testes were done. In ginseng treated group acid and alkaline phosphatases activity and LPO level did not show any significant alteration. In irradiated animals there was a significant increase in acid phosphatase activity and LPO level. However, significant decline in alkaline phosphatase activity was observed. The treatment of ginseng before irradiation causes significant decrease in acid phosphatase and LPO level and significant increase in alkaline phosphatase activity. One of the cause of radiation damage is lipid peroxidation. Due to lipid peroxidation, lysosomal membrane permeability alters and thus results in release of hydrolytic enzymes. So, an increase in acid phosphatase was noticed after radiation treatment. The alkaline phosphatase activity is associated with membrane permeability and different stages of spermatogenesis. Due to membrane damage and depletion of germ cells of testes after irradiation the enzyme activity was decreased. Ginseng markedly inhibits lipid peroxidation. It acts in indirect fashion to protect radical processes by inhibition of initiation of

  17. Diversity and Gene Expression of Phosphatase Genes Provide Insight into Soil Phosphorus Dynamics in a New Zealand Managed Grassland

    Science.gov (United States)

    Dunfield, K. E.; Gaiero, J. R.; Condron, L.

    2017-12-01

    Healthy and diverse communities of soil organisms influence key soil ecosystem services such as carbon sequestration, water quality protection, climate regulation and nutrient cycling. Microbially driven mineralization of organic phosphorus is an important contributor to plant available inorganic orthophosphates. In acidic soils, microbes produce non-specific acid phosphatases (NSAPs) which act on common forms of organic phosphorus (P). Our current understanding of P turnover in soils has been limited by lack of research tools capable of targeting these genes. Thus, we developed a set of oligonucleotide PCR primers that targeted bacteria with the genetic potential for acid phosphatase production. A long term randomized-block pasture trial was sampled following 22 years of continued aerial biomass removal and retention. Primers were used to target genes encoding alkaline phosphatase (phoD) and the three classes (CAAP, CBAP, CCAP) of non-specific acid phosphatases. PCR amplicons targeting total genes and gene transcripts were sequenced using Illumina MiSeq to understand the diversity of the bacterial phosphatase producing communities. In general, the majority of operational taxonomic units (OTUs) were shared across both treatments and across metagenomes and transcriptomes. However, analysis of DNA OTUs revealed significantly different communities driven by treatment differences (P reduced Olsen P levels (15 vs. 36 mg kg-1 in retained treatment). Acid phosphatase activity was measured in all samples, and found to be highest in the biomass retained treatment (16.8 vs. 11.4 µmol g-1 dry soil h-1), likely elevated due to plant-derived enzymes; however, was still correlated to bacterial gene abundances. Overall, the phosphatase producing microbial communities responded to the effect of consistent P limitation as expected, through alteration in the composition of the community structure and through increased levels of gene expression of the phosphatase genes.

  18. Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation

    Science.gov (United States)

    Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand

    2016-01-01

    ABSTRACT Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCE The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous

  19. Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

    Directory of Open Access Journals (Sweden)

    Hannes C A Drexler

    Full Text Available Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1

  20. In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Krebs, H O; Hoffschulte, H K; Müller, M

    1989-05-01

    We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.

  1. CLONING, SEQUENCE ANALYSIS, AND CHARACTERIZATION OF PUTATIVE BETA-LACTAMASE OF STENOTROPHOMONAS MALTOPHILIA

    Directory of Open Access Journals (Sweden)

    Chong Seng Shueh

    2012-10-01

    Full Text Available The main objective of current study was to explore the function of chromosomal putative beta-lactamase gene (smlt 0115 in clinical Stenotrophomonas maltophilia. Antibiotic susceptibility test (AST screening for current antimicrobial drugs was done and Minimum Inhibitory Concentration (MIC level towards beta-lactams was determined by E-test. Putative beta-lactamase gene of S. maltophilia was amplified via PCR, with specific primers, then cloned into pET-15 expression plasmid and transformed into Escherichia coli BL21. The gene was sequenced and analyzed. The expressed protein was purified by affinity chromatography and the kinetic assay was performed. S. maltophilia ATCC 13637 was included in this experiment. Besides, a hospital strain which exhibited resistant to a series of beta-lactams including cefepime was identified via AST and MIC, hence it was named as S2 strain and was considered in this study. Sequencing result showed that putative beta-lactamase gene obtained from ATCC 13637 and S2 strains were predicted to have cephalosporinase activity by National Center for Biotechnology Information (NCBI blast program. Differences in the sequences of both ATCC 13637 and S2 strains were found via ClustalW alignment software. Kinetic assay proved a cephalosporinase characteristic produced by E. coli BL21 clone that overexpressed the putative beta-lactamase gene cloned under the control of an external promoter. Yet, expressed protein purified from S2 strain had high catalytic activity against beta-lactam antibiotics which was 14-fold higher than expressed protein purified from ATCC 13637 strain. This study represents the characterization analysis of putative beta-lactamase gene (smlt 0115 of S. maltophilia. The presence of the respective gene in the chromosome of S. maltophilia suggested that putative beta-lactamase gene (smlt 0115 of S. maltophilia plays a role in beta-lactamase resistance.

  2. Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

    DEFF Research Database (Denmark)

    Nilsson, Martin; Chiang, Wen-Chi; Fazli, Mustafa

    2011-01-01

    We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P....... putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable...

  3. Probing the origins of catalytic discrimination between phosphate and sulfate monoester hydrolysis: comparative analysis of alkaline phosphatase and protein tyrosine phosphatases.

    Science.gov (United States)

    Andrews, Logan D; Zalatan, Jesse G; Herschlag, Daniel

    2014-11-04

    Catalytic promiscuity, the ability of enzymes to catalyze multiple reactions, provides an opportunity to gain a deeper understanding of the origins of catalysis and substrate specificity. Alkaline phosphatase (AP) catalyzes both phosphate and sulfate monoester hydrolysis reactions with a ∼10(10)-fold preference for phosphate monoester hydrolysis, despite the similarity between these reactions. The preponderance of formal positive charge in the AP active site, particularly from three divalent metal ions, was proposed to be responsible for this preference by providing stronger electrostatic interactions with the more negatively charged phosphoryl group versus the sulfuryl group. To test whether positively charged metal ions are required to achieve a high preference for the phosphate monoester hydrolysis reaction, the catalytic preference of three protein tyrosine phosphatases (PTPs), which do not contain metal ions, were measured. Their preferences ranged from 5 × 10(6) to 7 × 10(7), lower than that for AP but still substantial, indicating that metal ions and a high preponderance of formal positive charge within the active site are not required to achieve a strong catalytic preference for phosphate monoester over sulfate monoester hydrolysis. The observed ionic strength dependences of kcat/KM values for phosphate and sulfate monoester hydrolysis are steeper for the more highly charged phosphate ester with both AP and the PTP Stp1, following the dependence expected based on the charge difference of these two substrates. However, the dependences for AP were not greater than those of Stp1 and were rather shallow for both enzymes. These results suggest that overall electrostatics from formal positive charge within the active site is not the major driving force in distinguishing between these reactions and that substantial discrimination can be attained without metal ions. Thus, local properties of the active site, presumably including multiple positioned dipolar

  4. Involvement of protein tyrosine phosphatases BcPtpA and BcPtpB in regulation of vegetative development, virulence and multi-stress tolerance in Botrytis cinerea.

    Directory of Open Access Journals (Sweden)

    Qianqian Yang

    Full Text Available Tyrosine phosphorylation and dephosphorylation have emerged as fundamentally important mechanisms of signal transduction and regulation in eukaryotic cells, governing many processes, but little has been known about their functions in filamentous fungi. In this study, we deleted two putative protein tyrosine phosphatase (PTP genes (BcPTPA and BcPTPB in Botrytis cinerea, encoding the orthologs of Saccharomyces cerevisiae Ptp2 and Ptp3, respectively. Although BcPtpA and BcPtpB have opposite functions in conidiation, they are essential for sclerotial formation in B. cinerea. BcPTPA and BcPTPB deletion mutants ΔBcPtpA-10 and ΔBcPtpB-4 showed significantly increased sensitivity to osmotic and oxidative stresses, and to cell wall damaging agents. Inoculation tests showed that both mutants exhibited dramatically decreased virulence on tomato leaves, apples and grapes. In S. cerevisiae, it has been shown that Ptp2 and Ptp3 negatively regulate the high-osmolarity glycerol (HOG pathway and the cell wall integrity (CWI pathway. Although both BcPtpA and BcPtpB were able to inactive Hog1 and Mpk1 in S. cerevisiae, in contrast to S. cerevisiae, they positively regulate phosphorylation of BcSak1 (the homologue of Hog1 and BcBmp3 (the homologue of Mpk1 in B. cinerea under stress conditions. These results demonstrated that functions of PTPs in B. cinerea are different from those in S. cerevisiae, and BcPtpA and BcPtpB play important roles in regulation of vegetative development, virulence and in adaptation to oxidative, osmotic and cell-wall damage stresses in B. cinerea.

  5. Characterization of soluble and membrane-bound alkaline phosphatase in Nilaparvata lugens and their potential relation to development and insecticide resistance.

    Science.gov (United States)

    Wang, Zengxia; Liu, Shuhua; Yang, Baojun; Liu, Zewen

    2011-09-01

    Two forms (soluble and membrane-bound) of alkaline phosphatases (ALPs) were found in the brown planthopper, Nilaparvata lugens. In order to further study ALPs in N. lugens, two putative ALP genes (Nl-ALP1 and Nl-ALP2) were identified in this pest. Both Nl-ALP1 and Nl-ALP2 show approximately the same degree of sequence identity (40-50%) to other insect soluble and membrane-bound forms of ALP. Correlation of ALP activity and mRNA levels at different developmental stages, or following application of 20-hydroxyecdysone (20E) and insecticide fenvalerate, suggests that Nl-ALP1 and Nl-ALP2 might encode a soluble (sALP) and a membrane-bound ALP (mALP), respectively. Nl-ALP1-specific antibody Nl1-I detected only a specific band in soluble protein preparations and Nl-ALP2 specific antibody Nl2-I only detected a specific band in insoluble protein preparations, which provided conclusive linkages between Nl-ALP1 and a sALP and between Nl-ALP2 and a m ALP. Then, Nl-ALP1 was denoted as Nl-sALP for a sALP and Nl-ALP2 was denoted as Nl-mALP for a mALP. Only sALP activity and Nl-sALP mRNA level were induced by 20E and fenvalerate, which was confirmed by the density of specific band detected by Nl1-I in Sus strain with or without fenvalerate treatment. Additionally, the sALP activity, as well as Nl-sALP mRNA level, was significantly higher in a fenvalerate resistant population, compared with Sus strain. These results indicate that the sALP is more responsive to chemical stimulus, such as hormone and insecticide, and might play dual roles in development and insecticide tolerance. © 2011 Wiley Periodicals, Inc.

  6. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    OpenAIRE

    Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

    2016-01-01

    Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones, which include the xanthanolides. To date, the biogenesis of xanthanolides, especiallytheir downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes...

  7. Asymmetric total synthesis of a putative sex pheromone component from the parasitoid wasp Trichogramma turkestanica

    NARCIS (Netherlands)

    Geerdink, Danny; Buter, Jeffrey; van Beek, Teris A.; Minnaard, Adriaan J.

    2014-01-01

    Virgin females of the parasitoid wasp Trichogramma turkestanica produce minute amounts of a sex pheromone, the identity of which has not been fully established. The enantioselective synthesis of a putative component of this pheromone, (6S,8S,10S)-4,6,8,10-tetramethyltrideca-2E,4E-dien-1-ol (2), is

  8. Search strings for the study of putative occupational determinants of disease

    NARCIS (Netherlands)

    Mattioli, S.; Zanardi, F.; Baldasseroni, A.; Schaafsma, F.; Cooke, R.M.T.; Mancini, G.; Fierro, M.; Santangelo, C.; Farioli, A.; Fucksia, S.; Curti, S.; Violante, F.S.; Verbeek, J.

    2010-01-01

    Objective To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Methods Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms

  9. Search strings for the study of putative occupational determinants of disease

    NARCIS (Netherlands)

    Mattioli, Stefano; Zanardi, Francesca; Baldasseroni, Alberto; Schaafsma, Frederieke; Cooke, Robin M. T.; Mancini, Gianpiero; Fierro, Mauro; Santangelo, Chiara; Farioli, Andrea; Fucksia, Serenella; Curti, Stefania; Violante, Francesco S.; Verbeek, Jos

    2010-01-01

    To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms describing

  10. Putative contact ketoconazole shampoo-triggered pemphigus foliaceus in a dog.

    Science.gov (United States)

    Sung, Hyun-Jeong; Yoon, In-Hwa; Kim, Jung-Hyun

    2017-09-01

    A 10-year-old spayed female cocker spaniel dog was referred for an evaluation of acute-onset generalized pustular cutaneous lesions following application of ketoconazole shampoo. Cytologic and histopathologic examinations of the lesions revealed intra-epidermal pustules with predominantly neutrophils and acantholytic cells. This is the first description of putative contact ketoconazole shampoo-triggered pemphigus foliaceus in a dog.

  11. Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

    Science.gov (United States)

    Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Toranzos, Gary A; Marota, Isolina; Giuffra, Valentina; Cano, Raul J

    2017-11-07

    Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

  12. Cloning and sequence analysis of putative type II fatty acid synthase ...

    Indian Academy of Sciences (India)

    Prakash

    Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. ... acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP .... Helix II plays a dominant role in the interaction ... main distinguishing features of plant ACPs in plastids and ..... synthase component; J. Biol.

  13. Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies

    Directory of Open Access Journals (Sweden)

    Tasha M. Santiago-Rodriguez

    2017-11-01

    Full Text Available Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

  14. Total synthesis of the putative structure of the novel triquinane natural product isocapnellenone

    OpenAIRE

    Mehta, Goverdhan; Murthy, Sai Krishna A; Umarye, Jayant D

    2002-01-01

    A total synthesis of the ‘putative structure’ 7, attributed to the novel triquinane sesquiterpene isolated recently from two Buddelia species has been accomplished. The spectral data for 7 is a complete mismatch with those reported for the natural product and warrants a revision of the assigned structure.

