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Sample records for purine-nucleoside phosphorylase

  1. Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1978-01-01

    Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared...

  2. Purine nucleoside phosphorylase deficiency in two unrelated Saudi patients

    OpenAIRE

    Alangari, Abdullah; Al-Harbi, Abdullah; Al-Ghonaium, Abdulaziz; Santisteban, Ines; Hershfield, Michael

    2009-01-01

    Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder that results in combined immunodeficiency, neurologic dysfunction and autoimmunity. PNP deficiency has never been reported from Saudi Arabia or in patients with an Arabic ethnic background. We report on two Saudi girls with PNP deficiency. Both showed severe lymphopenia and neurological involvement. Sequencing of the PNP gene of one girl revealed a novel missense mutation Pro146>Leu in exon 4 due...

  3. Anopheles gambiae Purine Nucleoside Phosphorylase: Catalysis, Structure, and Inhibition

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    Taylor,E.; Rinaldo-Matthis, A.; Li, L.; Ghanem, M.; Hazleton, K.; Cassera, M.; Almo, S.; Schramm, V.

    2007-01-01

    The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast for AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.

  4. Can Crystal Symmetry and Packing Influence the Active Site Conformation of Homohexameric Purine Nucleoside Phosphorylases?

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    Marija Luić

    2016-06-01

    Full Text Available It is generaly believed that enzymes retain most of their functionality in the crystal form due to the large solvent content of protein crystals. This is facilitated by the fact that their natural environment in solution is not too far from the one found in the crystal form. Nevertheless, if the nature of the enzyme is such to require conformational changes, overcoming of the crystal packing constraints may prove to be too difficult. Such conformational change is present in one class of enzymes (purine nucleoside phosphorylases, that is the subject of our scientific interest for many years. The influence of crystal symmetry and crystal packing on the conformation of the active sites in the case of homohexameric purine nucleoside phosphorylases is presented and analysed. This work is licensed under a Creative Commons Attribution 4.0 International License.

  5. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

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    Abramchik, Yu. A.; Timofeev, V. I.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2015-07-01

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P21 and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  6. Purification, crystallization, and preliminary X-ray diffraction study of purine nucleoside phosphorylase from E. coli

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    Abramchik, Yu. A., E-mail: inna@ns.crys.ras.ru; Timofeev, V. I., E-mail: espiov@ibch.ru; Zhukhlistova, N. E., E-mail: tostars@mail.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Muravieva, T. I.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Kuranova, I. P. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-07-15

    Crystals of E. coli purine nucleoside phosphorylase were grown in microgravity by the capillary counter-diffusion method through a gel layer. The X-ray diffraction data set suitable for the determination of the three-dimensional structure at atomic resolution was collected from one crystal at the Spring-8 synchrotron facility to 0.99 Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unit-cell parameters: a = 74.1 Å, b = 110.2 Å, c = 88.2 Å, α = γ = 90°, β = 111.08°. The crystal contains six subunits of the enzyme comprising a hexamer per asymmetric unit. The hexamer is the biological active form of E. coli. purine nucleoside phosphorylase.

  7. Structural determinants of the 5'-methylthioinosine specificity of Plasmodium purine nucleoside phosphorylase.

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    Teraya M Donaldson

    Full Text Available Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.

  8. Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

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    Timofeev, V. I.; Abramchik, Yu. A.; Zhukhlistova, N. E.; Muravieva, T. I.; Esipov, R. S.; Kuranova, I. P.

    2016-03-01

    Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment of the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB_ID: 4RJ2).

  9. Structural characterization of purine nucleoside phosphorylase from human pathogen Helicobacter pylori.

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    Štefanić, Zoran; Mikleušević, Goran; Luić, Marija; Bzowska, Agnieszka; Leščić Ašler, Ivana

    2017-08-01

    Microaerophilic bacterium Helicobacer pylori is a well known human pathogen involved in the development of many diseases. Due to the evergrowing infection rate and increase of H. pylori antibiotic resistence, it is of utmost importance to find a new way to attack and eradicate H. pylori. The purine metabolism in H. pylori is solely dependant on the salvage pathway and one of the key enzymes in this pathway is purine nucleoside phosphorylase (PNP). In this timely context, we report here the basic biochemical and structural characterization of recombinant PNP from the H. pylori clinical isolate expressed in Escherichia coli. Structure of H. pylori PNP is typical for high molecular mass PNPs. However, its activity towards adenosine is very low, thus resembling more that of low molecular mass PNPs. Understanding the molecular mechanism of this key enzyme may lead to the development of new drug strategies and help in the eradication of H. pylori. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency

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    Aust, M.R.; Norby-Slycord, C.J.; Andrews, L.G.; Markert, M.L. (Duke Univ. Medical Center, Durham, NC (United States)); Barrett, M.J. (Sunnyside Hospital, Clackamas, OR (United States))

    1992-10-01

    Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency. 52 refs., 5 figs.

  11. Metabolites of purine nucleoside phosphorylase (NP in serum have the potential to delineate pancreatic adenocarcinoma.

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    Shaiju K Vareed

    Full Text Available Pancreatic Adenocarcinoma (PDAC, the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers that has motivated the use of high throughput molecular approaches to delineate potential biomarkers. To pursue identification of a distinct marker, this study profiled the secretory proteome in 16 PDAC, 2 carcinoma in situ (CIS and 7 benign patients using label-free mass spectrometry coupled to 1D-SDS-PAGE and Strong Cation-Exchange Chromatography (SCX. A total of 431 proteins were detected of which 56 were found to be significantly elevated in PDAC. Included in this differential set were Parkinson disease autosomal recessive, early onset 7 (PARK 7 and Alpha Synuclein (aSyn, both of which are known to be pathognomonic to Parkinson's disease as well as metabolic enzymes like Purine Nucleoside Phosphorylase (NP which has been exploited as therapeutic target in cancers. Tissue Microarray analysis confirmed higher expression of aSyn and NP in ductal epithelia of pancreatic tumors compared to benign ducts. Furthermore, extent of both aSyn and NP staining positively correlated with tumor stage and perineural invasion while their intensity of staining correlated with the existence of metastatic lesions in the PDAC tissues. From the biomarker perspective, NP protein levels were higher in PDAC sera and furthermore serum levels of its downstream metabolites guanosine and adenosine were able to distinguish PDAC from benign in an unsupervised hierarchical classification model. Overall, this study for the first time describes elevated levels of aSyn in PDAC as well as highlights the potential of evaluating NP protein expression and levels of its downstream metabolites to develop a multiplex panel for non-invasive detection of PDAC.

  12. Transition Path Sampling Study of the Reaction Catalyzed by Purine Nucleoside Phosphorylase

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    Saen-oon, Suwipa; Schramm, Vern L.; Schwartz, Steven D.

    2010-01-01

    The Transition Path Sampling (TPS) method is a powerful technique for studying rare events in complex systems, that allows description of reactive events in atomic detail without prior knowledge of reaction coordinates and transition states. We have applied TPS in combination with a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) method to study the enzyme human purine nucleoside phosphorylase (hPNP). This enzyme catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to generate the corresponding purine base and (deoxy)ribose 1-phosphate. Hundreds of reactive trajectories were generated. Analysis of this transition path ensembles provides insight into the detailed mechanistic dynamics of reaction in the enzyme. Our studies have indicated a reaction mechanism involving the cleavage of the N-ribosidic bond to form transition states with substantial ribooxacarbenium ion character, that is then followed by conformational changes in the enzyme and the ribosyl group leading to migration of the anomeric carbon of the ribosyl group toward phosphate to form the product ribose 1-phosphate. This latter process is crucial in PNP, because several strong H-bonds form between active site residues in order to capture and align the phosphate nucleophile. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. Analysis of these transition state structures showed that bond-breaking and bond-forming distances are not a good choice for the reaction coordinate, but that the pseudorotational phase of the ribose ring is also a significant variable. PMID:20664707

  13. Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases.

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    Wielgus-Kutrowska, B; Kulikowska, E; Wierzchowski, J; Bzowska, A; Shugar, D

    1997-01-15

    Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen). It is also a substrate of the enzyme from Escherichia coli. The Km values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino. The pseudo first-order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E. coli enzymes. The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP. Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with Ki values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots. Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir. Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E. coli PNP, with Ki values similar to those for inhibition of other natural substrates. The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates. The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.

  14. Thermodynamics of the Purine Nucleoside Phosphorylase Reaction Revealed by Computer Simulations.

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    Isaksen, Geir Villy; Åqvist, Johan; Brandsdal, Bjørn Olav

    2017-01-10

    Enzymes are able to catalyze chemical reactions by reducing the activation free energy, yielding significant increases in the reaction rates. This can thermodynamically be accomplished by either reducing the activation enthalpy or increasing the activation entropy. The effect of remote mutations on the thermodynamic activation parameters of human purine nucleoside phosphorylase is examined using extensive molecular dynamics and free energy simulations. More than 2700 independent reaction free energy profiles for six different temperatures have been calculated to obtain high-precision computational Arrhenius plots. On the basis of these, the activation enthalpies and entropies were computed from linear regression of the plots with ΔG(⧧) as a function of 1/T, and the obtained thermodynamic activation parameters are in very good agreement with those from experiments. The Arrhenius plots immediately show that the 6-oxopurines (INO and GUO) have identical slopes, whereas the 6-aminopurine (ADO) has a significantly different slope, indicating that the substrate specificity is related to the difference in thermodynamic activation parameters. Furthermore, the calculations show that the human PNP specificity for 6-oxopurines over 6-aminopurines originates from significant differences in electrostatic preorganization. The effect of the remote double mutation, K22E and H104R (E:R), has also been examined, as it alters human PNP toward the bovine PNP. These residues are situated on the protein surface, 28-35 Å from the active site, and the mutation alters the enthalpy-entropy balance with little effect on the catalytic rates. It is thus quite remarkable that the empirical valence bond method can reproduce the enthalpies and entropies induced by these long-range mutations.

  15. A purine nucleoside phosphorylase in Solanum tuberosum L. (potato) with specificity for cytokinins contributes to the duration of tuber endodormancy.

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    Bromley, Jennifer R; Warnes, Barbara J; Newell, Christine A; Thomson, Jamie C P; James, Celia M; Turnbull, Colin G N; Hanke, David E

    2014-03-01

    StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

  16. Inhibition and Structure of Trichomonas vaginalis Purine Nucleoside Phosphorylase with Picomolar Transition State Analogues

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    Rinaldo-Matthis,A.; Wing, C.; Ghanem, M.; Deng, H.; Wu, P.; Gupta, A.; Tyler, P.; Evans, G.; Furneaux, R.; et al.

    2007-01-01

    Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to the PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition stte mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min, and a K{sub m}/K{sub d} ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min, and a K{sub m}/K{sub d} ratio of 203,300. The tight binding of DADMe-ImmA supports a late S{sub N}1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP-ImmA{center_dot}PO{sub 4} and TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4} ternary complexes differ from previous structures with substrate anologues. The tight binding with DADMe-ImmA is in part due to a 2.7 {angstrom} ionic interaction between a PO{sub 4} oxygen and the N1 cation of the hydroxypyrrolidine and is weaker in the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure at 3.5 {angstrom}. However, the TvPNP{center_dot}ImmA{center_dot}PO{sub 4} structure includes hydrogen bonds between the 2'-hydroxyl and the protein that are not present in TvPNP{center_dot}DADMe-ImmA{center_dot}PO{sub 4}. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope

  17. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

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    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-02

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.

  18. Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

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    López-Cruz, Roberto I; Pérez-Milicua, Myrna Barjau; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal-Vertiz, Jaime A; de la Rosa, Alejandro; Vázquez-Medina, José P; Zenteno-Savín, Tania

    2014-05-01

    Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas.

  19. Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

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    de Giuseppe, Priscila O.; Martins, Nadia H.; Meza, Andreia N.; dos Santos, Camila R.; Pereira, Humberto D’Muniz; Murakami, Mario T.

    2012-01-01

    The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2′deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2′deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5′ hydroxyl group of adenosine and Arg43* side chain contributes for the ribosyl radical to adopt an unusual C3’-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl6 and Br8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr91) is responsible for the lack of negative cooperativity of phosphate binding in this

  20. Insights into phosphate cooperativity and influence of substrate modifications on binding and catalysis of hexameric purine nucleoside phosphorylases.

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    Priscila O de Giuseppe

    Full Text Available The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233 displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxyribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43* side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl(6 and Br(8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91 is responsible for the lack of negative cooperativity of phosphate binding

  1. Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma.

    Science.gov (United States)

    Giuliani, Patricia; Zuccarini, Mariachiara; Buccella, Silvana; Rossini, Margherita; D'Alimonte, Iolanda; Ciccarelli, Renata; Marzo, Matteo; Marzo, Antonio; Di Iorio, Patrizia; Caciagli, Francesco

    2016-01-15

    Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions.

  2. hTERT-targeted E. coli purine nucleoside phosphorylase gene/6-methylpurine deoxyribose therapy for pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    ZHOU Jia-hua; TANG Bo; LIU Xun-liang; HE Dao-wei; YANG De-tong

    2007-01-01

    Background Pancreatic cancer is one of the most common tumors and has a 5-year survival for all stages of less than 5%. Most patients with pancreatic cancer are diagnosed at an advanced stage and therefore are not candidates for surgical resection. In recent years, investigation into alternative treatment strategies for this aggressive disease has led to advances in the field of gene therapy for pancreatic cancer. E. coli purine nucleoside phosphorylase/6-methylpurine deoxyribose (ePNP/MePdR) is a suicide gene/prodrug system where PNP enzyme cleaves nontoxic MePdR into cytotoxic membrane-permeable compounds 6-methylpurine (MeP) with high bystander activity, hTERT is expressed in cell lines and tissues for telomerase activity. In this study we examined the efficacy of ePNP under the control of hTERT promoter sequences and assessed the selective killing effects of the ePNP/prodrug MePdR system on pancreatic tumors.Methods Recombinant pET-PNP was established. The protein of E. coli PNPase was expressed and an antibody to E.coli PNPase was prepared. Transcriptional activities of hTERT promoter sequences were analyzed using a luciferase reporter gene. A recombinant phTERT-ePNP vector was constructed. The ePNP/MePdR system affects SW1990 human pancreatic cancer cell lines in vitro.Results The hTERT promoter had high transcriptional activity and conferred specificity on cancer cell lines. The antibody to E. coli PNPase was demonstrated to be specific for the ePNP protein. The MePdR treatment induced a high in vitro cytotoxicity on the sole hTERT-ePNP-producing cell lines and affected SW1990 cells in a dose-dependent manner.Conclusions The hTERT promoter control of the ePNP/MePdR system can provide a beneficial anti-tumor treatment in pancreatic cancer cell lines including a good bystander killing effect.

  3. High-sensitivity capillary electrophoresis method for monitoring purine nucleoside phosphorylase and adenosine deaminase reactions by a reversed electrode polarity switching mode.

    Science.gov (United States)

    Iqbal, Jamshed; Müller, Christa E

    2011-07-22

    A simple, efficient, and highly sensitive in-line CE method was developed for the characterization and for inhibition studies of the nucleoside-metabolizing enzymes purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) present in membrane preparations of human 1539 melanoma cells. After filling the running buffer (50 mM borate buffer, 100 mM SDS, pH 9.10) into a fused-silica capillary (50 cm effective length × 75 μm), a large sample volume was loaded by hydrodynamic injection (5 psi, 36 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-20 kV). The current was monitored and the polarity was reversed when 95% of the current had been recovered. The separation of the neutral analytes (nucleosides and nucleobases) was performed by applying a voltage of 15 kV. An about 10-fold improvement of sensitivity for the five investigated analytes (adenosine, inosine, adenine, hypoxanthine, xanthine) was achieved by large-volume stacking with polarity switching when compared with CE without stacking. For inosine and adenine detection limits as low as 60 nM were achieved. To the best of our knowledge, this represents the highest sensitivity for nucleoside and nucleobase analysis using CE with UV detection reported so far. The Michaelis-Menten constants (K(m)) for PNP and ADA and the inhibition constants (K(i)) for standard inhibitors determined with the new method were consistent with literature data.

  4. The transition state analog inhibitor of Purine Nucleoside Phosphorylase (PNP) Immucillin-H arrests bone loss in rat periodontal disease models.

    Science.gov (United States)

    Deves, Candida; de Assunção, Thiago Milech; Ducati, Rodrigo Gay; Campos, Maria Martha; Basso, Luiz Augusto; Santos, Diogenes Santiago; Batista, Eraldo L

    2013-01-01

    Purine nucleoside phosphorylase (PNP) is a purine-metabolizing enzyme that catalyzes the reversible phosphorolysis of 6-oxypurine (deoxy)nucleosides to their respective bases and (deoxy)ribose-1-phosphate. It is a key enzyme in the purine salvage pathway of mammalian cells. The present investigation sought to determine whether the PNP transition state analog inhibitor (Immucillin-H) arrests bone loss in two models of induced periodontal disease in rats. Periodontal disease was induced in rats using ligature or LPS injection followed by administration of Immucillin-H for direct analysis of bone loss, histology and TRAP staining. In vitro osteoclast differentiation and activation of T CD4+ cells in the presence of Immucillin-H were carried out for assessment of RANKL expression, PNP and Cathepsin K activity. Immucillin-H inhibited bone loss induced by ligatures and LPS, leading to a reduced number of infiltrating osteoclasts and inflammatory cells. In vitro assays revealed that Immucillin-H could not directly abrogate differentiation of osteoclast precursor cells, but affected lymphocyte-mediated osteoclastogenesis. On the other hand, incubation of pre-activated T CD4+ with Immucillin-H decreased RANKL secretion with no compromise of cell viability. The PNP transition state analog Immucillin-H arrests bone loss mediated by T CD4+ cells with no direct effect on osteoclasts. PNP inhibitor may have an impact in the treatment of diseases characterized by the presence of pathogens and imbalances of bone metabolism.

  5. 6-Methylpurine derived sugar modified nucleosides: Synthesis and in vivo antitumor activity in D54 tumor expressing M64V-Escherichia coli purine nucleoside phosphorylase.

    Science.gov (United States)

    Hassan, Abdalla E A; Abou-Elkhair, Reham A I; Parker, William B; Allan, Paula W; Secrist, John A

    2016-01-27

    Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

  6. Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

    Science.gov (United States)

    Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

    2016-01-01

    Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a

  7. Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.

    Science.gov (United States)

    Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

    2016-01-01

    Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a

  8. 6-Methylpurine derived sugar modified nucleosides: Synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases.

    Science.gov (United States)

    Hassan, Abdalla E A; Abou-Elkhair, Reham A I; Parker, William B; Allan, Paula W; Secrist, John A

    2016-04-01

    6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

  9. Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT

    Directory of Open Access Journals (Sweden)

    Wu Anna M

    2009-12-01

    Full Text Available Abstract Background Antibody Directed Enzyme Prodrug Therapy (ADEPT can be used to generate cytotoxic agents at the tumor site. To date non-human enzymes have mainly been utilized in ADEPT. However, these non-human enzymes are immunogenic limiting the number of times that ADEPT can be administered. To overcome the problem of immunogenicity, a fully human enzyme, capable of converting a non-toxic prodrug to cytotoxic drug was developed and joined to a human tumor specific scFv yielding a fully human targeting agent. Methods A double mutant of human purine nucleoside phosphorylase (hDM was developed which unlike the human enzyme can cleave adenosine-based prodrugs. For tumor-specific targeting, hDM was fused to the human anti-HER2/neu single chain Fv (scFv, C6 MH3B1. Enzymatic activity of hDM with its natural substrates and prodrugs was determined using spectrophotomeric approaches. A cell proliferation assay was used to assess the cytotoxicity generated following conversion of prodrug to drug as a result of enzymatic activity of hDM. Affinity of the targeting scFv, C6 MH3B1 fused to hDM to Her2/neu was confirmed using affinity chromatography, surface plasmon resonance, and flow-cytometry. Results In vitro hDM-C6 MH3B1 binds specifically to HER2/neu expressing tumor cells and localizes hDM to tumor cells, where the enzymatic activity of hDM-C6 MH3B1, but not the wild type enzyme, results in phosphorolysis of the prodrug, 2-fluoro-2'-deoxyadenosine to the cytotoxic drug 2-fluoroadenine (F-Ade causing inhibition of tumor cell proliferation. Significantly, the toxic small drug diffuses through the cell membrane of HER2/neu expressing cells as well as cells that lack the expression of HER2/neu, causing a bystander effect. F-Ade is toxic to cells irrespective of their growth rate; therefore, both the slowly dividing tumor cells and the non-dividing neighboring stromal cells that support tumor growth should be killed. Analysis of potential novel MHCII

  10. Novel purine nucleoside analogues for hematological malignancies.

    Science.gov (United States)

    Korycka, Anna; Lech-Marańda, Ewa; Robak, Tadeusz

    2008-06-01

    Recently, the search for more effective and safer antineoplastic agents has led to synthesis and introduction into preclinical and clinical studies of a few new purine nucleoside analogues (PNA). Three of them: clofarabine (CAFdA), nelarabine, and forodesine (immucillin H, BCX-1777), despite belonging to the same group of drugs such as PNA, have shown some differences concerning their active forms, metabolic properties and mechanism of action. However, all these drugs have demonstrated promising activity in patients with relapsed and refractory acute lymphoblastic leukemia (ALL). CAFdA was approved for the therapy of relapsed or refractory ALL in the third line of treatment. It has proved promising in pediatric patients as well as in some patients who are able to proceed to allogenic hematopietic stem cell transplantation (HSCT). Moreover, the drug exhibits an efficacy in acute myeloid leukemia (AML), blast crisis of chronic myelogenous leukemia (CML-BP) and myelodysplastic syndrome (MDS). Nelarabine is recommended for T-ALL and T-cell lymphoblastic lymphoma (T-LBL) with the overall response rates ranging from 11 to 60%. However, the use of the drug is limited by potentially severe neurotoxicity. Forodesine is a purine nucleoside phosphorylase (PNP) inhibitor and it has shown activity in relapsed and refractory T- and B-cells leukemias as well as in cutaneous T-cell lymphoma (CTCL). Recently patented, a few of inventions in the field of pharmaceutical preparation of new PNA have also been published. Great hopes are currently set on the use of these drugs in the treatment of lymphoid and myeloid malignancies in adult and in pediatric patients, however ongoing studies will help to define their role in the standard therapy.

  11. Development, validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase.

    Science.gov (United States)

    Cattaneo, Giulia; Ubiali, Daniela; Calleri, Enrica; Rabuffetti, Marco; Höfner, Georg C; Wanner, Klaus T; De Moraes, Marcela C; Martinelli, Leonardo K B; Santos, Diógenes Santiago; Speranza, Giovanna; Massolini, Gabriella

    2016-11-02

    Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC 2.4.2.1) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The enzymatic assay (v = 0.5 mL, enzymeplate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met the criteria of the CDER guidance of FDA. Kinetic parameters were in agreement with those reported in literature (HsPNP KM = 0.150 ± 0.020 mM vs 0.133 ± 0.015 mM; MtbPNP KM = 0.060 ± 0.009 mM vs 0.040 ± 0.003 mM for Ino), thus demonstrating the reliability of the newly developed enzymatic assay. Preliminary inhibition assays confirmed the effects reported for Acyclovir (Acv) and Formycin A (FA) against HsPNP and MtbPNP. The validated enzymatic assay was applied to the evaluation of a set of 8-halo-, 8-amino-, 8-O-alkyl-substituted purine ribonucleosides synthesized on purpose as potential inhibitors against MtbPNP. The assayed 8-substituted ribonucleosides did not exert a significant inhibitory effect against the tested enzymes up to 1 mM.

  12. Genetics Home Reference: purine nucleoside phosphorylase deficiency

    Science.gov (United States)

    ... disorders that damage the immune system and cause severe combined immunodeficiency (SCID). People with SCID lack virtually all immune ... Management Formal Diagnostic Criteria (1 link) ACT Sheet: Severe Combined Immunodeficiency (SCID) and Conditions Associated with T Cell Lymphoneia ( ...

  13. An Efficient and Facile Methodology for Bromination of Pyrimidine and Purine Nucleosides with Sodium Monobromoisocyanurate (SMBI

    Directory of Open Access Journals (Sweden)

    Roger Stromberg

    2013-10-01

    Full Text Available An efficient and facile strategy has been developed for bromination of nucleosides using sodium monobromoisocyanurate (SMBI. Our methodology demonstrates bromination at the C-5 position of pyrimidine nucleosides and the C-8 position of purine nucleosides. Unprotected and also several protected nucleosides were brominated in moderate to high yields following this procedure.

  14. A high-yielding, strictly regioselective prebiotic purine nucleoside formation pathway.

    Science.gov (United States)

    Becker, Sidney; Thoma, Ines; Deutsch, Amrei; Gehrke, Tim; Mayer, Peter; Zipse, Hendrik; Carell, Thomas

    2016-05-13

    The origin of life is believed to have started with prebiotic molecules reacting along unidentified pathways to produce key molecules such as nucleosides. To date, a single prebiotic pathway to purine nucleosides had been proposed. It is considered to be inefficient due to missing regioselectivity and low yields. We report that the condensation of formamidopyrimidines (FaPys) with sugars provides the natural N-9 nucleosides with extreme regioselectivity and in good yields (60%). The FaPys are available from formic acid and aminopyrimidines, which are in turn available from prebiotic molecules that were also detected during the Rosetta comet mission. This nucleoside formation pathway can be fused to sugar-forming reactions to produce pentosides, providing a plausible scenario of how purine nucleosides may have formed under prebiotic conditions.

  15. Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36

    CSIR Research Space (South Africa)

    Visser, Daniel F

    2010-03-01

    Full Text Available medium was as follows: 14.6 g/l K2HPO4, 2 g/l (NH4)2SO4, 3.6 g/l Na2HPO4, 2.5 g/l citric acid, 1.2 g/l MgSO4, 5 g/l NH4NO3, and 20 g/l yeast extract. Glucose (17.5 g/l) and trace element solution (5 ml/l) was sterilized separately and added... reaction mixtures (1 ml) containing 1 mM guanosine in 50 mM universal buffer (50 mM Tris, 50 mM boric acid, 33 mM citric acid 50 mM Na2PO4), adjusted to pH values between 3 and 11 with either HCl or NaOH. PNPase (0.025 U) was added to ini- tiate...

  16. Enhancement of peripheral nerve regrowth by the purine nucleoside analog and cell cycle inhibitor, roscovitine

    Directory of Open Access Journals (Sweden)

    Vincent Law

    2016-10-01

    Full Text Available Peripheral nerve regeneration is a slow process that can be associated with limited outcomes and thus, a search for novel and effective therapy for peripheral nerve injury and disease is crucial. Here, we found that roscovitine, a synthetic purine nucleoside analog, enhances neurite outgrowth in neuronal-like PC12 cells. Furthermore, ex vivo analysis of pre-injured adult rat dorsal root ganglion neurons showed that roscovitine enhances neurite regrowth in these cells. Likewise, in vivo transected sciatic nerves in rats locally perfused with roscovitine had augmented repopulation of new myelinated axons beyond the transection zone. By mass spectrometry, we found that roscovitine interacts with tubulin and actin. It interacts directly with tubulin and causes a dose-dependent induction of tubulin polymerization as well as enhances GTP-dependent tubulin polymerization. Conversely, roscovitine interacts indirectly with actin and counteracts the inhibitory effect of Cdk5 on Arp2/3-dependent actin polymerization, and thus, causes actin polymerization. Moreover, in the presence of neurotrophic factors such as NGF, roscovitine-enhanced neurite outgrowth is mediated by increased activation of the ERK1/2 and p38 MAPK pathways. Since microtubule and F-actin dynamics are critical for axonal regrowth, the ability of roscovitine to activate the ERK1/2 and p38 MAPK pathways, and support polymerization of tubulin and actin indicate a major role for this purine nucleoside analog in the promotion of axonal regeneration. Together, our findings demonstrate a therapeutic potential for the purine nucleoside analog, roscovitine, in peripheral nerve injury.

  17. Enhancement of Peripheral Nerve Regrowth by the Purine Nucleoside Analog and Cell Cycle Inhibitor, Roscovitine.

    Science.gov (United States)

    Law, Vincent; Dong, Sophie; Rosales, Jesusa L; Jeong, Myung-Yung; Zochodne, Douglas; Lee, Ki-Young

    2016-01-01

    Peripheral nerve regeneration is a slow process that can be associated with limited outcomes and thus a search for novel and effective therapy for peripheral nerve injury and disease is crucial. Here, we found that roscovitine, a synthetic purine nucleoside analog, enhances neurite outgrowth in neuronal-like PC12 cells. Furthermore, ex vivo analysis of pre-injured adult rat dorsal root ganglion (DRG) neurons showed that roscovitine enhances neurite regrowth in these cells. Likewise, in vivo transected sciatic nerves in rats locally perfused with roscovitine had augmented repopulation of new myelinated axons beyond the transection zone. By mass spectrometry, we found that roscovitine interacts with tubulin and actin. It interacts directly with tubulin and causes a dose-dependent induction of tubulin polymerization as well as enhances Guanosine-5'-triphosphate (GTP)-dependent tubulin polymerization. Conversely, roscovitine interacts indirectly with actin and counteracts the inhibitory effect of cyclin-dependent kinases 5 (Cdk5) on Actin-Related Proteins 2/3 (Arp2/3)-dependent actin polymerization, and thus, causes actin polymerization. Moreover, in the presence of neurotrophic factors such as nerve growth factor (NGF), roscovitine-enhanced neurite outgrowth is mediated by increased activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways. Since microtubule and F-actin dynamics are critical for axonal regrowth, the ability of roscovitine to activate the ERK1/2 and p38 MAPK pathways and support polymerization of tubulin and actin indicate a major role for this purine nucleoside analog in the promotion of axonal regeneration. Together, our findings demonstrate a therapeutic potential for the purine nucleoside analog, roscovitine, in peripheral nerve injury.

  18. Enhancement of Peripheral Nerve Regrowth by the Purine Nucleoside Analog and Cell Cycle Inhibitor, Roscovitine

    Science.gov (United States)

    Law, Vincent; Dong, Sophie; Rosales, Jesusa L.; Jeong, Myung-Yung; Zochodne, Douglas; Lee, Ki-Young

    2016-01-01

    Peripheral nerve regeneration is a slow process that can be associated with limited outcomes and thus a search for novel and effective therapy for peripheral nerve injury and disease is crucial. Here, we found that roscovitine, a synthetic purine nucleoside analog, enhances neurite outgrowth in neuronal-like PC12 cells. Furthermore, ex vivo analysis of pre-injured adult rat dorsal root ganglion (DRG) neurons showed that roscovitine enhances neurite regrowth in these cells. Likewise, in vivo transected sciatic nerves in rats locally perfused with roscovitine had augmented repopulation of new myelinated axons beyond the transection zone. By mass spectrometry, we found that roscovitine interacts with tubulin and actin. It interacts directly with tubulin and causes a dose-dependent induction of tubulin polymerization as well as enhances Guanosine-5′-triphosphate (GTP)-dependent tubulin polymerization. Conversely, roscovitine interacts indirectly with actin and counteracts the inhibitory effect of cyclin-dependent kinases 5 (Cdk5) on Actin-Related Proteins 2/3 (Arp2/3)-dependent actin polymerization, and thus, causes actin polymerization. Moreover, in the presence of neurotrophic factors such as nerve growth factor (NGF), roscovitine-enhanced neurite outgrowth is mediated by increased activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways. Since microtubule and F-actin dynamics are critical for axonal regrowth, the ability of roscovitine to activate the ERK1/2 and p38 MAPK pathways and support polymerization of tubulin and actin indicate a major role for this purine nucleoside analog in the promotion of axonal regeneration. Together, our findings demonstrate a therapeutic potential for the purine nucleoside analog, roscovitine, in peripheral nerve injury.

  19. Hypoxanthine production by ischemic heart demonstrated by high pressure liquid chromatography of blood purine nucleosides and oxypurines

    NARCIS (Netherlands)

    E. Harmsen; J.W. de Jong (Jan Willem); P.W.J.C. Serruys (Patrick)

    1981-01-01

    textabstractAn isocratic high pressure liquid chromatographic system was developed for the estimation of purine nucleosides and oxypurines in blood. Use was made of a reversed-phase column. Nucleotides derived from erythrocytes affected the separation; these compounds were removed with A12O3. The

  20. Early Cellular Responses of Purine Nucleoside-mediated Protection of Hypoxia-induced Injuries of Neuronal PC12 Cells

    Directory of Open Access Journals (Sweden)

    Bettina Tomaselli

    2005-01-01

    Full Text Available Hypoxia in brain may lead to cell death by apoptosis and necrosis. In parallel adenosine, a powerful endogenous neuroprotectant is formed. We wanted to investigate the effect of adenosine and its purine nucleoside relatives, inosine and guanosine on early cellular responses to hypoxia. O2-sensitive neuronal PC12-cells were subjected to chemical hypoxia induced with rotenone, an inhibitor of mitochondrial complex I. Loss of viability after hypoxic insult was impressively rescued by adenosine, guanosine and inosine. PC12-cells mainly express the A2A adenosine receptor. Its inhibition with a specific antagonist (CSC induced cell death of PC12-cells, which could be salvaged by adenosine but not with guanosine or inosine. We have previously demonstrated the important role of mitogen activated protein kinases 1/2 (p42/44 MAPK in purine-mediated rescue. In this study we were interested in the involvement of protein kinases whose activities mediate these processes, including protein kinase A (PKA, phosphoinositide 3-kinase (PI3-K and protein kinase C-related kinases (PRK 1/2. Pharmacological inhibition of PKA and PI3-K increased hypoxia-induced toxicity and likewise also affected the rescue by purine nucleosides. Nerve growth factor (NGF and purine nucleosides induced an activation of PRK 1/2, which to our knowledge indicates for the first time that these kinases are potentially involved in purine nucleoside-mediated rescue of hypoxic neuronal cells. Results suggest that A2A receptor expressing cells are mainly dependent on the purine nucleoside adenosine for their rescue after hypoxic insult. In addition to PKA, PI3-K is an important effector molecule in A2A-mediated signaling and for the rescue of PC12-cells after hypoxic insult.

  1. Current Status of Older and New Purine Nucleoside Analogues in the Treatment of Lymphoproliferative Diseases

    Directory of Open Access Journals (Sweden)

    Pawel Robak

    2009-03-01

    Full Text Available For the past few years more and more new cytotoxic agents active in the treatment of hematological malignancies have been synthesized and become available for either in vitro studies or clinical trials. Among them the class of antineoplastic drugs belonging to the purine nucleoside analogues group (PNAs plays an important role. Three of them: pentostatin (DCF, cladribine (2-CdA and fludarabine (FA were approved by Food and Drug Administration (FDA for the treatment of hematological malignancies. Recently three novel PNAs: clofarabine (CAFdA, nelarabine (ara-G and forodesine (immucillin H, BCX-1777 have been synthesized and introduced into preclinical studies and clinical trials. These agents seem to be useful mainly for the treatment of human T-cell proliferative disorders and they are currently undergoing clinical trials in lymphoid malignancies. However, there are also several studies suggesting the role of these drugs in B-cell malignancies. This review will summarize current knowledge concerning the mechanism of action, pharmacologic properties, clinical activity and toxicity of PNAs accepted for use in clinical practice, as well as new agents available for clinical trials.

  2. Effect of purine nucleoside analogue-acyclovir on the sperm parameters and testosterone production in rats.

    Science.gov (United States)

    Movahed, Elham; Sadrkhanlou, Rajabali; Ahmadi, Abbas; Nejati, Vahid; Zamani, Zahra

    2013-04-01

    Acyclovir (ACV), a synthetic purine nucleoside analogue derived from guanosine, is known to be toxic to gonads and the aim of this study was to evaluate the effect of ACV on the sperm parameters and testosterone production in rat. In this experimental study, forty adult male Wistar rats (220 ± 20 g) were randomly divided into five groups (n=8 for each group). One group served as control and one group served as sham control [distilled water was intraperitoneally (i.p.) injected]. ACV was administered intraperitoneally in the drug treatment groups (4, 16 and 48 mg/kg/day) for 15 days. Eighteen days after the last injection, rats were sacrificed by CO2 inhalation. After that, cauda epididymides were removed surgically. At the end, sperm concentrations in the cauda epididymis, sperm motility, morphology, viability, chromatin quality and DNA integrity were analyzed. Serum testosterone concentrations were determined. The results showed that ACV did not affect sperm count, but decreased sperm motility and sperm viability at 16 and 48 mg/kg dose-levels. Sperm abnormalities increased at 48 mg/kg dose-level of ACV. Further, ACV significantly increases DNA damage at 16 and 48 mg/kg dose-levels and chromatin abnormality at all doses. Besides, a significant decrease in serum testosterone concentrations was observed at 16 and 48 mg/ kg doses. The present results highly support the idea that ACV induces testicular toxicity by adverse effects on the sperm parameters and serum level of testosterone in male rats.

  3. High yielding cascade enzymatic synthesis of 5-methyluridine using a novel Purine Nucleoside Phosphorylase, from Bacillus halodurans Alk36

    CSIR Research Space (South Africa)

    Vissser, D

    2010-09-01

    Full Text Available were amplified as previously described3,4. E.coli BL21(DE3) was used as the expression host. • Production of Enzymes in Batch Fermentation - E. coli UP and B. halodurans BHPNP1 were produced in batch fermentations using GMO media5. Crude extracts...

  4. Nicotinamide riboside phosphorylase from beef liver: purification and characterization.

    Science.gov (United States)

    Imai, T; Anderson, B M

    1987-04-01

    Nicotinamide riboside phosphorylase (NR phosphorylase) from beef liver has been purified to apparent homogeneity at 300-fold purification with a 35% yield. Kinetic constants for the enzyme-catalyzed phosphorolysis were as follows Knicotinamide riboside, 2.5 +/- 0.4 mM; Kinorganic phosphate, 0.50 +/- 0.12 mM; Vmax, 410 +/- 30 X 10(-6) mol min-1 mg protein-1, respectively. The molecular weights of the native enzyme and subunit structure were determined to be 131,000 and 32,000, respectively, suggesting the beef liver NR phosphorylase to be tetrameric in structure and consistent with the presence of identical subunits. The amino acid composition was shown to be very similar to that reported for human erythrocyte purine-nucleoside phosphorylase but differing considerably from that found for rat liver purine-nucleoside phosphorylase. In addition to catalytic activity with nicotinamide riboside, the beef liver enzyme catalyzed a phosphorolytic reaction with inosine and guanosine exhibiting activity ratios, nicotinamide riboside:inosine: guanosine of 1.00:0.35:0.29, respectively. These ratios of activity remained constant throughout purification of the beef liver enzyme and no separation of these activities was detected. Phosphorolysis of nicotinamide riboside was inhibited competitively by inosine (Ki = 75 microM) and guanosine (Ki = 75 microM). Identical rates of thermal denaturation of the beef liver enzyme were observed when determined for the phosphorolysis of either nicotinamide riboside or inosine. These observations coupled with studies of pH and specific buffer effects indicate the phosphorolysis of nicotinamide riboside, inosine, and guanosine to be catalyzed by the same enzyme.

  5. Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside.

    Science.gov (United States)

    Wei, Xiao-Kun; Ding, Qing-Bao; Zhang, Lu; Guo, Yong-Li; Ou, Lin; Wang, Chang-Lu

    2008-07-01

    Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.

