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Sample records for diphosphates

  1. Manganese dipyridoxyl diphosphate:

    DEFF Research Database (Denmark)

    H, Brurok; Ardenkjær-Larsen, Jan Henrik; G, Hansson

    1999-01-01

    Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent for magnetic resonance imaging (MRI) of the liver. Aims of the study were to examine if MnDPDP possesses superoxide dismutase (SOD) mimetic activity in vitro, and if antioxidant protection can be demonstrated in an ex vivo rat heart...

  2. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  3. Motesanib diphosphate in progressive differentiated thyroid cancer

    DEFF Research Database (Denmark)

    Sherman, Steven I; Wirth, Lori J; Droz, Jean-Pierre

    2008-01-01

    BACKGROUND: The expression of vascular endothelial growth factor (VEGF) is characteristic of differentiated thyroid cancer and is associated with aggressive tumor behavior and a poor clinical outcome. Motesanib diphosphate (AMG 706) is a novel oral inhibitor of VEGF receptors, platelet-derived gr...

  4. Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

    International Nuclear Information System (INIS)

    Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2010-01-01

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

  5. Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.

    Science.gov (United States)

    Schilmiller, Anthony L; Schauvinhold, Ines; Larson, Matthew; Xu, Richard; Charbonneau, Amanda L; Schmidt, Adam; Wilkerson, Curtis; Last, Robert L; Pichersky, Eran

    2009-06-30

    We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

  6. Uranium (Vi) sorption onto zirconium diphosphate chemically modified

    International Nuclear Information System (INIS)

    Garcia G, N.; Ordonez R, E.

    2010-10-01

    This work deals with the uranium (Vi) speciation after sorption onto zirconium diphosphate (ZrP 2 O 7 ) surface, hydrated and in a surface modified with organic acids. Oxalic and citric acids were chosen to modify the ZrP 2 O 7 surface because they have poly carboxylic groups and they mimic the organic matter in nature. Thus the interest of this work is to evaluate the uranium (Vi) sorption edge at different s ph values in natural and modified surfaces. The luminescence technique (fluorescence and phosphorescence, respectively) was used for the quantification and speciation of uranyl sorbed at the zirconium diphosphate interface. The fluorescence experiment, showed that adsorption of uranyl on surface of zirconium diphosphate tends to 100%. The speciation shows that there are different complexes in surface which were formed between zirconium diphosphate and uranyl, since it is produced a displacement of wavelength in fluorescence spectra of each system. (Author)

  7. Synthesis and behavior at heating of amorphous calcium diphosphate

    International Nuclear Information System (INIS)

    Levchenko, L.V.; Nurkina, Z.S.; Griggs, D.; Sinyayev, V.A.

    2005-01-01

    There is description of synthesis of amorphous calcium diphosphate in the article. The compound was received via exchange reaction between sodium diphosphate and calcium chloride in water solution. The results of investigation of behavior when heated of produced substance are presented here as well. Composition and structure of precipitated substances and products of its thermal convention were determined by methods of IR-, NMR 31 P and X-ray spectroscopy

  8. Properties of ribulose diphosphate carboxylase immobilized on porous glass

    Science.gov (United States)

    Shapira, J.; Hanson, C. L.; Lyding, J. M.; Reilly, P. J.

    1974-01-01

    Ribulose-1,5-diphosphate carboxylase from spinach has been bound to arylamine porous glass with a diazo linkage and to alklamine porous glass with glutaraldehyde. Stability at elevated temperatures and responses to changes of pH and ribulose-1,5-diphosphate, Mg(2+), and dithiothreitol concentrations were not significantly different from the soluble enzyme, though stability at 4 C was somewhat improved.

  9. Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

    Science.gov (United States)

    Sitthithaworn, W; Kojima, N; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Noji, M; Saito, K; Niwa, Y; Sankawa, U

    2001-02-01

    cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.

  10. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first path-way-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate...

  11. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a...

  12. Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion

    Czech Academy of Sciences Publication Activity Database

    Lai, D.-H.; Bontempi, E. J.; Lukeš, Julius

    2012-01-01

    Roč. 183, č. 2 (2012), s. 189-192 ISSN 0166-6851 R&D Projects: GA ČR(CZ) GAP305/11/2179 Institutional support: RVO:60077344 Keywords : Trypanosoma brucei * Sleeping sickness * Ubiquinone * Solanesyl-diphosphate synthase * Digitonin permeabilization * In situ tagging Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.734, year: 2012 http://www.sciencedirect.com/science/article/pii/S0166685112000539

  13. Mutational, Phylogeny and Evolution Analyses of Salvia Copalyl Diphosphate Synthase

    International Nuclear Information System (INIS)

    Hao, D. C.; Thimmappa, R. B.; Xiao, P. G.

    2016-01-01

    The cyclization of geranylgeranyl diphosphate (GGPP) is catalyzed by copalyl diphosphate synthase (CPS), a class II diterpene synthase (diTPS), to form copalyl diphosphate (CPP), which is an essential substrate of a variety of diterpenes in secondary metabolism of angiosperm including Salvia medicinal plants. The protein environment of the N-terminal class II active site stabilizes the carbocation intermediates and maintains the catalytic activity of angiosperm class II diTPS. The virtual modeling and mutagenesis of the class II diTPS of Salvia miltiorrhiza (SmCPS) were accomplished to illuminate the catalytic activity of SmCPS. Terminal truncations and point mutations established the role of the Beta-Gamma domain and Alpha domain, i.e., they facilitate the flexible conformational change of the class II active site after substrate binding. E203 and K238 in the N-ter Gamma domain of SmCPS1 are functional in the substrate binding and conformational transition and might be essential in catalysis. Similar to other CPSs, the ensuing protonation of the GGPP substrate and coordination of the diphosphate group are governed by highly conserved residues in the DxDD motif of SmCPS, e.g., D372 of CPS1. Moreover, F256 and Y505 stabilize the carbocation and control the enzymatic activity during CPP formation. The amino acids of the predicted active sites, despite under purifying selection, vary greatly, corresponding to the functional flexibility of angiosperm CPSs. Molecular phylogeny and evolution analyses suggest early and ongoing evolution of labdane-related diterpenoid metabolism in angiosperm. (author)

  14. Solid-Phase Synthesis of a New Diphosphate 5-Aminoimidazole-4-carboxamide Riboside (AICAR Derivative and Studies toward Cyclic AICAR Diphosphate Ribose

    Directory of Open Access Journals (Sweden)

    Gennaro Piccialli

    2011-09-01

    Full Text Available The solid-phase synthesis of the first example of a new diphosphate AICAR derivative is reported. The new substance is characterized by the presence of a 5'-phosphate group while a second phosphate moiety is installed on a 5-hydroxypentyl chain attached to the 4-N-position of AICAR. Cyclization of the diphosphate derivative by pyrophosphate bond formation allowed for the formation of a novel AICAR-based cyclic ADP-ribose (cADPR mimic.

  15. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...... an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x...... mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6....

  16. Evaluation of the sorption of Eu(III) in titanium diphosphate

    International Nuclear Information System (INIS)

    Ortiz O, H.B.; Ordonez R, E.; Fernandez V, S.M.

    2007-01-01

    In this work its are presented: the synthesis, physicochemical characterization and the surface parameters estimation that can be related with the retention properties of the titanium diphosphate for the actinides of valence III (Pu, Am, Cm among others), using the Eu 3+ like a chemical analog. The surface area, hydration time, zero charge point, density of active sites and the surface species distribution in the titanium diphosphate are reported. This information was used to explain the retention of the Eu(lll) in the surface of the titanium diphosphate. (Author)

  17. Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase.

    Science.gov (United States)

    Kojima, N; Sitthithaworn, W; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Sankaw, U

    2000-07-01

    cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.

  18. Modification of zirconium diphosphate with salicylic acid and its effect on the uranium (Vi) sorption

    International Nuclear Information System (INIS)

    Almazan T, M. G.; Garcia G, N.; Simoni, E.

    2014-10-01

    The surface of zirconium diphosphate (ZrP 2 O 7 ) was modified with salicylic acid and its effect was evaluated on the uranium (Vi) sorption. The modified surface of the material was analyzed with different analytical techniques among which are included the atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. This analysis allowed showing that the salicylic acid is being held on the surface of the zirconium diphosphate. The reactivity of modified zirconium diphosphate compared with uranium (Vi) was investigated using the classical method of batch sorption. The analysis of sorption isotherms shows that the salicylic acid has an important effect in the uranium (Vi) sorption. According to the study conducted, the interaction among the uranium (Vi) and the surface of zirconium diphosphate modified with the salicylic acid most likely leads to the complexes formation of binary (U(Vi)/ZrP 2 O 7 ) and ternary (U(Vi)/salicylate/ZrP 2 O 7 ) surface. (Author)

  19. The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from ...

    African Journals Online (AJOL)

    The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from Taxus media: Cloning, characterization and functional identification. Y Sun, M Chen, J Tang, W Liu, C Yang, Y Yang, X Lan, M Hsieh, Z Liao ...

  20. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  1. Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil, Anthonomus grandis.

    Science.gov (United States)

    Taban, A Huma; Tittiger, Claus; Blomquist, Gary J; Welch, William H

    2009-06-01

    Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full-length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three-dimensional structure of coleopteran FPPS was determined and compared to the X-ray crystal structure of avian FPPS. The alpha-helical fold is conserved in AgFPPS and the size of the active site cavity is consistent with the enzyme being a FPPS. (c) 2009 Wiley Periodicals, Inc.

  2. Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

    Science.gov (United States)

    Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

    2001-01-01

    An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

  3. Kinetic study of the thorium phosphate - diphosphate dissolution

    International Nuclear Information System (INIS)

    Dacheux, N.; Thomas, A.C.; Brandel, V.; Genet, M.

    2000-01-01

    The thorium phosphate-diphosphate Th 4 (PO 4 ) 4 P 2 O 7 (TPD) structure allows the replacement of large amounts of thorium by tetravalent actinides leading to the formation of solid solutions. This compound was obtained in powdered or sintered form after pressing at room temperature at 300-800 MPa then heating at 1250 deg. C for 10-30 hours. The resistance of this material to aqueous corrosion was determined by varying several parameters such as surface, leaching flow, acidity or temperature. It was thus possible to independently determine the influence of each parameter on the leaching rate provided that the saturation of the solution was not obtained. In acidic media, the partial order related to [H 3 O + ] was found to be in the 0.31-0.35 range while, in basic media, the partial order related to [OH - ] was almost the same (0.45). The activation energy (42 kJ/mol) was determined between 4 deg. C and 120 deg. C. Moreover, the addition of phosphate in the leachate slightly increased the TPD dissolution rate. When the saturation of the solution is reached, a gelatinous precipitate controls the thorium and phosphate concentrations. The complete characterization of this solid led to the proposed general formula Th 2 (PO 4 ) 2 (HPO 4 ). n H 2 O which conventional solubility product (at I = 0 M) is very low: K * S,0 10 -66.6±1.2 even in very acidic media. (authors)

  4. Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Kasparyan, Elena; Richter, Michael; Dresen, Carola; Walter, Lydia S; Fuchs, Georg; Leeper, Finian J; Wacker, Tobias; Andrade, Susana L A; Kolter, Geraldine; Pohl, Martina; Müller, Michael

    2014-12-01

    The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

  5. The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase.

    Science.gov (United States)

    Demissie, Zerihun A; Erland, Lauren A E; Rheault, Mark R; Mahmoud, Soheil S

    2013-03-01

    Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

  6. Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.; Steussy, Calvin Nicklaus; Stauffacher, Cynthia V. (Purdue)

    2017-10-12

    The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

  7. Kinetic study of the thorium phosphate - diphosphate dissolution

    Energy Technology Data Exchange (ETDEWEB)

    Dacheux, N.; Thomas, A.C.; Brandel, V.; Genet, M. [Paris-11 Univ., 91 - Orsay (France). Inst. de Physique Nucleaire; Aupiais, J. [CEA/DAM-Ile de France, Dept. Analyse Surveillance Environnement, DASE, Service Radioanalyses Chimie Environnement, 91 - Bruyeres-Le-Chatel (France)

    2000-07-01

    The thorium phosphate-diphosphate Th{sub 4}(PO{sub 4}){sub 4}P{sub 2}O{sub 7} (TPD) structure allows the replacement of large amounts of thorium by tetravalent actinides leading to the formation of solid solutions. This compound was obtained in powdered or sintered form after pressing at room temperature at 300-800 MPa then heating at 1250 deg. C for 10-30 hours. The resistance of this material to aqueous corrosion was determined by varying several parameters such as surface, leaching flow, acidity or temperature. It was thus possible to independently determine the influence of each parameter on the leaching rate provided that the saturation of the solution was not obtained. In acidic media, the partial order related to [H{sub 3}O{sup +}] was found to be in the 0.31-0.35 range while, in basic media, the partial order related to [OH{sup -}] was almost the same (0.45). The activation energy (42 kJ/mol) was determined between 4 deg. C and 120 deg. C. Moreover, the addition of phosphate in the leachate slightly increased the TPD dissolution rate. When the saturation of the solution is reached, a gelatinous precipitate controls the thorium and phosphate concentrations. The complete characterization of this solid led to the proposed general formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}). n H{sub 2}O which conventional solubility product (at I = 0 M) is very low: K{sup *}{sub S,0} 10{sup -66.6{+-}}{sup 1.2} even in very acidic media. (authors)

  8. Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate

    OpenAIRE

    Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

    2015-01-01

    Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transform...

  9. Recent Advances in the Development of Mammalian Geranylgeranyl Diphosphate Synthase Inhibitors

    Directory of Open Access Journals (Sweden)

    Staci L. Haney

    2017-05-01

    Full Text Available The enzyme geranylgeranyl diphosphate synthase (GGDPS catalyzes the synthesis of the 20-carbon isoprenoid geranylgeranyl diphosphate (GGPP. GGPP is the isoprenoid donor for protein geranylgeranylation reactions catalyzed by the enzymes geranylgeranyl transferase (GGTase I and II. Inhibitors of GGDPS result in diminution of protein geranylgeranylation through depletion of cellular GGPP levels, and there has been interest in GGDPS inhibitors as potential anti-cancer agents. Here we discuss recent advances in the development of GGDPS inhibitors, including insights gained by structure-function relationships, and review the preclinical data that support the continued development of this novel class of drugs.

  10. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits.

    Science.gov (United States)

    Gutensohn, Michael; Orlova, Irina; Nguyen, Thuong T H; Davidovich-Rikanati, Rachel; Ferruzzi, Mario G; Sitrit, Yaron; Lewinsohn, Efraim; Pichersky, Eran; Dudareva, Natalia

    2013-08-01

    Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co-expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS-SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co-expression of snapdragon GPPS-SSU with the O. basilicum α-zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities resulted in increased levels of ZIS-derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re-direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  11. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  12. Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1999-01-01

    Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2...

  13. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...

  14. ACTIVATION OF G-PROTEINS BY RECEPTOR-STIMULATED NUCLEOSIDE DIPHOSPHATE KINASE IN DICTYOSTELIUM

    NARCIS (Netherlands)

    Bominaar, Anthony A.; Molijn, Anco C.; Pestel, Martine; Veron, Michel; Haastert, Peter J.M. van

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC 2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase

  15. Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

    Science.gov (United States)

    Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

    2010-06-01

    Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

  16. Silver vanadium diphosphate Ag2VP2O8: Electrochemistry and characterization of reduced material providing mechanistic insights

    International Nuclear Information System (INIS)

    Takeuchi, Esther S.; Lee, Chia-Ying; Cheng, Po-Jen; Menard, Melissa C.; Marschilok, Amy C.; Takeuchi, Kenneth J.

    2013-01-01

    Silver vanadium phosphorous oxides (Ag w V x P y O z ) are notable battery cathode materials due to their high energy density and demonstrated ability to form in-situ Ag metal nanostructured electrically conductive networks within the cathode. While analogous silver vanadium diphosphate materials have been prepared, electrochemical evaluations of these diphosphate based materials have been limited. We report here the first electrochemical study of a silver vanadium diphosphate, Ag 2 VP 2 O 8 , where the structural differences associated with phosphorous oxides versus diphosphates profoundly affect the associated electrochemistry. Reminiscent of Ag 2 VO 2 PO 4 reduction, in-situ formation of silver metal nanoparticles was observed with reduction of Ag 2 VP 2 O 8 . However, counter to Ag 2 VO 2 PO 4 reduction, Ag 2 VP 2 O 8 demonstrates a significant decrease in conductivity upon continued electrochemical reduction. Structural analysis contrasting the crystallography of the parent Ag 2 VP 2 O 8 with that of the proposed Li 2 VP 2 O 8 reduction product is employed to gain insight into the observed electrochemical reduction behavior, where the structural rigidity associated with the diphosphate anion may be associated with the observed particle fracturing upon deep electrochemical reduction. Further, the diphosphate anion structure may be associated with the high thermal stability of the partially reduced Ag 2 VP 2 O 8 materials, which bodes well for enhanced safety of batteries incorporating this material. - Graphical abstract: Structure and galvanostatic intermittent titration-type test data for silver vanadium diphosphate, Ag 2 VP 2 O 8 . Highlights: ► First electrochemical study of a silver vanadium diphosphate, Ag 2 VP 2 O 8 . ► In-situ formation of Ag 0 nanoparticles was observed upon electrochemical reduction. ► Structural analysis used to provide insight of the electrochemical behavior

  17.   Adenosine-diphosphate (ADP) reduces infarct size and improves porcine heart function after myocardial infarction

    DEFF Research Database (Denmark)

    Bune, Laurids Touborg; Larsen, Jens Kjærgaard Rolighed; Thaning, Pia

    2013-01-01

    Acute myocardial infarction continues to be a major cause of morbidity and mortality. Timely reperfusion can substantially improve outcomes and the administration of cardioprotective substances during reperfusion is therefore highly attractive. Adenosine diphosphate (ADP) and uridine-5-triphoshat...... infusion during reperfusion reduces IS by ~20% independently from systemic release of t-PA. ADP-induced reduction in both preload and afterload could account for the beneficial myocardial effect....

  18. Influence of the temperature in the uranium (Vi) sorption in zirconium diphosphate

    International Nuclear Information System (INIS)

    Garcia G, N.; Solis, D.; Ordonez R, E.

    2012-10-01

    In the present work was evaluated the uranium (Vi) sorption at 10, 20, 30, 40 and 60 C on the zirconium diphosphate (ZrP 2 O 7 ). They were carried out kinetic and isotherms using the method by lots, these will allow to fix the sorption time (kinetic) and to explain the behavior of this sorption in different ph conditions and temperature (isotherm). The quantity of retained uranium in the surface was quantified by means of the fluorescence technique. (Author)

  19. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    Directory of Open Access Journals (Sweden)

    Jianrong Wang

    2014-12-01

    Full Text Available Poria cocos (P. cocos has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%. The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP from geranyl diphosphate (GPP and isopentenyl diphosphate (IPP. Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  20. Optimization of primaquine diphosphate tablet formulation for controlled drug release using the mixture experimental design.

    Science.gov (United States)

    Duque, Marcelo Dutra; Kreidel, Rogério Nepomuceno; Taqueda, Maria Elena Santos; Baby, André Rolim; Kaneko, Telma Mary; Velasco, Maria Valéria Robles; Consiglieri, Vladi Olga

    2013-01-01

    A tablet formulation based on hydrophilic matrix with a controlled drug release was developed, and the effect of polymer concentrations on the release of primaquine diphosphate was evaluated. To achieve this purpose, a 20-run, four-factor with multiple constraints on the proportions of the components was employed to obtain tablet compositions. Drug release was determined by an in vitro dissolution study in phosphate buffer solution at pH 6.8. The polynomial fitted functions described the behavior of the mixture on simplex coordinate systems to study the effects of each factor (polymer) on tablet characteristics. Based on the response surface methodology, a tablet composition was optimized with the purpose of obtaining a primaquine diphosphate release closer to a zero order kinetic. This formulation released 85.22% of the drug for 8 h and its kinetic was studied regarding to Korsmeyer-Peppas model, (Adj-R(2) = 0.99295) which has confirmed that both diffusion and erosion were related to the mechanism of the drug release. The data from the optimized formulation were very close to the predictions from statistical analysis, demonstrating that mixture experimental design could be used to optimize primaquine diphosphate dissolution from hidroxypropylmethyl cellulose and polyethylene glycol matrix tablets.

  1. Synthesis of a tritium labeled photolabile analogue of farnesyl diphosphate: (E,E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate (DATFP-GDP)

    International Nuclear Information System (INIS)

    Liu, J.; Benedict, C.R.

    1996-01-01

    Tritiated (E,E)-(2-diazo-3-trifluoropropionyloxy)geranyl disphosphate (DATFP-GDP) has been used as a photolabile analogue of (E,E)-farnesyl diphosphate (E,E-FDP) for an aid in isolating enzymes utilizing E,E-FDP as a substrate. We now report an alternative method of synthesizing this probe in which the tritium label is introduced in the step just before the introduction of the diphospate group. Thus, DATFP-geranial is oxidized to DATFP-geranial with activated manganese dioxide. The tritium label is introduced by reduction of the aldehyde with NaBT 4 . The DATFP-group successfully withstands both of these steps. The overall yield for these two steps is 69%. Diphosphorylation of the resulting alcohol afforded DATFP-[1- 3 H]-GDP in 8% yield with a specific activity of 48.6 μCi/μmol and radiochemical purity of 94%. (Author)

  2. Geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (FPS), Fps1, and an FPS-like protein, Spo9, essential for sporulation.

    Science.gov (United States)

    Ye, Yanfang; Fujii, Makoto; Hirata, Aiko; Kawamukai, Makoto; Shimoda, Chikashi; Nakamura, Taro

    2007-09-01

    Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

  3. Structure of trihydrated rare-earth acid diphosphates LnHP2O7·3H2O (Ln=La, Er)

    International Nuclear Information System (INIS)

    Ben Moussa, S.; Ventemillas, S.; Cabeza, A.; Gutierrez-Puebla, E.; Sanz, J.

    2004-01-01

    In trihydrated lanthanum acid-diphosphates LnHP 2 O 7 ·3H 2 O, prepared from acid LnCl 3 and Na 4 P 2 O 7 solutions (pH=1), two crystal forms were obtained. Layered structures of two representative members of this family have been determined by single-crystal X-ray diffraction (XRD) technique. In the case of orthorhombic LaHP 2 O 7 ·3H 2 O (type I), lanthanum cations are ninefold coordinated and diphosphate groups adopt a staggered (alternated) configuration. In the case of triclinic ErHP 2 O 7 ·3H 2 O (type II), erbium cations are eightfold coordinated and diphosphate groups adopt an eclipsed configuration. In agreement with Infrared (IR) spectroscopic data, a bended configuration for diphosphate groups has been deduced. In both structures, one-dimensional chains of edge-sharing rare-earth polyhedra are linked together by diphosphate groups to form the phosphate layers. In both diphosphates, PO 4 and HPO 4 environments have been identified by 31 P MAS-NMR technique. In the two compounds, OH groups of HPO 4 tetrahedra point out of diphosphate planes interacting with adjacent layers. In La-diphosphate, the interaction between HPO 4 groups and water molecules of adjacent layers is favored; however, in Er-diphosphate, the interaction between phosphate acid groups of contiguous layers is produced. Based on structural information deduced, differences detected in IR and NMR spectra of two disphosphates are discussed

  4. Co-expression of peppermint geranyl diphosphate synthase small subunit enhances monoterpene production in transgenic tobacco plants.

    Science.gov (United States)

    Yin, Jun-Lin; Wong, Woon-Seng; Jang, In-Cheol; Chua, Nam-Hai

    2017-02-01

    Monoterpenes are important for plant survival and useful to humans. In addition to their function in plant defense, monoterpenes are also used as flavors, fragrances and medicines. Several metabolic engineering strategies have been explored to produce monoterpene in tobacco but only trace amounts of monoterpenes have been detected. We investigated the effects of Solanum lycopersicum 1-deoxy-d-xylulose-5-phosphate synthase (SlDXS), Arabidopsis thaliana geranyl diphosphate synthase 1 (AtGPS) and Mentha × piperita geranyl diphosphate synthase small subunit (MpGPS.SSU) on production of monoterpene and geranylgeranyl diphosphate (GGPP) diversities, and plant morphology by transient expression in Nicotiana benthamiana and overexpression in transgenic Nicotiana tabacum. We showed that MpGPS.SSU could enhance the production of various monoterpenes such as (-)-limonene, (-)-linalool, (-)-α-pinene/β-pinene or myrcene, in transgenic tobacco by elevating geranyl diphosphate synthase (GPS) activity. In addition, overexpression of MpGPS.SSU in tobacco caused early flowering phenotype and increased shoot branching by elevating contents of GA 3 and cytokinins due to upregulated transcript levels of several plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway genes, geranylgeranyl diphosphate synthases 3 (GGPPS3) and GGPPS4. Our method would allow the identification of new monoterpene synthase genes using transient expression in N. benthamiana and the improvement of monoterpene production in transgenic tobacco plants. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  5. Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator

    International Nuclear Information System (INIS)

    Flentke, G.R.; Frey, P.A.

    1990-01-01

    UDPgalactose 4-epimerase from Escherichia coli is rapidly inactivated by the compounds uridine 5'-diphosphate chloroacetol (UDC) and uridine 5'-diphosphate bromoacetol (UCB). Both UDC and UDB inactivate the enzyme in neutral solution concomitant with the appearance of chromophores absorbing maximally at 325 and 328 nm, respectively. The reaction of UDC with the enzyme follows saturation kinetics characterized by a K D of 0.110 mM and k inact of 0.84 min -1 at pH 8.5 and ionic strength 0.2 M. The inactivation by UDC is competitively inhibited by competitive inhibitors of UDPgalactose 4-epimerase, and it is accompanied by the tight but noncovalent binding of UDC to the enzyme in a stoichiometry of 1 mol of UDC/mol of enzyme dimer, corresponding to 1 mol of UDC/mol of enzyme-bound NAD + . The inactivation of epimerase by uridine 5'-diphosphate [ 2 H 2 ]chloroacetol proceeds with a primary kinetic isotope effect (k H /k D ) of 1.4. The inactivation mechanism is proposed to involve a minimum of three steps: (a) reversible binding of UDC to the active site of UDPgalactose 4-epimerase; (b) enolization of the chloroacetol moiety of enzyme-bound UDC, catalyzed by an enzymic general base at the active site; (c) alkylation of the nicotinamide ring of NAD + at the active site by the chloroacetol enolate. The resulting adduct between UDC and NAD + is proposed to be the chromophore with λ max at 325 nm. The enzymic general base required to facilitate proton transfer in redox catalysis by this enzyme may be the general base that facilitates enolization of the chloroacetol moiety of UDC in the inactivation reaction

  6. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  7. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis....

  8. Oritavancin diphosphate.

    Science.gov (United States)

    Cada, Dennis J; Baker, Danial E

    2014-12-01

    Each month, subscribers to The Formulary Monograph Service receive 5 to 6 well-documented monographs on drugs that are newly released or are in late phase 3 trials. The monographs are targeted to Pharmacy & Therapeutics Committees. Subscribers also receive monthly 1-page summary monographs on agents that are useful for agendas and pharmacy/nursing in-services. A comprehensive target drug utilization evaluation/medication use evaluation (DUE/MUE) is also provided each month. With a subscription, the monographs are sent in print and are also available on-line. Monographs can be customized to meet the needs of a facility. A drug class review is now published monthly with The Formulary Monograph Service. Through the cooperation of The Formulary, Hospital Pharmacy publishes selected reviews in this column. For more information about The Formulary Monograph Service, call The Formulary at 800-322-4349. The December 2014 monograph topics are olodaterol, peginterferon beta-1a, testosterone nasal gel, ferric citrate corredination complex, and safinamide. The Safety MUE is on olodaterol.

  9. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms...... possesses very different thermal properties. The B. caldolyticus enzyme has optimal activity at 60-65 degrees C and a half-life of 26 min at 65 degrees C, compared to values of 46 degrees C and 60 s at 65 degrees C, respectively, for the B. subtilis enzyme. Chemical cross-linking shows that both enzymes...... are hexamers. Vmax is determined as 440 micromol.min(-1).mg protein(-1) and Km values for ATP and ribose 5-phosphate are determined as 310 and 530 microM, respectively, for the B. caldolyticus enzyme. The enzyme requires 50 mM Pi as well as free Mg2+ for maximal activity. Manganese ion substitutes for Mg2...

  10. Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Piotr Szymczyk

    2016-09-01

    Full Text Available The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.

  11. Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2014-07-01

    Full Text Available A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS. Five of the new compounds show IC50 values of less than 1 μM against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS. The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 μM concentration.

  12. Cloning and sequencing of cDNAs specifying a novel class of phosphoribosyl diphosphate synthase in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Krath, Britta N.; Eriksen, Tina A.; Poulsen, Tim S.

    1999-01-01

    cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison and ph...

  13. Analysis of quinocide in unprocessed primaquine diphosphate and primaquine diphosphate tablets using gas chromatography-mass spectrometry with supersonic molecular beams.

    Science.gov (United States)

    Brondz, Ilia; Fialkov, Alexander B; Amirav, Aviv

    2009-01-30

    Malaria is one of the most widespread and deadly diseases on the planet. Every year, about 500 million new cases are diagnosed, and the annual death toll is about 3 million. Primaquine has strong antiparasitic effects against gametocytes and can therefore prevent the spread of the parasite from treated patients to mosquitoes. It is also used in radical cures and prevents relapse. Consequently, primaquine is an often-used drug. In this study the separation of unprocessed primaquine from the contaminant quinocide based on gas chromatography-mass spectrometry with supersonic molecular beam (SMB) is presented and 7.5 mg primaquine diphosphate tablets were analyzed. We present a novel method for fast determination of quinocide which is an isomer of primaquine as the main contaminant in unprocessed primaquine and in its medical form as tablets by gas chromatography-mass spectrometry with SMB (also named supersonic GC-MS). Supersonic GC-MS provides enhanced molecular ion without any ion source related peak tailing plus extended range of compounds amenable for GC-MS analysis. In addition, major isomer mass spectral effects were revealed in the mass spectra of primaquine and quinocide which facilitated the unambiguous identification of quinocide in primaquine tablets. Fast GC-MS analysis is demonstrated with less then 2 min elution time of the drug and its main contaminants.

  14. Poly(adenosine 5'-diphosphate) ribose polymerase activation as a cause of metabolic dysfunction in critical illness.

    Science.gov (United States)

    Liaudet, Lucas

    2002-03-01

    Poly(adenosine 5'-diphosphate) ribose polymerase is a nuclear enzyme activated in response to genotoxic stress induced by a variety of DNA damaging agents. Several oxygen and nitrogen-centered free radicals, notably peroxynitrite, are strong inducers of DNA damage and poly(adenosine 5'-diphosphate) ribose polymerase activation in vitro and in vivo. Activation of this nuclear enzyme depletes the intracellular stores of its substrate nicotinamide adenine dinucleotide, slowing the rate of glycolysis, mitochondrial electron transport and adenosine triphosphate formation. This process triggers a severe energetic crisis within the cell, leading to acute cell dysfunction and cell necrosis. Poly(adenosine 5'-diphosphate) ribose polymerase also plays an important role in the regulation of inflammatory cascades, through a functional association with various transcription factors and transcription co-activators. Recent works identified this enzyme as a critical mediator of cellular metabolic dysfunction, inflammatory injury, and organ damage in conditions associated with overwhelming oxidative stress, including systemic inflammation, circulatory shock, and ischemia-reperfusion. Accordingly, pharmacological inhibitors of poly(adenosine 5'-diphosphate) ribose polymerase protect against cell death and tissue injury in such conditions, and may therefore represent novel therapeutic tools to limit multiple organ damage and dysfunction in critically ill patients.

  15. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    Science.gov (United States)

    Kumar, Hitesh; Kumar, Sanjay

    2013-09-15

    The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 araispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Early Cessation of Adenosine Diphosphate Receptor Inhibitors Among Acute Myocardial Infarction Patients Treated With Percutaneous Coronary Intervention

    DEFF Research Database (Denmark)

    Fosbøl, Emil L; Ju, Christine; Anstrom, Kevin J

    2016-01-01

    BACKGROUND: Guidelines recommend the use of adenosine diphosphate receptor inhibitor (ADPri) therapy for 1 year postacute myocardial infarction; yet, early cessation of therapy occurs frequently in clinical practice. METHODS AND RESULTS: We examined 11 858 acute myocardial infarction patients tre...

  17. Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

    Czech Academy of Sciences Publication Activity Database

    Manzano, D.; Busquets, A.; Closa, M.; Hoyerová, Klára; Schaller, H.; Kamínek, Miroslav; Arró, M.; Ferrer, A.

    2006-01-01

    Roč. 61, 1-2 (2006), s. 195-213 ISSN 0167-4412 R&D Projects: GA AV ČR(CZ) IAA600380507 Institutional research plan: CEZ:AV0Z50380511 Keywords : Arabidopsis thaliana * cytokinin * farnesyl diphosphate synthase * isoprenoid Subject RIV: EF - Botanics Impact factor: 3.577, year: 2006

  18. Density fluctuation in a screened Coulombic colloid dispersion: comparison of the liquid and cubic phases of lipid A-diphosphate

    International Nuclear Information System (INIS)

    Brown, Helen; Ross, D. Keith; Paradies, Henrich H.

    2004-01-01

    Light-, small-angle X-ray and neutron scattering measurements of the dynamic structure factor S(Q,t) of strong interacting dispersions of lipid A-diphosphate were recorded and analysed applying existing models of liquid state theory. Lipid A-diphosphate ordering was observed at low volume fractions (phi=2.2x10 -4 ) and at very low ionic strength (I=10 -5 M). Upon increasing the particle number density of lipid A-diphosphate a transformation of the lattices of the colloidal crystals from a BCC lattice (a=36.20 nm) to a FCC lattice (a=57.30 nm) occurred. This strongly suggests a similarity in the preformed liquid structure and the cubic colloidal phase. The fit of both S eff (Q) and the principle peak I p (Q) with the effective particle charge supports of the main conclusions drawn from the SANS experiments and the liquid state theory indicating the presence of long-range order for the dispersions of lipid A-diphosphate

  19. Dependence of the product chain-length on detergents for long-chain E-polyprenyl diphosphate synthases

    Science.gov (United States)

    Pan, Jian-Jung; Ramamoorthy, Gurusankar; Poulter, C. Dale

    2013-01-01

    Long-chain E-polyprenyl diphosphate synthases (E-PDS) catalyze repetitive addition of isopentenyl diphosphate (IPP) to the growing prenyl chain of an allylic diphosphate. The polyprenyl diphosphate products are required for the biosynthesis of ubiquinones and menaquinones required for electron transport during oxidative phosphorylation to generate ATP. In vitro, the long-chain PDSs require addition of phospholipids or detergents to the assay buffer to enhance product release and maintain efficient turnover. During preliminary assays of product chain-length with anionic, zwitterionic, and non-ionic detergents, we discovered considerable variability. Examination of a series of non-ionic PEG detergents with several long-chain E-PDSs from different organisms revealed that in vitro incubations with nonaethylene glycol monododecyl ether or Triton X-100 typically gave chain lengths that corresponded to those of the isoprenoid moieties in respiratory quinones synthesized in vivo. In contrast incubations in buffer with n-butanol, CHAPS, DMSO, n-octyl-β-glucopyranoside, or β-cyclodextrin or in buffer without detergent typically proceeded more slowly and gave a broad range of chain lengths. PMID:23802587

  20. Self irradiation effects on the thorium phosphate diphosphate dissolution (TPD): simulation by external irradiations

    International Nuclear Information System (INIS)

    Tamain, C.; Ozgumus, A.; Dacheux, N.; Garrido, F.; Thome, L.; Corbel, C.; Genet, M.

    2004-01-01

    The Thorium Phosphate Diphosphate (TPD), proposed as a ceramic for the long term immobilization of actinides, was externally irradiated with several ions and energies (but also with gamma rays) in order to simulate the self-irradiation. The influence of the electronic energy loss was first investigated. Thus, the XRD measurements have shown a complete amorphization of the material under 10 13 ions of Kr.cm -2 , while no significant structural change occurred after 5.10 13 S.cm -2 , 2.10 16 He.cm -2 or 320 kGy of dose of gamma rays. The dissolution of the raw and irradiated pellets was studied versus several parameters such as amorphized fraction, energy loss of incident ions, radiolytic species produced in situ in the leachate during irradiation (such as H 2 O 2 ), temperature and acidity. The results reveal an important increase of the dissolution kinetics for amorphized pellets compared to raw ceramic. (authors)

  1. Electron density reactivity indexes of the tautomeric/ionization forms of thiamin diphosphate.

    Science.gov (United States)

    Jaña, Gonzalo A; Delgado, Eduardo J

    2013-09-01

    The generation of the highly reactive ylide in thiamin diphosphate catalysis is analyzed in terms of the nucleophilicity of key atoms, by means of density functional calculations at X3LYP/6-31++G(d,p) level of theory. The Fukui functions of all tautomeric/ionization forms are calculated in order to assess their reactivity. The results allow to conclude that the highly conserved glutamic residue does not protonate the N1' atom of the pyrimidyl ring, but it participates in a strong hydrogen bonding, stabilizing the eventual negative charge on the nitrogen, in all forms involved in the ylide generation. This condition provides the necessary reactivity on key atoms, N4' and C2, to carry out the formation of the ylide required to initiate the catalytic cycle of ThDP-dependent enzymes. This study represents a new approach for the ylide formation in ThDP catalysis.

  2. Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

    Science.gov (United States)

    Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

    2017-08-26

    Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX 8 W) motifs, and enzyme assays showed that the presence of both RRX 8 W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX 8 W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  4. Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

    2016-05-01

    Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

  5. Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate

    Science.gov (United States)

    Samoudi, Mojtaba; Omid Yeganeh, Negar; Shahbani Zahiri, Hossein; Shariati, Parvin; Hajhosseini, Reza

    2015-01-01

    Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli). Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters. Results: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. Conclusion: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 . PMID:26306151

  6. Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

    Science.gov (United States)

    Despinasse, Yolande; Fiorucci, Sébastien; Antonczak, Serge; Moja, Sandrine; Bony, Aurélie; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis; Jullien, Frédéric

    2017-05-01

    Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221. Copyright © 2017. Published by Elsevier Ltd.

  7. A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice.

    Science.gov (United States)

    Zhou, Fei; Wang, Cheng-Yuan; Gutensohn, Michael; Jiang, Ling; Zhang, Peng; Zhang, Dabing; Dudareva, Natalia; Lu, Shan

    2017-06-27

    In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.

  8. Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase. Alanine-scanning mutagenesis of the flexible catalytic loop

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Bentsen, Ann-Kristin K; Harlow, Kenneth W

    2005-01-01

    Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197-208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced...... in an Escherichia coli strain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilis phosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail. The physical properties of the two enzymes were similar to those of the wildtype...

  9. Evaluation of the sorption of Eu(III) in titanium diphosphate; Evaluacion de la sorcion de Eu(III) en difosfato de titanio

    Energy Technology Data Exchange (ETDEWEB)

    Ortiz O, H.B.; Ordonez R, E.; Fernandez V, S.M. [ININ, Carretera Mexico-Toluca Km 36.5, Salazar, Estado de Mexico (Mexico)]. e-mail: hortiz@nuclear.inin.mx

    2007-07-01

    In this work its are presented: the synthesis, physicochemical characterization and the surface parameters estimation that can be related with the retention properties of the titanium diphosphate for the actinides of valence III (Pu, Am, Cm among others), using the Eu{sup 3+} like a chemical analog. The surface area, hydration time, zero charge point, density of active sites and the surface species distribution in the titanium diphosphate are reported. This information was used to explain the retention of the Eu(lll) in the surface of the titanium diphosphate. (Author)

  10. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and...

  11. Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    1996-01-01

    Phosphoribosyl diphosphate-lacking (Δprs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase...... reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth...... was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene...

  12. X-ray characteristics and thermal transformations of double diphosphates MLnP2O7 (M-K, Rb, Cs; Ln-REE of yttrium subgroup)

    International Nuclear Information System (INIS)

    Anisimova, N.Yu.; Chudinova, N.N.; Trunov, V.K.; AN SSSR, Moscow

    1993-01-01

    Results on studying double diphosphates of rare earth metals with K, Rb and Cs are studied by methods of X-ray analysis, high-temperature roentgenography and DTA. Double diphosphates of rare earth and alkali metals are crystallized in three structural types: monoclinic, rhombic and hexagonal, whereby rhombic form by heating inversely transforms into hexagonal one. MLnP 2 O 7 thermal transformations are studied

  13. Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis.

    Science.gov (United States)

    Rai, Avanish; Smita, Shachi S; Singh, Anup Kumar; Shanker, Karuna; Nagegowda, Dinesh A

    2013-09-01

    Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.

  14. Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate.

    Science.gov (United States)

    Gutensohn, Michael; Nguyen, Thuong T H; McMahon, Richard D; Kaplan, Ian; Pichersky, Eran; Dudareva, Natalia

    2014-07-01

    Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  15. Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses.

    Science.gov (United States)

    Manzano, David; Andrade, Paola; Caudepón, Daniel; Altabella, Teresa; Arró, Montserrat; Ferrer, Albert

    2016-09-01

    Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. © 2016

  16. Open reading frame 176 in the photosynthesis gene cluster of Rhodobacter capsulatus encodes idi, a gene for isopentenyl diphosphate isomerase.

    OpenAIRE

    Hahn, F M; Baker, J A; Poulter, C D

    1996-01-01

    Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway. A database search based on probes from the highly conserved regions in three eukaryotic IPP isomerases revealed substantial similarity with ORF176 in the photosynthesis gene cluster in Rhodobacter capsulatus. The open reading frame was cloned into an Escherichia coli expression vector. The encoded 20-kDa protein, which was purified in two steps by ion exchange and hydrophobic...

  17. Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non-diphosphate Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    O' Dowd, Bing [Department of Chemistry, University of Illinois, 600 South Mathews Avenue Urbana IL 61801 USA; Williams, Sarah [Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla CA 92093 USA; Wang, Hongxin [Department of Chemistry, University of California, 1 Shields Avenue Davis CA 95616 USA; Lawrence Berkeley National Laboratory, 1 Cyclotron Road Berkeley CA 94720 USA; No, Joo Hwan [Center for Biophysics and Computational Biology, Urbana, IL (United States); Rao, Guodong [Department of Chemistry, University of Illinois, 600 South Mathews Avenue Urbana IL 61801 USA; Wang, Weixue [Center for Biophysics and Computational Biology, Urbana, IL (United States); McCammon, J. Andrew [Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla CA 92093 USA; Howard Hughes Medical Institute, University of California at San Diego, La Jolla CA 92093 USA; National Biomedical Computation Resource, University of California at San Diego, La Jolla CA 92093 USA; Cramer, Stephen P. [Department of Chemistry, University of California, 1 Shields Avenue Davis CA 95616 USA; Lawrence Berkeley National Laboratory, 1 Cyclotron Road Berkeley CA 94720 USA; Oldfield, Eric [Department of Chemistry, University of Illinois, 600 South Mathews Avenue Urbana IL 61801 USA

    2017-04-07

    Isoprenoid biosynthesis is an important area for anti-infective drug development. One isoprenoid target described is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H + /2e - reduction. IspH contains a 4 Fe-4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe-4 S clusters by η 1 coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H 2 O, O 2 ) binding. Based on these ideas, we used in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a K i value of ≈500 nm against Pseudomonas aeruginosa IspH.

  18. Triclinic-cubic phase transition and negative expansion in the actinide IV (Th, U, Np, Pu) diphosphates

    International Nuclear Information System (INIS)

    Wallez, Gilles; Bregiroux, Damien; Raison, Philippe E.; Bykov, Denis; Konings, Rudy J.M.; Dacheux, Nicolas; Clavier, Nicolas; Delevoye, Laurent; Popa, Karin; Fitch, Andrew N.

    2012-01-01

    The AnP 2 O 7 diphosphates (An = Th, U, Np, Pu) have been synthesized by various routes depending on the stability of the An(IV) cation and its suitability for the unusual octahedral environment. Synchrotron and X-ray diffraction, thermal analysis, Raman spectroscopy, and 31 P nuclear magnetic resonance reveal them as a new family of diphosphates which probably includes the recently studied CeP 2 O 7 . Although they adopt at high temperature the same cubic archetypal cell as the other known MP 2 O 7 diphosphates, they differ by a very faint triclinic distortion at room temperature that results from an ordering of the P 2 O 7 units, as shown using high-resolution synchrotron diffraction for UP 2 O 7 . The uncommon triclinic-cubic phase transition is first order, and its temperature is very sensitive to the ionic radius of An(IV). The conflicting effects which control the thermal variations of the P-O-P angle are responsible for a strong expansion of the cell followed by a contraction at higher temperature. This inversion of expansion occurs at a temperature significantly higher than the phase transition, at variance with the parent compounds with smaller Mn(IV) cations in which the two phenomena coincide. As shown by various approaches, the P-O-b-P linkage remains bent in the cubic form. (authors)

  19. Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

    International Nuclear Information System (INIS)

    Gómez Barroso, J. A.; Pereira, H.; Miranda, M.; Pereira, C.; Garratt, R. C.; Aguilar, C. F.

    2010-01-01

    T. cruzi TcNDPK1 was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl 2 , 20% PEG 3350. Data were collected to 3.5 Å resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 Å. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease

  20. The uridine diphosphate glucuronosyltransferases: quantitative structure-activity relationships for hydroxyl polychlorinated biphenyl substrates

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Degao [Dalian University of Technology, Department of Environmental Science and Technology, Dalian (China)

    2005-10-01

    Quantitative structure-activity relationships (QSARs), which relate the glucuronidation of hydroxyl polychlorinated biphenyls (OH-PCBs) - catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs) - to their physicochemical properties and molecular structural parameters, can be used to predict the rate constants and interpret the mechanism of glucuronidation. In this study, QSARs have been developed that use 23 semi-empirical calculated quantum chemical descriptors to predict the logarithms of the constants 1/K{sub m} and V{sub max}, related to enzyme kinetics. A partial least squares regression method was used to select the optimal set of descriptors to minimize the multicollinearity between the descriptors, as well as to maximize the cross-validated coefficient (Q{sup 2} {sub cum}) values. The key descriptors affecting log(1/K{sub m}) were E{sub lumo}- E{sub homo} (the energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital) and q{sub C}{sup -} (the largest negative net atomic charge on a carbon atom), while the key descriptors affecting log V{sub max} were the polarizability {alpha}, the Connolly solvent-excluded volume (CSEV), and logP (the logarithm of the partition coefficient for octanol/water). From the results obtained it can be concluded that hydrophobic and electronic aspects of OH-PCBs are important in the glucuronidation of OH-PCBs. (orig.)

  1. Intestinal absorption of cytidine diphosphate choline and its changes in the digestive tract

    International Nuclear Information System (INIS)

    Yashima, Keisuke; Takamatsu, Masatoshi; Okuda, Kunio

    1975-01-01

    Intestinal absorption of cytidine diphosphate choline (CDP-choline), its structural changes in the digestive tract, and hepatic uptake have been investigated in rats using 14 C-labeled ( 14 CH 3 attached to N of choline) and 3 H-labeled (at C 5 of pyrimidine) compounds. The results indicate that: 1) CDP-choline is relatively stable in the stomach, but is quickly degraded into cytidine and choline in the intestine; 2) The hepatic uptakes of 14 C and 3 H reach the maximum in two to three hours after oral administration; 3) Whereas the amount of 14 C remaining in the gut is inversely related to the hepatic uptake, no similar correlation is seen with 3 H-labeled CDP-choline, and 4) Extrahepatic uptake of 14 C and 3 H is very small. The possibility of phosphorylation in the mucosa of choline and cytidine has been discussed, based on the differences in relative amount of radioactivity in individual broken-down products in the intestinal lumen and mucosa. (auth.)

  2. Chemical interaction of potassium diphosphate with cadmium nitrate in aqueous solution

    International Nuclear Information System (INIS)

    Kokhanovskij, V.V.

    1993-01-01

    Formation of low-soluble compounds in 1.5 mol/l isomolar cross section of K 4 P 2 O 7 -Cd(NO 3 ) 2 -H 2 O system was studied. Liquid phases are studied by the methods of refractometry and pH value measuring, an solid ones - by the methods of chemical and X-ray phase analysis, IR spectroscopy, chromatography and microscopy. Three individual chemical compounds K 2 CdP 2 O 7 x 4H 2 O, K 2 Cd 3 (P 2 O 7 ) 2 x 3H 2 O and Cd 2 P 2 O 7 x 3.5H 2 O and some their mixtures were isolated and investigated. It is shown that doulble diphosphate K 6 Cd(P 2 O 7 ) 2 x 6H 2 O does not precipitate spontanously, but instead of it in wide region of system K 2 CdP 2 O 7 x 4H 2 O crystallizes as elongated acicular crystals or as thin plates of improper form

  3. Surface complexation modeling of uranium (Vi) retained onto zirconium diphosphate in presence of organic acids

    International Nuclear Information System (INIS)

    Almazan T, M. G.; Garcia G, N.; Ordonez R, E.

    2010-10-01

    In the field of nuclear waste disposal, predictions regarding radionuclide migration through the geosphere, have to take account the effects of natural organic matter. This work presents an investigation of interaction mechanisms between U (Vi) and zirconium diphosphate (ZrP 2 O 7 ) in presence of organic acids (citric acid and oxalic acid). The retention reactions were previously examined using a batch equilibrium method. Previous results showed that U (Vi) retention was more efficient when citric acid or oxalic acid was present in solid surface at lower ph values. In order to determine the retention equilibria for both systems studied, a phosphorescence spectroscopy study was carried out. The experimental data were then fitted using the Constant Capacitance Model included in the FITEQL4.0 code. Previous results concerning surface characterization of ZrP 2 O 7 (surface sites density and surface acidity constants) were used to constraint the modeling. The best fit for U (Vi)/citric acid/ZrP 2 O 7 and U (Vi)/oxalic acid/ZrP 2 O 7 systems considered the formation of a ternary surface complex. (Author)

  4. Biphasic Elimination of Tenofovir Diphosphate and Nonlinear Pharmacokinetics of Zidovudine Triphosphate in a Microdosing Study

    Science.gov (United States)

    Chen, Jianmeng; Flexner, Charles; Liberman, Rosa G.; Skipper, Paul L.; Louissaint, Nicolette; Tannenbaum, Steven R.; Hendrix, Craig; Fuchs, Edward

    2012-01-01

    Objective Phase 0 studies can provide initial pharmacokinetics (PK) data in humans and help to facilitate early drug development, but their predictive value for standard dosing is controversial. To evaluate the prediction of microdosing for active intracellular drug metabolites, we compared the PK profile of two antiretroviral drugs, zidovudine (ZDV) and tenofovir (TFV), in microdose and standard dosing regimens. Study Design We administered a microdose (100 μg) of 14C-labeled drug (ZDV or tenofovir disoproxil fumarate (TDF)) with or without a standard unlabelled dose (300 mg) to healthy volunteers. Both the parent drug in plasma and the active metabolite, ZDV-triphosphate (ZDV-TP) or TFV-diphosphate (TFV-DP) in PBMCs and CD4+ cells were measured by AMS. Results The intracellular ZDV-TP concentration increased less than proportionally over the dose range studied (100 μg to 300 mg), while the intracellular TFV-DP PK were linear over the same dose range. ZDV-TP concentrations were lower in CD4+ cells versus total peripheral blood mononuclear cells (PBMCs), while TFV-DP concentrations were not different in CD4+ cells and PBMCs. Conclusion Our data were consistent with a rate-limiting step in the intracellular phosphorylation of ZDV but not TFV. AMS shows promise for predicting the PK of active intracellular metabolites of nucleosides, but nonlinearity of PK may be seen with some drugs. PMID:23187888

  5. Surface and micellar properties of Chloroquine Diphosphate and its interactions with surfactants and Human Serum Albumin

    International Nuclear Information System (INIS)

    Usman, Muhammad; Siddiq, Mohammad

    2013-01-01

    Highlights: ► Free energy of adsorption is more negative than free energy of micellization. ► Shifts in UV/Visible spectra in presence of SDS indicated interaction of CLQ with SDS. ► The decrease in fluorescence intensity of HSA by CLQ shows its binding with HSA. -- Abstract: This manuscript addresses the physicochemical behavior of an antimalarial drug Chloroquine Diphosphate (CLQ) as well as its interaction with anionic surfactants and Human Serum Albumin (HSA). Surface tension and specific conductivity were employed to detect the critical micelle concentration (CMC) and thus its surface and thermodynamic parameters were calculated. Solubilization of this drug within micelles of anionic surfactant sodium dodecyl sulfate (SDS) has also been studied. UV/Visible spectroscopy was used to calculate partition coefficient (K x ), free energy of partition and number of drug molecules per micelle. The complexation of drug with HSA at physiological conditions (pH 7.4) has also been analyzed by using UV/Visible and fluorescence spectroscopy. The values of drug-protein binding constant, number of binding sites and free energy of binding were calculated

  6. Crystallization and preliminary X ray analysis of nucleoside diphosphate kinase 1 from T. cruzi

    International Nuclear Information System (INIS)

    Gomez Barroso, J.A.; Aguilar, C.F.; Miranda, M.R.; Pereira, C.A.