  15. Cloning and characterization of prunus serotina AGAMOUS, a putative flower homeotic gene

    Science.gov (United States)

    Xiaomei Liu; Joseph Anderson; Paula Pijut

    2010-01-01

    Members of the AGAMOUS subfamily of MADS-box transcription factors play an important role in regulating the development of reproductive organs in flowering plants. To help understand the mechanism of floral development in black cherry (Prunus serotina), PsAG (a putative flower homeotic identity gene) was isolated...

  16. Sequence analysis of putative swrW gene required for surfactant ...

    African Journals Online (AJOL)

    owner

    2012-07-17

    Jul 17, 2012 ... These nucleotide and protein sequence analysis of the putative swrW gene provides vital information on the versatility .... chain reaction (PCR) products were stored at 4°C. Presence of ... identical to the same gene with an E-value of 0.0. .... The Prokaryotes-A Handbook on the Biol. of Bacteria:Ecophysiol.

  17. Distribution of putative virulence genes and antimicrobial drug resistance in Vibrio harveyi

    Digital Repository Service at National Institute of Oceanography (India)

    Parvathi, A.; Mendez, D.; Anto, C.

    zonula occludens toxin (Zot) and a hemolysin-coregulated protein gene (hcp) by polymerase chain reaction (PCR). Of the four putative reversible toxin genes, vhh-1 was detected in 31% of the isolates, vhh-2 in 46%, vhh-3 in 23% and vhh-4 was detected in 27...

  18. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    Science.gov (United States)

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera ( Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir , we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C ( V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  19. Identification of EhTIF-IA: The putative E. histolytica orthologue of the ...

    Indian Academy of Sciences (India)

    2016-02-04

    Feb 4, 2016 ... We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within ..... a putative EhTIF-IA with e-value (3e−25). Comparison of .... some biogenesis is correlated with altered rates of rDNA transcription ..... ylation by CK2 facilitates rDNA transcription by promoting dissociation of ...

  20. DETERMINATION OF ROCURONIUM AND ITS PUTATIVE METABOLITES IN BODY-FLUIDS AND TISSUE-HOMOGENATES

    NARCIS (Netherlands)

    KLEEF, UW; PROOST, JH; ROGGEVELD, J

    1993-01-01

    A sensitive and selective HPLC method was developed for the quantification of the neuromuscular blocking agent rocuronium and its putative metabolites (the 17-desacetyl derivative and the N-desallyl derivative of rocuronium) in plasma, urine, bile, tissue homogenates and stoma fluid. Samples were

  1. Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri.

    Science.gov (United States)

    Nigg, Jared C; Nouri, Shahideh; Falk, Bryce W

    2016-07-28

    Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera. Copyright © 2016 Nigg et al.

  2. Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri

    OpenAIRE

    Nigg, Jared C.; Nouri, Shahideh; Falk, Bryce W.

    2016-01-01

    Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri. Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera.

  3. Vasodilator effects and putative guanylyl cyclase stimulation by 2-nitro-1-phenylethanone and 2-nitro-2-phenyl-propane-1,3-diol on rat aorta.

    Science.gov (United States)

    Vasconcelos, Thiago Brasileiro de; Ribeiro-Filho, Helder Veras; Lahlou, Saad; Pereira, José Geraldo de Carvalho; Oliveira, Paulo Sérgio Lopes de; Magalhães, Pedro Jorge Caldas

    2018-07-05

    Compounds containing a nitro group may reveal vasodilator properties. Several nitro compounds have a NO 2 group in a short aliphatic chain connected to an aromatic group. In this study, we evaluated in rat aorta the effects of two nitro compounds, with emphasis on a putative recruitment of the soluble guanylate cyclase (sGC) pathway to induce vasodilation. Isolated aortic rings were obtained from male Wistar rats to compare the effects induced by 2-nitro-1-phenylethanone (NPeth) or 2-nitro-2-phenyl-propane-1,3-diol (NPprop). In aortic preparations contracted with phenylephrine or KCl, NPeth and NPprop induced vasorelaxant effects that did not depend on the integrity of vascular endothelium. NPeth had a lesser vasorelaxant efficacy than NPprop and only the NPprop effects were inhibited by pretreatment with the sGC inhibitors, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or methylene blue. In an ODQ-preventable manner, NPprop inhibited the contractile component of the phenylephrine-induced response mediated by intracellular Ca 2+ release or by extracellular Ca 2+ recruitment through receptor- or voltage-operated Ca 2+ channels. In contrast, NPprop was inert against the transient contraction induced by caffeine in Ca 2+ -free medium. In an ODQ-dependent manner, NPprop inhibited the contraction induced by the protein kinase C activator phorbol 12,13-dibutyrate or by the tyrosine phosphatase inhibitor sodium orthovanadate. In silico docking analysis of a sGC homologous protein revealed preferential site for NPprop. In conclusion, the nitro compounds NPeth and NPprop induced vasorelaxation in rat aortic rings. Aliphatic chain substituents selectively interfered in the ability of these compounds to induce vasorelaxant effects, and only NPprop relaxed aortic rings via a sGC pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914.

    Science.gov (United States)

    Attardi, Barbara J; Burgenson, Janet; Hild, Sheri A; Reel, Jerry R; Blye, Richard P

    2002-02-25

    To obtain selective antiprogestins, we have examined the in vitro antiprogestational/antiglucocorticoid properties of two novel compounds, CDB-4124 and the putative monodemethylated metabolite, CDB-4453, in transcription and receptor binding assays and compared them to CDB-2914 and mifepristone. All four antiprogestins bound with high affinity to rabbit uterine progestin receptors (PR) and recombinant human PR-A and PR-B (rhPR-A, rhPR-B) and were potent inhibitors of R5020-induced transactivation of the PRE2-tk-luciferase (PRE2-tk-LUC) reporter plasmid and endogenous alkaline phosphatase production in T47D-CO human breast cancer cells. None of these compounds exhibited agonist activity in these cells. Induction of luciferase activity was potentiated about five-fold by 8-Br-cAMP under basal conditions and to the same extent in the presence of the PR antagonists. Mifepristone bound to rabbit thymic glucocorticoid receptors (GR) with approximately twice the avidity of the CDB antiprogestins. Inhibition of GR-mediated transcription of PRE2-tk-LUC was assessed in HepG2 human hepatoblastoma cells. Mifepristone exhibited greater antiglucocorticoid activity than CDB-2914, 4124, and 4453, about 12-, 22-, and 185-fold, respectively. Thus, while there was a good correlation between binding to PR and functional activity of these antiprogestins, GR binding was not predictive of their glucocorticoid antagonist activity. In agreement with our in vivo results, CDB-4124 and CDB-4453, as well as CDB-2914, are potent antiprogestins in vitro, but show considerably less antiglucocorticoid activity than mifepristone.

  5. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    Science.gov (United States)

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

  6. The Association of Endothelin-1 Signaling with Bone Alkaline Phosphatase Expression and Protumorigenic Activities in Canine Osteosarcoma.

    Science.gov (United States)

    Neumann, Z L; Pondenis, H C; Masyr, A; Byrum, M L; Wycislo, K L; Fan, T M

    2015-01-01

    Canine osteosarcoma (OS) is an aggressive sarcoma characterized by pathologic skeletal resorption and pulmonary metastases. A number of negative prognostic factors, including bone alkaline phosphatase, have been identified in dogs with OS, but the underlying biologic factors responsible for such observations have not been thoroughly investigated. Endothelin-1-mediated signaling is active during bone repair, and is responsible for osteoblast migration, survival, proliferation, and bone alkaline phosphatase expression. The endothelin-1 signaling axis is active in canine OS cells, and this pathway is utilized by malignant osteoblasts for promoting cellular migration, survival, proliferation, and bone alkaline phosphatase activities. 45 dogs with appendicular OS. The expressions of endothelin-1 and endothelin A receptor were studied in OS cell lines and in samples from spontaneously occurring tumors. Activities mediated by endothelin-1 signaling were investigated by characterizing responses in 3 OS cell lines. In 45 dogs with OS, bone alkaline phosphatase concentrations were correlated with primary tumor osteoproductivity. Canine OS cells express endothelin-1 and endothelin A receptor, and this signaling axis mediates OS migration, survival, proliferation, and bone alkaline phosphatase activities. In OS-bearing dogs, circulating bone alkaline phosphatase activities were positively correlated with primary tumor relative bone mineral densities. Canine OS cells express endothelin-1 and functional endothelin A receptors, with the potential for a protumorigenic signaling loop. Increases in bone alkaline phosphatase activity are associated with osteoblastic OS lesions, and might be an epiphenomenon of active endothelin-1 signaling or excessive osteoproduction within the localized bone microenvironment. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  7. Phosphatase activity and culture conditions of the yeast Candida mycoderma sp. and analysis of organic phosphorus hydrolysis ability.

    Science.gov (United States)

    Yan, Mang; Yu, Liufang; Zhang, Liang; Guo, Yuexia; Dai, Kewei; Chen, Yuru

    2014-11-01

    Orthophosphate is an essential but limiting macronutrient for plant growth. About 67% cropland in China lacks sufficient phosphorus, especially that with red soil. Extensive soil phosphorus reserves exist in the form of organic phosphorus, which is unavailable for root uptake unless hydrolyzed by secretory acid phosphatases. Thus, many microorganisms with the ability to produce phosphatase have been exploited. In this work, the activity of an extracellular acid phosphatase and yeast biomass from Candida mycoderma was measured under different culture conditions, such as pH, temperature, and carbon source. A maximal phosphatase activity of 8.47×10(5)±0.11×10(5)U/g was achieved by C. Mycoderma in 36 hr under the optimal conditions. The extracellular acid phosphatase has high activity over a wide pH tolerance range from 2.5 to 5.0 (optimum pH3.5). The effects of different phosphorus compounds on the acid phosphatase production were also studied. The presence of phytin, lecithin or calcium phosphate reduced the phosphatase activity and biomass yield significantly. In addition, the pH of the culture medium was reduced significantly by lecithin. The efficiency of the strain in releasing orthophosphate from organic phosphorus was studied in red soil (used in planting trees) and rice soil (originating as red soil). The available phosphorus content was increased by 230% after inoculating 20 days in rice soil and decreased by 50% after inoculating 10 days in red soil. This work indicates that the yeast strain C. mycoderma has potential application for enhancing phosphorus utilization in plants that grow in rice soil. Copyright © 2014. Published by Elsevier B.V.

  8. Control of Acid Phosphatases Expression from Aspergillus niger by Soil Characteristics

    Directory of Open Access Journals (Sweden)

    Ely Nahas

    2015-10-01

    Full Text Available ABSTRACTThis work studied the acid phosphatase (APase activity from culture medium (extracellular, eAPase and mycelial extract (intracellular, iAPase ofAspergillus niger F111. The influence of fungus growth and phosphate concentration of the media on the synthesis and secretion of phosphatase was demonstrated. The effects of pH, substrate concentration and inorganic and organic compounds added to the reaction mixture on APase activity were also studied. Both enzymes were repressed by high concentrations of phosphate. Overexpression of iAPase in relation to eAPase was detected; iAPase activity was 46.1 times higher than eAPase. The maximal activity of eAPase was after 24h of fungus growth and for iAPase was after 96h. Optimal pH and substrate concentrations were 4.5 and 8.0 mM, respectively. Michaelis-Menten constant (Km for the hydrolysis of p-nitrophenyl phosphate was 0.57 mM with Vmax = 14,285.71 U mg-1 mycelium for the iAPase and 0.31 mM with V max = 147.06 U mg-1 mycelium for eAPase. Organic substances had little effect on acid phosphatases when compared with the salts. Both the APases were inhibited by 10 mM KH 2PO4 and 5 mM (NH42MoO4; eAPase was also inhibited by 1 mM CoCl2.

  9. Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures.

    Science.gov (United States)

    Becerra-Arteaga, Alejandro; Mason, Hugh S; Shuler, Michael L

    2006-01-01

    Tobacco NT1 cell suspension cultures secreting active human secreted alkaline phosphatase (SEAP) were generated for the first time as a model system to study recombinant protein production, secretion, and stability in plant cell cultures. The SEAP gene encodes a secreted form of the human placental alkaline phosphatase (PLAP). During batch culture, the highest level of active SEAP in the culture medium (0.4 U/mL, corresponding to approximately 27 mg/L) was observed at the end of the exponential growth phase. Although the level of active SEAP decreased during the stationary phase, the activity loss did not appear to be due to SEAP degradation (based on Western blots) but due to SEAP denaturation. The protein-stabilizing agents polyvinylpirrolidone (PVP) and bacitracin were added extracellularly to test for their ability to reduce the loss of SEAP activity during the stationary phase. Bacitracin (100 mg/L) was the most effective treatment at sustaining activity levels for up to 17 days post-subculture. Commercially available human placental alkaline phosphatase (PLAP) was used to probe the mechanism of SEAP deactivation. Experiments with PLAP in sterile and conditioned medium corroborated the denaturation of SEAP by factors generated by cell growth and not due to simple proteolysis. We also show for the first time that the factors promoting activity loss are heat labile at 95 degrees C but not at 70 degrees C, and they are not inactivated after a 5 day incubation period under normal culture conditions (27 degrees C). In addition, there were no significant changes in pH or redox potential when comparing sterile and cell-free conditioned medium during PLAP incubation, indicating that these factors were unimportant.