  6. Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside

    Institute of Scientific and Technical Information of China (English)

    Xiao-kun WEI; Qing-bao DING; Lu ZHANG; Yong-li GUO; Lin OU; Chang-lu WANG

    2008-01-01

    Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells ofEnterobacter aerogenes DCJO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.

  7. Nicotinamide Riboside and Nicotinic Acid Riboside Salvage in Fungi and Mammals: QUANTITATIVE BASIS FOR Urh1 AND PURINE NUCLEOSIDE PHOSPHORYLASE FUNCTION IN NAD+METABOLISM*S⃞

    OpenAIRE

    Belenky, Peter; Christensen, Kathryn C.; Gazzaniga, Francesca; Pletnev, Alexandre A.; Brenner, Charles

    2009-01-01

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiate...

  8. Inosine, an Endogenous Purine Nucleoside, Suppresses Immune Responses and Protects Mice from Experimental Autoimmune Encephalomyelitis: a Role for A2A Adenosine Receptor.

    Science.gov (United States)

    Junqueira, Stella Célio; Dos Santos Coelho, Igor; Lieberknecht, Vicente; Cunha, Mauricio Peña; Calixto, João B; Rodrigues, Ana Lúcia S; Santos, Adair Roberto Soares; Dutra, Rafael Cypriano

    2016-04-30

    Multiple sclerosis (MS) is a T cell autoimmune, inflammatory, and demyelinating disease of the central nervous system (CNS). Currently available therapies have partially effective actions and numerous side reactions. Inosine, an endogenous purine nucleoside, has immunomodulatory, neuroprotective, and analgesic properties. Herein, we evaluated the effect of inosine on the development and progression of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Inosine (1 or 10 mg/kg, i.p.) was administrated twice a day for 40 days. Immunological and inflammatory responses were evaluated by behavioral, histological, immunohistochemical, ELISA, RT-PCR, and Western blotting analysis. The administration of inosine exerted neuroprotective effects against EAE by diminishing clinical signs, including thermal and mechanical hyperalgesia, as well as weight loss typical of the disease. These beneficial effects of inosine seem to be associated with the blockade of inflammatory cell entry into the CNS, especially lymphocytes, thus delaying the demyelinating process and astrocytes activation. In particular, up-regulation of IL-17 levels in the secondary lymphoid tissues, a result of EAE, was prevented by inosine treatment in EAE mice. Additionally, inosine consistently prevented A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. Altogether, these results allow us to propose that this endogenous purine might be a putative novel and helpful tool for the prevention of autoimmune and neurodegenerative diseases, such as MS. Thus, inosine could have considerable implications for future therapies of MS, and this study may represent the starting point for further investigation into the role of inosine and adenosinergic receptors in neuroinflammation processes. Graphical Abstract Preventive treatment with inosine inhibits the development and progression of EAE in C57Bl/6 mice. Furthermore, neuroinflammation and demyelinating processes

  9. Rare 2-Substituted Purine Nucleosides

    Science.gov (United States)

    1989-05-01

    acetonyinosine, AVS-002159) has been found to have very high activity (TI > 1000) against the Sandfly Fever Virus (Phlebovirus). Another compound (2...propenyl group is occurring at the slow step of the reaction, i.e. the transmetalation stage. Temperature appears to be an important factor in...exclusive formation of the 1-propenyl compound 79 is realized when the temperature is raised to 110 °C. Deprotection of 79 with trimethyIsilyl iodide in

  10. Rare 2-Substituted Purine Nucleosides

    Science.gov (United States)

    1987-10-17

    LimiteA ten US/^oy3rnAnl /I 5 MONITORING ORGANIZATION REPORT NUMBER(S) 7a NAME OF MONITORING ORGANIZATION 7b A00RESS(Ofy. State, and ZIP Code...t). EtjN, CH,CN O Ac’° „ HN-^VN-\\ POCI OCH, OCH, H,C=CH PdCI^CHjCN), N»OCH, tolutnt, A CH.OH {^U HOCH, .. PS AcOCH, AcO OAc

  11. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A;

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  12. Glutathione-dependent reduction of arsenate by glycogen phosphorylase a reaction coupled to glycogenolysis.

    Science.gov (United States)

    Németi, Balázs; Gregus, Zoltán

    2007-11-01

    Arsenate (As(V)) is reduced in the body to the more toxic arsenite (As(III)). We have shown that two enzymes catalyzing phosphorolytic cleavage of their substrates, namely purine nucleoside phosphorylase and glyceraldehyde-3-phosphate dehydrogenase, can reduce As(V) in presence of an appropriate thiol and their substrates. Another phosphorolytic enzyme that may also reduce As(V) is glycogen phosphorylase (GP). With inorganic phosphate (P(i)), GP catalyzes the breakdown of glycogen to glucose-1-phosphate; however, it also accepts As(V). Testing the hypothesis that GP can reduce As(V), we incubated As(V) with the phosphorylated GPa or the dephosphorylated GPb purified from rabbit muscle and quantified the As(III) formed from As(V) by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. In the presence of adenosine monophosphate (AMP), glycogen, and glutathione (GSH), both GP forms reduced As(V) at rates increasing with enzyme and As(V) concentrations. The As(V) reductase activity of GPa was 10-fold higher than that of GPb. However, incubating GPb with GP kinase and ATP (that converts GPb to GPa) increased As(V) reduction by phosphorylase up to the rate produced by GPa incubated under the same conditions. High concentration of inorganic sulfate, which activates GPb like phosphorylation, also promoted reduction of As(V) by GPb. As(V) reduction by GPa (like As(V) reduction in rats) required GSH. It also required glycogen (substrate for GP) and was stimulated by AMP (allosteric activator of GP) even at low micromolar concentrations. P(i), substrate for GP competing with As(V), inhibited As(III) formation moderately at physiological concentrations. Glucose-1-phosphate, the product of GP-catalyzed glycogenolysis, also decreased As(V) reduction. Summarizing, GP is the third phosphorolytic enzyme identified capable of reducing As(V) in vitro. For reducing As(V) by GP, GSH and glycogen are indispensable, suggesting that the reduction is

  13. Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites.

    Science.gov (United States)

    Gero, A M; Scott, H V; O'Sullivan, W J; Christopherson, R I

    1989-04-01

    The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.

  14. The chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

    Directory of Open Access Journals (Sweden)

    Ilja V. Fateev

    2014-07-01

    Full Text Available Two approaches to the synthesis of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyladenine (1, clofarabine were studied. The first approach consists in the chemical synthesis of 2-deoxy-2-fluoro-α-D-arabinofuranose-1-phosphate (12a, 2FAra-1P via three step conversion of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranose (9 into the phosphate 12a without isolation of intermediary products. Condensation of 12a with 2-chloroadenine catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP resulted in the formation of clofarabine in 67% yield. The reaction was also studied with a number of purine bases (2-aminoadenine and hypoxanthine, their analogues (5-aza-7-deazaguanine and 8-aza-7-deazahypoxanthine and thymine. The results were compared with those of a similar reaction with α-D-arabinofuranose-1-phosphate (13a, Ara-1P. Differences of the reactivity of various substrates were analyzed by ab initio calculations in terms of the electronic structure (natural purines vs analogues and stereochemical features (2FAra-1P vs Ara-1P of the studied compounds to determine the substrate recognition by E. coli nucleoside phosphorylases. The second approach starts with the cascade one-pot enzymatic transformation of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a, followed by its condensation with 2-chloroadenine thereby affording clofarabine in ca. 48% yield in 24 h. The following recombinant E. coli enzymes catalyze the sequential conversion of 2-deoxy-2-fluoro-D-arabinose into the phosphate 12a: ribokinase (2-deoxy-2-fluoro-D-arabinofuranose-5-phosphate, phosphopentomutase (PPN; no 1,6-diphosphates of D-hexoses as co-factors required (12a, and finally PNP. The substrate activities of D-arabinose, D-ribose and D-xylose in the similar cascade syntheses of the relevant 2-chloroadenine nucleosides were studied and compared with the activities of 2-deoxy-2-fluoro-D-arabinose. As expected, D-ribose exhibited the best substrate

  15. Drug: D10432 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D10432 Drug Ulodesine succinate (USAN); Ulodesine hemisuccinate monohydrate (C12H16...N4O3)2. C4H6O4. 2H2O 682.2922 682.6795 D10432.gif Treatment of gout purine-nucleoside phosphorylase (PNP) in...gs [BR:br08310] Enzymes Transferases purine-nucleoside phosphorylase (PNP) [HSA:4860] [KO:K03783] Ulodesine D10432

  16. Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Abou Hachem, Maher; Petersen, Bent O.

    2010-01-01

    Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with gluco...

  17. Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

    NARCIS (Netherlands)

    Nakai, H.; Abou Hachem, M.; Petersen, B.O.; Westphal, Y.; Mannerstedt, K.; Baumann, M.J.; Dilokpimol, A.; Schols, H.A.; Duus, J.O.; Svensson, B.

    2010-01-01

    Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from alpha-D-glucose 1-phosphate as donor with gluc

  18. Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

    NARCIS (Netherlands)

    Nakai, H.; Abou Hachem, M.; Petersen, B.O.; Westphal, Y.; Mannerstedt, K.; Baumann, M.J.; Dilokpimol, A.; Schols, H.A.; Duus, J.O.; Svensson, B.

    2010-01-01

    Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from alpha-D-glucose 1-phosphate as donor with gluc

  19. Synthesis of acyclic purine nucleoside compounds: Dimethyl 2-( 2-( 2-(alkylthio) -6-amino-9H-purin-9-yl) ethyl) malonate%无环嘌呤核苷化合物2-(2-(6-氨基-2-烷硫基-9H-嘌呤-9-基)乙基)丙二酸二甲酯的合成

    Institute of Scientific and Technical Information of China (English)

    王馨悦; 李顺来; 杜洪光

    2011-01-01

    以腺嘌呤(1)为原料,经过氧化、水解、还原、环合和与S-烷基化反应得到2-烷硫基腺嘌呤(6);甲烷三羧酸三乙酯与1,2-二溴乙烷反应得到3-溴丙烷-1,1,1-三羧酸三乙酯(7);6和7在碱性条件下反应得到3-(6-氨基-2-烷硫基-9H-嘌呤-9-基)丙烷-1,1,1-三羧酸三乙酯(8);8在甲醇钠作用下发生脱酯基和酯交换反应,得到目标化合物2-(2-(6-氨基-2-烷硫基-9H-嘌呤-9-基)乙基)丙二酸二甲酯(9).通过FT-IR、1H- NMR、13C-NMR及HRMS对6种新的无环嘌呤核苷化合物8和9的结构进行了确证.%Three 2-alkylthioadenines have been prepared from adenine in five steps; oxidation, hydrolysis, reduction, cycle closing and S-alkylation. Triethyl methanetricarboxylate was treated with 1,2-dibromoethane to yield tri-ethyl 3-bromopropane-1 ,1,1-tricarboxylate, which was reacted with the 2-alkylthioadenines in the presence of potassium carbonate to yield triethyl 3-(2-( alkylthio)-6-amino-9H-purin-9-yl) propane-1,1,1-tricarboxylate. Treatment of this material with sodium methoxide in methanol gave dimethyl 2-(2-(2-( alkylthio)-6-amino-9H-purin-9-yl) ethyl) malonate. Six new acyclic purine nucleoside compounds have been obtained and their structures were determined by FT-IR, 'H-NMR, i3C-NMR and high resolution mass spectrometry.

  20. The relation of starch phosphorylases to starch metabolism in wheat.

    Science.gov (United States)

    Schupp, Nicole; Ziegler, Paul

    2004-10-01

    Tissues of wheat (Triticum aestivum L., var. Star) exhibit three starch phosphorylase activity forms resolved by non-denaturing polyacrylamide gel affinity electrophoresis (P1, P2 and P3). Compartmentation analysis of young leaf tissues showed that P3 is plastidic, whereas P1 and P2 are cytosolic. P1 exhibits a strong binding affinity to immobilized glycogen upon electrophoresis, whereas P2 and the chloroplastic P3 do not. Cytosolic leaf phosphorylase was purified to homogeneity by affinity chromatography. The single polypeptide product constituted both the P1 and P2 activity forms. Probes for the detection of phosphorylase transcripts were derived from cDNA sequences of cytosolic and plastidic phosphorylases, and these-together with activity assays and a cytosolic phosphorylase-specific antiserum-were used to monitor phosphorylase expression in leaves and seeds. Mature leaves contained only plastidic phosphorylase, which was also strongly evident in the endosperm of developing seeds at the onset of reserve starch accumulation. Germinating seeds contained only cytosolic phosphorylase, which was restricted to the embryo. Plastidic phosphorylase thus appears to be associated with transitory leaf starch metabolism and with the initiation of seed endosperm reserve starch accumulation, but it plays no role in the degradation of the reserve starch. Cytosolic phosphorylase may be involved in the processing of incoming carbohydrate during rapid tissue growth.

  1. Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase.

    Science.gov (United States)

    Nakai, Hiroyuki; Petersen, Bent O; Westphal, Yvonne; Dilokpimol, Adiphol; Abou Hachem, Maher; Duus, Jens Ø; Schols, Henk A; Svensson, Birte

    2010-10-01

    Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (alpha/alpha)(6)-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with beta-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (alpha/alpha)(6)-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413-Glu421, His413-Ile418 and His413-Glu415 from loop 3, that include His413 and Glu415 presumably recognising the alpha-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426-Ala431 in TP and Thr419-Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (alpha1, alpha1)- and alpha-(1,2)-regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.

  2. Recent development of phosphorylases possessing large potential for oligosaccharide synthesis

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Kitaoka, Motomitsu; Svensson, Birte

    2013-01-01

    catalysts for efficient synthesis of particular oligosaccharides from a donor sugar 1-phosphate and suitable carbohydrate acceptors with strict regioselectivity. Although utilization of phosphorylases for oligosaccharide synthesis has been limited because only few different enzymes are known, recently...... the number of reported phosphorylases has gradually increased, providing the variation making these enzymes useful tools for efficient synthesis of diverse oligosaccharides.......Phosphorylases are one group of carbohydrate active enzymes involved in the cleavage and formation of glycosidic linkages together with glycoside hydrolases and sugar nucleotide-dependent glycosyltransferases. Noticeably, the catalyzed phosphorolysis is reversible, making phosphorylases suitable...

  3. Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Petersen, B.O.; Westphal, Y.

    2010-01-01

    of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413-Glu421, His413-Ile418 and His413-Glu415 from loop 3, that include His413 and Glu415 presumably recognising the alpha-anomeric O-1...... in yields superior of maltose by reverse phosphorolysis with (alpha 1, alpha 1)- and alpha-(1,2)- regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific...

  4. Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis

    DEFF Research Database (Denmark)

    Sprogøe, Desiree; van den Broek, Lambertus A M; Mirza, Osman

    2004-01-01

    phosphorylase from Bifidobacterium adolescentis (BiSP) refined at 1.77 A resolution. It represents the first 3D structure of a sucrose phosphorylase and is the first structure of a phosphate-dependent enzyme from the glycoside hydrolase family 13. The structure of BiSP is composed of the four domains A, B, B...

  5. Glucose 6-phosphate regulates hepatic glycogenolysis through inactivation of phosphorylase.

    Science.gov (United States)

    Aiston, Susan; Andersen, Birgitte; Agius, Loranne

    2003-06-01

    High glucose concentration suppresses hepatic glycogenolysis by allosteric inhibition and dephosphorylation (inactivation) of phosphorylase-a. The latter effect is attributed to a direct effect of glucose on the conformation of phosphorylase-a. Although glucose-6-phosphate (G6P), like glucose, stimulates dephosphorylation of phosphorylase-a by phosphorylase phosphatase, its physiological role in regulating glycogenolysis in intact hepatocytes has not been tested. We show in this study that metabolic conditions associated with an increase in G6P, including glucokinase overexpression and incubation with octanoate or dihydroxyacetone, cause inactivation of phosphorylase. The latter conditions also inhibit glycogenolysis. The activity of phosphorylase-a correlated inversely with the G6P concentration within the physiological range. The inhibition of glycogenolysis and inactivation of phosphorylase-a caused by 10 mmol/l glucose can be at least in part counteracted by inhibition of glucokinase with 5-thioglucose, which lowers G6P. In conclusion, metabolic conditions that alter the hepatic G6P content affect glycogen metabolism not only through regulation of glycogen synthase but also through regulation of the activation state of phosphorylase. Dysregulation of G6P in diabetes by changes in activity of glucokinase or glucose 6-phosphatase may be a contributing factor to impaired suppression of glycogenolysis by hyperglycemia.

  6. The effect of fluoropyrimidines with or without thymidine phosphorylase inhibitor on the expression of thymidine phosphorylase.

    NARCIS (Netherlands)

    Bruin, de M.; Capel, van T; Smid, K.; Fukushima, M; Hoekman, K.; Pinedo, H.M.; Peters, G.J.

    2004-01-01

    Thymidine phosphorylase (platelet-derived-endothelial-cell-growth-factor) catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate, activates 5'-deoxy-5-fluorouridine (5'DFUR) and inactivates trifluorothymidine (TFT). The effect of 5'DFUR and TFT with or without

  7. Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase

    NARCIS (Netherlands)

    Nakai, H.; Petersen, B.O.; Westphal, Y.; Dilokpimol, A.; Hachem, M.A.; Duus, J.O.; Schols, H.A.; Svensson, B.

    2010-01-01

    Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (a/a)6-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with ß-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about

  8. Modified 5-fluorouracil: Uridine phosphorylase inhibitor

    Science.gov (United States)

    Lashkov, A. A.; Shchekotikhin, A. A.; Shtil, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2016-09-01

    5-Fluorouracil (5-FU) is a medication widely used in chemotherapy to treat various types of cancer. Being a substrate for the reverse reaction catalyzed by uridine phosphorylase (UPase), 5-FU serves as a promising prototype molecule (molecular scaffold) for the design of a selective UPase inhibitor that enhances the antitumor activity of 5-FU and exhibits intrinsic cytostatic effects on cancer cells. The chemical formula of the new compound, which binds to the uracil-binding site and, in the presence of a phosphate anion, to the phosphate-binding site of UPase, is proposed and investigated by molecular simulation methods.

  9. The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.; Parker, William B.; Piskur, Jure; Serra, I.; Terreni, M.; Ealick, Steven E. (Cornell); (Pavia); (Lund); (Southern Research)

    2011-09-20

    Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

  10. Murine norovirus (MNV-1) exposure in vitro to the purine nucleoside analog Ribavirin increases quasispecies diversity.

    Science.gov (United States)

    Julian, Timothy R; Baugher, Joseph D; Rippinger, Christine M; Pinekenstein, Rebecca; Kolawole, Abimbola O; Mehoke, Thomas S; Wobus, Christiane E; Feldman, Andrew B; Pineda, Fernando J; Schwab, Kellogg J

    2016-01-04

    Ribavirin is a pharmaceutical antiviral used for the treatment of RNA virus infections including norovirus, hepatitis C virus, hepatitis E virus, Lassa virus, respiratory syncytial virus, and rhinovirus. Despite the drug's history and documented efficacy, the antiviral mechanism of Ribavirin remains unclear. Mechanisms proposed include depletion of the intracellular GTP pool, immunomodulatory effects, induction of error catastrophe, inhibition of viral polymerase activity, and/or inhibition of viral capping. In the present study, we leveraged deep sequencing data to demonstrate that Ribavirin increases murine norovirus (MNV-1) viral diversity. By serial passaging MNV-1 in RAW 264.7 cells for twenty generations in the presence of Ribavirin, we demonstrated statistically significant increases in both the number of unique haplotypes and the average pairwise difference (APD). Based on statistically significant differences in the probability of nucleotide mutations based on Roche 454 sequencing, we also demonstrated that single nucleotide substitutions are increased in the presence of Ribavirin. Finally, we demonstrated Ribavirin's impact on statistically significantly reducing the relative proportion of the dominant sequence within the quasispecies.

  11. Crystal structure of sucrose phosphorylase from Bifidobacterium adolescentis.

    NARCIS (Netherlands)

    Sprogoe, D.; Broek, van den L.A.M.; Mirza, O.; Kastrup, J.S.; Voragen, A.G.J.; Gajhede, M.; Skov, L.K.

    2004-01-01

    Around 80 enzymes are implicated in the generic starch and sucrose pathways. One of these enzymes is sucrose phosphorylase, which reversibly catalyzes the conversion of sucrose and orthophosphate to d-Fructose and a-d-glucose 1-phosphate. Here, we present the crystal structure of sucrose phosphoryla

  12. Increased hepatic glycogen synthetase and decreased phosphorylase in trained rats

    DEFF Research Database (Denmark)

    Galbo, H; Saugmann, P; Richter, Erik

    1979-01-01

    Rats were either physically trained by a 12 wk swimming program or were freely eating or weight matched, sedentary controls. Trained rats had a higher relative liver weight and total hepatic glycogen synthetase (EC 2.4.1.11) activity and a lower phosphorylase (EC 2.4.1.1) activity than the other...

  13. Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum.

    Science.gov (United States)

    Nakai, Hiroyuki; Hachem, Maher Abou; Petersen, Bent O; Westphal, Yvonne; Mannerstedt, Karin; Baumann, Martin J; Dilokpimol, Adiphol; Schols, Henk A; Duus, Jens Ø; Svensson, Birte

    2010-12-01

    Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→2)-D-glucopyranose, and β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl](n)-(1→3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as

  14. Stimulating effect of phosphatidic acid on autophosphorylation of phosphorylase kinase.

    Science.gov (United States)

    Negami, A I; Sasaki, H; Yamamura, H

    1985-09-16

    Autophosphorylation of phosphorylase kinase from rabbit skeletal muscle was stimulated by acidic phospholipids such as phosphatidic acid (PA), phosphatidylinositol, and phosphatidyl-serine. PA stimulated an initial velocity of autophosphorylation 3.8-fold. When fully autophosphorylated, about 11 mol of phosphate per tetramer (alpha beta gamma delta) were incorporated in the presence of PA and about 6.5 mol in the absence of PA. In the presence of PA (100 micrograms/ml), there was a concomitant enhancement of its kinase activity about 25-fold at pH 6.8. PA (100 micrograms/ml) sharply decreased an apparent Ka for Ca2+ on autophosphorylation from 4.0 X 10(-5) M to 1.0 X 10(-6) M. Available evidence indicates that the Ca2+-activated, PA-dependent autophosphorylation of phosphorylase kinase shows an ability to stimulate glycogen breakdown.

  15. Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods.

    Science.gov (United States)

    Schneider, E M; Becker, J U; Volkmann, D

    1981-02-01

    Phosphorylase was purified from young and senescent potato tubers. Antibodies raised against the enzyme from young tubers crossreacted with phosphorylase from old tissue, although the latter exhibited different physico-chemical properties. In polyacrylamide gel electrophoresis it migrated with higher mobility, its subunit molecular weight was determined in the range of 40,000 in contrast to 100,000 of the phosphorylase in young tubers. The enzyme of senescent tubers displayed an isoelectric point of 5.4 different from the one of young tubers with 5.0, and the diffusion coefficients of the two enzymes varied. The appearance of the phosphorylase form typical for senescent tissue is connected with changes in the intracellular localization as revealed by immunofluorescence. Before massive starch accumulation is initiated, non-vacuolated subepidermal cells contain antigenically active material in their cytoplasm. During starch accumulation in fully differentiated storage parenchyma, only amyloplasts fluoresce, indicating the presence of adsorbed phosphorylase protein. Cytoplasmic phosphorylase can be detected in the continuance of senescence and, finally, after 16 months of tuber storage, the particle-bound enzyme had mostly disappeared. Simultaneously, we observed membrane destruction and decomposition on the ultrastructural level. The phosphorylase from senescent potatoes is a converted molecule and seems to be formed by proteolytic cleavage. The location of phosphorylase in the amyloplasts during starch synthesis indicates that it also plays a role in starch synthesis and not only in its degradation.

  16. A deoxyadenylate kinase activity associated with polynucleotide phosphorylase from Micrococcus luteus

    Science.gov (United States)

    Craine, Jonathan E.; Klee, Claude B.

    1976-01-01

    We report here the presence of two enzymatic activities associated with highly purified preparations of polynucleotide phosphorylase from Micrococcus luteus. The first, a nuclease activity, which is not separated from the phosphorylase on hydroxylapatite, may be due to substitution of H2O for phosphate in the phosphorolysis reaction. The second ac tivity, a deoxyadenylate kinase, the bulk of which is not resolved from the phosphorylase using gel filtration, sucrose density gradient centrifugation, DEAE-Sephadex, or hydroxylapatite chromatography, may represent a new activity of polynucleotide phosphorylase or be due to an enzyme which is tightly bound to the phosphorylase. Several properties of the kinase are described and its possible significance with respect to the overall enzyme mechanism is discussed. PMID:188014

  17. Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

    Science.gov (United States)

    Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-09-01

    Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

  18. Route of administration of pentobarbital affects activity of liver glycogen phosphorylase

    DEFF Research Database (Denmark)

    Mikines, K J; Sonne, B; Richter, Erik

    1986-01-01

    pentobarbital (5 mg/100 g body wt) either intraperitoneally, as a slow intravenous infusion, or as an intravenous or intracardial bolus. Times from administration of barbiturate to sampling of the liver were 10 min, 10 min, 85 +/- 32 s (mean +/- SE), and 53 +/- 10 s, respectively. Phosphorylase a activity...... in % of total phosphorylase activity was 40 +/- 2, 56 +/- 4, 82 +/- 3, and 92 +/- 2, respectively, all significantly different. Thus the route of administration of pentobarbital affects the phosphorylase a activity and should be considered when evaluating this activity. This fact can only be partially explained...

  19. Mitochondrial neurogastrointestinal encephalomyopathy caused by thymidine phosphorylase enzyme deficiency: From pathogenesis to emerging therapeutic options

    NARCIS (Netherlands)

    R. Yadak (Rana); P.S. Smitt; M.W. van Gisbergen; N.P. van Til (Niek); I.F.M. de Coo (René)

    2017-01-01

    textabstractMitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a progressive metabolic disorder caused by thymidine phosphorylase (TP) enzyme deficiency. The lack of TP results in systemic accumulation of deoxyribonucleosides thymidine (dThd) and deoxyuridine (dUrd). In these patients,

  20. Activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells and isolated hepatocytes by organophosphates.

    Science.gov (United States)

    Kauffman, F C; Davis, L H; Whittaker, M

    1990-01-15

    Several organophosphates including diisopropylfluorophosphonate (DPF) and a variety of compounds used as chemical warfare agents produced dose- and time-dependent increases in phosphorylase-a, the phosphorylated form of glycogen phosphorylase in rat pheochromocytoma cells, PC12, and isolated hepatocytes. Increases in phosphorylase-a did not occur in cells exposed to the carbamates, physostigmine or pyridostigmine, or to O-ethyl S-2-diisopropylaminoethylmethyl-phosphonathiolate (VX), an organophosphate which is protonated at physiological pH. When extracellular pH was increased to pH 8, VX acted like the other organophosphates and increased phosphorylase-a activity. The possibility that organophosphates increase phosphorylase-a in intact cells by releasing Ca2+ from intracellular binding sites is supported by the following findings: organophosphate-induced increases in phosphorylase-a did not correlate with changes in cyclic AMP in the two cell types studied; in PC12 cells, increases in this activity occurred in the absence of extracellular calcium and were not inhibited by the calcium channel blocker, verapamil; fluorescence of the calcium sensitive dye, Quin-2, in PC12 cells preloaded with the acetoxymethyl ester of the dye was increased by soman; finally, addition of the calcium ionophore, A23187, to PC12 cells maintained in calcium-free medium caused sarin-stimulated phosphorylase-a activity to return rapidly to basal levels. Collectively, these data argue strongly that organophosphates increase phosphorylase-a activity in intact cells via a novel mechanism involving release of calcium from intracellular binding sites.

  1. Precision Synthesis of Functional Polysaccharide Materials by Phosphorylase-Catalyzed Enzymatic Reactions

    Directory of Open Access Journals (Sweden)

    Jun-ichi Kadokawa

    2016-04-01

    Full Text Available In this review article, the precise synthesis of functional polysaccharide materials using phosphorylase-catalyzed enzymatic reactions is presented. This particular enzymatic approach has been identified as a powerful tool in preparing well-defined polysaccharide materials. Phosphorylase is an enzyme that has been employed in the synthesis of pure amylose with a precisely controlled structure. Similarly, using a phosphorylase-catalyzed enzymatic polymerization, the chemoenzymatic synthesis of amylose-grafted heteropolysaccharides containing different main-chain polysaccharide structures (e.g., chitin/chitosan, cellulose, alginate, xanthan gum, and carboxymethyl cellulose was achieved. Amylose-based block, star, and branched polymeric materials have also been prepared using this enzymatic polymerization. Since phosphorylase shows a loose specificity for the recognition of substrates, different sugar residues have been introduced to the non-reducing ends of maltooligosaccharides by phosphorylase-catalyzed glycosylations using analog substrates such as α-d-glucuronic acid and α-d-glucosamine 1-phosphates. By means of such reactions, an amphoteric glycogen and its corresponding hydrogel were successfully prepared. Thermostable phosphorylase was able to tolerate a greater variance in the substrate structures with respect to recognition than potato phosphorylase, and as a result, the enzymatic polymerization of α-d-glucosamine 1-phosphate to produce a chitosan stereoisomer was carried out using this enzyme catalyst, which was then subsequently converted to the chitin stereoisomer by N-acetylation. Amylose supramolecular inclusion complexes with polymeric guests were obtained when the phosphorylase-catalyzed enzymatic polymerization was conducted in the presence of the guest polymers. Since the structure of this polymeric system is similar to the way that a plant vine twines around a rod, this polymerization system has been named

  2. The cytosol activity of thymidine phosphorylase in endometrial cancer

    Directory of Open Access Journals (Sweden)

    Bieńkiewicz Andrzej

    2008-11-01

    Full Text Available Abstract Background Thymidine phosphorylase (TP is identical with platelet-derived endothelial cell growth factor (PD-ECGF which promotes angiogenesis. The aim of this study was to evaluate the cytosol activity of TP in tumor samples from patients with endometrial cancer. Methods The activity of TP was measured by the spectrophotometric method in the cytosol of endometrial tumor samples from 43 patients. Moreover, the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP protein and microvessel density (MD were examined in the same endometrial tumor samples by immunohistochemical staining. Normal endometrium from 16 women, treated surgically due to nononcological reasons served as a control. A relationship between the cytosol TP activity, PD-ECGF/TP protein expression, MD and clinicopathologic features was investigated. Results A significantly higher the cytosol TP activity, PD-ECGF/TP protein expression and MD was stated in malignant tumor samples when compared to the control (samples of normal endometrium. A positive statistically significant correlation between the cytosol enzyme activity and PD-ECGF/TP protein expression and MD was found, but weaker from the remaining ones between PD-ECGF/TP protein expression and MD was observed. Besides no correlation between the cytosol TP activity, PD-ECGF/TP protein expression as well as MD and grading or histopatological type of endometrial cancer was stated. Conclusion The cytosol TP activity in endometrial cancer is significantly higher than in normal endometrium, with no relation as to the stage and grade of tumors, but correlates with the PD-ECGF/TP protein expression and MD may therefore be associated with favorable prognosis in patients treated with chemo- or radiotherapy after surgery.

  3. X-ray structures of uridine phosphorylase from Vibrio cholerae in complexes with uridine, thymidine, uracil, thymine, and phosphate anion: Substrate specificity of bacterial uridine phosphorylases

    Science.gov (United States)

    Prokofev, I. I.; Lashkov, A. A.; Gabdulkhakov, A. G.; Balaev, V. V.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2016-11-01

    In many types of human tumor cells and infectious agents, the demand for pyrimidine nitrogen bases increases during the development of the disease, thus increasing the role of the enzyme uridine phosphorylase in metabolic processes. The rational use of uridine phosphorylase and its ligands in pharmaceutical and biotechnology industries requires knowledge of the structural basis for the substrate specificity of the target enzyme. This paper summarizes the results of the systematic study of the three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complexes with substrates of enzymatic reactions—uridine, phosphate anion, thymidine, uracil, and thymine. These data, supplemented with the results of molecular modeling, were used to consider in detail the structural basis for the substrate specificity of uridine phosphorylases. It was shown for the first time that the formation of a hydrogen-bond network between the 2'-hydroxy group of uridine and atoms of the active-site residues of uridine phosphorylase leads to conformational changes of the ribose moiety of uridine, resulting in an increase in the reactivity of uridine compared to thymidine. Since the binding of thymidine to residues of uridine phosphorylase causes a smaller local strain of the β-N1-glycosidic bond in this the substrate compared to the uridine molecule, the β-N1-glycosidic bond in thymidine is more stable and less reactive than that in uridine. It was shown for the first time that the phosphate anion, which is the second substrate bound at the active site, interacts simultaneously with the residues of the β5-strand and the β1-strand through hydrogen bonding, thus securing the gate loop in a conformation

  4. Interaction of muscle glycogen phosphorylase b reconstituted from apoenzyme and analogs of pyridoxal-5'-phosphate with specific ligands.

    Science.gov (United States)

    Chebotareva, N A; Sugrobova, N P; Bulanova, L N; Poznanskaya, A A; Kurganov, B I; Gunar, V I

    1996-04-01

    Phosphorylase b from rabbit skeletal muscles was reconstituted with analogs of PLP containing residues -CH(2)-CH(2)-COOH, trans-CH=CH-COOH or -C=-COOH at position 5. Replacing native coenzyme in the phosphorylase molecule with any PLP analog tested leads to the decrease in the enzyme affinity for the allosteric inhibitor, FMN. Phosphorylase b reconstituted with analogs of PLP shows the greater ability for association in tetramers in the presence of 1 mM AMP than native enzyme.

  5. Studies on responsiveness of hepatoma cells to catecholamines. IV. Lack of adrenergic activation of phosphorylase in rat ascites hepatoma cells.

    Science.gov (United States)

    Miyamoto, K; Yanaoka, T; Sanae, F; Wakusawa, S; Koshiura, R

    1986-10-01

    Glycogen phosphorylase a activity in 7 rat ascites hepatoma cell lines treated with adrenergic agents, phenylephrine, epinephrine and isoproterenol, was investigated as compared with that in freshly isolated rat hepatocytes. Basal phosphorylase activities in hepatoma cells except AH7974 cells were lower than that in hepatocytes. Phosphorylase in hepatoma cells was not activated by any of the agents, while the enzyme activity in hepatocytes was clearly increased in a dose- and time-dependent manner. Phosphorylase in hepatocytes was sensitive to glucagon, but it was found to be insensitive to glucagon in all hepatoma cells. The present results suggest that rat ascites hepatoma cells may escape the glycogenolytic regulation by catecholamines and glucagon.

  6. Two nucleoside uptake systems in Lactococcus lactis: Competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools

    DEFF Research Database (Denmark)

    Martinussen, Jan; Wadskov-Hansen, Steen Lyders Lerche; Hammer, Karin

    2003-01-01

    in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K. for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition...

  7. Localization of thymidine phosphorylase in advanced gastric and colorectal cancer.

    Science.gov (United States)

    Kobayashi, Michiya; Okamoto, Ken; Akimori, Toyokazu; Tochika, Naoshige; Yoshimoto, Tadashi; Okabayashi, Takehiro; Sugimoto, Takeki; Araki, Keijiro

    2004-01-01

    Thymidine phosphorylase (TP) is known to be more concentrated in human cancer tissues than in adjacent normal tissue based on findings using enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. However, the ultrastructural localization of TP in cancer tissues has not previously been demonstrated. We investigated the localization of TP in gastric cancer and colorectal cancer tissue by ELISA, immunohistochemistry, and immunoelectron microscopy. Between April 1997 and May 2000, we obtained surgically resected specimens from 42, 46, and 36 cases of advanced gastric, colon, and rectal cancer, respectively. ELISA demonstrated that the TP level was higher in cancer tissues than in adjacent normal tissue. Immunohistochemically, cancer cells were positive for the enzyme in some cases. However, in a number of cases immunopositive inflammatory cells were also present in cancerous tissues. At the electron microscope level, TP was diffusely distributed in the cytoplasm of cancer cells and in the mitochondria of the neutrophil in gastric cancer tissue. In rectal cancer tissues, cytoplasmic granules in macrophages in cancer tissues were immunoreactive for the TP. These findings suggest that TP is produced by macrophages and exists in neutrophils and cancer cells.

  8. Synthesis of branched polyglucans by the tandem action of potato phosphorylase and Deinococcus geothermalis glycogen branching enzyme

    NARCIS (Netherlands)

    van der Vlist, Jeroen; Reixach, Marta Palomo; van der Maarel, Marc; Dijkhuizen, Lubbert; Schouten, Arend Jan; Loos, Katja

    2008-01-01

    An enzymatic tandem reaction is described in which the enzymes phosphorylase and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) catalyze the synthesis of branched polyglucans from glucose-1-phosphate (G-1-P). Phosphorylase consumes G-1-P and polymerizes linear amylose while DR GBE intro

  9. Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

    Energy Technology Data Exchange (ETDEWEB)

    Ting, L; Shi, W; Lewandowicz, A; Singh, V; Mwakingwe, A; Birck, M R; Taylor Ringia, E A; Bench, G; Madrid, D C; Tyler, P C; Evans, G B; Furneaux, R H; Schramm, V L; Kim, K

    2004-05-19

    Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of the polyamine pathway are recycled in a novel pathway in which 5'-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5'-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5'-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not previously been described. 5'-methylthio-Immucillin-H, a transition state analogue inhibitor that is selective for malarial over human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may have application as antimalarials.

  10. Fetal-onset severe skeletal muscle glycogenosis associated with phosphorylase-b kinase deficiency.

    Science.gov (United States)

    Bührer, C; van Landeghem, F; Brück, W; Felderhoff-Müser, U; Vorgerd, M; Obladen, M

    2000-04-01

    We report on a premature newborn girl delivered after 32 weeks of gestation by cesarean section after sparse limb movements, fetal tachycardia and late heart rate decelerations had suggested fetal distress. Following 1 day of mechanical ventilation, adequate pulmonary gas exchange was achieved by spontaneous breathing. Main symptoms were virtually complete absence of spontaneous movements, increased flexor tonus of the extremities, and hypotonia of the trunk. Inability to suck or swallow required nasogastric gavage feeding. There were no hypoglycemic episodes. Echocardiography revealed normal myocardial function. Creatine kinase was 237 U/I at 2 days of life, declining to normal values thereafter. Muscle biopsy revealed increased glycogen storage with subsarcolemmal glycogen deposits and low phosphorylase-a activity while total phosphorylase was normal after in vitro activation, suggestive of phosphorylase-b kinase deficiency. No mutation was detected in exon 1 of the myophosphorylase gene. No psychomotor development was observed, and the infant died of central apnea at 3 months of age.