    2009-01-01

    Introduction: Trypanosoma cruzi is the etiologic agent of Chagas disease. The Nucleoside diphosphate kinases (NDPKs) are enzymes involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform. The objective of this work is obtaining protein's crystals, diffract and process the data for tridimensional structure resolution. Materials and Methods: TcNDPK1 was expressed in E. coli as a fusion protein with Nterminal His-tag. TcNDPK1 was overexpressed and purified by FPLC. Crystallization was assayed by sitting drop and hanging drop vapor diffusion method. Crystals was frozen and diffracted on synchrotron x-ray radiation in Campinas (Brasil). The data set collected was reduced and merged using MOSFLM and SCALA programs. Results and Discussion: His-TcNDPK was overexpressed, purified and crystallized. The crystals are diffracted and collected the data to 3.5A. The crystals belong to the trigonal space group P3, with unit cell parameters a=127.94, b=127.84, c=275.49. Structure determination is under way. These results will provide relevant information that could be the first step in rational drug design for treating Chagas disease.(authors)

  7. Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

    International Nuclear Information System (INIS)

    Hamel, E.; Batra, J.K.; Lin, C.M.

    1986-01-01

    Using highly purified calf brain tubulin bearing [8- 14 C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins, the authors have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg 2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly

  8. Fibrillin 5 Is Essential for Plastoquinone-9 Biosynthesis by Binding to Solanesyl Diphosphate Synthases in Arabidopsis

    Science.gov (United States)

    Kim, Eun-Ha; Lee, Yongjik

    2015-01-01

    Fibrillins are lipid-associated proteins in plastids and are ubiquitous in plants. They accumulate in chromoplasts and sequester carotenoids during the development of flowers and fruits. However, little is known about the functions of fibrillins in leaf tissues. Here, we identified fibrillin 5 (FBN5), which is essential for plastoquinone-9 (PQ-9) biosynthesis in Arabidopsis thaliana. Homozygous fbn5-1 mutations were seedling-lethal, and XVE:FBN5-B transgenic plants expressing low levels of FBN5-B had a slower growth rate and were smaller than wild-type plants. In chloroplasts, FBN5-B specifically interacted with solanesyl diphosphate synthases (SPSs) 1 and 2, which biosynthesize the solanesyl moiety of PQ-9. Plants containing defective FBN5-B accumulated less PQ-9 and its cyclized product, plastochromanol-8, but the levels of tocopherols were not affected. The reduced PQ-9 content of XVE:FBN5-B transgenic plants was consistent with their lower photosynthetic performance and higher levels of hydrogen peroxide under cold stress. These results indicate that FBN5-B is required for PQ-9 biosynthesis through its interaction with SPS. Our study adds FBN5 as a structural component involved in the biosynthesis of PQ-9. FBN5 binding to the hydrophobic solanesyl moiety, which is generated by SPS1 and SPS2, in FBN5-B/SPS homodimeric complexes stimulates the enzyme activity of SPS1 and SPS2. PMID:26432861

  9. Temperature effects on the interaction mechanisms between the europium (III) and uranyl ions and zirconium diphosphate

    International Nuclear Information System (INIS)

    Finck, N.

    2006-10-01

    Temperature should remain higher than 25 C in the near field environment of a nuclear waste repository for thousands years. In this context, the aim of this work is to study the temperature influence on the interaction mechanisms between europium (III) and uranyl ions and zirconium diphosphate, as well as the influence of a complexing medium (nitrate) on the sorption of the lanthanide. The experimental definition of the equilibria was achieved by combining a structural investigation with the macroscopic sorption data. Surface complexes were characterized at all temperatures (25 C to 90 C) by TRLFS experiments carried out on dry and in situ samples using an oven. This characterization was completed by XPS experiments carried out at 25 C on samples prepared at 25 C and 90 C. The reaction constants (surface hydration and cations sorption) were obtained by simulating the experimental data with the constant capacitance surface complexation model. The reaction constants temperature dependency allowed one to characterize thermodynamically the different reactions by application of the van't Hoff relation. The validity of this law was tested by performing microcalorimetric measurements of the sorption heat for both cations. (author)

  10. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    Science.gov (United States)

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  11. The effect of cytidine-diphosphate choline (CDP-choline) on brain lipid changes during aging

    International Nuclear Information System (INIS)

    De Medio, G.E.; Trovarelli, G.; Piccinin, G.L.; Porcellati, G.

    1984-01-01

    Lipid synthesis has been tested in vivo in different brain areas of 12-month-old male rats. Cortex, striatum, brainstem, and subcortex of brain have been examined. The cerebellum was discarded. Mixtures of (2- 3 H)glycerol and (Me- 14 C)choline were injected into the lateral ventricle of the brain as lipid precursors, and their incorporation into total lipid, water-soluble intermediates and choline-containing phospholipids was examined 1 hr after isotope injection. In another series of experiments cytidine-5'-diphosphate choline (CDP-choline) was injected intraventricularly to the aged rats 10 min before sacrifice with a simultaneous injection, and radioactivity assays were performed as above. Distribution of radioactivity content of CDP-choline among brain areas 10 min after its administration showed a noticeable enrichment of the nucleotide and water-soluble-related compounds in the examined areas, but to a lesser degree in the cerebral cortex. The incorporation of labelled glycerol, which is severely depressed in aged rats in all four areas [Gaiti et al, 1982, 1983], was increased only in the cortex, and apparently decreased in the other areas. This last result is probably due to a dilution effect brought about by the administered cold CDP-choline upon the ( 14 C)-containing water-soluble metabolites. As a consequence, the ( 3 H)/( 14 C) ratio in total lipid and in isolated phosphatidylcholine and choline plasmalogen increased after CDP-choline treatment

  12. The thorium phosphate diphosphate as matrix for radioactive waste conditioning: radionuclide immobilization and behavior under irradiation

    International Nuclear Information System (INIS)

    Pichot, Erwan

    1999-01-01

    The aim of this work was to perform successively the decontamination of liquid solutions and the final immobilization of radionuclide storage using the same matrix. For this, thorium phosphate-diphosphate (TPD) of the formula Th 4 P 6 O 23 , is proposed as a very resistant to water corrosion matrix. A new compound, thorium phosphate hydrogeno-phosphate (TPHP) of the formula Th 2 (PO 4 ) 2 (HPO 4 ), nH 2 O with n=3-7 was synthesized and characterized. Heated at 1100 deg.C it is transformed into the TDP. Ion exchange properties of TPHP were investigated. The exchange yields of imponderable caesium, strontium and americium ion onto TPHP (NaNO 3 0.1 M media at pH=6) are equal to 60% for the first one and 100% for the two others. The results interpreted in terms of ion-exchange led to determine selectivity coefficient values for each cation and suggested that only hydrated ions are exchanged. While the TPD is proposed for the high level nuclear waste storage, the irradiation effects, particularly structural modifications were studied using both γ irradiation and charged particle irradiation. ESR and TL methods were carried out in order to identify radicals created during gamma radiation exposure. Correlation between ESR and TL experiments performed at room temperature clearly show three of PO 3 2- species and one POO· species of free radicals. We have shown that Au-ion irradiation in the range of MeV energy involved TPD structure and chemical modifications. Important sputtering was interpreted in terms of local thermal chemical decomposition. We have shown, at room temperature, that the amorphization dose for heavy ion irradiation is between 0.1 to 0.4 dpa. (author)

  13. Cloning and expression analysis of two dehydrodolichyl diphosphate synthase genes from Tripterygium wilfordii

    Directory of Open Access Journals (Sweden)

    Lin-Hui Gao

    2018-01-01

    Full Text Available Objective: To clone and investigate two dehydrodolichyl diphosphate synthase genes of Tripterygium wilfordii by bioinformatics and tissue expression analysis. Materials and Methods: According to the T. wifordii transcriptome database, specific primers were designed to clone the TwDHDDS1 and TwDHDDS2 genes via PCR. Based on the cloned sequences, protein structure prediction, multiple sequence alignment and phylogenetic tree construction were performed. The expression levels of the genes in different tissues of T. wilfordii were measured by real-time quantitative PCR. Results: The TwDHDDS1 gene encompassed a 873 bp open reading frame (ORF and encoded a protein of 290 amino acids. The calculated molecular weight of the translated protein was about 33.46 kDa, and the theoretical isoelectric point (pI was 8.67. The TwDHDDS2 encompassed a 768 bp ORF, encoding a protein of 255 amino acids with a calculated molecular weight of about 21.19 kDa, and a theoretical isoelectric point (pI of 7.72. Plant tissue expression analysis indicated that TwDHDDS1 and TwDHDDS2 both have relatively ubiquitous expression in all sampled organ tissues, but showed the highest transcription levels in the stems. Conclusions: The results of this study provide a basis for further functional studies of TwDHDDS1 and TwDHDDS2. Most importantly, these genes are promising genetic targets for the regulation of the biosynthetic pathways of important bioactive terpenoids such as triptolide.

  14. Comparison of agrobacterium mediated wheat and barley transformation with nucleoside diphosphate kinase 2 (NDPK2) gene

    International Nuclear Information System (INIS)

    Waheed, U.; Shah, M.M.; Smedley, M.; Harwood, W.

    2016-01-01

    An efficient and reliable transformation system is imperative for improvement of important crop species like barley and wheat. Wheat transformation is complex due to larger genome size and polyploidy while barley has a limitation of genotypic dependency. The objective of current study was to compare the relative transformation efficiency of wheat and barley using specific expression vector pBRACT 214-NDPK2 constructed through gateway cloning carrying Nucleoside Diphosphate Kinase 2 (NDPK2) gene. The vector was used to compare the transformation response in both crops using immature embryos through Agrobacterium mediated transformation. Both wheat and barley showed different responses towards callus induction and regeneration. Immature embryos of 1.5 to 2 mm in diameter was found optimum for wheat callus induction while 1 to 1.5 mm for barley. Both embryogenic and non-embryogenic calli were found in wheat with significantly greater tendency for embryogenecity in barley. The overall regeneration response was found different for all transformed wheat and barley cultivars. Wheat cultivars showed good response initially that drastically slowed down in later stages with the exception of Fielder that reached to the green shoots with good roots. The barley transformed lines showed good regeneration response as compared to wheat. PCR analysis of putative transformants using genomic DNA showed a maximum of 27% transformation efficiency in barely. No true transformation response was obtained in all cultivars of wheat used in this study. The protocol developed for wheat and barley transformation will greatly be helpful in crop improvement programme through genetic engineering especially in diploid relatives of cereals. (author)

  15. Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

    Science.gov (United States)

    Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

    2013-12-01

    Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

  16. Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

    Science.gov (United States)

    Misra, Rajesh Chandra; Garg, Anchal; Roy, Sudeep; Chanotiya, Chandan Singh; Vasudev, Prema G; Ghosh, Sumit

    2015-11-01

    Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  18. Formation of nicotinamide ribose diphosphate ribose, a new metabolite of the NAD pathway, by growing mycelium of Aspergillus niger

    International Nuclear Information System (INIS)

    Kuwahara, Masaaki

    1976-01-01

    A new step of NAD metabolism was shown in Aspergillus niger. Radioactive nicotinic acid and nicotinamide were incorporated into nicotinamide ribose diphosphate ribose (NAm-RDPR), which had been isolated from the culture filtrate. Its content in the culture medium increased with an increase of culture time, and this compound was proved to be a terminal metabolite in the NAD pathway. The experimental results also showed that the Preiss-Handler pathway and the NAD cycling system function in the NAD biosynthesis in A. niger. A part of the radioactive precursors was also incorporated into an unknown compound. (auth.)

  19. Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation.

    Science.gov (United States)

    Sasaki, Daisuke; Fujihashi, Masahiro; Okuyama, Naomi; Kobayashi, Yukiko; Noike, Motoyoshi; Koyama, Tanetoshi; Miki, Kunio

    2011-02-04

    Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.

  20. Functional characterization of ent-copalyl diphosphate synthase from Andrographis paniculata with putative involvement in andrographolides biosynthesis.

    Science.gov (United States)

    Shen, Qinqin; Li, Lixia; Jiang, Yu; Wang, Qiang

    2016-01-01

    To characterize the ent-copalyl diphosphate (ent-CPP) synthase involved in the biosynthetic pathway of andrographolides in a medicinal plant, Andrographis paniculata. The ent-CPP synthase (ent-CPS) gene was cloned from A. paniculata and its encoded ApCPS was demonstrated to react with (E,E,E)-geranylgeranyl diphosphate to form ent-CPP through recombinant expression in Escherichia coli. Site-directed mutagenesis of the Asp to Ala in the conserved DXDD motif of ApCPS resulted in loss of function. One Arg is located in the conserved position close to DXDD motif indicating the involvement of ApCPS in specialized metabolism. In addition, RT-PCR analysis revealed that ApCPS was expressed in all tissues of A. paniculata at all growth stages, which is consistent with andrographolides accumulating in these organs. Methyl jasmonate induced ApCPS gene expression, matching inducible accumulation of andrographolides in vivo. ApCPS is the first ent-CPS characterized in A. paniculata and is suggested to be involved in biosynthesis of andrographolides that have high pharmaceutical values.

  1. Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major

    International Nuclear Information System (INIS)

    Tonoli, Celisa Caldana Costa; Vieira, Plinio Salmazo; Ward, Richard John; Arni, Raghuvir Krishnaswamy; Oliveira, Arthur Henrique Cavalcante de; Murakami, Mario Tyago

    2009-01-01

    Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the nucleoside diphosphate kinase b from Leishmania major are reported. The crystals belonged to the trigonal space group P3 2 21 and diffracted to 2.18 Å resolution. Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host–parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 Å resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 Å. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3 2 21

  2. Study of the thorium phosphate-diphosphate (TPD) dissolution: kinetic aspect - thermodynamic aspect: analysis of the neo-formed phases; Etude de la dissolution du phosphate diphosphate de thorium: - aspect cinetique - aspect thermodynamique: analyse des phases neoformees

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, A.Ch

    2000-10-06

    The aim of this work is to study the aqueous corrosion of the thorium phosphate-diphosphate (TPD), of the formula Th{sub 4}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, in the framework of the actinides immobilization. In order to complete the anterior studies concerning solid solutions where thorium is substituted by a tetravalent ion (uranium (IV) or plutonium (IV)) in the TPD structure, compounds of thorium and neptunium phosphate-diphosphate, of formula Th{sub 4-x}Np{sub x}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, have been prepared. Furthermore, a new chemical way of synthesis has been investigated in order to sinter solids solution of thorium and uranium phosphate-diphosphate (TUPD) in good conditions. The TPD dissolution study showed two principals steps. The first one corresponds to the control of element concentration by the material dissolution whereas the second corresponds to the formation of secondary precipitates for which thermodynamic equilibrium controls the concentration of the species in solution. Leaching tests have been performed varying several independent parameters in order to determine the TPD dissolution rate. The partial orders related to the protons or to the hydroxide ions have been found between 0.35 and 0.45 whereas the apparent dissolution rate constants are in the range 1.10{sup -5} for 9.10{sup -5} g.m{sup -2}.j{sup -1} for acidic and basic media. The neo-formed phases have been characterized after the dissolution of TPD and TUPD. We found that the TPD leaching in acidic medium leads to the formation of the crystallized thorium phosphate-hydrogen-phosphate (TPHP), of formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}), x H{sub 2}O, whereas the TUPD dissolution leads to the TPHP and an other compound, of formula (UO{sub 2}){sub 3}(PO{sub 4}){sub 2}, 5 H{sub 2}O. We calculated its solubility product which is in good agreement with those found in the literature. The phases formed during the leaching of solids containing plutonium; americium or curium (Th

  3. Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver

    Directory of Open Access Journals (Sweden)

    Chen Ren

    2012-10-01

    Full Text Available Abstract Background Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. Results To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629 encoding isopentenyl diphosphate isomerase (IPI was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line control. Conclusions Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our

  4. Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Rosenkrantz, Tina J; Haldimann, Andreas

    2003-01-01

    An enzymatic pathway for synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) without the participation of PRPP synthase was analyzed in Escherichia coli. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene, the gene...

  5. Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract.

    Science.gov (United States)

    Yamamura, Y; Mizuguchi, Y; Taura, F; Kurosaki, F

    2014-10-01

    A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells.

  6. Hepatic conversion of bilirubin monoglucuronide to diglucuronide in uridine diphosphate-glucuronyl transferase-deficient man and rat by bilirubin glucuronoside glucuronosyltransferase

    NARCIS (Netherlands)

    Chowdhury, J. R.; Jansen, P. L.; Fischberg, E. B.; Daniller, A.; Arias, I. M.

    1978-01-01

    The microsomal enzyme uridine diphosphate (UDP) glucuronate glucuronyltransferase (E.C. 2.4.1.17) catalyzes formation of bilirubin mono-glucuronide from bilirubin and UDPglucuronic acid. Bilirubin glucuronoside glucuronosyltransferase (E.C. 2.4.1.95), an enzyme concentrated in plasma

  7. Acquisition of wild-type HIV-1 infection in a patient on pre-exposure prophylaxis with high intracellular concentrations of tenofovir diphosphate: a case report.

    NARCIS (Netherlands)

    Hoornenborg, Elske; Prins, Maria; Achterbergh, Roel C A; Woittiez, Lycke R; Cornelissen, Marion; Jurriaans, Suzanne; Kootstra, Neeltje A; Anderson, Peter L; Reiss, Peter; de Vries, Henry J C; Prins, Jan M; de Bree, Godelieve J

    2017-01-01

    Pre-exposure prophylaxis (PrEP) with emtricitabine and tenofovir disoproxil fumarate is highly effective against acquisition of HIV infection, and only two cases of infection with a multidrug-resistant virus have been reported under adequate long-term adherence, as evidenced by tenofovir diphosphate

  8. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    Science.gov (United States)

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  9. Tenofovir-induced Fanconi syndrome and osteomalacia in two HIV-infected patients: Role of intracellular tenofovir diphosphate levels and review of the literature

    NARCIS (Netherlands)

    Haverkort, M.E.; van der Spek, B.W.; Lips, P.T.A.M.; Slieker, W.A.; ter Heine, R.; Huitema, A.D.; Bronsveld, W.

    2011-01-01

    We present 2 human immunodeficiency virus-infected patients with tenofovir disoproxil fumarate-induced Fanconi syndrome, leading to osteomalacia. Intracellular tenofovir diphosphate levels were measured in 1 patient and were found to be very high, with plasma tenofovir levels just slightly elevated.

  10. Theoretical pKa prediction of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc

    Science.gov (United States)

    Vipperla, Bhavaniprasad; Griffiths, Thomas M.; Wang, Xingyong; Yu, Haibo

    2017-01-01

    The pKa value of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc (UDP-GlcNAc) has been successfully calculated using density functional theory methods in conjunction with the Polarizable Continuum Models. Theoretical methods were benchmarked over a dataset comprising of alkyl phosphates. B3LYP/6-31+G(d,p) calculations using SMD solvation model provide excellent agreement with the experimental data. The predicted pKa for UDP-GlcNAc is consistent with most recent NMR studies but much higher than what it has long been thought to be. The importance of this study is evident that the predicted pKa for UDP-GlcNAc supports its potential role as a catalytic base in the substrate-assisted biocatalysis.

  11. Revisiting the Latency of Uridine Diphosphate-Glucuronosyltransferases (UGTs—How Does the Endoplasmic Reticulum Membrane Influence Their Function?

    Directory of Open Access Journals (Sweden)

    Yuejian Liu

    2017-08-01

    Full Text Available Uridine diphosphate-glucuronosyltransferases (UGTs are phase 2 conjugation enzymes mainly located in the endoplasmic reticulum (ER of the liver and many other tissues, and can be recovered in artificial ER membrane preparations (microsomes. They catalyze glucuronidation reactions in various aglycone substrates, contributing significantly to the body’s chemical defense mechanism. There has been controversy over the last 50 years in the UGT field with respect to the explanation for the phenomenon of latency: full UGT activity revealed by chemical or physical disruption of the microsomal membrane. Because latency can lead to inaccurate measurements of UGT activity in vitro, and subsequent underprediction of drug clearance in vivo, it is important to understand the mechanisms behind this phenomenon. Three major hypotheses have been advanced to explain UGT latency: compartmentation, conformation, and adenine nucleotide inhibition. In this review, we discuss the evidence behind each hypothesis in depth, and suggest some additional studies that may reveal more information on this intriguing phenomenon.

  12. Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

    2000-01-01

    A steady state kinetic investigation of the Pi activation of 5-phospho-D-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that Pi can bind randomly to the enzyme either before or after an ordered addition of free Mg2+ and substrates. Unsaturation with ribose 5-phosphate increased...... the apparent cooperativity of Pi activation. At unsaturating Pi concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with Pi directs the subsequent ordered binding of Mg2+ and substrates via a fast pathway, whereas...... saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...

  13. Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate for the investigation of biosynthesis of O-antigenic polysaccharides in Pseudomonas aeruginosa and Escherichia coli O104.

    Science.gov (United States)

    Torgov, Vladimir; Danilov, Leonid; Utkina, Natalia; Veselovsky, Vladimir; Brockhausen, Inka

    2017-12-01

    Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P 1 -(11-phenoxyundecyl)-P 2 - (2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P 1 -(11-phenoxyundecyl)-P 2 -(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Temperature effects on the interaction mechanisms between the europium (III) and uranyl ions and zirconium diphosphate; Effets de la temperature sur les mecanismes d'interaction entre les ions europium (3) et uranyle et le diphosphate de zirconium

    Energy Technology Data Exchange (ETDEWEB)

    Finck, N

    2006-10-15

    Temperature should remain higher than 25 C in the near field environment of a nuclear waste repository for thousands years. In this context, the aim of this work is to study the temperature influence on the interaction mechanisms between europium (III) and uranyl ions and zirconium diphosphate, as well as the influence of a complexing medium (nitrate) on the sorption of the lanthanide. The experimental definition of the equilibria was achieved by combining a structural investigation with the macroscopic sorption data. Surface complexes were characterized at all temperatures (25 C to 90 C) by TRLFS experiments carried out on dry and in situ samples using an oven. This characterization was completed by XPS experiments carried out at 25 C on samples prepared at 25 C and 90 C. The reaction constants (surface hydration and cations sorption) were obtained by simulating the experimental data with the constant capacitance surface complexation model. The reaction constants temperature dependency allowed one to characterize thermodynamically the different reactions by application of the van't Hoff relation. The validity of this law was tested by performing microcalorimetric measurements of the sorption heat for both cations. (author)

  15. Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase.

    Science.gov (United States)

    Rudolf, Jeffrey D; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2016-08-31

    Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg(2+)-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

  16. 7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

    Science.gov (United States)

    Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

    2012-10-09

    Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

  17. Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses1[OPEN

    Science.gov (United States)

    Andrade, Paola; Caudepón, Daniel; Arró, Montserrat

    2016-01-01

    Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the iron-deficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development. PMID

  18. Insights into the Thiamine Diphosphate Enzyme Activation Mechanism: Computational Model for Transketolase Using a Quantum Mechanical/Molecular Mechanical Method.

    Science.gov (United States)

    Nauton, Lionel; Hélaine, Virgil; Théry, Vincent; Hecquet, Laurence

    2016-04-12

    We propose the first computational model for transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, using a quantum mechanical/molecular mechanical method on the basis of crystallographic TK structures from yeast and Escherichia coli, together with experimental kinetic data reported in the literature with wild-type and mutant TK. This model allowed us to define a new route for ThDP activation in the enzyme environment. We evidenced a strong interaction between ThDP and Glu418B of the TK active site, itself stabilized by Glu162A. The crucial point highlighted here is that deprotonation of ThDP C2 is not performed by ThDP N4' as reported in the literature, but by His481B, involving a HOH688A molecule bridge. Thus, ThDP N4' is converted from an amino form to an iminium form, ensuring the stabilization of the C2 carbanion or carbene. Finally, ThDP activation proceeds via an intermolecular process and not by an intramolecular one as reported in the literature. More generally, this proposed ThDP activation mechanism can be applied to some other ThDP-dependent enzymes and used to define the entire TK mechanism with donor and acceptor substrates more accurately.

  19. Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

    Science.gov (United States)

    Chin, Young-Wook; Seo, Nari; Kim, Jae-Han; Seo, Jin-Ho

    2016-11-01

    2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

    Science.gov (United States)

    Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

    2017-10-01

    Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62mM and 77.38s -1 , respectively, and the k cat /K m value was 124.81mM -1 s -1 . The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

  1. The thorium phosphate diphosphate as matrix for radioactive waste conditioning: radionuclide immobilization and behavior under irradiation; Le phosphate diphosphate de thorium, matrice pour le conditionnement des dechets radioactifs: immobilisation de radionucleides, comportement sous irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pichot, Erwan [Inst. de Physique Nucleaire, Paris-11 Univ., 91 - Orsay (France)

    1999-04-13

    The aim of this work was to perform successively the decontamination of liquid solutions and the final immobilization of radionuclide storage using the same matrix. For this, thorium phosphate-diphosphate (TPD) of the formula Th{sub 4}P{sub 6}O{sub 23}, is proposed as a very resistant to water corrosion matrix. A new compound, thorium phosphate hydrogeno-phosphate (TPHP) of the formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}), nH{sub 2}O with n=3-7 was synthesized and characterized. Heated at 1100 deg.C it is transformed into the TDP. Ion exchange properties of TPHP were investigated. The exchange yields of imponderable caesium, strontium and americium ion onto TPHP (NaNO{sub 3} 0.1 M media at pH=6) are equal to 60% for the first one and 100% for the two others. The results interpreted in terms of ion-exchange led to determine selectivity coefficient values for each cation and suggested that only hydrated ions are exchanged. While the TPD is proposed for the high level nuclear waste storage, the irradiation effects, particularly structural modifications were studied using both {gamma} irradiation and charged particle irradiation. ESR and TL methods were carried out in order to identify radicals created during gamma radiation exposure. Correlation between ESR and TL experiments performed at room temperature clearly show three of PO{sub 3}{sup 2-} species and one POO{center_dot} species of free radicals. We have shown that Au-ion irradiation in the range of MeV energy involved TPD structure and chemical modifications. Important sputtering was interpreted in terms of local thermal chemical decomposition. We have shown, at room temperature, that the amorphization dose for heavy ion irradiation is between 0.1 to 0.4 dpa. (author) 146 refs., 46 figs., 21 tabs.

  2. Study of the irradiation effects on thorium phosphate diphosphate ({beta}-TPD): consequences on its chemical durability; Etude des effets d'irradiation sur le phosphate diphosphate de thorium ({beta}-PDT): consequences sur la durabilite chimique

    Energy Technology Data Exchange (ETDEWEB)

    Tamain, C

    2005-12-15

    Since Thorium Phosphate Diphosphate (beta-TPD) can be considered as a potential host matrix for long-term storage in underground repository, it is necessary to study the irradiation effects on the structure of this ceramics and the consequences on its chemical durability. Sintered samples of beta-TPD and of associated solid solutions of beta-TUPD were irradiated under ion beams and then altered in aqueous solutions. Depending on the electronic LET value, beta-TPD can be completely or partly amorphized. Furthermore, the ability of recrystallization of the amorphous material by thermal annealing was also demonstrated. Some leaching tests, realized on these irradiated samples, have shown a significant effect of the amorphous fraction on the normalized dissolution rate which was increased by a factor of 10 from the crystallized to the fully amorphized material. Correlatively, the amorphous fraction also modified the delay to reach the saturation conditions associated to the thermodynamic equilibria involved. On the other hand, it exhibited no influence neither on other kinetic parameters, such as activation energy of the dissolution process or partial order related to the proton concentration, nor on the nature of the neo-formed phase formed at the saturation of the leachate and identified as Thorium Phosphate Hydrogeno-Phosphate Hydrate (TPHPH). Beta-TUPD samples were also irradiated by gamma and alpha rays during leaching tests to study the effects of radiolysis in the leaching medium on the normalized leaching rate. It appeared that the radiolytic species occurring in the dissolution mechanism were unstable, disappearing quickly when stopping the irradiation. (author)

  3. A Dual Repeat Cis-Element Determines Expression of GERANYL DIPHOSPHATE SYNTHASE for Monoterpene Production in Phalaenopsis Orchids

    Directory of Open Access Journals (Sweden)

    Yu-Chen Chuang

    2018-06-01

    Full Text Available Phalaenopsis bellina is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis transcription factors (TFs cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids.

  4. Synthesis and structural characterisation of iron(II) and copper(II) diphosphates containing flattened metal oxotetrahedra

    International Nuclear Information System (INIS)

    Keates, Adam C.; Wang, Qianlong; Weller, Mark T.

    2014-01-01

    Single crystal and bulk polycrystalline forms of K 2 MP 2 O 7 (M=Fe(II), Cu(II)) have been synthesised and their structures determined from single crystal X-ray diffraction data. Both compounds crystallize in the tetragonal system, space group P-42 1 m. Their structures are formed from infinite sheets of linked oxopolyhedra of the stoichiometry [MP 2 O 7 ] 2− with potassium cations situated between the layers. The MO 4 tetrahedra share oxygen atoms with [P 2 O 7 ] 4− diphosphate groups and the potassium ions have KO 8 square prismatic geometry. In both compounds the M(II) centre has an unusual strongly flattened, tetrahedral coordination to oxygen, as a result of the Jahn–Teller (JT) effect for the high spin d 6 Fe(II) and p-orbital mixing or a second order JT effect for d 9 Cu(II) centres in four fold coordination. The uncommon transition metal ion environments found in these materials are reflected in their optical absorption spectra and magnetism data. - Graphical abstract: The structures of the tetragonal polymorphs of K 2 MP 2 O 7 , M=Cu(II), Fe(II), consist of infinite sheets of stoichiometry [MP 2 O 7 ] 2− , formed from linked pyrophosphate groups and MO 4 tetrahedra, separated by potassium ions. In both compounds the unusual tetrahedral coordination of the M(II) centre is strongly flattened as a result of Jahn–Teller (JT) effects for high spin, d 6 Fe(II) and p-orbital mixing and second-order JT effects for d 9 Cu(II). Display Omitted - Highlights: • Tetrahedral copper and iron(II) coordinated by oxygen. • New layered phosphate structure. • Jahn–Teller and d 10 distorted coordinations

  5. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors.

    Science.gov (United States)

    Bettendorff, Lucien; Wins, Pierre

    2009-06-01

    Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

  6. Study of Thorium Phosphate Diphosphate (TPD) formation in nitric medium for the decontamination of high activity actinides bearing effluents

    International Nuclear Information System (INIS)

    Rousselle, Jerome

    2004-01-01

    Considering several activities in the nuclear industry and research, several low-level liquids wastes (LLLW) containing actinides in nitric medium must be decontaminated before being released in the environment. These liquid wastes mainly contain significant amounts of uranium(VI), neptunium(V) and plutonium(IV). In this work, two chemical ways were studied to decontaminate LLLW then to incorporate simultaneously uranium, neptunium and plutonium in the Thorium Phosphate Diphosphate (TPD). Both ways started from a nitric solution containing thorium and the actinides considered, present at their lower stable oxidation state. The first way consisted in the initial precipitation of actinide and thorium mixed oxalate. After drying the mixture containing the powder and phosphoric acid under dried argon, a poly-phase system was obtained. It was mainly composed by a thorium-actinide oxalate-phosphate. This mixture was transformed into a TPDAn solid solution (An = U, Np and/or Pu) by heating treatment at 1200 deg. C under inert atmosphere. The second way consisted in the precipitation of a precursor of TPD, identified as the Thorium Phosphate Hydrogen Phosphate loaded with the actinides considered. The gel initially formed by mixing concentrated phosphoric acid solution with the nitric actinide solution was heated at 90 - 160 deg. C in a closed PTFE container for several weeks. It led to the TPDAn solid solutions after heating at 1100 deg. C in air or under inert argon. The efficiency of both processes was evaluated through the determination of the decontamination for each actinide considered. Considering the encouraging results obtained for both kinds of processes, some complementary studies are now required before performing the effective decontamination of real Low-Level Liquid Waste using one of the methods proposed. (author) [fr

  7. Incorporation of tetravalent actinides in three phosphated matrices: britholite, monazite/brabandite and thorium phosphate diphosphate (β-TPD)

    International Nuclear Information System (INIS)

    Terra, O.

    2005-03-01

    Three phosphate based ceramics were studied for the immobilization of tri- and tetravalent actinides: britholite Ca 9 Nd 1-x An x IV (PO 4 ) 5-x (SiO 4 ) 1+x F 2 , monazite/brabantite solid solutions Ln 1-2x III Ca x An x IP O 4 and Thorium Phosphate Diphosphate (β-TPD) Th 4- xAn x IV (PO 4 ) 4 P 2 O 7 . For each material, the incorporation of thorium and uranium (IV) was studied as a surrogate of plutonium. This work was the early beginning of the incorporation of 239 Pu and/or 238 Pu in order to evaluate the effects of α-decay on the three crystallographic structures. The incorporation of tetravalent cations was carried out by dry chemistry methods, using mechanical grinding to improve the reactivity of the initial mixture then the homogeneity of final solid prepared after calcination at high temperature (1200-1400 deg C). For britholites, the thorium incorporation was complete for weight loading up to 20 wt.%, leading to the preparation of homogeneous and single phase solid solutions when using the coupled substitution (Nd 3+ , PO 4 3- ) ↔ (Th 4+ , SiO 4 4- ). Due to redox problems, the incorporation of uranium was limited to 5 to 8 wt.% and always led to a two-phase mixture of U-britholite and CaU 2 O 5+y . The preparation of homogeneous solid solutions of β-TUPD and of brabantites containing thorium and uranium samples was successfully obtained using three steps of mechanical grinding/calcination. For each matrix, dense pellets were prepared prior to the study of their chemical behaviour during leaching tests. The chemical durability of brabantites and β-TUPD were found to be close to that reported in literature. The formation of neo-formed phases was also evidenced onto the surface of Th-britholite samples. (author)

  8. Control activity of yeast geranylgeranyl diphosphate synthase from dimer interface through H-bonds and hydrophobic interaction.

    Science.gov (United States)

    Chang, Chih-Kang; Teng, Kuo-Hsun; Lin, Sheng-Wei; Chang, Tao-Hsin; Liang, Po-Huang

    2013-04-23

    Previously we showed that yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive monomer when the first N-terminal helix involved in dimerization is deleted. This raises questions regarding why dimerization is required for GGPPS activity and which amino acids in the dimer interface are essential for dimerization-mediated activity. According to the GGPPS crystal structure, three amino acids (N101, N104, and Y105) located in the helix F of one subunit are near the active site of the other subunit. As presented here, when these residues were replaced individually with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A but not N104A/Y105A showed remarkably decreased k(cat) values (200-250-fold). The triple mutant N101A/N104A/Y105A displayed no detectable activity, although dimer was retained in these mutants. Because N101 and Y105 form H-bonds with H139 and R140 in the other subunit, respectively, we generated H139A/R140A double mutant and found it was inactive and became monomeric. Therefore, the multiple mutations apparently influence the integrity of the catalytic site due to the missing H-bonding network. Moreover, Met111, also on the highly conserved helix F, was necessary for dimer formation and enzyme activity. When Met111 was replaced with Glu, the negative-charged repulsion converted half of the dimer into a monomer. In conclusion, the H-bonds mainly through N101 for maintaining substrate binding stability and the hydrophobic interaction of M111 in dimer interface are essential for activity of yeast GGPPS.

  9. Study of the irradiation effects on thorium phosphate diphosphate (β-TPD): consequences on its chemical durability

    International Nuclear Information System (INIS)

    Tamain, C.

    2005-12-01

    Since Thorium Phosphate Diphosphate (beta-TPD) can be considered as a potential host matrix for long-term storage in underground repository, it is necessary to study the irradiation effects on the structure of this ceramics and the consequences on its chemical durability. Sintered samples of beta-TPD and of associated solid solutions of beta-TUPD were irradiated under ion beams and then altered in aqueous solutions. Depending on the electronic LET value, beta-TPD can be completely or partly amorphized. Furthermore, the ability of recrystallization of the amorphous material by thermal annealing was also demonstrated. Some leaching tests, realized on these irradiated samples, have shown a significant effect of the amorphous fraction on the normalized dissolution rate which was increased by a factor of 10 from the crystallized to the fully amorphized material. Correlatively, the amorphous fraction also modified the delay to reach the saturation conditions associated to the thermodynamic equilibria involved. On the other hand, it exhibited no influence neither on other kinetic parameters, such as activation energy of the dissolution process or partial order related to the proton concentration, nor on the nature of the neo-formed phase formed at the saturation of the leachate and identified as Thorium Phosphate Hydrogeno-Phosphate Hydrate (TPHPH). Beta-TUPD samples were also irradiated by gamma and alpha rays during leaching tests to study the effects of radiolysis in the leaching medium on the normalized leaching rate. It appeared that the radiolytic species occurring in the dissolution mechanism were unstable, disappearing quickly when stopping the irradiation. (author)

  10. Functional evidence for the critical amino-terminal conserved domain and key amino acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE.

    Science.gov (United States)

    Hsieh, Wei-Yu; Sung, Tzu-Ying; Wang, Hsin-Tzu; Hsieh, Ming-Hsiun

    2014-09-01

    The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes. © 2014

  11. Acid dissociation constants of uridine-5 Prime -diphosphate compounds determined by {sup 31}phosphorus nuclear magnetic resonance spectroscopy and internal pH referencing

    Energy Technology Data Exchange (ETDEWEB)

    Jancan, Igor [Louisiana State University, Department of Chemistry, Baton Rouge, LA 70803 (United States); Macnaughtan, Megan A., E-mail: macnau@lsu.edu [Louisiana State University, Department of Chemistry, Baton Rouge, LA 70803 (United States)

    2012-10-24

    Highlights: Black-Right-Pointing-Pointer The first reported phosphate and imide pK{sub a} values of UDP-GlcNAc and UDP-S-GlcNAc. Black-Right-Pointing-Pointer New role for the monosaccharide in the imide pK{sub a} of uridine-5 Prime -phosphate compounds. Black-Right-Pointing-Pointer UDP-S-GlcNAc and UDP-GlcNAc have the same phosphate pK{sub a}, unlike thioyl analogs. Black-Right-Pointing-Pointer The {sup 31}P chemical shift of inorganic phosphate is a viable internal pH reference. Black-Right-Pointing-Pointer Stability of the external {sup 31}P chemical shift reference is essential. - Abstract: The acid dissociation constant (pK{sub a}) of small, biological molecules is an important physical property used for investigating enzyme mechanisms and inhibitor design. For phosphorus-containing molecules, the {sup 31}P nuclear magnetic resonance (NMR) chemical shift is sensitive to the local chemical environment, particularly to changes in the electronic state of the molecule. Taking advantage of this property, we present a {sup 31}P NMR approach that uses inorganic phosphate buffer as an internal pH reference to determine the pK{sub a} values of the imide and second diphosphate of uridine-5 Prime -diphosphate compounds, including the first reported values for UDP-GlcNAc and UDP-S-GlcNAc. New methods for using inorganic phosphate buffer as an internal pH reference, involving mathematical correction factors and careful control of the chemical shift reference sample, are illustrated. A comparison of the newly determined imide and diphosphate pK{sub a} values of UDP, UDP-GlcNAc, and UDP-S-GlcNAc with other nucleotide phosphate and thio-analogs reveals the significance of the monosaccharide and sulfur position on the pK{sub a} values.

  12. Influence of the temperature in the uranium (Vi) sorption in zirconium diphosphate; Influencia de la temperatura en la sorcion de uranio (VI) en difosfato de circonio

    Energy Technology Data Exchange (ETDEWEB)

    Garcia G, N.; Solis, D. [Universidad Autonoma del Estado de Mexico, Facultad de Quimica, Paseo Colon y Paseo Tollocan, 50120 Toluca, Estado de Mexico (Mexico); Ordonez R, E., E-mail: nidgg@yahoo.com.mx [ININ, Departamento de Quimica, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2012-10-15

    In the present work was evaluated the uranium (Vi) sorption at 10, 20, 30, 40 and 60 C on the zirconium diphosphate (ZrP{sub 2}O{sub 7}). They were carried out kinetic and isotherms using the method by lots, these will allow to fix the sorption time (kinetic) and to explain the behavior of this sorption in different ph conditions and temperature (isotherm). The quantity of retained uranium in the surface was quantified by means of the fluorescence technique. (Author)

  13. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    Science.gov (United States)

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The defective phosphoribosyl diphosphate synthase in a temperature-sensitive prs-2 mutant of Escherichia coli is compensated by increased enzyme synthesis

    DEFF Research Database (Denmark)

    Post, David A.; Switzer, Robert L.; Hove-Jensen, Bjarne

    1996-01-01

    An Escherichia coli strain which is temperature-sensitive for growth due to a mutation (prs-2) causing a defective phosphoribosyl diphosphate (PRPP) synthase has been characterized. The temperature-sensitive mutation was mapped to a 276 bp HindIII-BssHII DNA fragment located within the open reading...... temperature shift to 42 degrees C. The other mutation was a C -> T transition located 39 bp upstream of the G -> A mutation, i.e. outside the coding sequence and close to the Shine-Dalgarno sequence. Cells harbouring only the C -> T mutation in a plasmid contained approximately three times as much PRPP...

  15. Pertussis toxin substrate is a guanosine 5'-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein.

    OpenAIRE

    Wong, S K; Martin, B R; Tolkovsky, A M

    1985-01-01

    We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guano...

  16. The nucleoside diphosphate kinase gene Nme3 acts as quantitative trait locus promoting non-Mendelian inheritance.

    Directory of Open Access Journals (Sweden)

    Hermann Bauer

    Full Text Available The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95% from heterozygous (t/+ males to their offspring. This phenotype is termed transmission ratio distortion (TRD and is caused by the interaction of the t-complex responder (Tcr with several quantitative trait loci (QTL, the t-complex distorters (Tcd1 to Tcd4, all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr, a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and

  17. Synthesis and structural characterisation of iron(II) and copper(II) diphosphates containing flattened metal oxotetrahedra

    Energy Technology Data Exchange (ETDEWEB)

    Keates, Adam C. [School of Chemistry, University of Southampton, Southampton, Hampshire SO17 1B,. UK (United Kingdom); Wang, Qianlong [Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY (United Kingdom); Weller, Mark T., E-mail: m.t.weller@bath.ac.uk [Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY (United Kingdom)

    2014-02-15

    Single crystal and bulk polycrystalline forms of K{sub 2}MP{sub 2}O{sub 7} (M=Fe(II), Cu(II)) have been synthesised and their structures determined from single crystal X-ray diffraction data. Both compounds crystallize in the tetragonal system, space group P-42{sub 1}m. Their structures are formed from infinite sheets of linked oxopolyhedra of the stoichiometry [MP{sub 2}O{sub 7}]{sup 2−} with potassium cations situated between the layers. The MO{sub 4} tetrahedra share oxygen atoms with [P{sub 2}O{sub 7}]{sup 4−} diphosphate groups and the potassium ions have KO{sub 8} square prismatic geometry. In both compounds the M(II) centre has an unusual strongly flattened, tetrahedral coordination to oxygen, as a result of the Jahn–Teller (JT) effect for the high spin d{sup 6} Fe(II) and p-orbital mixing or a second order JT effect for d{sup 9} Cu(II) centres in four fold coordination. The uncommon transition metal ion environments found in these materials are reflected in their optical absorption spectra and magnetism data. - Graphical abstract: The structures of the tetragonal polymorphs of K{sub 2}MP{sub 2}O{sub 7}, M=Cu(II), Fe(II), consist of infinite sheets of stoichiometry [MP{sub 2}O{sub 7}]{sup 2−}, formed from linked pyrophosphate groups and MO{sub 4} tetrahedra, separated by potassium ions. In both compounds the unusual tetrahedral coordination of the M(II) centre is strongly flattened as a result of Jahn–Teller (JT) effects for high spin, d{sup 6} Fe(II) and p-orbital mixing and second-order JT effects for d{sup 9} Cu(II). Display Omitted - Highlights: • Tetrahedral copper and iron(II) coordinated by oxygen. • New layered phosphate structure. • Jahn–Teller and d{sup 10} distorted coordinations.

  18. High thiamine diphosphate concentrations in erythrocytes can be achieved in dialysis patients by oral administration of benfontiamine.

    Science.gov (United States)

    Frank, T; Bitsch, R; Maiwald, J; Stein, G

    2000-06-01

    The influence of either orally administered S-benzoylthiamine-O-monophosphate (benfotiamine) or thiamine nitrate on the thiamine status was tested in a randomised, two-group comparison study in 20 end-stage renal disease (ESRD) patients. Main outcome measures were the pharmacokinetics of thiamine diphosphate (TDP) in blood, the in vitro erythrocyte transketolase activity, its activation coefficient (alpha-ETK) and the TDP concentration in erythrocytes. After ingestion of a single dose of either 100 mg thiamine nitrate (corresponding to 305 micromol thiamine) or 100 mg benfotiamine (corresponding to 214 micromol thiamine), the blood levels of thiamine phosphate esters were analysed by means of high-performance liquid chromatography for a 24-h period. The TDP concentration in erythrocytes was calculated using the haematocrit and TDP concentration in blood. Erythrocyte transketolase activity and alpha-ETK were measured before and 10 h after administration. The pharmacokinetics of TDP in blood were compared with healthy subjects of other studies retrieved from database query. Regarding the blood concentrations of TDP, the patients with ESRD had a 4.3 times higher area under the concentration time curve after benfotiamine administration than after thiamine nitrate. After benfotiamine administration, the peak plasma concentration of TDP exceeded that in healthy subjects by 51%. In the ESRD patients, after 24 h, the mean TDP concentration in erythrocytes increased from 158.7+/-30.9 ng/ml initially to 325.8+/-50.9 ng/ml after administration of benfotiamine and from 166.2+/-51.9 ng/ml to 200.5+/-50.0 ng/ml after thiamine nitrate administration. The ratio between the maximum erythrocyte TDP concentration and basal concentration was 2.66+/-0.6 in the benfotiamine group and 1.44+/-0.2 in the group receiving thiamine nitrate (P benfotiamine intake (P = 0.02) and from 3.71+/-0.8 microkat/l to 4.02+/-0.7 microkat/l after thiamine nitrate intake (P = 0.08). Likewise, alpha

  19. Effect of fructose diphosphate combined with large-dose vitamin C therapy on the myocardial oxidative stress injury after neonatal asphyxia

    Directory of Open Access Journals (Sweden)

    Chun-Hua Liang1

    2017-04-01

    Full Text Available Objective: To study the effect of fructose diphosphate combined with large-dose vitamin C therapy on the myocardial oxidative stress injury after neonatal asphyxia. Methods: 40 patients with neonatal asphyxia who were treated in our hospital between June 2013 and April 2016 were collected and divided into the control group (n=20 who received large-dose vitamin C therapy and the observation group (n=20 who received fructose diphosphate combined with large-dose vitamin C therapy according to the double-blind randomized control method, and the treatment lasted for 10 d. Immediately after admission and after 10 d of treatment, RIA method was used to detect the serum levels of oxidative stress indexes, color Doppler diasonograph was used to determine left cardiac function parameters, and the myocardial enzyme spectrum detector was used to determine myocardial enzyme spectrum index levels. Results: Immediately after admission, the differences in the systemic oxidative stress degree, the left cardiac function damage degree and the myocardial enzyme spectrum index levels were not statistically significant between two groups of patients (P>0.05. After 10 d of treatment, serum malondialdehyde (MDA, advanced oxidation protein products (AOPP, creatine kinase isoenzyme (CK-MB, N-terminal pro-brain natriuretic peptide (Nt-proBNP, heart-type fatty acid-binding protein (H-FABP and troponin I (cTnI contents of observation group were lower than those of control group (P<0.05 while superoxide dismutase (SOD content was higher than that of control group (P<0.05, and the left cardiac function parameter ejection time (ET level was higher than that of control group (P<0.05 while left ventricular isovolumetric contraction time (ICT and left ventricular isovolumetric relaxation time (IRT levels were lower than those of control group (P<0.05. Conclusion: Fructose diphosphate combined with large-dose vitamin C can reduce the systemic oxidative stress of neonatal asphyxia

  20. Tricistronic operon expression of the genes gcaD (tms), which encodes N-acetylglucosamine 1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphosphate synthetase, and ctc in vegetative cells of Bacillus subtilis

    DEFF Research Database (Denmark)

    Hilden, Ida; Krath, Britta N.; Hove-Jensen, Bjarne

    1995-01-01

    The gcaD, prs, and ctc genes were shown to be organized as a tricistronic operon. The transcription of the prs gene, measured as phosphoribosyl diphosphate synthetase activity, and of the ctc gene, measured as β-galactosidase activity specified by a ctc-lacZ protein fusion, were dependent...

  1. Modification of zirconium diphosphate with salicylic acid and its effect on the uranium (Vi) sorption; Modificacion del difosfato de circonio con acido salicilico y su efecto sobre la sorcion de uranio (VI)

    Energy Technology Data Exchange (ETDEWEB)

    Almazan T, M. G.; Garcia G, N. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico); Simoni, E., E-mail: guadalupe.almazan@inin.gob.mx [Universidad Paris Sud, Instituto de Fisica Nuclear, Georges Clemenceau No. 15, Orsay (France)

    2014-10-15

    The surface of zirconium diphosphate (ZrP{sub 2}O{sub 7}) was modified with salicylic acid and its effect was evaluated on the uranium (Vi) sorption. The modified surface of the material was analyzed with different analytical techniques among which are included the atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. This analysis allowed showing that the salicylic acid is being held on the surface of the zirconium diphosphate. The reactivity of modified zirconium diphosphate compared with uranium (Vi) was investigated using the classical method of batch sorption. The analysis of sorption isotherms shows that the salicylic acid has an important effect in the uranium (Vi) sorption. According to the study conducted, the interaction among the uranium (Vi) and the surface of zirconium diphosphate modified with the salicylic acid most likely leads to the complexes formation of binary (U(Vi)/ZrP{sub 2}O{sub 7}) and ternary (U(Vi)/salicylate/ZrP{sub 2}O{sub 7}) surface. (Author)

  2. SIMULTANEOUS ANALYSIS OF AZIDOTHYMIDINE AND ITS MONOPHOSPHATE, DIPHOSPHATE AND TRIPHOSPHATE DERIVATIVES IN BIOLOGICAL-FLUIDS, TISSUE AND CULTURED-CELLS BY A RAPID HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD

    NARCIS (Netherlands)

    MOLEMA, G; JANSEN, RW; Visser, Jan; MEIJER, DKF

    1992-01-01

    A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro

  3. Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts...... is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two...... in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4....

  4. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    Science.gov (United States)

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-09

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

  5. Structure of the ent -Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Rudolf, Jeffrey D.; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2016-08-31

    Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three alpha-helical domains (alpha beta gamma), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (alpha) and type II TSs (beta gamma). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtnaT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 angstrom, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg2+-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

  6. Chemical interaction of potassium diphosphate with cadmium nitrate in aqueous solution. Khimicheskie vzaimodejstviya difosfatov kaliya s azotnokislym kadmiem v vodnom rastvore

    Energy Technology Data Exchange (ETDEWEB)

    Kokhanovskij, V V [AN Belorusskoj SSR, Minsk (Belarus). Inst. Obshchej i Neorganicheskoj Khimii

    1993-06-01

    Formation of low-soluble compounds in 1.5 mol/l isomolar cross section of K[sub 4]P[sub 2]O[sub 7]-Cd(NO[sub 3])[sub 2]-H[sub 2]O system was studied. Liquid phases are studied by the methods of refractometry and pH value measuring, an solid ones - by the methods of chemical and X-ray phase analysis, IR spectroscopy, chromatography and microscopy. Three individual chemical compounds K[sub 2]CdP[sub 2]O[sub 7] x 4H[sub 2]O, K[sub 2]Cd[sub 3](P[sub 2]O[sub 7])[sub 2] x 3H[sub 2]O and Cd[sub 2]P[sub 2]O[sub 7] x 3.5H[sub 2]O and some their mixtures were isolated and investigated. It is shown that doulble diphosphate K[sub 6]Cd(P[sub 2]O[sub 7])[sub 2] x 6H[sub 2]O does not precipitate spontanously, but instead of it in wide region of system K[sub 2]CdP[sub 2]O[sub 7] x 4H[sub 2]O crystallizes as elongated acicular crystals or as thin plates of improper form.