  10. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin , Pascal D. (Novartis)

    2016-06-29

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

  11. Nuclear protein phosphatase-1: an epigenetic regulator of fear memory and amygdala long-term potentiation.

    Science.gov (United States)

    Koshibu, K; Gräff, J; Mansuy, I M

    2011-01-26

    Complex brain diseases and neurological disorders in human generally result from the disturbance of multiple genes and signaling pathways. These disturbances may derive from mutations, deletions, translocations or rearrangements of specific gene(s). However, over the past years, it has become clear that such disturbances may also derive from alterations in the epigenome affecting several genes simultaneously. Our work recently demonstrated that epigenetic mechanisms in the adult brain are in part regulated by protein phosphatase 1 (PP1), a protein Ser/Thr phosphatase that negatively regulates hippocampus-dependent long-term memory (LTM) and synaptic plasticity. PP1 is abundant in brain structures involved in emotional processing like the amygdala, it may therefore be involved in the regulation of fear memory, a form of memory related to post-traumatic stress disorder (PTSD) in human. Here, we demonstrate that PP1 is a molecular suppressor of fear memory and synaptic plasticity in the amygdala that can control chromatin remodeling in neurons. We show that the selective inhibition of the nuclear pool of PP1 in amygdala neurons significantly alters posttranslational modifications (PTMs) of histones and the expression of several memory-associated genes. These alterations correlate with enhanced fear memory, and with an increase in long-term potentiation (LTP) that is transcription-dependent. Our results underscore the importance of nuclear PP1 in the amygdala as an epigenetic regulator of emotional memory, and the relevance of protein phosphatases as potential targets for therapeutic treatment of brain disorders like PTSD. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Root phosphatase activity, plant growth and phosphorus accumulation of maize genotypes

    Directory of Open Access Journals (Sweden)

    Machado Cynthia Torres de Toledo

    2004-01-01

    Full Text Available The activity of the enzyme phosphatase (P-ase is a physiological characteristic related to plant efficiency in relation to P acquisition and utilization, and is genetically variable. As part of a study on maize genotype characterization in relation to phosphorus (P uptake and utilization efficiency, two experiments were set up to measure phosphatase (P-ase activity in intact roots of six local and improved maize varieties and two sub-populations. Plants were grown at one P level in nutrient solution (4 mg L-1 and the P-ase activity assay was run using 17-day-old plants for varieties and 24-day-old plants for subpopulations. Shoot and root dry matter yields and P concentrations and contents in plant parts were determined, as well as P-efficiency indexes. Root P-ase activity differed among varieties, and highest enzimatic activities were observed in two local varieties -'Catetão' and 'Caiano' -and three improved varieties -'Sol da Manhã', 'Nitrodente' and 'BR 106'. 'Carioca', a local variety, had the lowest activity. Between subpopulations, 'ND2', with low yielding and poorly P-efficient plants, presented higher root P-ase activity as compared to 'ND10', high yielding and highly P-efficient plants. In general, subpopulations presented lower P-ase activities as compared to varieties. Positive and/or negative correlations were obtained between P-ase activity and P-efficiency characteristics, specific for the genotypes, not allowing inference on a general and clear association between root-secreted phosphatase and dry matter production or P acquisition. Genotypic variability must be known and considered before using P-ase activity as an indicator of P nutritional status, or P tolerance, adaptation and efficiency under low P conditions.

  13. Changes phosphorus associated to phosphatase activity because of application of carbon, nitrogen and manure

    Science.gov (United States)

    Paredes, Cecilia; Gianfreda, Liliana; Mora, María de la Luz

    2015-04-01

    The Chilean Andisols are of great importance in the economy of southern Chile supporting the bulk of agricultural production. The major characteristics of Chilean volcanic soils are the high adsorption capacity of P with a concomitant low P availability to plants. Studies preliminary using dairy cattle dung suggest that we can improve P availability using organic P sources within the soil because of microorganism. Phosphorous solubilization by microorganisms is a complex phenomenon, which depends on many factors such as nutritional, physiological and growth condition of the culture. The principal mechanism for mineral phosphate solubilization is the production of organic acids where the enzyme phosphatases play a major role in the mineralization of organic phosphorous in soil. The objective of this study was to evaluate changes in soil phosphorus fractions due to application the cattle dung, glucose, nitrogen (N) and phosphorus (P). In this experiment we incubated soil samples with 300 g of cattle dung, 30 mg kg-1 of N and P and 1000 mg glucose kg-1. The soil samples were moistened to field capacity and incubated in plastic bags to room temperature by different time. The changes in P forms in soil were monitored through the Hedley fractionation procedure and phosphatase activity. Our preliminary results indicated that the application of cattle dung, glucose nitrogen and phosphorus, caused the increased phosphatase activity until to 7 days and then apparently return to normal values. Interestingly, we observed a rise in the inorganic P fraction extracted by NaHCO3 in the same period. In summary, the increase biological activity by carbon and nitrogen increase P availability. Acknowledgements: The authors thank Fondecyt 1141247 Project.

  14. Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyo-Deok; Lee, Joong-jae [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Kim, Yu Jung [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Hantschel, Oliver [School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne (Switzerland); Lee, Seung-Goo [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Kim, Hak-Sung, E-mail: hskim76@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of)

    2017-01-15

    Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL{sup −1} for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. - Highlights: • A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. • A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. • mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. • mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.

  15. Study of serum levels of calcium, phosphorus and alkaline phosphatase in chronic kidney disease

    Directory of Open Access Journals (Sweden)

    R. Freethi

    2016-03-01

    Full Text Available Chronic kidney disease (CKD is a worldwide public health problem, with increasing prevalence and lethal adverse outcomes like progressive loss of kidney function, cardiovascular disease and premature death. Disturbances in mineral metabolism and bone disease are common complications of CKD and an important cause of morbidity and decreased quality of life in patients with CKD. Patients with renal failure have an increased risk of cardiovascular mortality that may be due in part to vascular calcification. To measure serum levels of calcium, phosphorus and alkaline phosphatase in patients in various stages of CKD and to correlate the same with creatinine and estimated Glomerular Filtration Rate (eGFRvalues. This is a cross sectional study done at Thanjavur Medical College Hospital. 60 CKD patients and 50 healthy controls were enrolled in this study. Serum levels of creatinine, calcium, phosphorus and alkaline phosphatase were measured and eGFR values correlated with the serum creatinine. The mean values of creatinine (4.9 ± 2.23 mg/dl, cal cium (9.8 ± 0.456 mg/dl , phosphorus (4.19 ± 0.404 mg/dl and alkaline phosphatase (94.01 ± 15.10 U/L in the stu dy group are significantly higher than the control group in which the mean levels are 0.89 ± 0.102 mg/dl, 10.17 ± 0.37 mg/dl, 4.02 ± 0.16 mg/dl and 25.16 ± 4.65U/ L respectively. We have found that there is a significant difference in the above said parameters among patients in different stages of CKD (stage 3-5 indicating the progression of mineral bone disease with advancing stage of CKD.

  16. Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase.

    Science.gov (United States)

    Waymack, P P; Van Etten, R L

    1991-08-01

    An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

  17. Acid and Alkaline Phosphatase Levels in GCF during Orthodontic Tooth Movement

    Science.gov (United States)

    Farahani, Mohammad; Safavi, Seyed Mohammadreza; Dianat, Omid; Khoramian Tusi, Somayeh; Younessian, Farnaz

    2015-01-01

    Statement of the Problem The present constituents of gingival crevicular fluid (GCF) can reflect the changes occurring in underlying tissues. Considering variety of biologic bone markers, alkaline phosphatase and acid phosphatase have been examined as bone turn over markers in orthodontic tooth movement. Purpose The current study designed in a longitudinal pattern to determine the changes of acid and alkaline phosphatase (ACP & ALP) in GCF during orthodontic tooth movement. Materials and Method An upper canines from twelve patients (mean age: 14±2 years) undergoing extraction orthodontic treatment for distal movement served as the test tooth (DC), and its contralateral (CC) and antagonist (AC) canines were used as controls. The CC was included in orthodontic appliance without orthodontic force; the AC was free from any orthodontic appliance. The GCF around the experimental teeth was harvested from mesial and distal tooth sites immediately before appliance placement (T0), and 14 (T2) and 28 days (T3) after it and ALP and ACP concentration were determined spectrophotometrically. Results ALP concentration was elevated significantly in DC and CC groups at days 14 and 28 compared with the AC. In DC group, the ALP was significantly greater in mesial sites than distal site, while no significant changes were found between both sites of CC. The peak level of ALP was observed in mesial sites of DC at T2. Regarding ACP, significant elevation of this enzyme was seen in DC group both in mesial and distal sites at T2 and T3. The peak level of this enzyme was seen at T2. Conclusion Monitoring simultaneous changes of ALP and ACP levels in GCF can reflect the tissue responses occur in periodontium during bone formation and bone resorption during orthodontic tooth movement, respectively. PMID:26535403

  18. Finding the smoking gun: protein tyrosine phosphatases as tools and targets of unicellular microorganisms and viruses.

    Science.gov (United States)

    Heneberg, P

    2012-01-01

    Protein tyrosine phosphatases (PTPs) are increasingly recognized as important effectors of host-pathogen interactions. Since Guan and Dixon reported in 1990 that phosphatase YopH serves as an essential virulence determinant of Yersinia, the field shifted significantly forward, and dozens of PTPs were identified in various microorganisms and even in viruses. The discovery of extensive tyrosine signaling networks in non-metazoan organisms refuted the moth-eaten paradigm claiming that these organisms rely exclusively on phosphoserine/phosphothreonine signaling. Similarly to humans, phosphotyrosine signaling is thought to comprise a small fraction of total protein phosphorylation, but plays a disproportionately important role in cell-cycle control, differentiation, and invasiveness. Here we summarize the state-of-art knowledge on PTPs of important non-metazoan pathogens (Listeria monocytogenes, Staphylococcus aureus, Porphyromonas gingivalis, Caulobacter crescentus, Yersinia, Synechocystis, Leishmania, Plasmodium falciparum, Entamoeba histolytica, etc.), and focus also at the microbial proteins affecting directly or indirectly the PTPs of the host (Mycobacterium tuberculosis MTSA-10, Bacillus anthracis anthrax toxin, streptococcal β protein, Helicobacter pylori CagA and VacA, Leishmania GP63 and EF-1α, Plasmodium hemozoin, etc.). This is the first review summarizing the knowledge on biological activity and pharmacological inhibition of non-metazoan PTPs, with the emphasis of those important in host-pathogen interactions. Targeting of numerous non-metazoan PTPs is simplified by the fact that they act either as ectophosphatases or are secreted outside of the pathogen. Interfering with tyrosine phosphorylation represents a powerful pharmacologic approach, and even though the PTP inhibitors are difficult to develop, lifting the fog of phosphatase inhibition is of the great market potential and further clinical impact.

  19. Phosphatase activity in relation to key litter and soil properties in mature subtropical forests in China.

    Science.gov (United States)

    Hou, Enqing; Chen, Chengrong; Wen, Dazhi; Liu, Xian

    2015-05-15

    Phosphatase-mediated phosphorus (P) mineralization is one of the critical processes in biogeochemical cycling of P and determines soil P availability in forest ecosystems; however, the regulation of soil phosphatase activity remains elusive. This study investigated the potential extracellular activities of acid phosphomonoesterase (AcPME) and phosphodiesterase (PDE) and how they were related to key edaphic properties in the L horizon (undecomposed litter) and F/H horizon (fermented and humified litter) and the underlying mineral soil at the 0-15cm depth in eight mature subtropical forests in China. AcPME activity decreased significantly in the order of F/H horizon>L horizon>mineral soil horizon, while the order for PDE activity was L horizon=F/H horizon>mineral soil horizon. AcPME (X axis) and PDE (Y axis) activities were positively correlated in all horizons with significantly higher slope in the L and F/H horizons than in the mineral soil horizon. Both AcPME and PDE activities were positively related to microbial biomass C, moisture content and water-holding capacity in the L horizon, and were positively related to soil C:P, N:P and C:N ratios and fine root (diameter≤2mm) biomass in the mineral soil horizon. Both enzyme activities were also interactively affected by forest and horizon, partly due to the interactive effect of forest and horizon on microbial biomass. Our results suggest that modulator(s) of the potential extracellular activity of phosphatases vary with horizon, depending on the relative C, P and water availability of the horizon. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. PTEN phosphatase-independent maintenance of glandular morphology in a predictive colorectal cancer model system.

    Science.gov (United States)

    Jagan, Ishaan C; Deevi, Ravi K; Fatehullah, Aliya; Topley, Rebecca; Eves, Joshua; Stevenson, Michael; Loughrey, Maurice; Arthur, Kenneth; Campbell, Frederick Charles

    2013-11-01

    Organotypic models may provide mechanistic insight into colorectal cancer (CRC) morphology. Three-dimensional (3D) colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) coupling of cell division cycle 42 (cdc42) to atypical protein kinase C (aPKC). This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM) orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3) were ineffective. The isolated PTEN C2 domain (C2) accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1) in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.

  1. PTEN Phosphatase-Independent Maintenance of Glandular Morphology in a Predictive Colorectal Cancer Model System

    Directory of Open Access Journals (Sweden)

    Ishaan C. Jagan

    2013-11-01

    Full Text Available Organotypic models may provide mechanistic insight into colorectal cancer (CRC morphology. Three-dimensional (3D colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN coupling of cell division cycle 42 (cdc42 to atypical protein kinase C (aPKC. This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3 were ineffective. The isolated PTEN C2 domain (C2 accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na+/H+ exchanger regulatory factor-1 (NHERF-1 in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.

  2. [Activities and properties of glucose-6-phosphatase of turbellaria Phagocata sibirica and cestodes Bothriocephalus scorpii].

    Science.gov (United States)

    Burenina, E A

    2009-01-01

    There were studied activities and properties of mitochondrial and microsomal glucose-6-phosphatases (G6Pases) in free living turbellaria Phagocata sibirica and cestodes Bothriocephalus scorpii. Action of various effectors (sodium fluoride, glucose, HCO3-, citrate, Cu2+, DTT, EDTA, ATP, AFP) on the enzyme activity was studied. The obtained results and literature data demonstrate that G6Pase is present in various muscles of representatives of the animal kingdom. The conclusion can be made that invertebrate G6Pase releases glucose from glycogen and gluconeogenic precursors.