  11. Synthesis, screening and docking of small heterocycles as glycogen phosphorylase inhibitors.

    Science.gov (United States)

    Schweiker, Stephanie S; Loughlin, Wendy A; Lohning, Anna S; Petersson, Maria J; Jenkins, Ian D

    2014-09-12

    A series of morpholine substituted amino acids (phenylalanine, leucine, lysine and glutamic acid) was synthesized. A fragment-based screening approach was then used to evaluate a series of small heterocycles, including morpholine, oxazoline, dihydro-1,3-oxazine, tetrahydro-1,3-oxazepine, thiazoline, tetrahydro-1,3-pyrimidine, tetrahydro-1,3-diazepine and hexahydro-1H-benzimidazole, as potential inhibitors of Glycogen Phosphorylase a. Thiazoline 7 displayed an improved potency (IC50 of 25 μM) and had good LE and LELP values, as compared to heterocycles 1, 5, 9-13 and 19 (IC50 values of 1.1 mM-23.9 mM). A docking study using the crystal structure of human liver Glycogen Phosphorylase, provided insight into the interactions of heterocycles 5, 7, 9-13 and 19 with Glycogen Phosphorylase.

  12. Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway

    Science.gov (United States)

    Torini, Juliana Roberta; Brandão-Neto, José; DeMarco, Ricardo; Pereira, Humberto D'Muniz

    2016-01-01

    Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. 2.4.2.28) is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity. PMID:27935959

  13. Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5'-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway.

    Science.gov (United States)

    Torini, Juliana Roberta; Brandão-Neto, José; DeMarco, Ricardo; Pereira, Humberto D'Muniz

    2016-12-01

    Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. 2.4.2.28) is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity.

  14. In situ enzymatic removal of orthophosphate by the nucleoside phosphorylase catalyzed phosphorolysis of nicotinamide riboside.

    Science.gov (United States)

    Shriver, J W; Sykes, B D

    1982-09-01

    An enzymatic orthophosphate removal system is described which can be effectively used to continuously remove orthophosphate from biochemical samples. The phosphorolysis of nicotinamide riboside is catalyzed by calf spleen nucleoside phosphorylase to give ribose-1-PO4 and nicotinamide along with a proton. At pH 8 the production of ribose-1-PO4 from orthophosphate is essentially quantitative. This reaction can be monitored optically or by 31P nuclear magnetic resonance (NMR). Equations are given for determining the time required to remove a given amount of phosphate from a typical NMR sample with a known amount of nucleoside phosphorylase. The effects of a competing orthophosphate-producing reaction are considered.

  15. Active site conformational dynamics in human uridine phosphorylase 1.

    Directory of Open Access Journals (Sweden)

    Tarmo P Roosild

    Full Text Available Uridine phosphorylase (UPP is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 A resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an "induced-fit" association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.

  16. Autogenous regulation of Escherichia coli polynucleotide phosphorylase expression revisited.

    Science.gov (United States)

    Carzaniga, Thomas; Briani, Federica; Zangrossi, Sandro; Merlino, Giuseppe; Marchi, Paolo; Dehò, Gianni

    2009-03-01

    The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5' end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5' double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5' end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5' fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.

  17. Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase Expression Revisited▿ †

    Science.gov (United States)

    Carzaniga, Thomas; Briani, Federica; Zangrossi, Sandro; Merlino, Giuseppe; Marchi, Paolo; Dehò, Gianni

    2009-01-01

    The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5′-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5′ end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5′ double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5′ end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5′ fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript. PMID:19136586

  18. Polymer phosphorylases: clues to the emergence of non-replicative and replicative polymers.

    Science.gov (United States)

    Freire, Miguel Angel

    2011-12-01

    Polymer formation is arguably one of the essential factors that allowed the emergence, stabilisation and spread of life on Earth. Consequently, studies concerning biopolymers could shed light on the origins of life itself. Of particular interest are RNA and polysaccharide polymers, the archetypes of the contrasting proposed evolutionary scenarios and their respective polymerases. Nucleic acid polymerases were hypothesised, before their discovery, to have a functional similarity with glycogen phosphorylase. Further identification and characterisation of nucleic acid polymerases; particularly of polynucleotide phosphorylase (PNPase), provided experimental evidence for the initial premise. Once discovered, frequent similarities were found between PNPase and glycogen phosphorylase, in terms of catalytic features and biochemical properties. As a result, PNPase was seen as a model of primitive polymerase and used in laboratory precellular systems. Paradoxically, however, these similarities were not sufficient as an argument in favour of an ancestral common polymerisation mechanism prior to polysaccharides and polyribonucleotides. Here we present an overview of the common features shared by polymer phosphorylases, with new proposals for the emergence of polysaccharide and RNA polymers.

  19. Is muscle glycogenolysis impaired in X-linked phosphorylase b kinase deficiency?

    DEFF Research Database (Denmark)

    Orngreen, M.C.; Schelhaas, H.J.; Jeppesen, T.D.;

    2008-01-01

    OBJECTIVE: It is unclear to what extent muscle phosphorylase b kinase (PHK) deficiency is associated with exercise-related symptoms and impaired muscle metabolism, because 1) only four patients have been characterized at the molecular level, 2) reported symptoms have been nonspecific, and 3) lact...

  20. Role of phosphorylase in the mechanism of potato minituber storage cell changes during clinorotation

    Science.gov (United States)

    Nedukha, O.; Shnyukova, E.

    The differences between the cytochemical reaction intensity and activity of phosphorylase (EC 2.4.1.1) and carbohydrate content in storage parenchyma cells of Solanum tuberosum L. (cv Adreta) minitubers grown for 30 days in the horizontal clinostate (2 rev/min) and in the control have been studied by electroncytochemical and biochemical methods. It is established an acceleration of minitubers formation and storage parenchyma cell differentiation at clinorotation. Electroncytochemical investigation of phosphorylase activity localization in the storage parenchyma cells of minitubers grown in control and at clinorotation showed the product of the reaction as electron-dense precipitate was marked plastids. Intensity and density of precipitate was increase in stroma of plastids and on starch grain surface during of intensive growth of starch in amyloplast (on 10- and 20-days of the minituber formation) of clinorotated minitubers in comparison with that in the control. The precipitate amount was decreased in the plastids on 30 day of growth in both variants. Using biochemical methods it is found that activity of phosphorylase and content of mono- and disaccharide and also starch content changed in minitubers formed during clinorotation and in the control. Data obtained are discussed regarding the possible mechanism of phosphorylase activity change and the role of mono- and disaccharide in acceleration of storage organ formation during clinorotation.

  1. Indicator cell lines for the detection of hidden mycoplasma contamination, using an adenosine phosphorylase screening test

    NARCIS (Netherlands)

    Spierenburg, G.T.; Polak-Vogelzang, A.A.; Bast, B.J.E. G.

    Mycoplasmas are a major cause of cell culture contamination and are especially troublesome during HAT selection. The enzyme adenosine phosphorylase (adoP) is present in all common mycoplasma species but is considered to have a low activity in mammalian cells. However, using an adoP screening test,

  2. Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells

    NARCIS (Netherlands)

    Bijnsdorp, I. V.; Azijli, K.; Jansen, E. E.; Wamelink, M. M.; Jakobs, C.; Struys, E. A.; Fukushima, M.; Kruyt, F. A. E.; Peters, G. J.

    2010-01-01

    Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymi

  3. Structural rearrangements of sucrose phosphorylase from Bifidobacterium adolescentis during sucrose conversion

    DEFF Research Database (Denmark)

    Mirza, Osman; Henriksen, Lars Skov; Sprogøe, Desiree

    2006-01-01

    The reaction mechanism of sucrose phosphorylase from Bifidobacterium adolescentis (BiSP) was studied by site-directed mutagenesis and x-ray crystallography. An inactive mutant of BiSP (E232Q) was co-crystallized with sucrose. The structure revealed a substrate-binding mode comparable with that seen...

  4. Role of platelet-derived enclothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity

    NARCIS (Netherlands)

    de Bruin, M; van Capel, T; Van der Born, K; Kruyt, FA; Fukushinna, M; Hoekman, K; Pinedo, HM; Peters, GJ

    2003-01-01

    Platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (TP) catalyses the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate and is involved in the metabolism of fluoropyrimidines. It can also activate 5'-deoxyfluorouridine (5'DFUR) and possibl

  5. Is muscle glycogenolysis impaired in X-linked phosphorylase b kinase deficiency?

    NARCIS (Netherlands)

    Orngreen, M.C.; Schelhaas, H.J.; Jeppesen, T.D.; Akman, H.O.; Wevers, R.A.; Andersen, S.T.; Laak, H.J. ter; Diggelen, OP van; DiMauro, S.; Vissing, J.

    2008-01-01

    OBJECTIVE: It is unclear to what extent muscle phosphorylase b kinase (PHK) deficiency is associated with exercise-related symptoms and impaired muscle metabolism, because 1) only four patients have been characterized at the molecular level, 2) reported symptoms have been nonspecific, and 3) lactate

  6. Structure of the homodimer of uridine phosphorylase from Salmonella typhimurium in the native state at 1.9 Å resolution

    Science.gov (United States)

    Timofeev, V. I.; Pavlyuk, B. F.; Lashkov, A. A.; Seregina, T. A.; Gabdulkhakov, A. G.; Vaĭnshteĭn, B. K.; Mikhaĭlov, A. M.

    2007-11-01

    Uridine phosphorylase ( UPh) belongs to pyrimidine nucleoside phosphorylases. This enzyme catalyzes cleavage of the C-N glycoside bond in uridine to form uracil and ribose-1’-phosphate. Uridine phosphorylase supplies cells with nucleotide precursors by catalyzing the phosphorolysis of purine and pyrimidine nucleosides. This is an alternative to de novo nucleotide synthesis. The three-dimensional structure of native uridine phosphorylase from Salmonella typhimurium ( StUPh) in a new crystal form was solved and refined at 1.90 Å resolution ( R st = 20.37%; R free = 24.69%; the rmsd of bond lengths and bond angles are 0.009 Å and 1.223°, respectively). A homodimer containing two asynchronously functioning active sites was demonstrated to be the minimum structural unit necessary for function of the hexameric StUPh molecule ( L 33 L 2). Each active site is formed by amino acid residues of both subunits.

  7. RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression

    OpenAIRE

    Carzaniga, T.; Dehò, G; Briani, F.

    2015-01-01

    The complex posttranscriptional regulation mechanism of the Escherichia coli pnp gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for pnp autoregulation posit that the target of PNPase is a mature pnp mRNA previously processed at its 5′ end by RNase III, rather than the primary pnp transcript (RNase I...

  8. Recombinant production and biochemical characterization of a hyperthermostable α-glucan/maltodextrin phosphorylase from Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Rahman M. Mizanur

    2008-01-01

    Full Text Available Alpha-glucan phosphorylase catalyzes the reversible cleavage of α-1-4-linked glucose polymers into α-D-glucose-1-phosphate. We report the recombinant production of an α-glucan/maltodextrin phosphorylase (PF1535 from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 °C and pH 6.5–7. This archaeal protein retained its complete activity after 24 h at 80 °C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 °C in the presence of 10 mM maltoheptaose and at 10 mg ml–1 glycogen concentration was 52 U mg–1 and 31 U mg–1, respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 ± 2 mM and 0.60 ± 0.02 mM min–1 µg–1, respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.

  9. Engineering the specificity of trehalose phosphorylase as a general strategy for the production of glycosyl phosphates.

    Science.gov (United States)

    Chen, Chao; Van der Borght, Jef; De Vreese, Rob; D'hooghe, Matthias; Soetaert, Wim; Desmet, Tom

    2014-07-25

    A two-step process is reported for the anomeric phosphorylation of galactose, using trehalose phosphorylase as biocatalyst. The monosaccharide enters this process as acceptor but can subsequently be released from the donor side, thanks to the non-reducing nature of the disaccharide intermediate. A key development was the creation of an optimized enzyme variant that displays a strict specificity (99%) for β-galactose 1-phosphate as product.

  10. SOME RECENT FINDINGS IN THE BIOTECHNOLOGY OF BIOLOGICALLY IMPORTANT NUCLEOSIDES

    Directory of Open Access Journals (Sweden)

    A. Mikhailopulo

    2013-08-01

    Full Text Available Some recent findings in the biotechnology of biologically important nucleosides will be discussed, viz., (i a new strategy of the cascade one-pot transformation of D-pentoses into nucleosides based on the extension and deepening of the knowledge of the mechanism of functioning of the ribokinase, phosphopentomutase, and uridine, thymidine and purine nucleoside (PNP phosphorylases, and the role of different factors (structural, electronic, stereochemical in the glycoside bond formation, (ii the modern chemistries of the chemo-enzymatic syntheses of nucleosides, (iii the transglycosylation reaction using natural and sugar modified nucleosides as donors of carbohydrate residues and heterocyclic bases as acceptors catalyzed by nucleoside phosphorylases (NP.

  11. High phosphorylase activity is correlated with increased potato minituber formation and starch content during extended clinorotation

    Science.gov (United States)

    Nedukha, O. M.; Schnyukova, E. I.; Leach, J. E.

    2003-05-01

    The major purpose of these experiments were to investigate growth of potato storage organs and starch synthesis in minitubers at slow horizontal clinorotation (2 rpm), which partly mimics microgravity, and a secondary goal was to study the activity and localization of phosphorylase (EC 2.4.1.1) in storage parenchyma under these conditions. Miniplants of Solanum tuberosum L. (cv Adreta) were grown in culture for 30 days for both the vertical control and the horizontal clinorotation. During long-term clinorotation, an acceleration of minituber formation, and an increase of amyloplast number and size in storage parenchyma cells, as well as increased starch content, was observed in the minitubers. The differences among cytochemical reaction intensity, activity of phosphorylase, and carbohydrate content in storage parenchyma cells of minitubers grown in a horizontal clinostat were established by electron-cytochemical and biochemical methods. It is shown that high phosphorylase activity is correlated with increased starch content during extended clinorotation. The results demonstrate the increase in carbohydrate metabolism and possible accelerated growth of storage organs under the influence of microgravity, as mimicked by clinorotation; therefore, clinorotation can be used as a basis for future studies on mechanisms of starch synthesis under microgravity.

  12. 1,4-alpha-Glucan phosphorylase form Klebsiella pneumoniae covalently couple on porous glass.

    Science.gov (United States)

    Wengenmayer, F; Linder, D; Wallenfels, K

    1977-09-01

    A simplified procedure for the preparation of 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. An 80-fold purification is achieved in two steps with an overall yield of about 50%. The specific activity of the homogeneous enzyme protein is 17.7 units/mg. Compared with glycogen phosphorylase from rabbit muscle the enzyme from K. pneumoniae shows a markedly higher stability against deforming and chaotropic agents. The 1,4-alpha-glucan phosphorylase was covalently bound to porous glass particles by three different methods. Coupling with glutaraldehyde gave the highest specific activity, i.e., 5.6 units/mg of bound protein or 133 units/g of glass with maltodextrin as substrate. This corresponds to about 30% of the specific activity of the soluble enzyme. With substrates of higher molecular weight, such as glycogen or amylopectin, lower relative activity was observed. The immobilized enzyme preparations showed pH activity profiles which were slightly displaced to higher values and exhibited an increased temperature stability.

  13. 1, 4-alpha-Glucan phosphorylase from Klebsiella pneumoniae purification, subunit structure and amino acid composition.

    Science.gov (United States)

    Linder, D; Kurz, G; Bender, H; Wallenfels, K

    1976-11-01

    1. A 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae has been purified about 80-fold with an over-all yield greater than 35%. The purified enzyme has been shown to be homogeneous by gel electrophoresis at different pH-values, by isoelectric focusing, by dodecylsulfate electrophoresis and by ultracentrifugation. 2. The molecular weight of the native enzyme has been determined to be 180 000 by ultra-centrifugation studies, in good agreement with the value of 189 000 estimated by gel permeation chromatography. 3. The enzyme dissociates in the presence of 0.1% dodecylsulfate or 5 M guanidine hydrochloride into polypeptide chains. The molecular weight of these polypeptide chains has been found to be 88 000 by dodecylsulfate polyacrylamide gel electrophoresis and 99 000 by sedimentation equilibrium studies, indicating that the native enzyme is composed of two polypeptide chains. 4. The enzyme contains pyridoxalphosphate with a stoichiometry of two moles per 180 000 g protein, confirming that the 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is a dimeric enzyme. 5. The amino acid composition of the enzyme has been determined, and its correspondence to that of 1,4-alpha-glucan phosphorylases from other sources is discussed. 6. The pI of the enzyme has been shown to be 5.3 and its pH-optimum to be about pH 5.9. The enzyme is stable in the range from pH 5.9 to 10.5.

  14. [Properties of sucrose phosphorylase from recombinant Escherichia coli and enzymatic synthesis of alpha-arbutin].

    Science.gov (United States)

    Wan, Yuejia; Ma, Jiangfeng; Xu, Rong; He, Aiyong; Jiang, Min; Chen, Kequan; Jiang, Yin

    2012-12-01

    Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) can be produced by recombinant strain Escherichia coli Rosetta(DE3)/Pet-SPase. Crude enzyme was obtained from the cells by the high pressure disruption and centrifugation. Sucrose phosphorylase was purified by Ni-NTA affinity column chromatography and desalted by ultrafiltration. The specific enzyme activity was 1.1-fold higher than that of the crude enzyme, and recovery rate was 82.7%. The purified recombinant SPase had a band of 59 kDa on SDS-PAGE. Thermostability of the enzyme was shown at temperatures up to 37 degrees C, and pH stability between pH 6.0 and 6.7. The optimum temperature and pH were 37 degrees C and 6.7, respectively. The K(m) of SPase for sucrose was 7.3 mmol/L, and Vmax was 0.2 micromol/(min x mg). Besides, alpha-arbutin was synthesized from sucrose and hydroquinone by transglucosylation with recombinant SPase. The optimal conditions for synthesis of alpha-arbutin were 200 U/mL of recombinant SPase, 20% of sucrose, and 1.6% hydroquinone at pH 6-6.5 and 25 degrees C for 21 h. Under these conditions, alpha-arbutin was obtained with a 78.3% molar yield with respect to hydroquinone, and the concentration of alpha-arbutin was about 31 g/L.

  15. Glycogen phosphorylase is involved in stress endurance and biofilm formation in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Lerner, Anat; Castro-Sowinski, Susana; Lerner, Hadas; Okon, Yaacov; Burdman, Saul

    2009-11-01

    Here we report the identification of a glycogen phosphorylase (glgP) gene in the plant growth-promoting rhizobacterium Azospirillum brasilense, Sp7, and the characterization of a glgP marker exchange mutant of this strain. The glgP mutant showed a twofold reduction of glycogen phosphorylase activity and an increased glycogen accumulation as compared with wild-type Sp7, indicating that the identified gene indeed encodes a protein with glycogen phosphorylase activity. Interestingly, the glgP mutant had higher survival rates than the wild type after exposure to starvation, desiccation and osmotic pressure. The mutant was shown to be compromised in its biofilm formation ability. Analysis of the exopolysaccharide sugar composition of the glgP mutant revealed a decrease in the amount of glucose, accompanied by increases in rhamnose, fucose and ribose, as compared with the Sp7 exopolysaccharide. To the best of our knowledge, this is the first study that demonstrates GlgP activity in A. brasilense, and shows that glycogen accumulation may play an important role in the stress endurance of this bacterium.

  16. Architecture of Amylose Supramolecules in Form of Inclusion Complexes by Phosphorylase-Catalyzed Enzymatic Polymerization

    Science.gov (United States)

    Kadokawa, Jun-ichi

    2013-01-01

    This paper reviews the architecture of amylose supramolecules in form of inclusion complexes with synthetic polymers by phosphorylase-catalyzed enzymatic polymerization. Amylose is known to be synthesized by enzymatic polymerization using α-d-glucose 1-phosphate as a monomer, by phosphorylase catalysis. When the phosphorylase-catalyzed enzymatic polymerization was conducted in the presence of various hydrophobic polymers, such as polyethers, polyesters, poly(ester-ether), and polycarbonates as a guest polymer, such inclusion supramolecules were formed by the hydrophobic interaction in the progress of polymerization. Because the representation of propagation in the polymerization is similar to the way that a vine of a plant grows, twining around a rod, this polymerization method for the formation of amylose-polymer inclusion complexes was proposed to be named “vine-twining polymerization”. To yield an inclusion complex from a strongly hydrophobic polyester, the parallel enzymatic polymerization system was extensively developed. The author found that amylose selectively included one side of the guest polymer from a mixture of two resemblant guest polymers, as well as a specific range in molecular weights of the guest polymers poly(tetrahydrofuran) (PTHF) in the vine-twining polymerization. Selective inclusion behavior of amylose toward stereoisomers of chiral polyesters, poly(lactide)s, also appeared in the vine-twining polymerization. PMID:24970172

  17. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

    Science.gov (United States)

    Balestri, Francesco; Giannecchini, Michela; Sgarrella, Francesco; Carta, Maria Caterina; Tozzi, Maria Grazia; Camici, Marcella

    2007-02-01

    The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

  18. Isolation, crystallization and preliminary crystallographic analysis of Salmonella typhimurium uridine phosphorylase crystallized with 2,2′-anhydrouridine

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, Vladimir I.; Lashkov, Alexander A.; Gabdoulkhakov, Azat G.; Pavlyuk, Bogdan Ph. [A. V. Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninskiy Prospect 59, 119333 Moscow (Russian Federation); Kachalova, Galina S. [Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya Street 3, 142290 Pushchino, Moscow Region (Russian Federation); Betzel, Christian [Institute fur Biochemie und Lebensmittelchemie, University of Hamburg, c/o DESY, Building 22, Notkestrasse 85, 22604 Hamburg (Germany); Morgunova, Ekaterina Yu.; Zhukhlistova, Nadezhda E.; Mikhailov, Al’bert M., E-mail: amm@ns.crys.ras.ru [A. V. Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninskiy Prospect 59, 119333 Moscow (Russian Federation)

    2007-10-01

    S. typhimurium uridine phosphorylase has been isolated and crystallized in the presence of ligand. Uridine phosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleoside phosphorylase family of enzymes which catalyzes the phosphorolytic cleavage of the C—N glycoside bond of uridine, with the formation of ribose 1-phosphate and uracil. This enzyme has been shown to be important in the activation and catabolism of fluoropyrimidines. Modulation of its enzymatic activity may affect the therapeutic efficacy of chemotherapeutic agents. The structural investigation of the bacterial uridine phosphorylases, both unliganded and complexed with substrate/product analogues and inhibitors, may help in understanding the catalytic mechanism of the phosphorolytic cleavage of uridine. Salmonella typhimurium uridine phosphorylase has been crystallized with 2,2′-anhydrouridine. X-ray diffraction data were collected to 2.15 Å. Preliminary analysis of the diffraction data indicates that the crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 88.52, b = 123.98, c = 133.52 Å. The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit.

  19. A phase 1 study of the pharmacokinetics of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (components of TAS-102) vs trifluridine alone

    OpenAIRE

    Cleary, James M.; Rosen, Lee S.; Yoshida, Kenichiro; Rasco, Drew; Shapiro, Geoffrey I.; Sun, Weijing

    2017-01-01

    Summary Background Trifluridine, a thymidine-based chemotherapeutic, has limited bioavailability after clinical administration as it is rapidly degraded via thymidine phosphorylase. An oral combination tablet combines trifluridine with a potent thymidine phosphorylase inhibitor, tipiracil hydrochloride. This study?s objective was to evaluate whether trifluridine/tipiracil (TAS-102) administration increases trifluridine exposure vs trifluridine alone. Methods This open-label pharmacokinetic st...

  20. In silico binding analysis and SAR elucidations of newly designed benzopyrazine analogs as potent inhibitors of thymidine phosphorylase.

    Science.gov (United States)

    Taha, Muhammad; Ismail, Nor Hadiani; Imran, Syahrul; Rahim, Fazal; Wadood, Abdul; Al Muqarrabun, Laode Muhammad Ramadhan; Khan, Khalid Mohammed; Ghufran, Mehreen; Ali, Muhammad

    2016-10-01

    Thymidine phosphorylase (TP) is up regulated in wide variety of solid tumors and therefore presents a remarkable target for drug discovery in cancer. A novel class of extremely potent TPase inhibitors based on benzopyrazine (1-28) has been developed and evaluated against thymidine phosphorylase enzyme. Out of these twenty-eight analogs eleven (11) compounds 1, 4, 14, 15, 16, 17, 18, 19, 20, 24 and 28 showed potent thymidine phosphorylase inhibitory potentials with IC50 values ranged between 3.20±0.30 and 37.60±1.15μM when compared with the standard 7-Deazaxanthine (IC50=38.68±4.42μM). Structure-activity relationship was established and molecular docking studies were performed to determine the binding interactions of these newly synthesized compounds. Current studies have revealed that these compounds established stronger hydrogen bonding networks with active site residues as compare to the standard compound 7DX.

  1. Preliminary investigation of the three-dimensional structure of Salmonella typhimurium uridine phosphorylase in the crystalline state.

    Science.gov (United States)

    Dontsova, Maria V; Gabdoulkhakov, Azat G; Molchan, Olga K; Lashkov, Alexandr A; Garber, Maria B; Mironov, Alexandr S; Zhukhlistova, Nadegda E; Morgunova, Ekaterina Yu; Voelter, Wolfgang; Betzel, Christian; Zhang, Yang; Ealick, Steven E; Mikhailov, Al'bert M

    2005-04-01

    Uridine phosphorylase (UPh) catalyzes the phosphorolytic cleavage of the C-N glycosidic bond of uridine to ribose 1-phosphate and uracil in the pyrimidine-salvage pathway. The crystal structure of the Salmonella typhimurium uridine phosphorylase (StUPh) has been determined at 2.5 A resolution and refined to an R factor of 22.1% and an Rfree of 27.9%. The hexameric StUPh displays 32 point-group symmetry and utilizes both twofold and threefold non-crystallographic axes. A phosphate is bound at the active site and forms hydrogen bonds to Arg91, Arg30, Thr94 and Gly26 of one monomer and Arg48 of an adjacent monomer. The hexameric StUPh model reveals a close structural relationship to Escherichia coli uridine phosphorylase (EcUPh).

  2. X-ray structure of Salmonella typhimurium uridine phosphorylase complexed with 5-fluorouracil and molecular modelling of the complex of 5-fluorouracil with uridine phosphorylase from Vibrio cholerae.

    Science.gov (United States)

    Lashkov, Alexander A; Sotnichenko, Sergey E; Prokofiev, Igor I; Gabdulkhakov, Azat G; Agapov, Igor I; Shtil, Alexander A; Betzel, Christian; Mironov, Alexander S; Mikhailov, Al'bert M

    2012-08-01

    Uridine phosphorylase (UPh), which is a key enzyme in the reutilization pathway of pyrimidine nucleoside metabolism, is a validated target for the treatment of infectious diseases and cancer. A detailed analysis of the interactions of UPh with the therapeutic ligand 5-fluorouracil (5-FUra) is important for the rational design of pharmacological inhibitors of these enzymes in prokaryotes and eukaryotes. Expanding on the preliminary analysis of the spatial organization of the active centre of UPh from the pathogenic bacterium Salmonella typhimurium (StUPh) in complex with 5-FUra [Lashkov et al. (2009), Acta Cryst. F65, 601-603], the X-ray structure of the StUPh-5-FUra complex was analysed at atomic resolution and an in silico model of the complex formed by the drug with UPh from Vibrio cholerae (VchUPh) was generated. These results should be considered in the design of selective inhibitors of UPhs from various species.

  3. Thymidine esters as substrate analogue inhibitors of angiogenic enzyme thymidine phosphorylase in vitro.

    Science.gov (United States)

    Javaid, Sumaira; Ishtiaq, Marium; Shaikh, Muniza; Hameed, Abdul; Choudhary, M Iqbal

    2017-02-01

    Thymidine phosphorylase (TP) catalyzes the cleavage of thymidine into thymine and 2-deoxy-α-d-ribose-1-phosphate. Elevated activity of TP prevents apoptosis, and induces angiogenesis which ultimately leads to tumor growth and metastasis. Critical role of TP in cancer progression makes it a valid target in anti-cancer research. Discovery of small molecules as TP inhibitors is vigorously pursued in cancer therapy. In the present study, we functionalized thymidine as benzoyl ester to synthesize compounds 3-16. In vitro evaluation of thymidine esters for their thymidine phosphorylase inhibition activity was subsequently carried out. Compounds 4, 10, 14, and 15 showed good activities with lower IC50 values than the standard, 7-deazaxanthine (IC50=41.0±1.63μM). Among them, compound 14 showed five folds higher activity (IC50=7.5±0.8μM), while 4 (IC50=18.5±1.0μM) and 10 (IC50=18.8±1.2μM) showed two folds higher activity than the standard. Compound 15 showed slightly better activity (IC50=33.3±1.5μM) to the standard. Potent compounds were further subjected to kinetic and molecular docking studies to identify their mode of inhibition, and to study their interactions with the protein at atomic level, respectively. All active compounds were non-cytotoxic to mouse fibroblast 3T3 cell line. These results identify thymidine esters as substrate analogue (substrate-like) inhibitors of angiogenic enzyme thymidine phosphorylase for further studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Mitochondrial neurogastrointestinal encephalomyopathy: novel pathogenic mutations in thymidine phosphorylase gene in two Italian brothers.

    Science.gov (United States)

    Libernini, Laura; Lupis, Chiara; Mastrangelo, Mario; Carrozzo, Rosalba; Santorelli, Filippo Maria; Inghilleri, Maurizio; Leuzzi, Vincenzo

    2012-08-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE, MIM 603041) is an autosomal recessive multisystem disorder occurring due to mutations in a nuclear gene coding for the enzyme thymidine phosphorylase (TYMP). Clinical features of MNGIE include gastrointestinal dysmotility, cachexia, ptosis or ophthalmoparesis, peripheral neuropathy, diffuse leukoencephalopathy, and signs of mitochondrial dysfunction in tissues. We report the clinical and molecular findings in two brothers in whom novel TYMP gene mutations (c.215-13_215delinsGCGTGA; c.1159 + 2T > A) were associated with different clinical presentations and outcomes.

  5. Structures of native human thymidine phosphorylase and in complex with 5-iodouracil

    OpenAIRE

    Mitsiki, Eirini; Papageorgiou, Anastassios C.; Iyer, Shalini; Thiyagarajan, Nethaji; Prior, Steven H.; Sleep, Darrell; Finnis, Chris; Acharya, K. Ravi

    2009-01-01

    Thymidine phosphorylase (TP) first identified as platelet derived endothelial cell growth factor (PD-ECGF) plays a key role in nucleoside metabolism. Human TP (hTP) is implicated in angiogenesis and is overexpressed in several solid tumors. Here, we report the crystal structures of recombinant hTP and its complex with a substrate 5-iodouracil (5IUR) at 3.0 and 2.5 Å, respectively. In addition, we provide information on the role of specific residues in the enzymatic activity of hTP through mut...

  6. Amino acid anthranilamide derivatives as a new class of glycogen phosphorylase inhibitors.

    Science.gov (United States)

    Evans, Karen A; Li, Yue H; Coppo, Frank T; Graybill, Todd L; Cichy-Knight, Maria; Patel, Mehul; Gale, Jennifer; Li, Hu; Thrall, Sara H; Tew, David; Tavares, Francis; Thomson, Stephen A; Weiel, James E; Boucheron, Joyce A; Clancy, Daphne C; Epperly, Andrea H; Golden, Pamela L

    2008-07-15

    A series of amino acid anthranilamide derivatives identified from a high-throughput screening campaign as novel, potent, and glucose-sensitive inhibitors of human liver glycogen phosphorylase a are described. A solid-phase synthesis using Wang resin was also developed which provided efficient access to a variety of analogues, and resulted in the identification of key structure-activity relationships, and the discovery of a potent exemplar (IC(50)=80 nM). The SAR scope, synthetic strategy, and in vitro results for this series are presented herein.

  7. Iminosugars as potential inhibitors of glycogenolysis: structural insights into the molecular basis of glycogen phosphorylase inhibition.

    Science.gov (United States)

    Oikonomakos, Nikos G; Tiraidis, Costas; Leonidas, Demetres D; Zographos, Spyros E; Kristiansen, Marit; Jessen, Claus U; Nørskov-Lauritsen, Leif; Agius, Loranne

    2006-09-21

    Iminosugars DAB (5), isofagomine (9), and several N-substituted derivatives have been identified as potent inhibitors of liver glycogen phosphorylase a (IC(50) = 0.4-1.2 microM) and of basal and glucagon-stimulated glycogenolysis (IC(50) = 1-3 microM). The X-ray structures of 5, 9, and its N-3-phenylpropyl analogue 8 in complex with rabbit muscle glycogen phosphorylase (GPb) shows that iminosugars bind tightly at the catalytic site in the presence of the substrate phosphate and induce conformational changes that characterize the R-state conformation of the enzyme. Charged nitrogen N1 is within hydrogen-bonding distance with the carbonyl oxygen of His377 (5) and in ionic contact with the substrate phosphate oxygen (8 and 9). Our findings suggest that the inhibitors function as oxocarbenium ion transition-state analogues. The conformational change to the R state provides an explanation for previous findings that 5, unlike inhibitors that favor the T state, promotes phosphorylation of GPb in hepatocytes with sequential inactivation of glycogen synthase.

  8. Regio- and stereoselective glucosylation of diols by sucrose phosphorylase using sucrose or glucose 1-phosphate as glucosyl donor

    NARCIS (Netherlands)

    Renirie, R.; Pukin, A.; Lagen, van B.; Franssen, M.C.R.

    2010-01-01

    Previously it has been shown that glycerol can be regioselectively glucosylated by sucrose phosphorylase from Leuconostoc mesenteroides to form 2-O-alpha-D-glucopyranosyl-glycerol (Coedl et al., Angew. Chem. Int. Ed. 47 (2008) 10086-10089). A series of compounds related to glycerol were investigated

  9. Isolation, crystallization and preliminary crystallographic analysis of Salmonella typhimurium uridine phosphorylase crystallized with 2,2′-anhydrouridine

    Science.gov (United States)

    Timofeev, Vladimir I.; Lashkov, Alexander A.; Gabdoulkhakov, Azat G.; Pavlyuk, Bogdan Ph.; Kachalova, Galina S.; Betzel, Christian; Morgunova, Ekaterina Yu.; Zhukhlistova, Nadezhda E.; Mikhailov, Al’bert M.

    2007-01-01

    Uridine phosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleoside phosphorylase family of enzymes which catalyzes the phosphorolytic cleavage of the C—N glycoside bond of uridine, with the formation of ribose 1-­phosphate and uracil. This enzyme has been shown to be important in the activation and catabolism of fluoropyrimidines. Modulation of its enzymatic activity may affect the therapeutic efficacy of chemotherapeutic agents. The structural investigation of the bacterial uridine phosphorylases, both unliganded and complexed with substrate/product analogues and inhibitors, may help in understanding the catalytic mechanism of the phosphorolytic cleavage of uridine. Salmonella typhimurium uridine phosphorylase has been crystallized with 2,2′-anhydrouridine. X-ray diffraction data were collected to 2.15 Å. Preliminary analysis of the diffraction data indicates that the crystal belongs to space group P212121, with unit-cell parameters a = 88.52, b = 123.98, c = 133.52 Å. The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit. PMID:17909287

  10. Isolation, crystallization and preliminary crystallographic analysis of Salmonella typhimurium uridine phosphorylase crystallized with 2,2'-anhydrouridine.

    Science.gov (United States)

    Timofeev, Vladimir I; Lashkov, Alexander A; Gabdoulkhakov, Azat G; Pavlyuk, Bogdan Ph; Kachalova, Galina S; Betzel, Christian; Morgunova, Ekaterina Yu; Zhukhlistova, Nadezhda E; Mikhailov, Al'bert M

    2007-10-01

    Uridine phosphorylase (UPh; EC 2.4.2.3) is a member of the pyrimidine nucleoside phosphorylase family of enzymes which catalyzes the phosphorolytic cleavage of the C-N glycoside bond of uridine, with the formation of ribose 1-phosphate and uracil. This enzyme has been shown to be important in the activation and catabolism of fluoropyrimidines. Modulation of its enzymatic activity may affect the therapeutic efficacy of chemotherapeutic agents. The structural investigation of the bacterial uridine phosphorylases, both unliganded and complexed with substrate/product analogues and inhibitors, may help in understanding the catalytic mechanism of the phosphorolytic cleavage of uridine. Salmonella typhimurium uridine phosphorylase has been crystallized with 2,2'-anhydrouridine. X-ray diffraction data were collected to 2.15 A. Preliminary analysis of the diffraction data indicates that the crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 88.52, b = 123.98, c = 133.52 A. The solvent content is 45.51%, assuming the presence of one hexamer molecule per asymmetric unit.

  11. Screening and characterization of purine nucleoside degrading lactic acid bacteria isolated from Chinese sauerkraut and evaluation of the serum uric acid lowering effect in hyperuricemic rats.

    Directory of Open Access Journals (Sweden)

    Ming Li

    Full Text Available Hyperuricemia is well known as the cause of gout. In recent years, it has also been recognized as a risk factor for arteriosclerosis, cerebrovascular and cardiovascular diseases, and nephropathy in diabetic patients. Foods high in purine compounds are more potent in exacerbating hyperuricemia. Therefore, the development of probiotics that efficiently degrade purine compounds is a promising potential therapy for the prevention of hyperuricemia. In this study, fifty-five lactic acid bacteria isolated from Chinese sauerkraut were evaluated for the ability to degrade inosine and guanosine, the two key intermediates in purine metabolism. After a preliminary screening based on HPLC, three candidate strains with the highest nucleoside degrading rates were selected for further characterization. The tested biological characteristics of candidate strains included acid tolerance, bile tolerance, anti-pathogenic bacteria activity, cell adhesion ability, resistance to antibiotics and the ability to produce hydrogen peroxide. Among the selected strains, DM9218 showed the best probiotic potential compared with other strains despite its poor bile resistance. Analysis of 16S rRNA sequences showed that DM9218 has the highest similarity (99% to Lactobacillus plantarum WCFS1. The acclimated strain DM9218-A showed better resistance to 0.3% bile salt, and its survival in gastrointestinal tract of rats was proven by PCR-DGGE. Furthermore, the effects of DM9218-A in a hyperuricemia rat model were evaluated. The level of serum uric acid in hyperuricemic rat can be efficiently reduced by the intragastric administration of DM9218-A (P<0.05. The preventive treatment of DM9218-A caused a greater reduction in serum uric acid concentration in hyperuricemic rats than the later treatment (P<0.05. Our results suggest that DM9218-A may be a promising candidate as an adjunctive treatment in patients with hyperuricemia during the onset period of disease. DM9218-A also has potential as a probiotic in the prevention of hyperuricemia in the normal population.

  12. Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

    Science.gov (United States)

    Kyriakis, Efthimios; Stravodimos, George A; Kantsadi, Anastassia L; Chatzileontiadou, Demetra S M; Skamnaki, Vassiliki T; Leonidas, Demetres D

    2015-07-08

    We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 μM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency.

  13. 3D-QSAR studies on glycogen phosphorylase inhibitors by flexible comparative molecular field analysis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Canceling grids accommodating probes in comparative molecular field analysis (CoMFA), the idea of flexibleness is introduced into the CoMFA, and in combination with swarm intelligent algorithm which attempts to optimize distributions of diverse probes around drug molecules, a new 3D-QSAR method is proposed in this context as flexible comparative molecular field analysis (FCoMFA). In preliminary at-tempts to performing QSAR studies on 47 glycogen phosphorylase inhibitors, FCoMFA is employed and confirmed to be potent to exploring ligand-receptor interaction manners at active positions and thus to generating stable and predictable models. Simultaneously by an intuitive graphics regarding probe distribution patterns, impacts of different substituted groups on activities is also given an insight into.