  7. Chemical synthesis of guanosine diphosphate mannuronic acid (GDP-ManA) and its C-4-O-methyl and C-4-deoxy congeners.

    Science.gov (United States)

    Zhang, Qingju; Howell, P Lynne; Overkleeft, Herman S; Filippov, Dmitri V; van der Marel, Gijsbert A; Codée, Jeroen D C

    2017-10-10

    Described is the first synthesis of guanosine diphosphate mannuronic acid (GDP-ManA), the sugar donor used by algae and bacteria for the production of alginate, an anionic polysaccharide composed of β-d-mannuronic acid (ManA) and α-l-guluronic acid (GulA). Understanding the biosynthesis of these polyanionic polysaccharides on the molecular level, opens up avenues to use and modulate the biosynthesis machinery for biotechnological and therapeutic applications. The synthesis reported here delivers multi-milligram amounts of the GDP-ManA donor that can be used to study the polymerase (Alg8 in Pseudomonas aeruginosa) that generates the poly-ManA chain. Also reported is the assembly of two close analogues of GDP-ManA: the first bears a C-4-O-methyl group, while the second has been deoxygenated at this position. Both molecules may be used as "chain stoppers" in future enzymatic ManA polymerisation reactions. The crucial pyrophosphate linkage of the GDP-mannuronic acids has been constructed by the phosphorylation of the appropriate ManA-1-phosphates with a guanosine phosphoramidite. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Effect of temperature and pH on the actiity of ribulose 1,5-diphosphate carboxylase from the thermophilic hydrogen bacterium Pseudomonas thermophila

    Energy Technology Data Exchange (ETDEWEB)

    Romanova, A K; Emnova, E E; Zykalova, K A

    1980-01-01

    The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completey loses its activity at 80/sup 0/C. The activity of RDP carboxylase at 60/sup 0/C increases by 40% during the first 10 min of heating in the presence of Mg/sup 2 +/ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50 to 55/sup 0/C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 keal.mole/sup -1/, but 11.6 kcal.mole/sup -1/ in frozen preparations. The optimal pH is 7.0 to 7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps, thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temeprature (the enzyme activity is negligible at 30/sup 0/C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.

  9. Chemical, structural and magnetic studies of new mono- and diphosphates appearing in ternary diagrams AO-CuO-(P2O5); A= Mg, Ca, Sr, Ba

    International Nuclear Information System (INIS)

    Moqine, A.

    1990-01-01

    A systematic study of mixed mono- and diphosphates of AO-CuO-(P 2 O 5 ) systems (A= Mg, Ca, Sr, Ba) has been carried out. This study showed up, on one hand, continuous and wide ranges in magnesium system and on the other hand, eight new phases in the other systems: Sr sub 18 Cu sub 3 (PO 4 ) 14 , A 2 Cu (PO 4 ) 2 , (A= Sr, Ba), Sr 3 Cu 3 (PO 4 ) 4 , BaCu 2 (PO 4 ) 2 and ACu(P 2 O 7 ) (A= Ca, Sr, Ba). The crystalline structures of SrCu(P 2 O 7 ), BaCu(P 2 O 7 ),Sr 3 Cu 3 (PO 4 ) 4 and BaCu 2 (PO 4 ) 2 phosphates have been described and discussed. The first compound presents a Tunnel structure and the three others have Foliation structures. The magnetic behaviours of some Cu sup 2+ ions phos-phates have been correlated to their crystalline structures. The experimental data have been parametered using the Ising model and/or the Heisenberg model. In this work, three Ising theoretical models have been developed. This magnetic study showed up new chains of antiferro- or ferrimagnetic Cu sup 2+ ions. 59 figs., 32 tabs., 113 refs. (author)

  10. Uridine diphosphate glucuronide transferase 1A1FNx0128 gene polymorphism and the toxicity of irinotecan in recurrent and refractory small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Fan Yun

    2014-01-01

    Full Text Available Objective: The aim was to investigate the association between uridine diphosphate glucuronide transferase 1A1 (UGT1A1 gene promoter region polymorphism and irinotecan-related adverse effects and efficacy on recurrent and refractory small cell lung cancer (SCLC. Materials and Methods: A total of 31 patients with recurrent and refractory SCLC were enrolled in this study from June 2012 to August 2013 and received at least two cycles of single-agent irinotecan chemotherapy. The efficacy and adverse effects of irinotecan were evaluated. DNA was extracted from peripheral blood and direct sequencing method was employed to test UGT1A1FNx0128 polymorphism, thus analyzing the correlation between UGT1A1FNx0128 polymorphism and irinotecan-related side-effects and efficacy. Results: A total of 25 cases (80.6% were UGT1A1FNx0128 wild-type (TA 6 /(TA 6 ; 6 cases (19.4% were heterozygous mutant (TA 6 /(TA 7 , no homozygous mutant genotype (TA 7 /(TA 7 was found. The incidences of grade 3/4 neutropenia, diarrhea and thrombocytopenia were 35.5%, 25.8% and 22.6% in all the patients, respectively. The incidence of 3/4 adverse effects in patients with genotype (TA 6 /(TA 6 and heterozygous (TA 6 /(TA 7 had no statistical difference (P > 0.05 for all. The overall response rate (ORR was 32.3%. Median progression free survival (PFS and overall survival (OS were 4 months and 7.5 months in all patients, respectively. There was no statistical difference in ORR, PFS and OS between genotype (TA 6 /(TA 6 patients and heterozygous (TA 6 /(TA 7 patients. Conclusion: Irinotecan showed efficacy in patients with recurrent and refractory SCLC; UGT1A1 FNx01 28 polymorphism failed to predict the incidence of serious adverse effects and efficacy of irinotecan.

  11. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  12. Fructose-1, 6-diphosphate (FDP as a novel antidote for yellow oleander-induced cardiac toxicity: A randomized controlled double blind study

    Directory of Open Access Journals (Sweden)

    Dawson Andrew H

    2010-06-01

    Full Text Available Abstract Background Cardiac toxicity due to ingestion of oleander plant seeds in Sri Lanka and some other South Asian countries is very common. At present symptomatic oleander seed poisoning carries a mortality of 10% in Sri Lanka and treatment of yellow oleander poisoning is limited to gastric decontamination and atropine administration. The only proven effective antidote is digoxin antibodies but these are not available for routine use because of the high cost. The main objective of this study is to investigate the effectiveness of a new and inexpensive antidote for patients with life threatening arrhythmias due oleander poisoning. Method/design We set up a randomised double blind clinical trial to assess the effectiveness of Fructose 1, 6 diphosphate (FDP in acute yellow oleander poisoning patients admitted to the adult medical wards of a tertiary hospital in Sri Lanka. Patients will be initially resuscitated following the national guidelines and eligible patients will be randomised to receive either FDP or an equal amount of normal saline. The primary outcome measure for this study is the sustained reversion to sinus rhythm with a heart rate greater than 50/min within 2 hours of completion of FDP/placebo bolus. Secondary outcomes include death, reversal of hyperkalaemia on the 6, 12, 18 and 24 hour samples and maintenance of sinus rhythm on the holter monitor. Analysis will be on intention-to-treat. Discussion This trial will provide information on the effectiveness of FDP in yellow oleander poisoning. If FDP is effective in cardiac glycoside toxicity, it would provide substantial benefit to the patients in rural Asia. The drug is inexpensive and thus could be made available at primary care hospitals if proven to be effective. Trial Registration Current Controlled trial ISRCTN71018309

  13. Studies on the interactions of chloroquine diphosphate and phenelzine sulfate drugs with human serum albumin and human hemoglobin proteins by spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Tunç, Sibel, E-mail: stunc@akdeniz.edu.tr; Duman, Osman, E-mail: osmanduman@akdeniz.edu.tr; Bozoğlan, Bahar Kancı

    2013-08-15

    The interactions of chloroquine diphosphate (CQP) and phenelzine sulfate (PS) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins were investigated by various spectroscopic methods. It was found that CQP caused the fluorescence quenching of protein molecules through a static quenching mechanism, but PS did not. The values of Stern–Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were calculated for HSA–CQP and HMG–CQP systems at pH 7.4 and different temperatures. For CQP, there was only one binding site on HSA and HMG proteins and the binding affinity of HSA was higher than that of HMG. The binding constants decreased with increasing temperature. The values of negative enthalpy change and positive entropy change indicated that electrostatic interactions play an important role in the binding processes. In addition, the binding processes were spontaneous and carried out by exothermic reactions. According to Förster resonance energy transfer theory, the average binding distance between proteins and CQP was calculated as 3.72 nm for HSA–CQP system and 3.45 nm for HMG–CQP system. Circular dichroism analysis displayed that the addition of CQP led to a decrease in the α-helix amount of HSA and HMG proteins. -- Highlights: • Unlike PS, CQP was bounded by HSA and HMG proteins. • The fluorescence spectra of HSA and HMG were quenched by CQP through static mechanism. • HSA–CQP and HMG–CQP complexes were stabilized by electrostatic attraction forces. • Binding constants, thermodynamic parameters and binding distances were calculated. • The binding of CQP changed the conformational structure of HSA and HMG proteins.

  14. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Azad, Arun, E-mail: arun.azad@bccancer.bc.ca [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Pathology, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Bukczynska, Patricia; Jackson, Susan [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Haput, Ygal; Cullinane, Carleen [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia); McArthur, Grant A.; Solomon, Benjamin [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Division of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Medicine, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia)

    2014-02-01

    Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.

  15. Co-targeting Deoxyribonucleic Acid–Dependent Protein Kinase and Poly(Adenosine Diphosphate-Ribose) Polymerase-1 Promotes Accelerated Senescence of Irradiated Cancer Cells

    International Nuclear Information System (INIS)

    Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A.; Solomon, Benjamin

    2014-01-01

    Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination

  16. Investigation of the system ThO2-NpO2-P2O5. Solid solutions of thorium-neptunium (IV) phosphate-diphosphate

    International Nuclear Information System (INIS)

    Dacheux, N.; Thomas, A.C.; Brandel, V.; Genet, M.

    1998-01-01

    Considering that phosphate matrices could be potential candidates for the immobilization of actinides or for the final disposal of the excess plutonium from dismantled nuclear weapons, the chemistry of thorium phosphates has been re-examined. In the ThO 2 -P 2 O 5 system, the thorium phosphate-diphosphate Th 4 (PO 4 ) 4 P 2 O 7 (TPD) can be synthesized by wet and dry chemical processes. The substitution of thorium by other tetravalent actinides like uranium or plutonium can be obtained for 0 4-x Np x (PO 4 ) 4 P 2 O 7 (TNPD) with 0 4+ by Np 4+ in the TPD structure is evaluated to 2.08 (which corresponds to about 52 mol% of thorium replaced by neptunium (IV)). The field of existence of solid solutions Th 4-x U -x Np -x Pu U x U Np x Np Pu x Pu (PO 4 )4P 2 O 7 has been calculated. These solid solutions should be synthesized for 5x U +7x Np +9x Pu ≤15. In the NpO 2 -P 2 O 5 system, the unit cell parameters of Np 2 O(PO 4 ) 2 were refined by analogy with U 2 O(PO 4 ) 2 which crystallographic data have been published recently. For Np 2 O(PO 4 ) 2 the unit cell is orthorhombic with the following cell parameters: a=7.033(2)A, b=9.024(3)A, c=12.587(6)A and V=799(1)A 3 . The unit cell parameter obtained for α-NpP 2 O 7 (a=8.586(1)A) is in good agreement with those already reported in literature. (orig.)

  17. Crystal structures of human sulfotransferases SULT1B1 and SULT1C1 complexed with the cofactor product adenosine-3'- 5'-diphosphate (PAP)

    Energy Technology Data Exchange (ETDEWEB)

    Dombrovski, Luidmila; Dong, Aiping; Bochkarev, Alexey; Plotnikov, Alexander N. (Toronto)

    2008-09-17

    Cytosolic sulfotransferases (SULTs), often referred as Phase II enzymes of chemical defense, are a superfamily of enzymes that catalyze the transfer of a sulfonate group from 3{prime}-phosphoadenosine 5{prime}-phosphosulfate (PAPS) to an acceptor group of substrates. This reaction modulates the activities of a large array of small endogenous and foreign chemicals including drugs, toxic compounds, steroid hormones, and neurotransmitters. In some cases, however, SULTs activate certain food and environmental compounds to mutagenenic and carcinogenic metabolites. Twelve human SULTs have been identified, which are partitioned into three families: SULT1, SULT2 and SULT4. The SULT1 family is further divided in four subfamilies, A, B, C, and E, and comprises eight members (1A1, 1A2, 1A3, 1B1, 1C1, 1C2, 1C3, and 1E1). Despite sequence and structural similarity among the SULTs, the family and subfamily members appear to have different biological function. SULT1 family shows substrate-binding specificity for simple phenols, estradiol, and thyroid hormones, as well as environmental xenobiotics and drugs. Human SULT1B1 is expressed in liver, colon, small intestine, and blood leukocytes, and shows substrate-binding specificity to thyroid hormones and benzylic alcohols. Human SULT1C1 is expressed in the adult stomach, kidney, and thyroid, as well as in fetal kidney and liver. SULT1C1 catalyzes the sulfonation of p-nitrophenol and N-hydroxy-2-acetylaminofluorene in vitro. However, the in vivo function of the enzyme remains unknown. We intend to solve the structures for all of the SULTs for which structural information is not yet available, and compare the structural and functional features of the entire SULT superfamily. Here we report the structures of two members of SULT1 family, SULT1B1 and SULT1C1, both in complex with the product of the PAPS cofactor, adenosine-3{prime}-5{prime}-diphosphate (PAP).

  18. Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

    International Nuclear Information System (INIS)

    Wright, K.T.; Seabright, R.; Logan, A.; Lilly, A.J.; Khanim, F.; Bunce, C.M.; Johnson, W.E.B.

    2010-01-01

    Research highlights: → Extracellular Nm23H1 stimulates nerve growth. → Extracellular Nm23H1 provides pathfinding cues to growth cones. → The neurotrophic activity of Nm23H1 is independent of NDP kinase activity. → The neurotrophic activity of Nm23H1 is independent of NGF. -- Abstract: The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.

  19. Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Wright, K.T. [Keele University at the RJAH Orthopaedic Hospital, Oswestry, Shropshire (United Kingdom); Seabright, R.; Logan, A. [Neuropharmacology and Neurobiology, School of Clinical and Experimental Medicine, Birmingham University, Birmingham (United Kingdom); Lilly, A.J.; Khanim, F.; Bunce, C.M. [Biosciences, Birmingham University, Birmingham (United Kingdom); Johnson, W.E.B., E-mail: w.e.johnson@aston.ac.uk [Life and Health Sciences, Aston University, Birmingham (United Kingdom)

    2010-07-16

    Research highlights: {yields} Extracellular Nm23H1 stimulates nerve growth. {yields} Extracellular Nm23H1 provides pathfinding cues to growth cones. {yields} The neurotrophic activity of Nm23H1 is independent of NDP kinase activity. {yields} The neurotrophic activity of Nm23H1 is independent of NGF. -- Abstract: The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.

  20. Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms1[W][OA

    Science.gov (United States)

    Keeling, Christopher I.; Dullat, Harpreet K.; Yuen, Mack; Ralph, Steven G.; Jancsik, Sharon; Bohlmann, Jörg

    2010-01-01

    The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms. PMID:20044448

  1. Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-D-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-D-Rhamnosyltransferase WbpZ.

    Science.gov (United States)

    Wang, Shuo; Hao, Youai; Lam, Joseph S; Vlahakis, Jason Z; Szarek, Walter A; Vinnikova, Anna; Veselovsky, Vladimir V; Brockhausen, Inka

    2015-06-15

    The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of D-rhamnose (D-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-D-Rha and enzymatically synthesized GDP-D-[(3)H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one D-Rha residue from GDP-D-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring D-mannose (D-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-D-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-rhamnosyltransferase that has significant activity of GDP-D-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-D-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a D-Rha-transferase and a critical step in the development of CPA synthesis inhibitors. This is the first characterization of a D-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen. Copyright © 2015, American Society for

  2. Thermodynamic studies of thorium phosphate diphosphate and phase investigations of Th-P-O and Th-P-H2O systems

    International Nuclear Information System (INIS)

    Rawat, Deepak; Dash, Smruti; Joshi, A.R.

    2014-01-01

    Highlights: • Δ f H m o (298.15K)andC p,m o (T) of Th 2 P 3 O 13 H 3 (cr), Th 2 P 3 O 12 H(cr), α-Th 4 P 6 O 23 (cr) and β-Th 4 P 6 O 23 (cr) were measured. • Thermo-chemical reaction scheme was formulated to calculate the standard molar enthalpy of formation of β-Th 4 (PO 4 ) 4 P 2 O 7 (cr). • The thermodynamic functions of the compounds present in Th-P-O and Th-P-H 2 O systems have been estimated. • The Gibbs phase diagram and predominant area diagrams for Th-P-O system have been calculated. • E H –pH diagram of Th-P-H 2 O system has been computed to determine stability of β-Th 4 P 6 O 23 (cr) in the ground water. - Abstract: The standard molar enthalpy of formation of thorium phosphate diphosphate, β-Th 4 (PO 4 ) 4 P 2 O 7 (cr) has been determined using an isoperibol solution calorimeter, a differential scanning calorimeter and phase transition data of high temperature calorimeter. The enthalpy of precipitation of thorium phosphate-hydrogenphosphate hydrate, Th 2 (PO 4 ) 2 (HPO 4 )·H 2 O(cr), was measured at 298.15 K using an isoperibol solution calorimeter. Heat capacities of α-Th 4 (PO 4 ) 4 P 2 O 7 (cr) and β-Th 4 (PO 4 ) 4 P 2 O 7 (cr) were measured using a Differential Scanning Calorimeter. Combining these experimental data, and other auxiliary data from the literature, thermo-chemical reaction scheme was devised to calculate the standard molar enthalpy of formation of β-Th 4 (PO 4 ) 4 P 2 O 7 (cr) to be {−10565.5 ± 13.6} kJ mol −1 . The thermodynamic functions for β-Th 4 (PO 4 ) 4 P 2 O 7 (cr) have been computed using Δ f H m o (298.15K),C p,m o (T) and literature data. The Gibbs phase diagram and predominant area diagrams for Th-P-O system have been computed using the thermodynamic information of the various phase present in Th-P, P-O and ThO 2 -P 2 O 5 systems. E H –pH diagram of Th-P-H 2 O system has been computed to determine stability of Th 4 P 6 O 23 (cr) in ground water

  3. Altered expression profile of glycolytic enzymes during testicular ischemia reperfusion injury is associated with the p53/TIGAR pathway: effect of fructose 1,6-diphosphate

    Directory of Open Access Journals (Sweden)

    May Al-Maghrebi

    2016-07-01

    Full Text Available Background. Testicular ischemia reperfusion injury (tIRI is considered the mechanism underlying the pathology of testicular torsion and detorsion. Left untreated, tIRI can induce testis dysfunction, damage to spermatogenesis and possible infertility. In this study, we aimed to assess the activities and expression of glycolytic enzymes (GEs in the testis and their possible modulation during tIRI. The effect of fructose 1,6-diphosphate (FDP, a glycolytic intermediate, on tIRI was also investigated. Methods. Male Sprague-Dawley rats were divided into three groups: sham, unilateral tIRI, and tIRI + FDP (2 mg/kg. tIRI was induced by occlusion of the testicular artery for 1 h followed by 4 h of reperfusion. FDP was injected peritoneally 30 min prior to reperfusion. Histological and biochemical analyses were used to assess damage to spermatogenesis, activities of major GEs, and energy and oxidative stress markers. The relative mRNA expression of GEs was evaluated by real-time PCR. ELISA and immunohistochemistry were used to evaluate the expression of p53 and TP53-induced glycolysis and apoptosis regulator (TIGAR. Results. Histological analysis revealed tIRI-induced spermatogenic damage as represented by a significant decrease in the Johnsen biopsy score. In addition, tIRI reduced the activities of hexokinase 1, phosphofructokinase-1, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase C. However, mRNA expression downregulation was detected only for hexokinase 1, phosphoglycerate kinase 2, and lactate dehydrogenase C. ATP and NADPH depletion was also induced by tIRI and was accompanied by an increased Malondialdehyde concentration, reduced glutathione level, and reduced superoxide dismutase and catalase enzyme activities. The immunoexpression of p53 and TIGAR was markedly increased after tIRI. The above tIRI-induced alterations were attenuated by FDP treatment. Discussion. Our findings indicate that tIRI-induced spermatogenic damage is

  4. Thiamine and benfotiamine prevent stress-induced suppression of hippocampal neurogenesis in mice exposed to predation without affecting brain thiamine diphosphate levels.

    Science.gov (United States)

    Vignisse, Julie; Sambon, Margaux; Gorlova, Anna; Pavlov, Dmitrii; Caron, Nicolas; Malgrange, Brigitte; Shevtsova, Elena; Svistunov, Andrey; Anthony, Daniel C; Markova, Natalyia; Bazhenova, Natalyia; Coumans, Bernard; Lakaye, Bernard; Wins, Pierre; Strekalova, Tatyana; Bettendorff, Lucien

    2017-07-01

    Thiamine is essential for normal brain function and its deficiency causes metabolic impairment, specific lesions, oxidative damage and reduced adult hippocampal neurogenesis (AHN). Thiamine precursors with increased bioavailability, especially benfotiamine, exert neuroprotective effects not only for thiamine deficiency (TD), but also in mouse models of neurodegeneration. As it is known that AHN is impaired by stress in rodents, we exposed C57BL6/J mice to predator stress for 5 consecutive nights and studied the proliferation (number of Ki67-positive cells) and survival (number of BrdU-positive cells) of newborn immature neurons in the subgranular zone of the dentate gyrus. In stressed mice, the number of Ki67- and BrdU-positive cells was reduced compared to non-stressed animals. This reduction was prevented when the mice were treated (200mg/kg/day in drinking water for 20days) with thiamine or benfotiamine, that were recently found to prevent stress-induced behavioral changes and glycogen synthase kinase-3β (GSK-3β) upregulation in the CNS. Moreover, we show that thiamine and benfotiamine counteract stress-induced bodyweight loss and suppress stress-induced anxiety-like behavior. Both treatments induced a modest increase in the brain content of free thiamine while the level of thiamine diphosphate (ThDP) remained unchanged, suggesting that the beneficial effects observed are not linked to the role of this coenzyme in energy metabolism. Predator stress increased hippocampal protein carbonylation, an indicator of oxidative stress. This effect was antagonized by both thiamine and benfotiamine. Moreover, using cultured mouse neuroblastoma cells, we show that in particular benfotiamine protects against paraquat-induced oxidative stress. We therefore hypothesize that thiamine compounds may act by boosting anti-oxidant cellular defenses, by a mechanism that still remains to be unveiled. Our study demonstrates, for the first time, that thiamine and benfotiamine prevent

  5. Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.

    Science.gov (United States)

    Dixon, C Jane; Hall, John F; Webb, Tania E; Boarder, Michael R

    2004-10-01

    Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

  6. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots

    Directory of Open Access Journals (Sweden)

    Chung TW

    2014-03-01

    Full Text Available Tze-Wen Chung,1,3 Pei-Yi Lin,2 Shoei-Shen Wang,2 Yen-Fung Chen31Department of Biomedical Engineering, National Yang-Ming University, 2Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; 3Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Yunlin, Taiwan, Republic of ChinaAbstract: Chitosan nanoparticles (NPs decorated with adenosine diphosphate (ADP (ANPs or fibrinogen (FNPs were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP or FNP (clot/FNP were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1±14.0, 251.0±9.8, and 326.5±14.5 nm and zeta potentials of 24.1±0.5, 20.6±1.9, and 15.3±1.5 mV (n=4, respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP, clot/ANP, and clot/FNP (at 0.05 wt% were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM, were significantly (P<0.05 (n=4 shorter than that of a clot induced by a phosphate-buffered solution (PBS (clot/PBS (63.6%±3.1%, 48.3%±6.2%, and 63.2%±4.7%, respectively. The ∆F2

  7. A new strategy for the cloning, overexpression and one step purification of three DHAP-dependent aldolases: rhamnulose-1-phosphate aldolase, fuculose-1-phosphate aldolase and tagatose-1,6-diphosphate aldolase.

    Science.gov (United States)

    Garcia-Junceda, E; Shen, G J; Sugai, T; Wong, C H

    1995-07-01

    Three DHAP-dependent aldolases, rhamnulose-1-phosphate aldolase (Rham-1PA), fuculose-1-phosphate aldolase (Fuc-1PA) and tagatose-1,6-diphosphate aldolase (TDPA) have been cloned and overexpressed in Escherichia coli using two different expression vectors: pTrcHis for the expression of Rham-1PA and Fuc-1PA and pRSET for the expression of TDPA. In each case the recombinant enzyme is synthesized as a fusion protein with a hexahistidine tag on the N-terminus. The three enzymes have been purified in only one step by chelation affinity chromatography. The effects of cultivation temperature and concentration of inducer have been studied in order to optimize the expression of the recombinant proteins and to avoid the formation of inclusion bodies.

  8. Influence of the temperature in the uranyl sorption in zirconium diphosphate modified with salicylic acid; Influencia de la temperatura en la sorcion de uranilo en difosfato de circonio modificado con acido salicilico

    Energy Technology Data Exchange (ETDEWEB)

    Garcia G, N.; Solis C, D. A. [Universidad Autonoma del Estado de Mexico, Facultad de Quimica, Paseo Colon y Paseo Tollocan s/n, 50000 Toluca, Estado de Mexico (Mexico); Ordonez R, E., E-mail: nidgg@yahoo.com.mx [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (MX)

    2011-11-15

    In this work the experimental conditions were established to evaluate the uranium (Vi) sorption to 20 and 40 C on the surface of the zirconium diphosphate (ZrP{sub 2}O{sub 7}) modified with a solution of salicylic acid 0.1 M. The modification of the ZrP{sub 2}O{sub 7} was produced during the hydrate process, taking advantage that these are formed complexes between the carboxyl and hydroxyl groups of salicylic acid and amphoteric species of the interface solid/liquid. The method is used by lots to elaborate the isotherms that explain the behavior of this sorption in different ph conditions and temperature, the quantity of the uranium reaction is analyzed with the fluorescence technique. The results indicated that in the temperature increases the uranium sorption on the material and is more efficient to low ph values. (Author)

  9. Incorporation of tetravalent actinides in three phosphated matrices: britholite, monazite/brabandite and thorium phosphate diphosphate ({beta}-TPD); Incorporation d'actinides tetravalents dans trois matrices phosphatees: britholite, monazite/brabantite et phosphate - diphosphate de thorium ({beta}-PDT)

    Energy Technology Data Exchange (ETDEWEB)

    Terra, O

    2005-03-01

    Three phosphate based ceramics were studied for the immobilization of tri- and tetravalent actinides: britholite Ca{sub 9}Nd{sub 1-x}An{sub x}{sup IV}(PO{sub 4}){sub 5-x}(SiO{sub 4}){sub 1+x}F{sub 2}, monazite/brabantite solid solutions Ln{sub 1-2x}{sup III} Ca{sub x}An{sub x}{sup IP}O{sub 4} and Thorium Phosphate Diphosphate ({beta}-TPD) Th{sub 4-}xAn{sub x}{sup IV}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}. For each material, the incorporation of thorium and uranium (IV) was studied as a surrogate of plutonium. This work was the early beginning of the incorporation of {sup 239}Pu and/or {sup 238}Pu in order to evaluate the effects of {alpha}-decay on the three crystallographic structures. The incorporation of tetravalent cations was carried out by dry chemistry methods, using mechanical grinding to improve the reactivity of the initial mixture then the homogeneity of final solid prepared after calcination at high temperature (1200-1400 deg C). For britholites, the thorium incorporation was complete for weight loading up to 20 wt.%, leading to the preparation of homogeneous and single phase solid solutions when using the coupled substitution (Nd{sup 3+}, PO{sub 4}{sup 3-}) {r_reversible} (Th{sup 4+}, SiO{sub 4}{sup 4-}). Due to redox problems, the incorporation of uranium was limited to 5 to 8 wt.% and always led to a two-phase mixture of U-britholite and CaU{sub 2}O{sub 5+y}. The preparation of homogeneous solid solutions of {beta}-TUPD and of brabantites containing thorium and uranium samples was successfully obtained using three steps of mechanical grinding/calcination. For each matrix, dense pellets were prepared prior to the study of their chemical behaviour during leaching tests. The chemical durability of brabantites and {beta}-TUPD were found to be close to that reported in literature. The formation of neo-formed phases was also evidenced onto the surface of Th-britholite samples. (author)

  10. Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate (NAD(PH-quinone oxidoreductase in mice

    Directory of Open Access Journals (Sweden)

    Yune-Fang Ueng

    2015-09-01

    Full Text Available Ruta graveolens (the common rue has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP, uridine diphosphate (UDP-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate (NAD(PH:quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(PH:quinone oxidoreductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (Fmix increased Cyp2b activities in males. The mixture of rutin and Fmix increased Cyp1a and Cyp2b activities. These results revealed that rutin and Fmix contributed at least in part to the P450 induction by rue.

  11. Correlations between polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A and C-C motif chemokine receptor 5 genes and infection with the hepatitis B virus in three ethnic groups in China.

    Science.gov (United States)

    Zhang, Chan; He, Yan; Shan, Ke-Ren; Tan, Kui; Zhang, Ting; Wang, Chan-Juan; Guan, Zhi-Zhong

    2018-02-01

    Objective To determine whether genetic polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A ( UGT1A) and the C-C motif chemokine receptor 5 ( CCR5) genes are associated with hepatitis B virus (HBV) infection in Yi, Yao and Han ethnic groups in the Guizhou Province of China. Methods The study enrolled subjects with and without HBV infection. Whole blood was used for DNA genotyping using standard techniques. The study determined the frequencies of several polymorphic alleles ( UGT1A6 [rs2070959], UGT1A1 [rs8175347], CCR5-59029 [rs1799987] and CCR5Δ32 [rs333]) and then characterized their relationship with HBV infection. Results A total of 404 subjects were enrolled in the study: 138 from the Yao group, 101 from the Yi group and 165 from the Han group. There was a significant difference in the frequency of UGT1A1 rs8175347 polymorphisms among the three groups. The rates of 7TA carriers of UGT1A1 rs8175347 in all three groups were significantly higher than the other genotypes. Individuals with genotype AA of UGT1A6 rs2070959 in the Yi group had a higher risk for HBV infection than in the Yao and Han groups. The frequency of genotype GG in CCR5-59029 in the Yao group was significantly higher than in the Yi group. The genotypes of CCR5Δ32 were not associated with HBV infection. Conclusion These findings provide genetic and epidemiological evidence for an association of UGT1A and CCR5-59029 polymorphisms with HBV infection in Chinese Yi and Yao populations.

  12. A combined variable temperature 600 MHz NMR/MD study of the calcium release agent cyclic adenosine diphosphate ribose (cADPR): Structure, conformational analysis, and thermodynamics of the conformational equilibria.

    Science.gov (United States)

    Javornik, Uroš; Plavec, Janez; Wang, Baifan; Graham, Steven M

    2018-01-02

    A combined variable temperature 600 MHz NMR/molecular dynamics study of the Ca 2+ -release agent cyclic adenosine 5'-diphosphate ribose (cADPR) was conducted. In addition to elucidating the major and minor orientations of the conformationally flexible furanose rings, γ- (C4'-C5'), and β- (C5'-O5') bonds, the thermodynamics (ΔH o , ΔS o ) associated with each of these conformational equilibria were determined. Both furanose rings were biased towards a south conformation (64-74%) and both β-bonds heavily favored trans conformations. The R-ring γ-bond was found to exist almost exclusively as the γ + conformer, whereas the A-ring γ-bond was a mixture of the γ + and γ t conformers, with the trans conformer being slightly favored. Enthalpic factors accounted for most of the observed conformational preferences, although the R-ring furanose exists as its major conformation based solely on entropic factors. There was excellent agreement between the NMR and MD results, particularly with regard to the conformer identities, but the MD showed a bias towards γ + conformers. The MD results showed that both N-glycosidic χ-bonds are exclusively syn. Collectively the data allowed for the construction of a model for cADPR in which many of the conformationally flexible units in fact effectively adopt single orientations and where most of the conformational diversity resides in its A-ring furanose and γ-bond. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. The Treatment of Cancer by a Radioactive Drug: Tritium-Labelled Tetra-Sodium 2-Methyl-1:4-Naphthaquinol Diphosphate; Traitement du Cancer par un Remede Radioactif: Le Diphosphate Tetrasodique de Methyl-2 Naphtoquinone-1,4 Tritie; 041b 0435 0447 0435 043d 0414 ; Tratamiento del Cancer con la Sal Tetrasodica del Acido 2-Metil-1,4-Naftoquinon Difosforico Marcada con Tritio

    Energy Technology Data Exchange (ETDEWEB)

    Marrian, D. H.; Marshall, Barbara; Mitchell, J. S.; Simon-Reuss, I. [Department of Radiotherapeutics, University of Cambridge, Cambridge (United Kingdom)

    1962-02-15

    The action of tetra-sodium 2-methyl-1: 4-naphthaquinoI diphosphate (''Synkavit''-Roche Products) as a radiosensitizer will be reviewed together with earlier methods of labelling this and similar molecules. The synthesis of this compound labelled with tritium has been accomplished so as to yield a radiochemically pure drug of specific activity 28 c/mmole (1 atom of tritium/molecule). It will be shown that this compound enters the living cell very rapidly both in-vivo and in-vitro and that cultures of malignant cells incorporate the label to a much greater extent than cultures of normal cells under identical conditions. Preliminary investigations on the use of this drug on patients with advanced, and often otherwise, refractory, malignant conditions will be described; uptake by the bone marrow is less than by the tumour, sometimes by a factor of 5. (author) [French] Les auteurs examinent l'action du diphosphate tetrasodique de methyl-2- naphtoquinone-1,4 ('Synkavit'-Produits Roche) comme radiosensibilisateur, ainsi que les methodes plus anciennes de marquage de ces molecules et de molecules analogues. On a fait la synthese de ce compose marque au tritium, de facon a obtenir un produit pharmaceutique radiochimiquement pur, dont l'activite specifique est de 28 curies par millimole (1 atome de tritium par molecule). Les auteurs demontrent que ce compose penetre tres rapidement dans la cellule vivante, tant in vivo qu'in vitro, et que les cultures de cellules malignes fixent l'indicateur dans une mesure beaucoup plus large que ne le feront les cellules normales dans les memes conditions. Les auteurs exposent les resultats de recherches preliminaires sur l'emploi de ce remede pour soigner des malades atteints de tumeurs malignes avancees et souvent refractaires a tout autre traitement. L'absorption par la moelle osseuse est inferieure, parfois de 5 fois, a l'absorption par la tumeur. (author) [Spanish] Los autores describen la accion radiosensibilizadora de la sal

  14. Elaboration of thorium uranium phosphate-diphosphate({beta}-TUPD) and {beta}-TUPD/monazite composite materials from crystallized precursors: sintering and study of the long term behavior of the ceramics; Elaboration de phosphate-diphosphate de thorium et d'uranium ({beta}-PDTU) et de materiaux composites {beta}-PDTU/Monazite a partir de precurseurs cristallises. Etudes du frittage et de la durabilite chimique

    Energy Technology Data Exchange (ETDEWEB)

    Clavier, N

    2004-11-01

    Thorium Phosphate-Diphosphate ({beta}-TPD) is actually considered as potential host matrix for the immobilization of radionuclides, and especially actinides, in the field of an underground repository. The studies reported in this work are based on the precipitation of the Thorium Phosphate Hydrogen-Phosphate Hydrate (TPHPH) as a precursor of {beta}-TPD. The crystal structure of TPHPH was solved then the reactions involved during its transformation into {beta}-TPD were established. It allows us to put in evidence a new monoclinic variety of TPD, called {alpha}-TPD, acting as intermediate of reaction. Moreover, the existence of a complete solid solution between TPHPH and UPHPH was demonstrated.The experimental conditions of sintering leading to an optimal densification of the pellets were determined. The relative density of the samples was always between 95 and 100% of the calculated value while a significant improvement of the homogeneity of the samples was noted. By this way, the process based on the precipitation of low-temperature crystallized precursors followed by their heat treatment at high temperature was applied to the preparation of {beta}-TUPD/Monazite based composites in the aim to incorporate simultaneously tri- and tetravalent actinides. The chemical durability of {beta}-TUPD sintered samples was evaluated. The normalized leaching rates determined in several experimental conditions revealed the good resistance of the solids to aqueous alteration. Moreover, the normalized dissolution rates exhibited a low dependence to temperature, pH as well as to several ions present in the leachate. For all the samples, thorium was quickly precipitated as a neo-formed phosphate phase identified to TPHPH. (author)

  15. Meniscectomia parcial como modelo experimental de osteoartrite em coelhos e efeito protetor do difosfato de cloroquina Partial meniscectomy as an experimental model of osteoarthritis in rabbits and protector effect of chloroquine diphosphate

    Directory of Open Access Journals (Sweden)

    Ana Paula P. Velosa

    2007-12-01

    Full Text Available OBJETIVO: Estabelecer as alterações morfológicas e o remodelamento do tecido cartilaginoso na progressão da osteoartrite (OA experimental para estudar o efeito do difosfato de cloroquina na cartilagem osteoartrítica. MÉTODOS: A osteoartrite experimental foi induzida em coelhos por meio de meniscectomia parcial. Para analisar a evolução da doença experimental foram estudados três grupos de dez animais, sacrificados a 3, 14 e 22 semanas de indução da doença. Para avaliar o efeito do difosfato de cloroquina um grupo de animais (n = 6 foi tratado preventivamente com 3 mg/kg ao dia, iniciados um mês antes da indução da osteoartrite, e mantidos até o sacrifício (22 semanas. Realizou-se análise histológica das articulações (H&E, tricrômico de Masson e imunofluorescência para colágeno dos tipos I, II e XI. A intensidade da agressão articular foi quantificada pelo escore de Mankin. RESULTADOS: O modelo experimental reproduziu todas as alterações morfológicas observadas na osteoartrite humana. Animais que receberam difosfato de cloroquina não desenvolveram lesões histológicas observadas na OA. Neste grupo houve significante preservação da estrutura da cartilagem articular (p OBJECTIVE: The aim of this study was to develop an experimental model of osteoarthritis (OA that could reproduce morphologic alterations viewed in this disease and to study the effect of chloroquine diphosphate on cartilage remodeling. METHODS: osteoarthritis was induced in rabbits by performing partial meniscectomy. To establish the experimental disease evolution, three groups of ten animals were sacrificed at 3, 14, 22 weeks after disease induction. To evaluate the effect of chloroquine diphosfate in OA progression, a group of six animals was treated preventively with 3 mg/kg/day, started one month prior to osteoarthritis induction and kept until the day of sacrifice (22 weeks. Histopathological (Masson trychrome, H&E, biochemical and

  16. New alkali metal diphosphates how materials to preserve the security of the environment: CsNaCu(P2O7), Rb2Cu(P2O7) and CsNaCo(P2O7) synthesis and crystal structure determination

    Science.gov (United States)

    Chernyatieva, Anastasiya; Filatova, Alyona; Spiridonova, Dariya; Krivovichev, Sergey

    2013-04-01

    In this work we describe preliminary results of the synthesis and of a crystal-chemical study of synthetic phosphates with transition metals. Due to the increasing requirements for environmental safety specialists from various industries, we are searching for sustainable forms of immobilization of hazardous waste during storage. We are also developing a component-based waste for new materials. In our continued exploratory synthesis of compounds containing transition-metals, we were able to produce the new diphosphate phases CsNaCu(P2O7), Rb2Cu(P2O7) and CsNaCo(P2O7). A crystal chemical study has allowed us to identify new phosphates. Crystals of CsNaCu(P2O7) (Phase 1) is orthorhombic, crystallizes in space group Pmn21, with a = 5.147(8), b = 15.126(2), c = 9.717(2) Å, V = 756.20 Å3, R1 = 0.066 and Rb2Cu(P2O7) (Phase 2) is orthorhombic as well, crystallizes in space group Pmcn, with a = 5.183(8), b = 10.096(1), c = 15.146(3) Å, V = 793.55 Å3, R1 = 0.063, they have been obtained by high-temperature reaction of RbNO3, CsNO3, Cu(NO3)2, NaOH and (NH4)4P2O7. Synthetic crystals of the phosphate of copper and rubidium were studied in detail by us on the structures of Rb2Cu(P2O7) and Rb2Cu3(P2O7)2 - new alkali metal copper diphosphates (CHERNYATIEVA et al., 2008). Here we report the synthesis, the structure and the properties of the title compounds and we compare these phases with the previously discovered K2CuP2O7 (ELMAADI et al., 1995) and CsNaMnP2O7 (HUANG et al., 1998). These structures crystallize in other space groups, although their structures are also based on 2-D layers, formed by P2O7 groups combined with polyhedra of the transition metals (CHERNYATIEVA et al., 2012). A crystal chemical study has allowed us to identify even new diphosphates CsNaCu(P2O7) (Phase 3). Crystals of CsNaCoP2O7 is monoclinic, space group P 21/n, with a = 7,424(2), b = 7,648(1), c = 12,931(3)Å, β = 90,71(2)° , V = 734.2(3) Å3 and R1 = 0.060. The structure is based framework of Co

  17. Tumor affinity of technetium-99m labeled radiopharmaceuticals. II. Sup(99m)Tc-Sn-diphosphonate (sup(99m)Tc-EHDP), sup(99m)Tc-Sn-dimercaptosuccinic acid (sup(99m)Tc-DMSA), sup(99m)Tc-Sn-diethyl stilbestrol diphosphate (sup(99m)Tc-DSDP)

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, K; Kobayashi, S; Hisada, K; Tonami, N [Kanazawa Univ. (Japan). School of Medicine; Ando, A

    1976-10-01

    The authors have examined the tumor affinity of various sup(99m)Tc-labelled radiopharmaceuticals to Ehrlich's tumor for the purpose of delineating human malignant neoplasm positively. The biologic distributions of sup(99m)Tc-Sn-diphosphonate (sup(99m)Tc-EHDP), sup(99m)Tc-Sn-dimercaptosuccinic acid (sup(99m)Tc-DMSA) and sup(99m)Tc-Sn-diethyl stilbestrol diphosphate (sup(99m)Tc-DSDP, sup(99m)Tc-Honvan) are included as the second report on the tumor affinity of Ehrlich-bearing mice. Tumor concentration of sup(99m)Tc-EHDP was lowest and the positive delineation of implanted tumor with sup(99m)Tc-EHDP was poorest in sequential images, though active accumulation in some soft tissues malignant neoplasms, breast cancer, and thyroid cancer, has been reported. Tumor concentration and the tumor-to-blood ratio of sup(99m)Tc-DMSA were not so high, contrary to our expectation that /sup 197/Hg-DMSA might show high tumor concentration and high tumor-to-blood ratio the same as /sup 197/Hg chlormerodrin of the renal scanning radiopharmaceuticals. Tumor concentration of sup(99m)Tc-DSDP was highest. The tumor-to-blood concentration ratio was lower than that of the above mentioned radiopharmaceuticals but the tumor-to-liver ratio and/or tumor-to-lung ratio was over 1.0 at the earlier time. Biologic distribution of sup(99m)Tc-DSDP was similar to that of /sup 32/P labeled DSDP. It is presumed that sup(99m)Tc is labeled at the phosphate ester of DSDP which is dephospholytated immediately by phospholylase in vivo following intravenous injection. Although it is not known precisely it may be assumed that the mechanism of accumulating sup(99m)Tc-DSDP in Ehrlich's tumor is related to the phospholylase activity in neoplasms.

  18. Structures of bilirubin conjugates synthesized in vitro from bilirubin and uridine diphosphate glucuronic acid, uridine diphosphate glucose or uridine diphosphate xylose by preparations from rat liver

    NARCIS (Netherlands)

    Fevery, J.; Leroy, P.; van de Vijver, M.; Heirwegh, K. P.

    1972-01-01

    1. In incubation mixtures containing digitonin-activated or untreated preparations from rat liver, albumin-solubilized bilirubin as the acceptor substrate and (a) UDP-glucuronic acid, (b) UDP-glucose or (c) UDP-xylose as the sugar donor, formation of the following ester glycosides was demonstrated:

  19. Formation of a Tc(III)-adenosine diphosphate complex

    International Nuclear Information System (INIS)

    Torres, J.; Kremer, C.; Kremer, E.

    1995-01-01

    A 99 Tc-ADP complex was prepared when KTcO 4 was reduced in aqueous medium by SnCl 2 , Na 2 S 2 O 4 , NaBH 4 or Zn in the presence of ADP in excess. The resulting solution was studied by chromatography and spectrophotometry. Electrochemical reduction and substitution on [Tc III (tu) 6 ] 3+ were investigated as alternative synthetic routes. The anionic Tc-ADP complex was isolated as a solid. Cerimetric titrations confirmed the oxidation state +3 for the central atom. IR and 1 H-NMR data showed that the purine base is bonded to the Tc central atom but not the ribose moiety. No oxo groups seemed to be directly bonded to the Tc atom. The complex is rather stable in neutral solutions. However, it decomposes to pertechnetate and TcO 2 at extreme pH values. (author). 16 refs., 2 figs., 3 tabs

  20. Biodistribution and dosimetric evaluation of 186Re hydroxy ethylen diphosphate

    International Nuclear Information System (INIS)

    Noto, M.G.; Manzini, Alberto

    1987-01-01

    The pharmacokinetics and the dose of radiation absorbed in different body tissues by the administration of 186 Re HEDP (hydroxyethylendiphosphate). The radiation dose in the standard man was established between 5,5 and 25 rad/mCi for red marrow, and red marrow and bone respectively; the radiation dose in metastases would be of 125 rad/mCi. It is concluded that this radiopharmaceutical is suitable for palliative treatment for pain with the mentioned patollogy. (M.E.L.) [es

  1. Formation of diphosphates. A NMR study on the mechanism and stereochemistry of diphosphate formation from chiral dioxaphosphorinanes

    NARCIS (Netherlands)

    Hulst, R; Visser, J.M.; De Vries, N.K.; Zijlstra, R.W J; Kooijman, H.; Smeets, W.J.J.; Spek, A.L.; Feringa, B.L.

    2000-01-01

    During the use of chiral 2-oxo-1,3,2-dioxaphosphorinanes as derivatizing reagents in the enantiomeric excess determination of amines, alcohols, and unprotected amino acids, minor traces of side reaction products were observed by 1H and 31P NMR spectroscopy. Analysis of the reaction mixture showed

  2. Chemistry of tetravalent actinides phosphates. The thorium phosphate-diphosphate as immobilisation matrix of actinides

    International Nuclear Information System (INIS)

    Dacheux, N.

    2002-01-01

    The author presents in this document its scientific works from 1992 to 2001, in order to obtain the enabling to manage scientific and chemical researches at the university Paris Sud Orsay. The first part gives an abstract of the thesis on the characterizations, lixiviation and synthesis of uranium and thorium based phosphate matrix in the framework of the search for a ceramic material usable in the radioactive waste storage. The second part presents briefly the researches realized at the CEA, devoted to a reliable, independent and accurate measure of some isotopes activity. The last part presents the abstracts of researches activities from 1996 to 2001 on the tetravalent actinides phosphates chemistry, the sintering of PDT and solid solutions of PDTU and the kinetic and thermodynamical studies of the PDT dissolution. Many references and some publication in full text are provided. (A.L.B.)

  3. Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

    1990-01-01

    enzyme required Mg2+ and inorganic phosphate for activity; Mn2+ supported only 30% the activity seen with Mg2+. Michaelis constants for ATP and ribose 5-phosphate (Rib5P) were 0.66 mM and 0.48 mM, respectively. Of several end products tested, only ADP was strongly inhibitory; GDP was a weak inhibitor....... ADP inhibition displayed homotropic cooperativity and was enhanced by increasing saturation of the enzyme with ATP. These observations strongly suggest a specific allosteric site for ADP binding. A comparison of physical and kinetic properties of bacterial and mammalian PPRibP synthetases is presented....

  4. Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

    OpenAIRE

    Wang, Yen-Chih; Dozier, Jonathan K.; Beese, Lorena S.; Distefano, Mark D.

    2014-01-01

    Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequen...

  5. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Georgescauld, Florian; Moynie, Lucile; Habersetzer, Johann

    2013-01-01

    activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80) -Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein...... of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M...

  6. Alterations of serum potassium, serum magnesium and adenosine diphosphate due to various contrast media containing iodine

    International Nuclear Information System (INIS)

    Lehrberger, G.

    1979-01-01

    As an introduction of the chemical structure of contrast media is explained. Then follows a survey about the complication rates in examinations with intravascularly applicable iodine-containing contrast media. In the next part clinical symptoms and signs of general and localized contrast media incompatibility reactions, the contrast medium protein reaction and the relationship between allergic reaction and contrast medium are explained. It was tried to attribute the large amount of side-effects to one primary reaction. In this connection the three above-mentioned components were investigated. (orig./MG) [de

  7. Mutation K42E in dehydrodolichol diphosphate synthase (DHDDS) causes recessive retinitis pigmentosa.

    Science.gov (United States)

    Lam, Byron L; Züchner, Stephan L; Dallman, Julia; Wen, Rong; Alfonso, Eduardo C; Vance, Jeffery M; Peričak-Vance, Margaret A

    2014-01-01

    A single-nucleotide mutation in the gene that encodes DHDDS has been identified by whole exome sequencing as the cause of the non-syndromic recessive retinitis pigmentosa (RP) in a family of Ashkenazi Jewish origin in which three of the four siblings have early onset retinal degeneration. The peripheral retinal degeneration in the affected siblings was evident in the initial examination in 1992 and only one had detectable electroretinogram (ERG) that suggested cone-rod dysfunction. The pigmentary retinal degeneration subsequently progressed rapidly. The identified mutation changes the highly conserved residue Lys42 to Glu, resulting in lower catalytic efficiency. Patterns of plasma transferrin isoelectric focusing gel were normal in all family members, indicating no significant abnormality in protein glycosylation. Dolichols have been shown to influence the fluidity and of the membrane and promote vesicle fusion. Considering that photoreceptor outer segments contain stacks of membrane discs, we believe that the mutation may lead to low dolichol levels in photoreceptor outer segments, resulting in unstable membrane structure that leads to photoreceptor degeneration.

  8. The 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase gene from ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... Total RNAs from each organ were isolated using RNAplantTM, a plant RNA isolation kit (TIAGEN,. Beijing, China). Cloning of the core fragment of TmHDR. Single-strand cDNAs were synthesized from 5 µg of total RNAs with ..... ara hdr, the endogenous hdr gene was replaced by a kanamycin-resistant ...

  9. [Renal excretion of methylene-diphosphate-technium-99m. Preliminary observations].

    Science.gov (United States)

    Vattimo, A; Martini, G

    1983-11-30

    The purpose of this study is to elucidate the mechanism of the renal excretion of 99mTc-MDP in man. We compared the renal clearance of 99mTc-MDP and 51Cr-EDTA (glomerular filtration rate agent). Since the 99mTc-MDP is bound to the plasma protein, the free fraction was calculated by dialysis. The clearances were obtained by single-injection technique. The plasma disappearance of the tracers was resolved into three exponential functions and area was calculated. The clearance was calculated by dividing the amount of the tracers excreted during the first four hours and the plasma area. In this study no difference was found in the clearance of the two agents. These findings suggest that the renal excretion of diphosphonate is related to the glomerular filtration rate.

  10. Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

    International Nuclear Information System (INIS)

    Kao, K.J.

    1988-01-01

    To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I-125-labeled platelets showed that only beta 2-M but not other I-125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I-125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins

  11. RESIDUAL PLATELET REACTIVITY DURING THERAPY WITH INHIBITORS OF CYCLOOXIGENASE OR ADENOSINE DIPHOSPHATE RECEPTORS

    Directory of Open Access Journals (Sweden)

    A. A. Lomonosova

    2012-01-01

    Full Text Available Aim. To compare effects of acetylsalicylic acid (ASA and two clopidogrel drugs on residual platelet aggregative reactivity (RPAR. Material and methods. Patients (n=40 with ischemic heart disease aged under 70 years were involved into the crossover study. Clinical examination included questionnaire survey , blood pressure (BP measurement, ECG registration, 24-hour ECG and BP monitoring, determination of blood levels of total cholesterol, high density lipoproteins, triglycerides, transaminases, and creatinine, complete blood cell count, including platelets number and hemoglobin level. Besides evaluation of the platelet aggregation by optical aggregometry was performed initially , after one week ASA treatment and after every next 3 week clopidogrel treatment period.  Results. RPAR during ASA monotherapy was 56.4±0.3%. There were no significant differences in effects of original and generic clopidogrel on RPAR. Сlopidogrel therapy reduced RPAR more significantly (42.2±0.2% than ASA monotherapy did (p=0.0003. Authors proposed definition for high level of RPAR during therapy - it is platelet aggregation more than 46%. Data analysis taking into account this criterion showed that a number of patients with high RPAR was 70 and 30% among patients treated with enterosoluble ASA and clopidogrel, respectively. Conclusion. Study results show that a significant number of patients receiving antiplatelet monotherapy does not achieve the target level of RPAR(<46%. These results may be a rationale for combined therapy in patients of this type.