  3. Relation of Serum Alkaline Phosphatase to liver scintigram in patients with hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nishimura, H; Harada, T; Nawata, J; Hayakawa, M; Nishioka, M; Takemoto, T; Yokoyama, T; Takahashi, M

    1982-12-01

    Serum Alkaline Phosphatase (ALP) was studied in relation to liver scintigrams of 54 patients with hepatocellular carcinoma. The ALP activity was higher with larger tumors and in multiple tumors. Within the single tumor group, the activity was higher when the tumor was located in the hilum than in the periphery. The incidence of ALP-1 isoenzyme (bile ALP) roughly paralleled the total ALP activity. These results suggest that the variation of serum ALP seen in each individual patients with hepatocellular carcinoma reflects the volume of cholestatic liver tissue, which is changed by the number, size and localization of the tumor nodules in the liver.

  4. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Czech Academy of Sciences Publication Activity Database

    Macůrek, Libor; Benada, Jan; Müllers, E.; Halim, V.A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Soňa; Hodný, Zdeněk; Lindqvist, A.; Medema, R.H.; Bartek, Jiří

    2013-01-01

    Roč. 12, č. 2 (2013), s. 251-262 ISSN 1538-4101 R&D Projects: GA ČR GPP305/10/P420; GA ČR GAP301/10/1525 Grant - others:Netherlands Genomic Initiative of NWO(NL) CGC; EK(XE) 259893 Institutional support: RVO:68378050 Keywords : DNA damage response * Wip1 phosphatase * cell cycle * mitotic progression * γH2AX Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.006, year: 2013

  5. Detection of extracellular phosphatases in natural spring phytoplankton of a shallow eutrophic lake (Donghu, China)

    Czech Academy of Sciences Publication Activity Database

    Cao, X.; Štrojsová, Alena; Znachor, Petr; Zapomělová, Eliška; Liu, G.; Vrba, Jaroslav; Zhou, Y.

    2005-01-01

    Roč. 40, č. 3 (2005), s. 251-258 ISSN 0967-0262. [International symposium on river and lake environments /12./. Wuhan, 01.11.2004-12.11.2004] R&D Projects: GA AV ČR(CZ) IAA6017202; GA MŠk(CZ) ME 617 Grant - others:Chinese Academy of Sciences(CN) KZCX1-SW-12-II-02-02; National Science Foundation(CN) 39170165, 39670149, 20177033, 2002CB412300 Institutional research plan: CEZ:AV0Z60170517 Keywords : enzyme labelled fluorescence * phosphatase activity * phytoplankton Subject RIV: EH - Ecology, Behaviour Impact factor: 2.064, year: 2005

  6. Effect of perfluorooctane sulfonate on the conformation of wheat germ acid phosphatase.

    Science.gov (United States)

    Xu, Dongmei; Jin, Jianchang; Shen, Tong; Wang, Yanhua

    2013-11-01

    Fluorescence spectroscopy was used to study the quenching mechanism, the type of force and the binding sites of perfluorooctane sulfonate (PFOS) on wheat germ acid phosphatase (ACPase). The results showed that the quenching effect of PFOS on ACPase was mainly due to a static quenching mechanism that occurred via the formation of hydrogen bonds and van der Waals forces. The results from synchronous fluorescence spectroscopy demonstrated that PFOS interacts with ACPase close to the tryptophan residues. In addition, synchronous fluorescence spectroscopy also showed that PFOS increases the hydrophobicity of the microenvironment of the tyrosine residues, hence decreasing the local polarity.

  7. Structural comparisons of two allelic variants of human placental alkaline phosphatase.

    Science.gov (United States)

    Millán, J L; Stigbrand, T; Jörnvall, H

    1985-01-01

    A simple immunosorbent purification scheme based on monoclonal antibodies has been devised for human placental alkaline phosphatase. The two most common allelic variants, S and F, have similar amino acid compositions with identical N-terminal amino acid sequences through the first 13 residues. Both variants have identical lectin binding properties towards concanavalin A, lentil-lectin, wheat germ agglutinin, phytohemagglutinin and soybean agglutinin, and identical carbohydrate contents as revealed by methylation analysis. CNBr fragments of the variants demonstrate identical high performance liquid chromatography patterns. The carbohydrate containing fragment is different from the 32P-labeled active site fragment and the N-terminal fragment.

  8. Secretion of acid phosphatase by axenic Entamoeba histolytica NIH-200 and properties of the extracellular enzyme.

    Science.gov (United States)

    Agrawal, A; Pandey, V C; Kumar, S; Sagar, P

    1989-01-01

    Entamoeba histolytica (NIH-200) secreted large amounts of acid phosphatase in its external environment when grown axenically in modified TPS-II medium. Fractionation by DEAE-cellulose chromatography of the precipitate obtained from the cell-free medium at 60% ammonium sulfate saturation yielded 3 distinct peaks of enzyme activity. The enzyme in all the peaks showed resistance to tartrate but was inhibited by fluoride, cupric chloride, ethylene diamine-tetra acetic acid, ammonium molybdate and cysteine; however, enzyme associated with different peaks differed in its polyacrylamide gel electrophoretic profiles and behavior towards concanavalin A.

  9. Identification of rat serum alkaline phosphatase isoenzyme by means of wheat germ agglutinin.

    Science.gov (United States)

    Wada, H; Niwa, N; Hayakawa, T; Tsuge, H

    1997-01-01

    Wheat germ agglutinin (WGA) precipitates bone type serum alkaline phosphatase (sALP) isoenzyme specifically. The precipitates are composed of the macromolecules of WGA and "bone type sALP" (WGA-ALP complex). In order to use bone type sALP as a marker in polyacrylamide gel electrophoresis (PAGE), a method to separate "bone type sALP" from the "WGA-ALP complex" was established by using N-acetyl-D-glucosamine (GlcNAc)-Sepharose 6E column chromatography. It was concluded that this method is useful for clinical examination in the rat.

  10. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    OpenAIRE

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun

    2011-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, wh...

  11. Unique players in the BMP pathway: Small C-terminal domain phosphatases dephosphorylate Smad1 to attenuate BMP signaling

    Science.gov (United States)

    Knockaert, Marie; Sapkota, Gopal; Alarcón, Claudio; Massagué, Joan; Brivanlou, Ali H.

    2006-01-01

    Smad transcription factors are key signal transducers for the TGF-β/bone morphogenetic protein (BMP) family of cytokines and morphogens. C-terminal serine phosphorylation by TGF-β and BMP membrane receptors drives Smads into the nucleus as transcriptional regulators. Dephosphorylation and recycling of activated Smads is an integral part of this process, which is critical for agonist sensing by the cell. However, the nuclear phosphatases involved have remained unknown. Here we provide functional, biochemical, and embryological evidence identifying the SCP (small C-terminal domain phosphatase) family of nuclear phosphatases as mediators of Smad1 dephosphorylation in the BMP signaling pathway in vertebrates. Xenopus SCP2/Os4 inhibits BMP activity in the presumptive ectoderm and leads to neuralization. In Xenopus embryos, SCP2/Os4 and human SCP1, 2, and 3 cause selective dephosphorylation of Smad1 compared with Smad2, inhibiting BMP- and Smad1-dependent transcription and leading to the induction of the secondary dorsal axis. In human cells, RNAi-mediated depletion of SCP1 and SCP2 increases the extent and duration of Smad1 phosphorylation in response to BMP, the transcriptional action of Smad1, and the strength of endogenous BMP gene responses. The present identification of the SCP family as Smad C-terminal phosphatases sheds light on the events that attenuate Smad signaling and reveals unexpected links to the essential phosphatases that control RNA polymerase II in eukaryotes. PMID:16882717

  12. Spinal motor neuron neuroaxonal spheroids in chronic aluminum neurotoxicity contain phosphatase-resistant high molecular weight neurofilament (NFH).

    Science.gov (United States)

    Gaytan-Garcia, S; Kim, H; Strong, M J

    1996-04-15

    It has previously been shown that a single intracisternal inoculum of AlCl3 in young adult New Zealand white rabbits will induce a dose-dependent phosphatase resistance of high molecular weight neurofilament protein (NFH) that is proportionate to the extent of neurofilamentous inclusion formation (Strong and Jakowec, 1994). To determine if the potential for dissolution of aluminum-induced neurofilamentous inclusions was dependent on the degree of NFH phosphatase resistance, we have examined NFH phosphatase sensitivity in a reversible chronic model of aluminum neurotoxicity. Rabbits receiving repeated intracisternal inoculums of 100 microgram AlCl3 at 28 day intervals until day 267 develop spinal motor neuron perikaryal and neuroaxonal neurofilamentous aggregates in a stereotypic, dose-dependent fashion. In the rabbits receiving inoculums until day 156 with survival until day 267 without further aluminum exposure, neuroaxonal spheroids remained prominent while perikaryal inclusions largely resolved. Immunoreactivity to a monoclonal antibody recognizing phosphorylated NFH (SMI 31) was abolished in perikaryal aggregates at each time interval by dephosphorylation with bovine alkaline phosphatase. However, neuroaxonal spheroids maintained their immunoreactivity. Using time-course dephosphorylation studies of spinal cord homogenates, we observed a significant reduction in the rate of dephosphorylation of NFH following 267 days of AlCl3 exposure (P < 0.05). These observations suggest that neuroaxonal spheroids contain phosphatase-resistant NFH isoforms and that the potential for resolution of intraneuronal neurofilamentous inclusions correlates with the susceptibility of NF within these inclusions to enzymatic dephosphorylation.

  13. Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiae overexpressing alkaline phosphatase.

    Science.gov (United States)

    Semkiv, Marta V; Dmytruk, Kostyantyn V; Abbas, Charles A; Sibirny, Andriy A

    2014-05-15

    The production of ethyl alcohol by fermentation represents the largest scale application of Saccharomyces cerevisiae in industrial biotechnology. Increased worldwide demand for fuel bioethanol is anticipated over the next decade and will exceed 200 billion liters from further expansions. Our working hypothesis was that the drop in ATP level in S. cerevisiae cells during alcoholic fermentation should lead to an increase in ethanol production (yield and productivity) with a greater amount of the utilized glucose converted to ethanol. Our approach to achieve this goal is to decrease the intracellular ATP level via increasing the unspecific alkaline phosphatase activity. Intact and truncated versions of the S. cerevisiae PHO8 gene coding for vacuolar or cytosolic forms of alkaline phosphatase were fused with the alcohol dehydrogenase gene (ADH1) promoter. The constructed expression cassettes used for transformation vectors also contained the dominant selective marker kanMX4 and S. cerevisiae δ-sequence to facilitate multicopy integration to the genome. Laboratory and industrial ethanol producing strains BY4742 and AS400 overexpressing vacuolar form of alkaline phosphatase were characterized by a slightly lowered intracellular ATP level and biomass accumulation and by an increase in ethanol productivity (13% and 7%) when compared to the parental strains. The strains expressing truncated cytosolic form of alkaline phosphatase showed a prolonged lag-phase, reduced biomass accumulation and a strong defect in ethanol production. Overexpression of vacuolar alkaline phosphatase leads to an increased ethanol yield in S. cerevisiae.

  14. Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8

    NARCIS (Netherlands)

    Camargo, de Brenda R.; Claassens, Nico J.; Quirino, Betania Ferraz; Noronha, Eliane F.; Kengen, Servé W.M.

    2018-01-01

    An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose

  15. Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

    Science.gov (United States)

    The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

  16. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Witters, L.A.; Bacon, G.W.

    1985-01-01

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

  17. Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

    International Nuclear Information System (INIS)

    Hristov, K.; Mitev, V.; Knox, K.

    2006-01-01

    Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

  18. Depotentiation from potentiated synaptic strength in a tristable system of coupled phosphatase and kinase

    Directory of Open Access Journals (Sweden)

    Mengjiao Chen

    2016-10-01

    Full Text Available Long-term potentiation (LTP of synaptic strength is strongly implicated in learning and memory. On the other hand, depotentiation, the reversal of synaptic strength from potentiated LTP state to the pre-LTP level, is required in extinction of the obsolete memory. A generic tristable system, which couples the phosphatase and kinase switches, exclusively explains how moderate and high elevation of intracellular calcium concentration triggers long-term depression (LTD and LTP, respectively. The present study, introducing calcium influx and calcium release from internal store into the tristable system, further show that significant elevation of cytoplasmic calcium concentration switches activation of both kinase and phosphatase to their basal states, thereby depotentiate the synaptic strength. A phase-plane analysis of the combined model was employed to explain the previously reported depotentiation in experiments and predict a threshold-like effect with calcium concentration. The results not only reveal a mechanism of NMDAR- and mGluR-dependent depotentiation, but also predict further experiments about the role of internal calcium store in induction of depotentiation and extinction of established memories.

  19. A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

    Directory of Open Access Journals (Sweden)

    Sarah E Herlihy

    Full Text Available In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

  20. A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

    Science.gov (United States)

    Herlihy, Sarah E; Tang, Yitai; Gomer, Richard H

    2013-01-01

    In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

  1. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer.

    Science.gov (United States)

    Thompson, Joshua J; Williams, Christopher S

    2018-02-26

    Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a heterotrimeric serine-threonine phosphatase-composed of a structural, regulatory, and catalytic subunit-that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components-especially the multitude of regulatory subunits-has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  2. A family of metal-dependent phosphatases implicated in metabolite damage-control

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Lili; Khusnutdinova, Anna; Nocek, Boguslaw; Brown, Greg; Xu, Xiaohui; Cui, Hong; Petit, Pierre; Flick, Robert; Zallot, Rémi; Balmant, Kelly; Ziemak, Michael J.; Shanklin, John; de Crécy-Lagard, Valérie; Fiehn, Oliver; Gregory, Jesse F.; Joachimiak, Andrzej; Savchenko, Alexei; Yakunin, Alexander F.; Hanson, Andrew D.

    2016-06-20

    DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.