  14. 3D-QSAR studies on glycogen phosphorylase inhibitors by flexible comparative molecular field analysis

    Institute of Scientific and Technical Information of China (English)

    ZHOU Peng; LI ZhiLiang

    2007-01-01

    Canceling grids accommodating probes in comparative molecular field analysis (CoMFA), the idea of flexibleness is introduced into the CoMFA, and in combination with swarm intelligent algorithm which attempts to optimize distributions of diverse probes around drug molecules, a new 3D-QSAR method is proposed in this context as flexible comparative molecular field analysis (FCoMFA). In preliminary attempts to performing QSAR studies on 47 glycogen phosphorylase inhibitors, FCoMFA is employed and confirmed to be potent to exploring ligand-receptor interaction manners at active positions and thus to generating stable and predictable models. Simultaneously by an intuitive graphics regarding probe distribution patterns, impacts of different substituted groups on activities is also given an insight into.

  15. Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Ruzanski, Christian; Krucewicz, Katarzyna

    2017-01-01

    (HvPho1) for starch biosynthesis in barley endosperm, we analyzed HvPho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of HvPho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis......The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose...... and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1...

  16. Differential Expression of a Glycogen Phosphorylase Gene in Volvariella volvacea Mycelium Exposed to Low Temperature Stress

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yan; WANG Hong; LI Zhengpeng; LU Lianjing; CHEN Mingjie

    2014-01-01

    Relative expression of a glycogen phosphorylase gene (pyg)in mycelia of cold-sensitive (V23)and cold-tolerant (VH3)strains of Volvariella volvacea during exposure to low temperature (0 ℃)over time courses was quantified by real-time PCR using theα-tubulin gene as internal control.Pyg expression levels in strain V23 decreased after 4 h exposure and,although recovering after 6 h,still remained lower than untreated controls.Gene expression in strain VH3 decreased sharply after low temperature exposure for 2 h, reaching a minimum value after 8 h when the relative expression level was only 0.28 times that of untreated controls.Overall,although pyg expression decreased in both V23 and VH3 over prolonged exposure,the fall was less pronounced in strain V23 compared with VH3.

  17. Mapping of a liver phosphorylase kinase [alpha]-subunit gene on the mouse x chromosome

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Yan; Derry, J.M.J.; Barnard, P.J. (MRC Molecular Neurobiology Unit, Cambridge (United Kingdom)); Hendrickx, J.; Coucke, P.; Willems, P.R. (Univ. of Antwerp (Belgium))

    1993-01-01

    Phosphorylase kinase (PHK) is a regulatory enzyme of the glycogenolytic pathway composed of a complex of four subunits. We recently mapped the muscle [alpha]-subunit gene (Phka) to the mouse X chromosome in a region syntenic with the proximal long arm of the human X chromosome and containing the human homologue of this gene, PHKA. We now report the mapping of the liver [alpha]-subunit gene to the telomeric end of the mouse X chromosome. This mapping position would suggest a location for the human liver [alpha]-subunit gene on the proximal short arm of the X chromosome, a region recently implicated in X-linked liver glycogenosis (XLG). 20 refs., 2 figs.

  18. Purification, crystallization and preliminary X-ray analysis of uridine phosphorylase from Salmonella typhimurium.

    Science.gov (United States)

    Dontsova, Mariya V; Savochkina, Yulia A; Gabdoulkhakov, Azat G; Baidakov, Sergey N; Lyashenko, Andrey V; Zolotukhina, Maria; Errais Lopes, Liubov; Garber, Mariya B; Morgunova, Ekaterina Yu; Nikonov, Stanislav V; Mironov, Alexandr S; Ealick, Steven E; Mikhailov, Al 'Bert M

    2004-04-01

    The structural udp gene encoding uridine phosphorylase (UPh) was cloned from the Salmonella typhimurium chromosome and overexpressed in Escherichia coli cells. S. typhimurium UPh (StUPh) was purified to apparent homogeneity and crystallized. The primary structure of StUPh has high homology to the UPh from E. coli, but the enzymes differ substantially in substrate specificity and sensitivity to the polarity of the medium. Single crystals of StUPh were grown using hanging-drop vapor diffusion with PEG 8000 as the precipitant. X-ray diffraction data were collected to 2.9 A resolution. Preliminary analysis of the diffraction data indicated that the crystal belonged to space group P6(1(5)), with unit-cell parameters a = 92.3, c = 267.5 A. The solvent content is 37.7% assuming the presence of one StUPh hexamer per asymmetric unit.

  19. Structure of cellobiose phosphorylase from Clostridium thermocellum in complex with phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Bianchetti, Christopher M.; Elsen, Nathaniel L.; Fox, Brian G.; Phillips, Jr., George N. (UW)

    2012-03-27

    Clostridium thermocellum is a cellulosome-producing bacterium that is able to efficiently degrade and utilize cellulose as a sole carbon source. Cellobiose phosphorylase (CBP) plays a critical role in cellulose degradation by catalyzing the reversible phosphate-dependent hydrolysis of cellobiose, the major product of cellulose degradation, into -D-glucose 1-phosphate and D-glucose. CBP from C. thermocellum is a modular enzyme composed of four domains [N-terminal domain, helical linker, (/)6-barrel domain and C-terminal domain] and is a member of glycoside hydrolase family 94. The 2.4 {angstrom} resolution X-ray crystal structure of C. thermocellum CBP reveals the residues involved in coordinating the catalytic phosphate as well as the residues that are likely to be involved in substrate binding and discrimination.

  20. Purification and characterization of the maize amyloplast stromal 112-kDa starch phosphorylase.

    Science.gov (United States)

    Mu, H H; Yu, Y; Wasserman, B P; Carman, G M

    2001-04-01

    A plastidic 112-kDa starch phosphorylase (SP) has been identified in the amyloplast stromal fraction of maize. This starch phosphorylase was purified 310-fold from maize endosperm and characterized with respect to its enzymological and kinetic properties. The purification procedure included ammonium sulfate fractionation, Sephacryl 300 HR chromatography, affinity starch adsorption, Q-Sepharose, and Mono Q chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and SDS-polyacrylamide gel electrophoresis. Anti-SP antibodies recognized the purified 112-kDa SP enzyme and N-terminal amino acid sequence analysis confirmed that the purified enzyme is the amyloplast stromal 112-kDa SP. Analysis of the purified enzyme by Superose 6 gel filtration chromatography indicated that the native enzyme consisted of two identical subunits. The pH optimum for the enzyme was 6.0 in the synthetic direction and 5.5 in the phosphorolytic direction. SP activity was inhibited by thioreactive agents, diethyl pyrocarbonate, phenylglyoxal, and ADP-glucose. The activation energies for the synthetic and phosphorolytic reactions were 11.1 and 16.9 kcal/mol, respectively, and the enzyme was thermally labile above 50 degrees C. Results of kinetic experiments indicated that the enzyme catalyzes its reaction via a sequential Bi Bi mechanism. The Km value for amylopectin was eight-fold lower than that of glycogen. A kinetic analysis indicated that the phosphorolytic reaction was favored over the synthetic reaction when malto-oligosaccharides (4 to 7 units) were used as substrates. The specificity constants (Vmax/Km) of the enzyme measured in either the synthetic or the phosphorolytic directions increased with increasing chain length.

  1. [Reconstruction of muscle glycogen phosphorylase b from an apoenzyme and pyridoxal-5'-phosphate and its analogs. Interaction of apophosphorylase and the reconstructed enzyme with specific ligands].

    Science.gov (United States)

    Chebotareva, N A; Sugrobova, N P; Bulanova, L N; Poznanskaia, A A; Kurganov, B I; Gunar, V I

    1995-12-01

    Sedimentation methods were used to study the effects of modification of the pyridoxal-5'-phosphate (PLP) molecule at the 5th position on the affinity of reconstituted muscle glycogen phosphorylase b for the substrate (glycogen) and the allosteric inhibitor (FMN) as well as on the enzyme capacity to association induced by AMP. Reconstituted phosphorylase b was obtained with PLP analogs containing at the 5th position -CH2-CH2-COOH (analog I), trans-CH=CH-COOH (analog II) or -C identical to COOH (analog III) residues. Reconstitution of phosphorylase b is accompanied by the recovery of the enzyme quaternary structure. Phosphorylase b reconstituted with PLP or analogs I, II and III is not distinguished practically from the native enzyme in its affinity for glycogen. Substitution of the native coenzyme in the phosphorylase molecule with any tested PLP analog leads to lower enzyme affinity for FMN. Microscopic dissociation constants of the FMN-enzyme complexes increase in the following order: enzyme.I < enzyme.II < enzyme.III. Phosphorylase b reconstituted with analogs I, II and III differs substantially from the native enzyme in its capacity to association in the presence of 1 mM AMP: the reconstituted enzyme is represented practically by only the tetrameric form.

  2. Redesign of the Active Site of Sucrose Phosphorylase through a Clash-Induced Cascade of Loop Shifts.

    Science.gov (United States)

    Kraus, Michael; Grimm, Clemens; Seibel, Jürgen

    2016-01-01

    Sucrose phosphorylases have been applied in the enzymatic production of glycosylated compounds for decades. However, several desirable acceptors, such as flavonoids or stilbenoids, that exhibit diverse antimicrobial, anticarcinogenic or antioxidant properties, remain poor substrates. The Q345F exchange in sucrose phosphorylase from Bifidobacterium adolescentis allows efficient glucosylation of resveratrol, (+)-catechin and (-)-epicatechin in yields of up to 97 % whereas the wild-type enzyme favours sucrose hydrolysis. Three previously undescribed products are made available. The crystal structure of the variant reveals a widened access channel with a hydrophobic aromatic surface that is likely to contribute to the improved activity towards aromatic acceptors. The generation of this channel can be explained in terms of a cascade of structural changes arising from the Q345F exchange. The observed mechanisms are likely to be relevant for the design of other tailor-made enzymes.

  3. New synthesis of 3-(β-D-glucopyranosyl)-5-substituted-1,2,4-triazoles, nanomolar inhibitors of glycogen phosphorylase.

    Science.gov (United States)

    Kun, Sándor; Bokor, Éva; Varga, Gergely; Szőcs, Béla; Páhi, András; Czifrák, Katalin; Tóth, Marietta; Juhász, László; Docsa, Tibor; Gergely, Pál; Somsák, László

    2014-04-09

    O-Perbenzoylated 5-(β-D-glucopyranosyl)tetrazole was reacted with N-benzyl carboximidoyl chlorides to give the corresponding 4-benzyl-3-(β-D-glucopyranosyl)-5-substituted-1,2,4-triazoles. Removal of the O-benzoyl and N-benzyl protecting groups by base catalysed transesterification and catalytic hydrogenation, respectively, furnished a series of 3-(β-D-glucopyranosyl)-5-substituted-1,2,4-triazoles with aliphatic, mono- and bicyclic aromatic, and heterocyclic substituents in the 5-position. Enzyme kinetic studies revealed these compounds to inhibit rabbit muscle glycogen phosphorylase b: best inhibitors were the 5-(4-aminophenyl)- (Ki 0.67 μM) and the 5-(2-naphthyl)-substituted (Ki 0.41 μM) derivatives. This study uncovered the C-glucopyranosyl-1,2,4-triazoles as a novel skeleton for nanomolar inhibition of glycogen phosphorylase.

  4. Effects of /sup 45/Ca on murine skeletal muscle. 1. Alterations of glycogen, phosphorylase and phosphohexose isomerase levels

    Energy Technology Data Exchange (ETDEWEB)

    Asotra, K.; Katoch, S.S.; Krishan, K.; Malhotra, R.K. (Himachal Pradesh Univ., Simla (India). Dept. of Bio-sciences)

    1983-01-01

    Adult Swiss albino mice weighing 16+-1 g were injected with 3.7x10/sup 4/ Bq and 7.4x10/sup 4/ Bq/g body weight of /sup 45/Ca. Mice of both dose groups were autopsied on days 1, 3, 5, 7, 14 and 28 after /sup 45/Ca administration. Diaphragm and gastrocnemius in the /sup 45/Ca-treated and normal mice were analyzed for quantitation of glycogen as well as bioassay of phosphorylase and phosphohexose isomerase activities. Internal irradiation with the two doses of /sup 45/Ca resulted in glycogen accumulation in both the muscles. /sup 45/Ca-treated diaphragm showed greater radioresponse but a slower recovery than gastrocnemius with respect to glycogen accumulation. A decline in the rates of glycogenolysis and glycolysis indicated by decreased phosphorylase and phosphohexose isomerase activities appeared to be responsible for glycogen accumulation in skeletal muscle on account of /sup 45/Ca treatment.

  5. Prediction of potential antimalarial targets of artemisinin based on protein information from whole genome of Plasmodium falciparum

    Institute of Scientific and Technical Information of China (English)

    HAN LiPing; HUANG Qiang; NAN Peng; ZHONG Yang

    2009-01-01

    On the basis of the genomic data and protein pathway information about Plasmodium falciparum clone 3D7 from the NCBI taxonomy database and the KEGG database,eight key protein enzymes in the signal pathways were selected to perform molecular docking with artemisinin.The binding modes obtained from the molecular docking suggested that purine nucleoside phosphorylase (pfPNP),peptide deformylase (pfPDF),and ribose 5-phosphate isomerase (pfRpiA) may be involved in the antimalarial mode of action of artemisinin.Artemisinin exhibited its antimalarial activity probably by interfering with the metabolic pathways of purine,pyrimidine,methionine,glyoxylate and dicarboxylate,or pentose phosphate.

  6. RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression.

    Science.gov (United States)

    Carzaniga, Thomas; Dehò, Gianni; Briani, Federica

    2015-06-01

    The complex posttranscriptional regulation mechanism of the Escherichia coli pnp gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for pnp autoregulation posit that the target of PNPase is a mature pnp mRNA previously processed at its 5' end by RNase III, rather than the primary pnp transcript (RNase III-dependent models), and that PNPase activity eventually leads to pnp mRNA degradation by RNase E. However, some published data suggest that pnp expression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δpnp rnc(+) and Δpnp Δrnc strains with a chromosomal pnp-lacZ translational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition for pnp primary transcript between PNPase and the ribosomal protein S1. In Escherichia coli, polynucleotide phosphorylase (PNPase, encoded by pnp) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of the pnp primary transcript, creates the substrate for PNPase regulatory activity, eventually leading to pnp mRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III

  7. Glycogen stability and glycogen phosphorylase activities in isolated skeletal muscles from rat and toad.

    Science.gov (United States)

    Goodman, C A; Stephenson, G M

    2000-01-01

    There is increasing evidence that endogenous glycogen depletion may affect excitation-contraction (E-C) coupling events in vertebrate skeletal muscle. One approach employed in physiological investigations of E-C coupling involves the use of mechanically skinned, single fibre preparations obtained from tissues stored under paraffin oil, at room temperature (RT: 20-24 degrees C) and 4 degrees C for several hours. In the present study, we examined the effect of these storage conditions on the glycogen content in three muscles frequently used in research on E-C coupling: rat extensor digitorum longus (EDL) and soleus (SOL) and toad iliofibularis (IF). Glycogen content was determined fluorometrically in homogenates prepared from whole muscles, stored under paraffin oil for up to 6 h at RT or 4 degrees C. Control muscles and muscles stored for 0.5 and 6 h were also analysed for total phosphorylase (Phos(total)) and phosphorylase a (Phos a) activities. No significant change was observed in the glycogen content of EDL and SOL muscles stored at RT for 0.5 h. In rat muscles stored at RT for longer than 0.5 h, the glycogen content decreased to 67.6% (EDL) and 78.7% (SOL) of controls after 3 h and 25.3% (EDL) and 37.4% (SOL) after 6 h. Rat muscles stored at 4 degrees C retained 79.0% (EDL) and 92.5% (SOL) of glycogen after 3 h and 75.2% (EDL) and 61.1% (SOL) after 6 h. The glycogen content of IF muscles stored at RT or 4 degrees C for 6 h was not significantly different from controls. Phos(total) was unchanged in all muscles over the 6 h period, at both temperatures. Phos a was also unchanged in the toad IF muscles, but in rat muscles it decreased rapidly, particularly in EDL (4.1-fold after 0.5 h at RT). Taken together these results indicate that storage under paraffin oil for up to 6 h at RT or 4 degrees C is accompanied by minimal glycogen loss in toad IF muscles and by a time- and temperature-dependent glycogen loss in EDL and SOL muscles of the rat.

  8. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    Science.gov (United States)

    Balaev, V. V.; Lashkov, A. A.; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

    2015-03-01

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis ( YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability ( R work = 16.2, R free = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  9. Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

    Energy Technology Data Exchange (ETDEWEB)

    Balaev, V. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2015-03-15

    Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (R{sub work} = 16.2, R{sub free} = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

  10. Development of a conductometric phosphate biosensor based on tri-layer maltose phosphorylase composite films.

    Science.gov (United States)

    Zhang, Zhiqiang; Jaffrezic-Renault, Nicole; Bessueille, François; Leonard, Didier; Xia, Siqing; Wang, Xuejiang; Chen, Ling; Zhao, Jianfu

    2008-05-12

    A conductometric biosensor for phosphate detection was developed using maltose phosphorylase (MP) from recombinant Escherichia coli immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin (BSA). The process parameters for the fabrication of the mono-enzymatic sensor and various experimental variables such as the enzyme loading, time of immobilization in saturated GA vapour, working buffer solution and temperature were investigated with regard to their influence on sensitivity, detection limit, dynamic range, operational and storage stability. The biosensor can work well at the temperature between 20 degrees C and 50 degrees C, and reach 90% of steady-state conductance in about 10s. The sensor has two linear ranges, one is from 1.0 microM to 20 microM phosphate with a detection limit of 1.0 microM, and the other is between 20 microM and 400 microM phosphate. When stored in citrate buffer (0.1M, pH 6.0) at 4 degrees C, the biosensor showed good stability over two months. No obvious interference from other anionic species like SO(4)(2-), Cl(-), NO(3)(-), NO(2)(-) and HCO(3)(-) was detected. The biosensor is suitable for use in real water samples.

  11. A conserved loop in polynucleotide phosphorylase (PNPase) essential for both RNA and ADP/phosphate binding.

    Science.gov (United States)

    Carzaniga, Thomas; Mazzantini, Elisa; Nardini, Marco; Regonesi, Maria Elena; Greco, Claudio; Briani, Federica; De Gioia, Luca; Dehò, Gianni; Tortora, Paolo

    2014-02-01

    Polynucleotide phosphorylase (PNPase) reversibly catalyzes RNA phosphorolysis and polymerization of nucleoside diphosphates. Its homotrimeric structure forms a central channel where RNA is accommodated. Each protomer core is formed by two paralogous RNase PH domains: PNPase1, whose function is largely unknown, hosts a conserved FFRR loop interacting with RNA, whereas PNPase2 bears the putative catalytic site, ∼20 Å away from the FFRR loop. To date, little is known regarding PNPase catalytic mechanism. We analyzed the kinetic properties of two Escherichia coli PNPase mutants in the FFRR loop (R79A and R80A), which exhibited a dramatic increase in Km for ADP/Pi binding, but not for poly(A), suggesting that the two residues may be essential for binding ADP and Pi. However, both mutants were severely impaired in shifting RNA electrophoretic mobility, implying that the two arginines contribute also to RNA binding. Additional interactions between RNA and other PNPase domains (such as KH and S1) may preserve the enzymatic activity in R79A and R80A mutants. Inspection of enzyme structure showed that PNPase has evolved a long-range acting hydrogen bonding network that connects the FFRR loop with the catalytic site via the F380 residue. This hypothesis was supported by mutation analysis. Phylogenetic analysis of PNPase domains and RNase PH suggests that such network is a unique feature of PNPase1 domain, which coevolved with the paralogous PNPase2 domain. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. Defects in Polynucleotide phosphorylase impairs virulence in Escherichia coli O157:H7

    Directory of Open Access Journals (Sweden)

    Jia eHu

    2015-08-01

    Full Text Available Polynucleotide phosphorylase (PNPase is reported to regulate virulence in Salmonella, Yersinia spp. and Campylobacter jejuni, yet its role in Escherichia coli O157:H7 has not been investigated. To gain insights into its roles in E. coli O157:H7 virulence, pnp deletion mutants were generated and the major virulence factors were compared to their parental wild type strains. Deletion of pnp in E. coli O157:H7 dramatically decreased stx2 mRNA expression and Stx2 protein production, and impaired lambdoid prophage activation in E. coli O157:H7. Quantitative PCR further confirmed that the Stx2 phage lytic growth was repressed by pnp deletion. Consistent with reduced Stx2 production and Stx2 phage activation, the transcriptional levels of genes involved in phage lysis and replication were down-regulated. In addition, disruption of pnp in E. coli O157:H7 decreased its adhesion to intestinal epithelial cells as well as cattle colonic explant tissues. On the other hand, PNPase inactivation in E. coli O157:H7 enhanced Tir protein content and the transcription of type three secretion system components, including genes encoding intimin, Tir and EspB as well as LEE positive regulator, Ler. Collectively, data indicate that PNPase has pleiotropic effects on the virulence of E. coli O157:H7.

  13. The kinetic mechanism of Human Thymidine Phosphorylase - a molecular target for cancer drug development.

    Science.gov (United States)

    Deves, Candida; Rostirolla, Diana Carolina; Martinelli, Leonardo Kras Borges; Bizarro, Cristiano Valim; Santos, Diogenes Santiago; Basso, Luiz Augusto

    2014-03-04

    Human Thymidine Phosphorylase (HTP), also known as the platelet-derived endothelial cell growth factor (PD-ECGF) or gliostatin, catalyzes the reversible phosphorolysis of thymidine (dThd) to thymine and 2-deoxy-α-d-ribose-1-phosphate (2dR1P). HTP is a key enzyme in the pyrimidine salvage pathway involved in dThd homeostasis in cells. HTP is a target for anticancer drug development as its enzymatic activity promotes angiogenesis. Here, we describe cloning, expression, and purification to homogeneity of recombinant TYMP-encoded HTP. Peptide fingerprinting and the molecular mass value of the homogenous protein confirmed its identity as HTP assessed by mass spectrometry. Size exclusion chromatography showed that HTP is a dimer in solution. Kinetic studies revealed that HTP displayed substrate inhibition for dThd. Initial velocity and isothermal titration calorimetry (ITC) studies suggest that HTP catalysis follows a rapid-equilibrium random bi-bi kinetic mechanism. ITC measurements also showed that dThd and Pi binding are favorable processes. The pH-rate profiles indicated that maximal enzyme activity was achieved at low pH values. Functional groups with apparent pK values of 5.2 and 9.0 are involved in dThd binding and groups with pK values of 6.1 and 7.8 are involved in phosphate binding.

  14. Gene expression and molecular characterization of a thermostable trehalose phosphorylase from Thermoanaerobacter tengcongensis

    Institute of Scientific and Technical Information of China (English)

    REN Yuanyuan; DAI Xiuyu; ZHOU Jian; LIU Jingfang; PEI Huadong; XIANG Hua

    2005-01-01

    A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from a-glucose-1-phosphate (α-GIc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli.The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70℃ (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual activity after incubation at 50℃ for 7 h. Under the optimum reaction conditions, 50 μg crude enzyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-GIc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L trehalose is degraded within the same time period. Dot blotting revealed that the treP gene in T.tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of the T. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for industrial production of trehalose.

  15. Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

    Science.gov (United States)

    Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

    2014-03-01

    Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.

  16. Dual-action hypoglycemic and hypocholesterolemic agents that inhibit glycogen phosphorylase and lanosterol demethylase.

    Science.gov (United States)

    Harwood, H James; Petras, Stephen F; Hoover, Dennis J; Mankowski, Dayna C; Soliman, Victor F; Sugarman, Eliot D; Hulin, Bernard; Kwon, Younggil; Gibbs, E Michael; Mayne, James T; Treadway, Judith L

    2005-03-01

    Diabetic dyslipidemia requires simultaneous treatment with hypoglycemic agents and lipid-modulating drugs. We recently described glycogen phosphorylase inhibitors that reduce glycogenolysis in cells and lower plasma glucose in ob/ob mice (J. Med. Chem., 41: 2934, 1998). In evaluating the series prototype, CP-320626, in dogs, up to 90% reduction in plasma cholesterol was noted after 2 week treatment. Cholesterol reductions were also noted in ob/ob mice and in rats. In HepG2 cells, CP-320626 acutely and dose-dependently inhibited cholesterolgenesis without affecting fatty acid synthesis. Inhibition occurred together with a dose-dependent increase in the cholesterol precursor, lanosterol, suggesting that cholesterolgenesis inhibition was due to lanosterol 14alpha-demethylase (CYP51) inhibition. In ob/ob mice, acute treatment with CP-320626 resulted in a decrease in hepatic cholesterolgenesis with concomitant lanosterol accumulation, further implicating CYP51 inhibition as the mechanism of cholesterol lowering in these animals. CP-320626 and analogs directly inhibited rhCYP51, and this inhibition was highly correlated with HepG2 cell cholesterolgenesis inhibition (R2 = 0.77). These observations indicate that CP-320626 inhibits cholesterolgenesis via direct inhibition of CYP51, and that this is the mechanism whereby CP-320626 lowers plasma cholesterol in experimental animals. Dual-action glycogenolysis and cholesterolgenesis inhibitors therefore have the potential to favorably affect both the hyperglycemia and the dyslipidemia of type 2 diabetes.

  17. Structure-activity relationships of flavonoids as potential inhibitors of glycogen phosphorylase.

    Science.gov (United States)

    Kato, Atsushi; Nasu, Norio; Takebayashi, Kenji; Adachi, Isao; Minami, Yasuhiro; Sanae, Fujiko; Asano, Naoki; Watson, Alison A; Nash, Robert J

    2008-06-25

    Flavonoids are ubiquitous components in vegetables, fruits, tea, and wine. Therefore, they are often consumed in large quantities in our daily diet. Several flavonoids have been shown to have potential as antidiabetic agents. In the present study, we focused on inhibition of glycogen phosphorylase (GP) by flavonoids. 6-Hydroxyluteolin, hypolaetin, and quercetagetin were identified as good inhibitors of dephosphorylated GP (GPb), with IC 50 values of 11.6, 15.7, and 9.7 microM, respectively. Furthermore, a structure-activity relationship study revealed that the presence of the 3' and 4' OH groups in the B-ring and double bonds between C2 and C3 in flavones and flavonols are important factors for enzyme recognition and binding. Quercetagetin inhibited GPb in a noncompetitive manner, with a K i value of 3.5 microM. Multiple inhibition studies by Dixon plots suggested that quercetagetin binds to the allosteric site. In primary cultured rat hepatocytes, quercetagetin and quercetin suppressed glucagon-stimulated glycogenolysis, with IC 50 values of 66.2 and 68.7 microM, respectively. These results suggested that as a group of novel GP inhibitors, flavonoids have potential to contribute to the protection or improvement of control of diabetes type II.

  18. Kinetics and mechanistic study of competitive inhibition of thymidine phosphorylase by 5-fluoruracil derivatives.

    Science.gov (United States)

    Petaccia, Manuela; Gentili, Patrizia; Bešker, Neva; D'Abramo, Marco; Giansanti, Luisa; Leonelli, Francesca; La Bella, Angela; Gradella Villalva, Denise; Mancini, Giovanna

    2016-04-01

    In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N(3) position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3'-azido-2'-fluoro-dideoxyuridin. Theoretical and experimental results showed that all the derivatives exert the same inhibition activity of 5-FU either as monomer and when embedded in lipid bilayer.

  19. 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

  20. 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.

  1. Structures of yeast Apa2 reveal catalytic insights into a canonical AP₄A phosphorylase of the histidine triad superfamily.

    Science.gov (United States)

    Hou, Wen-Tao; Li, Wen-Zhe; Chen, Yuxing; Jiang, Yong-Liang; Zhou, Cong-Zhao

    2013-08-09

    The homeostasis of intracellular diadenosine 5',5″'-P(1),P(4)-tetraphosphate (Ap4A) in the yeast Saccharomyces cerevisiae is maintained by two 60% sequence-identical paralogs of Ap4A phosphorylases (Apa1 and Apa2). Enzymatic assays show that, compared to Apa1, Apa2 has a relatively higher phosphorylase activity towards Ap3A (5',5″'-P(1),P(3)-tetraphosphate), Ap4A, and Ap5A (5',5″'-P(1),P(5)-tetraphosphate), and Ap4A is the favorable substrate for both enzymes. To decipher the catalytic insights, we determined the crystal structures of Apa2 in the apo-, AMP-, and Ap4A-complexed forms at 2.30, 2.80, and 2.70Å resolution, respectively. Apa2 is an α/β protein with a core domain of a twisted eight-stranded antiparallel β-sheet flanked by several α-helices, similar to the galactose-1-phosphate uridylyltransferase (GalT) members of the histidine triad (HIT) superfamily. However, a unique auxiliary domain enables an individual Apa2 monomer to possess an intact substrate-binding cleft, which is distinct from previously reported dimeric GalT proteins. This cleft is perfectly complementary to the favorable substrate Ap4A, the AMP and ATP moieties of which are perpendicular to each other, leaving the α-phosphate group exposed at the sharp turn against the catalytic residue His161. Structural comparisons combined with site-directed mutagenesis and activity assays enable us to define the key residues for catalysis. Furthermore, multiple-sequence alignment reveals that Apa2 and homologs represent canonical Ap4A phosphorylases, which could be grouped as a unique branch in the GalT family. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer

    Institute of Scientific and Technical Information of China (English)

    王常玉; 翁艳洁; 王鸿雁; 石英; 马丁

    2010-01-01

    The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression a...

  3. Phytogenic Polyphenols as Glycogen Phosphorylase Inhibitors: The Potential of Triterpenes and Flavonoids for Glycaemic Control in Type 2 Diabetes.

    Science.gov (United States)

    Leonidas, Demetres D; Hayes, Joseph M; Kato, Atsushi; Skamnaki, Vassiliki T; Chatzileontiadou, Demetra S M; Kantsadi, Anastassia L; Kyriakis, Efthimios; Chetter, Ben A; Stravodimos, George A

    2017-01-01

    Glycogen phosphorylase (GP) is a validated pharmaceutical target for the development of antihyperglycaemic agents. Phytogenic polyphenols, mainly flavonoids and pentacyclic triterpenes, have been found to be potent inhibitors of GP. These compounds have both pharmaceutical and nutraceutical potential for glycemic control in diabetes type 2. This review focuses mainly on the most successful (potent) of these compounds discovered to date. The protein-ligand interactions that form the structural basis of their potencies are discussed, highlighting the potential for exploitation of their scaffolds in the future design of new GP inhibitors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. Molecular Mechanisms of Allosteric Inhibition of Brain Glycogen Phosphorylase by Neurotoxic Dithiocarbamate Chemicals.

    Science.gov (United States)

    Mathieu, Cécile; Bui, Linh-Chi; Petit, Emile; Haddad, Iman; Agbulut, Onnik; Vinh, Joelle; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2017-02-03

    Dithiocarbamates (DTCs) are important industrial chemicals used extensively as pesticides and in a variety of therapeutic applications. However, they have also been associated with neurotoxic effects and in particular with the development of Parkinson-like neuropathy. Although different pathways and enzymes (such as ubiquitin ligases or the proteasome) have been identified as potential targets of DTCs in the brain, the molecular mechanisms underlying their neurotoxicity remain poorly understood. There is increasing evidence that alteration of glycogen metabolism in the brain contributes to neurodegenerative processes. Interestingly, recent studies with N,N-diethyldithiocarbamate suggest that brain glycogen phosphorylase (bGP) and glycogen metabolism could be altered by DTCs. Here, we provide molecular and mechanistic evidence that bGP is a target of DTCs. To examine this system, we first tested thiram, a DTC pesticide known to display neurotoxic effects, observing that it can react rapidly with bGP and readily inhibits its glycogenolytic activity (kinact = 1.4 × 10(5) m(-1) s(-1)). Using cysteine chemical labeling, mass spectrometry, and site-directed mutagenesis approaches, we show that thiram (and certain of its metabolites) alters the activity of bGP through the formation of an intramolecular disulfide bond (Cys(318)-Cys(326)), known to act as a redox switch that precludes the allosteric activation of bGP by AMP. Given the key role of glycogen metabolism in brain functions and neurodegeneration, impairment of the glycogenolytic activity of bGP by DTCs such as thiram may be a new mechanism by which certain DTCs exert their neurotoxic effects.

  5. Affinity crystallography reveals the bioactive compounds of industrial juicing byproducts of Punica granatum for glycogen phosphorylase.

    Science.gov (United States)

    Stravodimos, George A; Kantsadi, Anastassia L; Apostolou, Anna; Kyriakis, Efthimios; Kafaski-Kanelli, Vassiliki-Nafsika; Solovou, Theodora G A; Gatzona, Pagona; Liggri, Panagiota Cv; Theofanous, Stavroula; Gorgogietas, Vyron A; Kissa, Apostolia; Psachoula, Chariklia; Chatzileontiadou, Demetra S M; Lemonakis, Angelos; Psarra, Anna-Maria G; Skamnaki, Vassiliki T; Haroutounian, Serkos; Leonidas, Demetres D

    2017-06-18

    Glycogen phosphorylase (GP) is a pharmaceutical target for the discovery of new antihyperglycaemic agents. Punica granatum is a well-known plant for its potent antioxidant and antimicrobial activities but so far has not been examined for antihyperglycaemic activity. To examine the inhibitory potency of eighteen polyphenolic extracts obtained from Punica granatum fruits and industrial juicing byproducts against GP and discover their most bioactive ingredients. Kinetic experiments were conducted to measure the IC50 values of the extracts while affinity crystallography was used to identify the most bioactive ingredient. The inhibitory effect of one of the polyphenolic extracts was also verified ex vivo, in HepG2 cells. All extracts exhibit significant in vitro inhibitory potency (IC50 values in the range of low μg/mL). Affinity crystallography revealed that the most bioactive ingredients of the extracts were chlorogenic and ellagic acids, found bound in the active and the inhibitor site of GP, respectively. While ellagic acid is an established GP inhibitor, the inhibition of chlorogenic acid is reported for the first time. Kinetic analysis indicated that chlorogenic acid is an inhibitor with Ki=2.5 x 10-3 M that acts synergistically with ellagic acid. Our study provides the first evidence for a potential antidiabetic usage of Punica granatum extracts as antidiabetic food supplements. Although, more in vivo studies have to be performed before these extracts reach the stage of antidiabetic food supplements our study provides a first positive step towards this process. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development.

    Science.gov (United States)

    Cuesta-Seijo, Jose A; Ruzanski, Christian; Krucewicz, Katarzyna; Meier, Sebastian; Hägglund, Per; Svensson, Birte; Palcic, Monica M

    2017-01-01

    The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 (HvPho1) for starch biosynthesis in barley endosperm, we analyzed HvPho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of HvPho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that HvPho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the de novo production of α-1,4-glucans using HvPho1 from G1P as the sole substrate. The structural properties of HvPho1 provide insights into the low affinity of HvPho1 for large polysaccharides like starch or amylopectin. Our results suggest that HvPho1 may play a role during the initiation of starch biosynthesis in barley.

  7. Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Nabila eHaddad

    2012-03-01

    Full Text Available Polynucleotide phosphorylase, encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As the two major ribonucleases (RNase E and RNase II of Escherichia coli are absent from Campylobacter genome, this study was focused on PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3, motility (NANA synthetase, stress-response (KatA, DnaK, Hsp90 and translation system (EF-Tu, EF-G were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay and the decrease of cell adhesion/invasion ability.

  8. Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase.

    Science.gov (United States)

    Firestone, Ross S; Cameron, Scott A; Karp, Jerome M; Arcus, Vickery L; Schramm, Vern L

    2017-02-17

    Human 5'-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5'-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (-ΔCp), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCp of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCp. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCp for binding. In addition, ΔCp values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (-ΔCp(‡)). A comparison of the ΔCp(‡) for MTAP presteady state chemistry and ΔCp for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP-inhibitor complexes indicate remarkable protein thermal stability (to Tm = 99 °C) in complexes with transition-state analogues.

  9. Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

    Science.gov (United States)

    Briani, Federica; Curti, Serena; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni

    2008-11-01

    The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

  10. Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli.

    Science.gov (United States)

    Carzaniga, Thomas; Sbarufatti, Giulia; Briani, Federica; Dehò, Gianni

    2017-04-04

    Polynucleotide phosphorylase (PNPase, encoded by pnp) is generally thought of as an enzyme dedicated to RNA metabolism. The pleiotropic effects of PNPase deficiency is imputed to altered processing and turnover of mRNAs and small RNAs, which in turn leads to aberrant gene expression. However, it has long since been known that this enzyme may also catalyze template-independent polymerization of dNDPs into ssDNA and the reverse phosphorolytic reaction. Recently, PNPase has been implicated in DNA recombination, repair, mutagenesis and resistance to genotoxic agents in diverse bacterial species, raising the possibility that PNPase may directly, rather than through control of gene expression, participate in these processes. In this work we present evidence that in Escherichia coli PNPase enhances both homologous recombination upon P1 transduction and error prone DNA repair of double strand breaks induced by zeocin, a radiomimetic agent. Homologous recombination does not require PNPase phosphorolytic activity and is modulated by its RNA binding domains whereas error prone DNA repair of zeocin-induced DNA damage is dependent on PNPase catalytic activity and cannot be suppressed by overexpression of RNase II, the other major enzyme (encoded by rnb) implicated in exonucleolytic RNA degradation. Moreover, E. coli pnp mutants are more sensitive than the wild type to zeocin. This phenotype depends on PNPase phosphorolytic activity and is suppressed by rnb, thus suggesting that zeocin detoxification may largely depend on RNA turnover. Our data suggest that PNPase may participate both directly and indirectly through regulation of gene expression to several aspects of DNA metabolism such as recombination, DNA repair and resistance to genotoxic agents.

  11. The exoribonuclease Polynucleotide Phosphorylase influences the virulence and stress responses of yersiniae and many other pathogens

    Directory of Open Access Journals (Sweden)

    Jason A. Rosenzweig

    2013-11-01

    Full Text Available Microbes are incessantly challenged by both biotic and abiotic stressors threatening their existence. Therefore, bacterial pathogens must possess mechanisms to successfully subvert host immune defenses as well as overcome the stress associated with host-cell encounters. To achieve this, bacterial pathogens typically experience a genetic re-programming whereby anti-host/stress factors become expressed and eventually translated into effector proteins. In that vein, the bacterial host-cell induced stress-response is similar to any other abiotic stress to which bacteria respond by up-regulating specific stress-responsive genes. Following the stress encounter, bacteria must degrade unnecessary stress responsive transcripts through RNA decay mechanisms. The 3 pathogenic yersiniae (Yersinia pestis, Y. pseudo-tuberculosis, and Y. enterocolitica are all psychrotropic bacteria capable of growth at 4˚C; however, cold growth is dependent on the presence of an exoribonuclease, polynucleotide phosphorylase (PNPase. PNPase has also been implicated as a virulence factor in several notable pathogens including the salmonellae, Helicobacter pylori, and the yersiniae (where it typically influences the type three secretion system. Further, PNPase has been shown to associate with ribonuclease E (endoribonuclease, RhlB (RNA helicase, and enolase (glycolytic enzyme in several Gram-negative bacteria forming a large, multi-protein complex known as the RNA degradosome. This review will highlight studies demonstrating the influence of PNPase on the virulence potentials and stress responses of various bacterial pathogens as well as focusing on the degradosome- dependent and -independent roles played by PNPase in yersiniae stress responses.