  12. Chemistry of tetravalent actinides phosphates. The thorium phosphate-diphosphate as immobilisation matrix of actinides; Chimie des phosphates d'actinides tetravalents. Le phosphate-diphosphate de thorium en tant que matrice d'imobilisation des actinides

    Energy Technology Data Exchange (ETDEWEB)

    Dacheux, N

    2002-07-01

    The author presents in this document its scientific works from 1992 to 2001, in order to obtain the enabling to manage scientific and chemical researches at the university Paris Sud Orsay. The first part gives an abstract of the thesis on the characterizations, lixiviation and synthesis of uranium and thorium based phosphate matrix in the framework of the search for a ceramic material usable in the radioactive waste storage. The second part presents briefly the researches realized at the CEA, devoted to a reliable, independent and accurate measure of some isotopes activity. The last part presents the abstracts of researches activities from 1996 to 2001 on the tetravalent actinides phosphates chemistry, the sintering of PDT and solid solutions of PDTU and the kinetic and thermodynamical studies of the PDT dissolution. Many references and some publication in full text are provided. (A.L.B.)

  13. Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma cells. : resistance of osteosarcoma to nitrogen bisphosphonates

    OpenAIRE

    Ory , Benjamin; Moriceau , Gatien; Trichet , Valérie; Blanchard , Frédéric; Berreur , Martine; Rédini , Françoise; Rogers , Michael; Heymann , Dominique

    2008-01-01

    International audience; We recently demonstrated original anti-tumor effects of zoledronic acid (Zol) on osteosarcoma cell lines independently of their p53 and Rb status. The present study investigated the potential Zol-resistance acquired by osteosarcoma cells after prolonged treatment. After 12 weeks of culture in the presence of 1 microm Zol, the effects of high doses of Zol (10-100 microm) were compared between the untreated rat (OSRGA, ROS) and human (MG63, SAOS2) osteosarcoma cells and ...

  14. Enhancement of Farnesyl Diphosphate Pool as Direct Precursor of Sesquiterpenes Through Metabolic Engineering of the Mevalonate Pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Asadollahi, Mohammadali; Maury, Jerome; Schalk, M.

    2010-01-01

    , resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous overexpression of tHMG1 and repression of ERG9 did...... not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids...

  15. Modification of surface of zirconium diphosphate in solution; Modificacion de la superficie del difosfato de circonio en solucion

    Energy Technology Data Exchange (ETDEWEB)

    Garcia G, N.; Ordonez R, E. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico); Barrera D, C. E. [Universidad Autonoma del Estado de Mexico, Facultad de Quimica, Paseo Colon y Paseo Tollocan, Toluca 50120, Estado de Mexico (Mexico); Simoni, E. [Universite de Paris-Sud-XI, Institut de Physique Nucleaire d' Orsay, Bat 100, 91406 Orsay (France)], e-mail: eduardo.ordonez@inin.gob.mx

    2009-10-15

    The hydration process of ZrP{sub 2}O{sub 7}, forms active places in the surface of material that depend on the time and of nature of means in that it is immersed. The objective of this work is to study the influence that affect the active places of ZrP{sub 2}O{sub 7} for the action of poly carboxylic acid solutions during the hydration process. The experimentation was realized hydrating the ZrP{sub 2}O{sub 7} with solutions of sodium perchlorate, citric acid/sodium perchlorate and oxalic acid/sodium perchlorate during 24 hours, subsequently it is evaluated if some change exists in active places density formed by area unit. The organic acids action also modifies the acidity constants of surface, evaluated by means of titration acid-base technique. With these results the distribution diagrams of amphoteric species were modeled for each studied system. The results of this study phase showed that places density of hydrated material with NaClO{sub 4} was of 5.3 s/nm{sup 2}, with citric acid 13.7 s/nm{sup 2} and oxalic acid 7.3 s/nm{sup 2}. We can conclude that when solutions are used with organic acids a significant change takes place in the acidity constants and of the species distribution in surface that are discussed along this work. (Auth0008.

  16. Solanesyl Diphosphate Synthase, an Enzyme of the Ubiquinone Synthetic Pathway, Is Required throughout the Life Cycle of Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Lai, De Hua; Poropat, E.; Pravia, C.; Landoni, M.; Couto, A.S.; Pérez Rojo, F.G.; Fuchs, A.G.; Dubin, M.; Elingold, I.; Rodríguez, J.B.; Ferella, M.; Esteva, M.I.; Bontempi, Esteban J.; Lukeš, Julius

    2014-01-01

    Roč. 13, č. 2 (2014), s. 320-328 ISSN 1535-9778 R&D Projects: GA ČR(CZ) GAP305/11/2179; GA MŠk LH12104; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : RNA interference * procyclic form * NADH dehydrogenase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.820, year: 2014

  17. Synthesis of analogues of acyclic nucleoside diphosphates containing a (phosphonomethyl)phosphanyl moiety and studies of their phosphorylation

    Czech Academy of Sciences Publication Activity Database

    Doláková, Petra; Dračínský, Martin; Fanfrlík, Jindřich; Holý, Antonín

    -, č. 7 (2009), s. 1082-1092 ISSN 1434-193X R&D Projects: GA AV ČR 1QS400550501 Grant - others:NIH(US) 1UC1AI062540-01 Institutional research plan: CEZ:AV0Z40550506 Keywords : antiviral agents * nucleotides * phosphorylation * dUTPase Subject RIV: CC - Organic Chemistry Impact factor: 3.096, year: 2009

  18. Interaction of phosphates of the acyclic nucleoside phosphonates with nucleoside diphosphate kinase from yeast and bovine liver

    Czech Academy of Sciences Publication Activity Database

    Horská, Květoslava; Votruba, Ivan; Holý, Antonín

    2006-01-01

    Roč. 71, č. 1 (2006), s. 35-43 ISSN 0010-0765 R&D Projects: GA AV ČR(CZ) IBS4055109 Institutional research plan: CEZ:AV0Z40550506 Keywords : enzymatic phosphorylation * acyclic nucleotide analogs * NTP analogues Subject RIV: CC - Organic Chemistry Impact factor: 0.881, year: 2006

  19. Deletion in the uridine diphosphate glucuronyltransferase 2B17 gene is associated with delayed pubarche in healthy boys

    DEFF Research Database (Denmark)

    Mouritsen, Annette Korsholm; Busch, Alexander Siegfried; Aksglaede, Lise

    2018-01-01

    (del/ins) vs. 12.06 years (11.79-12.33) in boys with the wildtype genotype (ins/ins) (p=0.029, corrected for BMI z-score). The effect accounted for 0.34 years delay per allele (95%CI: 0.03-0.64). A comparable trend was observed for onset of testicular enlargement >3ml but did not reach significance...... and Measures: 668 healthy boys (cross-sectional) aged 6.1-21.9 years (COPENHAGEN puberty study conducted from 2005 to 2006) were included. 65 of the boys where followed longitudinally every 6 months. Participants were genotyped for UGT2B17 copy number variation (CNV). Clinical pubertal staging including...... orchidometry, anthropometry and serum reproductive hormone levels. RESULTS: 59 of the 668 boys (8.8%) presented with a homozygous deletion of UGT2B17 (del/del). These boys experienced pubarche at a mean age of 12.73 years (12.00-13.46) vs. 12.40 years (12.11-12.68) in boys heterozygous for deletion of UGT2B17...

  20. The molecular origin of the thiamin diphosphate-induced spectral bands of ThDP-dependent enzymes

    NARCIS (Netherlands)

    Kovina, M.V.; Kok, A.; Sevostyanova, I.A.; Khailova, L.S.; Belkina, N.V.; Kochetov, G.A.

    2004-01-01

    New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP

  1. The molecular origin of the thiamine diphosphate-induced spectral bands of ThDP-dependent enzymes.

    Science.gov (United States)

    Kovina, Marina V; De Kok, Aart; Sevostyanova, Irina A; Khailova, Ludmila S; Belkina, Natalya V; Kochetov, German A

    2004-08-01

    New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1' atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1' and the 4'-nitrogen. Copyright 2004 Wiley-Liss, Inc.

  2. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  3. CD38 Structure-Based Inhibitor Design Using the N1-Cyclic Inosine 5'-Diphosphate Ribose Template.

    Directory of Open Access Journals (Sweden)

    Christelle Moreau

    Full Text Available Few inhibitors exist for CD38, a multifunctional enzyme catalyzing the formation and metabolism of the Ca(2+-mobilizing second messenger cyclic adenosine 5'-diphosphoribose (cADPR. Synthetic, non-hydrolyzable ligands can facilitate structure-based inhibitor design. Molecular docking was used to reproduce the crystallographic binding mode of cyclic inosine 5'-diphosphoribose (N1-cIDPR with CD38, revealing an exploitable pocket and predicting the potential to introduce an extra hydrogen bond interaction with Asp-155. The purine C-8 position of N1-cIDPR (IC50 276 µM was extended with an amino or diaminobutane group and the 8-modified compounds were evaluated against CD38-catalyzed cADPR hydrolysis. Crystallography of an 8-amino N1-cIDPR:CD38 complex confirmed the predicted interaction with Asp-155, together with a second H-bond from a realigned Glu-146, rationalizing the improved inhibition (IC50 56 µM. Crystallography of a complex of cyclic ADP-carbocyclic ribose (cADPcR, IC50 129 µM with CD38 illustrated that Glu-146 hydrogen bonds with the ligand N6-amino group. Both 8-amino N1-cIDPR and cADPcR bind deep in the active site reaching the catalytic residue Glu-226, and mimicking the likely location of cADPR during catalysis. Substantial overlap of the N1-cIDPR "northern" ribose monophosphate and the cADPcR carbocyclic ribose monophosphate regions suggests that this area is crucial for inhibitor design, leading to a new compound series of N1-inosine 5'-monophosphates (N1-IMPs. These small fragments inhibit hydrolysis of cADPR more efficiently than the parent cyclic compounds, with the best in the series demonstrating potent inhibition (IC50 = 7.6 µM. The lower molecular weight and relative simplicity of these compounds compared to cADPR make them attractive as a starting point for further inhibitor design.

  4. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

    Science.gov (United States)

    May, Bianca; Lange, B Markus; Wüst, Matthias

    2013-11-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Apyrase activity and adenosine diphosphate induced platelet aggregation inhibition by the salivary gland proteins of Culicoides variipennis, the North American vector of bluetongue viruses.

    Science.gov (United States)

    Pérez de León, A A; Tabachnick, W J

    1996-02-01

    Salivary gland homogenates of Culicoides variipennis, the primary vector of bluetongue (BLU) viruses in North America, were analyzed for apyrase activity. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is an anti-hemostatic and anti-inflammatory salivary enzyme of most hematophagous arthropods. The enzyme activity was measured by the release of orthophosphate using ATP, ADP, and AMP as substrates with Ca2+ as the divalent cation. ATPase (11.5 +/- 1 mU/pair of glands), ADPase (7.3 +/- 0.7 mU/pair of glands), and insignificant (P < 0.05) AMPase (0.07 mU/pair of glands) activities were detected in female salivary glands. Male salivary glands contained lower amounts of ATPase and ADPase activity (P < 0.05). The ATPase and ADPase activities were greatest at pH 8.5, and were similarly activated by Mg2+. Molecular sieving HPLC of salivary gland homogenates generated a single peak which coincided with ATPase and ADPase, but no AMPase, activity; the protein has an estimated molecular mass of 35,000 Da. ATPase and ADPase activity, and total protein concentration, were reduced (P < 0.05) in the salivary glands of females after taking a blood meal from a sheep. Salivary gland homogenates also inhibited ADP-induced platelet aggregation in vitro. It is concluded that the salivary ATPase and ADPase activities of C. variipennis reside in one enzyme, and that this enzyme is likely an apyrase. The apyrase activity is thought to be responsible for the inhibition of ADP-induced platelet aggregation, as indicated by the apparent discharge of apyrase from salivary glands into the host during blood feeding. This suggests that apyrase is one of the salivary proteins present in C. variipennis acting as antigens in the development of Culicoides hypersensitivity in ruminants and horses. Apyrase may inhibit an inflammatory response at the feeding site through the subsequent degradation of its end-product, AMP, to adenosine, a potent anti-inflammatory substance, by the ecto-5' nucleotidase activity of neutrophils.

  6. A novel GDP-D-glucose phosphorylase involved in quality control of the nucleoside diphosphate sugar pool in Caenorhabditis elegans and mammals.

    Science.gov (United States)

    Adler, Lital N; Gomez, Tara A; Clarke, Steven G; Linster, Carole L

    2011-06-17

    The plant VTC2 gene encodes GDP-L-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-D-glucose to GDP and D-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-D-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-D-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-D-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-D-glucose in the C10F3.4 mutant worms, suggesting that the GDP-D-glucose phosphorylase may function to remove GDP-D-glucose formed by GDP-D-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological D-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals.

  7. A Novel GDP-d-glucose Phosphorylase Involved in Quality Control of the Nucleoside Diphosphate Sugar Pool in Caenorhabditis elegans and Mammals*

    Science.gov (United States)

    Adler, Lital N.; Gomez, Tara A.; Clarke, Steven G.; Linster, Carole L.

    2011-01-01

    The plant VTC2 gene encodes GDP-l-galactose phosphorylase, a rate-limiting enzyme in plant vitamin C biosynthesis. Genes encoding apparent orthologs of VTC2 exist in both mammals, which produce vitamin C by a distinct metabolic pathway, and in the nematode worm Caenorhabditis elegans where vitamin C biosynthesis has not been demonstrated. We have now expressed cDNAs of the human and worm VTC2 homolog genes (C15orf58 and C10F3.4, respectively) and found that the purified proteins also display GDP-hexose phosphorylase activity. However, as opposed to the plant enzyme, the major reaction catalyzed by these enzymes is the phosphorolysis of GDP-d-glucose to GDP and d-glucose 1-phosphate. We detected activities with similar substrate specificity in worm and mouse tissue extracts. The highest expression of GDP-d-glucose phosphorylase was found in the nervous and male reproductive systems. A C. elegans C10F3.4 deletion strain was found to totally lack GDP-d-glucose phosphorylase activity; this activity was also found to be decreased in human HEK293T cells transfected with siRNAs against the human C15orf58 gene. These observations confirm the identification of the worm C10F3.4 and the human C15orf58 gene expression products as the GDP-d-glucose phosphorylases of these organisms. Significantly, we found an accumulation of GDP-d-glucose in the C10F3.4 mutant worms, suggesting that the GDP-d-glucose phosphorylase may function to remove GDP-d-glucose formed by GDP-d-mannose pyrophosphorylase, an enzyme that has previously been shown to lack specificity for its physiological d-mannose 1-phosphate substrate. We propose that such removal may prevent the misincorporation of glucosyl residues for mannosyl residues into the glycoconjugates of worms and mammals. PMID:21507950

  8. Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

    Science.gov (United States)

    El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

    2000-02-15

    We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

  9. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  10. Magnesium-adenosine diphosphate binding sites in wild-type creatine kinase and in mutants: role of aromatic residues probed by Raman and infrared spectroscopies.

    Science.gov (United States)

    Hagemann, H; Marcillat, O; Buchet, R; Vial, C

    2000-08-08

    Two distinct methods were used to investigate the role of Trp residues during Mg-ADP binding to cytosolic creatine kinase (CK) from rabbit muscle: (1) Raman spectroscopy, which is very sensitive to the environment of aromatic side-chain residues, and (2) reaction-induced infrared difference spectroscopy (RIDS) and photolabile substrate (ADP[Et(PhNO(2))]), combined with site-directed mutagenesis on the four Trp residues of CK. Our Raman results indicated that the environment of Trp and of Tyr were not affected during Mg-ADP binding to CK. Analysis of RIDS of wild-type CK, inactive W227Y, and active W210,217,272Y mutants suggested that Trp227 was not involved in the stacking interactions. Results are consistent with Trp227 being essential to prevent water molecules from entering in the active site [as suggested by Gross, M., Furter-Graves, E. M., Wallimann, T., Eppenberger, H. M., and Furter, R. (1994) Protein Sci. 3, 1058-1068] and that another Trp could in addition help to steer the nucleotide in the binding site, although it is not essential for the activity of CK. Raman and infrared spectra indicated that Mg-ADP binding does not involve large secondary structure changes. Only 3-4 residues absorbing in the amide I region are directly implicated in the Mg-ADP binding (corresponding to secondary structure changes less than 1%), suggesting that movement of protein domains due to Mg-nucleotide binding do not promote large secondary structure changes.

  11. A common deletion in the uridine diphosphate glucuronyltransferase (UGT) 2B17 gene is a strong determinant of androgen excretion in healthy pubertal boys

    DEFF Research Database (Denmark)

    Juul, A; Sørensen, K; Aksglaede, L

    2008-01-01

    2B17 genotypes on urinary excretion of androgen metabolites in pubertal boys. STUDY DESIGN: A clinical study of 116 healthy boys aged 8-19 yr. UGT2B17 genotyping was performed using quantitative PCR. Serum FSH, LH, T, estradiol (E2), and SHBG were analyzed by immunoassays, and urinary levels......BACKGROUND: Testosterone (T) is excreted in urine as water-soluble glucuronidated and sulfated conjugates. The ability to glucuronidate T and other steroids depends on a number of different glucuronidases (UGT) of which UGT2B17 is essential. The aim of the study was to evaluate the influence of UGT...... of androgen metabolites were quantitated by gas chromatography/mass spectrometry in all subjects. RESULTS: Ten of 116 subjects (9%) presented with a homozygote deletion of the UGT2B17 gene (del/del), whereas 52 and 54 boys were hetero- and homozygous carriers of the UGT2B17 gene (del/ins and ins...

  12. Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

    Science.gov (United States)

    Reddy, Vaishnavi Amarr; Wang, Qian; Dhar, Niha; Kumar, Nadimuthu; Venkatesh, Prasanna Nori; Rajan, Chakravarthy; Panicker, Deepa; Sridhar, Vishweshwaran; Mao, Hui-Zhu; Sarojam, Rajani

    2017-09-01

    Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  13. Investigations on the structures of sup(99m)Tc and 113Sn pyrophosphate complexes and of sup(99m)Tc and 113Sn ethane hydroxy diphosphate complexes

    International Nuclear Information System (INIS)

    Hohloch, M.

    1980-01-01

    The complex formation of double labelling of bivalent 113 Sn and reduced, quadrovalent sup(99m)Tc with pyrophosphate (PPi) or ethane hydroxy diphosphorate (EHDP) has been investigated by means of in vivo distribution in the rat. The molar rates of sup(99m)Tc and 113 Sn to PPi resp. EHDP, as well as the pH-value and the initial concentration is varied. Furthermore, both elements were oxidized with H 2 O 2 in the alkaline medium. Four typical sup(99m)Tc and two typical 113 Sn in-vivo distribution patterns can be differentiated: 1. Pertechnetate, characterized by a strong enrichment in the stomach, forms when all Sn-II has been oxidized to Sn-IV in the preparation. 2. One bone-seeking 113 Sn-II PPi (EHDP) complex and a sup(99m)Tc-IV PPi (EHDP) complex each, which are formed at least equimolar ratio of Sn to PPi (EHDP) and suffiently high concentration of PPi (EHDP) in the physiological pH-value. 3. A non-bone-seeking sup(99m)Tc-IV compound, which is enriched in the kidneys instead, is formed in the weakly alkaline medium or at low PPi (EHDP) concentration. This is probably monomeric technetium dioxide dihydrate. 4. A sup(99m)Tc as well as a Sn colloid is formed at deficient ligand concentration (PPi or EHDP to Sn). The chemical composition of the complexes is discussed the possible reaction courses are illustrated in the following diagrams. (orig./MG) [de

  14. Use of actin-bound adenosine 5'-diphosphate as a method to determine the specific 32P-radioactivity of the gamma-phosphoryl group of adenosine 5'-triphosphate in a highly compartmentalized cell, the platelet

    International Nuclear Information System (INIS)

    Verhoeven, A.J.; Cook, C.A.; Holmsen, H.

    1988-01-01

    Determination of the specific 32 P-radioactivity of cytoplasmic ATP in 32 P-Pi-labeled platelets is complicated by the presence of a large pool of metabolically inactive, granule-stored nucleotides. Moreover, our data show that the specific 32 P-radioactivity of cytoplasmic ATP is severely underestimated when determined in platelets after the complete secretion of granule-stored nucleotides, possibly due to isotopic dilution with granule-stored phosphate. As F-actin-bound ADP is ethanol-insoluble, this pool can be readily separated from the other nucleotide pools in platelets. Here we show that the specific 32 P-radioactivity of F-actin-bound ADP accurately reflects that of the gamma-phosphoryl group of cytoplasmic ATP. During uptake of 32 P-Pi by human platelets the specific 32 P-radioactivity of F-actin-bound ADP equals that of the monoester phosphates of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, which are in metabolic equilibrium with cytoplasmic ATP. Therefore, this method enables the determination of the specific 32 P-radioactivity of the gamma-phosphoryl group of cytoplasmic ATP in platelets even under short-term labeling conditions

  15. Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition.

    Science.gov (United States)

    Lawlis, V B; Roche, T E

    1981-04-28

    Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.

  16. Safety, tolerability, and initial efficacy of AZD6140, the first reversible oral adenosine diphosphate receptor antagonist, compared with clopidogrel, in patients with non-ST-segment elevation acute coronary syndrome: primary results of the DISPERSE-2 trial

    DEFF Research Database (Denmark)

    Cannon, Christopher P; Husted, Steen; Harrington, Robert A

    2007-01-01

    , or clopidogrel 300-mg loading dose plus 75 mg once daily for up to 12 weeks. RESULTS: The primary end point, the Kaplan-Meier rate of major or minor bleeding through 4 weeks, was 8.1% in the clopidogrel group, 9.8% in the AZD6140 90-mg group, and 8.0% in the AZD6140 180-mg group (p = 0.43 and p = 0.......96, respectively, vs. clopidogrel); the major bleeding rates were 6.9%, 7.1%, and 5.1%, respectively (p = 0.91 and p = 0.35, respectively, vs. clopidogrel). Although not statistically significant, favorable trends were seen in the Kaplan-Meier rates of myocardial infarction (MI) over the entire study period (MI: 5...

  17. Data Generated by Quantitative Liquid Chromatography-Mass Spectrometry Proteomics Are Only the Start and Not the Endpoint: Optimization of Quantitative Concatemer-Based Measurement of Hepatic Uridine-5'-Diphosphate-Glucuronosyltransferase Enzymes with Reference to Catalytic Activity.

    Science.gov (United States)

    Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Al-Majdoub, Zubida M; Goosen, Theunis C; Rostami-Hodjegan, Amin; Barber, Jill

    2018-06-01

    Quantitative proteomic methods require optimization at several stages, including sample preparation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and data analysis, with the final analysis stage being less widely appreciated by end-users. Previously reported measurement of eight uridine-5'-diphospho-glucuronosyltransferases (UGT) generated by two laboratories [using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT)] reflected significant disparity between proteomic methods. Initial analysis of QconCAT data showed lack of correlation with catalytic activity for several UGTs (1A4, 1A6, 1A9, 2B15) and moderate correlations for UGTs 1A1, 1A3, and 2B7 ( R s = 0.40-0.79, P data analysis, starting from unprocessed LC-MS/MS data, was undertaken, with the aim of improving accuracy, defined by correlation against activity. Three main criteria were found to be important: choice of monitored peptides and fragments, correction for isotope-label incorporation, and abundance normalization using fractional protein mass. Upon optimization, abundance-activity correlations improved significantly for six UGTs ( R s = 0.53-0.87, P data analysis strategy and indicates, using examples, the significance of systematic data processing following acquisition. The proposed strategy offers significant improvement on existing guidelines applicable to clinically relevant proteins quantified using QconCAT. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Métodos diagnósticos para retinopatia induzida pelo difosfato de cloroquina nos portadores de lúpus eritematoso sistêmico Diagnostic methods for chloroquine diphosphate induced retinopathy in systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Luciana Duarte Rodrigues

    2009-06-01

    Full Text Available OBJETIVOS: Avaliar diferentes métodos diagnósticos para a avaliação de pacientes portadores de lúpus eritematoso sistêmico, usuários crônicos do difosfato de cloroquina (DFC e, portanto, com alto risco para retinopatia tóxica. MÉTODOS: Foram analisados 72 olhos de 36 pacientes consecutivos, seguidos no Serviço de Reumatologia do Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, de julho de 2007 a abril de 2008. Dados demográficos e clínicos foram avaliados com o intuito de estudar os fatores de alto risco e comparar os seguintes métodos oftalmológicos: acuidade visual, biomicroscopia da córnea, biomicroscopia do fundo, retinografia, angiofluoresceinografia da retina, campo visual macular com mira branca. RESULTADOS: Dos 36 pacientes, 34 (94,4% eram mulheres. A média de idade foi 39,9 ± 9,8 anos, com tempo de doença igual a 13,9 ± 6,6 anos. Além do uso crônico da cloroquina, os pacientes apresentaram altas doses diárias (>3 mg/kg e cumulativas. Não foi observada relação entre estes fatores de alto risco e maior prevalência de retinopatia. Foi encontrada prevalência de retinopatia igual a 38,9%, confirmada por alterações bilaterais, centrais ou paracentrais e reprodutíveis no exame de campo visual. Outros exames indicados para seguimento, como acuidade visual, biomicroscopia de fundo e angiofluoresceinografia não foram capazes de diagnosticar a maioria das alterações confirmadas pelo campo visual. CONCLUSÃO: Foi observada alta prevalência de retinopatia por cloroquina entre os pacientes com alto risco, usuários crônicos do DFC, segundo os achados do campo visual. A avaliação desses pacientes deve considerar a realização do exame de campo visual em intervalos menores que os propostos, mesmo quando não há suspeita clínica.PURPOSE: To evaluate different diagnostic methods for high risk chloroquine retinopathy due to prolonged use of chloroquine (more than 5 years by systemic lupus erythematosus patients. Methods: Seventy-two eyes of 36 consecutive patients, followed in the Division of Rheumatology, School of Medicine, University of São Paulo, were analyzed from July 2007 to April 2008. Demographic and clinical data were evaluated in order to study risk factors and to compare the following different ophthalmological methods: visual acuity, biomicroscopy, fundus examination, retinography, fluorescein angiogram, visual field test and, color vision tests. RESULTS: From 36 patients, 34 (94.4% were female. The mean age was 39.9 ± 9.8 years and the disease duration was 13.9 ± 6.6 years. Besides chronic use of chloroquine, patients also showed high daily and cumulative doses. These high risk factors were not related to a higher retinopathy prevalence. Visual field showed 38.9% of retinopathy prevalence. Other ophthalmological methods failed in detecting most cases. CONCLUSION: High prevalence of retinopathy in high risk patients was observed by visual field test, but other ophthalmological methods failed in detecting alterations. Ophthalmological assessment of these patients should include visual field, even in the absence of clinical alterations.

  19. The toxin-antitoxin εζ system: Role of ζ toxin in regulating ATP, GTP, (p)ppGpp and uridine diphosphate N-acetylglucosamine pool to cope with stress

    OpenAIRE

    Tabone, Mariangela

    2015-01-01

    Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-10-2015 The toxin-antitoxin (TA) systems are compact modules, usually comprising a pair of genes coding for a toxin and its cognate antitoxin. These systems are present in the chromosomes of Bacteria, Archaea, in phages and in the large majority of low copy number plasmids. Basically, toxins are proteins whose activity usually leads to t...

  20. Fulltext PDF

    Indian Academy of Sciences (India)

    Prakash

    2010-04-30

    deoxy-D-xylulose 5-phosphate; DXR, DXP reductoisomerase; FPP, farnesyl diphosphate; FPPS, FPP synthase; GA-3P, glyceraldehyde-3-phosphate; GGPP, geranyl geranyl diphosphate;. GGPPS, GGPP synthase; GPP, geranyl ...

  1. Mechanistic aspects of carotenoid biosynthesis

    KAUST Repository

    Moï se, Alexander R.; Al-Babili, Salim; Wurtzel, Eleanore T.

    2014-01-01

    precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A recently developed concept that could explain the role of the poly-cis pathway in carotenoid synthesis is that the intermediates of this pathway have additional physiological

  2. ORF Alignment: NC_000962 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available inase (EC 2.7.4.6) - Mycobacterium tuberculosis (strain ... H37RV) pdb|1K44|F Chain F, Mycobacterium Tuberculosis...losis Nucleoside Diphosphate Kinase ... pdb|1K44|D Chain D, Mycobacterium Tuberculosis... ... Nucleoside Diphosphate Kinase pdb|1K44|C Chain C, ... Mycobacterium Tuberculosis... Nucleoside Diphosphate Kinase ... pdb|1K44|B Chain B, Mycobacterium Tuberculosis ... Nucleos...ide Diphosphate Kinase pdb|1K44|A Chain A, ... Mycobacterium Tuberculosis

  3. ORF Alignment: NC_002755 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available inase (EC 2.7.4.6) - Mycobacterium tuberculosis (strain ... H37RV) pdb|1K44|F Chain F, Mycobacterium Tuberculosis...losis Nucleoside Diphosphate Kinase ... pdb|1K44|D Chain D, Mycobacterium Tuberculosis... ... Nucleoside Diphosphate Kinase pdb|1K44|C Chain C, ... Mycobacterium Tuberculosis... Nucleoside Diphosphate Kinase ... pdb|1K44|B Chain B, Mycobacterium Tuberculosis ... Nucleos...ide Diphosphate Kinase pdb|1K44|A Chain A, ... Mycobacterium Tuberculosis

  4. ORF Alignment: NC_002945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available inase (EC 2.7.4.6) - Mycobacterium tuberculosis (strain ... H37RV) pdb|1K44|F Chain F, Mycobacterium Tuberculosis...losis Nucleoside Diphosphate Kinase ... pdb|1K44|D Chain D, Mycobacterium Tuberculosis... ... Nucleoside Diphosphate Kinase pdb|1K44|C Chain C, ... Mycobacterium Tuberculosis... Nucleoside Diphosphate Kinase ... pdb|1K44|B Chain B, Mycobacterium Tuberculosis ... Nucleos...ide Diphosphate Kinase pdb|1K44|A Chain A, ... Mycobacterium Tuberculosis

  5. Structure of active IspH enzyme from escherichia coli provides mechanistic insights into substrate reduction

    KAUST Repository

    Grä wert, Tobias; Rohdich, Felix; Span, Lngrid; Backer, Adelbert; Eisenreich, Wolfgang; Eppinger, Jö rg; Groll, Michael

    2009-01-01

    The terminal step of the non-mevalonate pathway of terpene biosynthesis is catalyzed by IspH (see scheme). In the crystal structure of IspH from E. coli, a bound inorganic diphosphate ligand occupies the position of the diphosphate residue

  6. Bulletin of the Chemical Society of Ethiopia - Vol 11, No 2 (1997)

    African Journals Online (AJOL)

    Intramolecular hydophobic and stacking interactions in mixed ligand complexes formed by copper (II), 2,2'-bipyridyl or 1,10-phenanthroline, and n-butyl diphosphate (BuDP3-) or phenyl diphosphate (PhDP3-), EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT.

  7. Isoprenoid-derived plant signaling molecules: biosynthesis and biological importance

    Czech Academy of Sciences Publication Activity Database

    Tarkowská, Danuše; Strnad, Miroslav

    2018-01-01

    Roč. 247, č. 5 (2018), s. 1051-1066 ISSN 0032-0935 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Dimethylallyl diphosphate * Isopentenyl diphosphate * Isoprenoids * Phytoecdysteroids * Plant hormones * Terpenoids Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemical research methods Impact factor: 3.361, year: 2016

  8. The Non-Mevalonate Pathway to Isoprenoid Biosynthesis : A Potential Source of New Drug Targets

    NARCIS (Netherlands)

    Hirsch, Anna K.H.; Diederich, François

    2008-01-01

    Isoprenoids are an essential class of natural products with a myriad of biological functions. All isoprenoids are assembled using two common five-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that are biosynthesized via two completely independent routes: the

  9. ORF Alignment: NC_002951 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002951 gi|57650779 >1to3A 5 286 10 317 1e-88 ... ref|YP_186994.1| tagatose 1,6-dip...hosphate aldolase [Staphylococcus aureus subsp. ... aureus COL] gb|AAW37059.1| tagatose 1,6-diphospha...product [Staphylococcus ... aureus] dbj|BAB58354.1| tagatose 1,6-diphospha...te ... aldolase [Staphylococcus aureus subsp. aureus Mu50] ... sp|P0A010|LACD_STAAN Tagatose 1...,6-diphosphate aldolase ... (Tagatose-bisphosphate aldolase) ... (D-tagatose-1,6-bisphosphate

  10. ORF Alignment: NC_002745 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002745 gi|15927772 >1to3A 5 286 10 317 1e-88 ... ref|YP_186994.1| tagatose 1,6-dip...hosphate aldolase [Staphylococcus aureus subsp. ... aureus COL] gb|AAW37059.1| tagatose 1,6-diphospha...product [Staphylococcus ... aureus] dbj|BAB58354.1| tagatose 1,6-diphospha...te ... aldolase [Staphylococcus aureus subsp. aureus Mu50] ... sp|P0A010|LACD_STAAN Tagatose 1...,6-diphosphate aldolase ... (Tagatose-bisphosphate aldolase) ... (D-tagatose-1,6-bisphosphate

  11. ORF Alignment: NC_002758 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002758 gi|15925182 >1to3A 5 286 10 317 1e-88 ... ref|YP_186994.1| tagatose 1,6-dip...hosphate aldolase [Staphylococcus aureus subsp. ... aureus COL] gb|AAW37059.1| tagatose 1,6-diphospha...product [Staphylococcus ... aureus] dbj|BAB58354.1| tagatose 1,6-diphospha...te ... aldolase [Staphylococcus aureus subsp. aureus Mu50] ... sp|P0A010|LACD_STAAN Tagatose 1...,6-diphosphate aldolase ... (Tagatose-bisphosphate aldolase) ... (D-tagatose-1,6-bisphosphate

  12. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  13. Coenzyme metabolism in rat liver transketolase

    International Nuclear Information System (INIS)

    Gorbach, Z.V.; Kubyshin, V.L.; Maglysh, S.S.; Zabrodskaya, S.V.

    1987-01-01

    On the basis of the results of kinetic investigations, two binding sites for hydroxythiamine diphosphate were determined in apotransketolase, with sharply differing values of K/sub i/: (7-22) x 10 -9 and (13.0-19.7) x 10 -8 M. A study was made of the turnover rate of thiamine diphosphate in holotransketolase in rat liver tissue by a radioisotope method, using [ 14 C] thiamine as the labeled precursor. The half-substitution time and rate constant of degradation of the coenzyme in transketolase are close in absolute values to the analogous indices for the protein portion of the enzyme and constitute 153 h and 0.108 day -1 , respectively. Rat liver transketolase exists in vivo in the form of a substituted α-carbanion. Replacement of thiamine diphosphate by hydroxythiamine diphosphate in the holoenzyme has no effect on the formation of the intermediate α-carbanion form of the enzyme

  14. PSI1 is responsible for the stearic acid enrichment that is characteristic of phosphatidylinositol in yeast

    NARCIS (Netherlands)

    Le Guédard, Marina; Bessoule, Jean-Jacques; Boyer, Valérie; Ayciriex, Sophie; Velours, Gisèle; Kulik, Willem; Ejsing, Christer S.; Shevchenko, Andrej; Coulon, Denis; Lessire, René; Testet, Eric

    2009-01-01

    In yeast, both phosphatidylinositol and phosphatidylserine are synthesized from cytidine diphosphate-diacylglycerol. Because, as in other eukaryotes, phosphatidylinositol contains more saturated fatty acids than phosphatidylserine (and other phospholipids), it has been hypothesized that either

  15. Effect of platelet inhibition with cangrelor during PCI on ischemic events

    NARCIS (Netherlands)

    Bhatt, D.L.; Stone, G.W.; Mahaffey, K.W.; Gibson, C.M.; Steg, P.G.; Hamm, C.W.; Price, M.J.; Leonardi, S.; Gallup, D.; Bramucci, E.; Radke, P.W.; Widimsky, P.; Tousek, F.; Tauth, J.; Spriggs, D.; McLaurin, B.T.; Angiolillo, D.J.; Genereux, P.; Liu, T.; Prats, J.; Todd, M.; Skerjanec, S.; White, H.D.; Harrington, R.A.; Verheugt, F.W.; et al.,

    2013-01-01

    BACKGROUND: The intensity of antiplatelet therapy during percutaneous coronary intervention (PCI) is an important determinant of PCI-related ischemic complications. Cangrelor is a potent intravenous adenosine diphosphate (ADP)-receptor antagonist that acts rapidly and has quickly reversible effects.

  16. Stereo-specific synthesis of (13r)-manoyl oxide

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a method for manufacturing enantiomerically pure (13R)-manoyl oxide, said method comprising the steps of contacting geranylgeranyl diphosphate (GGPP) with a class II diterpene synthase to obtain labd-13-en-8,15-diol diphosphate (LPP), and then contacting the LPP...... with a class I diterpene synthase to obtain (13R)-manoyl oxide. The invention further relates to (13R)-manoyl oxide obtained by the method of the invention....

  17. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  18. Structure of active IspH enzyme from escherichia coli provides mechanistic insights into substrate reduction

    KAUST Repository

    Gräwert, Tobias

    2009-07-20

    The terminal step of the non-mevalonate pathway of terpene biosynthesis is catalyzed by IspH (see scheme). In the crystal structure of IspH from E. coli, a bound inorganic diphosphate ligand occupies the position of the diphosphate residue of the substrate. Together with mutation studies and theoretical calculations, these data support a mechanism which is analogous to the Birch reduction of allylic alcohols. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

  19. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  20. Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry

    Science.gov (United States)

    Puts, Johan; de Groot, Monique; Haex, Martin; Jakobs, Bernadette

    2015-01-01

    Background Vitamin B1 (thiamine-diphosphate) and B6 (pyridoxal-5’phosphate) are micronutrients. Analysis of these micronutrients is important to diagnose potential deficiency which often occurs in elderly people due to malnutrition, in severe alcoholism and in gastrointestinal compromise due to bypass surgery or disease. Existing High Performance Liquid Chromatography (HPLC) based methods include the need for derivatization and long analysis time. We developed an Ultra High Performance Liquid Chromatography Tandem Mass spectrometry (UHPLC-MS/MS) assay with internal standards for simultaneous measurement of underivatized thiamine-diphosphate and pyridoxal-5’phosphate without use of ion pairing reagent. Methods Whole blood, deproteinized with perchloric acid, containing deuterium labelled internal standards thiamine-diphosphate(thiazole-methyl-D3) and pyridoxal-5’phosphate(methyl-D3), was analyzed by UHPLC-MS/MS. The method was validated for imprecision, linearity, recovery and limit of quantification. Alternate (quantitative) method comparisons of the new versus currently used routine HPLC methods were established with Deming regression. Results Thiamine-diphosphate and pyridoxal-5’phosphate were measured within 2.5 minutes instrumental run time. Limits of detection were 2.8 nmol/L and 7.8 nmol/L for thiamine-diphosphate and pyridoxal-5’phosphate respectively. Limit of quantification was 9.4 nmol/L for thiamine-diphosphate and 25.9 nmol/L for pyridoxal-5’phosphate. The total imprecision ranged from 3.5–7.7% for thiamine-diphosphate (44–157 nmol/L) and 6.0–10.4% for pyridoxal-5’phosphate (30–130 nmol/L). Extraction recoveries were 101–102% ± 2.5% (thiamine-diphosphate) and 98–100% ± 5% (pyridoxal-5’phosphate). Deming regression yielded slopes of 0.926 and 0.990 in patient samples (n = 282) and national proficiency testing samples (n = 12) respectively, intercepts of +3.5 and +3 for thiamine-diphosphate (n = 282 and n = 12) and slopes of

  1. Study of the temperature influence during the uranium (Vi) sorption on surface of ZrP2O7 in presence of oxalic and salicylic acid

    International Nuclear Information System (INIS)

    Garcia G, N.

    2013-01-01

    This work studies the effect of temperature on the uranium (Vi) sorption onto zirconium diphosphate in the presence of organic acids (oxalic and salicylic acids). Zirconium diphosphate was synthesized by a chemical condensation reaction and characterized using several analytical techniques, in order to check its purity. This point is very important because the presence of any impurities or secondary phases may interfere with the hydration and sorption process. Prior to the sorption experiments, three batches of zirconium diphosphate were pre-equilibrated with NaClO 4 , oxalic acid or salicylic acid solutions. The hydrated solids were washed and dried and then again characterized in order to study the interactions between organic acids and zirconium diphosphate surface. Uranium sorption onto zirconium diphosphate (pre-equilibrated with NaClO 4 , oxalic acid and salicylic acid solutions) was investigated as a function of ph, organic acid and temperature (20, 40 y 60 grades C). Thermodynamic parameters for the sorption reactions (enthalpy change, entropy change and Gibbs free energy change) were determined from temperature dependence of distribution coefficient by using the Vant Hoff equation. Solids characterization after hydration shows that exist an interaction between organic acids and ZrP 2 O 7 . This fact was confirmed with the microcalorimetry study, the reaction heat for hydration of zirconium diphosphate in NaClO 4 solution was exothermic (-269.59 mJ) and for hydration of zirconium diphosphate in oxalic acid solution was endothermic (53.64 mJ). The experimental results showed important differences in the sorption mechanisms for the reaction of Uranium with ZrP 2 O 7 in the presence and absence of organic acids. For the zirconium diphosphate hydrated with oxalic acid, the sorption percentage was 50% from lowest ph values. For the zirconium diphosphate hydrated with salicylic acid, the initial concentration of uranium was 6 x 10 -4 M and a percentage of 10% was

  2. Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Johan Puts

    Full Text Available Vitamin B1 (thiamine-diphosphate and B6 (pyridoxal-5'phosphate are micronutrients. Analysis of these micronutrients is important to diagnose potential deficiency which often occurs in elderly people due to malnutrition, in severe alcoholism and in gastrointestinal compromise due to bypass surgery or disease. Existing High Performance Liquid Chromatography (HPLC based methods include the need for derivatization and long analysis time. We developed an Ultra High Performance Liquid Chromatography Tandem Mass spectrometry (UHPLC-MS/MS assay with internal standards for simultaneous measurement of underivatized thiamine-diphosphate and pyridoxal-5'phosphate without use of ion pairing reagent.Whole blood, deproteinized with perchloric acid, containing deuterium labelled internal standards thiamine-diphosphate(thiazole-methyl-D3 and pyridoxal-5'phosphate(methyl-D3, was analyzed by UHPLC-MS/MS. The method was validated for imprecision, linearity, recovery and limit of quantification. Alternate (quantitative method comparisons of the new versus currently used routine HPLC methods were established with Deming regression.Thiamine-diphosphate and pyridoxal-5'phosphate were measured within 2.5 minutes instrumental run time. Limits of detection were 2.8 nmol/L and 7.8 nmol/L for thiamine-diphosphate and pyridoxal-5'phosphate respectively. Limit of quantification was 9.4 nmol/L for thiamine-diphosphate and 25.9 nmol/L for pyridoxal-5'phosphate. The total imprecision ranged from 3.5-7.7% for thiamine-diphosphate (44-157 nmol/L and 6.0-10.4% for pyridoxal-5'phosphate (30-130 nmol/L. Extraction recoveries were 101-102% ± 2.5% (thiamine-diphosphate and 98-100% ± 5% (pyridoxal-5'phosphate. Deming regression yielded slopes of 0.926 and 0.990 in patient samples (n = 282 and national proficiency testing samples (n = 12 respectively, intercepts of +3.5 and +3 for thiamine-diphosphate (n = 282 and n = 12 and slopes of 1.04 and 0.84, intercepts of -2.9 and +20 for

  3. Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthesis and Growth of Arabidopsis

    Science.gov (United States)

    vom Dorp, Katharina; Hölzl, Georg; Plohmann, Christian; Eisenhut, Marion; Abraham, Marion

    2015-01-01

    Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate. PMID:26452599

  4. Purification, crystallization and preliminary X-ray crystallographic analysis of human ppGpp hydrolase, Mesh1

    Directory of Open Access Journals (Sweden)

    Dawei Sun

    2010-09-01

    Full Text Available Bacterial SpoT is a Mn2+-dependent pyrophosphohydrolase to hydrolyze guanosine 3’-diphosphate-5’-diphosphate to guanosine diphosphate and pyrophosphate. In this study, SpoT ortholog from Homo sapiens (hMesh1, was over-expressed in Escherichia coli, purified and crystallized using hanging-drop vapour-diffusion method with polyethylene glycol and sodium citrate. The native crystal of hMesh1 was diffracted to 2.1 Å using a synchrotron-radiation source and belonged to the monoclinic space group P21 with cell dimensions of a = 53.27 Å, b = 62.61 Å, c = 52.45 Å and β= 94.96˚. The crystal contains two molecules in the asymmetric unit, with a solvent content of 44% and a Matthews coefficient VM value of 2.18 Å3/Da.

  5. Studies on the control of development: isolation of Bacillus subtilis mutants blocked early in sporulation and defective in synthesis of highly phosphorylated nucleotides.

    Science.gov (United States)

    Rhaese, H J; Hoch, J A; Groscurth, R

    1977-03-01

    To test our model on the mechanism of initiation of differentiation in Bacillus subtilis, we tested early blocked (stage 0) sporulation mutants for their ability to synthesize highly phosphorylated nucleotides. We also isolated early blocked asporogenous mutants with the aid of the intercalating drug tilorone. Among all mutants tested we found that the spo0F-bearing strain was unable to synthesize adenosine 3'(2')-triphosphate 5'-triphosphate, pppAppp. A revertant of this mutant regained the ability to both sporulate and synthesize pppAppp. Ribosomes of the asporogenous mutant isolated at T2 (2 hr after the end of logarithmic growth) of sporulation, in contrast to the wild type, do not synthesize adenosine 3'(2')-diphosphate 5'-diphosphate, ppApp, or adenosine 3'(2')-diphosphate 5'-triphosphate, pppApp, but synthesize guanosine 3'(2')-diphosphate 5'-diphosphate, ppGpp, and guanosine 3'(2')-diphosphate 5'-triphosphate, pppGpp. This behavior is characteristic of ribosomes from vegetative, not sporulating, cells. Ribosomes from the sporogenous revertant behave like those of the wild type. The results suggest that the spo0F mutation may be a mutation in the structural gene for pppAppp synthetase. The inability to synthesize pppAppp in this strain also prevents the formation of "sporulation-specific ribosomes," i.e., ribosomes that synthetize ppApp and pppApp. The present experiments suggest that the nucleotide pppAppp participates in the initiation of sporulation by triggering a sequencies of events required for the production of heat-resistant spores.

  6. A new radiochemical assay for fructose-1,6-diphosphatase in human leucocytes

    International Nuclear Information System (INIS)

    Janssen, A.J.M.; Trijbels, F.J.M.

    1982-01-01

    Fructose-1,6-diphosphatase (D-fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11, FDPase) is one of the key enzymes of the gluconeogenic pathway. Measuring the activity both in the presence and in the absence of AMP yields the true FDPase activity, corrected for non-specific phosphatase activity. In this paper the authors introduce a new radiochemical assay for FDPase, based on the decarboxylating activity of 6-phosphogluconate dehydrogenase. One molecule [U- 14 C]fructose-1,6-diphosphate yields one molecule 14 CO 2 which can be captured in strongly basic solutions and counted in a liquid scintillation counter. (Auth.)

  7. Biochemical studies on the effect of fluoride on higher plants. II. The effect of fluoride on sucrose-synthesizing enzymes from higher plants

    Energy Technology Data Exchange (ETDEWEB)

    Yang, S F; Miller, G W

    1963-01-01

    A study was initiated to characterize the properties of partially purified phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase and uridine diphosphate glucose-fructose transglucosyalse, from various plant sources, with respect to activation by metal ions and inhibition by fluoride. Of the three enzymes studied, only phosphoglucomutase was very sensitive to fluoride. It is likely that the inhibition of sucrose synthesis in fluoride-fumigated plants might be due to the inhibition of phosphoglucomutase, which plays an important role in carbohydrate metabolism. However, at present, there is insufficient evidence to show the inhibition of phosphoglucomutase in vivo by fumigation with hydrogen fluoride.

  8. The Biosynthetic Origin of Irregular Monoterpenes in Lavandula

    Science.gov (United States)

    Demissie, Zerihun A.; Erland, Lauren A. E.; Rheault, Mark R.; Mahmoud, Soheil S.

    2013-01-01

    Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s−1, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering. PMID:23306202

  9. Atomic resolution structures of discrete stages on the reaction coordinate of the [Fe4S4] enzyme IspG (GcpE)

    KAUST Repository

    Quitterer, Felix; Frank, Annika; Wang, Ke; Rao, Guodong; O'Dowd, Bing; Li, Jikun; Guerra, Francisco; Abdel-Azeim, Safwat; Bacher, Adelbert; Eppinger, Jö rg; Oldfield, Eric; Groll, Michael

    2015-01-01

    IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent as well as an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) to (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate (HMBPP). In addition, the enzyme’s structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism which describes all stages from initial ring opening to formation of HMBPP via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many pro- and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.

  10. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    NARCIS (Netherlands)

    Makarchikov, A.F.; Wins, P.; Janssen, E.E.W.; Wieringa, B.; Grisar, T.; Bettendorff, L.

    2002-01-01

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate

  11. Galactose Epimerase Deficiency: Expanding the Phenotype

    NARCIS (Netherlands)

    Dias Costa, Filipa; Ferdinandusse, Sacha; Pinto, Carla; Dias, Andrea; Keldermans, Liesbeth; Quelhas, Dulce; Matthijs, Gert; Mooijer, Petra A.; Diogo, Luísa; Jaeken, Jaak; Garcia, Paula

    2017-01-01

    Galactose epimerase deficiency is an inborn error of metabolism due to uridine diphosphate-galactose-4'-epimerase (GALE) deficiency. We report the clinical presentation, genetic and biochemical studies in two siblings with generalized GALE deficiency.Patient 1: The first child was born with a

  12. Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer

    NARCIS (Netherlands)

    Mirza, M. R.; Monk, B. J.; Herrstedt, J.; Oza, A. M.; Mahner, S.; Redondo, A.; Fabbro, M.; Ledermann, J. A.; Lorusso, D.; Vergote, I.; Ben-Baruch, N. E.; Marth, C.; Madry, R.; Christensen, R. D.; Berek, J. S.; Dorum, A.; Tinker, A. V.; du Bois, A.; Gonzalez-Martin, A.; Follana, P.; Benigno, B.; Rosenberg, P.; Gilbert, L.; Rimel, B. J.; Buscema, J.; Balser, J. P.; Agarwal, S.; Matulonis, U. A.; van der Zee, A.G.J.

    2016-01-01

    BACKGROUND Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive,

  13. Modified method for labeling human platelets with indium-111 oxine using albumin density-gradient separation

    International Nuclear Information System (INIS)

    Bunting, R.W.; Callahan, R.J.; Finkelstein, S.; Lees, R.S.; Strauss, H.W.

    1982-01-01

    When labeling platelets with indium-111 oxine, albumin density-gradient separation minimizes the time spent to resuspend those platelets that have been centrifuged against a hard surface. Labeling efficiency or platelet viability, as measured by platelet survival or aggregation with adenosine diphosphate, are not adversely affected

  14. Production of trichodiene by Trichoderma harzianum alters the perception of this biocontrol strain by plants and antagonized fungi

    Science.gov (United States)

    Trichothecenes are phytotoxic sesquiterpenoid compounds of fungal origin which can act as virulence factors in plant diseases. Harzianum A (HA) is a non-phytotoxic trichothecene produced by Trichoderma arundinaceum. The first step in the biosynthesis of HA is the conversion of farnesyl diphosphate t...

  15. Ophirapstanol trisulfate, a new biologically active steroid sulfate from the deep water marine sponge Topsentia ophiraphidites.

    Science.gov (United States)

    Gunasekera, S P; Sennett, S H; Kelly-Borges, M; Bryant, R W

    1994-12-01

    Ophirapstanol trisulfate [1], a new steroid trisulfate related to sokotrasterol trisulfate was isolated from a deep water marine sponge Topsentia ophiraphidites. Compound 1 exhibited significant inhibition in the guanosine diphosphate/G-protein RAS exchange assay. The structure elucidation of 1 and ophirapstanol [2] by nmr spectroscopy is described.

  16. Atomic resolution structures of discrete stages on the reaction coordinate of the [Fe4S4] enzyme IspG (GcpE)

    KAUST Repository

    Quitterer, Felix

    2015-04-11

    IspG is the penultimate enzyme in non-mevalonate biosynthesis of the universal terpene building blocks isopentenyl diphosphate and dimethylallyl diphosphate. Its mechanism of action has been the subject of numerous studies but remained unresolved due to difficulties in identifying distinct reaction intermediates. Using a moderate reducing agent as well as an epoxide substrate analogue, we were now able to trap and crystallographically characterize various stages in the IspG catalyzed conversion of 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate (MEcPP) to (E)-1-hydroxy-2-methylbut-2-enyl-4-diphosphate (HMBPP). In addition, the enzyme’s structure was determined in complex with several inhibitors. These results, combined with recent electron paramagnetic resonance data, allowed us to deduce a detailed and complete IspG catalytic mechanism which describes all stages from initial ring opening to formation of HMBPP via discrete radical and carbanion intermediates. The data presented in this article provide a guide for the design of selective drugs against many pro- and eukaryotic pathogens to which the non-mevalonate pathway is essential for survival and virulence.