  3. Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine

    Science.gov (United States)

    Zylka, Mark J.; Sowa, Nathaniel A.; Taylor-Blake, Bonnie; Twomey, Margaret A.; Herrala, Annakaisa; Voikar, Vootele; Vihko, Pirkko

    2008-01-01

    SUMMARY Thiamine monophosphatase (TMPase, also known as Fluoride-Resistant Acid Phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of Prostatic Acid Phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent anti-nociceptive, anti-hyperalgesic and anti-allodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5’-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain. PMID:18940592

  4. The Phosphatase Dusp7 Drives Meiotic Resumption and Chromosome Alignment in Mouse Oocytes

    Directory of Open Access Journals (Sweden)

    Thomas Tischer

    2016-10-01

    Full Text Available Mammalian oocytes are stored in the ovary, where they are arrested in prophase for prolonged periods. The mechanisms that abrogate the prophase arrest in mammalian oocytes and reinitiate meiosis are not well understood. Here, we identify and characterize an essential pathway for the resumption of meiosis that relies on the protein phosphatase DUSP7. DUSP7-depleted oocytes either fail to resume meiosis or resume meiosis with a significant delay. In the absence of DUSP7, Cdk1/CycB activity drops below the critical level required to reinitiate meiosis, precluding or delaying nuclear envelope breakdown. Our data suggest that DUSP7 drives meiotic resumption by dephosphorylating and thereby inactivating cPKC isoforms. In addition to controlling meiotic resumption, DUSP7 has a second function in chromosome segregation: DUSP7-depleted oocytes that enter meiosis show severe chromosome alignment defects and progress into anaphase prematurely. Altogether, these findings establish the phosphatase DUSP7 as an essential regulator of multiple steps in oocyte meiosis.

  5. Electro-chemical coupling in the voltage-dependent phosphatase Ci-VSP

    Science.gov (United States)

    Kohout, Susy C.; Bell, Sarah C.; Liu, Lijun; Xu, Qiang; Minor, Daniel L.; Isacoff, Ehud Y.

    2010-01-01

    In the voltage sensing phosphatase, Ci-VSP, a voltage sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we find that the inter-domain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays show that the linker is also needed for activity in the isolated PD. We identify a late step of VSD motion in the full-length protein that depends on the linker. Strikingly, this VSD motion is found to require PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme. PMID:20364128

  6. Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

    International Nuclear Information System (INIS)

    Dowling, J.N.; Saha, A.K.; Glew, R.H.

    1987-01-01

    Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP 3 ) was explored. When neutrophil phosphoinositides were labeled with 32 P, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP 2 ) over 2 h. Treatment of [ 3 H]inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP 2 . Following fMLP stimulation, the fractional reduction in PIP 2 and the fractional increase in IP 3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP 3 was reduced by ACP pre-treatment. The reduction in IP 3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP 2 available for hydrolysis. However, some loss of IP 3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP 2 , the prognitor of IP 3 , and by hydrolyzing IP 3 itself

  7. Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

    Science.gov (United States)

    Szumańska, G; Gadamski, R

    1992-01-01

    Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

  8. Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

    Science.gov (United States)

    Van Etten, R L; Waymack, P P

    1991-08-01

    The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

  9. Human prostatic acid phosphatase: purification, characterization, and optimization of conditions for radioimmunoassay

    International Nuclear Information System (INIS)

    McCarthy, R.C.; Jakubowski, H.V.; Markowitz, H.

    1983-01-01

    Prostatic acid phosphatase was isolated from benign hypertrophic prostate tissue by ammonium sulfate precipitation and affinity chromatography procedures. The purified enzyme was characterized by two-dimensional gel electrophoresis and shown to have a cluster of protein spots with an apparent molecular weight of 48000 at pI 5.9 to 6.3 in 9 mol/l urea. The specific activity of the purified enzyme was 723 and 659 U/mg protein with α-naphthyl phosphate at 30 0 C and para-nitrophenyl phosphate at 37 0 C respectively. An antibody to the purified enzyme was raised in rabbits and used in a radioimmunoassay (RIA). The use of a phosphate buffer, pH 6.6, and iodination of prostatic acid phosphatase (PAP) by the Bolton-Hunter procedure improved the precision of the assay when compared to RIA's using a phosphate buffer, pH 7.0 or 7.3, or PAP iodinated by a chloramine-T procedure. The former RIA displaced 50% of the tracer at 2 μg of enzyme per liter of serum. The between-run coefficient of variation for 11 assays ranged from 3.9-7.7% with serum at 1.3 to 5.6 μg PAP/l. (Auth.)

  10. Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

    Science.gov (United States)

    Rayaprolu, Vamseedhar; Royal, Perrine; Stengel, Karen; Sandoz, Guillaume; Kohout, Susy C

    2018-05-07

    Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P 3 or PI(3,4)P 2 Our results indicate that dimerization plays a significant role in Ci-VSP function. © 2018 Rayaprolu et al.

  11. Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

    Directory of Open Access Journals (Sweden)

    José Luis Carrasco

    Full Text Available Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6, a previously reported DBP1 interactor, and MAP kinase (MAPK MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV, and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

  12. Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase, with Potential for Biotechnological Application.

    Science.gov (United States)

    Souza, Amanda Araújo; Leitão, Vanessa Oliveira; Ramada, Marcelo Henrique; Mehdad, Azadeh; Georg, Raphaela de Castro; Ulhôa, Cirano José; de Freitas, Sonia Maria

    2016-01-01

    Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65 °C, respectively, and is stable at 55 °C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0 was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.

  13. Expression of yeast lipid phosphatase Sac1p is regulated by phosphatidylinositol-4-phosphate

    Directory of Open Access Journals (Sweden)

    Mayinger Peter

    2008-01-01

    Full Text Available Abstract Background Phosphoinositides play a central role in regulating processes at intracellular membranes. In yeast, a large number of phospholipid biosynthetic enzymes use a common mechanism for transcriptional regulation. Yet, how the expression of genes encoding lipid kinases and phosphatases is regulated remains unknown. Results Here we show that the expression of lipid phosphatase Sac1p in the yeast Saccharomyces cerevisiae is regulated in response to changes in phosphatidylinositol-4-phosphate (PI(4P concentrations. Unlike genes encoding enzymes involved in phospholipid biosynthesis, expression of the SAC1 gene is independent of inositol levels. We identified a novel 9-bp motif within the 5' untranslated region (5'-UTR of SAC1 that is responsible for PI(4P-mediated regulation. Upregulation of SAC1 promoter activity correlates with elevated levels of Sac1 protein levels. Conclusion Regulation of Sac1p expression via the concentration of its major substrate PI(4P ensures proper maintenance of compartment-specific pools of PI(4P.

  14. Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

    Directory of Open Access Journals (Sweden)

    Anoushka Davé

    2014-04-01

    Full Text Available The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.

  15. Characterization of a unique class C acid phosphatase from Clostridium perfringens.

    Science.gov (United States)

    Reilly, Thomas J; Chance, Deborah L; Calcutt, Michael J; Tanner, John J; Felts, Richard L; Waller, Stephen C; Henzl, Michael T; Mawhinney, Thomas P; Ganjam, Irene K; Fales, William H

    2009-06-01

    Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

  16. Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens▿

    Science.gov (United States)

    Reilly, Thomas J.; Chance, Deborah L.; Calcutt, Michael J.; Tanner, John J.; Felts, Richard L.; Waller, Stephen C.; Henzl, Michael T.; Mawhinney, Thomas P.; Ganjam, Irene K.; Fales, William H.

    2009-01-01

    Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was ∼31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3′ and 5′ nucleoside monophosphates at pH 6. Calculated Kms ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 μmol of Pi/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class. PMID:19363079

  17. Prognostic role of serum prostatic acid phosphatase for 103Pd-based radiation for prostatic carcinoma

    International Nuclear Information System (INIS)

    Dattoli, Michael; Wallner, Kent; True, Lawrence; Sorace, Richard; Koval, John; Cash, Jennifer; Acosta, Rudolph; Biswas, Mohendra; Binder, Michael; Sullivan, Brent; Lastarria, Emilio; Kirwan, Novelle; Stein, Douglas

    1999-01-01

    Purpose: To establish the prognostic role of serum enzymatic prostatic acid phosphatase (PAP) in patients treated with palladium ( 103 Pd) and supplemental external beam irradiation (EBRT) for clinically localized, high-risk prostate carcinoma. Methods and Materials: One hundred twenty-four consecutive patients with Stage T2a-T3 prostatic carcinoma were treated from 1992 through 1995. Each patient had at least one of the following risk factors for extracapsular disease extension: Stage T2b or greater (100 patients), Gleason score 7-10 (40 patients), pretreatment prostate specific antigen (PSA) > 15 ng/ml (32 patients), or elevated serum PAP (25 patients). Patients received 41 Gy conformal EBRT to a limited pelvic field, followed 4 weeks later by a 103 Pd boost (prescription dose 80 Gy). Biochemical failure was defined as a PSA greater than 1 ng/ml (normal < 4 ng/ml). Results: The overall, actuarial freedom from biochemical failure at 4 years after treatment was 79%. In Cox-proportional hazard multivariate analysis, the strongest predictor of failure was elevated pretreatment acid phosphatase (p = 0.02), followed by Gleason score (p = 0.1), and PSA (p = 0.14). Conclusion: PAP was the strongest predictor of long-term biochemical failure. It may be a more accurate indicator of micrometastatic disease than PSA, and as such, we suggest that it be reconsidered for general use in radiation-treated patients

  18. SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Sung Jun [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Ni, Lisheng [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Osinski, Adam [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Tomchick, Diana R. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Brautigam, Chad A. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States; Luo, Xuelian [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

    2017-10-24

    The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1–MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.

  19. Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

    Science.gov (United States)

    Ma, Guang Xu; Zhou, Rong Qiong; Hu, Shi Jun; Huang, Han Cheng; Zhu, Tao; Xia, Qing You

    2014-06-01

    Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Robotic implementation of assays: tissue-nonspecific alkaline phosphatase (TNAP) case study.

    Science.gov (United States)

    Chung, Thomas D Y

    2013-01-01

    Laboratory automation and robotics have "industrialized" the execution and completion of large-scale, enabling high-capacity and high-throughput (100 K-1 MM/day) screening (HTS) campaigns of large "libraries" of compounds (>200 K-2 MM) to complete in a few days or weeks. Critical to the success these HTS campaigns is the ability of a competent assay development team to convert a validated research-grade laboratory "benchtop" assay suitable for manual or semi-automated operations on a few hundreds of compounds into a robust miniaturized (384- or 1,536-well format), well-engineered, scalable, industrialized assay that can be seamlessly implemented on a fully automated, fully integrated robotic screening platform for cost-effective screening of hundreds of thousands of compounds. Here, we provide a review of the theoretical guiding principles and practical considerations necessary to reduce often complex research biology into a "lean manufacturing" engineering endeavor comprising adaption, automation, and implementation of HTS. Furthermore we provide a detailed example specifically for a cell-free in vitro biochemical, enzymatic phosphatase assay for tissue-nonspecific alkaline phosphatase that illustrates these principles and considerations.

  1. Phosphatase of Regenerating Liver-3 Promotes Motility and Metastasis of Mouse Melanoma Cells

    Science.gov (United States)

    Wu, Xiaopeng; Zeng, Hu; Zhang, Xianming; Zhao, Ying; Sha, Haibo; Ge, Xiaomei; Zhang, Minyue; Gao, Xiang; Xu, Qiang

    2004-01-01

    Recent reports suggested that phosphatase of regenerating liver (PRL)-3 might be involved in colorectal carcinoma metastasis with an unknown mechanism. Here we demonstrated that PRL-3 expression was up-regulated in human liver carcinoma compared with normal liver. PRL-3 was also highly expressed in metastatic melanoma B16-BL6 cells but not in its lowly metastatic parental cell line, B16 cells. B16 cells transfected with PRL-3 cDNA displayed morphological transformation from epithelial-like shape to fibroblast-like shape. PRL-3-overexpressed cells showed much higher migratory ability, which could be reversed by specific anti-sense oligodeoxynucleotide and the phosphatase inhibitors sodium orthovanadate or potassium bisperoxo oxovanadate V. Meanwhile, the expression of the catalytically inactive PRL-3 mutations (D72A or C104S) significantly reduced the cell migratory capability. In addition, PRL-3 transfectants demonstrated altered extracellular matrix adhesive property and up-regulated integrin-mediated cell spreading efficiency. Furthermore, we confirmed that PRL-3 could facilitate lung and liver metastasis of B16 cells in an experimental metastasis model in mice, consistent with accelerated proliferation and growth rate both in vitro and in vivo. Together, these observations provide convincing evidence that PRL-3 truly plays a causal role in tumor metastasis. PMID:15161639

  2. Expression, prognostic significance and mutational analysis of protein tyrosine phosphatase SHP-1 in chronic myeloid leukemia.

    Science.gov (United States)

    Papadopoulou, Vasiliki; Kontandreopoulou, Elina; Panayiotidis, Panayiotis; Roumelioti, Maria; Angelopoulou, Maria; Kyriazopoulou, Lydia; Diamantopoulos, Panagiotis T; Vaiopoulos, George; Variami, Eleni; Kotsianidis, Ioannis; Athina Viniou, Nora

    2016-05-01

    The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone.

  3. Alkaline phosphatase binds tenaciously to titanium; implications for biological surface evaluation following bone implant retrieval.

    Science.gov (United States)

    Mansell, J P; Shiel, A I; Harwood, C; Stephens, D

    2017-07-01

    Enhancing the performance and longevity of titanium (Ti) implants continues to be a significant developmental theme in contemporary biomaterials design. Our specific focus pertains to the surface functionalisation of Ti using the bioactive lipid, lysophosphatidic acid (LPA) and certain phosphatase-resistant analogues of LPA. Coating survivorship to a plethora of testing regimens is required to align with due regulatory process before novel biomaterials can enter clinical trials. One of the key acceptance criteria is coating retention to the physical stresses experienced during implantation. In assessing coating stability to insertion into porcine bone we found that a subsequent in vitro assessment to confirm coating persistence was masked by abundant alkaline phosphatase (ALP) contamination adsorbed to the metal surface. Herein we report that ALP can bind to Ti in a matter of minutes by simply immersing Ti samples in aqueous solutions of the enzyme. We strongly discourage the in vitro monitoring of osteoblast and stromal cell ALP expression when assessing bioactive coating survivorship following Ti implant retrieval form native bone tissue. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Phosphotyrosine phosphatase and tyrosine kinase inhibition modulate airway pressure-induced lung injury.