  12. Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes: 1. Identification of 1-amino-1-cycloalkyl carboxylic acid headgroups

    Energy Technology Data Exchange (ETDEWEB)

    Sparks, Steven M.; Banker, Pierette; Bickett, David M.; Carter, H. Luke; Clancy, Daphne C.; Dickerson, Scott H.; Dwornik, Kate A.; Garrido, Dulce M.; Golden, Pamela L.; Nolte, Robert T.; Peat, Andrew J.; Sheckler, Lauren R.; Tavares, Francis X.; Thomson, Stephen A.; Wang, Liping; Weiel, James E.; (GSKNC)

    2009-05-15

    Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.

  13. Three-dimensional structure of thymidine phosphorylase from E. coli in complex with 3'-azido-2'-fluoro-2',3'-dideoxyuridine

    Science.gov (United States)

    Timofeev, V. I.; Abramchik, Yu. A.; Fateev, I. V.; Zhukhlistova, N. E.; Murav'eva, T. I.; Kuranova, I. P.; Esipov, R. S.

    2013-11-01

    The three-dimensional structures of thymidine phosphorylase from E. coli containing the bound sulfate ion in the phosphate-binding site and of the complex of thymidine phosphorylase with sulfate in the phosphate-binding site and the inhibitor 3'-azido-2'-fluoro-2',3'-dideoxyuridine (N3F-ddU) in the nucleoside-binding site were determined at 1.55 and 1.50 Å resolution, respectively. The amino-acid residues involved in the ligand binding and the hydrogen-bond network in the active site occupied by a large number of bound water molecules are described. A comparison of the structure of thymidine phosphorylase in complex with N3F-ddU with the structure of pyrimidine nucleoside phosphorylase from St. Aureus in complex with the natural substrate thymidine (PDB_ID: 3H5Q) shows that the substrate and the inhibitor in the nucleoside-binding pocket have different orientations. It is suggested that the position of N3F-ddU can be influenced by the presence of the azido group, which prefers a hydrophobic environment. In both structures, the active sites of the subunits are in the open conformation.

  14. Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

    Science.gov (United States)

    Wang, Hong; Brautigan, David L

    2006-11-01

    Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

  15. Guanine nucleotide exchange factor αPIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells

    DEFF Research Database (Denmark)

    Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea

    2015-01-01

    Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the sig...

  16. [Effect of low-intensity 900 MHz frequency electromagnetic radiation on rat liver and blood serum enzyme activities].

    Science.gov (United States)

    Nersesova, L S; Petrosian, M S; Gazariants, M G; Mkrtchian, Z S; Meliksetian, G O; Pogosian, L G; Akopian, Zh I

    2014-01-01

    The comparative analysis of the rat liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and purine nucleoside phosphorylase post-radiation activity levels after a total two-hour long single and fractional exposure of the animals to low-intensity 900 MHz frequency electromagnetic field showed that the most sensitive enzymes to the both schedules of radiation are the liver creatine kinase, as well as the blood serum creatine kinase and alkaline phosphatase. According to the comparative analysis of the dynamics of changes in the activity level of the liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase and purine nucleoside phosphorylase, both single and fractional radiation schedules do not affect the permeability of a hepatocyte cell membrane, but rather cause changes in their energetic metabolism. The correlation analysis of the post-radiation activity level changes of the investigated enzymes did not reveal a clear relationship between them. The dynamics of post-radiation changes in the activity of investigated enzyme levels following a single and short-term fractional schedules of radiation did not differ essentially.

  17. Selective killing of tumors deficient in methylthioadenosine phosphorylase: a novel strategy.

    Directory of Open Access Journals (Sweden)

    Martin Lubin

    Full Text Available BACKGROUND: The gene for methylthioadenosine phosphorylase (MTAP lies on 9p21, close to the gene CDKN2A that encodes the tumor suppressor proteins p16 and p14ARF. MTAP and CDKN2A are homozygously co-deleted, with a frequency of 35 to 70%, in lung and pancreatic cancer, glioblastoma, osteosarcoma, soft-tissue sarcoma, mesothelioma, and T-cell acute lymphoblastic leukemia. In normal cells, but not in tumor cells lacking MTAP, MTAP cleaves the natural substrate, 5'-deoxy-5'-methylthioadenosine (MTA, to adenine and 5-methylthioribose-1-phosphate (MTR-1-P, which are then converted to adenine nucleotides and methionine. This distinct difference between normal MTAP-positive cells and tumor MTAP-negative cells led to several proposals for therapy. We offer a novel strategy in which both MTA and a toxic adenine analog, such as 2,6-diaminopurine (DAP, 6-methylpurine (MeP, or 2-fluoroadenine (F-Ade, are administered. In MTAP-positive cells, abundant adenine, generated from supplied MTA, competitively blocks the conversion of an analog, by adenine phosphoribosyltransferase (APRT, to its active nucleotide form. In MTAP-negative tumor cells, the supplied MTA cannot generate adenine; hence conversion of the analog is not blocked. PRINCIPAL FINDINGS: We show that this combination treatment--adenine analog plus MTA--kills MTAP-negative A549 lung tumor cells, while MTAP-positive human fibroblasts (HF are protected. In co-cultures of the breast tumor cell line, MCF-7, and HF cells, MCF-7 is inhibited or killed, while HF cells proliferate robustly. 5-Fluorouracil (5-FU and 6-thioguanine (6-TG may also be used with our strategy. Though neither analog is activated by APRT, in MTAP-positive cells, adenine produced from supplied MTA blocks conversion of 5-FU and 6-TG to their toxic nucleotide forms by competing for 5-phosphoribosyl-1-pyrophosphate (PRPP. The combination of MTA with 5-FU or 6-TG, in the treatment of MTAP-negative tumors, may produce a significantly

  18. Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs

    Directory of Open Access Journals (Sweden)

    Venkatesh KV

    2005-05-01

    Full Text Available Abstract Background Signaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state. Results The glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase. Conclusion The highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different

  19. Ion mobility-mass spectrometry of phosphorylase B ions generated with supercharging reagents but in charge-reducing buffer.

    Science.gov (United States)

    Hogan, Christopher J; Ogorzalek Loo, Rachel R; Loo, Joseph A; de la Mora, Juan Fernandez

    2010-11-01

    We investigate whether "supercharging" reagents able to shift the charge state distributions (CSDs) of electrosprayed protein ions upward also influence gas-phase protein structure. A differential mobility analyzer and a mass spectrometer are combined in series (DMA-MS) to measure the mass and mobility of monomer and multimeric phosphorylase B ions (monomer molecular weight ∼97 kDa) in atmospheric pressure air. Proteins are electrosprayed from charge-reducing triethylammonium formate in water (pH = 6.8) with and without the addition of the supercharging reagent tetramethylene sulfone (sulfolane). Because the DMA measures ion mobility prior to collisional heating or declustering, it probes the structure of supercharged protein ions immediately following solvent (water) evaporation. As in prior studies, the addition of sulfolane is found to drastically increase both the mean and maximum charge state of phosphorylase B ions. Ions from all protein n-mers were found to yield mobilities that, for a given charge state, were ∼6-10% higher in the absence of sulfolane. We find that the mobility decrease which arises with sulfolane is substantially smaller than that typically observed for folded-to-unfolded transitions in protein ions (where a ∼60% decrease in mobility is typical), suggesting that supercharging reagents do not cause structural protein modifications in solution as large as noted recently by Williams and colleagues [E. R. Williams et al., J. Am. Soc. Mass Spectrom., 2010, 21, 1762-1774]. In fact, the measurements described here indicate that the modest mobility decrease observed can be partly attributed to sulfolane trapping within the protein ions during DMA measurements, and probably also in solution. As the most abundant peaks in measured mass-mobility spectra for ions produced with and without sulfolane correspond to non-covalently bound phosphorylase B dimers, we find that in spite of a change in mobility/cross section, sulfolane addition does not

  20. Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12.

    Science.gov (United States)

    Mikhailov, A M; Smirnova, E A; Tsuprun, V L; Tagunova, I V; Vainshtein, B K; Linkova, E V; Komissarov, A A; Siprashvili, Z Z; Mironov, A S

    1992-03-01

    Uridine phosphorylase (UPH) from Escherichia coli K-12 has been purified to near homogeneity from a strain harbouring the udp gene, encoding UPH, on a multicopy plasmid. UPH was purified to electrophoretic homogeneity with the specific activity 230 units/mg with a recovery of 80%, yielding 120 mg of enzyme from 3g cells. Crystals of enzyme suitable for X-ray diffraction analysis were obtained in a preparative ultracentrifuge. The packing of the molecules in the crystals may be described by the space group P2(1)2(1)2(1) with the unit cell constants a = 90.4; b = 128.8; c = 136.8 A. There is one molecule per asymmetric unit, Vm = 2.4. These crystals diffract to at least 2.5-2.7 A resolution. The hexameric structure of UPH was directly demonstrated by electron microscopy study and image processing.

  1. Crystallization and preliminary X-ray diffraction analysis of Salmonella typhimurium uridine phosphorylase complexed with 5-fluorouracil.

    Science.gov (United States)

    Lashkov, A A; Gabdoulkhakov, A G; Shtil, A A; Mikhailov, A M

    2009-06-01

    Uridine phosphorylase (UPh; EC 2.4.2.3) catalyzes the phosphorolytic cleavage of the N-glycosidic bond of uridine to form ribose 1-phosphate and uracil. This enzyme also activates pyrimidine-containing drugs, including 5-fluorouracil (5-FU). In order to better understand the mechanism of the enzyme-drug interaction, the complex of Salmonella typhimurium UPh with 5-FU was cocrystallized using the hanging-drop vapour-diffusion method at 294 K. X-ray diffraction data were collected to 2.2 A resolution. Analysis of these data revealed that the crystal belonged to space group C2, with unit-cell parameters a = 158.26, b = 93.04, c = 149.87 A, alpha = gamma = 90, beta = 90.65 degrees . The solvent content was 45.85% assuming the presence of six hexameric molecules of the complex in the unit cell.

  2. Two-step enzymatic synthesis of guanine arabionside%两步酶法合成阿糖鸟苷

    Institute of Scientific and Technical Information of China (English)

    魏晓琨; 丁庆豹; 欧伶; 张鹭; 王昌禄

    2008-01-01

    The chemical synthesis of Guanine arabinoside (ara-G) is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine arabinoside (ara-DA) was first synthesized with purine nucleoside phosphorylase and pyrimidine nucleoside phos-phorylase produced by Enterobacter aerogenes DGW-07. The conversion yield of ara-DA could reach above 90% when the reaction liquid contained 30 mmol·L-1 uracil arabinoside as arabinose donor, 10 mmol·L-1 2,6-diaminopurine as arabinose acceptor in pH 7.0 20 mmol·L-1 phosphate buffer, and reacted at 60℃ for 48h. Then, ara-DA was effectively transformed into ara-G with adenylate deaminase produced by Aspergillus oryzae DAW-01. The total process had no complex separation and purification.

  3. Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization, core and urea optimization and in vivo efficacy

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, Stephen A.; Banker, Pierette; Bickett, D. Mark; Boucheron, Joyce A.; Carter, H. Luke; Clancy, Daphne C.; Cooper, Joel P.; Dickerson, Scott H.; Garrido, Dulce M.; Nolte, Robert T.; Peat, Andrew J.; Sheckler, Lauren R.; Sparks, Steven M.; Tavares, Francis X.; Wang, Liping; Wang, Tony Y.; Weiel, James E.; (GSKNC)

    2009-05-15

    Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.

  4. Three-dimensional structures of unligated uridine phosphorylase from Yersinia pseudotuberculosis at 1.4 Å resolution and its complex with an antibacterial drug

    Science.gov (United States)

    Balaev, V. V.; Lashkov, A. A.; Gabdulkhakov, A. G.; Dontsova, M. V.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

    2015-07-01

    Uridine phosphorylases play an essential role in the cellular metabolism of some antibacterial agents. Acute infectious diseases (bubonic plague, yersiniosis, pseudotuberculosis, etc., caused by bacteria of the genus Yersinia) are treated using both sulfanilamide medicines and antibiotics, including trimethoprim. The action of an antibiotic on a bacterial cell is determined primarily by the character of its interactions with cellular components, including those which are not targets (for example, with pyrimidine phosphorylases). This type of interaction should be taken into account in designing drugs. The three-dimensional structure of uridine phosphorylase from the bacterium Yersinia pseudotuberculosis ( YptUPh) with the free active site was determined for the first time by X-ray crystallography and refined at 1.40 Å resolution (DPI = 0.062 Å; ID PDB: 4OF4). The structure of the complex of YptUPh with the bacteriostatic drug trimethoprim was studied by molecular docking and molecular dynamics methods. The trimethoprim molecule was shown to be buffered by the enzyme YptUPh, resulting in a decrease in the efficiency of the treatment of infectious diseases caused by bacteria of the genus Yersinia with trimethoprim.

  5. Effects of eugenol-reduced clove extract on glycogen phosphorylase b and the development of diabetes in db/db mice.

    Science.gov (United States)

    Sanae, Fujiko; Kamiyama, Ogusa; Ikeda-Obatake, Kyoko; Higashi, Yasuhiko; Asano, Naoki; Adachi, Isao; Kato, Atsushi

    2014-02-01

    We found that the 50% aqueous EtOH extract of clove (Syzygium aromaticum) had potent dose-dependent inhibitory activity toward glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes. Among the components, eugeniin inhibited glycogen phosphorylase b and glucagon-stimulated glucose production in primary rat hepatocytes, with IC50 values of 0.14 and 4.7 μM, respectively. In sharp contrast, eugenol showed no significant inhibition toward glycogen phosphorylase b, even at a concentration of 400 μM. Eugenol-reduced clove extracts (erCE) were prepared and when fed to a db/db mouse they clearly suppressed the blood glucose and HbA1c levels. Furthermore, plasma triglyceride and non-esterified fatty acid levels in 5% and 10% erCE-fed db/db mice were significantly lowered, compared with control db/db mice without erCE supplementation. These results suggested that dietary supplementation with the erCE could beneficially modify glucose and lipid metabolism and contribute to the prevention of the progress of hyperglycemia and metabolic syndrome.

  6. Inhibition of uptake of adenosine into human blood platelets

    NARCIS (Netherlands)

    Lips, J.P.M.; Sixma, J.J.; Trieschnigg, A.C.

    1980-01-01

    Adenosine transport into human blood platelets is mediated by two independent systems with different affinities. Both systems transport only purine nucleosides and no pyrimidine nucleosides. In experiments with differently substituted purine nucleosides, purines and analogues, differences in carrier

  7. Synthesis, enzyme kinetics and computational evaluation of N-(β-D-glucopyranosyl) oxadiazolecarboxamides as glycogen phosphorylase inhibitors.

    Science.gov (United States)

    Polyák, Mária; Varga, Gergely; Szilágyi, Bence; Juhász, László; Docsa, Tibor; Gergely, Pál; Begum, Jaida; Hayes, Joseph M; Somsák, László

    2013-09-15

    All possible isomers of N-β-D-glucopyranosyl aryl-substituted oxadiazolecarboxamides were synthesised. O-Peracetylated N-cyanocarbonyl-β-D-glucopyranosylamine was transformed into the corresponding N-glucosyl tetrazole-5-carboxamide, which upon acylation gave N-glucosyl 5-aryl-1,3,4-oxadiazole-2-carboxamides. The nitrile group of the N-cyanocarbonyl derivative was converted to amidoxime which was ring closed by acylation to N-glucosyl 5-aryl-1,2,4-oxadiazole-3-carboxamides. A one-pot reaction of protected β-D-glucopyranosylamine with oxalyl chloride and then with arenecarboxamidoximes furnished N-glucosyl 3-aryl-1,2,4-oxadiazole-5-carboxamides. Removal of the O-acetyl protecting groups by the Zemplén method produced test compounds which were evaluated as inhibitors of glycogen phosphorylase. Best inhibitors of these series were N-(β-D-glucopyranosyl) 5-(naphth-1-yl)-1,2,4-oxadiazol-3-carboxamide (Ki = 30 μM), N-(β-D-glucopyranosyl) 5-(naphth-2-yl)-1,3,4-oxadiazol-2-carboxamide (Ki =33 μM), and N-(β-D-glucopyranosyl) 3-phenyl-1,2,4-oxadiazol-5-carboxamide (Ki = 104 μM). ADMET property predictions revealed these compounds to have promising oral drug-like properties without any toxicity.

  8. Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly.

    Science.gov (United States)

    Hardwick, Steven W; Gubbey, Tobias; Hug, Isabelle; Jenal, Urs; Luisi, Ben F

    2012-04-01

    Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

  9. An Imprinted Cross-Linked Enzyme Aggregate (iCLEA of Sucrose Phosphorylase: Combining Improved Stability with Altered Specificity

    Directory of Open Access Journals (Sweden)

    Tom Desmet

    2012-09-01

    Full Text Available The industrial use of sucrose phosphorylase (SP, an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA. In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme’s specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C. This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA.

  10. Methylthioadenosine phosphorylase gene is silenced by promoter hypermethylation in human lymphoma cell line DHL-9: another mechanism of enzyme deficiency.

    Science.gov (United States)

    Ishii, Masaaki; Nakazawa, Keiko; Wada, Hideo; Nishioka, Junji; Nakatani, Kaname; Yamada, Yasuaki; Kamihira, Shimeru; Kusunoki, Masato; Nobori, Tsutomu

    2005-04-01

    Methylthioadenosine phosphorylase (MTAP) involved in the metabolism of purine and polyamine has been known to be deficient in a variety of tumors. Although this enzyme deficiency was reportedly caused by partial or total deletion of the MTAP gene, human MTAP-deficient lymphoma cell line DHL-9 has the intact MTAP gene. In order to determine the mechanism of MTAP deficiency in DHL-9, we carried out methylation-specific PCR analysis of sodium bisulfite-treated genomic DNA followed by DNA sequence analysis. Following incubation with various concentrations of 5-Aza-2'-deoxycytidine, DHL-9 cells were subjected to RT-PCR and an immunoblot analysis for MTAP expression. MTAP promoter in DHL-9 cells was methylated at cytosine of all CpG dinucleotides analyzed. Moreover, 5-Aza-2'-deoxycytidine treatment induced DHL-9 cells to express MTAP mRNA and protein. Taken together, MTAP deficiency in DHL-9 was caused by transcriptional silencing due to promoter methylation. Promoter methylation of the MTAP gene was also found in DNA samples from adult T-cell leukemia patients. These results indicated that promoter hypermethylation is another mechanism of MTAP deficiency in human malignancy. Thus, immunological diagnostics will be needed for an accurate evaluation of MTAP expression at the protein level.

  11. Activity of glycogen synthase and glycogen phosphorylase in normal and cirrhotic rat liver during glycogen synthesis from glucose or fructose.

    Science.gov (United States)

    Bezborodkina, Natalia N; Chestnova, Anna Yu; Okovity, Sergey V; Kudryavtsev, Boris N

    2014-03-01

    Cirrhotic patients often demonstrate glucose intolerance, one of the possible causes being a decreased glycogen-synthesizing capacity of the liver. At the same time, information about the rates of glycogen synthesis in the cirrhotic liver is scanty and contradictory. We studied the dynamics of glycogen accumulation and the activity of glycogen synthase (GS) and glycogen phosphorylase (GP) in the course of 120min after per os administration of glucose or fructose to fasted rats with CCl4-cirrhosis or fasted normal rats. Blood serum and liver pieces were sampled for examinations. In the normal rat liver administration of glucose/fructose initiated a fast accumulation of glycogen, while in the cirrhotic liver glycogen was accumulated with a 20min delay and at a lower rate. In the normal liver GS activity rose sharply and GPa activity dropped in the beginning of glycogen synthesis, but 60min later a high synthesis rate was sustained at the background of a high GS and GPa activity. Contrariwise, in the cirrhotic liver glycogen was accumulated at the background of a decreased GS activity and a low GPa activity. Refeeding with fructose resulted in a faster increase in the GS activity in both the normal and the cirrhotic liver than refeeding with glucose. To conclude, the rate of glycogen synthesis in the cirrhotic liver is lower than in the normal one, the difference being probably associated with a low GS activity.

  12. The development and validation of an immunoassay for the measurement of anti-thymidine phosphorylase antibodies in mouse and dog sera.

    Science.gov (United States)

    Gasson, Charlotte; Levene, Michelle; Bax, Bridget E

    2013-01-01

    Erythrocyte encapsulated thymidine phosphorylase (EE-TP) is under development as an enzyme replacement therapy for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a fatal metabolic disorder resulting from an inherited deficiency of the enzyme thymidine phosphorylase. We report here the development and validation of a sensitive electrochemiluminescent (ECL) bridging immunoassay to support Good Laboratory Practice (GLP)-compliant preclinical safety studies of EE-TP in the mouse and dog. Affinity-purified rabbit anti-E. coli thymidine phosphorylase (TP) antibody was used as a calibrator standard with an effective working range of 2.5-7500 ng/mL. The minimum required dilution (MRD) for both mouse and dog sera was 1:10. The mean analytical recoveries for anti-TP antibodies spiked into serum at 70 ng/mL and 7000 ng/mL were 117.9% and 93.2%, respectively for mouse, and 112.0% and 104.3%, respectively for dog. The intra-assay precision (coefficient of variation, CV) ranged between 1.1% and 8.0% in mouse serum, and 1.9% and 2.5% in dog serum. Inter-assay precision ranged between -1.6% and 6.7% in mouse serum, and -13.0% and -2.5% in dog serum. Assay cut-point/screening cut-point correction factors were 201.37 and 44.4, respectively for mouse and dog sera. For future analysis of positive test samples, less than 37.12% (mouse) and 31.41% (dog) inhibition of the assay signal in the confirmation assay will confer anti-TP antibody specificity. Assay drift and hook effects (prozone) were not observed. The intra-assay and inter-assay accuracy for robustness were within ±20%.

  13. Function of pyridoxal 5'-phosphate in glycogen phosphorylase: a model study using 6-fluoro-5'-deoxypyridoxal- and 5'-deoxypyridoxal-reconstituted enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Y.C.; Scott, R.D.; Graves, D.J.

    1987-01-27

    A new vitamin B/sub 6/ analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear /sup 1/H-/sup 18/F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDLP showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL except the K/sub m/ of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower V/sub max/ value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. /sup 19/F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the /sup 19/F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate. Analysis of the activation parameters, activation enthalpy and activation entropy, of the reaction of glycogen degradation catalyzed by phosphorylase containing pyridoxal phosphate, 6-FDPL, pyridoxal, or DPL showed that modifications of the coenzyme molecule affected only the activation entropy, not the activation enthalpy. Results of this study indicate that the protein structure surrounding the coenzyme molecule, as well as the coenzyme configuration, is altered upon the binding of ligands.

  14. 3'-Azidothymidine in the active site of Escherichia coli thymidine phosphorylase: the peculiarity of the binding on the basis of X-ray study.

    Science.gov (United States)

    Timofeev, Vladimir; Abramchik, Yulia; Zhukhlistova, Nadezda; Muravieva, Tatiana; Fateev, Ilya; Esipov, Roman; Kuranova, Inna

    2014-04-01

    The structural study of complexes of thymidine phosphorylase (TP) with nucleoside analogues which inhibit its activity is of special interest because many of these compounds are used as chemotherapeutic agents. Determination of kinetic parameters showed that 3'-azido-3'-deoxythymidine (3'-azidothymidine; AZT), which is widely used for the treatment of human immunodeficiency virus, is a reversible noncompetitive inhibitor of Escherichia coli thymidine phosphorylase (TP). The three-dimensional structure of E. coli TP complexed with AZT was solved by the molecular-replacement method and was refined at 1.52 Å resolution. Crystals for X-ray study were grown in microgravity by the counter-diffusion technique from a solution of the protein in phosphate buffer with ammonium sulfate as a precipitant. The AZT molecule was located with full occupancy in the electron-density maps in the nucleoside-binding pocket of TP, whereas the phosphate-binding pocket of the enzyme was occupied by phosphate (or sulfate) ion. The structure of the active-site cavity and conformational changes of the enzyme upon AZT binding are described in detail. It is found that the position of AZT differs remarkably from the positions of the pyrimidine bases and nucleoside analogues in other known complexes of pyrimidine phosphorylases, but coincides well with the position of 2'-fluoro-3'-azido-2',3'-dideoxyuridine (N3FddU) in the recently investigated complex of E. coli TP with this ligand (Timofeev et al., 2013). The peculiarities of the arrangement of N3FddU and 3'-azidothymidine in the nucleoside binding pocket of TP and correlations between the arrangement and inhibitory properties of these compounds are discussed.

  15. Age-associated tyrosine nitration of rat skeletal muscle glycogen phosphorylase b: characterization by HPLC-nanoelectrospray-tandem mass spectrometry.

    Science.gov (United States)

    Sharov, Victor S; Galeva, Nadezhda A; Kanski, Jaroslaw; Williams, Todd D; Schöneich, Christian

    2006-04-01

    We identified age-dependent post-translational modifications of skeletal muscle glycogen phosphorylase b (Ph-b), isolated from F1 hybrids of Fisher 344 x Brown Norway rats. Ph-b isolated from 34 months old rats showed a statistically significant decrease in specific activity compared to 6 months old animals: 13.8+/-0.7 vs. 20.6+/-0.8 U mg(-1) protein, respectively. Western blot analysis of the purified Ph-b with anti-3-NT antibodies revealed an age-dependent accumulation of 3-nitrotyrosine (3-NT), quantified by reverse-phase HPLC-UV analysis to increase from 0.05+/-0.03 to 0.34+/-0.11 (mol 3-NT/mol Ph-b) for 6 vs. 34 months old rats, respectively. HPLC-nanoelectrospray ionization-tandem mass spectrometry revealed the accumulation of 3-NT on Tyr113, Tyr161 and Tyr573. While nitration of Tyr113 was detected for both young and old rats, 3-NT at positions 161 and 573 was identified only for Ph-b isolated from 34 months old rats. The sequence of the rat muscle Ph-b was corrected based on our protein sequence mapping and a custom rat PHS2 sequence containing 17 differently located amino acid residues was used instead of the database sequence. The in vitro reaction of peroxynitrite with Ph-b resulted in the nitration of multiple Tyr residues at positions 51, 52, 113, 155, 185, 203, 262, 280, 404, 473, 731, and 732. Thus, the in vitro nitration conditions only mimic the nitration of a single Tyr residue observed in vivo suggesting alternative pathways controlling the accumulation of 3-NT in vivo. Our data show a correlation of age-dependent 3-NT accumulation with Ph-b inactivation.

  16. Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y: STRUCTURAL MAPPING AND EFFECT ON mRNA TURNOVER.

    Science.gov (United States)

    Salvo, Elizabeth; Alabi, Shanique; Liu, Bo; Schlessinger, Avner; Bechhofer, David H

    2016-03-25

    Polynucleotide phosphorylase (PNPase), a 3'-to-5' phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover ofBacillus subtilismRNA. The role of PNPase inB. subtilismRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strainversusa strain that is deleted forpnpA, the gene encoding PNPase. Recent studies have provided evidence for a degradosome-like complex inB. subtilisthat is built around the major decay-initiating endonuclease, RNase Y, and there is ample evidence for a strong interaction between PNPase and RNase Y. The role of the PNPase-RNase Y interaction in the exonucleolytic function of PNPase needs to be clarified. We sought to construct aB. subtilisstrain containing a catalytically active PNPase that could not interact with RNase Y. Mapping studies of the PNPase-RNase Y interaction were guided by a homology model ofB. subtilisPNPase based on the known structure of theEscherichia coliPNPase in complex with an RNase E peptide. Mutations inB. subtilisresidues predicted to be involved in RNase Y binding showed a loss of PNPase-RNase Y interaction. Two mRNAs whose decay is dependent on RNase Y and PNPase were examined in strains containing full-length PNPase that was either catalytically active but unable to interact with RNase Y, or catalytically inactive but able to interact with RNase Y. At least for these two mRNAs, disruption of the PNPase-RNase Y interaction did not appear to affect mRNA turnover.

  17. Ca2+-induced structural changes in phosphorylase kinase detected by small-angle x-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Priddy, Timothy S. [University of Missouri and University of Kansas Medical Center; Macdonald, Brian A. [Los Alamos National Laboratory (LANL); Heller, William T [ORNL; Nadeau, Owen W. [University of Kansas Medical Center; Trewhella, Jill [Los Alamos National Laboratory (LANL); Carlson, Gerald M. [University of Kansas Medical Center

    2005-01-01

    Phosphorylase kinase (PhK), a 1.3-MDa ({alpha}{beta}{gamma}{delta}){sub 4} hexadecameric complex, is a Ca{sup 2+}-dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched ({alpha}{beta}{gamma}{delta}){sub 2} octameric lobes that are oriented back-to-back with overall D{sub 2} symmetry and joined by connecting bridges. From chemical cross-linking and electron microscopy, it is known that the binding of Ca{sup 2+} by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca{sup 2+} in the PhK complex in solution, small-angle X-ray scattering was performed on nonactivated and Ca{sup 2+}-activated PhK. Although the overall dimensions of the complex were not affected by Ca{sup 2+}, the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca{sup 2+}-induced conformational change. Computer-generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca{sup 2+}-activated conformers of PhK are consistent with Ca{sup 2+}-induced conformational changes in both the lobes and the interlobal bridges.

  18. Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase.

    Science.gov (United States)

    Vidal-Meireles, André; Neupert, Juliane; Zsigmond, Laura; Rosado-Souza, Laise; Kovács, László; Nagy, Valéria; Galambos, Anikó; Fernie, Alisdair R; Bock, Ralph; Tóth, Szilvia Z

    2017-04-01

    Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and (1) O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

  19. Overexpression of the starch phosphorylase-like gene (PHO3 in Lotus japonicus has a profound effect on the growth of plants and reduction of transitory starch accumulation

    Directory of Open Access Journals (Sweden)

    Shanshan Qin

    2016-08-01

    Full Text Available Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1, plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied.

  20. Localization of a new type of X-linked liver glycogenosis to the chromosomal region Xp22 containing the liver {alpha}-subunit of phosphorylase kinase (PHKA2)

    Energy Technology Data Exchange (ETDEWEB)

    Hendrickx, J.; Coucke, P.; Willems, P.J. [Univ. of Antwerp (Belgium)] [and others

    1994-06-01

    The authors describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in constrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at {theta}=0, relative to DXS987. As both the classical XLG gene and the liver {alpha}-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, they propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG). 28 refs., 2 figs., 3 tabs.

  1. Overexpression of the Starch Phosphorylase-Like Gene (PHO3) in Lotus japonicus has a Profound Effect on the Growth of Plants and Reduction of Transitory Starch Accumulation

    Science.gov (United States)

    Qin, Shanshan; Tang, Yuehui; Chen, Yaping; Wu, Pingzhi; Li, Meiru; Wu, Guojiang; Jiang, Huawu

    2016-01-01

    Two isoforms of starch phosphorylase (PHO; EC 2.4.1.1), plastidic PHO1 and cytosolic PHO2, have been found in all plants studied to date. Another starch phosphorylase-like gene, PHO3, which is an ortholog of Chlamydomonas PHOB, has been detected in some plant lineages. In this study, we identified three PHO isoform (LjPHO) genes in the Lotus japonicus genome. Expression of the LjPHO3 gene was observed in all tissues tested in L. japonicus, and the LjPHO3 protein was located in the chloroplast. Overexpression of LjPHO3 in L. japonicus resulted in a drastic decline in starch granule sizes and starch content in leaves. The LjPHO3 overexpression transgenic seedlings were smaller, and showed decreased pollen fertility and seed set rate. Our results suggest that LjPHO3 may participate in transitory starch metabolism in L. japonicus leaves, but its catalytic properties remain to be studied. PMID:27630651

  2. Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-l-galactose Phosphorylase*

    Science.gov (United States)

    Urzica, Eugen I.; Adler, Lital N.; Page, M. Dudley; Linster, Carole L.; Arbing, Mark A.; Casero, David; Pellegrini, Matteo; Merchant, Sabeeha S.; Clarke, Steven G.

    2012-01-01

    The l-galactose (Smirnoff-Wheeler) pathway represents the major route to l-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-l-galactose phosphorylases converting GDP-l-galactose to l-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of l-ascorbate. Here we report that the l-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the l-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-l-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and l-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the l-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells. PMID:22393048

  3. A phase 1 study of the pharmacokinetics of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (components of TAS-102) vs trifluridine alone.

    Science.gov (United States)

    Cleary, James M; Rosen, Lee S; Yoshida, Kenichiro; Rasco, Drew; Shapiro, Geoffrey I; Sun, Weijing

    2017-04-01

    Background Trifluridine, a thymidine-based chemotherapeutic, has limited bioavailability after clinical administration as it is rapidly degraded via thymidine phosphorylase. An oral combination tablet combines trifluridine with a potent thymidine phosphorylase inhibitor, tipiracil hydrochloride. This study's objective was to evaluate whether trifluridine/tipiracil (TAS-102) administration increases trifluridine exposure vs trifluridine alone. Methods This open-label pharmacokinetic study randomly assigned patients with advanced solid tumors into two groups. On the morning of day 1, one group received a single 35 mg/m(2) dose of trifluridine/tipiracil and the other group received a single 35-mg/m(2) dose of trifluridine. Both groups received trifluridine/tipiracil 35 mg/m(2) on the evening of day 1, then twice daily on days 2-5 and 8-12 in a 28-day cycle. Results Twenty patients received an initial one-time dose of trifluridine alone and 19 other patients received an initial dose of trifluridine/tipiracil. Trifluridine area under the curve (AUC0-last) and maximum observed plasma concentrations (Cmax) were approximately 37- and 22-fold higher, respectively, with trifluridine/tipiracil vs trifluridine alone. Plasma concentrations of the major metabolite of trifluridine were lower following the administration of trifluridine/tipiracil vs trifluridine alone. Conclusion Tipiracil administered in combination with trifluridine significantly increased exposure to trifluridine compared with trifluridine alone.

  4. Elucidating the evolutionary history and expression patterns of nucleoside phosphorylase paralogs (vegetative storage proteins) in Populus and the plant kingdom.

    Science.gov (United States)

    Pettengill, Emily A; Pettengill, James B; Coleman, Gary D

    2013-08-19

    Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously uncharacterized NP-like proteins may

  5. Thymidine phosphorylase influences [(18)F]fluorothymidine uptake in cancer cells and patients with non-small cell lung cancer.

    Science.gov (United States)

    Lee, Seung Jin; Yeo, Jeong Seok; Lee, Haeng Jung; Lee, Eun Jung; Kim, Seog Young; Jang, Se Jin; Lee, Jong Jin; Ryu, Jin-Sook; Moon, Dae Hyuk

    2014-07-01

    Thymidine phosphorylase (TP), a key enzyme in the pyrimidine nucleoside salvage pathway, catalyses the reversible phosphorylation of thymidine, thereby generating thymine and 2-deoxy-D-ribose-1-phosphate. By regulating the levels of endogenous thymidine, TP may influence [(18)F]fluorothymidine ([(18)F]FLT) uptake. We investigated the effect of TP activity on [(18)F]FLT uptake by tumours. Uptake of [(3)H]FLT and [(3)H]thymidine ([(3)H]Thd) and the activities of TP, thymidine kinase 1 (TK1), and equilibrative nucleoside transporter 1 (ENT1) were determined in exponentially growing A431, A549, HT29, HOP92, ACHN, and SKOV3 cells in the presence or absence of tipiracil hydrochloride, a TP inhibitor. Eighty-five non-small cell lung cancer tissues from a patient cohort that was previously studied with [(18)F]FLT positron emission tomography (PET) were retrieved and subjected to immunohistochemical analysis of TP expression. Factors that affected the maximum standardised uptake value (SUVmax) of [(18)F]FLT-PET were identified by multiple linear regression analysis. A431 cells had the highest TP activity; A549 and HT29 cells had moderate TP activity; and ACHN, SKOV3, and HOP92 cells had little detectable TP activity. Cell lines with high TP activity took up more [(3)H]FLT than [(3)H]Thd, whereas cells with little TP activity took up more [(3)H]Thd than [(3)H]FLT. In cells with high TP activity, TP inhibition decreased [(3)H]FLT uptake and increased [(3)H]Thd uptake. However, TP inhibition had no effect on ACHN, SKOV3, and HOP92 cells. TP inhibition did not change TK1 or ENT1 activity, but did increase the intracellular level of thymidine. The SUVmax of [(18)F]FLT was affected by three independent factors: Ki-67 expression (P < 0.001), immunohistochemical TP score (P < 0.001), and tumour size (P = 0.015). TP activity influences [(18)F]FLT uptake, and may explain preferential uptake of [(18)F]FLT over [(3)H]Thd. These results provide important insights into the

  6. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    Energy Technology Data Exchange (ETDEWEB)

    Lashkov, A. A., E-mail: alashkov83@gmail.com; Sotnichenko, S. E.; Mikhailov, A. M. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2013-03-15

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis (YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to {alpha}/{beta} proteins, and its topology is a three-layer {alpha}/{beta}/{alpha} sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% {beta} strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium (StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli (EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  7. In silico analysis of the three-dimensional structures of the homodimer of uridine phosphorylase from Yersinia Pseudotuberculosis in the ligand-free state and in a complex with 5-fluorouracil

    Science.gov (United States)

    Lashkov, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

    2013-03-01

    Pseudotuberculosis is an acute infectious disease characterized by a lesion of the gastrointestinal tract. A positive therapeutic effect can be achieved by selectively suppressing the activity of uridine phosphorylase from the causative agent of the disease Yersinia pseudotuberculosis. The synergistic effect of a combination of the chemotherapeutic agent 5-fluorouracil and antimicrobial drugs, which block the synthesis of pyrimidine bases, on the cells of pathogenic protozoa and bacteria is described in the literature. The three-dimensional structures of uridine phosphorylase from Yersinia pseudotuberculosis ( YptUPh) both in the ligand-free state and in complexes with pharmacological agents are unknown, which hinders the search for and design of selective inhibitors of YptUPh. The three-dimensional structure of the ligand-free homodimer of YptUPh was determined by homology-based molecular modeling. The three-dimensional structure of the subunit of the YptUPh molecule belongs to α/β proteins, and its topology is a three-layer α/β/α sandwich. The subunit monomer of the YptUPh molecule consists of 38% helices and 24% β strands. A model of the homodimer structure of YptUPh in a complex with 5-FU was obtained by the molecular docking. The position of 5-FU in the active site of the molecule is very consistent with the known data on the X-ray diffraction structures of other bacterial uridine phosphorylases (the complex of uridine phosphorylase from Salmonella typhimurium ( StUPh) with 5-FU, ID PDB: 4E1V and the complex of uridine phosphorylase from Escherichia coli ( EcUPh) with 5-FU and ribose 1-phosphate, ID PDB: 1RXC).