  17. Cloning and characterization of the Yarrowia lipolytica squalene synthase (SQS1) gene and functional complementation of the Saccharomyces cerevisiae erg9 mutation

    NARCIS (Netherlands)

    Merkulov, S.; Assema, van F.; Springer, J.; Carmen, del A.F.; Mooibroek, H.

    2000-01-01

    The squalene synthase (SQS) gene encodes a key regulatory enzyme, farnesyl-diphosphate farnesyltransferase (EC 2.5.1.21), in sterol biosynthesis. The SQS1 gene was isolated from a subgenomic library of the industrially important yeast Yarrowia lipolytica, using PCR-generated probes. Probes were

  18. Catalytic site interactions in yeast OMP synthase

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Barr, Eric W.; Jensen, Kaj Frank

    2014-01-01

    45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal...

  19. Development of inhibitors of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway enzymes as potential anti-infective agents

    NARCIS (Netherlands)

    Masini, Tiziana; Hirsch, Anna K H

    2014-01-01

    Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl

  20. (C.sub.2./sub.N.sub.2./sub.H.sub.10./sub.) .sub.2./sub.Mg(HP.sub.2./sub.O.sub.7./sub.) .sub.2./sub..2H.sub.2./sub.O synthesis and crystal structure

    Czech Academy of Sciences Publication Activity Database

    Essehli, R.; El Bali, B.; Lachkar, M.; Dušek, Michal; Fejfarová, Karla; Bolte, M.

    2006-01-01

    Roč. 36, č. 10 (2006), s. 655-660 ISSN 1074-1542 Institutional research plan: CEZ:AV0Z10100521 Keywords : magnesium ructure * diphosphate * layered structure Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 0.658, year: 2006

  1. Bacterial origin of a diverse family of UDP-glycosyltransferase genes in the Tetranychus urticae genome

    NARCIS (Netherlands)

    Ahn, S.J.; Dermauw, W.; Wybouw, N.; Heckel, D.G.; Van Leeuwen, T.

    2014-01-01

    UDP-glycosyltransferases (UGTs) catalyze the conjugation of a variety of small lipophilic molecules with uridine diphosphate (UDP) sugars, altering them into more water-soluble metabolites. Thereby, UGTs play an important role in the detoxification of xenobiotics and in the regulation of

  2. Read-through transcript from NM23-H1 into the neighboring NM23-H2 gene encodes a novel protein, NM23-LV

    NARCIS (Netherlands)

    Valentijn, Linda J.; Koster, Jan; Versteeg, Rogier

    2006-01-01

    NM23-H1 and NM23-H2 are neighboring genes on chromosome 17q. They encode nucleoside diphosphate kinases that have additional roles in signal transduction, transcription, and apoptosis. NM23-H1 expression is a strong marker for prognosis and metastatic behavior in many tumor types. A new

  3. Activities of UDP-glucuronyltransferase, beta-glucuronidase and deiodinase types I and II in hyper- and hypothyroid rats

    NARCIS (Netherlands)

    Heide, S.M. van der; Joosten, B.H.G.M.; Everts, M.E.; Klaren, P.H.M.

    2004-01-01

    We have investigated the hypothesis that uridine 5'-diphosphate (UDP)-glucuronyltransferases (UGTs) and beta-glucuronidase are jointly involved in a mechanism for the storage and mobilization of iodothyronine metabolites in liver, kidney, heart and brain. Specifically, we predicted UGT activities to

  4. West African Journal of Pharmacology and Drug Research - Vol 25 ...

    African Journals Online (AJOL)

    Etude de l'amidon d'igname (dioscorea cayenensisrotundata) variete kponan en granulation par vobe humide dans la formulation de comprimes a base de chloroqueve diphosphate. EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. I.I Dally, A Koffi, S Coulibaly.

  5. Detergent inhibited, heat labile nucleoside triphosphatase in cores of avian myeloblastosis virus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Endogenous DNA synthesis was studied in isolated core particles of avian myeloblastosis virus. It was found that cores contained an enzymatic activity which rapidly converted the added nucleoside triphosphates to diphosphates (but not further) at 0 degrees C, thus inhibiting DNA synthesis...

  6. Labeling of thymine with 99m technetium: a suggestion of a chemical model

    International Nuclear Information System (INIS)

    Gutfilen, Bianca; Silva, Claudia Ribeiro da; Bernardo Filho, Mario; Ribeiro, Barbara Luzia Almeida; Mattos, Maura Ferreira

    1996-01-01

    Successful targeting of diagnose but also to stage cancer. It has been shown that certain tumor cells are permeable to low level of exogenous adenosine-diphosphate and adenosine-triphosphate nucleotides, that are incorporated into intracellular pools. We present the labeling of a nucleotide precursor, a base, thymine technetium-99m ( 99m Tc). (author)

  7. Labeling of thymine with {sup 99m} technetium: a suggestion of a chemical model

    Energy Technology Data Exchange (ETDEWEB)

    Gutfilen, Bianca; Silva, Claudia Ribeiro da; Bernardo Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria; Ribeiro, Barbara Luzia Almeida [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil). Centro de Pesquisa Basica; Mattos, Maura Ferreira [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Quimica

    1996-03-01

    Successful targeting of diagnose but also to stage cancer. It has been shown that certain tumor cells are permeable to low level of exogenous adenosine-diphosphate and adenosine-triphosphate nucleotides, that are incorporated into intracellular pools. We present the labeling of a nucleotide precursor, a base, thymine technetium-99m ({sup 99m} Tc). (author)

  8. The upregulation of thiamine (vitamin B1 biosynthesis in Arabidopsis thaliana seedlings under salt and osmotic stress conditions is mediated by abscisic acid at the early stages of this stress response

    Directory of Open Access Journals (Sweden)

    Rapala-Kozik Maria

    2012-01-01

    Full Text Available Abstract Background Recent reports suggest that vitamin B1 (thiamine participates in the processes underlying plant adaptations to certain types of abiotic and biotic stress, mainly oxidative stress. Most of the genes coding for enzymes involved in thiamine biosynthesis in Arabidopsis thaliana have been identified. In our present study, we examined the expression of thiamine biosynthetic genes, of genes encoding thiamine diphosphate-dependent enzymes and the levels of thiamine compounds during the early (sensing and late (adaptation responses of Arabidopsis seedlings to oxidative, salinity and osmotic stress. The possible roles of plant hormones in the regulation of the thiamine contribution to stress responses were also explored. Results The expression of Arabidopsis genes involved in the thiamine diphosphate biosynthesis pathway, including that of THI1, THIC, TH1 and TPK, was analyzed for 48 h in seedlings subjected to NaCl or sorbitol treatment. These genes were found to be predominantly up-regulated in the early phase (2-6 h of the stress response. The changes in these gene transcript levels were further found to correlate with increases in thiamine and its diphosphate ester content in seedlings, as well as with the enhancement of gene expression for enzymes which require thiamine diphosphate as a cofactor, mainly α-ketoglutarate dehydrogenase, pyruvate dehydrogenase and transketolase. In the case of the phytohormones including the salicylic, jasmonic and abscisic acids which are known to be involved in plant stress responses, only abscisic acid was found to significantly influence the expression of thiamine biosynthetic genes, the thiamine diphosphate levels, as well as the expression of genes coding for main thiamine diphosphate-dependent enzymes. Using Arabidopsis mutant plants defective in abscisic acid production, we demonstrate that this phytohormone is important in the regulation of THI1 and THIC gene expression during salt stress

  9. Study of the temperature influence during the uranium (Vi) sorption on surface of ZrP{sub 2}O{sub 7} in presence of oxalic and salicylic acid; Estudio de la influencia de la temperatura durante la sorcion de uranio (VI) en la superficie del ZrP{sub 2}O{sub 7} en presencia de acidos oxalico y salicilico

    Energy Technology Data Exchange (ETDEWEB)

    Garcia G, N.

    2013-07-01

    This work studies the effect of temperature on the uranium (Vi) sorption onto zirconium diphosphate in the presence of organic acids (oxalic and salicylic acids). Zirconium diphosphate was synthesized by a chemical condensation reaction and characterized using several analytical techniques, in order to check its purity. This point is very important because the presence of any impurities or secondary phases may interfere with the hydration and sorption process. Prior to the sorption experiments, three batches of zirconium diphosphate were pre-equilibrated with NaClO{sub 4}, oxalic acid or salicylic acid solutions. The hydrated solids were washed and dried and then again characterized in order to study the interactions between organic acids and zirconium diphosphate surface. Uranium sorption onto zirconium diphosphate (pre-equilibrated with NaClO{sub 4}, oxalic acid and salicylic acid solutions) was investigated as a function of ph, organic acid and temperature (20, 40 y 60 grades C). Thermodynamic parameters for the sorption reactions (enthalpy change, entropy change and Gibbs free energy change) were determined from temperature dependence of distribution coefficient by using the Vant Hoff equation. Solids characterization after hydration shows that exist an interaction between organic acids and ZrP{sub 2}O{sub 7}. This fact was confirmed with the microcalorimetry study, the reaction heat for hydration of zirconium diphosphate in NaClO{sub 4} solution was exothermic (-269.59 mJ) and for hydration of zirconium diphosphate in oxalic acid solution was endothermic (53.64 mJ). The experimental results showed important differences in the sorption mechanisms for the reaction of Uranium with ZrP{sub 2}O{sub 7} in the presence and absence of organic acids. For the zirconium diphosphate hydrated with oxalic acid, the sorption percentage was 50% from lowest ph values. For the zirconium diphosphate hydrated with salicylic acid, the initial concentration of uranium was 6 x 10

  10. Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum.

    Science.gov (United States)

    Morandim, Andreia de A; Bergamo, Débora Cristina B; Kato, Massuo Jorge; Cavalheiro, Alberto José; Bolzani, Vanderlan da S; Furlan, Maysa

    2005-01-01

    Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and 1 was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed.

  11. Terpenoids and Their Biosynthesis in Cyanobacteria

    Directory of Open Access Journals (Sweden)

    Bagmi Pattanaik

    2015-01-01

    Full Text Available Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids.

  12. Terpenoids and Their Biosynthesis in Cyanobacteria

    Science.gov (United States)

    Pattanaik, Bagmi; Lindberg, Pia

    2015-01-01

    Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids. PMID:25615610

  13. Studies on enzymes of C-4 pathway : Part V - Comparative studies of RUP2 carboxylase/oxygenase from maize and spinach

    International Nuclear Information System (INIS)

    Ramakrishna, J.; Bhagwat, A.S.; Sane, P.V.

    1978-01-01

    RuP 2 carboxylase (EC 4.1.1.39) isolated from maize, a C-4 plant possessed oxygenase activity. The ratio of carboxylase/oxygenase in the case of maize enzyme was more than 2-fold as compared to that of spinach. Fructose-1 6-diphosphate preferentially inhibited oxygenase function of the RuP 2 carboxylase/oxygenase in both the species when both the activities were assayed under identical conditions of pH, temperature, MgCl 2 , O 2 and RuP 2 concentration. Frutose-1, 6-diphosphate showed a fully competitive inhibition with respect to RuP 2 in the case of spinach, however the maize enzyme was inhibited seminoncompetitively. ( 14 C)-HCO 3 was used in the carboxylase assay. (author)

  14. Radiosensitization effects of nicotinamide on malignant and normal mouse tissue

    International Nuclear Information System (INIS)

    Jonsson, G.G.; Kjellen, E.; Pero, R.W.; Cameron, R.

    1985-01-01

    Inhibitors of the chromatin-associated enzyme adenosine diphosphate ribosyltransferase have been found to inhibit DNA strand rejoining and to potentiate lethality of DNA-damaging agents both in vivo and in vitro. The authors have in this work examined the radiosensitizing potential of one such inhibitor, nicotinamide, on tumor tissue by using transplanted C3H mouse mammary adenocarcinomas and on normal tissue in a tail-stunting experiment using BALB/cA mice. The data indicate a radiosensitizing effect of nicotinamide on tumor cells as well as on normal tissue. The data indicate a possible role of adenosine diphosphate ribosyltransferase inhibitors as a sensitizing agent in the radiotherapy of malignant tumors

  15. Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer

    DEFF Research Database (Denmark)

    Mirza, Mansoor R; Monk, Bradley J; Herrstedt, Jørn

    2016-01-01

    Background Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive, ......Background Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum...... or 4 adverse events that were reported in the niraparib group were thrombocytopenia (in 33.8%), anemia (in 25.3%), and neutropenia (in 19.6%), which were managed with dose modifications. Conclusions Among patients with platinum-sensitive, recurrent ovarian cancer, the median duration of progression...

  16. Bone scanning in severe external otitis

    International Nuclear Information System (INIS)

    Levin, W.J.; Shary, J.H. III; Nichols, L.T.; Lucente, F.E.

    1986-01-01

    Technetium99 Methylene Diphosphate bone scanning has been considered an early valuable tool to diagnose necrotizing progressive malignant external otitis. However, to our knowledge, no formal studies have actually compared bone scans of otherwise young, healthy patients with severe external otitis to scans of patients with clinical presentation of malignant external otitis. Twelve patients with only severe external otitis were studied with Technetium99 Diphosphate and were compared to known cases of malignant otitis. All scans were evaluated by two neuroradiologists with no prior knowledge of the clinical status of the patients. Nine of the 12 patients had positive bone scans with many scans resembling those reported with malignant external otitis. Interestingly, there was no consistent correlation between the severity of clinical presentation and the amount of Technetium uptake. These findings suggest that a positive bone scan alone should not be interpreted as indicative of malignant external otitis

  17. Biosynthesis of 2-methyl-3-buten-2-ol emitted from needles of Pinus ponderosa via the non-mevalonate DOXP/MEP pathway of isoprenoid formation.

    Science.gov (United States)

    Zeidler, J; Lichtenthaler, H K

    2001-06-01

    The volatile hemiterpene 2-methyl-3-buten-2-ol (MBO) is emitted from the needles of several pine species from the Western United States and contributes to ozone formation in the atmosphere. It is synthesised enzymatically from dimethylallyl diphosphate (DMAPP). We show here that needles of Pinus ponderosa Laws. incorporated [1-2H1]-1-deoxy-D-xylulose (d-DOX) into the emitted MBO, but not D,L-[2-13C]mevalonic acid lactone. Furthermore, MBO emission was inhibited by fosmidomycin, a specific inhibitor of the second enzyme of the mevalonate-independent pathway of isopentenyl diphosphate and DMAPP formation, i.e. the 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway. We thus prove that MBO emitted from needles of P. ponderosa is primarily formed via the DOXP/MEP pathway.

  18. Mechanistic aspects of carotenoid biosynthesis

    KAUST Repository

    Moïse, Alexander R.

    2014-01-08

    Carotenoid synthesis is based on the analysis of the phenotype of several mutant strains of tomato lacking carotenoid synthetic genes. Carotenoids are tetraterpenes derived through the condensation of the five-carbon (C5) universal isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A recently developed concept that could explain the role of the poly-cis pathway in carotenoid synthesis is that the intermediates of this pathway have additional physiological roles that extend beyond serving as precursors of lycopene. This concept is based on the analysis of the phenotype of several mutant strains of tomato lacking carotenoid synthetic genes. The feedback regulation of early carotenoid synthetic genes in response to a block in upstream metabolism represents a paradigm shift in our understanding of the mechanism and regulation of carotenoid synthesis and of metabolic regulation in general. The molecular details of a signaling pathway that regulates carotenogenesis in response to the levels of carotenoid precursors are still unclear.

  19. Crystal Structures of Glycosyltransferase UGT78G1 Reveal the Molecular Basis for Glycosylation and Deglycosylation of (Iso)flavonoids

    Energy Technology Data Exchange (ETDEWEB)

    Modolo, Luzia V.; Li, Lenong; Pan, Haiyun; Blount, Jack W.; Dixon, Richard A.; Wang, Xiaoqiang; (SRNF)

    2010-09-21

    The glycosyltransferase UGT78G1 from Medicago truncatula catalyzes the glycosylation of various (iso)flavonoids such as the flavonols kaempferol and myricetin, the isoflavone formononetin, and the anthocyanidins pelargonidin and cyanidin. It also catalyzes a reverse reaction to remove the sugar moiety from glycosides. The structures of UGT78G1 bound with uridine diphosphate or with both uridine diphosphate and myricetin were determined at 2.1 {angstrom} resolution, revealing detailed interactions between the enzyme and substrates/products and suggesting a distinct binding mode for the acceptor/product. Comparative structural analysis and mutagenesis identify glutamate 192 as a key amino acid for the reverse reaction. This information provides a basis for enzyme engineering to manipulate substrate specificity and to design effective biocatalysts with glycosylation and/or deglycosylation activity.

  20. A high-performance liquid chromatography-based radiometric assay for sucrose-phosphate synthase and other UDP-glucose requiring enzymes

    International Nuclear Information System (INIS)

    Salvucci, M.E.; Crafts-Brandner, S.J.

    1991-01-01

    A method for product analysis that eliminates a problematic step in the radiometric sucrose-phosphate synthase assay is described. The method uses chromatography on a boronate-derivatized high-performance liquid chromatography column to separate the labeled product, [14C]sucrose phosphate, from unreacted uridine 5'-diphosphate-[14C]glucose (UDP-Glc). Direct separation of these compounds eliminates the need for treatment of the reaction mixtures with alkaline phosphatase, thereby avoiding the problem of high background caused by contaminating phosphodiesterase activity in alkaline phosphatase preparations. The method presented in this paper can be applied to many UDP-Glc requiring enzymes; here the authors show its use for determining the activities of sucrose-phosphate synthase, sucrose synthase, and uridine diphosphate-glucose pyrophosphorylase in plant extracts

  1. Pentobarbital Sleep Time in Mouse Lines Selected for Resistance and Susceptibility to Fescue Toxicosis

    OpenAIRE

    Arthur, Kimberly Ann

    2002-01-01

    In previous work with mouse lines selected for resistance (R) and susceptibility (S) to fescue toxicosis, R mice had higher activities of Phase II liver enzymes glutathione S-transferase and uridine diphosphate glucuronosyl-transferase than S mice. Objectives of this study were: 1. to determine whether selection for toxicosis response had also caused divergence between lines in hepatic Phase I enzyme activity (as assessed by sleep time following sodium pentobarbital anesthesia), 2. to determi...

  2. Characterization and evolutionary analysis of ent-kaurene synthase like genes from the wild rice species Oryza rufipogon.

    Science.gov (United States)

    Toyomasu, Tomonobu; Miyamoto, Koji; Shenton, Matthew R; Sakai, Arisa; Sugawara, Chizu; Horie, Kiyotaka; Kawaide, Hiroshi; Hasegawa, Morifumi; Chuba, Masaru; Mitsuhashi, Wataru; Yamane, Hisakazu; Kurata, Nori; Okada, Kazunori

    2016-11-18

    Cultivated rice (Oryza sativa) possesses various labdane-related diterpene synthase genes, homologs of ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) that are responsible for the biosynthesis of phytohormone gibberellins. The CPS homologs and KS like (KSL) homologs successively converted geranylgeranyl diphosphate to cyclic diterpene hydrocarbons via ent-copalyl diphosphate or syn-copalyl diphosphate in O. sativa. Consequently, a variety of labdane-related diterpenoids, including phytoalexin phytocassanes, momilactones and oryzalexins, have been identified from cultivated rice. Our previous report indicated that the biosynthesis of phytocassanes and momilactones is conserved in Oryza rufipogon, the progenitor of Asian cultivated rice. Moreover, their biosynthetic gene clusters, containing OsCPS2 and OsKSL7 for phytocassane biosynthesis and OsCPS4 and OsKSL4 for momilactone biosynthesis, are also present in the O. rufipogon genome. We herein characterized O. rufipogon homologs of OsKSL5, OsKSL6, OsKSL8 responsible for oryzalexin S biosynthesis, and OsKSL10 responsible for oryzalexins A-F biosynthesis, to obtain more evolutionary insight into diterpenoid biosynthesis in O. sativa. Our phytoalexin analyses showed that no accumulation of oryzalexins was detected in extracts from O. rufipogon leaf blades. In vitro functional analyses indicated that unlike OsKSL10, O. rufipogon KSL10 functions as an ent-miltiradiene synthase, which explains the lack of accumulation of oryzalexins A-F in O. rufipogon. The different functions of KSL5 and KSL8 in O. sativa japonica to those in indica are conserved in each type of O. rufipogon, while KSL6 functions (ent-isokaurene synthases) are well conserved. Our study suggests that O. sativa japonica has evolved distinct specialized diterpenoid metabolism, including the biosynthesis of oryzalexins. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Man in the Cold Environment. A Bibliography with Informative Abstracts. Updated Version,

    Science.gov (United States)

    1982-09-01

    of the breathing regulator to malfunction due to cold. Two-hose regu- lators cause less trouble than single-hose regulators. Rubber "arctic cups ...by 5’-guanilyl imido- diphosphate was significantly decreased in rats adapted to both environmental changes. 304. MENSE , S. Effects of temperature on...Acclimatization mechanisms are the same. Women generally have lower sweat rates, an appropriate adjustment to lesser cooling needs. The menstrual cycle

  4. Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

    OpenAIRE

    Pandey, Ramesh Prasad; Parajuli, Prakash; Shin, Ju Yong; Lee, Jisun; Lee, Seul; Hong, Young-Soo; Park, Yong Il; Kim, Joong Su; Sohng, Jae Kyung

    2014-01-01

    A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratr...

  5. Ablation of the Galnt3 Gene Leads to Low-Circulating Intact Fibroblast Growth Factor 23 (Fgf23) Concentrations and Hyperphosphatemia Despite Increased Fgf23 Expression

    OpenAIRE

    Ichikawa, Shoji; Sorenson, Andrea H.; Austin, Anthony M.; Mackenzie, Donald S.; Fritz, Timothy A.; Moh, Akira; Hui, Siu L.; Econs, Michael J.

    2009-01-01

    Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generat...

  6. Metabolic engineering of the Stevia rebaudiana ent-kaurene biosynthetic pathway in recombinant Escherichia coli.

    Science.gov (United States)

    Kong, Min Kyung; Kang, Hyun-Jun; Kim, Jin Ho; Oh, Soon Hwan; Lee, Pyung Cheon

    2015-11-20

    The ent-kaurene is a dedicated precursor pool and is responsible for synthesizing natural sweeteners such as steviol glycosides. In this study, to produce ent-kaurene in Escherichia coli, we modularly constructed and expressed two ent-kaurene genes encoding ent-copalyl diphosphate synthase (CPPS) and ent-kaurene synthase (KS) from Stevia rebaudiana known as a typical plant producing steviol glycoside. The CPPS and KS from S. rebaudiana were functionally expressed in a heterologous host E. coli. Furthermore, in order to enhance ent-kaurene production in E. coli, six geranylgeranyl diphosphate synthases (GGPPS) from various microorganisms and eight strains of E. coli as host were compared by measuring ent-kaurene production. The highest ent-kaurene production of approximately 41.1mg/L was demonstrated in E. coli strain MG1655 co-expressing synthetic CPPS-KS module and GGPPS from Rhodobacter sphaeroides. The ent-kaurene production was further increased up to 179.6 mg/L by overexpression of the three key enzymes for isoprenoid precursor, 1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphate synthase (IspA) and isopentenyl diphosphate isomerase (IDI) from E. coli. Finally, the highest titer of ent-kaurene (578 mg/L) with a specific yield of ent-kaurene of 143.5mg/g dry cell weight was obtained by culturing E. coli strain MG1655 co-expressing the ent-kaurene module, DXS, IDI and IspA in 1L bioreactor containing 20 g/L glycerol. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Extraosseous uptake of 99sup(m)technetium methylene diphosphonate

    International Nuclear Information System (INIS)

    Sty, J.R.; Kun, L.; Casper, J.; Babbitt, D.P.

    1980-01-01

    A child with a ganglioneuroblastoma and tumor uptake of 99 sup(m)technetium methylene diphosphate ( 99 sup(m)Tc-MDP) is presented. After surgical removal of an encapsulated tumor and radiation therapy, an interval bone scan demonstrated the same presurgical abnormality. Awareness of abnormal uptake of 99 sup(m)Tc-MDP in irradiated renal tissue prevents interpreting radiation nephritis as recurrent tumor. (orig.) [de

  8. Using optical tweezers to relate the chemical and mechanical cross-bridge cycles.

    OpenAIRE

    Steffen, Walter; Sleep, John

    2004-01-01

    In most current models of muscle contraction there are two translational steps, the working stroke, whereby an attached myosin cross-bridge moves relative to the actin filament, and the repriming step, in which the cross-bridge returns to its original orientation. The development of single molecule methods has allowed a more detailed investigation of the relationship of these mechanical steps to the underlying biochemistry. In the normal adenosine triphosphate cycle, myosin.adenosine diphosph...

  9. Extraosseous uptake of /sup 99/sup(m)technetium methylene diphosphonate. Neuroblastoma or radiation therapy

    Energy Technology Data Exchange (ETDEWEB)

    Sty, J R; Kun, L; Casper, J; Babbitt, D P

    1980-01-01

    A child with a ganglioneuroblastoma and tumor uptake of /sup 99/sup(m)technetium methylene diphosphate (/sup 99/sup(m)Tc-MDP) is presented. After surgical removal of an encapsulated tumor and radiation therapy, an interval bone scan demonstrated the same presurgical abnormality. Awareness of abnormal uptake of /sup 99/sup(m)Tc-MDP in irradiated renal tissue prevents interpreting radiation nephritis as recurrent tumor.

  10. Methods for high yield production of terpenes

    Energy Technology Data Exchange (ETDEWEB)

    Kutchan, Toni; Higashi, Yasuhiro; Feng, Xiaohong

    2017-01-03

    Provided are enhanced high yield production systems for producing terpenes in plants via the expression of fusion proteins comprising various combinations of geranyl diphosphate synthase large and small subunits and limonene synthases. Also provided are engineered oilseed plants that accumulate monoterpene and sesquiterpene hydrocarbons in their seeds, as well as methods for producing such plants, providing a system for rapidly engineering oilseed crop production platforms for terpene-based biofuels.

  11. Freeze-Dried Human Red Blood Cells

    Science.gov (United States)

    1992-04-15

    period in the liquid state. 2. The levels of glycolytic intermediates (ATP, adenosine 5’triphosphate; 2,3-DPG 2, 3- diphosphoglycerate ) in rehydrated...8217 diphosphate, ADP; adenosine 5 monophosphate, AMP; 2,3- diphosphoglycerate . 2.3-DPG and lactate: (2) measurement of cell indices (mean cell volume (MCV), mean...monophosphate: 2,3-DPG. 2.3- diphosphoglycerate : MCV. Mean Cell Volume: MCH, Mean Cell Hemoglobin: MCHC, Mean Cell Hemoglobin Concentrations. ** Lactate levels

  12. Fonctionnalisation de la bentonite traitement et applications à léxtraction des colorants et colorants pour mordants.

    OpenAIRE

    MOULESSEHOUL, Hidayet

    2015-01-01

    L'objectif de notre étude est d'améliorer les propriétés de désorption de la bentonite vis à vis les colorants qui sont classés parmi les plus toxiques. La première partie du travail concerne la préparation et la caractérisation des bentonites modifiées avec les sels de diphosphate (porte-parapluie)TPhPMB.

  13. Control of Hepatic Glucose Metabolism by the Oral Hypoglycemic Sulfonylureas

    Science.gov (United States)

    1984-05-11

    diphosphate, 0.2 mM; 2,3 diphosphoglycerate , 0.1 mM; NADH, 0.2 mM; and 0.60 ml of deprotelnized sample. Pyruvate was measured by following the oxidation...J,; Rodgrlgues, L,M,; Whitton, P,A, and Hems, D,A, (1980) Control mechanisms in the acceleration of hepatic glycogen degradation during anoxia

  14. Novel fluorescent probe for highly sensitive bioassay using sequential enzyme-linked immunosorbent assay-capillary isoelectric focusing (ELISA-cIEF).

    Science.gov (United States)

    Henares, Terence G; Uenoyama, Yuta; Nogawa, Yuto; Ikegami, Ken; Citterio, Daniel; Suzuki, Koji; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-06-07

    This paper presents a novel rhodamine diphosphate molecule that allows highly sensitive detection of proteins by employing sequential enzyme-linked immunosorbent assay and capillary isoelectric focusing (ELISA-cIEF). Seven-fold improvement in the immunoassay sensitivity and a 1-2 order of magnitude lower detection limit has been demonstrated by taking advantage of the combination of the enzyme-based signal amplification of ELISA and the concentration of enzyme reaction products by cIEF.

  15. Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

    DEFF Research Database (Denmark)

    George, Kevin W; Thompson, Mitchell; Kim, Joonhoon

    2018-01-01

    evidence that IPP can be transported by E. coli, findings that might be broadly relevant for the study of isoprenoid biosynthesis. Finally, we discover that IPP accumulation leads to the formation of ApppI, a nucleotide analog of IPP that may contribute to observed toxicity phenotypes. This comprehensive...... assessment of IPP stress suggests potential strategies for the alleviation of prenyl diphosphate toxicity and highlights possible engineering targets for improved IPP flux and high titer isoprenoid production....

  16. Activities of UDP-glucuronyltransferase, beta-glucuronidase and deiodinase types I and II in hyper- and hypothyroid rats

    OpenAIRE

    Heide, S.M. van der; Joosten, B.H.G.M.; Everts, M.E.; Klaren, P.H.M.

    2004-01-01

    We have investigated the hypothesis that uridine 5'-diphosphate (UDP)-glucuronyltransferases (UGTs) and beta-glucuronidase are jointly involved in a mechanism for the storage and mobilization of iodothyronine metabolites in liver, kidney, heart and brain. Specifically, we predicted UGT activities to decrease and increase respectively, and beta-glucuronidase activity to increase and decrease respectively in hypo- and hyperthyroidism. To this end we have studied the effects of thyroid status on...

  17. Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes

    International Nuclear Information System (INIS)

    Boulias, C.; Moscarello, M.A.

    1989-01-01

    Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides

  18. The Antithrombotic and Fibrinolytic Effect of Natto in Hypercholesterolemia Rats

    OpenAIRE

    Park, Kum-Ju; Kang, Jung Il; Kim, Tae-Seok; Yeo, Ik-Hyun

    2012-01-01

    Antithrombotic and fibrinolytic activity of natto was evaluated on platelet aggregation in vitro and in vivo. Natto showed inhibitory effects on platelet aggregation induced by adenosine 5′diphosphate (ADP) and collagen. Orally administered natto also showed fibrinolytic activity in hypercholesterolemia rats. Normal levels of natto, when administered for four weeks, shortened euglobulin clot lysis time (ECLT) and prolonged partial thromboplastin time (PATT) significantly compared to non-treat...

  19. Biosynthetic elongation of isolated teichuronic acid polymers via glucosyl- and N-acetylmannosaminuronosyltransferases from solubilized cytoplasmic membrane fragments of Micrococcus luteus.

    Science.gov (United States)

    Hildebrandt, K M; Anderson, J S

    1990-01-01

    Cytoplasmic membrane fragments of Micrococcus luteus catalyze in vitro biosynthesis of teichuronic acid from uridine diphosphate D-glucose (UDP-glucose), uridine diphosphate N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA), and uridine diphosphate N-acetyl-D-glucosamine. Membrane fragments solubilized with Thesit (dodecyl alcohol polyoxyethylene ether) can utilize UDP-glucose and UDP-ManNAcA to effect elongation of teichuronic acid isolated from native cell walls. When UDP-glucose is the only substrate supplied, the detergent-solubilized glucosyltransferase incorporates a single glucosyl residue onto each teichuronic acid acceptor. When both UDP-glucose and UDP-ManNAcA are supplied, the glucosyltransferase and the N-acetylmannosaminuronosyltransferase act cooperatively to elongate the teichuronic acid acceptor by multiple additions of the disaccharide repeat unit. As shown by polyacrylamide gel electrophoresis, low-molecular-weight fractions of teichuronic acid are converted to higher-molecular-weight polymers by the addition of as many as 17 disaccharide repeat units. Images PMID:2118507

  20. Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.).

    Science.gov (United States)

    Yamaguchi, S; Saito, T; Abe, H; Yamane, H; Murofushi, N; Kamiya, Y

    1996-08-01

    The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene. ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB). A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent-kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region.

  1. Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway*

    Science.gov (United States)

    Onono, Fredrick; Subramanian, Thangaiah; Sunkara, Manjula; Subramanian, Karunai Leela; Spielmann, H. Peter; Morris, Andrew J.

    2013-01-01

    Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition. PMID:23908355

  2. Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia).

    Science.gov (United States)

    Landmann, Christian; Fink, Barbara; Festner, Maria; Dregus, Márta; Engel, Karl-Heinz; Schwab, Wilfried

    2007-09-15

    The essential oil of lavender (Lavandula angustifolia) is mainly composed of mono- and sesquiterpenes. Using a homology-based PCR strategy, two monoterpene synthases (LaLIMS and LaLINS) and one sesquiterpene synthase (LaBERS) were cloned from lavender leaves and flowers. LaLIMS catalyzed the formation of (R)-(+)-limonene, terpinolene, (1R,5S)-(+)-camphene, (1R,5R)-(+)-alpha-pinene, beta-myrcene and traces of alpha-phellandrene. The proportions of these products changed significantly when Mn(2+) was supplied as the cofactor instead of Mg(2+). The second enzyme LaLINS produced exclusively (R)-(-)-linalool, the main component of lavender essential oil. LaBERS transformed farnesyl diphosphate and represents the first reported trans-alpha-bergamotene synthase. It accepted geranyl diphosphate with higher affinity than farnesyl diphosphate and also produced monoterpenes, albeit at low rates. LaBERS is probably derived from a parental monoterpene synthase by the loss of the plastidial signal peptide and by broadening its substrate acceptance spectrum. The identification and description of the first terpene synthases from L. angustifolia forms the basis for the biotechnological modification of essential oil composition in lavender.

  3. Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes.

    Science.gov (United States)

    Sarker, Lukman S; Demissie, Zerihun A; Mahmoud, Soheil S

    2013-11-01

    The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.

  4. Crystal structure, magnetic and infrared spectroscopy studies of the LiCryFe1-yP2O7 solid solution

    International Nuclear Information System (INIS)

    Bih, Hssain; Saadoune, Ismael; Ehrenberg, Helmut; Fuess, Hartmut

    2009-01-01

    The lithium double diphosphates LiCr y Fe 1-y P 2 O 7 have been investigated by X-ray diffraction, SQUID measurements and vibrational spectroscopy. The Rietveld refinements based on the XRD patterns show the existence of a continuous solid solution over the whole composition range (0≤y≤1.0) with a continuous evolution of the monoclinic unit cell parameters (S.G. P2 1 ). The transition metal ions connect the diphosphate anions forming a three-dimensional network with channels filled by Li + cations expected to exhibit high mobility. All compounds order magnetically at low temperatures due the Fe-Fe interactions. The ordering temperature decreases with increasing Cr content. The slope in Curie-Weiss fits to the 1/χ vs T data in the paramagnetic domain clearly shows the existence of Fe 3+ and Cr 3+ in their high spin states, and a ferromagnetic component is clearly detected for y=0, 0.2 and 0.4. IR spectra have been interpreted using factor group analysis. The small shift of the frequencies is due to the influence of the chromium amount. The POP angles were estimated using the Lazarev's relationship. - Graphical abstract: Crystal structure of LiCr y Fe 1-y P 2 O 7 diphosphates, view along the 'a' axis. Red circles denote the lithium atoms. For interpretation of this figure legend, the reader is referred to the web version of this article.

  5. Synthesis of condensed double rubidium manganese phosphates in melts of polyphosphoric acids

    International Nuclear Information System (INIS)

    Guzeeva, L.S.; Tananaev, I.V.

    1988-01-01

    The aim of this work was to study the character of the reaction of MnO 2 and Rb 2 O with melts of polyphosphoric acids, and to establish the composition and the conditions of formation of the condensed double rubidium manganese phosphates. In the reaction of manganese and rubidium oxides with melts of polyphosphoric acids at 140-350 degree C, three types of trivalent manganese and rubidium condensed double phosphates are formed - diphosphates RbMn(H 2 P 2 O 7 ) and Rb 2 MnH 3 (P 2 O 7 ) 2 , triphosphate RbMnHP 3 O 10 and diphosphate RbMnP 2 O 7 - in addition to a double cyclotriphosphate of divalent manganese with rubidium RbMnP 3 O 9 . The thermal transformations of the compound isolated were studied. It was shown that rubidium manganese cyclotriphosphate can be obtained by the thermal decomposition of the diphosphate RbMn(H 2 P 2 O 7 ) 2

  6. Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds.

    Science.gov (United States)

    Keim, Verónica; Manzano, David; Fernández, Francisco J; Closa, Marta; Andrade, Paola; Caudepón, Daniel; Bortolotti, Cristina; Vega, M Cristina; Arró, Montserrat; Ferrer, Albert

    2012-01-01

    Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.

  7. Near-UV stress in salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response

    International Nuclear Information System (INIS)

    Kramer, G.F.; Baker, J.C.; Ames, B.N.

    1988-01-01

    We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenoisine 5', 5'''-triphosphoguanosine-3'''-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation

  8. Platelet aggregation and quality control of platelet concentrates produced in the Amazon Blood Bank

    Directory of Open Access Journals (Sweden)

    Maria José Dantas Coêlho

    2011-01-01

    Full Text Available BACKGROUND: The study of platelet aggregation is essential to assess in vitro platelet function by different platelet activation pathways. OBJECTIVE: To assess aggregation and biochemical parameters of random platelet concentrates produced at the Fundação HEMOAM using the quality control tests defined by law. METHODS: Whole blood samples from 80 donors and the respective platelet concentrate units were tested. Platelet concentrates were tested (platelet count, aggregation and pH on days 1, 3 and 5 of storage. Additionally a leukocyte count was done only on day 1 and microbiological tests on day 5 of storage. Collagen and adenosine diphosphate were used as inducing agonists for platelet aggregation testing. RESULTS: Donor whole blood had normal aggregation (aggregation with adenosine diphosphate = 67% and with collagen = 78%. The median aggregation in platelet concentrates with adenosine diphosphate was low throughout storage (18% on day 1, 7% on day 3 and 6% on day 5 and the median aggregation with collagen was normal only on day 1 and low thereafter (54.4% on day 1, 20.5% on day 3 and 9% on day 5. CONCLUSION: Although the results were within the norms required by law, platelet concentrates had low aggregation rates. We suggest the inclusion of a functional assessment test for the quality control of platelet concentrates for a more effective response to platelet replacement therapy.

  9. Condensation of the isoprenoid and amino precursors in the biosynthesis of domoic acid.

    Science.gov (United States)

    Savage, Thomas J; Smith, G Jason; Clark, Amy T; Saucedo, Portia N

    2012-01-01

    Understanding how environmental signals regulate production of domoic acid in blooms of Pseudo-nitzschia spp. at a molecular level requires description of the biochemical pathway to this kainoid neurotoxin. Precursor feeding studies have suggested domoic acid arises from the condensation of the C(10) isoprenoid geranyl diphosphate with glutamate, but the specific reactions leading to domoic acid from these precursors remain undescribed. Here, we develop a method to derivatize domoic acid with propyl chloroformate that enables gas chromatography-mass spectrometry (GC-MS) analysis to measure incorporation of stable isotopes into domoic acid generated in cultures incubated with isotopically-labeled substrates. We apply this method to demonstrate that both (2)H from [1-(2)H(2)]geraniol are incorporated into domoic acid, suggesting that the condensation of geranyl diphosphate with an amino group occurs by nucleophilic substitution of the diphosphate rather than by oxidation of geraniol to the aldehyde before reaction with an amino group to form an imine. Ultimately, these and similar studies will facilitate the identification of DA biosynthetic enzymes and genes which will enable the study of how environmental factors regulate DA biosynthesis at the molecular level. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Light-regulation of enzyme activity in anacystis nidulans (Richt.).

    Science.gov (United States)

    Duggan, J X; Anderson, L E

    1975-01-01

    The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

  11. Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds.

    Directory of Open Access Journals (Sweden)

    Verónica Keim

    Full Text Available Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP synthase (FPS, the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP. In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.

  12. Natural abundance deuterium nuclear magnetic resonance spectroscopy: Study of the biosynthesis of monoterpenes

    International Nuclear Information System (INIS)

    Leopold, M.F.

    1990-01-01

    Deuterium NMR spectroscopy at natural abundance (D NMR-na) is a new technique for exploring the biosynthesis of small molecules such as monoterpenes. The analysis of relative site-specific deuterium integration values is an effective means of measuring isotope effects, and examining the regio- and stereochemistry of biosynthetic reactions. The deuterium integration values of linalyl acetate and limonene isolated from the same source were consistent and showed that proton abstraction from the postulated α-terpinyl cation intermediate to form limonene is regioselective from the methyl derived from the Cs methyl of the precursor, geranyl diphosphate. This regiochemistry was observed in limonene samples from different sources and the measured primary kinetic isotope effect ranged from 0.25 to in excess of 100 (no deuterium was removed within experimental error). Various α- and β-pinene samples were isolated and D NMR-na analysis showed evidence of isotopically sensitive partitioning of the pinylcation in the formation of these products. This spectral analysis supported published radiolabeling studies but did not require synthesis of substrates or enzyme purification. The formation of 3-carene occurs without isomerization of the double bond which was previously postulated. The olefinic deuterium of the bicyclic compound was traced to the depleted deuterium at C 2 of isopentyl diphosphate by D NMR-na data and this supported unpublished radiolabeling studies. Study of irregular monoterpenes, chrysanthemyl acetate and lyratyl acetate, showed partitioning of dimethylallyl diphosphate (DMAPP) by chrysanthemyl cyclase. The α-secondary kinetic isotope effect of 1.06-1.12, obtained from relative deuterium integration values, suggested that S N 1 ionization of one molecule of DMAPP is the first step in the condensation reaction

  13. Germacrene A Synthase in Yarrow (Achillea millefolium Is an Enzyme with Mixed Substrate Specificity: Gene Cloning, Functional Characterization and Expression Analysis

    Directory of Open Access Journals (Sweden)

    Leila ePazouki

    2015-03-01

    Full Text Available Terpenoid synthases constitute a highly diverse gene family producing a wide range of cyclic and acyclic molecules consisting of isoprene (C5 residues. Often a single terpene synthase produces a spectrum of molecules of given chain length, but some terpene synthases can use multiple substrates, producing products of different chain length. Only a few such enzymes has been characterized, but the capacity for multiple-substrate use can be more widespread than previously thought. Here we focused on germacrene A synthase (GAS that is a key cytosolic enzyme in the sesquiterpene lactone biosynthesis pathway in the important medicinal plant Achillea millefolium (AmGAS. The full length encoding gene was heterologously expressed in Escherichia coli BL21 (DE3, functionally characterized, and its in vivo expression was analyzed. The recombinant protein catalyzed formation of germacrene A with the C15 substrate farnesyl diphosphate (FDP, while acyclic monoterpenes were formed with the C10 substrate geranyl diphosphate (GDP and cyclic monoterpenes with the C10 substrate neryl diphosphate (NDP. Although monoterpene synthesis has been assumed to be confined exclusively to plastids, AmGAS can potentially synthesize monoterpenes in cytosol when GDP or NDP become available. AmGAS enzyme had high homology with GAS sequences from other Asteraceae species, suggesting that multi-substrate use can be more widespread among germacrene A synthases than previously thought. Expression studies indicated that AmGAS was expressed in both autotrophic and heterotrophic plant compartments with the highest expression levels in leaves and flowers. To our knowledge, this is the first report on the cloning and characterization of germacrene A synthase coding gene in A. millefolium, and multi-substrate use of GAS enzymes.

  14. Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.

    Science.gov (United States)

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.

  15. Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases1[W][OA

    Science.gov (United States)

    Hall, Dawn E.; Zerbe, Philipp; Jancsik, Sharon; Quesada, Alfonso Lara; Dullat, Harpreet; Madilao, Lina L.; Yuen, Macaire; Bohlmann, Jörg

    2013-01-01

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs. PMID:23370714

  16. Evolution of conifer diterpene synthases: diterpene resin acid biosynthesis in lodgepole pine and jack pine involves monofunctional and bifunctional diterpene synthases.

    Science.gov (United States)

    Hall, Dawn E; Zerbe, Philipp; Jancsik, Sharon; Quesada, Alfonso Lara; Dullat, Harpreet; Madilao, Lina L; Yuen, Macaire; Bohlmann, Jörg

    2013-02-01

    Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs.

  17. Probing the reaction mechanism of IspH protein by x-ray structure analysis

    KAUST Repository

    Gräwert, Tobias

    2009-12-28

    Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovereddeoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl- 2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

  18. Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae▿

    Science.gov (United States)

    Tokuhiro, Kenro; Muramatsu, Masayoshi; Ohto, Chikara; Kawaguchi, Toshiya; Obata, Shusei; Muramoto, Nobuhiko; Hirai, Masana; Takahashi, Haruo; Kondo, Akihiko; Sakuradani, Eiji; Shimizu, Sakayu

    2009-01-01

    (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering. PMID:19592534

  19. Assessment of platelet function in healthy cats in response to commonly prescribed antiplatelet drugs using three point-of-care platelet function tests.

    Science.gov (United States)

    Ho, Kimberly K; Abrams-Ogg, Anthony Cg; Wood, R Darren; O'Sullivan, M Lynne; Kirby, Gordon M; Blois, Shauna L

    2017-06-01

    Objectives The objective was to determine if decreased platelet function could be detected after treatment with aspirin and/or clopidogrel in healthy cats using three point-of-care platelet function tests that evaluate platelet function by different methods: Multiplate (by impedance), Platelet Function Analyzer 100 (by mechanical aperture closure) and Plateletworks (by platelet counting). Methods Thirty-six healthy cats were randomly assigned to receive one of three oral treatments over an 8 day period: (1) aspirin 5 mg q72h; (2) aspirin 20.25 mg q72h; or (3) clopidogrel 18.75 mg q24h. Cats treated with 5 and 20.25 mg aspirin also received clopidogrel on days 4-8. Platelet aggregation in response to adenosine diphosphate and collagen ± arachidonic acid was assessed on days 1 (baseline), 4 and 8. Aspirin and clopidogrel metabolites were measured by high-performance liquid chromatography. Platelet function in response to treatment was analyzed by ANCOVA, linear regression and Spearman correlation. Results The only solitary aspirin effect was detected using Plateletworks with collagen in cats treated with 20.25 mg. The only effect detected by Multiplate was using arachidonic acid in cats treated with both aspirin 20.25 mg and clopidogrel. All clopidogrel treatment effects were detected by Platelet Function Analyzer 100, Plateletworks (adenosine diphosphate) and Plateletworks (collagen). Drug metabolites were present in all cats, but concentrations were minimally correlated to platelet function test results. Conclusions and relevance Platelet Function Analyzer 100 and Plateletworks using adenosine diphosphate ± collagen agonists may be used to detect decreased platelet function in response to clopidogrel treatment. Either aspirin is not as effective an antiplatelet drug as clopidogrel, or the tests used were not optimal to measure aspirin effect. Cats with heart disease are commonly prescribed antiplatelet drugs to decrease the risk of aortic thromboembolism

  20. Probing the reaction mechanism of IspH protein by x-ray structure analysis

    KAUST Repository

    Grä wert, Tobias; Span, Ingrid; Eisenreich, Wolfgang; Rohdich, Felix; Eppinger, Jö rg; Bacher, Adelbert; Groll, Michael

    2009-01-01

    Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) represent the two central intermediates in the biosynthesis of isoprenoids. The recently discovereddeoxyxylulose 5-phosphate pathway generates a mixture of IPP and DMAPP in its final step by reductive dehydroxylation of 1-hydroxy-2-methyl- 2-butenyl 4-diphosphate. This conversion is catalyzed by IspH protein comprising a central iron-sulfur cluster as electron transfer cofactor in the active site. The five crystal structures of IspH in complex with substrate, converted substrate, products and PPi reported in this article provide unique insights into the mechanism of this enzyme. While IspH protein crystallizes with substrate bound to a [4Fe-4S] cluster, crystals of IspH in complex with IPP, DMAPP or inorganic pyrophosphate feature [3Fe-4S] clusters. The IspH:substrate complex reveals a hairpin conformation of the ligand with the C(1) hydroxyl group coordinated to the unique site in a [4Fe-4S] cluster of aconitase type. The resulting alkoxide complex is coupled to a hydrogen-bonding network, which serves as proton reservoir via a Thr167 proton relay. Prolonged x-ray irradiation leads to cleavage of the C(1)-O bond (initiated by reducing photo electrons). The data suggest a reaction mechanism involving a combination of Lewis-acid activation and proton coupled electron transfer. The resulting allyl radical intermediate can acquire a second electron via the iron-sulfur cluster. The reaction may be terminated by the transfer of a proton from the β-phosphate of the substrate to C(1) (affording DMAPP) or C(3) (affording IPP).

  1. Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

    Directory of Open Access Journals (Sweden)

    Ben Buelow

    2009-07-01

    Full Text Available Poly adenosine diphosphate-ribose polymerase-1 (PARP-1 is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR, and formation of the soluble 2(nd messenger monomeric adenosine diphosphate-ribose (mADPR. Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

  2. Taxadiene Synthase Structure and Evolution of Modular Architecture in Terpene Biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    M Köksal; Y Jin; R Coates; R Croteau; D Christianson

    2011-12-31

    With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 {angstrom} resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 {angstrom} resolution). The TXS structure reveals a modular assembly of three {alpha}-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third 'insertion' domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis.

  3. A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

    Science.gov (United States)

    Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew; Chandrasekhar, Krishnamoorthy; Nemeria, Natalia; Zhang, Sheng; Jordan, Frank; Furey, William

    2006-06-02

    The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

  4. MTN-001: randomized pharmacokinetic cross-over study comparing tenofovir vaginal gel and oral tablets in vaginal tissue and other compartments.

    Directory of Open Access Journals (Sweden)

    Craig W Hendrix

    Full Text Available Oral and vaginal preparations of tenofovir as pre-exposure prophylaxis (PrEP for human immunodeficiency virus (HIV infection have demonstrated variable efficacy in men and women prompting assessment of variation in drug concentration as an explanation. Knowledge of tenofovir concentration and its active form, tenofovir diphosphate, at the putative vaginal and rectal site of action and its relationship to concentrations at multiple other anatomic locations may provide key information for both interpreting PrEP study outcomes and planning future PrEP drug development.MTN-001 was designed to directly compare oral to vaginal steady-state tenofovir pharmacokinetics in blood, vaginal tissue, and vaginal and rectal fluid in a paired cross-over design.We enrolled 144 HIV-uninfected women at 4 US and 3 African clinical research sites in an open label, 3-period crossover study of three different daily tenofovir regimens, each for 6 weeks (oral 300 mg tenofovir disoproxil fumarate, vaginal 1% tenofovir gel [40 mg], or both. Serum concentrations after vaginal dosing were 56-fold lower than after oral dosing (p<0.001. Vaginal tissue tenofovir diphosphate was quantifiable in ≥90% of women with vaginal dosing and only 19% of women with oral dosing. Vaginal tissue tenofovir diphosphate was ≥130-fold higher with vaginal compared to oral dosing (p<0.001. Rectal fluid tenofovir concentrations in vaginal dosing periods were higher than concentrations measured in the oral only dosing period (p<0.03.Compared to oral dosing, vaginal dosing achieved much lower serum concentrations and much higher vaginal tissue concentrations. Even allowing for 100-fold concentration differences due to poor adherence or less frequent prescribed dosing, vaginal dosing of tenofovir should provide higher active site concentrations and theoretically greater PrEP efficacy than oral dosing; randomized topical dosing PrEP trials to the contrary indicates that factors beyond tenofovir

  5. Transcriptome Sequencing Analysis Reveals a Difference in Monoterpene Biosynthesis between Scented Lilium ‘Siberia’ and Unscented Lilium ‘Novano’

    Directory of Open Access Journals (Sweden)

    Zenghui Hu

    2017-08-01

    Full Text Available Lilium is a world famous fragrant bulb flower with high ornamental and economic values, and significant differences in fragrance are found among different Lilium genotypes. In order to explore the mechanism underlying the different fragrances, the floral scents of Lilium ‘Sibeia’, with a strong fragrance, and Lilium ‘Novano’, with a very faint fragrance, were collected in vivo using a dynamic headspace technique. These scents were identified using automated thermal desorption—gas chromatography/mass spectrometry (ATD-GC/MS at different flowering stages. We used RNA-Seq technique to determine the petal transcriptome at the full-bloom stage and analyzed differentially expressed genes (DEGs to investigate the molecular mechanism of floral scent biosynthesis. The results showed that a significantly higher amount of Lilium ‘Siberia’ floral scent was released compared with Lilium ‘Novano’. Moreover, monoterpenes played a dominant role in the floral scent of Lilium ‘Siberia’; therefore, it is believed that the different emissions of monoterpenes mainly contributed to the difference in the floral scent between the two Lilium genotypes. Transcriptome sequencing analysis indicated that ~29.24 Gb of raw data were generated and assembled into 124,233 unigenes, of which 35,749 unigenes were annotated. Through a comparison of gene expression between these two Lilium genotypes, 6,496 DEGs were identified. The genes in the terpenoid backbone biosynthesis pathway showed significantly different expression levels. The gene expressions of 1-deoxy-D-xylulose 5-phosphate synthase (DXS, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR, isopentenyl diphosphate isomerase (IDI, and geranyl diphosphate synthase (GPS/GGPS, were upregulated in Lilium ‘Siberia’ compared to Lilium ‘Novano’, and two monoterpene synthase genes

  6. PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses.

    Science.gov (United States)

    Magnard, Jean-Louis; Roccia, Aymeric; Caissard, Jean-Claude; Vergne, Philippe; Sun, Pulu; Hecquet, Romain; Dubois, Annick; Hibrand-Saint Oyant, Laurence; Jullien, Frédéric; Nicolè, Florence; Raymond, Olivier; Huguet, Stéphanie; Baltenweck, Raymonde; Meyer, Sophie; Claudel, Patricia; Jeauffre, Julien; Rohmer, Michel; Foucher, Fabrice; Hugueney, Philippe; Bendahmane, Mohammed; Baudino, Sylvie

    2015-07-03

    The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta. Copyright © 2015, American Association for the Advancement of Science.