    Science.gov (United States)

    Parker, J C; Ivey, C L; Tucker, A

    1998-11-01

    We determined whether drugs which modulate the state of protein tyrosine phosphorylation could alter the threshold for high airway pressure-induced microvascular injury in isolated perfused rat lungs. Lungs were ventilated for successive 30-min periods with peak inflation pressures (PIP) of 7, 20, 30, and 35 cmH2O followed by measurement of the capillary filtration coefficient (Kfc), a sensitive index of hydraulic conductance. In untreated control lungs, Kfc increased by 1.3- and 3.3-fold relative to baseline (7 cmH2O PIP) after ventilation with 30 and 35 cmH2O PIP. However, in lungs treated with 100 microM phenylarsine oxide (a phosphotyrosine phosphatase inhibitor), Kfc increased by 4.7- and 16.4-fold relative to baseline at these PIP values. In lungs treated with 50 microM genistein (a tyrosine kinase inhibitor), Kfc increased significantly only at 35 cmH2O PIP, and the three groups were significantly different from each other. Thus phosphotyrosine phosphatase inhibition increased the susceptibility of rat lungs to high-PIP injury, and tyrosine kinase inhibition attenuated the injury relative to the high-PIP control lungs.

  5. Field-Evolved Mode 1 Resistance of the Fall Armyworm to Transgenic Cry1Fa-Expressing Corn Associated with Reduced Cry1Fa Toxin Binding and Midgut Alkaline Phosphatase Expression

    Science.gov (United States)

    Jakka, Siva R. K.; Gong, Liang; Hasler, James; Banerjee, Rahul; Sheets, Joel J.; Narva, Kenneth; Blanco, Carlos A.

    2015-01-01

    Insecticidal protein genes from the bacterium Bacillus thuringiensis (Bt) are expressed by transgenic Bt crops (Bt crops) for effective and environmentally safe pest control. The development of resistance to these insecticidal proteins is considered the most serious threat to the sustainability of Bt crops. Resistance in fall armyworm (Spodoptera frugiperda) populations from Puerto Rico to transgenic corn producing the Cry1Fa insecticidal protein resulted, for the first time in the United States, in practical resistance, and Bt corn was withdrawn from the local market. In this study, we used a field-collected Cry1Fa corn-resistant strain (456) of S. frugiperda to identify the mechanism responsible for field-evolved resistance. Binding assays detected reduced Cry1Fa, Cry1Ab, and Cry1Ac but not Cry1Ca toxin binding to midgut brush border membrane vesicles (BBMV) from the larvae of strain 456 compared to that from the larvae of a susceptible (Ben) strain. This binding phenotype is descriptive of the mode 1 type of resistance to Bt toxins. A comparison of the transcript levels for putative Cry1 toxin receptor genes identified a significant downregulation (>90%) of a membrane-bound alkaline phosphatase (ALP), which translated to reduced ALP protein levels and a 75% reduction in ALP activity in BBMV from 456 compared to that of Ben larvae. We cloned and heterologously expressed this ALP from susceptible S. frugiperda larvae and demonstrated that it specifically binds with Cry1Fa toxin. This study provides a thorough mechanistic description of field-evolved resistance to a transgenic Bt crop and supports an association between resistance and reduced Cry1Fa toxin binding and levels of a putative Cry1Fa toxin receptor, ALP, in the midguts of S. frugiperda larvae. PMID:26637593

  6. Cloning of soluble alkaline phosphatase cDNA and molecular basis of the polymorphic nature in alkaline phosphatase isozymes of Bombyx mori midgut.

    Science.gov (United States)

    Itoh, M; Kanamori, Y; Takao, M; Eguchi, M

    1999-02-01

    A cDNA coding for soluble type alkaline phosphatase (sALP) of Bombyx mori was isolated. Deduced amino acid sequence showed high identities to various ALPs and partial similarities to ATPase of Manduca sexta. Using this cDNA sequence as a probe, the molecular basis of electrophoretic polymorphism in sALP and membrane-bound type ALP (mALP) was studied. As for mALP, the result suggested that post-translational modification was important for the proteins to express activity and to represent their extensive polymorphic nature, whereas the magnitude of activities was mainly regulated by transcription. On the other hand, sALP zymogram showed poor polymorphism, but one exception was the null mutant, in which the sALP gene was largely lost. Interestingly, the sALP gene was shown to be transcribed into two mRNAs of different sizes, 2.0 and 2.4 Kb. In addition to the null mutant of sALP, we found a null mutant for mALP. Both of these mutants seem phenotypically silent, suggesting that the functional differentiation between these isozymes is not perfect, so that they can still work mutually and complement each other as an indispensable enzyme for B. mori.

  7. [Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

    Science.gov (United States)

    Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

    2012-01-01

    In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

  8. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  9. Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

    International Nuclear Information System (INIS)

    Morrow, Peter W.; Tung, H.Y. Lim; Hemmings, Hugh C.

    2004-01-01

    Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A 1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD 4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA

  10. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David  S.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard  J.; Ferguson, David  J.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan  H.; Mohamed, Alyaa  M.A.H.; Radhakrishnan, Anand; Arold, Stefan T.; Tate, Edward  W.; Holder, Anthony  A.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-01-01

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  11. Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima.

    Science.gov (United States)

    Koller, E; Wolfbeis, O S

    1984-11-15

    A direct and continuous kinetic method for the photometric and fluorometric determination of various acid phosphatases is described. It is based on new coumarin-derived phosphates, which after enzymatic hydrolysis undergo dissociation to form intensely colored and strongly fluorescent phenolate anions. The latter have absorption maxima ranging from 385 to 505 nm, and fluorescence maxima between 470 and 595 nm. The new substrates were compared with respect to their rate of enzymatic hydrolysis, optimum pH, and detection limits of acid phosphatase from potato and wheat germ. Detection limits of 0.001 unit/ml were found by photometry, and as low as 0.00006 unit/ml by fluorometry. The principal advantages of the new substrates over existing ones are longwave absorptions and emissions, large Stokes shifts, and the low pKa values of the corresponding phenols, thus allowing a direct and continuous assay of acid phosphatase even in weakly acidic solutions.

  12. Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: synthesis, phosphatase inhibition activity and cellular uptake studies.

    Science.gov (United States)

    Collins, Juliet; Cilibrizzi, Agostino; Fedorova, Marina; Whyte, Gillian; Mak, Lok Hang; Guterman, Inna; Leatherbarrow, Robin; Woscholski, Rudiger; Vilar, Ramon

    2016-04-28

    Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligands. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration than the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.

  13. Putative Biomarkers and Targets of Estrogen Receptor Negative Human Breast Cancer

    Directory of Open Access Journals (Sweden)

    Stephen W. Byers

    2011-07-01

    Full Text Available Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER, progesterone receptor (PR, and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.

  14. The solution structure of ChaB, a putative membrane ion antiporter regulator from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Iannuzzi Pietro

    2004-08-01

    Full Text Available Abstract Background ChaB is a putative regulator of ChaA, a Na+/H+ antiporter that also has Ca+/H+ activity in E. coli. ChaB contains a conserved 60-residue region of unknown function found in other bacteria, archaeabacteria and a series of baculoviral proteins. As part of a structural genomics project, the structure of ChaB was elucidated by NMR spectroscopy. Results The structure of ChaB is composed of 3 α-helices and a small sheet that pack tightly to form a fold that is found in the cyclin-box family of proteins. Conclusion ChaB is distinguished from its putative DNA binding sequence homologues by a highly charged flexible loop region that has weak affinity to Mg2+ and Ca2+ divalent metal ions.

  15. Exploring Universal Partnerships and Putative Marriages as Tools for Awarding Partnership Property in Contemporary Family Law

    Directory of Open Access Journals (Sweden)

    Elsje Bonthuys

    2016-12-01

    Full Text Available Following upon the Supreme Court of Appeal's judgment in Butters v Mncora 2012 4 SA 1 (SCA, which broadened the criteria and consequences of universal partnerships in cohabitation relationships, this article investigates the potential of universal partnerships and putative marriages to allocate rights to share in partnership property in other intimate relationships. It traverses several instances in which marriages are not recognised - bigamous marriages, Muslim and Hindu religious marriages and invalid customary marriages – examining whether the wives in these marriages could use universal partnerships and putative marriages to claim a share in property. It then considers the use of universal partnerships to obtain a share of property in civil marriages out of community of property. It concludes by pointing out several issues which are in need of clarification and where the common law should be developed to give effect to fundamental constitutional rights.

  16. Crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from Thermus thermophilus

    International Nuclear Information System (INIS)

    Nakamura, Tsutomu; Kashima, Yasuhiro; Mine, Shouhei; Oku, Takashi; Uegaki, Koichi

    2011-01-01

    In this study, a putative glucokinase/hexokinase from T. thermophilus was purified and crystallized. Diffraction data were collected and processed to 2.02 Å resolution. Glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. The open reading frame TTHA0299 of the extreme thermophile Thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. In this study, the glucokinase/hexokinase from T. thermophilus was purified and crystallized using polyethylene glycol 8000 as a precipitant. Diffraction data were collected and processed to 2.02 Å resolution. The crystal belonged to space group P2 1 , with unit-cell parameters a = 70.93, b = 138.14, c = 75.16 Å, β = 95.41°

  17. A putative ABC transporter is involved in negative regulation of biofilm formation by Listeria monocytogenes

    DEFF Research Database (Denmark)

    Zhu, Xinna; Long, Fei; Chen, Yonghui

    2008-01-01

    Listeria monocytogenes may persist for long periods in food processing environments. In some instances, this may be due to aggregation or biofilm formation. To investigate the mechanism controlling biofilm formation in the food-borne pathogen L. monocytogenes, we characterized LM-49, a mutant...... with enhanced ability of biofilm-formation generated via transposon Tn917 mutagenesis of L. monocytogenes 4b G. In this mutant, a Tn917 insertion has disrupted the coding region of the gene encoding a putative ATP binding cassette (ABC) transporter permease identical to Lmof2365_1771 (a putative ABC...... the same amount of biofilm biomass as the wild-type strain. Furthermore, transcription of the downstream lm.G_1770 was not influenced by the upstream Tn917 insertion, and the presence of Tn917 has no effect on biofilm formation. These results suggest that lm.G_1771 was solely responsible for the negative...

  18. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi

    DEFF Research Database (Denmark)

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar

    2015-01-01

    Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim...... of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug......-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874...

  19. Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13

    OpenAIRE

    Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J.; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J.; Dimitrov, Kiril M.; Afonso, Claudio L.

    2016-01-01

    Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13.

  20. Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13

    Science.gov (United States)

    Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J.; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J.; Dimitrov, Kiril M.

    2016-01-01

    Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13. PMID:27469958

  1. Evaluation of two putative susceptibility loci for oral clefts in the Danish population

    DEFF Research Database (Denmark)

    Mitchell, L E; Murray, J C; O'Brien, S

    2001-01-01

    . The present study evaluated potential associations between CL+/-P and CP and two putative clefting susceptibility loci, MSX1 and TGFB3, using data from a nationwide case-control study conducted in Denmark from 1991 to 1994. The potential effects of interactions between these genes and two common environmental......-environment interactions involving MSX1 or TGFB3 and either first trimester exposure to maternal cigarette smoke or alcohol consumption....

  2. Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

    Science.gov (United States)

    Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A; Barry, Kerrie W; Spatafora, Joseph; Grigoriev, Igor V; Martin, Francis M; Pujade-Renaud, Valérie

    2018-01-01

    Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species ( Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca , and Botrosphaeria dothidea ) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

  3. Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action

    DEFF Research Database (Denmark)

    Lucas, Guillaume; Rymar, Vladimir V; Du, Jenny

    2007-01-01

    parameters considered to be key markers of antidepressant action, but that are observed only after 2-3 week treatments with classical molecules: desensitization of 5-HT(1A) autoreceptors, increased tonus on hippocampal postsynaptic 5-HT(1A) receptors, and enhanced phosphorylation of the CREB protein...... intake consecutive to a chronic mild stress. These findings point out 5-HT(4) receptor agonists as a putative class of antidepressants with a rapid onset of action. Udgivelsesdato: 2007-Sep-6...

  4. Memory-guided sensory comparisons in the prefrontal cortex: contribution of putative pyramidal cells and interneurons.

    Science.gov (United States)

    Hussar, Cory R; Pasternak, Tatiana

    2012-02-22

    Comparing two stimuli that occur at different times demands the coordination of bottom-up and top-down processes. It has been hypothesized that the dorsolateral prefrontal (PFC) cortex, the likely source of top-down cortical influences, plays a key role in such tasks, contributing to both maintenance and sensory comparisons. We examined this hypothesis by recording from the PFC of monkeys comparing directions of two moving stimuli, S1 and S2, separated by a memory delay. We determined the contribution of the two principal cell types to these processes by classifying neurons into broad-spiking (BS) putative pyramidal cells and narrow-spiking (NS) putative local interneurons. During the delay, BS cells were more likely to exhibit anticipatory modulation and represent the remembered direction. While this representation was transient, appearing at different times in different neurons, it weakened when direction was not task relevant, suggesting its utility. During S2, both putative cell types showed comparison-related activity modulations. These modulations were of two types, each carried by different neurons, which either preferred trials with stimuli moving in the same direction or trials with stimuli of different directions. These comparison effects were strongly correlated with choice, suggesting their role in circuitry underlying decision making. These results provide the first demonstration of distinct contributions made by principal cell types to memory-guided perceptual decisions. During sensory stimulation both cell types represent behaviorally relevant stimulus features contributing to comparison and decision-related activity. However in the absence of sensory stimulation, putative pyramidal cells dominated, carrying information about the elapsed time and the preceding direction.