  8. Hemoglobin interacting proteins and implications of spectrin hemoglobin interaction.

    Science.gov (United States)

    Basu, Avik; Chakrabarti, Abhijit

    2015-10-14

    In this report we have analyzed interacting partners of hemoglobin inside erythrocyte and sought possible implications of hemoglobin-spectrin interaction. Our list of identified cytosolic hemoglobin interacting proteins includes redox regulators like peroxiredoxin-2, Cu-Zn superoxide dismutase, catalase, aldehyde dehydrogenase-1, flavin reductase and chaperones like HSP70, α-hemoglobin stabilizing protein. Others include metabolic enzymes like carbonic anhydrase-1, selenium binding protein-1, purine nucleoside phosphorylase and nucleoside diphosphate kinase. Additionally, various membrane proteins like α and β spectrin, ankyrin, band3, protein4.1, actin and glyceraldehyde 3 phosphate dehydrogenase have been shown to interact with hemoglobin. Our result indicates that major membrane skeleton protein spectrin, that also has a chaperone like activity, helps to fold the unstable alpha-globin chains in vitro. Taken together our results could provide insight into a protein network evolved around hemoglobin molecule inside erythrocyte that may add a new perspective in understanding the hemoglobin function and homeostasis.

  9. Genomic evidence for complementary purine metabolism in the pea aphid, Acyrthosiphon pisum, and its symbiotic bacterium Buchnera aphidicola.

    Science.gov (United States)

    Ramsey, J S; MacDonald, S J; Jander, G; Nakabachi, A; Thomas, G H; Douglas, A E

    2010-03-01

    The purine salvage pathway recycles purines to nucleotides, promoting efficient utilization of purine nucleotides. Exceptionally among animals with completely sequenced genomes, the pea aphid lacks key purine recycling genes that code for purine nucleoside phosphorylase and adenosine deaminase, indicating that the aphid can neither metabolize nucleosides to the corresponding purines, nor adenosine to inosine. Purine metabolism genes in the symbiotic bacterium Buchnera complement aphid genes, and Buchnera can meet its nucleotide requirement from aphid-derived guanosine. Buchnera demand for nucleosides may have relaxed the selection for purine recycling in the aphid, leading to the loss of key aphid purine salvage genes. Further, the coupled purine metabolism of aphid and Buchnera could contribute to the dependence of the pea aphid on this symbiosis.

  10. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Hazelton, Keith Z. [Yeshiva Univ., New York, NY (United States); Ho, Meng-Chaio [Yeshiva Univ., New York, NY (United States); Cassera, Maria B. [Yeshiva Univ., New York, NY (United States); Clinch, Keith [Industrial Research Ltd., Lower Hutt (New Zealand); Crump, Douglas R. [Industrial Research Ltd., Lower Hutt (New Zealand); Rosario Jr., Irving [Yeshiva Univ., New York, NY (United States); Merino, Emilio F. [Yeshiva Univ., New York, NY (United States); Almo, Steve C. [Yeshiva Univ., New York, NY (United States); Tyler, Peter C. [Industrial Research Ltd., Lower Hutt (New Zealand); Schramm, Vern L. [Yeshiva Univ., New York, NY (United States)

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  11. Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation.

    Science.gov (United States)

    Yuen, A H Y; Khalpey, Z; Lavitrano, M; McGregor, C G A; Kalsi, K K; Yacoub, M H; Smolenski, R T

    2006-01-01

    Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.

  12. An Efficient Chemoenzymatic Process for Preparation of Ribavirin

    Directory of Open Access Journals (Sweden)

    Vladimir Sakharov

    2015-01-01

    Full Text Available Ribavirin is an important antiviral drug, which is used for treatment of many diseases. The pilot-scale chemoenzymatic process for synthesis of the active pharmaceutical ingredient Ribavirin was developed with 32% overall yield and more than 99.5% purity. The described method includes the chemical synthesis of 1,2,4-triazole-3-carboxamide, which is a key intermediate and enzyme-catalyzed transglycosylation reaction for preparation of the desired product. 1,2,4-Triazole-3-carboxamide was synthesized from 5-amino-1,2,4-triazole-3-carboxylic acid by classical Chipen-Grinshtein method. Isolated from E. сoli BL21(DE3/pERPUPHHO1 strain the purine nucleoside phosphorylase was used as a biocatalytical system. All steps of this process were optimized and scaled.

  13. Synthesis and enzymatic evaluation of the guanosine analogue 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG): insights into the phosphorolysis reaction mechanism based on the blueprint transition state: S{sub N}1 or S{sub N}2?

    Energy Technology Data Exchange (ETDEWEB)

    DaSilveira Neto, Brenno A. [University of Brasilia (UnB), DF (Brazil). Lab. of Medicinal and Technological Chemistry; Lapis, Alexandre A.M. [Universidade Federal do Pampa (Unipampa), Bage, RS (Brazil); Netz, Paulo A.; Dias, Silvio L. P.; Tamborim, Silvia M.; Dupont, Jairton [Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS (Brazil). Inst. of Chemistry. Lab. of Molecular Catalysis; Spencer, John [University of Greenwich at Medway (United Kingdom). School of Science; Basso, Luiz A.; Santos, Digenes S. [Pontificia Universidade Catolica do Rio Grande do Sul (PUC-RS), Porto Alegre, RS (Brazil). Centro de Pesquisas em Biologia Molecular e Funcional

    2010-07-01

    A modified experimental procedure for the synthesis of MESG (2-amino-6-mercapto-7- methylpurine ribonucleoside) 1 has been successfully performed and its full characterization is presented. High resolution ESI(+)-MSMS indicates both the nucleoside bond cleavage as the main fragmentation in the gas phase and a possible S{sub N}1 mechanism. Ab initio transition state calculations based on the blue print transition state support this mechanistic rationale and discard an alternative S{sub N}2 mechanism. Assays using purine nucleoside phosphorylase (PNP) enzyme (human and M. tuberculosis sources) indicate its efficiency in the phosphorolysis of MESG and allow the quantitative determination of inorganic phosphate in real time assay. (author)

  14. Natural Products as New Treatment Options for Trichomoniasis: A Molecular Docking Investigation

    Directory of Open Access Journals (Sweden)

    Mary Snow Setzer

    2017-01-01

    Full Text Available Trichomoniasis, caused by the parasitic protozoan Trichomonas vaginalis, is the most common non-viral sexually-transmitted disease, and there can be severe complications from trichomoniasis. Antibiotic resistance in T. vaginalis is increasing, but there are currently no alternatives treatment options. There is a need to discover and develop new chemotherapeutic alternatives. Plant-derived natural products have long served as sources for new medicinal agents, as well as new leads for drug discovery and development. In this work, we have carried out an in silico screening of 952 antiprotozoal phytochemicals with specific protein drug targets of T. vaginalis. A total of 42 compounds showed remarkable docking properties to T. vaginalis methionine gamma-lyase (TvMGL and to T. vaginalis purine nucleoside phosphorylase (TvPNP. The most promising ligands were polyphenolic compounds, and several of these showed docking properties superior to either co-crystallized ligands or synthetic enzyme inhibitors.

  15. Determination of phosphate in soil extracts in the field: A green chemistry enzymatic method

    Directory of Open Access Journals (Sweden)

    Ellen R. Campbell

    2015-01-01

    First, the soil sample is extracted with deionized water and filtered. Next, an aliquot of the soil extract (0.5 mL is transferred to a disposable cuvette, containing 0.5 mL of reaction mixture [200 mM HEPES, pH 7.6, 20 mM MgCl2, with 80 nmol 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG and 1 unit of recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1], mixed, and incubated for 10 min at field temperature. Absorbance of the completed reaction is measured at 360 nm in open-source, portable photometer linked by bluetooth to a smartphone. The phosphate and phosphorus content of the soil is determined by comparison of its absorbance at 360 nm to a previously prepared standard phosphate curve, which is stored in the smartphone app.

  16. Crystal growth of phosphopantetheine adenylyltransferase, carboxypeptidase t, and thymidine phosphorylase on the international space station by the capillary counter-diffusion method

    Energy Technology Data Exchange (ETDEWEB)

    Kuranova, I. P., E-mail: inna@ns.crys.ras.ru; Smirnova, E. A.; Abramchik, Yu. A. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation); Chupova, L. A.; Esipov, R. S. [Russian Academy of Sciences, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation); Akparov, V. Kh. [Research Institute for Genetics and Selection of Industrial Microorganisms, Scientific Center of Russian Federation (Russian Federation); Timofeev, V. I.; Kovalchuk, M. V. [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2011-09-15

    Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis, thymidine phosphorylase from Escherichia coli, carboxypeptidase T from Thermoactinomyces vulgaris and its mutant forms, and crystals of complexes of these proteins with functional ligands and inhibitors were grown by the capillary counter-diffusion method in the Japanese Experimental Module Kibo on the International Space Station. The high-resolution X-ray diffraction data sets suitable for the determination of high-resolution three-dimensional structures of these proteins were collected from the grown crystals on the SPring-8 synchrotron radiation facility. The conditions of crystal growth for the proteins and the data-collection statistics are reported. The crystals grown in microgravity diffracted to a higher resolution than crystals of the same proteins grown on Earth.

  17. Phosphorylase re-expression, increase in the force of contraction and decreased fatigue following notexin-induced muscle damage and regeneration in the ovine model of McArdle disease.

    Science.gov (United States)

    Howell, J McC; Walker, K R; Creed, K E; Dunton, E; Davies, L; Quinlivan, R; Karpati, G

    2014-02-01

    McArdle disease is caused by a deficiency of myophosphorylase and currently a satisfactory treatment is not available. The injection of notexin into, or the layering of notexin onto, the muscles of affected sheep resulted in necrosis followed by regeneration of muscle fibres with the expression of both non-muscle isoforms of phosphorylase within the fibres and a reduction of the amount of glycogen in the muscle with an increase in the strength of contraction and a decrease in fatiguability in the muscle fibres. The sustained re-expression of both the brain and liver isoforms of phosphorylase within the muscle fibres provides further emphasis that strategies to enhance the re-expression of these isoforms should be investigated as a possible treatment for McArdle disease.

  18. Hasil In Silico Senyawa Z12501572, Z00321025, SCB5631028 dan SCB13970547 dibandingkan Turunan Zerumbon terhadap Human Liver Glycogen Phosphorylase (1l5Q sebagai Antidiabetes

    Directory of Open Access Journals (Sweden)

    Fitri Kusvila Aziz

    2016-12-01

    Full Text Available Abstrak Human Liver Glycogen Phosphorylase (HLGP, suatu katalis glikogen yang mengontrol pelepasan glukosa-1-fosfat glikogen dari hati. Enzim ini mempunyai peran sentral dalam luaran glukosa hati sehingga menjadi target obat antidiabetik. Kajian docking dilakukan pada komputer dengan prosesor Intel Pentium, RAM 1 GB dan Windows 7. Ligan yang digunakan adalah senyawa obat (Z12501572, Z00321025, SCB5631028 dan SCB13970547, dataset pembanding aktif glycogen phosphorylase outer dimer site (PYGL-out dan decoysdari www.dekois.com dan turunan zerumbon. Protein dipisahkan dari ligan nativ dan semua ligan beserta protein dikonversi menggunakan PyRx. Visualisasi interaksi ligan-protein dihasilkan dengan program Protein-Ligand Interaction Profiler (PLIP dan PyMOL. Senyawa ZER11 memiliki binding energy terbaik, yaitu -7.11 kkal/mol (untuk metode LGA dan GA dan -4.08 kkal/mol untuk metode SA. Nilai binding energy tersebut lebih rendah dari pada nilai untuk ligan native dan satu dari keempat senyawa obat, terlebih jika dibandingkan dengan bindingaffinity dari dataset dan decoys. Interaksi ligan-protein pada ketiga metode tersebut ditemukan sangat bervariasi. Hal berbeda terjadi untuk metode Vina, bindingenergy ZER11 (-9.9 kkal/mol lebih baik dibandingkan dengan ligan native dan keempat senyawa obat. Senyawa ZER11 memiliki residu interaksi yang sama dengan ligan native pada TRP67 dan LYS191 untuk metode Vina. Kata kunci: PDBID-1L5Q, AutoDock, docking molekuler, vina, antidiabetes   Abstract Human Liver Glycogen Phosphorylase (HLGP can catalyze glycogen and control the release of glucose-1-phosphate of glycogen from the liver. This enzyme has a central role in output rule of liver glucose as it can be used as an antidiabetic drug targets. Docking studies were carried out on PC with Intel Pentium, 1 GB RAM, in environment of Windows 7. Ligands used are drug compounds (Z12501572, Z00321025, SCB5631028 and SCB13970547, the active dataset comparator

  19. Clinical significance of the thymidylate synthase, dihydropyrimidine dehydrogenase, and thymidine phosphorylase mRNA expressions in hepatocellular carcinoma patients receiving 5-fluorouracil-based transarterial chemoembolization treatment

    Directory of Open Access Journals (Sweden)

    Zhao H

    2013-07-01

    Full Text Available Hongyun Zhao,1,* Yuanyuan Zhao,2,* Ying Guo,1 Yan Huang,2 Suxia Lin,3 Cong Xue,2 Fei Xu,2 Yang Zhang,1 Liping Zhao,2 Zhihuang Hu,2 Li Zhang1,2 1State Key Laboratory of Oncology in South China and National Anti-Cancer Drug Clinical Research Centre, 2State Key Laboratory of Oncology in South China and Department of Medical Oncology, 3Department of Pathological Oncology, Sun Yat-sen University Cancer Center, Guangzhou, People's Republic of China*These authors contributed equally to this workPurpose: To determine whether 5-fluorouracil (5-FU sensitivity is associated with the mRNA expressions of thymidylate synthase (TS, dihydropyrimidine dehydrogenase (DPD, and thymidine phosphorylase (TP in patients with hepatocellular carcinoma (HCC treated with 5-FU-based transarterial chemoembolization (TACE.Methods: Formalin-fixed, paraffin-embedded tumor specimens from 40 patients treated with 5-FU-based TACE were selected for the examination of TS, DPD, and TP expression level by a quantitative real-time reverse transcription- polymerase chain reaction (PCR technique. Patients were categorized into high and low expression groups according to the median expression level of each enzyme. Associations between the mRNA expression levels of TS, DPD, and TP and clinical parameters including treatment efficacies, clinicopathological factors, and prognosis were assessed.Results: High DPD expression was associated with worse treatment outcome, including intrahepatic disease progression rate (hazard ratio [HR] for high DPD versus low DPD, 2.212; 95% confidence interval [CI], 1.030–4.753; P = 0.042, extrahepatic disease progression rate (HR for high versus low DPD, 3.171; 95% CI, 1.003–10.023; P = 0.049, and progression-free survival (HR for high versus low DPD, 2.308; 95% CI, 1.102–4.836; P = 0.027. No correlation was found between the mRNA expression of TS/TP and treatment outcome.Conclusion: DPD mRNA expression level was negatively correlated with the clinical

  20. The RNA processing enzyme polynucleotide phosphorylase negatively controls biofilm formation by repressing poly-N-acetylglucosamine (PNAG production in Escherichia coli C

    Directory of Open Access Journals (Sweden)

    Carzaniga Thomas

    2012-11-01

    Full Text Available Abstract Background Transition from planktonic cells to biofilm is mediated by production of adhesion factors, such as extracellular polysaccharides (EPS, and modulated by complex regulatory networks that, in addition to controlling production of adhesion factors, redirect bacterial cell metabolism to the biofilm mode. Results Deletion of the pnp gene, encoding polynucleotide phosphorylase, an RNA processing enzyme and a component of the RNA degradosome, results in increased biofilm formation in Escherichia coli. This effect is particularly pronounced in the E. coli strain C-1a, in which deletion of the pnp gene leads to strong cell aggregation in liquid medium. Cell aggregation is dependent on the EPS poly-N-acetylglucosamine (PNAG, thus suggesting negative regulation of the PNAG biosynthetic operon pgaABCD by PNPase. Indeed, pgaABCD transcript levels are higher in the pnp mutant. Negative control of pgaABCD expression by PNPase takes place at mRNA stability level and involves the 5’-untranslated region of the pgaABCD transcript, which serves as a cis-element regulating pgaABCD transcript stability and translatability. Conclusions Our results demonstrate that PNPase is necessary to maintain bacterial cells in the planktonic mode through down-regulation of pgaABCD expression and PNAG production.

  1. Cloning and expression patterns of the brine shrimp (Artemia sinica) glycogen phosphorylase (GPase) gene during development and in response to temperature stress.

    Science.gov (United States)

    Zhao, Na; Hou, Ming; Wang, Ting; Chen, Yifei; Lv, Ying; Li, Zengrong; Zhang, Rui; Xin, Wenting; Zou, Xiangyang; Hou, Lin

    2014-01-01

    Glycogen serves as a metabolic reserve and is involved in macromolecular synthesis. Glycogen phosphorylase (GPase) is a key enzyme involved in intracellular glycogen catabolism, catalyzing the first step in glycogen degradation. In the diapause, GPase catalyzes glycogen into the closely related molecule, sorbitol. In this study, the full-length cDNA of the GPase gene (2,790 bp) was isolated from Artemia sinica for the first time by rapid amplification of cDNA ends technology. The GPase gene encoded a protein of 853 amino acids belonging to the Glycosyltransferase GTB type superfamily. The expression pattern and location of GPase were investigated at various stages during the embryonic development of A. sinica using real-time PCR and in situ hybridization. High GPase expression was detected at the 0 and 5 h stages. Subsequently, expression declined and was maintained at a low level during the stages from 10 to 40 h following by a small increase at day 3. Expression was downregulated at temperatures ranging from 25 to 20 °C and was subsequently upregulated in the range 15-5 °C. In situ hybridization assays showed wide distribution of the GPase gene during different developmental stages. From the results of this study, we conclude that the GPase gene expression is stress-related and might play an important role in Artemia development and metabolism.

  2. Down-Regulation of Methylthioadenosine Phosphorylase (MTAP) Induces Progression of Hepatocellular Carcinoma via Accumulation of 5′-Deoxy-5′-Methylthioadenosine (MTA)

    Science.gov (United States)

    Kirovski, Georgi; Stevens, Axel P.; Czech, Barbara; Dettmer, Katja; Weiss, Thomas S.; Wild, Peter; Hartmann, Arndt; Bosserhoff, Anja K.; Oefner, Peter J.; Hellerbrand, Claus

    2011-01-01

    Recently, we have shown that down-regulation of methylthioadenosine phosphorylase (MTAP) in hepatocellular carcinoma (HCC) cells enhances the invasive potential and the resistance against cytokines. Here, we aimed at investigating the molecular mechanism underlying this tumor-promoting effect and expanded the analysis to a large series of human HCC tissues. Liquid chromatography tandem mass spectrometry revealed that reduced MTAP expression resulted in higher intra- and extracellular concentrations of 5′-deoxy-5′-methylthioadenosine (MTA) in cultivated HCC cells and, concordantly, higher levels of MTA in HCC tissue. MTA induced matrix metalloproteinase (MMP) and interleukin-8 transcription in HCC cells in vitro, accompanied by enhanced proliferation and activation of the transcription factor NFκB. In addition, MTA secreted by HCC cells induced expression of fibroblast growth factor-2 and MMP1 in stromal myofibroblasts. In human HCC tissues, MTAP mRNA correlated inversely with MTA levels, and immunohistochemical analysis of a tissue microarray of 140 human HCCs revealed that low MTAP protein expression correlated with advanced tumor stages. In conclusion, MTAP deficiency results in accumulation of MTA, which is associated with increased tumorigenicity. These data further indicate MTAP as a tumor suppressor in HCC, and MTA as a potential biomarker for HCC progression. PMID:21356366

  3. Sourcing the affinity of flavonoids for the glycogen phosphorylase inhibitor site via crystallography, kinetics and QM/MM-PBSA binding studies: comparison of chrysin and flavopiridol.

    Science.gov (United States)

    Tsitsanou, Katerina E; Hayes, Joseph M; Keramioti, Maria; Mamais, Michalis; Oikonomakos, Nikos G; Kato, Atsushi; Leonidas, Demetres D; Zographos, Spyros E

    2013-11-01

    Flavonoids have been discovered as novel inhibitors of glycogen phosphorylase (GP), a target to control hyperglycemia in type 2 diabetes. To elucidate the mechanism of inhibition, we have determined the crystal structure of the GPb-chrysin complex at 1.9 Å resolution. Chrysin is accommodated at the inhibitor site intercalating between the aromatic side chains of Phe285 and Tyr613 through π-stacking interactions. Chrysin binds to GPb approximately 15 times weaker (Ki=19.01 μM) than flavopiridol (Ki=1.24 μM), exclusively at the inhibitor site, and both inhibitors display similar behavior with respect to AMP. To identify the source of flavopiridols' stronger affinity, molecular docking with Glide and postdocking binding free energy calculations using QM/MM-PBSA have been performed and compared. Whereas docking failed to correctly rank inhibitor binding conformations, the QM/MM-PBSA method employing M06-2X/6-31+G to model the π-stacking interactions correctly reproduced the experimental results. Flavopiridols' greater binding affinity is sourced to favorable interactions of the cationic 4-hydroxypiperidin-1-yl substituent with GPb, with desolvation effects limited by the substituent conformation adopted in the crystallographic complex. Further successful predictions using QM/MM-PBSA for the flavonoid quercetagetin (which binds at the allosteric site) leads us to propose the methodology as a useful and inexpensive tool to predict flavonoid binding. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Synthesis of chitin and chitosan stereoisomers by thermostable α-glucan phosphorylase-catalyzed enzymatic polymerization of α-D-glucosamine 1-phosphate.

    Science.gov (United States)

    Kadokawa, Jun-ichi; Shimohigoshi, Riko; Yamashita, Kento; Yamamoto, Kazuya

    2015-04-14

    The relationship between two aminopolysaccharide stereoisomers, namely α-(1→4)- and β-(1→4)-linked (N-acetyl)-D-glucosamine polymers, is of significant interest within the field of polysaccharide science, as they correspond to amino analogs of the representative abundant natural polysaccharides, viz. amylose and cellulose. While the latter glucosamine polymer is the basis of well-known natural polysaccharides, chitin and chitosan (linear polysaccharides composed of β-(1→4)-linked N-acetyl-D-glucosamine and D-glucosamine), to the best of our knowledge, the former (α-(1→4)-linked) has not been observed in nature. For the purpose of these studies, the synthesis of such non-natural aminopolysaccharides was performed by the thermostable α-glucan phosphorylase (from Aquifex aeolicus VF5)-catalyzed enzymatic polymerization of α-D-glucosamine 1-phosphate (GlcN-1-P), via successive α-glucosaminylations, in ammonia buffer containing Mg(2+) ions, resulting in the production of the α-(1→4)-linked D-glucosamine polymers, corresponding to the structure of the chitosan stereoisomer. Subsequent N-acetylation of the products gave the aminopolysaccharides, corresponding to the chitin stereoisomer.

  5. Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature.

    Science.gov (United States)

    Orawetz, Tom; Malinova, Irina; Orzechowski, Slawomir; Fettke, Joerg

    2016-03-01

    Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Pho1; EC 2.4.1.1) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of α-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Pho1 has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Pho1 activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures.

  6. Correlation of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2'-deoxyuridine

    Institute of Scientific and Technical Information of China (English)

    Tao Ma; Zheng-Gang Zhu; Yu-Bao Ji; Yi Zhang; Ying-Yan Yu; Bing-Ya Liu; Hao-Ran Yin; Yan-Zhen Lin

    2004-01-01

    AIM: To determine the expression levels of three metabolic enzymes of fluoropyrimidines: thymidylate synthase (TS),thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines, and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd).METHODS: TS, TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR, TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50), representing the sensitivityto drugs, were determined by MTT assay.RESULTS: IC50 values ranged from 1.28 to 12.26 uM for 5-FU, and from 5.02 to 24.21 uM for FdUrd, respectively.Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P<0.05), but neither TS nor TP correlated with 5-FU IC50 (P>0.05). Only TS mRNA level was sharply related with FdUrd sensitivity (P<0.05), but TP and DPD were not (P>0.05). A correlation was found between mRNA and protein levels of DPD (P<0.05), but not TP (P<0.05).CONCLUSION: DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd, respectively.

  7. Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins.

    Science.gov (United States)

    Cardenas, Paula P; Carzaniga, Thomas; Zangrossi, Sandro; Briani, Federica; Garcia-Tirado, Esther; Dehò, Gianni; Alonso, Juan C

    2011-11-01

    Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3' → 5' polarity in the presence of Mn(2+) and low inorganic phosphate (Pi) concentration, or to extend a 3'-OH end in the presence dNDP · Mn(2+). Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3'-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.

  8. The RNA processing enzyme polynucleotide phosphorylase negatively controls biofilm formation by repressing poly-N-acetylglucosamine (PNAG) production in Escherichia coli C.

    Science.gov (United States)

    Carzaniga, Thomas; Antoniani, Davide; Dehò, Gianni; Briani, Federica; Landini, Paolo

    2012-11-21

    Transition from planktonic cells to biofilm is mediated by production of adhesion factors, such as extracellular polysaccharides (EPS), and modulated by complex regulatory networks that, in addition to controlling production of adhesion factors, redirect bacterial cell metabolism to the biofilm mode. Deletion of the pnp gene, encoding polynucleotide phosphorylase, an RNA processing enzyme and a component of the RNA degradosome, results in increased biofilm formation in Escherichia coli. This effect is particularly pronounced in the E. coli strain C-1a, in which deletion of the pnp gene leads to strong cell aggregation in liquid medium. Cell aggregation is dependent on the EPS poly-N-acetylglucosamine (PNAG), thus suggesting negative regulation of the PNAG biosynthetic operon pgaABCD by PNPase. Indeed, pgaABCD transcript levels are higher in the pnp mutant. Negative control of pgaABCD expression by PNPase takes place at mRNA stability level and involves the 5'-untranslated region of the pgaABCD transcript, which serves as a cis-element regulating pgaABCD transcript stability and translatability. Our results demonstrate that PNPase is necessary to maintain bacterial cells in the planktonic mode through down-regulation of pgaABCD expression and PNAG production.

  9. Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using D-glucal as donor substrate.

    Science.gov (United States)

    Wildberger, Patricia; Brecker, Lothar; Nidetzky, Bernd

    2012-07-15

    Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize D-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α-D-glucose 1-phosphate as the product. When performed in D(2)O, enzymatic phosphorolysis of D-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of D-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered D-glucose next to D-glucal and phosphate, CuCPase produces 2-deoxy-β-D-glucosyl-(1→4)-D-glucose and 2-deoxy-α-D-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-β-D-glucosyl-(1→4)-D-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, D-glucal, phosphate, and D-glucose. Conversion of D-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α-D-glucose 1-phosphate that is difficult to prepare chemically.

  10. Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the gamma2-subunit of AMP-activated protein kinase.

    Science.gov (United States)

    Akman, Hasan O; Sampayo, James N; Ross, Fiona A; Scott, John W; Wilson, Gregory; Benson, Lee; Bruno, Claudio; Shanske, Sara; Hardie, D Grahame; Dimauro, Salvatore

    2007-10-01

    A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the gamma2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and "pseudo PHK deficiency" prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patient's father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.

  11. Comparative analysis of three-dimensional structures of homodimers of uridine phosphorylase from Salmonella typhimurium in the unligated state and in a complex with potassium ion

    Science.gov (United States)

    Lashkov, A. A.; Zhukhlistova, N. E.; Gabdulkhakov, A. G.; Mikhailov, A. M.

    2009-03-01

    The spatial organization of the homodimer of unligated uridine phosphorylase from Salmonella typhimurium ( St UPh) was determined with high accuracy. The structure was refined at 1.80 Å resolution to R work = 16.1% and R free = 20.0%. The rms deviations for the bond lengths, bond angles, and chiral angles are 0.006 Å, 1.042°, and 0.071°, respectively. The coordinate error estimated by the Luzzati plot is 0.166 Å. The coordinate error based on the maximum likelihood is 0.199 Å. A comparative analysis of the spatial organization of the homodimer in two independently refined structures and the structure of the homodimer St UPh in the complex with a K+ ion was performed. The substrate-binding sites in the homodimers StUPhs in the unligated state were found to act asynchronously. In the presence of a potassium ion, the three-dimensional structures of the subunits in the homodimer are virtually identical, which is apparently of importance for the synchronous action of both substrate-binding sites. The atomic coordinates of the refined structure of the homodimer and structure factors have been deposited in the Protein Data Bank (PDB ID code 3DPS).

  12. Comparative analysis of three-dimensional structures of homodimers of uridine phosphorylase from Salmonella typhimurium in the unligated state and in a complex with potassium ion

    Energy Technology Data Exchange (ETDEWEB)

    Lashkov, A. A.; Zhukhlistova, N. E.; Gabdulkhakov, A. G.; Mikhailov, A. M., E-mail: amm@ns.crys.ras.ru [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2009-03-15

    The spatial organization of the homodimer of unligated uridine phosphorylase from Salmonella typhimurium (St UPh) was determined with high accuracy. The structure was refined at 1.80 A resolution to R{sub work} = 16.1% and R{sub free} = 20.0%. The rms deviations for the bond lengths, bond angles, and chiral angles are 0.006 A, 1.042{sup o}, and 0.071{sup o}, respectively. The coordinate error estimated by the Luzzati plot is 0.166 A. The coordinate error based on the maximum likelihood is 0.199 A. A comparative analysis of the spatial organization of the homodimer in two independently refined structures and the structure of the homodimer St UPh in the complex with a K{sup +} ion was performed. The substrate-binding sites in the homodimers StUPhs in the unligated state were found to act asynchronously. In the presence of a potassium ion, the three-dimensional structures of the subunits in the homodimer are virtually identical, which is apparently of importance for the synchronous action of both substrate-binding sites. The atomic coordinates of the refined structure of the homodimer and structure factors have been deposited in the Protein Data Bank (PDB ID code 3DPS).

  13. Thymidine phosphorylase and angiogenesis in breast carcinoma%胸苷磷酸化酶与乳腺癌血管新生

    Institute of Scientific and Technical Information of China (English)

    李爱玲; 修瑞娟

    2005-01-01

    乳腺癌具有血管新生依赖性,促血管新生因子胸苷磷酸化酶(thymidine phosphorylase, TP)在乳腺癌组织中的表达明显高于周围正常组织,可促进乳腺癌血管新生活性表型的转化.其作用机制主要与TP特异作用于胸苷后的产物2-脱氧-D-核糖(2-dDR)有关.2-dDR可通过诱导浓度依赖性的内皮细胞迁移,促进血管网状结构形成;诱导血氧酶-1(HO-1)表达增强,通过一氧化碳(CO)的产生保护内皮细胞,增强其抗凋亡能力;诱导癌细胞的氧化应激并促进血管新生因子如VEGF、IL-8、MMP-1的释放等多种途径促进肿瘤血管新生.缺氧及降低微环境pH可以诱导TP的表达;细胞因子或生长因子如IL-1、TNF-α、IFN-γ可以提高TP的浓度和酶催化水平.针对TP的靶向性抗血管新生治疗是个体化性的,根据TP的表达情况可预测化疗药物疗效并选择合理的治疗,改善并提高高表达TP患者的预后.

  14. Glycogen phosphorylase isoenzyme BB, creatine kinase isoenzyme MB and troponin I for monitoring patients with percutaneous coronary intervention - a pilot study.

    Science.gov (United States)

    Skitek, Milan; Kranjec, Igor; Jerin, Aleš

    2014-02-01

    The glycogen phosphorylase isoenzyme BB (GPBB), as an ischemic marker, has not yet been investigated after elective percutaneous coronary intervention (PCI). ose aim of the study was to monitor GPBB, creatine kinase myocardial isoform (CK-MB) mass) and troponin I (TnI) value after PCI in correlation with ischemic incidents. Forty-two consecutive patients undergoing elective PCI were included in the study. Baseline blood samples and two more after the PCI (3 and 24 hours) were taken. The significance of cardiac markers in twenty-ththe stable patients with baseline values of CK-MB mass and TnI below the upper reference limit (URL) was evaluated based on ischemic incidents after PCI. TnI value was the only biomarker that was statistically significant at 3 and 24 hours after PCI in group of 23 stable patients. An overall comparisonthe biomarkers of 18 patients without and five patients with ischemic incidents displayed significant differences only for the baseline GPBB (p=0.019) and CK-MB mass 24 hours after PCI (p=0.034). Ischemic incidents were independently predictable only based on overall CK-MB mass measurements (OR=1.680, p=0.041) and particularly GPBB at baseline (OR=1.899, p=0.008) and CK-MB mass 24 hours after PCI (OR=2.111, p=0.022). Only significant increases in TnI were observed after elective PCI with ischemic incidents predicted using GPBB and CK-MB mass measurements.

  15. Determining the specific activity of thymidine phosphorylase in leukocytes of patients with MNGIE and the plasma thymidine level by RP-HPLC

    Directory of Open Access Journals (Sweden)

    Rezaei Sh

    2011-06-01

    Full Text Available "nBackground: Thymidine phosphorylase (TP catalyses the conversion of thymidine into thymine. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE is an autosomal recessive disease which is caused by mutations in the nuclear gene encoding TP, bringing about severe impairment of TP-enzyme specific activity and accumulation of thymidine in plasma. The clinical manifestations of MNGIE are recognizable and homogenous, but not in the early stages of the disease. In patients who are suspected of having MNGIE, determination of TP-specific activity in leukocytes and thymidine levels in plasma are diagnostic. The methods that are usually used for the measurement of TP activity and plasma thymidine are not rapid or accurate enough and lack sensitivity."n "nMethods: The specific activity of TP was measured by RP-HPLC in leukocytes of both the controls and the patients exhibiting clinical features suggestive of MNGIE. Moreover, plasma thymidine was assessed by the same method."n "nResults: The patients had detectable plasma thymidine (>3 µmol/L but it was undetectable in the healthy controls. The patients' TP-specific activity decreased to less than 5% relative to the controls (14±4 nmol/h/mg vs. 525±165 nmol/h/mg, P<0.05. A diagnostic algorithm for the definitive diagnosis of MNGIE is suggestible based on the results of this study which relies on the measurement of plasma thymidine, TP-specific activity in leukocytes, or both."n "nConclusion: In this study, we set up a sensitive and rapid assay for the evaluation of TP-specific activity by using RP-HPLC in Iran. In addition, we established reference values for TP-specific activity and plasma thymidine in the Iranian patients.

  16. Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum.

    Science.gov (United States)

    Maeda, Tomoya; Tanaka, Yuya; Wachi, Masaaki; Inui, Masayuki

    2017-03-01

    Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum, SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum The mature form of 4.5S RNA was inefficiently formed in ΔrneG Δpnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δpnp, ΔrneG, and ΔybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex.IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum, which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in this

  17. The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase.

    Science.gov (United States)

    Nakai, Hiroyuki; Baumann, Martin J; Petersen, Bent O; Westphal, Yvonne; Schols, Henk; Dilokpimol, Adiphol; Hachem, Maher A; Lahtinen, Sampo J; Duus, Jens Ø; Svensson, Birte

    2009-12-01

    A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mmbeta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1-->4)-Glcp (maltose), alpha-Glcp-(1-->4)-GlcNp (maltosamine), alpha-Glcp-(1-->4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1-->4)-Manp, alpha-Glcp-(1-->4)-Xylp and alpha-Glcp-(1-->4)- L-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

  18. Thymidine phosphorylase influences [{sup 18}F]fluorothymidine uptake in cancer cells and patients with non-small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Jin [Gachon University, College of Pharmacy, Incheon (Korea, Republic of); Yeo, Jeong Seok [Dongguk University Ilsan Hospital, Department of Nuclear Medicine, Goyang-si (Korea, Republic of); Lee, Haeng Jung; Lee, Eun Jung; Kim, Seog Young [Asan Medical Center, Institute for Innovative Cancer Research, Seoul (Korea, Republic of); Jang, Se Jin [University of Ulsan College of Medicine, Department of Pathology, Asan Medical Center, Seoul (Korea, Republic of); Lee, Jong Jin; Ryu, Jin-Sook; Moon, Dae Hyuk [University of Ulsan College of Medicine, Department of Nuclear Medicine, Asan Medical Center, Seoul (Korea, Republic of)

    2014-07-15

    Thymidine phosphorylase (TP), a key enzyme in the pyrimidine nucleoside salvage pathway, catalyses the reversible phosphorylation of thymidine, thereby generating thymine and 2-deoxy-D-ribose-1-phosphate. By regulating the levels of endogenous thymidine, TP may influence [{sup 18}F]fluorothymidine ([{sup 18}F]FLT) uptake. We investigated the effect of TP activity on [{sup 18}F]FLT uptake by tumours. Uptake of [{sup 3}H]FLT and [{sup 3}H]thymidine ([{sup 3}H]Thd) and the activities of TP, thymidine kinase 1 (TK1), and equilibrative nucleoside transporter 1 (ENT1) were determined in exponentially growing A431, A549, HT29, HOP92, ACHN, and SKOV3 cells in the presence or absence of tipiracil hydrochloride, a TP inhibitor. Eighty-five non-small cell lung cancer tissues from a patient cohort that was previously studied with [{sup 18}F]FLT positron emission tomography (PET) were retrieved and subjected to immunohistochemical analysis of TP expression. Factors that affected the maximum standardised uptake value (SUVmax) of [{sup 18}F]FLT-PET were identified by multiple linear regression analysis. A431 cells had the highest TP activity; A549 and HT29 cells had moderate TP activity; and ACHN, SKOV3, and HOP92 cells had little detectable TP activity. Cell lines with high TP activity took up more [{sup 3}H]FLT than [{sup 3}H]Thd, whereas cells with little TP activity took up more [{sup 3}H]Thd than [{sup 3}H]FLT. In cells with high TP activity, TP inhibition decreased [{sup 3}H]FLT uptake and increased [{sup 3}H]Thd uptake. However, TP inhibition had no effect on ACHN, SKOV3, and HOP92 cells. TP inhibition did not change TK1 or ENT1 activity, but did increase the intracellular level of thymidine. The SUVmax of [{sup 18}F]FLT was affected by three independent factors: Ki-67 expression (P < 0.001), immunohistochemical TP score (P < 0.001), and tumour size (P = 0.015). TP activity influences [{sup 18}F]FLT uptake, and may explain preferential uptake of [{sup 18}F]FLT over [{sup 3

  19. [Production and study of Bacillus subtilis mutants for genes involved in nucleoside catabolism].

    Science.gov (United States)

    Rumiantseva, E V; Sukhodolets, V V; Smirnov, Iu V

    1979-01-01

    By means of selection for a low thymine requirement the mutants fo thymine auxotrophs for deoxyriboaldolase (dra) and phosphodeoxyribomutase (drm) genes were obtained. Besides the mutants for pyrimidinenucleoside phosphorylase gene (pdp) were olso isolated using selection on the fluorodeoxyuridine resistance. The latter enzyme provides for pyrimidine nucleosides catabolism (thymidine, uridine) in Bacilli, as well as the conversion of exogenous thymine to thymidine in thymine auxotrophs. The data obtained when studying the deo-enzymes activities in various types of the mutants and also under the condition of induction by thymidine and acetoaldehyde are in accordance with the assumption that deoxyriboso-5-phosphate is an inductor of the deo-enzymes in Bacillus subtilis. The genes dra and pdp were tightly linked as it had been shown by the transformation experiments; in contrast, no linkage was revealed between dra and drm or pdp and drm. A secondary mutation (adn), not linked with dra and blocking the ability of bacteria to catabolise adenosine (purine nucleoside phosphorylase activity remains constant) was found in some dra-mutants.