  7. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    International Nuclear Information System (INIS)

    Peters, J.

    1977-01-01

    Some experimental work is described showing that near-U.V. irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-U.V., and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex. (U.K.)

  8. Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

    Science.gov (United States)

    Plaga, W; Lottspeich, F; Oesterhelt, D

    1992-04-01

    An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

  9. Genetic-Metabolic Coupling for Targeted Metabolic Engineering

    DEFF Research Database (Denmark)

    Cardinale, Stefano; Tueros Farfan, Felipe Gonzalo; Sommer, Morten Otto Alexander

    2017-01-01

    pertur-bations, thus limiting their relevance to specific conditions. Here, we address this issue by coupling cell fitness to the production of thiamine diphosphate in Escherichia coli using a synthetic RNA biosensor. We use this strategy to interrogate a library of transposon mutants and elucidate...... the native gene network influencing both cell fitness and thiamine production. Ultimately, we identify effectors of the OxyR-Fur stress response that limit thiamine biosynthesis via alternative regulation of iron storage and Fe-S cluster inclusion in enzymes. This study presents a new approach...

  10. Hermansky-Pudlak syndrome; a Case Report

    Directory of Open Access Journals (Sweden)

    Abbas Bagheri

    2010-01-01

    Full Text Available Purpose: To report a case of Hermansky-Pudlak syndrome. Case Report: A seven-year-old boy presented with marked generalized hypopigmentation, ocular exodeviation and nystagmus. He had history of easy bruising. Examination revealed green irides with marked transillumination, hypopigmented fundi and foveal hypoplasia. Further investigations disclosed platelet storage defect with adenosine diphosphate deficiency and abnormal aggregation compatible with Hermansky-Pudlak syndrome. The patient underwent strabismus surgery taking necessary precautions such as reserving platelet concentrates in case of a hemorrhagic event. Conclusion: Patients with albinism should be evaluated for Hermansky-Pudlak syndrome especially before surgery to prevent life-threatening complications.

  11. Deoxynucleotide-interconverting enzymes and the quantification of deoxynucleoside triphosphates in mammalian cells

    OpenAIRE

    Fuller, Steven A.; Hutton, John J.; Meier, John; Coleman, Mary Sue

    1982-01-01

    We have demonstrated that methanol extracts of human cells are heterogeneous with regard to content of dNDP (deoxynucleoside diphosphate) and dNMP (deoxynucleoside monophosphate) kinases. The presence of these enzymes can affect the reliability of techniques used to measure intracellular pools of deoxynucleotides. An optimized extraction procedure and enzymic assay for dNTP species in haematopoietic cells are described which provide sensitivity to measure 0.1–40pmol of dATP, dTTP and dGTP, an...

  12. Campylobacter fetus subspecies: Comparative genomics and prediction of potential virulence targets

    DEFF Research Database (Denmark)

    Ali, Amjad; Soares, Siomar C.; Santos, Anderson R.

    2012-01-01

    . The potential candidate factors identified for attenuation and/or subunit vaccine development against C. fetus subspecies contain: nucleoside diphosphate kinase (Ndk), type IV secretion systems (T4SS), outer membrane proteins (OMP), substrate binding proteins CjaA and CjaC, surface array proteins, sap gene......, and cytolethal distending toxin (CDT). Significantly, many of those genes were found in genomic regions with signals of horizontal gene transfer and, therefore, predicted as putative pathogenicity islands. We found CRISPR loci and dam genes in an island specific for C. fetus subsp. fetus, and T4SS and sap genes...

  13. Pseudopterosin Biosynthesis: Aromatization of the Diterpene Cyclase Product, Elisabethatriene

    Directory of Open Access Journals (Sweden)

    Amber C. Kohl

    2003-11-01

    Full Text Available Abstract: Putative precursors in pseudopterosin biosynthesis, the hydrocarbons isoelisabethatriene (10 and erogorgiaene (11, have been identified from an extract of Pseudopterogorgia elisabethae collected in the Florida Keys. Biosynthetic experiments designed to test the utilization of these compounds in pseudopterosin production revealed that erogorgiaene is transformed to pseudopterosins A-D. Together with our previous data, it is now apparent that early steps in pseudopterosin biosynthesis involve the cyclization of geranylgeranyl diphosphate to elisabethatriene followed by the dehydrogenation and aromatization to erogorgiaene.

  14. mRNA decapping enzyme from ribosomes of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Stevens, A.

    1980-01-01

    By use of [ 3 H]methyl-5'-capped [ 14 C]mRNA from yeast as a substrate, a decapping enzyme activity has been detected in enzyme fractions derived from a high salt wash of ribosomes of Saccharomyces cerevisiae. The product of the decapping reaction is [ 3 H]m 7 GDP. That the enzyme is not a non-specific pyrophosphatase is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed

  15. Thermal oxidation of InP surfaces modified with NiO + PbO mixtures

    International Nuclear Information System (INIS)

    Mittova, I.Ya.; Tomina, E.V.; Samsonov, A.A.; Lukin, A.N.; Simonov, S.P.

    2005-01-01

    The oxidation kinetics of (NiO + PbO)/InP, NiO/InP and PbO/InP structures in an oxygen flow is studied in the temperature range of 400-550 deg C. It is shown by IR spectroscopy that the thermal oxidation of (NiO + PbO)/InP structures leads to the formation of nickel and lead polyphosphates and indium ortho- and metaphosphates. The nickel phosphates may then gradually transform into diphosphates, depending on the oxidation temperature, whereas the lead phosphates undergo no changes [ru

  16. Comparison of biomolecule desorption yields for low and high energy primary ions

    International Nuclear Information System (INIS)

    Kamensky, I.; Hakansson, P.; Sundqvist, B.; McNeal, C.J.; MacFarlane, R.

    1982-01-01

    Ion induced desorption yields of molecular ions from samples of cesium iodide, glycylglycine, ergosterol, bleomycin and a trinucleoside diphosphate have been studied using primary beams of 54 MeV 63 Cu 9+ and 3 keV 133 Cs + . Mass analysis was performed with a time-of-flight technique. Each sample was studied with the same spectrometer for both low and high energy primary ions and without opening of the vacuum chamber in between the measurements. The results show that fast heavy ions give larger yields for all samples studied and that the yield ratios for high to low energy desorption increase with the mass of the sample molecule. (orig.)

  17. Electron attachment to DNA single strands: gas phase and aqueous solution.

    Science.gov (United States)

    Gu, Jiande; Xie, Yaoming; Schaefer, Henry F

    2007-01-01

    The 2'-deoxyguanosine-3',5'-diphosphate, 2'-deoxyadenosine-3',5'-diphosphate, 2'-deoxycytidine-3',5'-diphosphate and 2'-deoxythymidine-3',5'-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3',5'-dTDP (0.17 eV) and 3',5'-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3',5'-dTDP > 3',5'-dCDP > 3',5'-dGDP > 3',5'-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3'-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3'-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3',5'-dADP(-) (0.26 eV) and 3',5'-dGDP(-) (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers

  18. Malignant external otitis: The diagnostic value of bone scintigraphy

    International Nuclear Information System (INIS)

    Ostfeld, E.; Aviel, A.; Pelet, D.

    1981-01-01

    Technetium99m Methylene Diphosphate bone scintigraphy (BS) of the skull was performed in three patients with malignant external otitis (MEO). Pathological uptake of the radioisotope in the mastoid region was found during the early stages of MEO updating radiologic findings. The extent of the radioisotope accumulation during the early stages of MEO indicates that the actual tissue damage exceeds the clinical estimation. The follow-up BS findings correlate well with the clinical course of MEO indicating either healing or extension to the base of skull

  19. Carrier-free 8-azidoadenosine 5'-[γ-32P]triphosphate

    International Nuclear Information System (INIS)

    Sabbatini, G.P.; Holt, C. von

    1987-01-01

    The authors found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed the synthesis in a single-step procedure carrier-free 8-azidoadenosine 5'-[γ- 32 P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[γ- 32 P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue. 14 refs.; 4 figs.; 1 table

  20. Evaluation of the TEG® platelet mappingTM assay in blood donors

    DEFF Research Database (Denmark)

    Bochsen, Louise; Wiinberg, Bo; Kjelgaard-Hansen, Mads Jens

    2007-01-01

    for quantification of platelet function, including the contribution of the adenosine diphosphate (ADP) and thromboxane A2 (TxA2) receptors to clot formation. Methods In 43 healthy blood donors, the analytical (CVa) and inter-individual variability (CVg) of the TEG® Platelet MappingTM assay were determined together......Background Monitoring of antiplatelet therapy in patients at cardiovascular risk is difficult because existing platelet function tests are too sophisticated for clinical routine. The whole blood TEG® Platelet MappingTM assay measures clot strength as maximal amplitude (MA) and enables...

  1. A novel β-lactam derivative, albactam from the flowers of Albizia lebbeck with platelets anti-aggregatory activity in vitro.

    Science.gov (United States)

    El-Gamal, Ali Ali; Abd-El-Halim, Mohamed Farag; Kalil, Ashraf Taha; Basudan, Omer Ahmed; Al-Rehaily, Adnan Jathlan; Ahmad, Mohamed Shamim; El-Tahir, Kamal Hussin; Al-Massarani, Shaza Mohamed; Abdel-Mageed, Wael Moustafa

    2015-03-01

    A novel β-lactam derivative, albactam, was isolated from the alcoholic extract of the flowers of Albizia lebbeck. It showed a significant anti-aggregatory activity against adenosine diphosphate and arachidonic acid induced guinea-pigs' platelets aggregation in vitro. Six more known compounds were also isolated and fully characterized by measuring 1D and 2D NMR, two of them are the triterpenes β-amyrin and 11α, 12α-oxidotaraxerol, two ceramide derivatives and two flavonoids, kampferol 3-O-rutinoside and rutin.

  2. The effects of ascorbic acid on diphtheria toxin and intoxicated hela cells

    International Nuclear Information System (INIS)

    Clark, C.E.; Smith, T.J.

    1976-01-01

    Ascorbic acid (vitamin C) prevented diphtheria toxin from inhibiting the incorporation of [U- 14 C]-alanine into trichloroacetic acid precipitable material in HeLa cells. Ascorbic acid did not exhibit an effect on the adenosine diphosphate-ribosylation of amino acyl transferase 2 nor did it separate fragment A from fragment B in ''nicked'' toxin. A non-specific reducing agent, para-methylaminophenol sulfate, exhibited an effect of HeLa cells very similar to the results of ascorbic acid. Citric acid, a tricarboxylic acid, had no effect on HeLa cells. (auth.)

  3. High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

    DEFF Research Database (Denmark)

    Kang, Aram; Meadows, Corey W.; Canu, Nicolas

    2017-01-01

    Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high...... ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass� isopentenol pathway using the promiscuous activity...

  4. Loss of ATM kinase activity leads to embryonic lethality in mice

    DEFF Research Database (Denmark)

    Daniel, J.A.; Pellegrini, M.; Filsuf, D.

    2012-01-01

    whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice......, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate...

  5. Carbon Dioxide Fixation in Isolated Kalanchoe Chloroplasts 1

    Science.gov (United States)

    Levi, Carolyn; Gibbs, Martin

    1975-01-01

    Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO2 per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO2 fixation were primarily those of the photosynthetic carbon reduction cycle. PMID:16659249

  6. Carbon dioxide fixation in isolated Kalanchoe chloroplasts

    Energy Technology Data Exchange (ETDEWEB)

    Levi, C.; Gibbs, M.

    1975-07-01

    Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 ..mu..moles of CO/sub 2/ per milligram of chlorophyll per hour. The dark rate of fixation was about 1 percent of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1, 6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO/sub 2/ fixation were primarily those of the photosynthetic carbon reduction cycle. (auth)

  7. Perioperative Anaesthetic Management of a Patient of Gilbert’s Syndrome with Adult Congenital Heart Disease - A Rare Presentation

    Directory of Open Access Journals (Sweden)

    Sambhunath Das

    2014-11-01

    Full Text Available Gilbert's syndrome is a hereditary condition with the genetic mutation of the enzyme uridine diphosphate glucuronosyltransferase, characterized by intermittent jaundice in the absence of hemolysis or underlying liver disease. These patients develop jaundice when subjected to fasting, stress and exercise. Majority of anaesthetics are metabolized by liver. Anaesthesia, surgery and cardiopulmonary bypass (CPB can act as triggers to hepatic injury. The successful perioperative management of an adult congenital heart disease patient for atrial septal defect closure under cardiopulmonary bypass was discussed in this report.

  8. 1-O-alkyl-2-(omega-oxo)acyl-sn-glycerols from shark oil and human milk fat are potential precursors of PAF mimics and GHR

    DEFF Research Database (Denmark)

    Hartvigsen, Karsten; Ravandi, A.; Harkewicz, R.

    2006-01-01

    This study examines the feasibility that peroxidation and lipolysis of 1-O-alkyl-2,3-diacyl-sn-glycerols (DAGE) found in shark liver oil and human milk fat constitutes a potential source of dietary precursors of platelet activating factor (PAF) mimics and of gamma-hydroxybutyrate (GHB). Purified...... yielded 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-glycerol, as the major core aldehyde. Because diradylglycerols with short fatty chains are absorbed in the intestine and react with cytidine diphosphate-choline in the enterocytes, it is concluded that formation of such PAF mimics as 1-O-alkyl-2-(omega...

  9. Preparation of C-II labeled pyruvic acid for use in assessment of hypoxia in tumors. Project 4

    International Nuclear Information System (INIS)

    Anon.

    1986-01-01

    Of the three methods of synthesis of C-II-labeled pyruvic acid that we had proposed to investigate in order to determine the best and most appropriate synthesis of C-II-labeled pyruvate, the cold chemistry of Method A, via an isocyanide intermediate, has been verified. Similarly, the cold chemistry of Method B, via the 1,3-dithiane derivative, has been verified up to the deprotection and last step of the synthesis. The difficulties which have been encountered with the biochemistry of Method C from ribulose 1,5-diphosphate, have yet to be resolved. 12 refs., 6 figs

  10. In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Peters, J [California Univ., Irvine (USA)

    1977-06-09

    Some experimental work is described showing that near-uv irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-uv, and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex.

  11. Mass-spectral investigations of vaporization process of the condensed zinc phosphates

    International Nuclear Information System (INIS)

    Lopatin, S.L; Sinyayev, V.A.; Shugurov, S.M.

    2005-01-01

    There are the data of high temperature mass-spectrum experiment concerning of thermal decomposition of zinc cyclotriphosphate and zinc diphosphate presented in the given article. It is shown the both salts dissociate into phosphorus oxides, oxygen, and atomic zinc. Correlation between partial pressure of vapor components and composition of condensed phase are described. Effects of temperature and duration of the vaporization process on vapor composition are presented as well. Standard enthalpy of ZnPO 3 molecule decomposition into atoms is calculated. [author

  12. Monoterpene biosynthesis potential of plant subcellular compartments.

    Science.gov (United States)

    Dong, Lemeng; Jongedijk, Esmer; Bouwmeester, Harro; Van Der Krol, Alexander

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  13. Purinergic signaling pathways in endocrine system.

    Science.gov (United States)

    Bjelobaba, Ivana; Janjic, Marija M; Stojilkovic, Stanko S

    2015-09-01

    Adenosine-5'-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5'-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5'-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5'-triphosphate hydrolysis to adenosine-5'-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. Published by Elsevier B.V.

  14. Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP and terpenoid pathways leading to carotenoid biosynthesis.

    Directory of Open Access Journals (Sweden)

    Natália Corniani

    Full Text Available The 2-C-methyl-D-erythritol 4-phosphate (MEP pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

  15. In-silico Leishmania Target Selectivity of Antiparasitic Terpenoids

    Directory of Open Access Journals (Sweden)

    Ifedayo Victor Ogungbe

    2013-07-01

    Full Text Available Neglected Tropical Diseases (NTDs, like leishmaniasis, are major causes of mortality in resource-limited countries. The mortality associated with these diseases is largely due to fragile healthcare systems, lack of access to medicines, and resistance by the parasites to the few available drugs. Many antiparasitic plant-derived isoprenoids have been reported, and many of them have good in vitro activity against various forms of Leishmania spp. In this work, potential Leishmania biochemical targets of antiparasitic isoprenoids were studied in silico. Antiparasitic monoterpenoids selectively docked to L. infantum nicotinamidase, L. major uridine diphosphate-glucose pyrophosphorylase and methionyl t-RNA synthetase. The two protein targets selectively targeted by germacranolide sesquiterpenoids were L. major methionyl t-RNA synthetase and dihydroorotate dehydrogenase. Diterpenoids generally favored docking to L. mexicana glycerol-3-phosphate dehydrogenase. Limonoids also showed some selectivity for L. mexicana glycerol-3-phosphate dehydrogenase and L. major dihydroorotate dehydrogenase while withanolides docked more selectively with L. major uridine diphosphate-glucose pyrophosphorylase. The selectivity of the different classes of antiparasitic compounds for the protein targets considered in this work can be explored in fragment- and/or structure-based drug design towards the development of leads for new antileishmanial drugs.

  16. Genetic Evidence for the Physiological Significance of the d-Tagatose 6-Phosphate Pathway of Lactose and d-Galactose Degradation in Staphylococcus aureus1

    Science.gov (United States)

    Bissett, Donald L.; Anderson, Richard L.

    1974-01-01

    Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain. PMID:4277494

  17. Identification of a Plastid-Localized Bifunctional Nerolidol/Linalool Synthase in Relation to Linalool Biosynthesis in Young Grape Berries

    Directory of Open Access Journals (Sweden)

    Bao-Qing Zhu

    2014-12-01

    Full Text Available Monoterpenoids are a diverse class of natural products and contribute to the important varietal aroma of certain Vitis vinifera grape cultivars. Among the typical monoterpenoids, linalool exists in almost all grape varieties. A gene coding for a nerolidol/linalool (NES/LINS synthase was evaluated in the role of linalool biosynthesis in grape berries. Enzyme activity assay of this recombinant protein revealed that it could convert geranyl diphosphate and farnesyl diphosphate into linalool and nerolidol in vitro, respectively, and thus it was named VvRILinNer. However, localization experiment showed that this enzyme was only localized to chloroplasts, which indicates that VvRILinNer functions in the linalool production in vivo. The patterns of gene expression and linalool accumulation were analyzed in the berries of three grape cultivars (“Riesling”, “Cabernet Sauvignon”, “Gewurztraminer” with significantly different levels of monoterpenoids. The VvRILinNer was considered to be mainly responsible for the synthesis of linalool at the early developmental stage. This finding has provided us with new knowledge to uncover the complex monoterpene biosynthesis in grapes.

  18. Utilization of biodiesel by-product as substrate for high-production of β-farnesene via relatively balanced mevalonate pathway in Escherichia coli.

    Science.gov (United States)

    You, Shengping; Yin, Qingdian; Zhang, Jianye; Zhang, Chengyu; Qi, Wei; Gao, Lan; Tao, Zhiping; Su, Rongxin; He, Zhimin

    2017-11-01

    Farnesene has been identified as suitable jet fuel substitutes and metabolic engineering for microbial production of farnesene is an alternative and attractive route. In this study, due to accumulation of toxic intermediate isopentenyl pyrophosphate (IPP), an engineered Escherichia coli strain harboring heterologous mevalonate pathway produced only 4.11mg/L β-farnesene. Through higher-level expression of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase to minimize the accumulated IPP, another engineered strain with relatively balanced mevalonate pathway was constructed and had the highest production of β-farnesene to date (8.74g/L) by Escherichia coli in a lab bioreactor. Furthermore, this is the first report on utilization of biodiesel by-product (simple purification) as substrate for high-production of β-farnesene by the engineered strain optimized and β-farnesene concentration reached 2.83g/L in a lab bioreactor. Therefore, the engineered strain optimized could be used as a platform host for high-production of other terpenoids using biodiesel by-product as substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Raman and impedance spectroscopy methods of P{sub 2}O{sub 5}–Li{sub 2}O–Al{sub 2}O{sub 3} glass system doped with MgO

    Energy Technology Data Exchange (ETDEWEB)

    Jlassi, I., E-mail: ifa.jlassi@fst.rnu.tn [Laboratoire Physico-chimie des Matériaux Minéraux et leurs Applications, Centre National de Recherches en Sciences des Matériaux, B.P. 95, Hammam-Lif 2050 (Tunisia); Département de Physique, Faculté des Sciences de Tunis, Université de Tunis ElManar, Campus Universitaire Farhat Hachad, ElManar 2092 (Tunisia); Sdiri, N. [Laboratoire Physico-chimie des Matériaux Minéraux et leurs Applications, Centre National de Recherches en Sciences des Matériaux, B.P. 95, Hammam-Lif 2050 (Tunisia); Elhouichet, H. [Laboratoire Physico-chimie des Matériaux Minéraux et leurs Applications, Centre National de Recherches en Sciences des Matériaux, B.P. 95, Hammam-Lif 2050 (Tunisia); Département de Physique, Faculté des Sciences de Tunis, Université de Tunis ElManar, Campus Universitaire Farhat Hachad, ElManar 2092 (Tunisia); Ferid, M. [Laboratoire Physico-chimie des Matériaux Minéraux et leurs Applications, Centre National de Recherches en Sciences des Matériaux, B.P. 95, Hammam-Lif 2050 (Tunisia)

    2015-10-05

    Highlights: • We have prepared a new lithium diphosphate glasses doped MgO. • Investigate structural and electrical properties at room temperature. • Investigate relation between structure and electrical conductivity of the glass. - Abstract: Lithium diphosphate glasses doped MgO was prepared via a melt quenching technique. The samples were characterized by X-ray diffraction (XRD), Raman and impedance spectroscopy. XRD spectra reflected the amorphous nature of the glasses Raman spectra show structural network modifications with the composition variations of the studied glasses. Raman spectra of the studied glasses contain also typical phosphate glasses bands. Thus the band at ∼698 cm{sup −1} assigned to symmetric stretching vibrations of P−O−P groups and that from ∼1168 cm{sup −1} is attributed to symmetric stretching motions of the non-bridging oxygen (NBO) atoms bonded to phosphorous atoms (PO{sub 2}) in phosphate tetrahedron. Electric properties were investigated using complex impedance spectroscopy in a frequency range from 40 Hz to 6 MHz at room temperature. The impedance spectra were analyzed in terms of equivalent circuits involving resistors, capacitors and constant phase elements (CPE). Constant-phase elements (CPE) are used in equivalent electrical circuits for the fitting of experimental impedance data. The AC conductivity exhibited a Jonscher’s universal power law according with the relation σ(ω) = σ(0) + Aω{sup s} and it is observed that as the MgO content increases, frequency exponent (s) decreases.

  20. Progranulin inhibits platelet aggregation and prolongs bleeding time in rats.

    Science.gov (United States)

    Al-Yahya, A M; Al-Masri, A A; El Eter, E A; Hersi, A; Lateef, R; Mawlana, O

    2018-05-01

    Several adipokines secreted by adipose tissue have an anti-thrombotic and anti-atherosclerotic function. Recently identified adipokine progranulin was found to play a protective role in atherosclerosis. Bearing in mind the central role of platelets in inflammation and atherosclerosis, we aimed, in this study, to examine the effect of progranulin on platelet function and coagulation profile in rats. Healthy male albino Wistar rats weighing (250-300 g) were divided into 4 groups. Three groups were given increasing doses of progranulin (0.001 µg, 0.01 µg, and 0.1 µg) intraperitoneally, while the control group received phosphate-buffered saline (PBS). Bleeding time, prothrombin time, activated partial thromboplastin time and platelet aggregation responses to adenosine diphosphate and arachidonic acid were assessed. Administration of progranulin resulted in a significant inhibition of platelet aggregation in response to both adenosine diphosphate, and arachidonic acid. Bleeding time, prothrombin time and activated partial thromboplastin time were significantly prolonged in all groups that received progranulin, in particular, the 0.1 µg dose, in comparison to the control group. This preliminary data is first suggesting that the antiplatelet and anticoagulant action of progranulin could have a physiological protective function against thrombotic disorders associated with obesity and atherosclerosis. However, these results merit further exploration.

  1. Dynamics of Monoterpene Formation in Spike Lavender Plants

    Directory of Open Access Journals (Sweden)

    Isabel Mendoza-Poudereux

    2017-12-01

    Full Text Available The metabolic cross-talk between the mevalonate (MVA and the methylerythritol phosphate (MEP pathways was analyzed in spike lavender (Lavandula latifolia Med on the basis of 13CO2-labelling experiments using wildtype and transgenic plants overexpressing the 3-hydroxy-3-methylglutaryl CoA reductase (HMGR, the first and key enzyme of the MVA pathway. The plants were labelled in the presence of 13CO2 in a gas chamber for controlled pulse and chase periods of time. GC/MS and NMR analysis of 1,8-cineole and camphor, the major monoterpenes present in their essential oil, indicated that the C5-precursors, isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP of both monoterpenes are predominantly biosynthesized via the MEP pathway. Surprisingly, overexpression of HMGR did not have significant impact upon the crosstalk between the MVA and MEP pathways indicating that the MEP route is the preferred pathway for the synthesis of C5 monoterpene precursors in spike lavender.

  2. Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems.

    Science.gov (United States)

    Kemper, Katarina; Hirte, Max; Reinbold, Markus; Fuchs, Monika; Brück, Thomas

    2017-01-01

    With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli , this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

  3. Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism.

    Science.gov (United States)

    Crow, V L; Davey, G P; Pearce, L E; Thomas, T D

    1983-01-01

    The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway. Images PMID:6294064

  4. Cooperation and competition between adenylate kinase, nucleoside diphosphokinase, electron transport, and ATP synthase in plant mitochondria studied by 31P-nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Roberts, J.K.M.; Aubert, S.; Gout, E.; Bligny, R.; Douce, R.

    1997-01-01

    Nucleotide metabolism in potato (Solanum tuberosum) mitochondria was studied using 31P-nuclear magnetic resonance spectroscopy and the O2 electrode. Immediately following the addition of ADP, ATP synthesis exceeded the rate of oxidative phosphorylation, fueled by succinate oxidation, due to mitochondrial adenylate kinase (AK) activity two to four times the maximum activity of ATP synthase. Only when the AK reaction approached equilibrium was oxidative phosphorylation the primary mechanism for net ATP synthesis. A pool of sequestered ATP in mitochondria enabled AK and ATP synthase to convert AMP to ATP in the presence of exogenous inorganic phosphate. During this conversion, AK activity can indirectly influence rates of oxidation of both succinate and NADH via changes in mitochondrial ATP. Mitochondrial nucleoside diphosphokinase, in cooperation with ATP synthase, was found to facilitate phosphorylation of nucleoside diphosphates other than ADP at rates similar to the maximum rate of oxidative phosphorylation. These results demonstrate that plant mitochondria contain all of the machinery necessary to rapidly regenerate nucleoside triphosphates from AMP and nucleoside diphosphates made during cellular biosynthesis and that AK activity can affect both the amount of ADP available to ATP synthase and the level of ATP regulating electron transport

  5. Cladribine and Fludarabine Nucleotides Induce Distinct Hexamers Defining a Common Mode of Reversible RNR Inhibition.

    Science.gov (United States)

    Wisitpitthaya, Somsinee; Zhao, Yi; Long, Marcus J C; Li, Minxing; Fletcher, Elaine A; Blessing, William A; Weiss, Robert S; Aye, Yimon

    2016-07-15

    The enzyme ribonucleotide reductase (RNR) is a major target of anticancer drugs. Until recently, suicide inactivation in which synthetic substrate analogs (nucleoside diphosphates) irreversibly inactivate the RNR-α2β2 heterodimeric complex was the only clinically proven inhibition pathway. For instance, this mechanism is deployed by the multifactorial anticancer agent gemcitabine diphosphate. Recently reversible targeting of RNR-α-alone coupled with ligand-induced RNR-α-persistent hexamerization has emerged to be of clinical significance. To date, clofarabine nucleotides are the only known example of this mechanism. Herein, chemoenzymatic syntheses of the active forms of two other drugs, phosphorylated cladribine (ClA) and fludarabine (FlU), allow us to establish that reversible inhibition is common to numerous drugs in clinical use. Enzyme inhibition and fluorescence anisotropy assays show that the di- and triphosphates of the two nucleosides function as reversible (i.e., nonmechanism-based) inhibitors of RNR and interact with the catalytic (C site) and the allosteric activity (A site) sites of RNR-α, respectively. Gel filtration, protease digestion, and FRET assays demonstrate that inhibition is coupled with formation of conformationally diverse hexamers. Studies in 293T cells capable of selectively inducing either wild-type or oligomerization-defective mutant RNR-α overexpression delineate the central role of RNR-α oligomerization in drug activity, and highlight a potential resistance mechanism to these drugs. These data set the stage for new interventions targeting RNR oligomeric regulation.

  6. Evaluation of the antioxidant properties of N-acetylcysteine in human platelets: prerequisite for bioconversion to glutathione for antioxidant and antiplatelet activity.

    Science.gov (United States)

    Gibson, Kyle R; Neilson, Ilene L; Barrett, Fiona; Winterburn, Tim J; Sharma, Sushma; MacRury, Sandra M; Megson, Ian L

    2009-10-01

    N-Acetylcysteine (NAC) is a frequently used "antioxidant" in vitro, but the concentrations applied rarely correlate with those encountered with oral dosing in vivo. Here, we investigated the in vitro antioxidant and antiplatelet properties of NAC at concentrations (10-100 microM) that are achievable in plasma with tolerable oral dosing. The impact of NAC pretreatment (2 hours) on aggregation of platelets from healthy volunteers in response to thrombin and adenosine diphosphate and on platelet-derived nitric oxide (NO) was examined. NAC was found to be a weak reducing agent and a poor antioxidant compared with glutathione (reduced form) (GSH). However, platelets treated with NAC showed enhanced antioxidant activity and depression of reactive oxygen species generation associated with increases in intraplatelet GSH levels. An approximately 2-fold increase in NO synthase-derived nitrite was observed with 10 microM NAC treatment, but the effect was not concentration dependent. Finally, NAC significantly reduced both thrombin-induced and adenosine diphosphate-induced platelet aggregation. NAC should be considered a weak antioxidant that requires prior conversion to GSH to convey antioxidant and antithrombotic benefit at therapeutically relevant concentrations. Our results suggest that NAC might be an effective antiplatelet agent in conditions where increased oxidative stress contributes to heightened risk of thrombosis but only if the intraplatelet machinery to convert it to GSH is functional.

  7. Purinergic Signaling Pathways in Endocrine System

    Science.gov (United States)

    Bjelobaba, Ivana; Janjic, Marija M.; Stojilkovic, Stanko S.

    2015-01-01

    Adenosine-5′-triphosphate is released by neuroendocrine, endocrine, and other cell types and acts as an extracellular agonist for ligand-gated P2X cationic channels and G protein-coupled P2Y receptors in numerous organs and tissues, including the endocrine system. The breakdown of ATP by ectonucleotidases not only terminates its extracellular messenger functions, but also provides a pathway for the generation of two additional agonists: adenosine 5′-diphosphate, acting via some P2Y receptors, and adenosine, a native agonist for G protein-coupled adenosine receptors, also expressed in the endocrine system. This article provides a review of purinergic signaling pathways in the hypothalamic magnocellular neurosecretory cells and neurohypophysis, hypothalamic parvocellular neuroendocrine system, adenohypophysis, and effector glands organized in five axes: hypothalamic-pituitary-gonadal, hypothalamic-pituitary-thyroid, hypothalamic-pituitary-adrenal, hypothalamic-pituitary-growth hormone, and hypothalamic-pituitary-prolactin. We attempted to summarize current knowledge of purinergic receptor subtypes expressed in the endocrine system, including their roles in intracellular signaling, hormone secretion, and other cell functions. We also briefly review the release mechanism for adenosine-5′-triphosphate by neuroendocrine, endocrine and surrounding cells, the enzymes involved in adenosine-5′-triphosphate hydrolysis to adenosine-5′-diphosphate and adenosine, and the relevance of this pathway for sequential activation of receptors and termination of signaling. PMID:25960051

  8. Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis.

    Science.gov (United States)

    Yamamura, Yoshimi; Kurosaki, Fumiya; Lee, Jung-Bum

    2017-03-07

    Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.

  9. Identification of a Plastid-Localized Bifunctional Nerolidol/Linalool Synthase in Relation to Linalool Biosynthesis in Young Grape Berries

    Science.gov (United States)

    Zhu, Bao-Qing; Cai, Jian; Wang, Zhi-Qun; Xu, Xiao-Qing; Duan, Chang-Qing; Pan, Qiu-Hong

    2014-01-01

    Monoterpenoids are a diverse class of natural products and contribute to the important varietal aroma of certain Vitis vinifera grape cultivars. Among the typical monoterpenoids, linalool exists in almost all grape varieties. A gene coding for a nerolidol/linalool (NES/LINS) synthase was evaluated in the role of linalool biosynthesis in grape berries. Enzyme activity assay of this recombinant protein revealed that it could convert geranyl diphosphate and farnesyl diphosphate into linalool and nerolidol in vitro, respectively, and thus it was named VvRILinNer. However, localization experiment showed that this enzyme was only localized to chloroplasts, which indicates that VvRILinNer functions in the linalool production in vivo. The patterns of gene expression and linalool accumulation were analyzed in the berries of three grape cultivars (“Riesling”, “Cabernet Sauvignon”, “Gewurztraminer”) with significantly different levels of monoterpenoids. The VvRILinNer was considered to be mainly responsible for the synthesis of linalool at the early developmental stage. This finding has provided us with new knowledge to uncover the complex monoterpene biosynthesis in grapes. PMID:25470020

  10. Chemical test for mammalian feces in grain products: collaborative study.

    Science.gov (United States)

    Gerber, H R

    1989-01-01

    A collaborative study was conducted to validate the use of the AOAC alkaline phosphatase method for mammalian feces in corn meal, 44.B01-44.B06, for 7 additional products: brown rice cream, oat bran, grits, semolina, pasta flour, farina, and barley plus (a mixture of barley, oat bran, and brown rice). The proposed method determines the presence of alkaline phosphatase, an enzyme contained in mammalian feces, by using phenolphthalein diphosphate as the enzyme substrate in a test agar medium. Fecal matter is separated from the grain products by specific gravity differences in 1% test agar. As the product is distributed on liquid test agar, fecal fragments float while the grain products sink. The alkaline phosphatase cleaves phosphate radicals from phenolphthalein diphosphate, generating free phenolphthalein, which produces a pink to red-purple color around the fecal particles in the previously colorless medium. Collaborators' recovery averages ranged from 21.7 particles (72.3%) for oat bran to 25.3 particles (84.3%) for semolina at the 30 particle spike level. Overall average background was 0.4 positive reactions per food type. The collaborators reported that the method was quick, simple, and easy to use. The method has been approved interim official first action for all 7 grain products.

  11. Extraction and radiochromatographic division of the early photosynthetic products of C3 plants

    International Nuclear Information System (INIS)

    Manolov, P.; Rangelov, B.; Borichenko, N.

    1978-01-01

    A complete method for extraction, radiochromatographic separation and 14 C balance of the early photosynthetic products in C 3 plants (peach, apple, plum, grapevine and beans) was worked out on the basis of comparative tests of methods presented in literature and of results obtained by investigations carried out. It was established that in view of accomplishing high quality chromatogram an appropriate way to purify the extracts is to eliminate the lipids, pigments, soluble proteins and high molecular carbohydrates in a two-phase system of methanol (chlorophorm)-water (6:5, 5:5) and to block the cations by 0.1 M EDTA. Two-directional ascending chromatography was applied on FN 4 paper rinsed with 0,01 M EDTA and 2 M CH 3 COOH and solvents: for the first direction - 98% ethamol 1 M ammonium acetate pH 7,5: 0,1 M EDTA (75/30 l) by repeated ascending and for the second direction - butanol (propyonic acid) water (10/5/7) by threefold rinsing. Twenty-four 14 C compounds were separated and identified, namely: sucrose diphosphates, uridine diphosphate-glucose, glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, phosphoglyceric acid, phosphoglycolic acid, phosphoenolpyruvic acid, dihydroacetone phosphate, aspartate glutamate, glycine, serine, alanine, citrate, malate, glycerate, glycolate, sorbitol, fructose, glucose, sucrose, maltose and rafinose. For a full 14 C balance of the samples the radioactivity of starch, α-1,4-glucosyleglucans, lipids, pigments and residues was determined. (author)

  12. Reactions of metal oxides with molten NaPO3 + NaCl mixtures

    International Nuclear Information System (INIS)

    Kovarskaya, E.N.; Mityakhina, V.S.; Rodionov, Yu.I.; Silin, M.Yu.

    1988-01-01

    We consider the dissolution mechanism for iron (III), europium(III), and tin(IV) oxides in molten NaPO 3 + NaCl that are responsible for the peak solubilities. We chose Fe 2 O 3 as the basic material since this occurs in large amounts around damaged metal structures in rock salt mines in a proposed zone for storing vitrified radioactive wastes. Solubility measurement and paper chromatography show that Fe 2 O 3 dissolves in molten NaPO 3 + NaCl in air by reaction with the solvent to give double iron and sodium diphosphates and monophosphates in accordance with the initial solution-in-the-melt composition, the degree of equilibration, and the temperature. The elevated solubilities for initial NaCl contents close to 30 mole % are due to sodium triphosphates and tricyclophosphates present in these melts. Moessbauer spectroscopy confirms that double iron, europium and tin diphosphates and monophosphates containing sodium occur in these chloride-polyphosphate melts

  13. Oxygen-18 as a tool for studying photorespiration. Oxygen uptake and incorporation into glycolate, glycine and serine

    International Nuclear Information System (INIS)

    Gerster, R.; Dimon, B.; Tournier, P.; Peybernes, A.

    1977-01-01

    The intensity of photosynthesis and photorespiration has been determined by measuring 16 O 2 evolvement and 18 O 2 uptake on algae and leaves. In the case of algae, there is still an important oxygen uptake even when ribulose diphosphate oxygenase is inhibited by 10 -3 M cyanide. Oxygen-18 incorporation into glycolate, glycine and serine of photorespiring algae and leaves exposed to atmospheres containing 18 O 2 has also been measured. Only one of the two atoms present in molecular oxygen was incorporated into the carboxyl group of the glycolate excreted from algae; the rate of 18 O incorporation was important (65 to 80% according to experimental conditions), even in the presence of 10 -3 M cyanide. Thus, oxidation of ribulose diphosphate is not the sole reaction leading to 18 O glycolate synthesis. In the case of maize, there was a rapid and important 18 O incorporation into the carboxyl group of glycine and serine, the kinetics of which was determined as a function of CO 2 presence in the atmosphere. These results suggest that photorespiration is also operating in C 4 species. Furthermore, in vitro experiments showed that phosphorylated ceto-acids of the Calvin cycle were very sensitive to H 2 O 2 ; the corresponding reaction can explain O 2 uptake and 18 O labelling of glycolate. (author)

  14. Actions of p-synephrine on hepatic enzyme activities linked to carbohydrate metabolism and ATP levels in vivo and in the perfused rat liver.

    Science.gov (United States)

    Maldonado, Marcos Rodrigues; Bracht, Lívia; de Sá-Nakanishi, Anacharis Babeto; Corrêa, Rúbia Carvalho Gomes; Comar, Jurandir Fernando; Peralta, Rosane Marina; Bracht, Adelar

    2018-01-01

    p-Synephrine is one of the main active components of the fruit of Citrus aurantium (bitter orange). Extracts of the bitter orange and other preparations containing p-synephrine have been used worldwide to promote weight loss and for sports performance. The purpose of the study was to measure the action of p-synephrine on hepatic enzyme activities linked to carbohydrate and energy metabolism and the levels of adenine mononucleotides. Enzymes and adenine mononucleotides were measured in the isolated perfused rat liver and in vivo after oral administration of the drug (50 and 300 mg/kg) by using standard techniques. p-Synephrine increased the activity of glycogen phosphorylase in vivo and in the perfused liver. It decreased, however, the activities of pyruvate kinase and pyruvate dehydrogenase also in vivo and in the perfused liver. p-Synephrine increased the hepatic pools of adenosine diphosphate and adenosine triphosphate. Stimulation of glycogen phosphorylase is consistent with the reported increased glycogenolysis in the perfused liver and increased glycemia in rats. The decrease in the pyruvate dehydrogenase activity indicates that p-synephrine is potentially capable of inhibiting the transformation of carbohydrates into lipids. The capability of increasing the adenosine triphosphate-adenosine diphosphate pool indicates a beneficial effect of p-synephrine on the cellular energetics. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Efficacy of Tenofovir 1% Vaginal Gel in Reducing the Risk of HIV-1 and HSV-2 Infection

    Directory of Open Access Journals (Sweden)

    Christopher McConville

    2014-01-01

    Full Text Available Human Immunodeficiency Virus (HIV is a retrovirus that can result in rare opportunistic infections occurring in humans. The onset of these infections is known as Acquired Immune Deficiency Syndrome (AIDS. Sexual transmission is responsible for the majority of infections 1, resulting in transmission of HIV due to infected semen or vaginal and cervical secretions containing infected lymphocytes. HIV microbicides are formulations of chemical or biological agents that can be applied to the vagina or rectum with the intention of reducing the acquisition of HIV. Tenofovir is an NRTI that is phosphorylated by adenylate kinase to tenofovir diphosphate, which in turn competes with deoxyadeosine 5′-triphosphate for incorporation into newly synthesized HIV DNA. Once incorporated, tenofovir diphosphate results in chain termination, thus inhibiting viral replication. Tenofovir has been formulated into a range of vaginal formulations, such as rings, tablets gels and films. It has been shown to safe and effective in numerous animal models, while demonstrating safety and acceptability in numerous human trials. The most encouraging results came from the CAPRISA 004 clinical trial which demonstrated that a 1% Tenofovir vaginal gel reduced HIV infection by approximately 39%.

  16. Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

    Directory of Open Access Journals (Sweden)

    Katarina Kemper

    2017-05-01

    Full Text Available With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli, this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

  17. Ecto-ATPase CD39 Inactivates Isoprenoid-Derived Vγ9Vδ2 T Cell Phosphoantigens

    Directory of Open Access Journals (Sweden)

    Georg Gruenbacher

    2016-07-01

    Full Text Available In humans, Vγ9Vδ2 T cells respond to self and pathogen-associated, diphosphate-containing isoprenoids, also known as phosphoantigens (pAgs. However, activation and homeostasis of Vγ9Vδ2 T cells remain incompletely understood. Here, we show that pAgs induced expression of the ecto-ATPase CD39, which, however, not only hydrolyzed ATP but also abrogated the γδ T cell receptor (TCR agonistic activity of self and microbial pAgs (C5 to C15. Only mevalonate-derived geranylgeranyl diphosphate (GGPP, C20 resisted CD39-mediated hydrolysis and acted as a regulator of CD39 expression and activity. GGPP enhanced macrophage differentiation in response to the tissue stress cytokine interleukin-15. In addition, GGPP-imprinted macrophage-like cells displayed increased capacity to produce IL-1β as well as the chemokine CCL2 and preferentially activated CD161-expressing CD4+ T cells in an innate-like manner. Our studies reveal a previously unrecognized immunoregulatory function of CD39 and highlight a particular role of GGPP among pAgs.

  18. Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73-like, from Cerastes cerastes venom.

    Science.gov (United States)

    Saoud, Samah; Chérifi, Fatah; Benhassine, Traki; Laraba-Djebari, Fatima

    2017-05-01

    The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid sequence of Cc-5'NTase displayed high homology with many nucleotidases. Its activity was optimal at pH 7 with a specific hydrolytic activity toward mono-, di-, and triphosphate adenylated nucleotides. Cc-5'NTase preferentially hydrolyzed ADP and obeyed Michaelis-Menten kinetics. Among the metals and inhibitors tested, Ni 2+ and Mg 2+ completely potentiated enzyme activity, whereas EGTA, PMSF, iodoacetamide, vanillic acid, vanillyl mandelic acid, and 1,10-phenanthroline partially abolished its activity. Cc-5'NTase was not lethal for mice at 5 mg/kg and exhibited in vivo anticoagulant effect. It also dose-dependently inhibited adenosine diphosphate-induced platelet aggregation by converting adenosine diphosphate to adenosine and prohibited arachidonic acid-induced aggregation but was not effective on fibrinogen-induced aggregation. Cc-5'NTase could be a good tool as pharmacological molecule in thrombosis diagnostic and/or therapy. © 2016 Wiley Periodicals, Inc.

  19. Influence of casein hydrolysates on exopolysaccharide synthesis by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

    Science.gov (United States)

    Zhang, Qingli; Yang, Bao; Brashears, Mindy M; Yu, Zhimin; Zhao, Mouming; Liu, Ning; Li, Yinjuan

    2014-05-01

    A lot of interesting research has been undertaken to enhance the yield of exopolysaccharides (EPS) produced by lactic acid bacteria (LAB). The objective of this study was to determine the influence of casein hydrolysates (CH) with molecular weight less than 3 kDa on cell viability, EPS synthesis and the enzyme activity involved in EPS synthesis during the co-culturing of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus in MRS broth for 72 h at 37 ± 0.1 °C. The highest EPS yield (150.1 mg L⁻¹) was obtained on CH prepared with papain (CHP) at 48 h. At 24 h, EPS were composed of galactose, glucose and rhamnose in a molar ratio of 1.0:2.4:1.5. The monosaccharide composition changed with extension of the fermentation time. The activities of α-phosphoglucomutase, uridine 5'-diphosphate (UDP)-glucose pyrophosphorylase and UDP-galactose 4-epimerase were associated with EPS synthesis. Moreover, the activities of β-phosphoglucomutase and deoxythymadine 5'-diphosphate (dTDP)-glucose pyrophosphorylase involved in rhamnose synthesis were very low at the exponential growth phase and could not be detected during other given periods. The influence of different CH (<3 kDa) on LAB viability, EPS production, EPS monomeric composition and activity levels of key metabolic enzymes was distinct. Besides, their influence was related to the distribution of amino acids. © 2013 Society of Chemical Industry.

  20. ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

    Science.gov (United States)

    Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

    2012-01-01

    Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

  1. ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

    Directory of Open Access Journals (Sweden)

    Venkateswarlu Kanamarlapudi

    Full Text Available Adenosine diphosphate (ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1 and P2Y(12 purinoceptors. Recently, we demonstrated that P2Y(1 and P2Y(12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6 in the internalization and function of P2Y(1 and P2Y(12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1 or P2Y(12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

  2. Preliminary X-ray crystallographic analysis of the d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis

    International Nuclear Information System (INIS)

    Petrareanu, Georgiana; Balasu, Mihaela C.; Zander, Ulrich; Scheidig, Axel J.; Szedlacsek, Stefan E.

    2010-01-01

    The expression, purification, preliminary crystallization and crystallographic analysis of phosphoketolase from L. lactis ssp. lactis (strain IL 1403) are reported. Phosphoketolases are thiamine diphosphate-dependent enzymes which play a central role in the pentose-phosphate pathway of heterofermentative lactic acid bacteria. They belong to the family of aldehyde-lyases and in the presence of phosphate ion cleave the carbon–carbon bond of the specific substrate d-xylulose 5-phosphate (or d-fructose 6-phosphate) to give acetyl phosphate and d-glyceraldehyde 3-phosphate (or d-erythrose 4-phosphate). Structural information about phosphoketolases is particularly important in order to fully understand their mechanism as well as the steric course of phosphoketolase-catalyzed reactions. Here, the purification, preliminary crystallization and crystallographic characterization of d-xylulose 5-phosphate phosphoketolase from Lactococcus lactis are reported. The presence of thiamine diphosphate during purification was essential for the enzymatic activity of the purified protein. The crystals belonged to the monoclinic space group P2 1 . Diffraction data were obtained to a resolution of 2.2 Å

  3. Insights Into the Bifunctional Aphidicolan-16-ß-ol Synthase Through Rapid Biomolecular Modeling Approaches.

    Science.gov (United States)

    Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B

    2018-01-01

    Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modeling techniques offer an alternative route to study the enzyme's reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modeling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modeling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789, and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modeling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of

  4. Insights Into the Bifunctional Aphidicolan-16-ß-ol Synthase Through Rapid Biomolecular Modeling Approaches

    Directory of Open Access Journals (Sweden)

    Max Hirte

    2018-04-01

    Full Text Available Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modeling techniques offer an alternative route to study the enzyme's reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modeling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modeling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789, and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modeling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially

  5. Insights into the bifunctional Aphidicolan-16-ß-ol synthase through rapid biomolecular modelling approaches

    Science.gov (United States)

    Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B.

    2018-04-01

    Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modelling techniques offer an alternative route to study the enzyme’s reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modelling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modelling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789 and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modelling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location

  6. The effect of 24S-hydroxycholesterol on cholesterol homeostasis in neurons: quantitative changes to the cortical neuron proteome.

    Science.gov (United States)

    Wang, Yuqin; Muneton, Sabina; Sjövall, Jan; Jovanovic, Jasmina N; Griffiths, William J

    2008-04-01

    In humans, the brain represents only about 2% of the body's mass but contains about one-quarter of the body's free cholesterol. Cholesterol is synthesized de novo in brain and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study, we investigated the effect of 24S-hydroxycholesterol on the proteome of rat cortical neurons. With the use of two-dimensional liquid chromatography linked to nanoelectrospray tandem mass spectrometry, over 1040 proteins were identified including members of the cholesterol, isoprenoid and fatty acid synthesis pathways. With the use of stable isotope labeling technology, the protein expression patterns of enzymes in these pathways were investigated. 24S-Hydroxycholesterol was found to down-regulate the expression of members of the cholesterol/isoprenoid synthesis pathways including 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (EC 2.3.3.10), diphosphomevalonate decarboxylase (EC 4.1.1.33), isopentenyl-diphosphate delta isomerase (EC 5.3.3.2), farnesyl-diphosphate synthase (Geranyl trans transferase, EC 2.5.1.10), and dedicated sterol synthesis enzymes, farnesyl-diphosphate farnesyltransferase 1 (squalene synthase, EC 2.5.1.21) and methylsterol monooxygenase (EC 1.14.13.72). The expression of many enzymes in the cholesterol/isoprenoid and fatty acid synthesis pathways are regulated by the membrane-bound transcription factors named sterol regulatory element-binding proteins (SREBPs), which themselves are both transcriptionally and post-transcriptionally regulated. The current proteomic data indicates that 24S-hydroxycholesterol down-regulates cholesterol synthesis in neurons, possibly, in a post-transcriptional manner through SREBP-2. In contrast to cholesterol metabolism, enzymes responsible for the synthesis of fatty acids were not

  7. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L. and their relevance for perfume manufacture

    Directory of Open Access Journals (Sweden)

    Caniard Anne

    2012-07-01

    Full Text Available Abstract Background Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Results Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S-copalyl diphosphate. A class I diTPS (SsSS then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae. Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. Conclusions SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid

  8. Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture.