  5. Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

    Directory of Open Access Journals (Sweden)

    David Lopez

    2018-03-01

    Full Text Available Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

  6. Identification of a putative nuclear export signal motif in human NANOG homeobox domain

    International Nuclear Information System (INIS)

    Park, Sung-Won; Do, Hyun-Jin; Huh, Sun-Hyung; Sung, Boreum; Uhm, Sang-Jun; Song, Hyuk; Kim, Nam-Hyung; Kim, Jae-Hwan

    2012-01-01

    Highlights: ► We found the putative nuclear export signal motif within human NANOG homeodomain. ► Leucine-rich residues are important for human NANOG homeodomain nuclear export. ► CRM1-specific inhibitor LMB blocked the potent human NANOG NES-mediated nuclear export. -- Abstract: NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ( 125 MQELSNILNL 134 ) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-ΔNLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

  7. IMMOBILIZATION OF ACID PHOSPHATASE (TYPE I) FROM WHEAT GERM ON GLUTARALDEHYDE ACTIVATED CHITOSAN BEADS: OPTIMIZATION AND CHARACTERIZATION

    OpenAIRE

    K. Belho; S.R. Nongpiur; P.K. Ambasht

    2014-01-01

    Acid phosphatase from wheat germ (specific activity 1.327 U/mg protein) was used for immobilization on glutaraldehyde activated chitosan beads. Upon activation of chitosan beads, elongated fibers with pores were observed. The optimum percent immobilization obtained was 81.25%. The pH optimum of immobilized acid phosphatase was 5.5 with a shift of 0.5 units from the pH optimum of soluble enzyme (5.0). The values of Km for p-nitrophenylphosphate with soluble and immobilized acid pho...

  8. Effects of drugs of abuse on putative rostromedial tegmental neurons, inhibitory afferents to midbrain dopamine cells.

    Science.gov (United States)

    Lecca, Salvatore; Melis, Miriam; Luchicchi, Antonio; Ennas, Maria Grazia; Castelli, Maria Paola; Muntoni, Anna Lisa; Pistis, Marco

    2011-02-01

    Recent findings have underlined the rostromedial tegmental nucleus (RMTg), a structure located caudally to the ventral tegmental area, as an important site involved in the mechanisms of aversion. RMTg contains γ-aminobutyric acid neurons responding to noxious stimuli, densely innervated by the lateral habenula and providing a major inhibitory projection to reward-encoding midbrain dopamine (DA) neurons. One of the key features of drug addiction is the perseverance of drug seeking in spite of negative and unpleasant consequences, likely mediated by response suppression within neural pathways mediating aversion. To investigate whether the RMTg has a function in the mechanisms of addicting drugs, we studied acute effects of morphine, cocaine, the cannabinoid agonist WIN55212-2 (WIN), and nicotine on putative RMTg neurons. We utilized single unit extracellular recordings in anesthetized rats and whole-cell patch-clamp recordings in brain slices to identify and characterize putative RMTg neurons and their responses to drugs of abuse. Morphine and WIN inhibited both firing rate in vivo and excitatory postsynaptic currents (EPSCs) evoked by stimulation of rostral afferents in vitro, whereas cocaine inhibited discharge activity without affecting EPSC amplitude. Conversely, nicotine robustly excited putative RMTg neurons and enhanced EPSCs, an effect mediated by α7-containing nicotinic acetylcholine receptors. Our results suggest that activity of RMTg neurons is profoundly influenced by drugs of abuse and, as important inhibitory afferents to midbrain DA neurons, they might take place in the complex interplay between the neural circuits mediating aversion and reward.

  9. Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study.

    Science.gov (United States)

    Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

    2016-06-15

    Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis [Pg], Aggregatibacer actinomycetemcomitans [Aa], Fusobacterium nucleatum [Fn], Prevotella intermedia [Pi], Tannerella forsythia [Tf], and Treponema denticola [Td]) and three herpesviruses (Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], and herpes simplex virus [HSV]) were detected. Socio-demographic data and oral health related behaviors, and salivary estradiol and progesterone levels were also collected. The results showed no significant differences in socio-demographic background, oral health related behaviors, and salivary estradiol and progesterone levels between the two groups (all P > 0.05). The detection rates of included periodontopathic microorganisms were not significantly different between the two groups (all P > 0.05), but the coinfection rate of EBV and Pg was significantly higher in the case group than in the control group (P = 0.028). EBV and Pg coinfection may promote the development of chronic periodontitis among pregnant women.

  10. [Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

    Science.gov (United States)

    Petrova, L P; Prilipov, A G; Katsy, E I

    2017-01-01

    It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

  11. Improvement of Student Understanding of How Kinetic Data Facilitates the Determination of Amino Acid Catalytic Function through an Alkaline Phosphatase Structure/Mechanism Bioinformatics Exercise

    Science.gov (United States)

    Grunwald, Sandra K.; Krueger, Katherine J.

    2008-01-01

    Laboratory exercises, which utilize alkaline phosphatase as a model enzyme, have been developed and used extensively in undergraduate biochemistry courses to illustrate enzyme steady-state kinetics. A bioinformatics laboratory exercise for the biochemistry laboratory, which complements the traditional alkaline phosphatase kinetics exercise, was…

  12. Histochemical study on effects of low dose γ-irradiation on acid phosphatase in liver of the pigeons Columbus livia intermedia Strickland

    International Nuclear Information System (INIS)

    Gadhia, P.K.; Shah, V.C.

    1982-01-01

    Effects of total body γ-irradiation with sub-lethal dose (400 rads) on acid phosphatase have been studied in the liver of pigeon. The histochemical study showed increased activity of acid phosphatase in liver after 48 hr and 72 hr of irradiation. (author)

  13. Caged xanthones displaying protein tyrosine phosphatase 1B (PTP1B) inhibition from Cratoxylum cochinchinense.

    Science.gov (United States)

    Li, Zuo Peng; Lee, Hyeong-Hwan; Uddin, Zia; Song, Yeong Hun; Park, Ki Hun

    2018-08-01

    Four new caged xanthones (1-4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1-6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1-6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC 50 values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining K m , V max , and the ratio of K ik and K iv , which revealed that all the compounds behaved as competitive inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Localization of acid phosphatase activity in the apoplast of root nodules of pea (Pisum sativum

    Directory of Open Access Journals (Sweden)

    Marzena Sujkowska

    2011-01-01

    Full Text Available Changes in the activity of acid phosphatase (AcPase in the apoplast of pea root nodule were investigated. The activity was determined using lead and cerium methods. The results indicated a following sequence of AcPase activity appearance during the development of the infection thread: 1 low AcPase activity appears in the outer part of cells of symbiotic bacteria; 2 bacteria show increased AcPase activity, and the enzyme activity appears in the thread walls; 3 activity exhibits also matrix of the infection thread; 4 bacteria just before their release from the infection threads show high AcPase activity; 5 AcPase activity ceases after bacteria transformation into bacteroids. The increase in bacterial AcPase activity may reflect a higher demand for inorganic phosphorus necessary for propagation of the bacteria within the infection threads and/or involved in bacteria release from the infection threads.

  15. A ten-week biochemistry lab project studying wild-type and mutant bacterial alkaline phosphatase.

    Science.gov (United States)

    Witherow, D Scott

    2016-11-12

    This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzymes. The experiments are designed to build on students' work from week to week in a way that requires them to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):555-564, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

  16. A Disposable Alkaline Phosphatase-Based Biosensor for Vanadium Chronoamperometric Determination

    Directory of Open Access Journals (Sweden)

    Ana Lorena Alvarado-Gámez

    2014-02-01

    Full Text Available A chronoamperometric method for vanadium ion determination, based on the inhibition of the enzyme alkaline phosphatase, is reported. Screen-printed carbon electrodes modified with gold nanoparticles were used as transducers for the immobilization of the enzyme. The enzymatic activity over 4-nitrophenyl phosphate sodium salt is affected by vanadium ions, which results in a decrease in the chronoamperometric current registered. The developed method has a detection limit of 0.39 ± 0.06 µM, a repeatability of 7.7% (n = 4 and a reproducibility of 8% (n = 3. A study of the possible interferences shows that the presence of Mo(VI, Cr(III, Ca(II and W(VI, may affect vanadium determination at concentration higher than 1.0 mM. The method was successfully applied to the determination of vanadium in spiked tap water.

  17. [High gene conversion frequency between genes encoding 2-deoxyglucose-6-phosphate phosphatase in 3 Saccharomyces species].

    Science.gov (United States)

    Piscopo, Sara-Pier; Drouin, Guy

    2014-05-01

    Gene conversions are nonreciprocal sequence exchanges between genes. They are relatively common in Saccharomyces cerevisiae, but few studies have investigated the evolutionary fate of gene conversions or their functional impacts. Here, we analyze the evolution and impact of gene conversions between the two genes encoding 2-deoxyglucose-6-phosphate phosphatase in S. cerevisiae, Saccharomyces paradoxus and Saccharomyces mikatae. Our results demonstrate that the last half of these genes are subject to gene conversions among these three species. The greater similarity and the greater percentage of GC nucleotides in the converted regions, as well as the absence of long regions of adjacent common converted sites, suggest that these gene conversions are frequent and occur independently in all three species. The high frequency of these conversions probably result from the fact that they have little impact on the protein sequences encoded by these genes.

  18. Further characterization of serum alkaline phosphatase from male and female beagle dogs.

    Science.gov (United States)

    Amacher, D E; Higgins, C V; Schomaker, S J; Clay, R J

    1989-01-01

    Alkaline phosphatase (AP) from the sera of both male and female beagle dogs was partially purified and then analyzed for the presence of AP isoenzymes having intestinal or osseous characteristics as detected by bromotetramisole inhibition or wheat germ lectin agarose electrophoresis, respectively. The sera from both sexes were similar in regard to the presence of AP isoenzymes with intestinal (16 vs. 20%) or osseous (19 vs. 23%) characteristics, but serum AP from the male had a greater sialic acid content and only the male serum contained a detectable constitutive acidic (pI = 3.4) AP isoenzyme. This was similar to a serum AP isoenzyme previously found elevated in the sera of dogs afflicted with hyperadrenocorticalism or of dogs treated with certain corticosteroids.

  19. Hemagglutinating and acid phosphatase (AcPASE activities in developing seedlings of four species of Cucurbitaceae

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2014-01-01

    Full Text Available The acid phosphatase and hemagglutinating activities of four species of Cucurbitaceae were determined during seeds germination and seedlings development. In all cases traces of enzyme and hemagglutinating activities were found in dry and imbibided seeds. In developing seedlings of Cucumis sativus the activities increased to maximum on the 3rd day while in other species on the 6th day of germination and than fell down. Dot blot and Western blot techniques have shown that in seeds and seedlings of all investigated species present were proteins which cross-reacted with antibodies raised against lectins: CLBa and Con A. It has been shown that proteins from seeds and seedlings of Cucurbita maxima var. bambino, Cucurbita pepo var. giromontia and Cucumis sativus had more pronounced antigenical similarity to lectin CLBa (from Cucurbitaceae than Con A, while proteins from cotyledons of Cucurbita pepo var. patissonina reacted better with antibodies raised against Con A (the lectin from Papilionaceae than with CLBa.

  20. Relationship between serum heat-stable alkaline phosphatase level and pregnancy

    International Nuclear Information System (INIS)

    Cao Guoxian; Xiao Weihong; Yu Huixin; Li Weiyi; Huang Xuquan

    1998-01-01

    Serum heat-stable alkaline phosphatase (HSAP) level in 649 cases of normal pregnancy and 164 cases of high-risk pregnancy is measured by radioimmunoassay (RIA). The results indicate that the HSAP level in normal pregnancy increased proportionally with gestation weeks (r = 0.9843). In 33 cases of pregnancy induced hypertension and 21 cases of intrauterine fetal growth retardation, the HSAP level is significantly low. In 7 cases of neonatal asphyxia and 26 cases of fetal distress, the HSAP level in the mother's serum is also low. In 53 cases of intrahepatic cholestasis of pregnancy, the HSAP level is similar to those of normal pregnancy. This study illustrates that HSAP RIA can play an important role in the evaluation of placental function and fetal prognosis for cases of high-risk pregnancy

  1. The voltage-sensing domain of a phosphatase gates the pore of a potassium channel.

    Science.gov (United States)

    Arrigoni, Cristina; Schroeder, Indra; Romani, Giulia; Van Etten, James L; Thiel, Gerhard; Moroni, Anna

    2013-03-01

    The modular architecture of voltage-gated K(+) (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv(Synth1), a functional voltage-gated, outwardly rectifying K(+) channel. Kv(Synth1) displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V(1/2) = +56 mV; z of ~1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

  2. Marine Longilenes, Oxasqualenoids with Ser-Thr Protein Phosphatase 2A Inhibition Activity

    Directory of Open Access Journals (Sweden)

    Francisco Cen-Pacheco

    2018-04-01

    Full Text Available The red seaweed Laurencia viridis is a rich source of oxygenated secondary metabolites that were derived from squalene. We report here the structures of three novel compounds, (+-longilene peroxide (1, longilene (2, and (+-prelongilene (3 that were isolated from this alga, in addition to other substances, 4 and 5, resulting from their acid-mediated degradation. The effect of compounds 1 and 3 against Ser-Thr protein phosphatase type 2A (PP2A was evaluated, showing that (+-longilene peroxide (1 inhibited PP2A (IC50 11.3 μM. In order to explain the interaction between PP2A and compounds 1 and 3, molecular docking simulations onto the PP2A enzyme-binding region were used.