  20. Novel trypanocidal analogs of 5'-(methylthio)-adenosine.

    Science.gov (United States)

    Sufrin, Janice R; Spiess, Arthur J; Marasco, Canio J; Rattendi, Donna; Bacchi, Cyrus J

    2008-01-01

    The purine nucleoside 5'-deoxy-5'-(hydroxyethylthio)-adenosine (HETA) is an analog of the polyamine pathway metabolite 5'-deoxy-5'-(methylthio)-adenosine (MTA). HETA is a lead structure for the ongoing development of selectively targeted trypanocidal agents. Thirteen novel HETA analogs were synthesized and examined for their in vitro trypanocidal activities against bloodstream forms of Trypanosoma brucei brucei LAB 110 EATRO and at least one drug-resistant Trypanosoma brucei rhodesiense clinical isolate. New compounds were also assessed in a cell-free assay for their activities as substrates of trypanosome MTA phosphorylase. The most potent analog in this group was 5'-deoxy-5'-(hydroxyethylthio)-tubercidin, whose in vitro cytotoxicity (50% inhibitory concentration [IC50], 10 nM) is 45 times greater than that of HETA (IC50, 450 nM) against pentamidine-resistant clinical isolate KETRI 269. Structure-activity analyses indicate that the enzymatic cleavage of HETA analogs by trypanosome MTA phosphorylase is not an absolute requirement for trypanocidal activity. This suggests that additional biochemical mechanisms are associated with the trypanocidal effects of HETA and its analogs.

  1. Induction of Recombinant Uridine Phosphorylase and Its Application in Biosynthesis of Pyrimidine Nucleosides%重组尿苷磷酸化酶的诱导及其在嘧啶核苷生物合成中的应用

    Institute of Scientific and Technical Information of China (English)

    丁庆豹; 欧伶; 魏东芝; 魏晓琨; 许彦梅; 张春艳

    2011-01-01

    Recombinant Escherichia coli pUDP, which overexpressed ufidine phosphorylase (UPase), was constructed. 0.5 mmol. L-1 lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth. The lactose-induced UPase was stable at 50 ℃. Wet cells of pUDP was used as catalyst to biosynthesize 5-fiuorouridine from 30 mmol. L-1 uridine and 5-fiuorouracil in phosphate buffer (pH 7.0) catalyzed at 50 ℃for 1.5 h and the yield of 5-fiuorouridine was higher than 68%. Under the optimum reaction conditions for production of 5-fiuoroufidine, 5-methyluridine and azauridine were synthesized from uridine by pUDP, the yield was 61.7% and 47.2% respectively. Deoxynucleosides were also synthesized by pUDP, but the yield was only about 20%.

  2. Synthesis and applications of selectively {sup 13}C-labeled RNA

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    SantaLucia, J. Jr.; Shen, L.X.; Lewis, H.; Cai, Z.; Tinoci, I. Jr. [Univ. of California, Berkeley, CA (United States)

    1994-12-01

    Spectral overlap is a substantial problem in NMR studies of RNA molecules >30 nucleotides. To overcome this difficulty, we synthesized selectively {sup 13}C-labeled RNAs and adapted several isotope-edited two- and three-dimensional NMR experiments originally developed for protein studies. We optimized protocols for synthesis of multi-gram quantities of CTP, UTp, ATP, and GTP using a combination of synthetic organic and enzymatic methods. Uracil is prepared in 40 to 50% yield from {sup 13}C-cyanide in two steps. Using acetyl- tribenzoyl-ribose and standard chemistry uracil is then attached to the sugar (90% yield). The tribenzoyl-uridine intermediate is converted into uridine or cytidine quantitatively, depending on the deblocking protocol. Labeled purines are synthesized using simple pyrimidine precursors and reacting with {sup 13}C-formic acid (80% yield). Purine nucleosides are then synthesized using uridine phosphorylase and purine nucleoside phosphorylase. The nucleosides were converted to NMPs by treatment with POC1{sub 3} in triethylphosphate. We converted NMPs to NTPs by standard enzymatic methods. Selectively labeled RNAs were synthesized by run-off transcription using {sup 13}C-labeled NTPs. Several different strategies help solve over-lap problems in larger RNAs. Isotope-edited two-dimensional NMR experiments such as {omega}1-1/2 X-filtered NOESY simplify NMR spectra by dividing the normal NOESY spectrum into two subspectra-one involving NOEs from protons bound to {sup 12}C and one from protons bound to {sup 13}C. For example, we labeled A and U residues of a 34-nucleotide pseudoknot, and the {sup 12}C subspectrum of the 1/2 X-filtered NOESY contained NOEs only from G and C residues (along with adenine 2H); the {sup 13}C subspectrum contained NOEs only from A and U residues. Each subspectrum has less overlap than the NOESY of an unlabeled sample; the editing strategy allows each resonance to be identified by residue type (A, C, G, or U).

  3. Ethenoguanines undergo glycosylation by nucleoside 2'-deoxyribosyltransferases at non-natural sites.

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    Wenjie Ye

    Full Text Available Deoxyribosyl transferases and functionally related purine nucleoside phosphorylases are used extensively for synthesis of non-natural deoxynucleosides as pharmaceuticals or standards for characterizing and quantitating DNA adducts. Hence exploring the conformational tolerance of the active sites of these enzymes is of considerable practical interest. We have determined the crystal structure at 2.1 Å resolution of Lactobacillus helveticus purine deoxyribosyl transferase (PDT with the tricyclic purine 8,9-dihydro-9-oxoimidazo[2,1-b]purine (N2,3-ethenoguanine at the active site. The active site electron density map was compatible with four orientations, two consistent with sites for deoxyribosylation and two appearing to be unproductive. In accord with the crystal structure, Lactobacillus helveticus PDT glycosylates the 8,9-dihydro-9-oxoimidazo[2,1-b]purine at N7 and N1, with a marked preference for N7. The activity of Lactobacillus helveticus PDT was compared with that of the nucleoside 2'-deoxyribosyltransferase enzymes (DRT Type II from Lactobacillus leichmannii and Lactobacillus fermentum, which were somewhat more effective in the deoxyribosylation than Lactobacillus helveticus PDT, glycosylating the substrate with product profiles dependent on the pH of the incubation. The purine nucleoside phosphorylase of Escherichia coli, also commonly used in ribosylation of non-natural bases, was an order of magnitude less efficient than the transferase enzymes. Modeling based on published active-site structures as templates suggests that in all cases, an active site Phe is critical in orienting the molecular plane of the purine derivative. Adventitious hydrogen bonding with additional active site residues appears to result in presentation of multiple nucleophilic sites on the periphery of the acceptor base for ribosylation to give a distribution of nucleosides. Chemical glycosylation of O9-benzylated 8,9-dihydro-9-oxoimidazo[2,1-b]purine also resulted

  4. Association of right-sided tumors with high thymidine phosphorylase gene expression levels and the response to oral uracil and tegafur/leucovorin chemotherapy among patients with colorectal cancer.

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    Sadahiro, Sotaro; Suzuki, Toshiyuki; Tanaka, Akira; Okada, Kazutake; Nagase, Hideki; Uchida, Junji

    2012-08-01

    To identify useful predictive factors for the response to 5-fluorouracil (5-FU)/leucovorin (LV) and oral uracil and tegafur (UFT)/LV chemotherapy among patients with colorectal cancer, we investigated the association between the gene expression levels of pyrimidine and folate metabolism-related enzymes in colorectal cancer (CRC) tissues and the response to UFT/LV neoadjuvant chemotherapy. The subjects were 76 CRC patients who were scheduled to undergo surgery. UFT (300 mg/m(2)/day) and LV (75 mg/body/day) were administered for 2 weeks before surgery. Biopsy samples were endoscopically obtained before drug administration. The gene expression levels of 14 genes in the biopsy samples were quantitatively evaluated using a real-time polymerase chain reaction (RT-PCR) assay. Fifteen patients (19.7 %) with marked pathological regression were judged to be responders. Thymidine phosphorylase (TP) gene expression levels among the responders were significantly higher than those among the non-responders. Right-sided tumors with high TP gene expression levels were associated with a significantly higher response rate to UFT/LV chemotherapy than left-sided tumors. TP gene expression levels in primary CRC tissues and the primary tumor site may be useful predictors of the efficacy of oral UFT/LV chemotherapy.

  5. Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

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    Dangoudoubiyam, Sriveny; Zhang, Zijing; Howe, Daniel K

    2014-09-01

    Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite.

  6. The identification of (3R,4S)-5-fluoro-5-deoxy-D-ribulose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces cattleya.

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    Onega, Mayca; McGlinchey, Ryan P; Deng, Hai; Hamilton, John T G; O'Hagan, David

    2007-10-01

    (3R,4S)-5-Fluoro-5-deoxy-D-ribulose-1-phosphate (5-FDRulP) has been identified as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. 5-FDRulP is generated after formation of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and then phosphorolysis of 5'-FDA to 5-fluoro-5-deoxy-D-ribose-1-phosphate (5-FDRP) by the action of a purine nucleoside phosphorylase. An isomerase mediates the conversion of 5-FDRP to 5-FDRulP. The identity of the (3R,4S) diastereoisomer of 5-FDRulP was established by comparative (19)F{(1)H} NMR studies whereby 5-FDRulP that accumulated in a cell free extract of S. cattleya, was treated with a phytase to generate the non-phosphorylated sugar, 5-fluoro-5-deoxy-D-ribulose (5-FDRul). This S. cattleya product was compared to the product of an in-vitro biotransformation where separately 5-fluoro-5-deoxy-D-ribose and 5-fluoro-5-deoxy-D-xylose were converted to 5-fluoro-5-deoxy-D-ribulose and 5-fluoro-5-deoxy-D-xylulose respectively by the action of glucose isomerase. It was demonstrated that 5-fluoro-5-deoxy-D-ribose gave the identical diastereoisomer to that observed from 5-FDRulP.

  7. In vitro reconstituted biotransformation of 4-fluorothreonine from fluoride ion: application of the fluorinase.

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    Deng, Hai; Cross, Stuart M; McGlinchey, Ryan P; Hamilton, John T G; O'Hagan, David

    2008-12-22

    In this paper, we report that fluoride ion is converted to the amino acid/antibiotic 4-fluorothreonine 2 in a biotransformation involving five (steps a-e) overexpressed enzymes. The biotransformation validates the biosynthetic pathway to 4-fluorothreonine in the bacterium Streptomyces cattleya (Schaffrath et al., 2002). To achieve an in vitro biotransformation, the fluorinase and the purine nucleoside phosphorylase (PNP) enzymes (steps a and b), which are coded for by the flA and flB genes of the fluorometabolite gene cluster in S. cattleya, were overexpressed. Also, an isomerase gene product that can convert 5-FDRP 6 to 5-FDRibulP 7 (step c) was identified in S. cattleya, and the enzyme was overexpressed for the biotransformation. A fuculose aldolase gene from S. coelicolor was overexpressed in E. coli and was used as a surrogate aldolase (step d) in these experiments. To complete the complement of enzymes, an ORF coding the PLP-dependent transaldolase, the final enzyme of the fluorometabolite pathway, was identified in genomic DNA by a reverse genetics approach, and the S. cattleya gene/enzyme was then overexpressed in S. lividans. This latter enzyme is an unusual PLP-dependent catalyst with some homology to both bacterial serine hydroxymethyl transferases (SHMT) and C5 sugar isomerases/epimerases. The biotransformation demonstrates the power of the fluorinase to initiate C-F bond formation for organo-fluorine synthesis.

  8. Activation of guanine-{beta}-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

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    Leanza, Luigi; Miazzi, Cristina; Ferraro, Paola; Reichard, Peter; Bianchi, Vera, E-mail: vbianchi@bio.unipd.it

    2010-12-10

    The deoxyguanosine (GdR) analog guanine-ss-D-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

  9. Clinical Trials and Treatment of ATL

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    Kunihiro Tsukasaki

    2012-01-01

    Full Text Available ATL is a distinct peripheral T-lymphocytic malignancy associated with human T-cell lymphotropic virus type I (HTLV-1. The diversity in clinical features and prognosis of patients with this disease has led to its subtype-classification into four categories, acute, lymphoma, chronic, and smoldering types, defined by organ involvement, and LDH and calcium values. In case of acute, lymphoma, or unfavorable chronic subtypes (aggressive ATL, intensive chemotherapy like the LSG15 regimen (VCAP-AMP-VECP is usually recommended if outside of clinical trials, based on the results of a phase 3 trial. In case of favorable chronic or smoldering ATL (indolent ATL, watchful waiting until disease progression has been recommended, although the long-term prognosis was inferior to those of, for instance, chronic lymphoid leukemia. Retrospective analysis suggested that the combination of interferon alpha and zidovudine was apparently promising for the treatment of ATL, especially for types with leukemic manifestation. Allogeneic hematopoietic stem cell transplantation (allo-HSCT is also promising for the treatment of aggressive ATL possibly reflecting graft versus ATL effect. Several new agent trials for ATL are ongoing and in preparation, including a defucosylated humanized anti-CC chemokine receptor 4 monoclonal antibody, IL2-fused with diphtheria toxin, histone deacetylase inhibitors, a purine nucleoside phosphorylase inhibitor, a proteasome inhibitor, and lenalidomide.

  10. pnp4a Is the Causal Gene of the Medaka Iridophore Mutant guanineless

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    Tetsuaki Kimura

    2017-04-01

    Full Text Available See-through medaka lines are suitable for observing internal organs throughout life. They were bred by crossing multiple color mutants. However, some of the causal genes for these mutants have not been identified. The medaka has four pigment cell types: black melanophores, yellow xanthophores, white leucophores, and silvery iridophores. The causal genes of melanophore, xanthophore, and leucophore mutants have been elucidated, but the causal gene for the iridophore mutant remains unknown. Here, we describe the iridophore mutant, guanineless (gu, which exhibits a strong reduction in visible iridophores throughout its larval to adult stages. The gu locus was previously mapped to chromosome 5, but was located near the telomeric region, making it difficult to integrate into the chromosome. We sought the causal gene of gu using synteny analysis with the zebrafish genome and found a strong candidate, purine nucleoside phosphorylase 4a (pnp4a. Gene targeting and complementation testing showed that pnp4a is the causal gene of gu. This result will allow the establishment of inbred medaka strains or other useful strains with see-through phenotypes without major disruption in the genetic background of each strain.

  11. Determination of phosphate in soil extracts in the field: A green chemistry enzymatic method.

    Science.gov (United States)

    Campbell, Ellen R; Warsko, Kayla; Davidson, Anna-Marie; Bill Campbell, Wilbur H

    2015-01-01

    Measurement of ortho-phosphate in soil extracts usually involves sending dried samples of soil to a laboratory for analysis and waiting several weeks for the results. Phosphate determination methods often involve use of strong acids, heavy metals, and organic dyes. To overcome limitations of this approach, we have developed a phosphate determination method which can be carried out in the field to obtain results on the spot. This new method uses: •Small volumes.•An enzymatic reaction.•Green chemistry. First, the soil sample is extracted with deionized water and filtered. Next, an aliquot of the soil extract (0.5 mL) is transferred to a disposable cuvette, containing 0.5 mL of reaction mixture [200 mM HEPES, pH 7.6, 20 mM MgCl2, with 80 nmol 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) and 1 unit of recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1)], mixed, and incubated for 10 min at field temperature. Absorbance of the completed reaction is measured at 360 nm in open-source, portable photometer linked by bluetooth to a smartphone. The phosphate and phosphorus content of the soil is determined by comparison of its absorbance at 360 nm to a previously prepared standard phosphate curve, which is stored in the smartphone app.

  12. Multiple ligand simultaneous docking: orchestrated dancing of ligands in binding sites of protein.

    Science.gov (United States)

    Li, Huameng; Li, Chenglong

    2010-07-30

    Present docking methodologies simulate only one single ligand at a time during docking process. In reality, the molecular recognition process always involves multiple molecular species. Typical protein-ligand interactions are, for example, substrate and cofactor in catalytic cycle; metal ion coordination together with ligand(s); and ligand binding with water molecules. To simulate the real molecular binding processes, we propose a novel multiple ligand simultaneous docking (MLSD) strategy, which can deal with all the above processes, vastly improving docking sampling and binding free energy scoring. The work also compares two search strategies: Lamarckian genetic algorithm and particle swarm optimization, which have respective advantages depending on the specific systems. The methodology proves robust through systematic testing against several diverse model systems: E. coli purine nucleoside phosphorylase (PNP) complex with two substrates, SHP2NSH2 complex with two peptides and Bcl-xL complex with ABT-737 fragments. In all cases, the final correct docking poses and relative binding free energies were obtained. In PNP case, the simulations also capture the binding intermediates and reveal the binding dynamics during the recognition processes, which are consistent with the proposed enzymatic mechanism. In the other two cases, conventional single-ligand docking fails due to energetic and dynamic coupling among ligands, whereas MLSD results in the correct binding modes. These three cases also represent potential applications in the areas of exploring enzymatic mechanism, interpreting noisy X-ray crystallographic maps, and aiding fragment-based drug design, respectively.

  13. Determination of phosphate in soil extracts in the field: A green chemistry enzymatic method

    Science.gov (United States)

    Campbell, Ellen R.; Warsko, Kayla; Davidson, Anna-Marie; (Bill) Campbell, Wilbur H.

    2015-01-01

    Measurement of ortho-phosphate in soil extracts usually involves sending dried samples of soil to a laboratory for analysis and waiting several weeks for the results. Phosphate determination methods often involve use of strong acids, heavy metals, and organic dyes. To overcome limitations of this approach, we have developed a phosphate determination method which can be carried out in the field to obtain results on the spot. This new method uses: • Small volumes. • An enzymatic reaction. • Green chemistry. First, the soil sample is extracted with deionized water and filtered. Next, an aliquot of the soil extract (0.5 mL) is transferred to a disposable cuvette, containing 0.5 mL of reaction mixture [200 mM HEPES, pH 7.6, 20 mM MgCl2, with 80 nmol 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) and 1 unit of recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1)], mixed, and incubated for 10 min at field temperature. Absorbance of the completed reaction is measured at 360 nm in open-source, portable photometer linked by bluetooth to a smartphone. The phosphate and phosphorus content of the soil is determined by comparison of its absorbance at 360 nm to a previously prepared standard phosphate curve, which is stored in the smartphone app. PMID:26150991

  14. Transition-state-guided drug design for treatment of parasitic neglected tropical diseases.

    Science.gov (United States)

    Murkin, A S; Moynihan, M M

    2014-01-01

    Many of the deadliest neglected tropical diseases are caused by protozoan and helminthic parasites. These organisms have evolved several enzymes to exploit their host's metabolic resources and evade immune responses. Because these essential proteins are absent in humans, they are targets for antiparasitic drug development. Despite decades of investigation, no therapy has been successful in the eradication of these diseases, so new approaches are desired. Chemically stable analogues of the transition states of enzymatic reactions are often potent inhibitors, and several examples of clinically effective compounds are known for other diseases. The design of transition-state analogues is aided by structural models of the transition state, which are obtained by complementing experimental measurement of kinetic isotope effects with theoretical calculations. Such transition-state-guided inhibitor design has been demonstrated for human, bovine, malarial, and trypanosomal enzymes of the purine salvage pathway, including purine nucleoside phosphorylase, nucleoside hydrolases, and adenosine deaminase. Cysteine proteases, trans-sialidase, 1-deoxy-d-xylulose-5-phosphate reductoisomerase, and trypanothione synthetase are presented as additional candidates for application of transition-state analysis with the goal of identifying new leads for the treatment of parasitic neglected tropical diseases.

  15. Purine metabolism in Toxoplasma gondii

    Energy Technology Data Exchange (ETDEWEB)

    Krug, E.C.; Marr, J.J.; Berens, R.L.

    1989-06-25

    We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.

  16. Changes in the activities of some membrane-associated enzymes during in vivo ageing of the normal human erythrocyte.

    Science.gov (United States)

    Kadlubowski, M; Agutter, P S

    1977-09-01

    Human erythrocytes from healthy male donors were fractionated with respect to in vivo age by simple centrifugation in order to characterize changes in the functional integrity of the membrane during the life-span of the cell. The three enzymes, Na/K-ATPase, glyceraldehyde-3-phosphate dehydrogenase and NADH-ferricyanide reductase, were found not to change with age, but significant age-dependent decreases were observed in the cases of acetylcholinesterase, phosphoglycerate kinase, purine nucleoside phosphorylase, adenylate kinase, Mg-ATPase and alkaline phosphatase. The possibility that these changes were attributable to mechanisms other than age-related inactivation, such as reticulocyte contamination, differential resealing and crypticity, was investigated. Only the decrease in acetylcholinesterase could be explained wholly in terms of reticulocyte contamination. A decrease in membrane integrity on ageing was observed, which accounted for approximately half the change in alkaline phosphatase and may have contributed to the other enzyme activity changes. This membrane integrity effect masked a real decrease in the highly cryptic NADH-ferricyanide reductase, this decrease being apparent only after total disaggregation of the membrane with nonionic surfactant.

  17. Thymidylate synthase, dihydropyrimidine dehydrogenase, ERCC1, and thymidine phosphorylase gene expression in primary and metastatic gastrointestinal adenocarcinoma tissue in patients treated on a phase I trial of oxaliplatin and capecitabine

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    Danenberg Kathleen D

    2008-12-01

    Full Text Available Abstract Background Over-expression of thymidylate synthase (TS and dihydropyrimidine dehydrogenase (DPD in tumor tissue is associated with insensitivity to 5-fluorouracil (5-FU. Over-expression of ERCC1 correlates with insensitivity to oxaliplatin (OX therapy, while high thymidine phosphorylase (TP levels predict for increased sensitivity to capecitabine (Xel. Methods Biopsies of metastatic tumor were taken before OX (130 mg/m2 day 1 given with Xel (1200–3000 mg/m2 in two divided doses days 1–5 and 8–12 every 3-weeks. Micro-dissected metastatic and primary tumors were analyzed for relative gene expression by real-time quantitative polymerase chain reaction. The clinical protocol prospectively identified the molecular targets of interest that would be tested. Endpoints for the molecular analyses were correlation of median, first and third quartiles for relative gene expression of each target with response, time to treatment failure (TTF, and survival. Results Among 91 patients participating in this trial; 97% had colorectal cancer. The median number of prior chemotherapy regimens was 2, and most had prior 5-FU and irinotecan. In paired samples, median mRNA levels were significantly higher in metastatic versus primary tumor (-fold: TS (1.9, DPD (3.8, ERCC1 (2.1 and TP (1.6. A strong positive correlation was noted between DPD and TP mRNA levels in both primary (r = 0.693, p Conclusion Target gene expression in primary tumor was significantly lower than that in paired metastatic tissue. High ERCC1 mRNA levels in metastatic tumor was associated with a shorter TTF. Lower expression of TS mRNA correlated with a lower chance of early PD with XelOX therapy and improved overall survival.

  18. Diagnostic accuracy of heart fatty acid binding protein (H-FABP) and glycogen phosphorylase isoenzyme BB (GPBB) in diagnosis of acute myocardial infarction in patients with acute coronary syndrome.

    Science.gov (United States)

    Cubranic, Zlatko; Madzar, Zeljko; Matijevic, Sanja; Dvornik, Stefica; Fisic, Elizabeta; Tomulic, Vjekoslav; Kunisek, Juraj; Laskarin, Gordana; Kardum, Igor; Zaputovic, Luka

    2012-01-01

    This study aimed to assess whether heart fatty acid-binding protein (H-FABP) and glycogen phosphorylase isoenzyme BB (GPBB) could be used for the accurate diagnosis of acute myocardial infarction (AMI) in acute coronary syndrome (ACS) patients. The study included 108 ACS patients admitted to a coronary unit within 3 h after chest pain onset. AMI was distinguished from unstable angina (UA) using a classical cardiac troponin I (cTnI) assay. H-FABP and GPBB were measured by ELISA on admission (0 h) and at 3, 6, 12, and 24 h after admission; their accuracy to diagnose AMI was assessed using statistical methods. From 92 patients with ACS; 71 had AMI. H-FABP and GPBB had higher peak value after 3 h from admission than cTnI (P = 0.001). Both markers normalized at 24 h. The area under the receiver operating characteristic curves was significantly greater for both markers in AMI patients than in UA patients at all time points tested, including admission (P < 0.001). At admission, the H-FABP (37%) and GPBB (40%) sensitivities were relatively low. They increased at 3 and 6 h after admission for both markers and decreased again after 24 h. It was 40% for H-FABP and approximately 2-times lower for GPBB (P < 0.01). In AMI patients, both biomarkers had similar specificities, positive- and negative-predictive values, positive and negative likelihood ratios, and risk ratios for AIM. H-FABP and GPBB can contribute to early AMI diagnosis and can distinguish AMI from UA.

  19. Sodium valproate increases the brain isoform of glycogen phosphorylase: looking for a compensation mechanism in McArdle disease using a mouse primary skeletal-muscle culture in vitro

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    Noemí de Luna

    2015-05-01

    Full Text Available McArdle disease, also termed ‘glycogen storage disease type V’, is a disorder of skeletal muscle carbohydrate metabolism caused by inherited deficiency of the muscle-specific isoform of glycogen phosphorylase (GP-MM. It is an autosomic recessive disorder that is caused by mutations in the PYGM gene and typically presents with exercise intolerance, i.e. episodes of early exertional fatigue frequently accompanied by rhabdomyolysis and myoglobinuria. Muscle biopsies from affected individuals contain subsarcolemmal deposits of glycogen. Besides GP-MM, two other GP isoforms have been described: the liver (GP-LL and brain (GP-BB isoforms, which are encoded by the PYGL and PYGB genes, respectively; GP-BB is the main GP isoform found in human and rat foetal tissues, including the muscle, although its postnatal expression is dramatically reduced in the vast majority of differentiated tissues with the exception of brain and heart, where it remains as the major isoform. We developed a cell culture model from knock-in McArdle mice that mimics the glycogen accumulation and GP-MM deficiency observed in skeletal muscle from individuals with McArdle disease. We treated mouse primary skeletal muscle cultures in vitro with sodium valproate (VPA, a histone deacetylase inhibitor. After VPA treatment, myotubes expressed GP-BB and a dose-dependent decrease in glycogen accumulation was also observed. Thus, this in vitro model could be useful for high-throughput screening of new drugs to treat this disease. The immortalization of these primary skeletal muscle cultures could provide a never-ending source of cells for this experimental model. Furthermore, VPA could be considered as a gene-expression modulator, allowing compensatory expression of GP-BB and decreased glycogen accumulation in skeletal muscle of individuals with McArdle disease.

  20. Glycogen Phosphorylase Isoenzyme BB:A New Biomarker of Myocardial Injury%糖原磷酸化酶同工酶脑型:心肌损伤的新标志物

    Institute of Scientific and Technical Information of China (English)

    王宇竞

    2011-01-01

    Glycogen phosphorylase isoenzyme BB ( GPBB) is a key enzyme of glycogenolysis, as an enzyme for early laboratory detection of ischaemia.At present, GPBB is the most promising because it increases as early as one to four hours from the onset of chest pain.Its early release appears to be essentially dependent on ischemic myoeardial injury.Compared with the established cardiac markers, GPBB is a sensitive marker for the early diagnosis of acute coronary syndromes.Both multiple biomarkers and enzyme linked immunosorbent assay are used for the determination GPBB.GPBB is not only a marker of monitoring acute coronary syndromes,but also may be a marker of myocardial necrosis and ischemia caused by other myocardial injury factors.%糖原磷酸化酶同工酶脑型(GPBB)是糖原分解的关键酶,最早在实验室因缺血而被检测到.目前,GPBB成为最具潜质的心肌损伤生物标记,其在胸痛开始的1~4 h内浓度开始增加,并且释放本质上依赖于心肌缺血损伤.对比其他已知心肌标志物,GPBB对于诊断冠状动脉综合征中不同形式的心肌缺血损伤具有高度敏感性和特异性.近期随着酶联免疫吸附法、多种生物标记技术测定GPBB含量的应用,GPBB还可能应用于其他原因所致的心肌损害.

  1. Kinetic studies of HPr, HPr(H15D), HPr(H15E), and HPr(His approximately P) phosphorylation by the Streptococcus salivarius HPr(Ser) kinase/phosphorylase.

    Science.gov (United States)

    Casabon, Israël; Couture, Manon; Vaillancourt, Katy; Vadeboncoeur, Christian

    2009-11-17

    HPr is a central protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In streptococci, HPr can be phosphorylated at His(15) at the expense of PEP by enzyme I (EI) of the PTS, producing HPr(His approximately P). HPr can also be phosphorylated at Ser(46) by the ATP-dependent HPr(Ser) kinase/phosphorylase (HprK/P), producing HPr(Ser-P). Lastly, HPr can be phosphorylated on both residues, producing HPr(Ser-P)(His approximately P) (HPr-P2). We report here a study on the phosphorylation of Streptococcus salivarius HPr, HPr(H15D), HPr(H15E), and HPr(His approximately P) by HprK/P to assess the involvement of HprK/P in the synthesis of HPr-P2 in streptococcal cells. We first developed a spectrophotometric method for measuring HprK/P kinase activity. Using this assay, we found that the K(m) of HprK/P for HPr at pH 7.4 and 37 degrees C was approximately 110 muM, with a specificity constant (k(cat)/K(m)) of 1.7 x 10(4) M(-1) s(-1). The specificity constants for HPr(H15D) and HPr(H15E) were approximately 13 times lower. Kinetic studies conducted under conditions where HPr(His approximately P) was stable (i.e., pH 8.6 and 15 degrees C) showed that HPr(His approximately P) was a poorer substrate for HprK/P than HPr(H15D), the k(cat)/K(m) for HPr(H15D) and HPr(His approximately P) being approximately 9 and 26 times lower than that for HPr, respectively. Our results suggested that (i) the inefficiency of the phosphorylation of HPr(His approximately P) by HprK/P results from the presence of a negative charge at position 15 as well as from other structural elements and (ii) the contribution of streptococcal HprK/P to the synthesis of HPr-P2 in vivo is marginal.

  2. 原发性肝癌组织中胸苷磷酸化酶的表达及其临床意义%Expression of thymidine phosphorylase in hepatocellular carcinoma and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    刘维藩; 柯昌能; 李荣江

    2009-01-01

    Objective To determine the significance of thymidine phosphorylase for prognosis of HCC.Methods The clinical data and pathological finding of 65 patients with HCC treated in our hospital were retrospectively analyzed.Immunohistochemical staining was performed to evaluate the expression of TP and MVD count.Statistical analysis was used to analyze the relationship between TP expression and other pathological parameters.Survival curves in 54 patients were plotted using the Kaplan-Meier method.Results The positive expressed rate of HCC tissues was significantly higher than that of normal liver tissues (P<0.01).MVD count in positive-TP group and negative-TP group was respectively 16.14±5.11,10.11±4.55.The difference between two groups had statistical significance (P<0.01).TP expression was positively correlated with tumor size,Edmondson-Steiner grade,tumor capsule status,microsatellite marker and tumor thrombi in portal vein (TTPV).The rate of postoperational recurrence after one,two and three years in TP-positive group was respectively 40%,66.7%,79.2% and it was respectively 27.4%,31.3% and 45.7% in TP-negative group.The postoperative recurrence rate after two years and three years in poitive-TP group was significantly higher than that in negative-TP group (P<0.01).In 54 patients with HCC followed up successfully,the total survival rate in TP-positive group was significantly lower than in TP-negative group (P<0.01).Conclusion TP plays an important role in neovascularization of HCC and TP may be a new prognosis index of HCC.%目的 探讨胸苷磷酸化酶(TP)与原发性肝癌(HCC)术后预后的关系.方法 收集65例HCC病人围手术期一般临床资料和病理学资料,并进行随访.分析TP的免疫组织化学表达与微血管密度(MVD)计数的关系;对随访的54例病人作生存分析.结果 (1)肝癌组织TP阳性表达率明显高于正常肝组织(P<0.05).(2)肝癌组织TP阳性表达组、阴性表达组MVD计数分别为16.14±5.11、10.11±4

  3. Purine metabolism in response to hypoxic conditions associated with breath-hold diving and exercise in erythrocytes and plasma from bottlenose dolphins (Tursiops truncatus).

    Science.gov (United States)

    del Castillo Velasco-Martínez, Iris; Hernández-Camacho, Claudia J; Méndez-Rodríguez, Lía C; Zenteno-Savín, Tania

    2016-01-01

    In mammalian tissues under hypoxic conditions, ATP degradation results in accumulation of purine metabolites. During exercise, muscle energetic demand increases and oxygen consumption can exceed its supply. During breath-hold diving, oxygen supply is reduced and, although oxygen utilization is regulated by bradycardia (low heart rate) and peripheral vasoconstriction, tissues with low blood flow (ischemia) may become hypoxic. The goal of this study was to evaluate potential differences in the circulating levels of purine metabolism components between diving and exercise in bottlenose dolphins (Tursiops truncatus). Blood samples were taken from captive dolphins following a swimming routine (n=8) and after a 2min dive (n=8). Activity of enzymes involved in purine metabolism (hypoxanthine guanine phosphoribosyl transferase (HGPRT), inosine monophosphate deshydrogenase (IMPDH), xanthine oxidase (XO), purine nucleoside phosphorylase (PNP)), and purine metabolite (hypoxanthine (HX), xanthine (X), uric acid (UA), inosine monophosphate (IMP), inosine, nicotinamide adenine dinucleotide (NAD(+)), adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, guanosine diphosphate (GDP), guanosine triphosphate (GTP)) concentrations were quantified in erythrocyte and plasma samples. Enzymatic activity and purine metabolite concentrations involved in purine synthesis and degradation, were not significantly different between diving and exercise. Plasma adenosine concentration was higher after diving than exercise (p=0.03); this may be related to dive-induced ischemia. In erythrocytes, HGPRT activity was higher after diving than exercise (p=0.007), suggesting an increased capacity for purine recycling and ATP synthesis from IMP in ischemic tissues of bottlenose dolphins during diving. Purine recycling and physiological adaptations may maintain the ATP concentrations in bottlenose dolphins after diving and exercise.

  4. Phosphorylation of 3-deazaguanosine by nicotinamide riboside kinase in Chinese hamster ovary cells.

    Science.gov (United States)

    Saunders, P P; Tan, M T; Spindler, C D; Robins, R K

    1989-12-01

    The growth inhibitory activity of 3-deazaguanosine toward a mutant line (TGR-3) of Chinese hamster ovary cells deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was substantially reversed by the simultaneous addition of nicotinamide riboside. The activities of most other ribonucleoside analogues tested were unaffected. The formation of cellular 3-deazaGMP and 3-deazaGTP from the ribonucleoside analogue, as measured by high-pressure liquid chromatography, was inhibited by the presence of nicotinamide riboside. The inhibition was dependent on concentration of 3-deazaguanosine and could also be demonstrated by following the metabolism of 3-deazaguanosine, labeled with 14C in the ribose moiety, to [14C]3-deazaGTP. In the presence of 100 microM nicotinamide riboside formation of the labeled triphosphate derivative of 3-deazaguanosine was undetectable. A 3-deazaguanosine phosphorylating activity was separated from other cellular kinases by DEAE-cellulose chromatography. Contaminating purine nucleoside phosphorylase (EC 2.4.2.1) was subsequently removed by sucrose density gradient centrifugation. The resulting enzyme preparation demonstrated the greatest activities with nicotinamide riboside and 3-deazaguanosine and, in addition, could also phosphorylate tiazofurin and guanosine to lesser, but significant, degrees. These and other observations suggest that 3-deazaguanosine, and perhaps other agents such as tiazofurin, may, at least in part, be phosphorylated by a nicotinamide ribonucleoside kinase in these cells. If so, it is possible that the activity of this agent in other types of cells in vivo could be dependent upon the presence of this enzyme and that it could be influenced by cellular concentrations of the natural pyridine nucleoside.

  5. Circulating Human Neonatal Naïve B cells are Deficient in CD73 Impairing Purine Salvage

    Directory of Open Access Journals (Sweden)

    Matthew Aaron Pettengill

    2016-03-01

    Full Text Available Background: Extracellular purines, in particular adenosine (Ado and adenosine-triphosphate (ATP, are critical immunoregulatory molecules. Expression and activity of purine ecto-enzymes on B cells in neonatal and adult blood may influence their function and has been incompletely characterized. Methods: Mononuclear cells were isolated from human neonatal (cord blood or adult (peripheral blood subjects and evaluated directly by flow cytometry for expression of purine ecto-enzymes. Additionally, B cell subsets were isolated from mononuclear cell fractions by fluorescence-activated cell sorting and gene transcription of purine ecto-enzymes (CD39 and CD73, adenosine deaminase (ADA1, purine nucleoside phosphorylase (PNP and select purine receptors (A2a were evaluated by reverse transcription followed by qRT-PCR. Immuno-magnetic-bead isolated naïve B cells were evaluated for enzymatic activity by incubation with radio-labeled purines followed by thin-layer chromatography, and subsequent B cell Ado acquisition was evaluated by liquid scintillation quantitation of radio-labeled Ado uptake.Results: Relative to their adult counterparts, neonatal circulating naïve B cells were markedly and selectively deficient in CD73 as observed by gene transcription, surface protein expression, and enzyme activity. Neonatal naïve B cell deficiency of CD73 expression significantly impaired their capacity to acquire extracellular purines for purine salvage.Conclusions: Human neonatal circulating naïve B cells are selectively deficient in CD73, impairing extracellular purine acquisition and potentially contributing to impaired B cell responses in early life.

  6. Relation of polymorphism of arsenic metabolism genes to arsenic methylation capacity and developmental delay in preschool children in Taiwan.