    Science.gov (United States)

    Caniard, Anne; Zerbe, Philipp; Legrand, Sylvain; Cohade, Allison; Valot, Nadine; Magnard, Jean-Louis; Bohlmann, Jörg; Legendre, Laurent

    2012-07-26

    Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They

  9. Crystal Structure of (+)-[delta]-Cadinene Synthase from Gossypium arboreum and Evolutionary Divergence of Metal Binding Motifs for Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Gennadios, Heather A.; Gonzalez, Veronica; Di Costanzo, Luigi; Li, Amang; Yu, Fanglei; Miller, David J.; Allemann, Rudolf K.; Christianson, David W.; (UPENN); (Cardiff); (UC)

    2009-09-11

    (+)-{delta}-Cadinene synthase (DCS) from Gossypium arboreum (tree cotton) is a sesquiterpene cyclase that catalyzes the cyclization of farnesyl diphosphate in the first committed step of the biosynthesis of gossypol, a phytoalexin that defends the plant from bacterial and fungal pathogens. Here, we report the X-ray crystal structure of unliganded DCS at 2.4 {angstrom} resolution and the structure of its complex with three putative Mg{sup 2+} ions and the substrate analogue inhibitor 2-fluorofarnesyl diphosphate (2F-FPP) at 2.75 {angstrom} resolution. These structures illuminate unusual features that accommodate the trinuclear metal cluster required for substrate binding and catalysis. Like other terpenoid cyclases, DCS contains a characteristic aspartate-rich D{sup 307}DTYD{sup 311} motif on helix D that interacts with Mg{sub A}{sup 2+} and Mg{sub C}{sup 2+}. However, DCS appears to be unique among terpenoid cyclases in that it does not contain the 'NSE/DTE' motif on helix H that specifically chelates Mg{sub B}{sup 2+}, which is usually found as the signature sequence (N,D)D(L,I,V)X(S,T)XXXE (boldface indicates Mg{sub B}{sup 2+} ligands). Instead, DCS contains a second aspartate-rich motif, D{sup 451}DVAE{sup 455}, that interacts with Mg{sub B}{sup 2+}. In this regard, DCS is more similar to the isoprenoid chain elongation enzyme farnesyl diphosphate synthase, which also contains two aspartate-rich motifs, rather than the greater family of terpenoid cyclases. Nevertheless, the structure of the DCS-2F-FPP complex shows that the structure of the trinuclear magnesium cluster is generally similar to that of other terpenoid cyclases despite the alternative Mg{sub B}{sup 2+} binding motif. Analyses of DCS mutants with alanine substitutions in the D{sup 307}DTYD{sup 311} and D{sup 451}DVAE{sup 455} segments reveal the contributions of these segments to catalysis.

  10. Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex - Linkage of protein dynamics to catalysis

    Science.gov (United States)

    Jordan, Frank; Arjunan, Palaniappa; Kale, Sachin; Nemeria, Natalia S.; Furey, William

    2009-01-01

    The region encompassing residues 401–413 on the E1 component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli comprises a loop (the inner loop) which was not seen in the X-ray structure in the presence of thiamin diphosphate, the required cofactor for the enzyme. This loop is seen in the presence of a stable analogue of the pre-decarboxylation intermediate, the covalent adduct between the substrate analogue methyl acetylphosphonate and thiamin diphosphate, C2α-phosphonolactylthiamin diphosphate. It has been shown that the residue H407 and several other residues on this loop are required to reduce the mobility of the loop so electron density corresponding to it can be seen once the pre-decarboxylation intermediate is formed. Concomitantly, the loop encompassing residues 541–557 (the outer loop) appears to work in tandem with the inner loop and there is a hydrogen bond between the two loops ensuring their correlated motion. The inner loop was shown to: a) sequester the active center from carboligase side reactions; b) assist the interaction between the E1 and the E2 components, thereby affecting the overall reaction rate of the entire multienzyme complex; c) control substrate access to the active center. Using viscosity effects on kinetics it was shown that formation of the pre-decarboxylation intermediate is specifically affected by loop movement. A cysteine-less variant was created for the E1 component, onto which cysteines were substituted at selected loop positions. Introducing an electron spin resonance spin label and an 19F NMR label onto these engineered cysteines, the loop mobility was examined: a) both methods suggested that in the absence of ligand, the loop exists in two conformations; b) line-shape analysis of the NMR signal at different temperatures, enabled estimation of the rate constant for loop movement, and this rate constant was found to be of the same order of magnitude as the turnover number for the enzyme under the

  11. Microchannel conductivity measurements in microchip for on line monitoring of dephosphorylation rates of organic phosphates using paramagnetic-beads linked alkaline phosphatase.

    Science.gov (United States)

    Kechadi, Mohammed; Sotta, Bruno; Gamby, Jean

    2015-01-01

    This paper presents the use of polymer coated microelectrodes for the realtime conductivity monitoring in a microchannel photoablated through the polymer without contact. Based on this strategy, a small conductometry sensor has been developed to record in time conductivity variation when an enzymatic reaction occurs through the channel. The rate constant determination, k2, for the dephosphorylation of organic phosphate-alkaline phosphatase-superparamagnetic beads complex using chemically different substrates such as adenosine monoesterphosphate, adenosine diphosphate and adenosine triphosphate was taken as an example to demonstrate selectivity and sensivity of the detection scheme. The k2 value measured for each adenosine phosphate decreases from 39 to 30 s(-1) in proportion with the number (3, 2 and 1) of attached phosphate moiety, thus emphasizing the steric hindrance effect on kinetics. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. A unifying paradigm for naphthoquinone-based meroterpenoid (bio)synthesis

    Science.gov (United States)

    Miles, Zachary D.; Diethelm, Stefan; Pepper, Henry P.; Huang, David M.; George, Jonathan H.; Moore, Bradley S.

    2017-12-01

    Bacterial meroterpenoids constitute an important class of natural products with diverse biological properties and therapeutic potential. The biosynthetic logic for their production is unknown and defies explanation via classical biochemical paradigms. A large subgroup of naphthoquinone-based meroterpenoids exhibits a substitution pattern of the polyketide-derived aromatic core that seemingly contradicts the established reactivity pattern of polyketide phenol nucleophiles and terpene diphosphate electrophiles. We report the discovery of a hitherto unprecedented enzyme-promoted α-hydroxyketone rearrangement catalysed by vanadium-dependent haloperoxidases to account for these discrepancies in the merochlorin and napyradiomycin class of meroterpenoid antibiotics, and we demonstrate that the α-hydroxyketone rearrangement is potentially a conserved biosynthetic reaction in this molecular class. The biosynthetic α-hydroxyketone rearrangement was applied in a concise total synthesis of naphthomevalin, a prominent member of the napyradiomycin meroterpenes, and sheds further light on the mechanism of this unifying enzymatic transformation.

  13. Effect of 3-aminobenzamide on the rate of ligation during repair of alkylated DNA in human fibroblasts

    International Nuclear Information System (INIS)

    Morgan, W.F.; Cleaver, J.E.

    1983-01-01

    3-Aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase, produced rapid reversible changes in single-strand break frequencies in DNA from primary human fibroblasts damaged by alkylating agents, but it did not cause such changes in the DNA of cells damaged by ultraviolet light. The increase in single-strand peak frequencies was not due to an accumulation of blocked repair sites, such as occurs with DNA polymerase inhibitors, but to a delay in the rejoining of induced breaks. 3-Aminobenzamide increases the net break frequency that results from a dynamic balance between excision and ligation. This balance appears to be regulated at the ligation step by adenosine diphosphate ribosylation, which is rapidly altered by addition or removal of 3-aminobenzamide. The rapidity with which strand break frequencies change in the presence of 3-aminobenzamide implies that individual strand breaks resulting from excision at any time after exposure have a lifetime of no more than about 30 min in the cell

  14. (+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

    Science.gov (United States)

    Prosser, Ian; Phillips, Andy L; Gittings, Simon; Lewis, Mervyn J; Hooper, Antony M; Pickett, John A; Beale, Michael H

    2002-08-01

    Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

  15. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

    DEFF Research Database (Denmark)

    Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

    2006-01-01

    Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere...... light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse...

  16. EFFECT OF SODIUM PHOSPHATES ON SELECTED FOOD GRADE BACTERIA

    Directory of Open Access Journals (Sweden)

    Stanislav Kráčmar

    2011-04-01

    Full Text Available The aim of this study was to examine the inhibitory effect in vitro of selected sodium phosphates (under the corporate names Hexa 68, Hexa 70, Trikrystal, FST, Pyro 52, KPS, Didi on selected gram-positive and gram-negative bacteria. Seven different concentrations of each phosphate were used. Sensitivity of the bacterial strains to phosphates was observed in broth supplemented with salts. In vitro was showed a negative effect of various phosphates on growth of selected gram-positive bacteria. Orthophosphates and diphosphates (pyrophosphates did not have significant inhibitory effect on tested bacteria at neutral pH. With the exception of phosphate Trikrystal has not been found in vitro significant inhibitory effects on gram-negative bacteria.doi:10.5219/141

  17. Carbon nanopipettes characterize calcium release pathways in breast cancer cells

    International Nuclear Information System (INIS)

    Schrlau, Michael G; Brailoiu, Eugen; Dun, Nae J; Patel, Sandip; Gogotsi, Yury; Bau, Haim H

    2008-01-01

    Carbon-based nanoprobes are attractive for minimally invasive cell interrogation but their application in cell physiology has thus far been limited. We have developed carbon nanopipettes (CNPs) with nanoscopic tips and used them to inject calcium-mobilizing messengers into cells without compromising cell viability. We identify pathways sensitive to cyclic adenosine diphosphate ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) in breast carcinoma cells. Our findings demonstrate the superior utility of CNPs for intracellular delivery of impermeant molecules and, more generally, for cell physiology studies. The CNPs do not appear to cause any lasting damage to cells. Their advantages over commonly used glass pipettes include smaller size, breakage and clogging resistance, and potential for multifunctionality such as in concurrent injection and electrical measurements

  18. Carbon nanopipettes characterize calcium release pathways in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Schrlau, Michael G [Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, PA 19104 (United States); Brailoiu, Eugen; Dun, Nae J [Department of Pharmacology, Temple University, Philadelphia, PA 19104 (United States); Patel, Sandip [Department of Physiology, University College London, London WC1E 6BT (United Kingdom); Gogotsi, Yury [Department of Materials Science and Engineering, Drexel University, Philadelphia, PA 19104 (United States); Bau, Haim H [Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, 229 Towne Building, 220 S. 33rd Street, Philadelphia, PA 19104 (United States)], E-mail: mschrlau@seas.upenn.edu, E-mail: ebrailou@temple.edu, E-mail: patel.s@ucl.ac.uk, E-mail: yg36@drexel.edu, E-mail: ndun@temple.edu, E-mail: bau@seas.upenn.edu

    2008-08-13

    Carbon-based nanoprobes are attractive for minimally invasive cell interrogation but their application in cell physiology has thus far been limited. We have developed carbon nanopipettes (CNPs) with nanoscopic tips and used them to inject calcium-mobilizing messengers into cells without compromising cell viability. We identify pathways sensitive to cyclic adenosine diphosphate ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) in breast carcinoma cells. Our findings demonstrate the superior utility of CNPs for intracellular delivery of impermeant molecules and, more generally, for cell physiology studies. The CNPs do not appear to cause any lasting damage to cells. Their advantages over commonly used glass pipettes include smaller size, breakage and clogging resistance, and potential for multifunctionality such as in concurrent injection and electrical measurements.

  19. Microbial Cell Factories for the Production of Terpenoid Flavor and Fragrance Compounds.

    Science.gov (United States)

    Schempp, Florence M; Drummond, Laura; Buchhaupt, Markus; Schrader, Jens

    2018-03-14

    Terpenoid flavor and fragrance compounds are of high interest to the aroma industry. Microbial production offers an alternative sustainable access to the desired terpenoids independent of natural sources. Genetically engineered microorganisms can be used to synthesize terpenoids from cheap and renewable resources. Due to its modular architecture, terpenoid biosynthesis is especially well suited for the microbial cell factory concept: a platform host engineered for a high flux toward the central C 5 prenyl diphosphate precursors enables the production of a broad range of target terpenoids just by varying the pathway modules converting the C 5 intermediates to the product of interest. In this review typical terpenoid flavor and fragrance compounds marketed or under development by biotech and aroma companies are given, and the specificities of the aroma market are discussed. The main part of this work focuses on key strategies and recent advances to engineer microbes to become efficient terpenoid producers.

  20. Optimization time synthesis of nucleotide labelled [γ-32P]-ATP

    International Nuclear Information System (INIS)

    Rahman, Wira Y; Sarmini, Endang; Herlina; Lubis, Hotman; Triyanto; Hambali

    2013-01-01

    Adenosine triphosphate-labelled with γ- 32 P([γ- 32 p]-ATP) has been widely used in the biotechnology research, usually as a tracer to study aspects of physiological and pathological processes. In order to support biotechnology research in Indonesia, a process for production of [γ- 32 P]-ATP with enzymatic reaction was used as precursors DL-glyceraldehydde 3-phosphate, Adenosine Diphosphate (ADP) and H 3 32 PO 4 , and enzyme glyceraldehid 3-phosphate dehydrogenase, 3-phosphoglyceryc phosphokinase and lactate dehydrogenase. Optimization of incubation time labeled nucleotide synthesis process is performed to find the optimum conditions, in terms of the most advantageous time in the synthesis process. With the success of the synthesis and optimization is done incubation time of synthesis labeled nucleotide, the result suggested can be used for producing [γ- 32 P] -ATP to support the provision of radiolabeled nucleotide for biotechnology research in Indonesia. (author)

  1. Identification of UDPG-binding polypeptides and purified (1,3)-β-glucan synthase by photoaffinity labelling with 5-azido-UDPG

    International Nuclear Information System (INIS)

    Frost, D.J.; Wu, A.; Read, S.M.; Wasserman, B.P.; Drake, R.R.; Haley, B.E.

    1989-01-01

    The photoaffinity probe 5-azido-uridine 5'-β-[ 32 P]-diphosphate glucose was used to identify the major UDPG-binding polypeptide of red beet (1,3)-β-glucan synthase. Glucan synthase was purified from plasma membranes by sequential solubilization with CHAPS followed by product entrapment. Two major polypeptides at 72 and 54 kD were labelled by probe. Labelling of both was abolished with increasing levels of cold UDPG. However, labelling of the 54 kD polypeptide was dependent upon the presence of divalent cations. These data suggest that the 54 kD polypeptide is a substrate-binding and cation-regulated component of the glucan synthase complex

  2. Does bipolar pacemaker current activate blood platelets?

    DEFF Research Database (Denmark)

    Gjesdal, Grunde; Hansen, Annebirthe Bo; Brandes, Axel

    2009-01-01

    OBJECTIVE: The aim of this study was to investigate whether bipolar pacemaker current lead can activate blood platelets. The null hypothesis was that 1 minute of electrical stimulation of platelets would not influence their subsequent reactivity to adenosine diphosphate (ADP). BACKGROUND: Both...... platelets and muscle cells contain actin and myosin filaments, and both cells are activated following calcium influx. Muscle cells open their calcium channels and contract when exposed to an electric current. Current through a bipolar pacemaker lead will expose a small volume of blood, including platelets......, to the depolarizing current. Platelet activation may ensue, resulting in aggregation, release reaction, and contraction. In contrast, a unipolar pacemaker system will not depolarize blood, but transmit current directly into the myocardium, and the current afterward passes through other tissues before returning...

  3. Synthesis of tritium labelled nucleoside triphosphates by enzymatic phosphorylation

    International Nuclear Information System (INIS)

    Shen Decun; Ji Linzhen; Liao Sha

    1986-01-01

    [5- 3 H]UMP, [5- 3 H]CMP, [8- 3 H]AMP and [8- 3 H]GMP were prepared from 5BrUMP 5BrCMP 8BrAMP and 8BrGMP by catalytic halogentritium replacement at the same time. [5- 3 H]UTP, [5- 3 H]CTP, [8- 3 H]ATP and [8- 3 H]GTP were subsequently synthesized from [5- 3 H]UMP, [5- 3 H]CMP, [8- 3 H]AMP and [8- 3 H]GMP by enzymatic phosphorylation with the crude enzyme prepared from brewer's yeasts and purified by paper chromatography simultaneously. In addition, four kinds of tritium labelled nucleoside monophosphates and four kinds of tritium labelled nucleoside diphosphates were obtained as the by-products. The specific activity of these products is between 14-19 Ci/mmol and the radiochemical purity is more than 98%

  4. High prevalence of thiamine (vitamin B1) deficiency in early childhood among a nationally representative sample of Cambodian women of childbearing age and their children

    DEFF Research Database (Denmark)

    Whitfield, Kyly C; Smith, Geoffry; Chamnan, Chhoun

    2017-01-01

    the thiamine status (measured as erythrocyte thiamine diphosphate concentrations; eThDP) among a representative sample of Cambodian women of childbearing age (15-49 y) and their young children (6-69 mo). METHODOLOGY/PRINCIPLE FINDINGS: Samples for this cross-sectional analysis were collected as part...... of a national micronutrient survey linked to the Cambodian Demographic and Health Survey (CDHS) 2014. One-sixth of households taking part in the CDHS were randomly selected and re-visited for additional blood sampling for eThDP analysis (719 women and 761 children). Thiamine status was assessed using different...... cut-offs from literature. Women were mean (SD) 30 (6) y, and children (46% girls) were 41 (17) mo. Women had lower mean (95% CI) eThDP of 150 nmol/L (146-153) compared to children, 174 nmol/L (171-179; P

  5. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

    Science.gov (United States)

    Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

    1993-06-25

    Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

  6. [Genetic diversity analysis of Andrographis paniculata in China based on SRAP and SNP].

    Science.gov (United States)

    Chen, Rong; Wang, Xiao-Yun; Song, Yu-Ning; Zhu, Yun-feng; Wang, Peng-liang; Li, Min; Zhong, Guo-Yue

    2014-12-01

    In order to reveal genetic diversity of domestic Andrographis paniculata and its impact on quality, genetic backgrounds of 103 samples from 7 provinces in China were analyzed using SRAP marker and SNP marker. Genetic structures of the A. paniculata populations were estimated with Powermarker V 3.25 and Mega 6.0 software, and polymorphic SNPs were identified with CodonCode Aligner software. The results showed that the genetic distances of domestic A. paniculata germplasm ranged from 0. 01 to 0.09, and no polymorphic SNPs were discovered in coding sequence fragments of ent-copalyl diphosphate synthase. A. paniculata germplasm from various regions in China had poor genetic diversity. This phenomenon was closely related to strict self-fertilization and earlier introduction from the same origin. Therefore, genetic background had little impact on variable qualities of A. paniculata in domestic market. Mutation breeding, polyploid breeding and molecular breeding were proposed as promising strategies in germplasm innovation.

  7. Effect of gamma radiation on levels of adenine nucleotides in erythrocytes of healthy individuals after submaximum physical exertion

    International Nuclear Information System (INIS)

    Zagorski, T.; Dudek, I.; Mazurek, M.; Berkan, L.; Chmielewski, H.; Kedziora, J.

    1994-01-01

    The authors studied the effect of gamma radiation and submaximum physical exercise on adenosine-5'-triphosphate (ATP), adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) contents in erythrocytes of healthy males. Twenty one men aged 20-22 years were examined. They underwent physical exercise at doses of 2 w/kg body weight for 15 min. Erythrocytes were exposed to gamma radiation (500 Gy doses) from 60 Co source. The concentration of adenine nucleotides in erythrocytes was measured by the Boehringer Mannheim tests. The submaximum physical exercise was found to decrease ATP content and to increase ADP and AMP in erythrocytes. Gamma radiation at 500 Gy dose was found to decrease ATP concentration in erythrocytes both at rest and after submaximum exercise and to increase AD content. It was revealed that AMP content increased at rest and decreased after submaximum exercise in irradiated erythrocytes. (author). 20 refs, 1 tab

  8. Aluminium cyclohexaphosphate

    Directory of Open Access Journals (Sweden)

    Daniel Avignant

    2010-03-01

    Full Text Available Single crystals of the title compound, Al2P6O18, were obtained by solid-state reaction. The monoclinic structure is isotypic with its CrIII, GaIII and RuIII analogues and is built up of six-membered phosphate ring anions, P6O186−, isolated from each other and further linked by isolated AlO6 octahedra by sharing corners. Each AlO6 octahedron is linked to four P6O186− rings. More accurately, two rings are linked through bidentate diphosphate groups attached in the cis-positions to the AlO6 octahedron. The other two rings are linked to the two remaining corners, also in cis-positions of the AlO6 octahedron.

  9. Change in the aldolase activity in rats' hearts after irradiation with. gamma. and. beta. rays

    Energy Technology Data Exchange (ETDEWEB)

    Kukulyanskaya, M F; Borodina, G N

    1973-01-01

    The activity of aldolase fructoso-1-phosphate (I) and aldolase fructoso-1, 6-diphosphate (II) has been studied at various periods after total ..gamma.. and ..beta.. irradiation of rats at a dose of 42 rads. It has been shown that after ..gamma.. irradiation the activity of I increases in the supernatant liquid of the heart muscle homogenate, but drops sharply in the nuclei. In total, the activity of the homogenate did not change. The activity of II dropped for seven days, and after 1 hour and 30 days it was above the normal level. After ..beta.. radiation the activity of II is slowed down after 1, 7, and 15 days. These changes occur in all subcellular fractions. The authors note that the changes in the activity of aldolases are more sharply demarcated after ..gamma.. irradiation and are more substantial for II. (JPRS)

  10. Physiology of inactivation of microbial cells by near-ultraviolet light: mode of action and application for the enrichment of mutants of Escherichia coli and saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Peters, J.

    1976-01-01

    The mode of action of near-ultraviolet (NUV) light was studied in Escherichia coli. NUV light (maximum emission at 365 nm) caused the photodestruction of ribonucleoside diphosphate (RDP) reductase activity in vivo. Evidence was presented for a model suggesting that the loss of RDP-reductase resulted in a metabolic state analogous to that produced during starvation for thymine. Some important properties of cells irradiated by NUV light, cell death, loss of the ability to support the replication of DNA phages and a delay in the onset of cell division in sublethally irradiated cells, were accounted for in terms of photoinactivation of RDP-reductase. Conditions were described under which NUV light was an effective counterselective agent for the enrichment of mutants of Escherichia coli and Saccharomyces cerevisiae

  11. Update: Acute coronary syndromes (V). Personalized antiplatelet therapy.

    Science.gov (United States)

    Gurbel, Paul A; Rafeedheen, Rahil; Tantry, Udaya S

    2014-06-01

    It is well established that high on-treatment platelet reactivity to adenosine diphosphate during clopidogrel therapy is an independent risk factor for ischemic event occurrences in a postpercutaneous coronary intervention patients. However, the precise role of platelet function testing remains debated. Platelet function testing to ensure optimal platelet inhibition has been recommended by some authorities to improve outcomes in patients treated with clopidogrel. Recent prospective, randomized trials of personalized antiplatelet therapy have failed to demonstrate a benefit of platelet function testing in improving outcomes. In this review article, we discuss the mechanisms responsible for clopidogrel nonreponsiveness, recent trials of platelet function testing, and other new developments in the field of personalized antiplatelet therapy. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier Espana. All rights reserved.

  12. Characterization of ZrP{sub 2}O{sub 7} modified with oxalic acid and thermodynamic analysis associated to the uranyl (Vi) sorption; Caracterizacion de ZrP{sub 2}O{sub 7} modificado con acido oxalico y analisis termodinamico asociado a la sorcion de uranilo (VI)

    Energy Technology Data Exchange (ETDEWEB)

    Garcia G, N.; Ordonez R, E. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico); Solis C, D. A. [Universidad Autonoma del Estado de Mexico, Facultad de Quimica, Paseo Colon y Paseo Tollocan, 50120 Toluca, Estado de Mexico (Mexico); Simoni, E.; Drot, R.; Jeanson, A., E-mail: eduardo.ordonez@inin.gob.mx [Universite Paris-Sud, Institut de Physique Nucleaire, Georges Clemenceau No. 15, Orsay (France)

    2013-10-15

    Several physical and chemical tests were carried out to evaluate the influence of the oxalic acid in the uranium (Vi) sorption on the surface of the zirconium diphosphate. The physical analyses consist of atomic force microscopy (AFM) and total organic coal, and the chemists are the calculation of the reaction heat for the hydrate and sorption processes, the reaction speed constant and the sorption yield in the sodium perchlorate systems and of oxalic acid; these tests allow to corroborate that the oxalic acid influences positively in the uranium (Vi) sorption forming a ternary system of ZrP{sub 2}O{sub 7}/(C{sub 2}O{sub 4})/U(Vi), with a single configuration along the ph interval studied. (Author)

  13. Positiv effekt af intermitterende over- og undertryk (Vacusac) ved claudicatio intermittens

    DEFF Research Database (Denmark)

    Mehlsen, J; Himmelstrup, H; Himmelstrup, B

    1994-01-01

    Twenty-two patients with intermittent claudication were included in a double-blinded, randomized trial comparing the effects of 25 treatments with intermittent suction and pressure (Vacusac treatment) to 25 placebo applications given over a period of two months. Twelve patients participated in an...... treatment caused significant increments in ADP-induced platelet aggregation, while the effects on fibrinolysis were uncertain. It is concluded that intermittent suction and pressure treatment offers a new approach for conservative treatment of intermittent claudication....... in an open trial investigating the effects of the same treatment on adenosine diphosphate (ADP)-induced platelet aggregation and fibrinolysis. Active treatment resulted in significant improvements in pain-free and maximal walking distances, whereas no changes could be found during placebo application. Active...

  14. Beneficial effects of intermittent suction and pressure treatment in intermittent claudication

    DEFF Research Database (Denmark)

    Mehlsen, J; Himmelstrup, H; Himmelstrup, Bodil

    1993-01-01

    administration. The treatment caused significant increments in the ADP thresholds for platelet aggregation, while the effects on fibrinolysis were uncertain. It is concluded that intermittent suction and pressure treatment offers a new approach for conservative treatment of intermittent claudication....... participated in an open trial investigating the possible effects of the treatment on platelet aggregation and fibrinolysis. Pain-free and maximal walking distances were measured on a treadmill, and systolic blood pressure was measured on the upper limb, the ankle, and the first toe bilaterally. The threshold...... for adenosine diphosphate (ADP)-induced platelet aggregation was tested, and the fibrinolytic activity was estimated from the euglobulin clot lysis time. Active treatment resulted in significant improvements in pain-free and maximal walking distances, whereas no changes could be found during placebo...

  15. Assessing a traceability technique in fresh oranges (Citrus sinensis L. Osbeck) with an HS-SPME-GC-MS method. Towards a volatile characterisation of organic oranges.

    Science.gov (United States)

    Cuevas, Francisco Julián; Moreno-Rojas, José Manuel; Ruiz-Moreno, María José

    2017-04-15

    A targeted approach using HS-SPME-GC-MS was performed to compare flavour compounds of 'Navelina' and 'Salustiana' orange cultivars from organic and conventional management systems. Both varieties of conventional oranges showed higher content of ester compounds. On the other hand, higher content of some compounds related with the geranyl-diphosphate pathway (neryl and geranyl acetates) and some terpenoids were found in the organic samples. Furthermore, the partial least square discriminant analysis (PLS-DA) achieved an effective classification for oranges based on the farming system using their volatile profiles (90 and 100% correct classification). To our knowledge, it is the first time that a comparative study dealing with farming systems and orange aroma profile has been performed. These new insights, taking into account local databases, cultivars and advanced analytical tools, highlight the potential of volatile composition for organic orange discrimination. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Validation of a homology model of Mycobacterium tuberculosis DXS: rationalization of observed activities of thiamine derivatives as potent inhibitors of two orthologues of DXS.

    Science.gov (United States)

    Masini, T; Lacy, B; Monjas, L; Hawksley, D; de Voogd, A R; Illarionov, B; Iqbal, A; Leeper, F J; Fischer, M; Kontoyianni, M; Hirsch, A K H

    2015-12-14

    The enzyme DXS catalyzes the first, rate-limiting step of the 2-C-methyl-d-erythritol-4-phosphate (MEP, 1) pathway using thiamine diphosphate (ThDP) as cofactor; the DXS-catalyzed reaction constitutes also the first step in vitamin B1 and B6 metabolism in bacteria. DXS is the least studied among the enzymes of this pathway in terms of crystallographic information, with only one complete crystal structure deposited in the Protein Data Bank (Deinococcus radiodurans DXS, PDB: ). We synthesized a series of thiamine and ThDP derivatives and tested them for their biochemical activity against two DXS orthologues, namely D. radiodurans DXS and Mycobacterium tuberculosis DXS. These experimental results, combined with advanced docking studies, led to the development and validation of a homology model of M. tuberculosis DXS, which, in turn, will guide medicinal chemists in rationally designing potential inhibitors for M. tuberculosis DXS.

  17. Spin-Center Shift-Enabled Direct Enantioselective α-Benzylation of Aldehydes with Alcohols.

    Science.gov (United States)

    Nacsa, Eric D; MacMillan, David W C

    2018-03-07

    Nature routinely engages alcohols as leaving groups, as DNA biosynthesis relies on the removal of water from ribonucleoside diphosphates by a radical-mediated "spin-center shift" (SCS) mechanism. Alcohols, however, remain underused as alkylating agents in synthetic chemistry due to their low reactivity in two-electron pathways. We report herein an enantioselective α-benzylation of aldehydes using alcohols as alkylating agents based on the mechanistic principle of spin-center shift. This strategy harnesses the dual activation modes of photoredox and organocatalysis, engaging the alcohol by SCS and capturing the resulting benzylic radical with a catalytically generated enamine. Mechanistic studies provide evidence for SCS as a key elementary step, identify the origins of competing reactions, and enable improvements in chemoselectivity by rational photocatalyst design.

  18. [Experiments with sausage meat on the formation of N epsilon-carboxymethyllysine].

    Science.gov (United States)

    Hartkopf, J; Erbersdobler, H F

    1995-07-01

    In model experiments the influence of ingredients normally used for sausage production to a meat homogenate on the formation of N epsilon-carboxymethyllysine (CML) was investigated. The formation of CML is obviously more promoted from the reaction of ascorbate with lysine than from that of glucose with lysine. The addition of ascorbate in a practical concentration yielded 35 mg, the addition of glucose only 23 mg compared to 17 mg CML/kg protein in the control sample. The addition of diphosphate in a practical concentration besides glucose significantly increased the CML values from 23 mg to 30 mg CML/kg protein. On the other hand, nitrite did not enhance the formation of CML (21 mg/kg protein) in the sausage when used in concentrations usually applied in meat processing. Generally the values found in the meat products are quite low compared to data in other foods like milk products.

  19. Synthesis of binary condensed cesium-manganese phosphates in melts of polyphosphoric acids

    International Nuclear Information System (INIS)

    Guzeeva, L.S.; Tananaev, I.V.

    1988-01-01

    Cuts of Cs 2 O-MnO 2 -P 2 O 5H2 O system are investigated within 150 - 400 d egC temperature interval at P three initial relations: Mn(n), equal to 5; 7.5; 10 and P constant relation, Mn (n), equal to 15. It is shown, that interaction of manganese and cesium oxides with melts of polyphosphoric acids within 150 - 400 d egC temperature interval leads to release of three types of binary condenced phosphates of trivalent manganese and cesium: Cs 2 MnH 3 (P 2 O 7 ) 2 diphosphate, CsMnHP 3 O 10 triphosphate, Cs 3 Mn 3 (P 6 O-1 8 ) 2 cyclohexaphosphate and CsMnP 3 O 9 binary cyclotriphosphate of bivalent manganese. The released compounds are characterized by chemical, roentgenophase analyses, IR-spectrometry and thermogravimetry

  20. Cellular applications of 31P and 13C nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Shulman, R.G.; Brown, T.R.; Ugurbil, K.; Ogawa, S.; Cohen, S.M.; den Hollander, J.A.

    1979-01-01

    High-resolution nuclear magnetic resonance (NMR) studies of cells and purified mitochondria are discussed to show the kind of information that can be obtained in vivo. In suspensions of Escherichia coli both phosphorus-31 and carbon-13 NMR studies of glycolysis of bioenergetics are presented. In rat liver cells the pathways of gluconeogenesis from carbon-13-labeled glycerol are followed by carbon-13 NMR. In the intact liver cells cytosolic and mitochondrial pH's were separately measured by phosphorus-31 NMR. In purified mitochondria the internal and external concentrations of inorganic phosphate, adenosine diphosphate, and adenosine triphosphate were determined by phosphorus-31 while the pH difference across the membrane was measured simultaneously

  1. Reconsideration of the sequence of rigor mortis through postmortem changes in adenosine nucleotides and lactic acid in different rat muscles.

    Science.gov (United States)

    Kobayashi, M; Takatori, T; Iwadate, K; Nakajima, M

    1996-10-25

    We examined the changes in adenosine triphosphate (ATP), lactic acid, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in five different rat muscles after death. Rigor mortis has been thought to occur simultaneously in dead muscles and hence to start in small muscles sooner than in large muscles. In this study we found that the rate of decrease in ATP was significantly different in each muscle. The greatest drop in ATP was observed in the masseter muscle. These findings contradict the conventional theory of rigor mortis. Similarly, the rates of change in ADP and lactic acid, which are thought to be related to the consumption or production of ATP, were different in each muscle. However, the rate of change of AMP was the same in each muscle.

  2. Determination of adenosine phosphates in rat gastrocnemius at various postmortem intervals using high performance liquid chromatography.

    Science.gov (United States)

    Huang, Hong; Yan, Youyi; Zuo, Zhong; Yang, Lin; Li, Bin; Song, Yu; Liao, Linchuan

    2010-09-01

    Although the change in adenosine phosphate levels in muscles may contribute to the development of rigor mortis, the relationship between their levels and the onset and development of rigor mortis has not been well elucidated. In the current study, levels of the adenosine phosphates including adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in gastrocnemius at various postmortem intervals of 180 rats from different death modes were detected by high performance liquid chromatography. The results showed that the levels of ATP and ADP significantly decreased along with the postmortem period of rats from different death mode whereas the AMP level remained the same. In addition, it was found that changes in the ATP levels in muscles after death correlated well with the development of rigor mortis. Therefore, the ATP level could serve as a reference parameter for the deduction of rigor mortis in forensic science.

  3. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  4. Biochemical reasoning for radiation protection and screening methods for radiation sensitivity and potential carcinogenicity

    International Nuclear Information System (INIS)

    Riklis, Emanuel; Emerit, Ingrid

    1994-01-01

    Cells of different genetic characteristics respond differently to agents that modify radiation effects. When the modification is a result of chemical repair, reduction of the amount of damage by radical scavenging, production of hypoxia, or any other such mechanism, then the modification of the response will be the same for all types of cells, but not the same when biological or biochemical parameters are involved, because the differences between the cells affect the final outcome, and the genetic traits obviously become affected by chemical modifying agents. Some of these agents directly affect the repair of deoxyribonucleic acid (DNA) by mechanisms not yet understood. Another agent nicotinamide (NA), is directly linked to a repair pathway. Thus, a system that uses NA as a precursor of nicotinamide adenine dinucleotide (NAD) + , and uses NAD + to produce the polymer polyadenosine diphosphate ribose (PADPR) appears to be an interesting and important factor in the biochemical events that may be linked to improved radioprotection. (author). 36 refs., 5 figs

  5. Two residues determine the product profile of the class II diterpene synthases TPS14 and TPS21 of Tripterygium wilfordii

    DEFF Research Database (Denmark)

    Hansen, Nikolaj Lervad; Nissen, Jakob N.; Hamberger, Björn Robert

    2017-01-01

    residue gave mixed product profiles. Two mutants, TwTPS14:Y265H and TwTPS21:A325V, also produced ent-copalyl diphosphate, highlighting the evolutionary potential of enzymes of this family to drive rapid diversification of plant diterpene biosynthesis through neo-functionalization. Our study contributes......The medicinal plant Tripterygium wilfordii (Celastraceae) contains a pair of class II diterpene synthases (diTPS) of specialized labdane-type metabolism that, despite remarkably close homology, form strikingly different products. TwTPS21 catalyzes bicyclization of the linear C20 precursor......-directed mutagenesis, we generated a panel of six variants, where one, or both positions were exchanged between the enzymes. In coupled heterologous assays with a corresponding class I diTPS, TwTPS2, complete product interchange was observed in variants with both reciprocal mutations, while substitutions of either...

  6. Antiparasitic Activity of Sulfur- and Fluorine-Containing Bisphosphonates against Trypanosomatids and Apicomplexan Parasites

    Directory of Open Access Journals (Sweden)

    Tamila Galaka

    2017-01-01

    Full Text Available Based on crystallographic data of the complexes 2-alkyl(aminoethyl-1,1-bisphosphonates–Trypanosoma cruzi farnesyl diphosphate synthase, some linear 1,1-bisphosphonic acids and other closely related derivatives were designed, synthesized and biologically evaluated against T. cruzi, the responsible agent of Chagas disease and against Toxoplasma gondii, the etiologic agent of toxoplasmosis and also towards the target enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS and T gondii (TgFPPS, respectively. The isoprenoid-containing 1,1-bisphosphonates exhibited modest antiparasitic activity, whereas the linear α-fluoro-2-alkyl(aminoethyl-1,1-bisphosphonates were unexpectedly devoid of antiparasitic activity. In spite of not presenting efficient antiparasitic activity, these data turned out to be very important to establish a structural activity relationship.

  7. Pharmacokinetic drug interactions of morphine, codeine, and their derivatives: theory and clinical reality, Part II.

    Science.gov (United States)

    Armstrong, Scott C; Cozza, Kelly L

    2003-01-01

    Pharmacokinetic drug-drug interactions with codeine, dihydrocodeine, hydrocodone, oxycodone, and buprenorphine are reviewed in this column. These compounds have a very similar chemical structure to morphine. Unlike morphine, which is metabolized chiefly through conjugation reactions with uridine diphosphate glucuronosyl transferase (UGT) enzymes, these five drugs are metabolized both through oxidative reactions by the cytochrome P450 (CYP450) enzyme and conjugation by UGT enzymes. There is controversy as to whether codeine, dihydrocodeine, and hydrocodone are actually prodrugs requiring activation by the CYP450 2D6 enzyme or UGT enzymes. Oxycodone and buprenorphine, however, are clearly not prodrugs and are metabolized by the CYP450 2D6 and 3A4 enzymes, respectively. Knowledge of this metabolism assists in the understanding for the potential of drug-drug interactions with these drugs. This understanding is important so that clinicians can choose the proper dosages for analgesia and anticipate potential drug-drug interactions.

  8. Pharmacokinetic drug interactions of morphine, codeine, and their derivatives: theory and clinical reality, part I.

    Science.gov (United States)

    Armstrong, Scott C; Cozza, Kelly L

    2003-01-01

    Pharmacokinetic drug-drug interactions with morphine, hydromorphone, and oxymorphone are reviewed in this column. Morphine is a naturally occurring opiate that is metabolized chiefly through glucuronidation by uridine diphosphate glucuronosyl transferase (UGT) enzymes in the liver. These enzymes produce an active analgesic metabolite and a potentially toxic metabolite. In vivo drug-drug interaction studies with morphine are few, but they do suggest that inhibition or induction of UGT enzymes could alter morphine and its metabolite levels. These interactions could change analgesic efficacy. Hydromorphone and oxymorphone, close synthetic derivatives of morphine, are also metabolized primarily by UGT enzymes. Hydromorphone may have a toxic metabolite similar to morphine. In vivo drug-drug interaction studies with hydromorphone and oxymorphone have not been done, so it is difficult to make conclusions with these drugs.

  9. Enzymatic Synthesis of Acylphloroglucinol 3-C-Glucosides from 2-O-Glucosides using a C-Glycosyltransferase from Mangifera indica.

    Science.gov (United States)

    Chen, Dawei; Sun, Lili; Chen, Ridao; Xie, Kebo; Yang, Lin; Dai, Jungui

    2016-04-18

    A green and cost-effective process for the convenient synthesis of acylphloroglucinol 3-C-glucosides from 2-O-glucosides was exploited using a novel C-glycosyltransferase (MiCGTb) from Mangifera indica. Compared with previously characterized CGTs, MiCGTb exhibited unique de-O-glucosylation promiscuity and high regioselectivity toward structurally diverse 2-O-glucosides of acylphloroglucinol and achieved high yields of C-glucosides even with a catalytic amount of uridine 5'-diphosphate (UDP). These findings demonstrate for the first time the significant potential of a single-enzyme approach to the synthesis of bioactive C-glucosides from both natural and unnatural acylphloroglucinol 2-O-glucosides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Study design and rationale of a comparison of prasugrel and clopidogrel in medically managed patients with unstable angina/non-ST-segment elevation myocardial infarction: the TaRgeted platelet Inhibition to cLarify the Optimal strateGy to medicallY manage Acute Coronary Syndromes (TRILOGY ACS

    DEFF Research Database (Denmark)

    Chin, Chee Tang; Roe, Matthew T; Fox, Keith A A

    2010-01-01

    Practice guidelines recommend dual antiplatelet therapy with aspirin and clopidogrel for patients with non-ST-segment elevation acute coronary syndromes (NSTE ACS) regardless of in-hospital management strategy. Prasugrel-a thienopyridine adenosine diphosphate receptor antagonist that provides...... higher and less variable levels of platelet inhibition than clopidogrel-has demonstrated benefit when used to treat ACS patients undergoing percutaneous coronary intervention. However, the optimal approach to antiplatelet therapy for high-risk, medically managed NSTE ACS patients remains uncertain...... revascularization procedures for their index event. Patients will be randomly allocated to prasugrel + aspirin versus clopidogrel + aspirin for a median duration of 18 months. A reduction in the maintenance dose of prasugrel for elderly patients (age >or=75 years) and those with body weight or=75 years). TRILOGY...

  11. Disturbance of inorganic phosphate metabolism in diabetes mellitus: clinical manifestations of phosphorus-depletion syndrome during recovery from diabetic ketoacidosis

    Directory of Open Access Journals (Sweden)

    Lervang H

    2010-09-01

    Full Text Available Jørn Ditzel, Hans-Henrik LervangDepartment of Endocrinology, and Center for Prevention of Struma and Metabolic Diseases, Aalborg University Hospital, Aarhus University, DenmarkAbstract: The acute effects of intracellular phosphate depletion and hypophosphatemia on organs and tissues in and during recovery from diabetic ketoacidosis (DKA have been reviewed. When insufficient phosphate and/or oxygen are available for high energy phosphate synthesis, cell homeostasis cannot be maintained and cell integrity may be impaired. The clinical consequences are recognized as occasional cause of morbidity and mortality. Although phosphate repletion has not been routinely recommended in the treatment of DKA, physicians should be aware of these clinical conditions and phosphate repletion in such situations should be considered.Keywords: high energy phosphates, hypoxia, fructose 1,6-diphosphate

  12. Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

    Science.gov (United States)

    Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2017-02-22

    Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

  13. Nootkatone.

    Science.gov (United States)

    Leonhardt, Robin-Hagen; Berger, Ralf G

    2015-01-01

    The continuing interest in the sesquiterpene ketone (+)-nootkatone is stimulated by its strong grapefruit-like odor and numerous further bioactivities. Also numerous were the attempts to chemosynthesize or biotechnologically produce the compound. Cytochrome P450 enzymes from bacteria and fungi were intensively studied and expressed in Escherichia coli and in more food compatible hosts, such as Saccharomyces cerevisiae. The lipoxygenase-catalyzed generation was demonstrated using an enzyme from several Pleurotus species. Laccases required artificial mediators for an efficient catalysis. More recently, plant valencene synthases were expressed in microbial hosts. Combined with an endogenous farnesyl diphosphate delivery pathway and a valencene oxidase, this approach opened access to high yields of nootkatone possessing the appreciated attribute of "natural" according to present food legislation. Little biochemical engineering was carried out on the novel recombinant strains, leaving many options for future improved bioprocesses.

  14. Biosensor reveals multiple sources for mitochondrial NAD⁺.

    Science.gov (United States)

    Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

    2016-06-17

    Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations. Copyright © 2016, American Association for the Advancement of Science.

  15. A conserved NAD+ binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  16. Novel pathway of NAD metabolism in Aspergillus niger

    International Nuclear Information System (INIS)

    Kuwahara, Masaaki

    1977-01-01

    New steps of NAD metabolism were shown in Aspergillus niger. Radioactive nicotinic acid and nicotinamide were incorporated into nicotinamide ribose diphosphate ribose (NAmRDPR), which had been isolated from the culture filtrate. The enzyme preparation of the mold degraded NAmRDPR to form nicotinamide mononucleotide and nicotinic acid under the neutral and alkaline conditions. In the acid extracts of the mycelia grown on the radioactive precursors, high level of radioactivity was detected on NAD. The experimental results showed that the Preiss-Handler pathway and the NAD cycling system function in the NAD biosynthesis in A. niger. A part of the radioactive precursors was also incorporated into nicotinic acid ribonucleoside, which was thought to be formed from nicotinic acid mononucleotide. (auth.)

  17. Computer-assisted study on the reaction between pyruvate and ylide in the pathway leading to lactyl-ThDP.

    Science.gov (United States)

    Alvarado, Omar; Jaña, Gonzalo; Delgado, Eduardo J

    2012-08-01

    In this study the formation of the lactyl-thiamin diphosphate intermediate (L-ThDP) is addressed using density functional theory calculations at X3LYP/6-31++G(d,p) level of theory. The study includes potential energy surface scans, transition state search, and intrinsic reaction coordinate calculations. Reactivity is analyzed in terms of Fukui functions. The results allow to conclude that the reaction leading to the formation of L-ThDP occurs via a concerted mechanism, and during the nucleophilic attack on the pyruvate molecule, the ylide is in its AP form. The calculated activation barrier for the reaction is 19.2 kcal/mol, in agreement with the experimental reported value.

  18. Overexpression, crystallization and preliminary X-ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve

    International Nuclear Information System (INIS)

    Suzuki, Ryuichiro; Kim, Byung-Jun; Shibata, Tsuyoshi; Iwamoto, Yuki; Katayama, Takane; Ashida, Hisashi; Wakagi, Takayoshi; Shoun, Hirofumi; Fushinobu, Shinya; Yamamoto, Kenji

    2010-01-01

    Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from B. breve was overexpressed and crystallized. The crystals belonged to the tetragonal space group I422 and diffracted to beyond 1.7 Å resolution. The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl 2 , 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution

  19. ADP-ribosyl-N₃: A Versatile Precursor for Divergent Syntheses of ADP-ribosylated Compounds.

    Science.gov (United States)

    Li, Lingjun; Li, Qianqian; Ding, Shengqiang; Xin, Pengyang; Zhang, Yuqin; Huang, Shenlong; Zhang, Guisheng

    2017-08-14

    Adenosine diphosphate-ribose (ADP-ribose) and its derivatives play important roles in a series of complex physiological procedures. The design and synthesis of artificial ADP-ribosylated compounds is an efficient way to develop valuable chemical biology tools and discover new drug candidates. However, the synthesis of ADP-ribosylated compounds is currently difficult due to structural complexity, easily broken pyrophosphate bond and high hydrophilicity. In this paper, ADP-ribosyl-N₃ was designed and synthesized for the first time. With ADP-ribosyl-N₃ as the key precursor, a divergent post-modification strategy was developed to prepare structurally diverse ADP-ribosylated compounds including novel nucleotides and peptides bearing ADP-ribosyl moieties.

  20. In situ visualization of Li/Ag2VP2O8 batteries revealing rate-dependent discharge mechanism

    Science.gov (United States)

    Kirshenbaum, Kevin; Bock, David C.; Lee, Chia-Ying; Zhong, Zhong; Takeuchi, Kenneth J.; Marschilok, Amy C.; Takeuchi, Esther S.

    2015-01-01

    The functional capacity of a battery is observed to decrease, often quite dramatically, as discharge rate demands increase. These capacity losses have been attributed to limited ion access and low electrical conductivity, resulting in incomplete electrode use. A strategy to improve electronic conductivity is the design of bimetallic materials that generate a silver matrix in situ during cathode reduction. Ex situ x-ray absorption spectroscopy coupled with in situ energy-dispersive x-ray diffraction measurements on intact lithium/silver vanadium diphosphate (Li/Ag2VP2O8) electrochemical cells demonstrate that the metal center preferentially reduced and its location in the bimetallic cathode are rate-dependent, affecting cell impedance. This work illustrates that spatial imaging as a function of discharge rate can provide needed insights toward improving realizable capacity of bimetallic cathode systems.

  1. Autoradiographic imaging of phosphoinositide turnover in the brain

    International Nuclear Information System (INIS)

    Hwang, P.M.; Bredt, D.S.; Snyder, S.H.

    1990-01-01

    With [ 3 H]cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product [ 3 H]cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways

  2. Studies on screening, isolation and purification of a fibrinolytic protease from an isolate (VK12) of Ganoderma lucidum and evaluation of its antithrombotic activity.

    Science.gov (United States)

    Kumaran, Sekar; Palani, Perumal; Nishanthi, Ramasami; Kaviyarasan, Venkatesan

    2011-01-01

    Antithrombotic activity of a protease purified from a medicinal mushroom, Ganoderma lucidum, has been evaluated platelet aggregation in vitro and pulmonary thrombosis in vivo. The purified protease exhibited concentration dependent inhibitory effects on platelet aggregation induced by ADP (adenosine diphosphate), with an IC(50) value of 2.4 mg/mL. The purified protease protected mice against thrombotic death or paralysis induced by collagen and epinephrine in a dose dependent manner when administered orally. It produced a significant inhibition of thrombotic death or paralysis at 60 µg/kg body weight, while aspirin produced a significant inhibition of thrombosis at 10-20 mg/kg body weight. The purified protease also has showed fibrinolytic activity and alters coagulation parameters such as activated partial thromboplastin time (APTT), and thrombin time (TT) in rat platelet. These results suggested that the antithrombotic activity of Ganoderma lucidum protease might be due to antiplatelet activity rather than anticoagulation activity.

  3. The use of bone scintigraphy to establish cancer extension records: application to prostate and breast cancers

    International Nuclear Information System (INIS)

    Methlin, G.; Grob, J.C.; Moyses, B.; Deschler, J.M.; Ott, G.; Berthelen, B.

    1975-01-01

    The recent development of the technique described, especially in the systematic search for metastases was due to the introduction of stannous diphosphate complexed with sup(99m)Tc well suited to bone scintigraphy. The documents are obtained in two stages. First a whole-body scintigraph is taken by means of an omniview device fitted to a Picker dynacamera, and this scanning phase is followed immediately by static views centred mainly on abnormal or doubtful regions. The results are given for 136 breast cancers and 61 prostate cancers, two cancers particularly liable to affect the bone, and show that scintigraphy offers positive advantages over radiological investigations. These advantages are offset to some extent by the non-specific nature of the hyperfixing regions since different diseases involving changes in the bone structure can give similar images, but for breast and prostate cancers bone scintigraphy presents distinct possibilities in the search for metastases of the skeleton [fr

  4. Biosynthesis of sesquiterpene lactones in pyrethrum (Tanacetum cinerariifolium)

    DEFF Research Database (Denmark)

    Ramirez, Aldana M; Saillard, Nils; Yang, Ting

    2013-01-01

    The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent...... these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation...... of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (α or β) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum...

  5. 7-epizingiberene, a novel bisabolane sesquiterpene from wild tomato leaves

    International Nuclear Information System (INIS)

    Breeden, D.C.; Coates, R.M.

    1994-01-01

    A C 15 hydrocarbon isolated from the leaves of 2 wild tomato species, Lycopersicon hirsutum f glabratum PI 199381 and Lycopersicon hirsutum PI 365906, has been identified as 7 - epizingiberene (2), a diastereomer of zingiberene (1) that occurs in essential oil of ginger. The structure assignment for 2 is based upon its 1 H NMR, 13 C NMR, IR, UV, and mass spectral characteristics. All spectral data for zingiberene and epizingiberene are identical except for 9 of 15 13C NMR resonances, which establish the diastereomeric relationship of these sesquiterpenes. The 4S, 7R stereochemistry of epizingiberene was proven by dehydrogenation to (7R)- ar - curcumene (4). The opposite 7R and 7S stereochemistry of the zingiberenes implicates the probable occurrence of opposite sidechain rotations of a common (S)-bisabolyl carbocation intermediate (1OA) to allow stereoelectronically favorable hydride shifts in their respective biosyntheses from (E, E)-farnesyl diphosphate. (author)

  6. Allosteric regulation of the GTP activated and CTP inhibited uracil phosphoribosyltransferase from the thermophilic archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Arent, Susan; Larsen, Sine

    2005-01-01

    The upp gene, encoding uracil phosphoribosyltransferase (UPRTase) from the thermoacidophilic archaeon Sulfolobus solfataricus, was cloned and expressed in Escherichia coli. The enzyme was purified to homogeneity. It behaved as a tetramer in solution and showed optimal activity at pH 5.5 when...... assayed at 60 °C. Enzyme activity was strongly stimulated by GTP and inhibited by CTP. GTP caused an approximately 20-fold increase in the turnover number kcat and raised the Km values for 5-phosphoribosyl-1-diphosphate (PRPP) and uracil by two- and >10-fold, respectively. The inhibition by CTP...... was complex as it depended on the presence of the reaction product UMP. Neither CTP nor UMP were strong inhibitors of the enzyme, but when present in combination their inhibition was extremely powerful. Ligand binding analyses showed that GTP and PRPP bind cooperatively to the enzyme and that the inhibitors...

  7. Biotechnological storage and utilization of entrapped solar energy.

    Science.gov (United States)

    Bhattacharya, Sumana; Schiavone, Marc; Nayak, Amiya; Bhattacharya, Sanjoy K

    2005-03-01

    Our laboratory has recently developed a device employing immobilized F0F1 adenosine triphosphatase (ATPase) that allows synthesis of adenosine triphosphate (ATP) from adenosine 5'-diphosphate and inorganic phosphate using solar energy. We present estimates of total solar energy received by Earth's land area and demonstrate that its efficient capture may allow conversion of solar energy and storage into bonds of biochemicals using devices harboring either immobilized ATPase or NADH dehydrogenase. Capture and storage of solar energy into biochemicals may also enable fixation of CO2 emanating from polluting units. The cofactors ATP and NADH synthesized using solar energy could be used for regeneration of acceptor D-ribulose-1,5-bisphosphate from 3-phosphoglycerate formed during CO2 fixation.