  3. Lipid phosphate phosphatase activity regulates dispersal and bilateral sorting of embryonic germ cells in Drosophila

    Science.gov (United States)

    Renault, Andrew D.; Kunwar, Prabhat S.; Lehmann, Ruth

    2010-01-01

    In Drosophila, germ cell survival and directionality of migration are controlled by two lipid phosphate phosphatases (LPP), wunen (wun) and wunen-2 (wun2). wun wun2 double mutant analysis reveals that the two genes, hereafter collectively called wunens, act redundantly in primordial germ cells. We find that wunens mediate germ cell-germ cell repulsion and that this repulsion is necessary for germ cell dispersal and proper transepithelial migration at the onset of migration and for the equal sorting of the germ cells between the two embryonic gonads during their migration. We propose that this dispersal function optimizes adult fecundity by assuring maximal germ cell occupancy of both gonads. Furthermore, we find that the requirement for wunens in germ cell survival can be eliminated by blocking germ cell migration. We suggest that this essential function of Wunen is needed to maintain cell integrity in actively migrating germ cells. PMID:20431117

  4. Cloning and Characterization of Purple Acid Phosphatase Phytases from Wheat, Barley, Maize and Rice

    DEFF Research Database (Denmark)

    Dionisio, Giuseppe; Madsen, Claus Krogh; Holm, Preben Bach

    2011-01-01

    development and germination. In wheat, it was demonstrated that a and b isogene expression is driven by different promoters (approximately 31% identity). TaPAPhy_a/b promoter reporter gene expression in transgenic grains and peptide mapping of TaPAPhy purified from wheat bran and germinating grains confirmed......Barley (Hordeum vulgare) and wheat (Triticum aestivum) possess significant phytase activity in the mature grains. Maize (Zea mays) and rice (Oryza sativa) possess little or virtually no preformed phytase activity in the mature grain and depend fully on de novo synthesis during germination. Here......, it is demonstrated that wheat, barley, maize, and rice all possess purple acid phosphatase (PAP) genes that, expressed in Pichia pastoris, give fully functional phytases (PAPhys) with very similar enzyme kinetics. Preformed wheat PAPhy was localized to the protein crystalloid of the aleurone vacuole. Phylogenetic...

  5. Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte

    2011-01-01

    this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture...... mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked...... enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from...

  6. Phospholipid metabolism and nuclear function: roles of the lipin family of phosphatidic acid phosphatases.

    Science.gov (United States)

    Siniossoglou, Symeon

    2013-03-01

    Phospholipids play important roles in nuclear function as dynamic building blocks for the biogenesis of the nuclear membrane, as well as signals by which the nucleus communicates with other organelles, and regulate a variety of nuclear events. The mechanisms underlying the nuclear roles of phospholipids remain poorly understood. Lipins represent a family of phosphatidic acid (PA) phosphatases that are conserved from yeasts to humans and perform essential functions in lipid metabolism. Several studies have identified key roles for lipins and their regulators in nuclear envelope organization, gene expression and the maintenance of lipid homeostasis in yeast and metazoans. This review discusses recent advances in understanding the roles of lipins in nuclear structure and function. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Modulation of Tight Junction Structure and Function by Kinases and Phosphatases Targeting Occludin

    Directory of Open Access Journals (Sweden)

    Max Johannes Dörfel

    2012-01-01

    Full Text Available Tight junctions (TJs typically represent the most apical contacts in epithelial and endothelial cell layers where they play an essential role in the separation of extracellular or luminal spaces from underlying tissues in the body. Depending on the protein composition, TJs define the barrier characteristics and in addition maintain cell polarity. Two major families of integral membrane proteins form the typical TJ strand network, the tight junction-associated MARVEL protein (TAMP family members occludin, tricellulin, and MarvelD3 as well as a specific set of claudins. Occludin was the first identified member of these tetraspanins and is now widely accepted as a regulator of TJ assembly and function. Therefore, occludin itself has to be tightly regulated. Phosphorylation of occludin appears to be of central importance in this context. Here we want to summarize current knowledge on the kinases and phosphatases directly modifying occludin, and their role in the regulation of TJ structure, function, and dynamics.

  8. A sandwich-type optical immunosensor based on the alkaline phosphatase enzyme for Salmonella thypimurium detection

    Science.gov (United States)

    Widyastuti, E.; Puspitasari Schonherr, M. F.; Masruroh, A.; Anggraeni, R. A.; Nisak, Y. K.; Mursidah, S.

    2018-03-01

    Salmonella is pathogenic bacteria that caused foodborne diseases which being called Salmonellosis. Prevalence of Salmonellosis that being caused by Salmonella thypimurium in Indonesia is quite high. However, detection of Salmonella bacteria in food still limited, complicated, and required a lot time. Sensitive optical assay for Salmonella thypimurium paper based detection has been developed by integrating sandwich assay between antibody-antigen complex and alkaline phosphatase enzyme that produce visible bluish-purple colour with presence of NBT-BCIP substrate. The results showed that Limit of Quantitation of detection is 105 CFU mL-1 with detection time 15 minutes. Linearity test between Colour intensity that produced from Salmonella concentration presence on samples showed that detection has good linearity. Selectivity test exhibited excellent sensitivity with good discrimination against Escherichia coli.

  9. Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

    Directory of Open Access Journals (Sweden)

    Ardizzone Michele

    2008-08-01

    Full Text Available Abstract Background The present study has investigated the protein tyrosine phosphatase H1 (PTPH1 expression pattern in mouse brain and its impact on CNS functions. Methods We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype were also behaviorally tested for CNS functions. Results In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females. Conclusion These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.

  10. Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B.

    Science.gov (United States)

    Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A M; Imhoff, Johannes F

    2017-06-21

    In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B ( 1 ) contains an additional phenolic hydroxy function at C-6 and exhibits an IC 50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin ( 2 ) did not show any inhibition of this enzymatic activity. Asperentin B ( 1 ) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness.

  11. Defects in the acid phosphatase ACPT cause recessive hypoplastic amelogenesis imperfecta.

    Science.gov (United States)

    Smith, Claire El; Whitehouse, Laura LE; Poulter, James A; Brookes, Steven J; Day, Peter F; Soldani, Francesca; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

    2017-08-01

    We identified two homozygous missense variants (c.428C>T, p.(T143M) and c.746C>T, p.(P249L)) in ACPT, the gene encoding acid phosphatase, testicular, which segregates with hypoplastic amelogenesis imperfecta in two unrelated families. ACPT is reported to play a role in odontoblast differentiation and mineralisation by supplying phosphate during dentine formation. Analysis by computerised tomography and scanning electron microscopy of a primary molar tooth from an individual homozygous for the c.746C>T variant revealed an enamel layer that was hypoplastic, but mineralised with prismatic architecture. These findings implicate variants in ACPT as a cause of early failure of amelogenesis during the secretory phase.

  12. Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells.

    Science.gov (United States)

    Sheremet, Ya A; Yemets, A I; Azmi, A; Vissenberg, K; Verbelen, J P; Blume, Ya B

    2012-01-01

    To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.

  13. Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP)

    Science.gov (United States)

    Li, Hui; Li, Kang-shuai; Su, Jing; Chen, Lai-Zhong; Xu, Yun-Fei; Wang, Hong-Mei; Gong, Zheng; Cui, Guo-Ying; Yu, Xiao; Wang, Kai; Yao, Wei; Xin, Tao; Li, Min-Yong; Xiao, Kun-Hong; An, Xiao-fei; Huo, Yuqing; Xu, Zhi-gang; Sun, Jin-Peng; Pang, Qi

    2013-01-01

    Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. PMID:24117863

  14. Coordinated Regulation of Insulin Signaling by the Protein Tyrosine Phosphatases PTP1B and TCPTP

    Science.gov (United States)

    Galic, Sandra; Hauser, Christine; Kahn, Barbara B.; Haj, Fawaz G.; Neel, Benjamin G.; Tonks, Nicholas K.; Tiganis, Tony

    2005-01-01

    The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. Our previous studies have shown that the closely related tyrosine phosphatase TCPTP might also contribute to the regulation of insulin receptor (IR) signaling in vivo (S. Galic, M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis, Mol. Cell. Biol. 23:2096-2108, 2003). Here we show that PTP1B and TCPTP function in a coordinated and temporally distinct manner to achieve an overall regulation of IR phosphorylation and signaling. Whereas insulin-induced phosphatidylinositol 3-kinase/Akt signaling was prolonged in both TCPTP−/− and PTP1B−/− immortalized mouse embryo fibroblasts (MEFs), mitogen-activated protein kinase ERK1/2 signaling was elevated only in PTP1B-null MEFs. By using phosphorylation-specific antibodies, we demonstrate that both IR β-subunit Y1162/Y1163 and Y972 phosphorylation are elevated in PTP1B−/− MEFs, whereas Y972 phosphorylation was elevated and Y1162/Y1163 phosphorylation was sustained in TCPTP−/− MEFs, indicating that PTP1B and TCPTP differentially contribute to the regulation of IR phosphorylation and signaling. Consistent with this, suppression of TCPTP protein levels by RNA interference in PTP1B−/− MEFs resulted in no change in ERK1/2 signaling but caused prolonged Akt activation and Y1162/Y1163 phosphorylation. These results demonstrate that PTP1B and TCPTP are not redundant in insulin signaling and that they act to control both common as well as distinct insulin signaling pathways in the same cell. PMID:15632081

  15. The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi.

    Science.gov (United States)

    Kück, Ulrich; Beier, Anna M; Teichert, Ines

    2016-05-01

    The striatin-interacting phosphatases and kinases (STRIPAK) complex is a highly conserved eukaryotic protein complex that was recently described for diverse animal and fungal species. Here, we summarize our current knowledge about the composition and function of the STRIPAK complex from the ascomycete Sordaria macrospora, which we discovered by investigating sexually sterile mutants (pro), having a defect in fruiting body development. Mass spectrometry and yeast two-hybrid analysis defined core subunits of the STRIPAK complex, which have structural homologs in animal and other fungal organisms. These subunits (and their mammalian homologs) are PRO11 (striatin), PRO22 (STRIP1/2), SmMOB3 (Mob3), PRO45 (SLMAP), and PP2AA, the structural, and PP2Ac, the catalytic subunits of protein phosphatase 2A (PP2A). Beside fruiting body formation, the STRIPAK complex controls vegetative growth and hyphal fusion in S. macrospora. Although the contribution of single subunits to diverse cellular and developmental processes is not yet fully understood, functional analysis has already shown that mammalian homologs are able to substitute the function of distinct fungal STRIPAK subunits. This underscores the view that fungal model organisms serve as useful tools to get a molecular insight into cellular and developmental processes of eukaryotes in general. Future work will unravel the precise localization of single subunits within the cell and decipher their STRIPAK-related and STRIPAK-independent functions. Finally, evidence is accumulating that there is a crosstalk between STRIPAK and various signaling pathways, suggesting that eukaryotic development is dependent on STRIPAK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Protection against gamma-radiation injury by protein tyrosine phosphatase 1B

    Directory of Open Access Journals (Sweden)

    Marina Mojena

    2018-07-01

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic β-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-β signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant. Keywords: Protein tyrosine phosphatase, Cell viability, Irradiation sensitivity, Lethality, p53

  17. Protein phosphatase 5 is necessary for ATR-mediated DNA repair

    International Nuclear Information System (INIS)

    Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

    2011-01-01

    Research highlights: → Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. → However, it is not clear exactly how PP5 participates in this process. → Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

  18. Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

    Science.gov (United States)

    Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

    2017-11-01

    Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. X-ray structure reveals a new class and provides insight into evolution of alkaline phosphatases.

    Directory of Open Access Journals (Sweden)

    Subhash C Bihani

    Full Text Available The alkaline phosphatase (AP is a bi-metalloenzyme of potential applications in biotechnology and bioremediation, in which phosphate monoesters are nonspecifically hydrolysed under alkaline conditions to yield inorganic phosphate. The hydrolysis occurs through an enzyme intermediate in which the catalytic residue is phosphorylated. The reaction, which also requires a third metal ion, is proposed to proceed through a mechanism of in-line displacement involving a trigonal bipyramidal transition state. Stabilizing the transition state by bidentate hydrogen bonding has been suggested to be the reason for conservation of an arginine residue in the active site. We report here the first crystal structure of alkaline phosphatase purified from the bacterium Sphingomonas. sp. Strain BSAR-1 (SPAP. The crystal structure reveals many differences from other APs: 1 the catalytic residue is a threonine instead of serine, 2 there is no third metal ion binding pocket, and 3 the arginine residue forming bidentate hydrogen bonding is deleted in SPAP. A lysine and an aspargine residue, recruited together for the first time into the active site, bind the substrate phosphoryl group in a manner not observed before in any other AP. These and other structural features suggest that SPAP represents a new class of APs. Because of its direct contact with the substrate phosphoryl group, the lysine residue is proposed to play a significant role in catalysis. The structure is consistent with a mechanism of in-line displacement via a trigonal bipyramidal transition state. The structure provides important insights into evolutionary relationships between members of AP superfamily.

  20. Fabrication of hydrogels with elasticity changed by alkaline phosphatase for stem cell culture.

    Science.gov (United States)

    Toda, Hiroyuki; Yamamoto, Masaya; Uyama, Hiroshi; Tabata, Yasuhiko

    2016-01-01

    The objective of this study is to design hydrogels whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture and evaluate the effect of hydrogel elasticity on an osteogenic gene expression of cells. Hydrogels were prepared by the radical polymerization of acrylamide (AAm), N,N'-methylenebisacrylamide (BIS), and Phosmer™M containing phosphate groups (PE-PAAm hydrogels). The storage modulus of PE-PAAm hydrogels prepared was changed by the preparation conditions. When human mesenchymal stem cells (hMSC) were cultured on the ALP-responsive PE-PAAm hydrogels in the presence or absence of ALP, the morphology of hMSC was observed and one of the osteogenic differentiation markers, Runx2, was evaluated. By ALP addition into the culture medium, the morphology of hMSC was changed into an elongated shape without cell damage. ALP addition modified the level of Runx2 gene expression, which was influenced by the modulus of PE-PAAm hydrogels. It is concluded that the elasticity change of hydrogel substrates in cell culture had an influence on the Runx2 gene expression of hMSC. Stem cells sense the surface elasticity of culture substrates, and their differentiation fate is biologically modified by substrate properties. Most of experiments have been performed in static conditions during cell culture, while the in vivo microenvironment is dynamically changed. In this study, we established to design an enzyme-responsive hydrogel whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture to mimic in vivo conditions. As a result, the cells were deformed and the gene expression level of an osteogenic maker, Runx2, was modified by ALP treatment. This is the novel report describing to demonstrate that the dynamic alteration of hydrogel substrate elasticity could modulate the osteoblastic gene expression of human MSC in vitro. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.