  8. The effects of long-term oral benfotiamine supplementation on peripheral nerve function and inflammatory markers in patients with type 1 diabetes: a 24-month, double-blind, randomized, placebo-controlled trial.

    Science.gov (United States)

    Fraser, David A; Diep, Lien M; Hovden, Inger Anette; Nilsen, Kristian B; Sveen, Kari Anne; Seljeflot, Ingebjørg; Hanssen, Kristian F

    2012-05-01

    To study the effects of long-term oral benfotiamine supplementation on peripheral nerve function and soluble inflammatory markers in patients with type 1 diabetes. The study randomly assigned 67 patients with type 1 diabetes to receive 24-month benfotiamine (300 mg/day) or placebo supplementation. Peripheral nerve function and levels of soluble inflammatory variables were assessed at baseline and at 24 months. Fifty-nine patients completed the study. Marked increases in whole-blood concentrations of thiamine and thiamine diphosphate were found in the benfotiamine group (both P benfotiamine (300 mg/day) supplementation over 24 months has no significant effects upon peripheral nerve function or soluble markers of inflammation in patients with type 1 diabetes.

  9. The Effects of Long-Term Oral Benfotiamine Supplementation on Peripheral Nerve Function and Inflammatory Markers in Patients With Type 1 Diabetes

    Science.gov (United States)

    Fraser, David A.; Diep, Lien M.; Hovden, Inger Anette; Nilsen, Kristian B.; Sveen, Kari Anne; Seljeflot, Ingebjørg; Hanssen, Kristian F.

    2012-01-01

    OBJECTIVE To study the effects of long-term oral benfotiamine supplementation on peripheral nerve function and soluble inflammatory markers in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS The study randomly assigned 67 patients with type 1 diabetes to receive 24-month benfotiamine (300 mg/day) or placebo supplementation. Peripheral nerve function and levels of soluble inflammatory variables were assessed at baseline and at 24 months. RESULTS Fifty-nine patients completed the study. Marked increases in whole-blood concentrations of thiamine and thiamine diphosphate were found in the benfotiamine group (both P benfotiamine (300 mg/day) supplementation over 24 months has no significant effects upon peripheral nerve function or soluble markers of inflammation in patients with type 1 diabetes. PMID:22446172

  10. Bis(2,6-dimethylanilinium diaquabis(dihydrogen diphosphato-κ2O,O′cobaltate(II

    Directory of Open Access Journals (Sweden)

    Ahlem Ben Saad

    2014-03-01

    Full Text Available In the title compound, (C8H12N2[Co(H2P2O72(H2O2], the Co2+ ion lies on a crystallographic inversion centre and adopts a slightly distorted octahedral CoO6 coordination geometry arising from two chelating diphosphate [H2P2O7]2− ligands and two trans water molecules. In the crystal, the components are linked by O—H...O, N—H...O and C—H...O hydrogen bonds and weak aromatic π–π stacking [shortest centroid–centroid separation = 3.778 (2 Å] interactions. (001 layers of alternating organic cations and complex inorganic anions are apparent.

  11. Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

    Science.gov (United States)

    Abu-Taha, Issam H; Heijman, Jordi; Feng, Yuxi; Vettel, Christiane; Dobrev, Dobromir; Wieland, Thomas

    2018-02-01

    Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

  12. The Path of Carbon in Photosynthesis. XXI. The Cyclic Regenerationof Carbon Dioxide Acceptor

    Energy Technology Data Exchange (ETDEWEB)

    Bassham, J.A.; Benson, A.A.; Kay, Lorel D.; Harris, Anne Z.; Wilson, A.T.; Calvin, M.

    1953-10-01

    Photosynthesizing plants have been exposed to C{sup 14}O{sub 2} for short periods of time (0.4 to 15 sec.) and the products of carbon dioxide reduction analyzed by paper chromatography and radio autography. Methods have been developed for the degradation of ribulose and sedoheptulose. These sugars, obtained as their phosphate esters from the above C{sup 14}O{sub 2} exposures and from other experiments, have been degraded and their distribution of radiocarbon determined. The distribution of radiocarbon in these sugars, and other data, indicate that sedoheptulose phosphate and ribulose diphosphates are formed during photosynthesis from triose and hexose phosphates, the latter being synthesized, in turn, by the reduction of 3-phosphoglyceric acid.

  13. Effects of drugs on platelet function.

    Science.gov (United States)

    Morse, E E

    1977-01-01

    Numerous drugs and chemicals affect the function of human blood platelets. The mechanism of action of some medications is partly understood. Aspirin is the most frequently involved drug. It appears to interfere with the platelet release reaction by acetylation of a platelet membrane protein which may be involved in the synthesis of prostaglandins. Other anti-inflammatory drugs, including indomethacin, phenylbutazone, ibuprophen (Motrin) and clonixin, also interfere with the release reaction but have a shorter acting course than aspirin. Some drugs stimulate adenylcyclase (gliclazide) or block phosphodiesterase, (dipyridamole, caffeine) both of which actions lead to an increase in adenosine cyclic 3':5' monophosphate (cAMP) and decrease aggregation by adenosine diphosphate (ADP). These interactions should be known to clinical scientists since patients using these medicaments may manifest abnormal platelet function tests in the laboratory and mild hemorrhagic syndromes in the clinic.

  14. Programming adaptive control to evolve increased metabolite production.

    Science.gov (United States)

    Chou, Howard H; Keasling, Jay D

    2013-01-01

    The complexity inherent in biological systems challenges efforts to rationally engineer novel phenotypes, especially those not amenable to high-throughput screens and selections. In nature, increased mutation rates generate diversity in a population that can lead to the evolution of new phenotypes. Here we construct an adaptive control system that increases the mutation rate in order to generate diversity in the population, and decreases the mutation rate as the concentration of a target metabolite increases. This system is called feedback-regulated evolution of phenotype (FREP), and is implemented with a sensor to gauge the concentration of a metabolite and an actuator to alter the mutation rate. To evolve certain novel traits that have no known natural sensors, we develop a framework to assemble synthetic transcription factors using metabolic enzymes and construct four different sensors that recognize isopentenyl diphosphate in bacteria and yeast. We verify FREP by evolving increased tyrosine and isoprenoid production.

  15. Method for enzyme synthesis of radioactive thymine 5'-deoxyribonucleotides

    International Nuclear Information System (INIS)

    Nejedly, Z.; Ekl, J.; Hybs, K.; Kolina, J.; Filip, J.; Votruba, I.; Skoda, J.

    1978-01-01

    The enzyme synthesis is described for thymidine-5'-monophosphate, thymidine-5'-diphosphate and thymidine-5'-triphosphate specifically or nonspecifically labelled with 14 C or 3 H. The anabolic transformation of radioactive thymine to radioactive thymine 5'-deoxyribonucleotides is catalyzed by the action of enzyme preparations separated from Escherichia coli bacteria. It is achieved by the action of nonpurified cell-free extracts on special auxotrophic mutants of the thymine-dependent Escherichia coli SPT - strain in the presence of deoxyriboso-1-phosphate and adenosine-5'-triphosphate. The radioactive thymidine-5'-monophosphate may further be phosphorylated. In reaction mixtures, radioactive thymine, deoxyriboso-1-phosphate and adenosine-5'-triphosphate are used in molar ratios of 1:1:2 to 1:10:100, the optimum molar ratio being 1:5:10. (B.S.)

  16. DNA damage and repair activity after broccoli intake in young healthy smokers

    DEFF Research Database (Denmark)

    Riso, Patrizia; Martini, Daniela; Møller, Peter

    2010-01-01

    compounds, including smokers. The aim of the study was to evaluate the effect of broccoli intake on biomarkers of DNA damage and repair. Twenty-seven young healthy smokers consumed a portion of steamed broccoli (250 g/day) or a control diet for 10 days each within a crossover design with a washout period...... mRNA expression levels of repair and defence enzymes: 8-oxoguanine DNA glycosylase (OGG1), nucleoside diphosphate linked moiety X-type motif 1 (NUDT1) and heme oxygenase 1 (HO-1). After broccoli consumption, the level of oxidised DNA lesions decreased by 41% (95% confidence interval: 10%, 72......%) and the resistance to H(2)O(2)-induced DNA strand breaks increased by 23% (95% CI: 13%, 34%). Following broccoli intake, a higher protection was observed in subjects with glutathione S-transferase (GST) M1-null genotype. The expression level and activity of repair enzymes was unaltered. In conclusion, broccoli...

  17. A Simple and Rapid Determination of ATP, ADP and AMP Concentrations in Pericarp Tissue of Litchi Fruit by High Performance Liquid Chromatography

    Directory of Open Access Journals (Sweden)

    Weibo Jiang

    2006-01-01

    Full Text Available A simple and rapid method using high performance liquid chromatography (HPLC was developed to determine levels of adenosine triphosphate (ATP, adenosine diphosphate (ADP and adenosine monophosphate (AMP in litchi fruit pericarp tissue. This HPLC method used acetonitrile gradient elution and shortened the time required for determinations of adenosine phosphates. This analysis exhibited good repeatability (coefficients of variation 1.28–1.80 % and recovery rate (94.7–97.1 %. The correlation coefficients of ATP, ADP and AMP with their peak areas at a range of 0–80 ng were 0.9946, 0.9994 and 0.9974, respectively. This method was applied to determine levels of adenosine phosphates in pericarp tissue of litchi fruit at harvest. There were 27.4 μg/g of ATP, 35.4 μg/g of ADP and 7.9 μg/g of AMP on a fresh mass basis.

  18. Novel vitamin E forms in leaves of Kalanchoe daigremontiana and Phaseolus coccineus.

    Science.gov (United States)

    Kruk, Jerzy; Pisarski, Adam; Szymańska, Renata

    2011-11-15

    In the present study, we isolated novel tocochromanols from green leaves of Kalanchoe daigremontiana and primary leaves of etiolated seedlings of Phaseolus coccineus that were identified as β-, γ-, and δ-tocomonoenols with unsaturation at the terminal isoprene unit of the side chain. The content of γ-tocomonoenol in leaves of etiolated bean increased gradually with the age of seedlings, reaching 50% of the γ-tocopherol level in 40-day-old plants. The content of this compound in leaves was increased by short illumination of etiolated plants and by addition of homogentisic acid, a biosynthetic precursor of tocopherols. These data indicated that γ-tocomonoenol is synthesized de novo from homogentisic acid and tetrahydro-geranylgeraniol diphosphate, a phytol precursor. Based on these results, a biosynthetic pathway of tocomonoenols is proposed. Copyright © 2011 Elsevier GmbH. All rights reserved.

  19. Noncanonical Reactions of Flavoenzymes

    Directory of Open Access Journals (Sweden)

    Pablo Sobrado

    2012-11-01

    Full Text Available Enzymes containing flavin cofactors are predominantly involved in redox reactions in numerous cellular processes where the protein environment modulates the chemical reactivity of the flavin to either transfer one or two electrons. Some flavoenzymes catalyze reactions with no net redox change. In these reactions, the protein environment modulates the reactivity of the flavin to perform novel chemistries. Recent mechanistic and structural data supporting novel flavin functionalities in reactions catalyzed by chorismate synthase, type II isopentenyl diphosphate isomerase, UDP-galactopyranose mutase, and alkyl-dihydroxyacetonephosphate synthase are presented in this review. In these enzymes, the flavin plays either a direct role in acid/base reactions or as a nucleophile or electrophile. In addition, the flavin cofactor is proposed to function as a “molecular scaffold” in the formation of UDP-galactofuranose and alkyl-dihydroxyacetonephosphate by forming a covalent adduct with reaction intermediates.

  20. Comparison of the Chemical Properties of Forest Soil from the Silesian Beskid, Poland

    Directory of Open Access Journals (Sweden)

    Maria Zołotajkin

    2014-01-01

    Full Text Available There is spruce forests degradation observed in the Silesian Beskid. The aim of the work was the assessment of parameters diversifying organic layers of soils in two forest areas: degraded and healthy spruce forests of Silesian Beskid. 23 soil samples were collected from two fields—14 soil samples from a degraded forest and 9 soil samples from a forest, where pandemic dying of spruce is not observed. Implementation of hierarchical clustering to experimental data analysis allowed drawing a conclusion that the two forest areas vary significantly in terms of content of aluminium extracted with solutions of barium chloride (Alexch, sodium diphosphate (Alpyr, and pHKCl and in the amount of humus in soil.

  1. Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

    DEFF Research Database (Denmark)

    Barba Espin, Gregorio; Dedvisitsakul, Plaipol; Hägglund, Per

    2014-01-01

    respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping...... and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat...... shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions...

  2. NME2 reduces proliferation, migration and invasion of gastric cancer cells to limit metastasis.

    Directory of Open Access Journals (Sweden)

    Yan-fei Liu

    Full Text Available Gastric cancer is one of the most common malignancies and has a high rate of metastasis. We hypothesize that NME2 (Nucleoside Diphosphate Kinase 2, which has previously been considered as an anti-metastatic gene, plays a role in the invasiveness of gastric cancer cells. Using a tissue chip technology and immunohistochemistry, we demonstrated that NME2 expression was associated with levels of differentiation of gastric cancer cells and their metastasis into the lymph nodes. When the NME2 gene product was over-expressed by ;in vitro stable transfection, cells from BGC823 and MKN45 gastric cancer cell lines had reduced rates of proliferation, migration, and invasion through the collagen matrix, suggesting an inhibitory activity of NME2 in the propagation and invasion of gastric cancer. NME2 could, therefore, severe as a risk marker for gastric cancer invasiveness and a potential new target for gene therapy to enhance or induce NME2 expression.

  3. Effects of High-Pressure Treatment on the Muscle Proteome of Hake by Bottom-Up Proteomics.

    Science.gov (United States)

    Carrera, Mónica; Fidalgo, Liliana G; Saraiva, Jorge A; Aubourg, Santiago P

    2018-05-02

    A bottom-up proteomics approach was applied for the study of the effects of high-pressure (HP) treatment on the muscle proteome of fish. The performance of the approach was established for a previous HP treatment (150-450 MPa for 2 min) on frozen (up to 5 months at -10 °C) European hake ( Merluccius merluccius). Concerning possible protein biomarkers of quality changes, a significant degradation after applying a pressure ≥430 MPa could be observed for phosphoglycerate mutase-1, enolase, creatine kinase, fructose bisphosphate aldolase, triosephosphate isomerase, and nucleoside diphosphate kinase; contrary, electrophoretic bands assigned to tropomyosin, glyceraldehyde-3-phosphate dehydrogenase, and beta parvalbumin increased their intensity after applying a pressure ≥430 MPa. This repository of potential protein biomarkers may be very useful for further HP investigations related to fish quality.

  4. Hydrogen peroxide-induced pericarp browning of harvested longan fruit in association with energy metabolism.

    Science.gov (United States)

    Lin, Yifen; Lin, Yixiong; Lin, Hetong; Ritenour, Mark A; Shi, John; Zhang, Shen; Chen, Yihui; Wang, Hui

    2017-06-15

    Energy metabolism of "Fuyan" longan fruit treated with hydrogen peroxide (H 2 O 2 ), the most stable of the reactive oxygen, and its relationship to pericarp browning were investigated in this work. The results displayed that H 2 O 2 significantly decreased contents of adenosine triphosphate (ATP) and adenosine diphosphate (ADP). It also inhibited activities of H + -ATPase, Ca 2+ -ATPase and Mg 2+ -ATPase in membranes of plasma, vacuole and mitochondria during the early-storage and mid-storage (except for mitochondrial membrane Mg 2+ -ATPase). These results gave convincing evidence that the treatment of H 2 O 2 accelerating pericarp browning in harvested longans was due to a decrease of ATPase activity and available ATP content. This might break the ion homeostasis and the integrity of mitochondria, which might reduce energy charge and destroy the function and compartmentalization of cell membrane. These together aggravated browning incidence in pericarp of harvested longan fruit. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Localization of phosphatidylinositol signaling components in rat taste cells: Role in bitter taste transduction

    International Nuclear Information System (INIS)

    Hwang, P.M.; Verma, A.; Bredt, D.S.; Snyder, S.H.

    1990-01-01

    To assess the role of phosphatidylinositol turnover in taste transduction we have visualized, in rat tongue, ATP-dependent endoplasmic reticular accumulation of 45 Ca 2+ , inositol 1,4,5-trisphosphate receptor binding sites, and phosphatidylinositol turnover monitored by autoradiography of [ 3 H]cytidine diphosphate diacylglycerol formed from [ 3 H]cytidine. Accumulated 45 Ca 2+ , inositol 1,4,5-trisphosphate receptors, and phosphatidylinositol turnover are selectively localized to apical areas of the taste buds of circumvallate papillae, which are associated with bitter taste. Further evidence for a role of phosphatidylinositol turnover in bitter taste is our observation of a rapid, selective increase in mass levels of inositol 1,4,5-trisphosphate elicited by low concentrations of denatonium, a potently bitter tastant

  6. A newly validated high-performance liquid chromatography method with diode array ultraviolet detection for analysis of the antimalarial drug primaquine in the blood plasma.

    Science.gov (United States)

    Carmo, Ana Paula Barbosa do; Borborema, Manoella; Ribeiro, Stephan; De-Oliveira, Ana Cecilia Xavier; Paumgartten, Francisco Jose Roma; Moreira, Davyson de Lima

    2017-01-01

    Primaquine (PQ) diphosphate is an 8-aminoquinoline antimalarial drug with unique therapeutic properties. It is the only drug that prevents relapses of Plasmodium vivax or Plasmodium ovale infections. In this study, a fast, sensitive, cost-effective, and robust method for the extraction and high-performance liquid chromatography with diode array ultraviolet detection (HPLC-DAD-UV ) analysis of PQ in the blood plasma was developed and validated. After plasma protein precipitation, PQ was obtained by liquid-liquid extraction and analyzed by HPLC-DAD-UV with a modified-silica cyanopropyl column (250mm × 4.6mm i.d. × 5μm) as the stationary phase and a mixture of acetonitrile and 10mM ammonium acetate buffer (pH = 3.80) (45:55) as the mobile phase. The flow rate was 1.0mL·min-1, the oven temperature was 50OC, and absorbance was measured at 264nm. The method was validated for linearity, intra-day and inter-day precision, accuracy, recovery, and robustness. The detection (LOD) and quantification (LOQ) limits were 1.0 and 3.5ng·mL-1, respectively. The method was used to analyze the plasma of female DBA-2 mice treated with 20mg.kg-1 (oral) PQ diphosphate. By combining a simple, low-cost extraction procedure with a sensitive, precise, accurate, and robust method, it was possible to analyze PQ in small volumes of plasma. The new method presents lower LOD and LOQ limits and requires a shorter analysis time and smaller plasma volumes than those of previously reported HPLC methods with DAD-UV detection. The new validated method is suitable for kinetic studies of PQ in small rodents, including mouse models for the study of malaria.

  7. Characterization of two monoterpene synthases involved in floral scent formation in Hedychium coronarium.

    Science.gov (United States)

    Yue, Yuechong; Yu, Rangcai; Fan, Yanping

    2014-10-01

    Hedychium coronarium, a perennial herb belonging to the family Zingiberaceae, is cultivated as a garden plant or cut flower as well as for medicine and aromatic oil. Its flowers emit a fresh and inviting scent, which is mainly because of monoterpenes present in the profile of the floral volatiles. However, fragrance produced as a result of monoterpenes has not been well studied. In the present study, two novel terpene synthase (TPS) genes (HcTPS7 and HcTPS8) were isolated to study the biosynthesis of monoterpenes in H. coronarium. In vitro characterization showed that the recombinant HcTPS7 was capable of generating sabinene as its main product, in addition to nine sub-products from geranyl diphosphate (GPP). Recombinant HcTPS8 almost specifically catalyzed the formation of linalool from GPP, while it converted farnesyl diphosphate (FPP) to α-bergamotene, cis-α-bisabolene, β-farnesene and other ten sesquiterpenes. Subcellular localization experiments revealed that HcTPS7 and HcTPS8 were located in plastids. Real-time PCR analyses showed that HcTPS7 and HcTPS8 genes were highly expressed in petals and sepals, but were almost undetectable in vegetative organs. The changes of their expression levels in petals were positively correlated with the emission patterns of sabinene and linalool, respectively, during flower development. The results indicated that HcTPS7 and HcTPS8 were involved in the biosynthesis of sabinene and linalool in H. coronarium flowers. Results on these two TPSs first characterized from H. coronarium provide new insights into molecular mechanisms of terpene biosynthesis in this species and also lay the basis for biotechnological modification of floral scent profile in Hedychium.

  8. The Tomato Terpene Synthase Gene Family1[W][OA

    Science.gov (United States)

    Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T.H.; Spyropoulou, Eleni A.; Bleeker, Petra M.; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E.; Schilmiller, Anthony L.; Last, Robert L.; Schuurink, Robert C.; Pichersky, Eran

    2011-01-01

    Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

  9. Characterization of enzymatic properties of human ribonucleotide reductase holoenzyme reconstituted in vitro from hRRM1, hRRM2, and p53R2 subunits.

    Science.gov (United States)

    Qiu, Weihua; Zhou, Bingsen; Darwish, Dana; Shao, Jimin; Yen, Yun

    2006-02-10

    Ribonucleotide reductase (RR) is a highly regulated enzyme in the deoxyribonucleotide synthesis pathway. RR is responsible for the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which are essential for DNA synthesis and repair. Besides two subunits, hRRM1 and hRRM2, p53R2 is a newly identified member of RR family that is induced by ultraviolet light in a p53-dependent manner. To understand the molecular interaction of RR subunits, we employed a eukaryotic expression system to express and purify all three subunits. After in vitro reconstitution, the results of [(3)H]CDP reduction assay showed that both eukaryotic recombinant hRRM2 and p53R2 proteins could interact with hRRM1 to form functional RR holoenzyme. The reconstituted RR activity was time-dependent and the reaction rate reached the plateau phase after 40min incubation. No matter the concentration, RR holoenzyme reconstituted from p53R2 and hRRM1 could only achieve about 40-75% kinetic activity of that from hRRM2 and hRRM1. The synthetic C-terminal heptapeptide competition assays confirmed that hRRM2 and p53R2 share the same binding site on hRRM1, but the binding site on hRRM1 demonstrated higher affinity for hRRM2 than for p53R2. In allosteric regulation assay, the effect of activation or inhibition of hRRM1 with ATP or dATP suggested that these effectors could regulate RR activity independent of different RR small subunits. Taken together, the eukaryotic expression system RR holoenzyme will provide a very useful tool to understand the molecular mechanisms of RR activity and the interactions of its subunits.

  10. Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis.

    Science.gov (United States)

    Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E; Chen, Ming; Zhou, Yongming; Yu, Bin; Cahoon, Edgar B

    2015-08-01

    Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. © 2015 American Society of

  11. Energy status of pig donor organs after ischemia is independent of donor type.

    Science.gov (United States)

    Stadlbauer, Vanessa; Stiegler, Philipp; Taeubl, Philipp; Sereinigg, Michael; Puntschart, Andreas; Bradatsch, Andrea; Curcic, Pero; Seifert-Held, Thomas; Zmugg, Gerda; Stojakovic, Tatjana; Leopold, Barbara; Blattl, Daniela; Horki, Vera; Mayrhauser, Ursula; Wiederstein-Grasser, Iris; Leber, Bettina; Jürgens, Günther; Tscheliessnigg, Karlheinz; Hallström, Seth

    2013-04-01

    Literature is controversial whether organs from living donors have a better graft function than brain dead (BD) and non-heart-beating donor organs. Success of transplantation has been correlated with high-energy phosphate (HEP) contents of the graft. HEP contents in heart, liver, kidney, and pancreas from living, BD, and donation after cardiac death in a pig model (n=6 per donor type) were evaluated systematically. BD was induced under general anesthesia by inflating a balloon in the epidural space. Ten hours after confirmation, organs were retrieved. Cardiac arrest was induced by 9V direct current. After 10min of ventricular fibrillation without cardiac output, mechanical and medical reanimation was performed for 30min before organ retrieval. In living donors, organs were explanted immediately. Freeze-clamped biopsies were taken before perfusion with Celsior solution (heart) or University of Wisconsin solution (abdominal organs) in BD and living donors or with Histidine-Tryptophan-Ketoglutaric solution (all organs) in non-heart-beating donors, after perfusion, and after cold ischemia (4h for heart, 6h for liver and pancreas, and 12h for kidney). HEPs (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and phosphocreatine), xanthine, and hypoxanthine were measured by high-performance liquid chromatography. Energy charge and adenosine triphosphate-to-adenosine diphosphate ratio were calculated. After ischemia, organs from different donor types showed no difference in energy status. In all organs, a decrease of HEP and an increase in hypoxanthine contents were observed during perfusion and ischemia, irrespective of the donor type. Organs from BD or non-heart-beating donors do not differ from living donor organs in their energy status after average tolerable ischemia. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  13. The effect of prophylaxis with chloroquine and proguanil on delayed-type hypersensitivity and antibody production following vaccination with diphtheria, tetanus, polio, and pneumococcal vaccines.

    Science.gov (United States)

    Gyhrs, A; Pedersen, B K; Bygbjerg, I; Henrichsen, J; Heron, I; Petersen, I; Skinhoj, P

    1991-11-01

    In vitro studies have shown that anti-malarial drugs suppress immunity. In this study, the effects of chloroquine and proguanil (Paludrine) on the cellular and humoral immune system were measured by two in vivo methods: 1) cell-mediated immunity (delayed cutaneous hypersensitivity) i.e., skin tests with seven delayed-type common antigens (Multitest) and 2) humoral immunity by measurement of specific antibody response to vaccination. Sixty healthy young individuals were randomized into four groups and given 1) no treatment (controls), 2) chloroquine diphosphate (500 mg/week), 3) chloroquine diphosphate (1,000 mg/week), or 4) proguanil hydrochloride (200 mg/day) for six weeks. Skin testing was performed on days 0 and 28. Vaccinations with diphtheria, tetanus, polio, and pneumococcal polysaccharide antigen vaccines were performed on day 28, and the presence of specific antibodies was determined on days 0, 28, and 42. The skin tests induced a significant increase in skin reactive areas from day 0 to day 28 in all groups. Furthermore, the skin test induced an increase in the level of specific IgG for diphtheria and tetanus, but had no effect on antibodies to antigens not included in the skin test. The results showed that there were no significant differences among the four groups regarding skin test areas and increases in antibody titers following vaccination. Therefore, it is concluded that in healthy persons, six weeks intake of chloroquine, even in double doses, or proguanil in chemoprophylactic dosages, does not induce any detectable suppression of delayed-type hypersensitivity or vaccination responses to diphtheria, tetanus, polio, or pneumococcal polysaccharide antigens.

  14. Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH.

    Science.gov (United States)

    Zhou, Jia; Yang, Liyang; Wang, Chonglong; Choi, Eui-Sung; Kim, Seon-Won

    2017-04-20

    The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4mg/L of protoilludene after 48h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4mg/L. Overexpression of the Fpr and FldA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7mg/L. The results indicate that IDI, Fpr-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib

    Science.gov (United States)

    Lipsky, Andrew H.; Farooqui, Mohammed Z.H.; Tian, Xin; Martyr, Sabrina; Cullinane, Ann M.; Nghiem, Khanh; Sun, Clare; Valdez, Janet; Niemann, Carsten U.; Herman, Sarah E. M.; Saba, Nakhle; Soto, Susan; Marti, Gerald; Uzel, Gulbu; Holland, Steve M.; Lozier, Jay N.; Wiestner, Adrian

    2015-01-01

    Ibrutinib is associated with bleeding-related adverse events of grade ≤2 in severity, and infrequently with grade ≥3 events. To investigate the mechanisms of bleeding and identify patients at risk, we prospectively assessed platelet function and coagulation factors in our investigator-initiated trial of single-agent ibrutinib for chronic lymphocytic leukemia. At a median follow-up of 24 months we recorded grade ≤2 bleeding-related adverse events in 55% of 85 patients. No grade ≥3 events occurred. Median time to event was 49 days. The cumulative incidence of an event plateaued by 6 months, suggesting that the risk of bleeding decreases with continued therapy. At baseline, von Willebrand factor and factor VIII levels were often high and normalized on treatment. Platelet function measured via the platelet function analyzer (PFA-100™) was impaired in 22 patients at baseline and in an additional 19 patients on ibrutinib (often transiently). Collagen and adenosine diphosphate induced platelet aggregation was tested using whole blood aggregometry. Compared to normal controls, response to both agonists was decreased in all patients with chronic lymphocytic leukemia, whether on ibrutinib or not. Compared to untreated chronic lymphocytic leukemia patients, response to collagen showed a mild further decrement on ibrutinib, while response to adenosine diphosphate improved. All parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time (HR 2.74, P=0.012), lower levels of von Willebrand factor activity (HR 2.73, P=0.009) and factor VIII (HR 3.73, P=0.0004). In conclusion, both disease and treatment-related factors influence the risk of bleeding. Patients at greater risk for bleeding of grade ≤2 can be identified by clinical laboratory tests and counseled to avoid aspirin, non-steroidal anti-inflammatory drugs and fish oils. ClinicalTrials.gov identifier NCT01500733 PMID

  16. Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production1

    Science.gov (United States)

    Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi

    2015-01-01

    Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites. PMID:26438788

  17. Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production.

    Science.gov (United States)

    Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi; Wong, Limsoon; Chua, Nam-Hai; Jang, In-Cheol

    2015-12-01

    Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites. © 2015 American Society of Plant Biologists. All Rights Reserved.

  18. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  19. Chlorophyll Synthase under Epigenetic Surveillance Is Critical for Vitamin E Synthesis, and Altered Expression Affects Tocopherol Levels in Arabidopsis1[OPEN

    Science.gov (United States)

    Zhang, Chunyu; Zhang, Wei; Ren, Guodong; Li, Delin; Cahoon, Rebecca E.; Chen, Ming; Zhou, Yongming; Yu, Bin

    2015-01-01

    Chlorophyll synthase catalyzes the final step in chlorophyll biosynthesis: the esterification of chlorophyllide with either geranylgeranyl diphosphate or phytyl diphosphate (PDP). Recent studies have pointed to the involvement of chlorophyll-linked reduction of geranylgeranyl by geranylgeranyl reductase as a major pathway for the synthesis of the PDP precursor of tocopherols. This indirect pathway of PDP synthesis suggests a key role of chlorophyll synthase in tocopherol production to generate the geranylgeranyl-chlorophyll substrate for geranylgeranyl reductase. In this study, contributions of chlorophyll synthase to tocopherol formation in Arabidopsis (Arabidopsis thaliana) were explored by disrupting and altering expression of the corresponding gene CHLOROPHYLL SYNTHASE (CHLSYN; At3g51820). Leaves from the homozygous chlysyn1-1 null mutant were nearly devoid of tocopherols, whereas seeds contained only approximately 25% of wild-type tocopherol levels. Leaves of RNA interference lines with partial suppression of CHLSYN displayed marked reductions in chlorophyll but up to a 2-fold increase in tocopherol concentrations. Cauliflower mosaic virus35S-mediated overexpression of CHLSYN unexpectedly caused a cosuppression phenotype at high frequencies accompanied by strongly reduced chlorophyll content and increased tocopherol levels. This phenotype and the associated detection of CHLSYN-derived small interfering RNAs were reversed with CHLSYN overexpression in rna-directed rna polymerase6 (rdr6), which is defective in RNA-dependent RNA polymerase6, a key enzyme in sense transgene-induced small interfering RNA production. CHLSYN overexpression in rdr6 had little effect on chlorophyll content but resulted in up to a 30% reduction in tocopherol levels in leaves. These findings show that altered CHLSYN expression impacts tocopherol levels and also, show a strong epigenetic surveillance of CHLSYN to control chlorophyll and tocopherol synthesis. PMID:26048882

  20. Adenine and guanine nucleotide metabolism during platelet storage at 22 degree C

    International Nuclear Information System (INIS)

    Edenbrandt, C.M.; Murphy, S.

    1990-01-01

    Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C-adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation

  1. PARP inhibitor rucaparib induces changes in NAD levels in cells and liver tissues as assessed by MRS.

    Science.gov (United States)

    Almeida, Gilberto S; Bawn, Carlo M; Galler, Martin; Wilson, Ian; Thomas, Huw D; Kyle, Suzanne; Curtin, Nicola J; Newell, David R; Maxwell, Ross J

    2017-09-01

    Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy ( 1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD + ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD + levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD + levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD + levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD + depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD + levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD + would be valuable. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Escherichia coli class Ib ribonucleotide reductase contains a dimanganese(III)-tyrosyl radical cofactor in vivo†

    Science.gov (United States)

    Cotruvo, Joseph A.; Stubbe, JoAnne

    2011-01-01

    Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (FeIII2-Y•) and dimanganese(III)-Y• (MnIII2-Y•) cofactors in the β2 subunit, NrdF [Cotruvo J.A., Jr. and Stubbe J., Biochemistry (2010) 49, 1297–1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the MnIII2-Y• cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ~95% purity, with 0.2 Y•/β2, 0.9 Mn/β2, and a specific activity of 720 nmol/min/mg. In these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the MnIII2-Y• cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo. PMID:21250660

  3. A newly validated high-performance liquid chromatography method with diode array ultraviolet detection for analysis of the antimalarial drug primaquine in the blood plasma

    Directory of Open Access Journals (Sweden)

    Ana Paula Barbosa do Carmo

    Full Text Available Abstract INTRODUCTION: Primaquine (PQ diphosphate is an 8-aminoquinoline antimalarial drug with unique therapeutic properties. It is the only drug that prevents relapses of Plasmodium vivax or Plasmodium ovale infections. In this study, a fast, sensitive, cost-effective, and robust method for the extraction and high-performance liquid chromatography with diode array ultraviolet detection (HPLC-DAD-UV analysis of PQ in the blood plasma was developed and validated. METHODS: After plasma protein precipitation, PQ was obtained by liquid-liquid extraction and analyzed by HPLC-DAD-UV with a modified-silica cyanopropyl column (250mm × 4.6mm i.d. × 5μm as the stationary phase and a mixture of acetonitrile and 10mM ammonium acetate buffer (pH = 3.80 (45:55 as the mobile phase. The flow rate was 1.0mL·min-1, the oven temperature was 50OC, and absorbance was measured at 264nm. The method was validated for linearity, intra-day and inter-day precision, accuracy, recovery, and robustness. The detection (LOD and quantification (LOQ limits were 1.0 and 3.5ng·mL-1, respectively. The method was used to analyze the plasma of female DBA-2 mice treated with 20mg.kg-1 (oral PQ diphosphate. RESULTS: By combining a simple, low-cost extraction procedure with a sensitive, precise, accurate, and robust method, it was possible to analyze PQ in small volumes of plasma. The new method presents lower LOD and LOQ limits and requires a shorter analysis time and smaller plasma volumes than those of previously reported HPLC methods with DAD-UV detection. CONCLUSIONS: The new validated method is suitable for kinetic studies of PQ in small rodents, including mouse models for the study of malaria.

  4. Combining Vγ9Vδ2 T Cells with a Lipophilic Bisphosphonate Efficiently Kills Activated Hepatic Stellate Cells

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    Xiaoying Zhou

    2017-10-01

    Full Text Available Activated hepatic stellate cells (aHSCs are now established as a central driver of fibrosis in human liver injury. In the presence of chronic or repeated injury, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC can occur, so there is interest in down-regulating aHSCs activity in order to treat these diseases. Here, we report that Vγ9Vδ2 T cells are reduced in patients with liver cirrhosis, stimulating us to investigate possible interactions between Vγ9Vδ2 T cells and aHSCs. We find that Vγ9Vδ2 T cells kill aHSCs and killing is enhanced when aHSCs are pretreated with BPH-1236, a lipophilic analog of the bone resorption drug zoledronate. Cytotoxicity is mediated by direct cell-to-cell contact as shown by Transwell experiments and atomic force microscopy, with BPH-1236 increasing the adhesion between aHSCs and Vγ9Vδ2 T cells. Mechanistically, BPH-1236 functions by inhibiting farnesyl diphosphate synthase, leading to accumulation of the phosphoantigen isopentenyl diphosphate and recognition by Vγ9Vδ2 T cells. The cytolytic process is largely dependent on the perforin/granzyme B pathway. In a Rag2−/−γc−/− immune-deficient mouse model, we find that Vγ9Vδ2 T cells home-in to the liver, and when accompanied by BPH-1236, kill not only orthotopic aHSCs but also orthotopic HCC tumors. Collectively, our results provide the first proof-of-concept of a novel immunotherapeutic strategy for the treatment of fibrosis–cirrhosis–HCC diseases using adoptively transferred Vγ9Vδ2 T cells, combined with a lipophilic bisphosphonate.

  5. Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media.

    Science.gov (United States)

    Willrodt, Christian; David, Christian; Cornelissen, Sjef; Bühler, Bruno; Julsing, Mattijs K; Schmid, Andreas

    2014-08-01

    The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)-limonene from glycerol and glucose as carbon sources using recombinant Escherichia coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel of biological origin. Key parameters that limit the (S)-limonene yield, related to genetics, physiology, and reaction engineering, were identified. The growth-dependent production of (S)-limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain, as it showed low acetate formation, fast growth, and high productivity. A two-liquid phase fed-batch fermentation with glucose as the sole carbon and energy source resulted in the formation of 700 mg L(org) (-1) (S)-limonene. Specific activities of 75 mU g(cdw) (-1) were reached, but decreased relatively quickly. The use of glycerol as a carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU g(cdw) (-1) ) leading to a final (S)-limonene concentration of 2,700 mg L(org) (-1) . Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)-limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation also did not limit (S)-limonene production. The two-liquid phase fed-batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Biosynthesis of the oxygenated diterpene nezukol in the medicinal plant Isodon rubescens is catalyzed by a pair of diterpene synthases.

    Directory of Open Access Journals (Sweden)

    Kyle A Pelot

    Full Text Available Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP synthase and a (+-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.

  7. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    International Nuclear Information System (INIS)

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells

  8. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

    Directory of Open Access Journals (Sweden)

    Chen Jiang

    2017-01-01

    Full Text Available Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP, a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.

  9. Ibandronate metal complexes: solution behavior and antiparasitic activity.

    Science.gov (United States)

    Demoro, Bruno; Rostán, Santiago; Moncada, Mauricio; Li, Zhu-Hong; Docampo, Roberto; Olea Azar, Claudio; Maya, Juan Diego; Torres, Julia; Gambino, Dinorah; Otero, Lucía

    2018-03-01

    To face the high costs of developing new drugs, researchers in both industry and academy are looking for ways to repurpose old drugs for new uses. In this sense, bisphosphonates that are clinically used for bone diseases have been studied as agents against Trypanosoma cruzi, causative parasite of Chagas disease. In this work, the development of first row transition metal complexes (M = Co 2+ , Mn 2+ , Ni 2+ ) with the bisphosphonate ibandronate (iba, H 4 iba representing the neutral form) is presented. The in-solution behavior of the systems containing iba and the selected 3d metal ions was studied by potentiometry. Mononuclear complexes [M(H x iba)] (2-x)- (x = 0-3) and [M(Hiba) 2 ] 4- together with the formation of the neutral polynuclear species [M 2 iba] and [M 3 (Hiba) 2 ] were detected for all studied systems. In the solid state, complexes of the formula [M 3 (Hiba) 2 (H 2 O) 4 ]·6H 2 O were obtained and characterized. All obtained complexes, forming [M(Hiba)] - species under the conditions of the biological studies, were more active against the amastigote form of T. cruzi than the free iba, showing no toxicity in mammalian Vero cells. In addition, the same complexes were selective inhibitors of the parasitic farnesyl diphosphate synthase (FPPS) enzyme showing poor inhibition of the human one. However, the increase of the anti-T. cruzi activity upon coordination could not be explained neither through the inhibition of TcFPPS nor through the inhibition of TcSPPS (T. cruzi solanesyl-diphosphate synthase). The ability of the obtained metal complexes of catalyzing the generation of free radical species in the parasite could explain the observed anti-T. cruzi activity.

  10. Analysis of the enzymatic formation of citral in the glands of sweet basil.

    Science.gov (United States)

    Iijima, Yoko; Wang, Guodong; Fridman, Eyal; Pichersky, Eran

    2006-04-15

    Basil glands of the Sweet Dani cultivar contain high levels of citral, a mixture of geranial and its cis-isomer neral, as well as low levels of geraniol and nerol. We have previously reported the identification of a cDNA from Sweet Dani that encodes an enzyme responsible for the formation of geraniol from geranyl diphosphate in the glands, and that these glands cannot synthesize nerol directly from geranyl diphosphate. Here, we report the identification of two basil cDNAs encoding NADP+-dependent dehydrogenases that can use geraniol as the substrate. One cDNA, designated CAD1, represents a gene whose expression is highly specific to gland cells of all three basil cultivars examined, regardless of their citral content, and encodes an enzyme with high sequence similarity to known cinnamyl alcohol dehydrogenases (CADs). The enzyme encoded by CAD1 reversibly oxidizes geraniol to produce geranial (which reversibly isomerizes to neral via keto-enol tautomerization) at half the efficiency compared with its activity with cinnamyl alcohol. CAD1 does not use nerol and neral as substrates. A second cDNA, designated GEDH1, encodes an enzyme with sequence similarity to CAD1 that is capable of reversibly oxidizing geraniol and nerol in equal efficiency, and prolonged incubation of geraniol with GEDH1 in vitro produces not only geranial and neral, but also nerol. GEDH1 is also active, although at a lower efficiency, with cinnamyl alcohol. However, GEDH1 is expressed at low levels in glands of all cultivars compared with its expression in leaves. These and additional data presented indicate that basil glands may contain additional dehydrogenases capable of oxidizing geraniol.

  11. Synthesis, crystal structure, and vibrational spectroscopic and UV-visible studies of Cs{sub 2}MnP{sub 2}O{sub 7}

    Energy Technology Data Exchange (ETDEWEB)

    Kaoua, Saida; Krimi, Saida [LPCMI, Faculte des Sciences Aien Chok, UH2C, Casablanca (Morocco); Pechev, Stanislav; Gravereau, Pierre; Chaminade, Jean-Pierre [CNRS, Universite de Bordeaux, ICMCB, 87, Avenue du Dr. A. Schweitzer, Pessac (France); Couzi, Michel [CNRS, Universite de Bordeaux, ISM, UMR 5255, F-33400 Talence (France); El Jazouli, Abdelaziz, E-mail: eljazouli_abdelaziz@yahoo.fr [LCMS, URAC 17, Faculte des Sciences Ben M' Sik, UH2MC, Casablanca (Morocco)

    2013-02-15

    A new member of the A{sub 2}MP{sub 2}O{sub 7} diphosphate family, Cs{sub 2}MnP{sub 2}O{sub 7}, has been synthesized and structurally characterized. The crystal structure was determined by single crystal X-Ray diffraction. Cs{sub 2}MnP{sub 2}O{sub 7} crystallizes in the orthorhombic system, space group Pnma ( Music-Sharp-Sign 62), with the unit cell parameters a=16.3398(3), b=5.3872(1), c=9.8872(2) A, Z=4 and V=870.33(3) A{sup 3}. The structure parameters were refined to a final R{sub 1}/wR{sub 2}=0.0194/0.0441 for 1650 observed reflections. The 2D framework of Cs{sub 2}MnP{sub 2}O{sub 7} structure consists of P{sub 2}O{sub 7} and MnO{sub 5} units. The corner-shared MnO{sub 5} and P{sub 2}O{sub 7} units are alternately arranged along the b axis to form [(MnO)P{sub 2}O{sub 7}]{sub {infinity}} chains. These chains are interconnected by an oxygen atom to form sheets parallel to the (b, c) plane. The cesium atoms are located between the sheets in 9- and 10-fold coordinated sites. The infrared and Raman vibrational spectra have been investigated. A factor group analysis leads to the determination of internal modes of (P{sub 2}O{sub 7}) groups. UV-visible spectrum consists of weak bands, between 340 and 700 nm, assigned to the forbidden d-d transitions of Mn{sup 2+} ion, and of a strong band around 250 nm, attributed to the O--Mn charge transfer. - Graphical abstract: Structure of Cs{sub 2}MnP{sub 2}O{sub 7}: The 2D structure of Cs{sub 2}MnP{sub 2}O{sub 7} is built from P{sub 2}O{sub 7} diphosphate groups and MnO{sub 5} square pyramids which share corners and form [(MnO)P{sub 2}O{sub 7}]{sub {infinity}} chains along b axis. These chains are interconnected by an oxygen atom to form wavy (MnP{sub 2}O{sub 7}){sup 2-} sheets parallel to the (b, c) plane. The cesium ions are located between these sheets in the inter-layers space, in zigzag positions. Highlights: Black-Right-Pointing-Pointer A new diphosphate, Cs{sub 2}MnP{sub 2}O{sub 7}, has been synthesized and structurally

  12. Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels.

    Science.gov (United States)

    Pissarek, M; Garcia de Arriba, S; Schäfer, M; Sieler, D; Nieber, K; Illes, P

    1998-10-01

    In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95%O2-5%CO2 with 95%N2-5%CO2) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K(ATP)) channels, diazoxide (3-300 microM). The effect of 30 but not 300 microM diazoxide was reversed by washout. Tolbutamide (300 microM), an antagonist of K(ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 microM) on the HD. Neither diazoxide (3-300 microM) nor tolbutamide (300 microM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 microM) or tolbutamide (300 microM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between -130 and -50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 microM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri

  13. Design and Synthesis of a Novel Class of Flavonoid Derivatives via Sequential Phosphorylation and its Application for Greener Nanoparticle Synthesis

    Science.gov (United States)

    Osonga, Francis Juma

    Flavonoids exhibit arrays of biological effects that are beneficial to humans, including anti-viral, anti-oxidative, anti-inflammatory and anti-carcinogenic effects. However, these applications have been hindered by their poor stability and solubility in common solvents. Consequently, there is significant interest in the modification of flavonoids to improve their solubility. This poor solubility is also believed to be responsible for its permeability and bioavailability. Hence the central goal of this work is to design synthetic strategies for the sequential protection of the -OH groups in order to produce phosphorylated quercetin and apigenin derivatives. This work is divided into two parts: the first part presents the design, synthesis, and characterization of novel flavonoid derivatives via global and sequential phosphorylation. The second part focuses on the application of the synthesized derivatives for greener nanoparticle synthesis. This work shows for the first time that sequential phosphorylation of Quercetin is feasible through the design of 4 new derivatives namely: 5,4'-O-Quercetin Diphosphate (QDPI), 4'-O-phosphate Quercetin (4'-QPI), 5,4'-Quercetin Diphosphate (5,4'-QDP) and monophosphate 4-QP. The synthesis of 4'-QP and 5, 4'-QDP was successful with 85% and 60.5% yields respectively. In addition, the progress towards the total synthesis of apigenin phosphate derivatives (7, 4'-ADP and 7-AP) is presented. The synthesized derivatives were characterized using 1H, 13C, and 31P NMR. The phosphorylated derivatives were subsequently explored as reducing agents for sustainable synthesis of gold, silver and copper nanoparticles. We have successfully demonstrated the photochemical synthesis of gold nanoplates of sizes ranging from 10 - 200 nm using water soluble QDP in the presence of sunlight. This work contributes immensely in promoting the ideals of green nanosynthesis by (i) eliminating the use of organic solvents in the nanosynthesis, (ii) exploiting the

  14. Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

    Science.gov (United States)

    Garg, Anchal; Agrawal, Lalit; Misra, Rajesh Chandra; Sharma, Shubha; Ghosh, Sumit

    2015-09-02

    Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs. De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent

  15. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus

    Directory of Open Access Journals (Sweden)

    Clark Shawn M

    2013-01-01

    Full Text Available Abstract Background Bitter acids (e.g. humulone are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP pathway. We used RNA sequencing (RNA-seq to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic and reverse (catabolic reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial and

  16. 5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

    Science.gov (United States)

    Ogino, Minako; Ogino, Tomoaki

    2017-03-15

    The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as

  17. 5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

    Science.gov (United States)

    Ogino, Minako

    2017-01-01

    ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups

  18. Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

    Directory of Open Access Journals (Sweden)

    Pathan AAK

    2016-05-01

    Full Text Available Akbar Ali Khan Pathan,1,2,* Bhavana Panthi,3,* Zahid Khan,1 Purushotham Reddy Koppula,4–6 Mohammed Saud Alanazi,1 Sachchidanand,3 Narasimha Reddy Parine,1 Mukesh Chourasia3,* 1Genome Research Chair (GRC, Department of Biochemistry, College of Science, King Saud University, 2Integrated Gulf Biosystems, Riyadh, Kingdom of Saudi Arabia; 3Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Hajipur, India; 4Department of Internal Medicine, School of Medicine, 5Harry S. Truman Memorial Veterans Affairs Hospital, 6Department of Radiology, School of Medicine, Columbia, MO, USA *These authors contributed equally to this work Objective: Kirsten rat sarcoma (K-Ras protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods: In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results: Interestingly, the designed compounds exhibit a binding preference for the

  19. Nicotine Component of Cigarette Smoke Extract (CSE) Decreases the Cytotoxicity of CSE in BEAS-2B Cells Stably Expressing Human Cytochrome P450 2A13.

    Science.gov (United States)

    Ji, Minghui; Zhang, Yudong; Li, Na; Wang, Chao; Xia, Rong; Zhang, Zhan; Wang, Shou-Lin

    2017-10-13

    Cytochrome P450 2A13 (CYP2A13), an extrahepatic enzyme mainly expressed in the human respiratory system, has been reported to mediate the metabolism and toxicity of cigarette smoke. We previously found that nicotine inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by CYP2A13, but its influence on other components of cigarette smoke remains unclear. The nicotine component of cigarette smoke extract (CSE) was separated, purified, and identified using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), splitting CSE into a nicotine section (CSE-N) and nicotine-free section (CSE-O). Cell viability and apoptosis by Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted on immortalized human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13) or vector (B-V), respectively. Interestingly, CSE and CSE-O were toxic to BEAS-2B cells whereas CSE-N showed less cytotoxicity. CSE-O was more toxic to B-2A13 cells than to B-V cells (IC 50 of 2.49% vs. 7.06%), which was flatted by 8-methoxypsoralen (8-MOP), a CYP inhibitor. CSE-O rather than CSE or CSE-N increased apoptosis of B-2A13 cells rather than B-V cells. Accordingly, compared to CSE-N and CSE, CSE-O significantly changed the expression of three pairs of pro- and anti-apoptotic proteins, Bcl-2 Associated X Protein/B cell lymphoma-2 (Bax/Bcl-2), Cleaved Poly (Adenosine Diphosphate-Ribose) Polymerase/Poly (Adenosine Diphosphate-Ribose) Polymerase (C-PARP/PARP), and C-caspase-3/caspase-3, in B-2A13 cells. In addition, recombination of CSE-N and CSE-O (CSE-O/N) showed similar cytotoxicity and apoptosis to the original CSE. These results demonstrate that the nicotine component decreases the metabolic activation of CYP2A13 to CSE and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking.

  20. Novel microfilaricidal activity of nanosilver

    Directory of Open Access Journals (Sweden)

    Singh SK

    2012-02-01

    Full Text Available Sunil K Singh1, Kalyan Goswami2, Richa D Sharma2, Maryada VR Reddy2, Debabrata Dash11Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 2Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, IndiaPurpose: The currently available drug repertoire against lymphatic filariasis, a major health hazard in the developing world, is inadequate and is fraught with serious limitations. Thus, the development of an effective antifilarial strategy has become a global research thrust mandated by the World Health Organization. Nanoparticles of silver endowed with antibacterial potency are known to induce apoptosis in eukaryotic cells. The present study was designed to investigate the possible microfilaricidal efficacy of silver nanoparticles and to establish the validity of apoptotic rationale in antifilarial drug designing.Methods: This report analyzed the effect of nanoparticles of silver as well as gold (size range: 10–15 nm on the microfilariae of Brugia malayi obtained from the lavage of peritoneal cavities of infected jirds (Meriones unguiculatus. The study included a microfilarial motility assay, a trypan blue exclusion test, a poly(adenosine diphosphate-ribose polymerase activity study, ethidium bromide/acridine orange differential staining, and transmission, as well as scanning electron microscopic evaluation of ultrastructural changes in microfilariae.Results: The study demonstrates that nanoparticles of silver, but not of gold, elicited significant loss in microfilarial motility. Differential staining of parasites with ethidium bromide and acridine orange, poly(adenosine diphosphate-ribose polymerase activity in microfilarial lysate, and electron microscopic findings underscored apoptotic death of parasites attributable to nanosilver. In a trypan blue exclusion test, the 50% lethal dose of nanosilver was measured to be 101.2 µM, which was higher than the recorded complete