    Science.gov (United States)

    Hsieh, Ru-Lan; Su, Chien-Tien; Shiue, Horng-Sheng; Chen, Wei-Jen; Huang, Shiau-Rung; Lin, Ying-Chin; Lin, Ming-I; Mu, Shu-Chi; Chen, Ray-Jade; Hsueh, Yu-Mei

    2017-04-15

    Inefficient arsenic methylation capacity has been associated with developmental delay in children. The present study was designed to explore whether polymorphisms and haplotypes of arsenic methyltransferase (AS3MT), glutathione-S-transferase omegas (GSTOs), and purine nucleoside phosphorylase (PNP) affect arsenic methylation capacity and developmental delay. A case-control study was conducted from August 2010 to March 2014. All participants were recruited from the Shin Kong Wu Ho-Su Memorial Teaching Hospital. In total, 179 children with developmental delay and 88 children without delay were recruited. Urinary arsenic species, including arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)) were measured using a high-performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. The polymorphisms of AS3MT, GSTO, and PNP were performed using the Sequenom MassARRAY platform with iPLEX Gold chemistry. Polymorphisms of AS3MT genes were found to affect susceptibility to developmental delay in children, but GSTO and PNP polymorphisms were not. Participants with AS3MT rs3740392 A/G+G/G genotype, compared with AS3MT rs3740392 A/A genotype, had a significantly lower secondary methylation index. This may result in an increased OR for developmental delay. Participants with the AS3MT high-risk haplotype had a significantly higher OR than those with AS3MT low-risk haplotypes [OR and 95% CI, 1.59 (1.08-2.34)]. This is the first study to show a joint dose-response effect of this AS3MT high-risk haplotype and inefficient arsenic methylation capacity on developmental delay. Our data provide evidence that AS3MT genes are related to developmental delay and may partially influence arsenic methylation capacity. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Single nucleotide polymorphisms in glycogen phosphorylase gene and their association with growth performance and glycogen content in Pacific oyster Crassostrea gigas%长牡蛎糖原磷酸化酶基因SNPs与生长性状和糖原含量的相关性分析

    Institute of Scientific and Technical Information of China (English)

    刘思玮; 李琪; 于红; 孔令锋

    2013-01-01

      为研究糖原磷酸化酶基因多态性与长牡蛎(Crassostrea gigas)生长和糖原含量的相关性,本研究对长牡蛎糖原磷酸化酶基因(Cg-GPH)编码区单核苷酸多态性(SNPs)与来自5个家系322个长牡蛎个体的生长性状(包括壳高、壳长、壳宽、总体质量以及软体部质量)和糖原含量进行了关联分析。结果表明,在1940 bp的长牡蛎糖原磷酸化酶基因中共检测到82个SNPs位点,其中63个SNPs位点位于外显子区域,1个SNPs位点位于5′-UTR区,18个SNPs位点位于3′-UTR区,编码区SNPs平均密度为1/25 bp;5个SNPs位点与生长性状存在显著相关(P<0.05),未检测到与糖原含量相关SNPs。根据以上5个SNPs位点共构建得到6个SNP单倍型,其中,Cg-GPH基因的H6(CTGAT)单倍型在总体质量性状方面均显著高于其他5种单倍型(P<0.05),表明H6单倍型可能是对长牡蛎体质量增加最有利的单倍型。研究结果为今后长牡蛎生长性状的遗传改良提供了基础资料。%Glycogen phosphorylase plays a key role in the metabolism of glycogen, a molecule critical for the growth and development of oysters. We evaluated the effect of polymorphisms in the Crassostrea gigas glycogen phosphorylase gene (Cg-GPH) on growth and glycogen content. We tested the association between single nucleo-tide polymorphisms (SNPs) in coding regions of the gene and growth performance (shell height, shell length, shell depth, total weight and soft-tissue weight) and glycogen content using 322 oysters from five full-sib families. We identified 82 SNPs in the 1 940 bp Cg-GPH gene, including 63 SNPs in the exon region, one SNP locus located in the 5′-UTR, and 18 SNPs in the 3′-UTR. The average density of SNPs in the coding region was one per 25 bp. Five SNPs were significantly associated with growth performance (P<0.05), but there was no association between the SNPs and glycogen content. Furthermore, among the six SNP haplotypes constructed using

  8. 纳米磁粉固定化酶催化合成α-D-葡萄糖-1-磷酸%Efficient Synthesis of α-D-Glucose-1-Phosphate by Maltodextrin Phosphorylase Immobilized on Amino-functionalized Magnetic Nanoparticles

    Institute of Scientific and Technical Information of China (English)

    董青; 欧阳立明; 刘建文; 许建和

    2010-01-01

    α-D-Glucose-1-phosphate(Gle-1-P) is an expensive intermediate in the biosynthesis of nuclcotide glucose.This paper describes a biocataiytic system for the efficient synthesis of Glc-1-P from the low cost raw materials:maltodextrin and phosphate at ordinary temperatares.After molecular cloning and the expression of a maltodextrin phosphorylase (MalPase) gone from E.coli (Escherichia coli) K12,the resultant recombinant enzyme was immobilized on amino-fanctionalized magnetic nanoparticles for recycling and repeated use.Conditions for the biocatalytic reaction were optimized and the immobilized MalPase could be easily recovered and reused over eight cycles in the repeated synthesis of Glc-1-P.After simple purification steps approximately 440 mg of crude product Was obtained with a moderate isolation yield of 70.5%.%建立了以麦芽糊精和磷酸盐为底物,在常温下合成α-D-葡萄糖-1-磷酸的生物催化体系.从大肠杆菌K12中克隆表达了麦芽糊精磷酸化酶,并固定化在氨基修饰的磁性纳米颗粒上,以便于酶的回收和重复利用.在优化的反应条件下,于200ml体系中连续使用该固定化酶8批次,催化合成了α-D-葡萄糖-1-磷酸.经过简单的纯化步骤,最终得到440mg产品,分离产率为70.5%.

  9. Expression of Pyrimidine-Nucleoside Phosphorylase Gene from Enterobacter Aerogenes in Escherichia Coli BL21%来源于产气肠杆菌的嘧啶核苷磷酸化酶基因在大肠杆菌BL21中的表达

    Institute of Scientific and Technical Information of China (English)

    魏元刚; 倪孟祥; 葛亚文; 吴梧桐

    2007-01-01

    实现产气肠杆菌(Enterobacter aerogenes)嘧啶核苷磷酸化酶(Pyrimidine-nucleoside phosphorylase,PyPNase)基因在大肠杆菌中的高效表达.用来源于产气肠杆菌的核苷磷酸化酶N末端序列与NCBI中公布的已知序列进行比对,设计一对引物,以该菌株基因组DNA为模板扩增出PyPNase基因.定向克隆到PET-28a(+)载体后转化到表达宿主大肠杆菌BL21中,筛选阳性克隆并测序,然后在LB培养基中优化发酵条件.结果:目的基因片断长度为1 302 bp.表达产物经SDS-PAGE和酶活测定表明PyPNase基因在大肠杆菌中获得活性表达,43 ku处有表达蛋白.37℃培养2 h至对数生长中期,降温到31℃,0.1mmol/L IPTG诱导5 h获得最大酶活力为512.3 U/mg,为出发菌株的17.2倍.重组PyPNase的成功表达为今后大规模生产5-FUR打下了基础.

  10. Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization

    CSIR Research Space (South Africa)

    Visser, Daniel F

    2010-08-01

    Full Text Available debris, the expressed protein was further purified by ultrafiltration through a 100 kDa membrane (Amicon). The resultant protein solutions were then incubated at temperatures between 40 and 80 C degrees for 60 min to determine the temperature at which... characteristics on any prokaryotic UPs. It is only therefore possible to compare this mutant UP to other wild type enzymes, as listed in Table 5. Very few PyNPs and particularly UPs from prokaryote sources have been fully characterised. Other than those...

  11. Purification of Potato Glycogen Phosphorylase by Affinity Electrophoresis

    OpenAIRE

    Takeo, Kazusuke; Suzuno, Ryosuke; Fujimoto, Masanori; Kuwahara, Akira

    1980-01-01

    The feasibility that membrane action potential in mammalian myocardium can be produced by means of field-stimulation was investigated. Guinea-pig papillary muscle was driven by either point-or field-stimulation and transmembrane potentials were recorded simultaneously by two glass microelectrodes positioned at two different sites of the preparation. Both point-stimulation and weak field-stimulation produced a propagated response, while strong field-stimulation produced a membrane action poten...

  12. Ischemic nucleotide breakdown increases during cardiac development due to drop in adenosine anabolism/catabolism ratio

    NARCIS (Netherlands)

    J.W. de Jong (Jan Willem); E. Keijzer (Elisabeth); T. Huizer (Tom); B. Schoutsen

    1990-01-01

    markdownabstractAbstract Our earlier work on reperfusion showed that adult rat hearts released almost twice as much purine nucleosides and oxypurines as newborn hearts did [Am J Physiol 254 (1988) H1091]. A change in the ratio anabolism/catabolism of adenosine could be responsible for this effect.

  13. Synthesis and Bioactivity of Novel Trisubstituted Triazole Nucleosides.

    Science.gov (United States)

    Wen, Yi-ning; Zhang, Zhi-feng; Liu, Ning-ning; Xiang, Yu-hong; Zhang, Zhuo-yong; Andrei, Graciela; Snoeck, Robert; Schols, Dominique; Zhang, Qing-shan; Wu, Qin-pei

    2016-01-01

    A series of novel trisubstituted 1,2,3-triazole purine nucleosides were efficiently synthesized via Huisgen 1,3-dipolar cycloaddition in good yields. Bioactivity against cytomegalovirus (CMV) and varicella-zoster virus (VZV) in human embryonic lung cell cultures was evaluated and all compounds show low antiviral activity.

  14. Suppression of feline immunodeficiency virus infection in vivo by 9-(2-phosphonomethoxyethyl)adenine

    NARCIS (Netherlands)

    Horzinek, M.C.; Egberink, H.F.; Borst, M.; Niphuis, H.; Balzarini, J.; Neu, H.; Schellekens, H.; Clercq, H. de; Koolen, M.J.M.

    1990-01-01

    The acyclic purine nucleoside analogue 9-(2-phosphonomethoxyethyl)adenine [PMEA; formerly referred to as 9-(2-phosphonylmethoxyethyl)adenine] is a potent and selective inhibitor of human immunodeficiency virus replication in vitro and of Moloney murine sarcoma virus-induced tumor formation in mice.

  15. 1.45 A resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

    Energy Technology Data Exchange (ETDEWEB)

    Chojnowski, Grzegorz [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw (Poland); International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland); Breer, Katarzyna; Narczyk, Marta; Wielgus-Kutrowska, Beata [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw (Poland); Czapinska, Honorata [International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland); Hashimoto, Mariko; Hikishima, Sadao; Yokomatsu, Tsutomu [School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Bochtler, Matthias [International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw (Poland); Schools of Chemistry and Biosciences, Park Place, CF10 3AT Cardiff (United Kingdom); Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01309 Dresden (Germany); Girstun, Agnieszka; Staron, Krzysztof [Department of Molecular Biology, Institute of Biochemistry, University of Warsaw, Miecznikowa 1, 02-096 Warsaw (Poland); Bzowska, Agnieszka, E-mail: abzowska@biogeo.uw.edu.pl [Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw (Poland)

    2010-01-01

    Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K{sub d} ({approx}190 pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45 A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position).

  16. Significantly increased risk of carotid atherosclerosis with arsenic exposure and polymorphisms in arsenic metabolism genes

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, Yi-Chen [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Lien, Li-Ming [Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); School of Medicine, Taipei Medical University, Taipei, Taiwan (China); Department of Neurology, Shin Kong WHS Memorial Hospital, Taipei, Taiwan (China); Chung, Wen-Ting [Department of Neurology, Wanfang Hospital, Taipei Medical University, Taipei, Taiwan (China); Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan (China); Hsieh, Fang-I; Hsieh, Pei-Fan [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Wu, Meei-Maan [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Graduate Institute of Basic Medicine, College of Medicine, Fu-Jen Catholic University, Taipei, Taiwan (China); Tseng, Hung-Pin [Department of Neurology, Lotung Poh-Ai Hospital, I-Lan, Taiwan (China); Chiou, Hung-Yi, E-mail: hychiou@tmu.edu.tw [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, 250 Wusing St., Taipei 11031, Taiwan (China); Chen, Chien-Jen [Genomics Research Center, Academia Sinica, Taipei, Taiwan (China)

    2011-08-15

    Individual susceptibility to arsenic-induced carotid atherosclerosis might be associated with genetic variations in arsenic metabolism. The purpose of this study is to explore the interaction effect on risk of carotid atherosclerosis between arsenic exposure and risk genotypes of purine nucleoside phosphorylase (PNP), arsenic (+3) methyltransferase (As3MT), and glutathione S-transferase omega 1 (GSTO1) and omega 2 (GSTO2). A community-based case-control study was conducted in northeastern Taiwan to investigate the arsenic metabolic-related genetic susceptibility to carotid atherosclerosis. In total, 863 subjects, who had been genotyped and for whom the severity of carotid atherosclerosis had been determined, were included in the present study. Individual well water was collected and arsenic concentration determined using hydride generation combined with flame atomic absorption spectrometry. The result showed that a significant dose-response trend (P=0.04) of carotid atherosclerosis risk associated with increasing arsenic concentration. Non-significant association between genetic polymorphisms of PNP Gly51Ser, Pro57Pro, As3MT Met287Thr, GSTO1 Ala140Asp, and GSTO2 A-183G and the risk for development of carotid atherosclerosis were observed. However, the significant interaction effect on carotid atherosclerosis risk was found for arsenic exposure (>50 {mu}g/l) and the haplotypes of PNP (p=0.0115). A marked elevated risk of carotid atherosclerosis was observed in subjects with arsenic exposure of >50 {mu}g/l in drinking water and those who carried the PNP A-T haplotype and at least either of the As3MT risk polymorphism or GSTO risk haplotypes (OR, 6.43; 95% CI, 1.79-23.19). In conclusion, arsenic metabolic genes, PNP, As3MT, and GSTO, may exacerbate the formation of atherosclerosis in individuals with high levels of arsenic concentration in well water (>50 {mu}g/l). - Highlights: {yields}Arsenic metabolic genes might be associated with carotid atherosclerosis. {yields

  17. 转染胸苷磷酸化酶基因对5'-脱氧氟尿苷抑制结肠癌细胞的影响%Effect of thymidine phosphorylase cDNA transfection on the inhibition of human colon carcinoma cell line by 5'-deoxy-5-fluorouridine

    Institute of Scientific and Technical Information of China (English)

    高庆; 张继民; 刘剑; 王绮雯; 叶钿均; 刘影

    2013-01-01

    目的 探讨转染胸苷磷酸化酶(TP)cDNA对5’-脱氧氟尿苷(5’-DFUR)抑制人结肠癌细胞LOVO作用的影响.方法 将TP cDNA序列克隆以慢病毒表达载体包装后转染人结肠癌细胞LOVO(LOVO-TP组),另设空白对照组和LOVO-载体组.以流式细胞仪检测3组细胞转染效率,RTPCR法检测3组细胞TP mRNA表达;Western blot法检测转染前后TP蛋白水平;MTT法检测转染前后LOVO对5’-DFUR药物敏感性的变化;高效液相色谱法(HPLC)检测3组细胞转化不同浓度5'-DFUR生成5-FU量的情况.结果 转染TP基因并传代5代后,LOVO细胞的转染效率在95%左右.转染TP基因后,LOVO-TP组的TP mRNA表达水平为空白对照组的(282.5±86.8)倍(P<0.01),而LOVO-载体组与对照组相比差异无统计学意义(P>0.05);Western blot检测显示,LOVO-TP组的TP蛋白表达明显高于对照组和LOVO-载体组.5’-DFUR对LOVO细胞半数有效剂量(IC50)对照组为(1607.3±56.8) μmol/L,明显高于LOVO-TP组的(1087.7±89.1) μmol/L(P<0.01);而LOVO-染载体组为(1699.5±38.7)tμmol/L,与对照组相比差异无统计学意义(P>0.05).培养基中分别加入0、500、1000和2000 μmol/L的5'-DFUR后,对照组培养基中分别检出0、2.10、3.13和7.19 μmol/L的氟尿嘧啶(5-FU);而在LOVO-TP组的细胞培养基中则分别检出0、22.16、30.94和40.02 μmol/L的5-FU;而LOVO-载体组的培养基中则几乎无5-FU检出.结论 转染TP cDNA能够明显提高LOVO细胞的TP mRNA及TP蛋白表达水平,使细胞外5’-DFUR转化为5-FU增多,明显提高5’-DFUR对LOVO的细胞毒性作用.%Objective To investigate the inhibiting impact of 5'-deoxy-5-fluorouridine(5'-DFUR)on human colon carcinoma cell line LOVO after transfection of thymidine phosphorylase (TP) cDNA.Methods TP cDNA was transfected into human colon carcinoma cell line LOVO with lentiviral vector pLenti6.3_MCS_IRES2-EGFP,and the transfection efficiency was analyzed by flow cytometry.TP mRNA and protein

  18. Glycogen phosphorylase isoenzyme BB in the diagnosis of acute myocardial infarction in early stage%糖原磷酸化酶同工酶脑型在急性心肌梗死早期诊断中的临床意义

    Institute of Scientific and Technical Information of China (English)

    周昭仑; 温少磊; 许兆延; 杨希立; 张健瑜; 肖长华

    2008-01-01

    Objective To evaluate the value of glycogen phosphorylase isoenzyme BB (GPBB) in the diagnosis of early acute myocardial infarction (AMI). Methods The plasma levels of GPBB were mea-sured by sandwich ELISA in 115 patients with suspected AMI at admission within 6 hours after onset of chest pain and 55 normal healthy subjects. The plasma concent of cardiac troponin-Ⅰ (cTnI), creatine kinase-MB (CK-MB) and myoglobin (MYO) was assayed at the same time by using corpuscle chemiluminescence. The patients were classified retrospectively into AMI group (n = 45) , unstable angina pectoris (UAP) group (n =40) , stable angina pectoris (SAP) group (n = 13) and non-cardiac chest pain (NCCP) group (n =17).The diagnostic validity was evaluated in terms of sensitivity and specificity. Results The diagnostic sensitivity of GPBB for AMI was 64.29 % within 3 hours and 88.89 % within 6 hours after onset of chest pain,which is significantly higher than that of cTnI (28.57 %, 60.00 %) and CK-MB (21.43 %, 64.44 % ). There was no significant difference in specificity among the four markers. The diagnostic accuracy of GPBB within 3hours and 6 hours (80.77 %, 89.57% ) was significantly higher than that of cTnI (61.54%, 81.74% ),CK-MB (50.00%, 75.65%) and MYO (73.08% ,73.91%). Conclusions GPBB seems to be a sensitive and specific biochemical cardiac marker for AMI in the early stage. Its diagnostic accuracy is higher than that of cTnI, CK-MB, MYO.%目的 探讨糖原磷酸化酶同工酶脑型(GPBB)在急性心肌梗死(AMI)早期诊断中的价值.方法 选择115例发病6 h内的胸痛患者,其中AMI患者45例(AMI组),非AMI患者70例(非AMI组).非AMI组中不稳定型心绞痛(UAP)40例、稳定型心绞痛(SAP)13例、非心源性胸痛17例;选择55例健康体检者作为健康对照组.检测发病0~6 h的血浆GPBB水平,并与心肌肌钙蛋白Ⅰ(cTnI)、肌酸激酶同工酶-MB(CK-MB)和肌红蛋白(MYO)的检测结果进行比较,分析上述指标诊断发病0~3 h和0

  19. Transfection of thymidine phosphorylase cDNA with lentiviral vector enhances the anticancer effect of 5'-deoxy-5-fluorouridine on colorectal cancer cell lines HT29 and LS174T%慢病毒转染胸苷磷酸化酶基因增强5'-脱氧氟尿苷抗结肠癌细胞活性

    Institute of Scientific and Technical Information of China (English)

    叶钿均; 王绮雯; 张继民; 刘奇

    2015-01-01

    Objective To explore the changes of anticancer efficiency of 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-fluorouracil (5-Fu) in colorectal cancer cell line HT29 and LS174T cells after transfection of thymidine phosphorylase (TP) cDNA with a lentiviral vector.Methods TP cDNA was transfected into human colorectal cancer cell lines HT29 and LS174T with the lentiviral vector pLenti6.3-MCS-IRES2-EGFP,and the transfection efficiency of the two cell lines passed 5 generations was analyzed by flow cytometry.The expression of TP protein and the relative quantitative expression of TP mRNA in these 2 cell lines were detected by Western blot and RT-PCR,respectively.The 50% inhibitory concentration (IC50) of 5'-DFUR and 5-Fu in both HT29 and LS174T parent cells and TP-transfected cells were assessed by MTS assay.Finally,the concentration of converted 5-Fu was detected by high performance liquid chromatography (HPLC) either in the medium containing a series of concentrations of 5'-DFUR,in which HT29/HT29-TP or LS174T/LS174T-TP cells were cultured,or in the cell culture lysates.Results The HT29 and LS174T cells transfected with human TP cDNA were monitored for 5 generations,and their transfection efficiency was about 95.0%.Immunohistochemical staining showed that both the parent cells and TP-transfected HT29 and LS174T cells were TP-positive,while vector-transfected cells were TPnegative.Western blotting showed that the TP protein expression in HT29-TP and LS174T-TP cells were significantly increased compared with that in their parents cells.The relative quantity (RQ) values of TP mRNA in HT29-TP and LS174T-TP cells were 8.45 ± 0.15 and 2 615.02 ± 253.97,respectively,which were significantly higher than that in their parents cells (P <0.01).The IC50 values of 5'-DFUR on HT29-TP cells and its parents cell were (14.33 ± 0.74) μmol/L and (707.66 ± 5.66) μmol/L,respectively (P<0.05),while (0.59 ±0.11) μmol/L in LS174T-TP cells and (239.20 ±21.83) μmol/L in its parent

  20. Influence of interferon-α2a on thymidine phosphorylase expression and anticancer activity of 5′-deoxy-fluorouridine in human colon carcinoma cell lines LOVO and SW480%干扰素α2a对人结肠癌细胞胸苷磷酸化酶表达和5′-脱氧氟尿苷抗癌活性的影响

    Institute of Scientific and Technical Information of China (English)

    夏强; 王绮雯; 张继民

    2012-01-01

    Objective To detect the effect of interferon-α2a (IFN-α2a) on thymidine phosphorylase(TP) mRNA expression levels and the anticancer activity of 5-fluorouracil (5-FU) and 5′-deoxy-fluorouridine(5′-DFUR) in human colon carcinoma cell lines LOVO and SW480.Methods Two human colon cancer cell lines LOVO and SW480 were cultured and treated with IFN-α2a at a series of dosage,and fluorescence quantitative PCR was carried out to detect the TP mRNA expression levels in these 2 cell lines.Then MTT assay and software Templet were used to determine the change of 50% inhibition concentration of 5-FU or 5′-DFUR combined with IFN-α2a on the two cell lines.Results The TP mRNA expressions were up-regulated significantly by IFN-α2a at the doses of 500 U/ml and 5000 U/ml in LOVO(P<0.01 ).Compared with untreated cells(IFN-α2a 0 U/ml),no significance was found for TP mRNA expression levels in LOVO and SW480 treated by IFN-α2a at the dose of 50 U/ml (P>0.05).There was no significant difference for TP mRNA expression in SW480 between the dose of 0U/ml and 500 U/ml of IFN-α2a (P>0.05),while a significant increace was detected at the dose of 5000 U/ml (P<0.01).No significant difference was found for the IC50 values after treatment of 5-FU combined with IFN-α2a (20 U/ml) on LOVO and SW480 compared with 5-FU alone,while the IC50values after treatment of 5′-DFUR combined with IFN-α2a decreased significantly compared with 5′-DFUR alone (P<0.05).Conclusion There is no direct inhibition effect of IFN-α2a on LOVO and SW480 in vitro,while it can up-regulate TP mRNA expression levels both in LOVO and SW480,and enhance the anticancer effect of 5′-DFUR on these 2 cell lines.%目的 探讨干扰素-α2a(IFN-α2a)对人结肠癌细胞胸苷磷酸化酶(TP)mRNA表达水平以及5′-脱氧氟尿苷(5′-DFUR)和氟尿嘧啶(5-FU)抗癌效应的影响.方法 不同剂量IFN-α2a分别处理人结肠癌细胞LOVO和SW480,荧光定量PCR检测TP mRNA表达水平

  1. Transfected thymidine phosphorylase cDNA with lentiviral vector and INF-α2a enhance the anticancer effects of 5'-deoxy-5-fluorouridine on colorectal cancer cell line LOVO%转染胸苷磷酸化酶基因联合干扰素α2a增强5'-脱氧氟尿苷对结肠癌细胞株的细胞毒作用

    Institute of Scientific and Technical Information of China (English)

    刘奇; 王绮雯; 张继民; 方耿

    2015-01-01

    目的 利用慢病毒载体将胸苷磷酸化酶(TP)基因转染人结肠癌细胞株LOVO,然后应用干扰素-α2a(INF-α2a)处理转染前后细胞,分别测定5'-脱氧氟尿苷(5'-DFUR)和5-氟尿嘧啶(5-FU)对LOVO抗肿瘤效应的影响.方法 构建包含TP基因的慢病毒载体,转染结肠癌细胞株LOVO,以流式细胞技术和免疫荧光法检测在第5代LOVO中的转染效率;加INF-α2a培养24h后分别应用RT-PCR和Wester blot检测细胞的TP mRNA和TP蛋白表达;MTS法检测5'-DFUR和5-FU对各组细胞的半数抑制浓度(IC50);高效液相色谱法检测各组细胞转化5'-DFUR为5-FU的量.结果 转染TP基因的效率达95%.LOVO+INF-α2a组的TP mRNA表达是LOVO组的1.87倍,而LOVO-TP组和LOVO-TP+ INF-α2a组TPmRNA表达则比LOVO组分别增加了18.56倍和59.61倍(P<0.01).LOVO-TP+ INF-α2a组和LOVO-TP组蛋白表达水平比LOVO组分别增加242.23倍和197.69倍(P<0.01).5'-DFUR对LOVO-TP组细胞的IC50值仅为LOVO组细胞的37.2%,而LOVO-TP+INF-α2a组的IC50则是LOVO组的18.2%.LOVO-TP组在加入100、200、400 μmol/L5'-DFUR的培养基中检测的5-FU浓度比LOVO组分别增高了26.13%、33.23%、32.95%,而LOVO-TP+ INF-α2a组比LOVO组分别增高了49.94%、61.66%、72.14%. 结论 转染TP eDNA能够明显提高LOVO细胞的TP mRNA及TP蛋白的表达水平,而INF-α2a则进一步增强这种上调作用.增高的TP表达使细胞外5'-DFUR转化为5-FU含量增加,明显提高5'-DFUR的细胞毒性.%Objective Thymidine phosphorylase (TP) cDNA was transfected into colorectal cancer cell lines LOVO with the lentiviral vector,the anticancer effeciency of 5'-deoxy-5-fluorouridine (5'-DFUR)and 5-fluorouracil (5-FU) on LOVO cells were evaluated.Methods TP cDNA were transfected into LOVO cell line with the lentiviral vector pLenti6.3_MCS_IRES2-EGFP,and the transfection efficiency was analyzed by flow cytometer and immunohistochemistry.Cells were divided into six groups

  2. Suppression of feline immunodeficiency virus infection in vivo by 9-(2-phosphonomethoxyethyl)adenine

    OpenAIRE

    Horzinek, M.C.; Egberink, H F; Borst, M.; Niphuis, H; Balzarini, J; Neu, H.; Schellekens, H.; De Clercq, H; Koolen, M.J.M.

    1990-01-01

    The acyclic purine nucleoside analogue 9-(2-phosphonomethoxyethyl)adenine [PMEA; formerly referred to as 9-(2-phosphonylmethoxyethyl)adenine] is a potent and selective inhibitor of human immunodeficiency virus replication in vitro and of Moloney murine sarcoma virus-induced tumor formation in mice. In the latter system PMEA has stronger antiretroviral potency and selectivity than 3'-azido-3'-thymidine (AZT). We have now investigated the effect of the drug in cats infected with the feline immu...

  3. Metabolic profiles and free radical scavenging activity of Cordyceps bassiana fruiting bodies according to developmental stage.

    Directory of Open Access Journals (Sweden)

    Sun-Hee Hyun

    Full Text Available The metabolic profiles of Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana.

  4. Metabolic Profiles and Free Radical Scavenging Activity of Cordyceps bassiana Fruiting Bodies According to Developmental Stage

    Science.gov (United States)

    Hyun, Sun-Hee; Lee, Seok-Young; Sung, Gi-Ho; Kim, Seong Hwan; Choi, Hyung-Kyoon

    2013-01-01

    The metabolic profiles of Cordyceps bassiana according to fruiting body developmental stage were investigated using gas chromatography-mass spectrometry. We were able to detect 62 metabolites, including 48 metabolites from 70% methanol extracts and 14 metabolites from 100% n-hexane extracts. These metabolites were classified as alcohols, amino acids, organic acids, phosphoric acids, purine nucleosides and bases, sugars, saturated fatty acids, unsaturated fatty acids, or fatty amides. Significant changes in metabolite levels were found according to developmental stage. Relative levels of amino acids, purine nucleosides, and sugars were higher in development stage 3 than in the other stages. Among the amino acids, valine, isoleucine, lysine, histidine, glutamine, and aspartic acid, which are associated with ABC transporters and aminoacyl-tRNA biosynthesis, also showed higher levels in stage 3 samples. The free radical scavenging activities, which were significantly higher in stage 3 than in the other stages, showed a positive correlation with purine nucleoside metabolites such as adenosine, guanosine, and inosine. These results not only show metabolic profiles, but also suggest the metabolic pathways associated with fruiting body development stages in cultivated C. bassiana. PMID:24058459

  5. Effect of Chicory on Uric Acid and Uricopoiesis Metabolic Enzymes Activities of Hyperuricemia Quail%菊苣干预高尿酸血症鹌鹑尿酸及相关代谢酶活性研究

    Institute of Scientific and Technical Information of China (English)

    黄胜男; 林志健; 张冰; 赓迪; 牛红娟; 朱春胜; 王雪洁; 孙博喻

    2015-01-01

    Objective To investigate the effect of chicory on uric acid and uricopoiesis metabolic enzymes activities in hyperuricemia quail and to explore the possible mechanism of chicory in preventing and treating hyperuricemia. Methods Sixty quails were evenly randomized into 5 groups according to the body weight, namely normal group, model group, benzbromarone(20 mg·kg-1·d-1) group, high- and low-dosage chicory groups(10, 5 g·kg-1·d-1 respectively). Except for the normal group, the quails in other groups were given high purine diet(ordinary forage mixed with 15 g·kg-1·d-1 of yeast powder) to induce hyperuricemia(HUA)model. And then we observed the changes of serum uric acid level and activities of uricopoiesis metabolic enzymes dynamically during the chicory treatment for 28 days. Results On the chicory administration day 7, 14, 21 and 28, serum uric acid level of quail was decreased obviously, and the activities of uricopoiesis metabolic enzymes of 5'-nucleotidase(5'-NT), adenosine deaminase (ADA),purine nucleoside phosphorylase(PNP),guanine deaminase(GD),xanthine oxidase(XO)were inhibited to various degrees in chicory groups(P<0.05, P<0.01 compared with the model group). Conclusion Chicory has the effect on lowering serum uric acid level in quail hyperuricemia model, which may be associated with reducing the activities of 5'-NT,ADA,PNP,GD,and XO.%目的:探讨菊苣对高尿酸血症(HUA)鹌鹑尿酸及相关代谢酶活性的影响,阐释菊苣防治HUA的可能机制。方法将60只鹌鹑,按体质量随机分为5组,即正常组,模型组,苯溴马隆组(20 mg·kg-1·d-1),菊苣高、低剂量组(10,5 g·kg-1·d-1),每组12只。除正常组喂饲鹌鹑普通饲料外,其余各组均给予高嘌呤饲料(普通饲料拌入酵母干粉15 g·kg-1·d-1)复制HUA模型。以菊苣治疗28 d,动态观察鹌鹑尿酸(uric acid,UA)水平及UA生成代谢酶活性的变化。结果给药7,14,21,28 d,菊苣高、低

  6. QSAR Studies of 6-Amino Uracil Base Analogues: A Thymidine Phosphorylase Inhibitor in Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Surya Prakash B. N. Gupta

    2008-01-01

    Full Text Available A novel series of 6-amino uracil base analogue were synthesized. QSAR study was used to relate the selective nonsubstrate inhibitory activity of 6-amino uracil base analogue with various physicochemical descriptors. Stepwise multiple regression analysis was performed to find out the correlation between various physicochemical descriptors and biological activity of the compounds by using Openstat 2 version 6.5.1 and valstat statistical software. Out of the several equations developed, the best equation having the highest significance was selected for further study. The equation is able to explain 60% of total variance and are more than 95% significant as revealed by the F value.

  7. Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083.

    NARCIS (Netherlands)

    Broek, van den L.A.M.; Boxtel, van E.L.; Kievit, R.P.; Verhoef, R.P.; Beldman, G.; Voragen, A.G.J.

    2004-01-01

    Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that

  8. Isoform-selective regulation of glycogen phosphorylase by energy deprivation and phosphorylation in astrocytes

    DEFF Research Database (Denmark)

    Müller, Margit S; Pedersen, Sofie E; Walls, Anne B;

    2015-01-01

    by determination of glycogen content showing an increase in glycogen levels following knockdown of either GPMM or GPBB. NE triggered glycogenolysis within 15 min in control cells and after GPBB knockdown. However, astrocytes in which expression of GPMM had been silenced showed a delay in response to NE...

  9. Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity.

    OpenAIRE

    Bruin,, Henk; Capel, van, T; Born, van der, T.; Kruyt, F.A.E.; Fukushima, M.(High Energy Accelerator Research Organization (KEK), 305-0801, Tsukuba, Japan); Hoekman, K.; Pinedo, H. M.; Peters, G. J.

    2003-01-01

    0.01) in our cell panel. To determine the role of TP in the sensitivity to 5FU, 5'DFUR, Ft and TFT, cells were cultured with the various fluoropyrimidines with or without TPI and differences in IC(50)'s were established. TPI modified 5'DFUR, increasing the IC(50)'s 2.5- to 1396-fold in WiDR and Colo320TP1, respectively. 5-Fluorouracil could be modified by inhibiting TP but to a lesser extent than 5'DFUR: IC(50)'s increased 1.9- to 14.7-fold for WiDR and Colo320TP1, respectively. There was no ...

  10. Role of platelet-derived endothelial cell growth factor/thymidine phosphorylase in fluoropyrimidine sensitivity.

    NARCIS (Netherlands)

    Bruin, de M.; Capel, van T; Born, van der K.; Kruyt, F.A.E.; Fukushima, M; Hoekman, K.; Pinedo, H.M.; Peters, G.J.

    2003-01-01

    0.01) in our cell panel. To determine the role of TP in the sensitivity to 5FU, 5'DFUR, Ft and TFT, cells were cultured with the various fluoropyrimidines with or without TPI and differences in IC(50)'s were established. TPI modified 5'DFUR, increasing the IC(50)'s 2.5- to 1396-fold in WiDR and Colo

  11. 3-Glucosylated 5-amino-1,2,4-oxadiazoles: synthesis and evaluation as glycogen phosphorylase inhibitors

    Directory of Open Access Journals (Sweden)

    Marion Donnier-Maréchal

    2015-04-01

    Full Text Available Glycogen phosporylase (GP is a promising target for the control of glycaemia. The design of inhibitors binding at the catalytic site has been accomplished through various families of glucose-based derivatives such as oxadiazoles. Further elaboration of the oxadiazole aromatic aglycon moiety is now reported with 3-glucosyl-5-amino-1,2,4-oxadiazoles synthesized by condensation of a C-glucosyl amidoxime with N,N’-dialkylcarbodiimides or Vilsmeier salts. The 5-amino group introduced on the oxadiazole scaffold was expected to provide better inhibition of GP through potential additional interactions with the enzyme’s catalytic site; however, no inhibition was observed at 625 µM.

  12. High-yielding cascade enzymatic synthesis of 5-methyluridine using a novel combination of nucleoside phosphorylases

    CSIR Research Space (South Africa)

    Visser, Daniel F

    2010-07-01

    Full Text Available ml scale using defined growth media (K2HPO4, 14.6 g.l-1; (NH4)2SO4 2 g.l-1; Na2HPO4, 3.6 g.l-1; Citric Acid, 2.5 g.l-1; MgSO4, 0.25 g.l-1; NH4NO3, 5 g.l-1; yeast extract, 10 g.l-1; glucose 30 g.l-1 and ampicillin, 100 µg.ml- 1). An overnight... was reached at 4 h. Enzyme Recovery. After fermentation, the broth was harvested and allowed to settle overnight at 4°C. The biomass was separated from the supernatant by decanting, and subjected to a freeze-thaw cycle alternating between +20oC and – 20...

  13. Consensus engineering of sucrose phosphorylase: The outcome reflects the sequence input

    NARCIS (Netherlands)

    Aerts, D.; Verhaeghe, T.; Joosten, H.J.; Vriend, G.; Soetaert, W.; Desmet, T.

    2013-01-01

    Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tri

  14. Molecular modeling study of the thymidine phosphorylase inhibitor by SBDD and classical QSAR analysis

    National Research Council Canada - National Science Library

    多田, 幸雄; 数野, 秀樹; 佐藤, 勉; 鈴木, 則彦; 江村, 智弘; 矢野, 伸吾

    2017-01-01

    トリフルオロチミジン(TFT)は抗腫瘍活性を有するが、チミジンホスホリラーゼ(TP)により不活性なチミンに容易に代謝されるので、TFT単独では臨床で満足な抗腫瘍活性を示すことができない。ヒトのTP (HTP)...

  15. Biosynthesis of anti-HCV compounds using thermophilic microorganisms.

    Science.gov (United States)

    Rivero, Cintia W; De Benedetti, Eliana C; Sambeth, Jorge E; Lozano, Mario E; Trelles, Jorge A

    2012-10-01

    This work describes the application of thermophilic microorganisms for obtaining 6-halogenated purine nucleosides. Biosynthesis of 6-chloropurine-2'-deoxyriboside and 6-chloropurine riboside was achieved by Geobacillus stearothermophilus CECT 43 with a conversion of 90% and 68%, respectively. Furthermore, the selected microorganism was satisfactorily stabilized by immobilization in an agarose matrix. This biocatalyst can be reused at least 70 times without significant loss of activity, obtaining 379mg/L of 6-chloropurine-2'-deoxyriboside. The obtained compounds can be used as antiviral agents.

  16. High performance liquid chromatographic profiles of nucleosides, bases and tryptophan in the plasma of the Tasmanian devil and four other marsupial species.

    Science.gov (United States)

    Sallis, J D; Nicol, S C; Perrone, P; Brown, P R

    1984-01-01

    Plasma profiles of nucleosides, bases and trytophan of five marsupial species were established using the reversed-phase mode of high performance liquid chromatography (RHPLC). Within each species, the profiles were highly reproducible and between species there were distinct differences. In the Tasmanian devil, the circulating levels of constituents examined with one exception, were generally lower than in the other marsupials. The exception was a constituent present in large amounts and having the characteristics of a purine nucleoside derivative which was found only in the plasma of the devil.

  17. N2-(1-Methoxycarbonylethyl)guanosine, a new nucleoside coupled with an amino acid derivative from Amanita exitialis

    Institute of Scientific and Technical Information of China (English)

    Yu Lang Chi; Hui Ye Zhang; Jing Hua Xue; Jing Hao; Mei Fang Liu; Xiao Yi Wei

    2009-01-01

    A new purine nucleoside coupled with an amino acid derivative, N2-(1-methoxycarbonylethyl)guanosine 1, along with βearboline and russulaceramide was isolated from the fruiting bodies ofAmanita exitialis, a newly described poisonous mushroom. Its structure was elucidated by spectroscopic methods. This is the first report of naturally occurring nucleosides in which an α-amino acid derivative is bonded through its a-amino nitrogen to a nucleobase aglycone by a C-N bond. The new compound was found to be toxic in brine shrimp lethality test (BST).

  18. Prognostic significance of numeric aberrations of genes for thymidylate synthase, thymidine phosphorylase and dihydrofolate reductase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Søren Astrup; Vainer, B.; Witton, C.J.

    2008-01-01

    ) in colorectal cancer, and to evaluate its prognostic significance following adjuvant chemotherapy, since these enzymes are closely related to efficacy of 5-fluorouracil (5FU). PATIENTS AND METHODS: Consecutive patients (n = 314), who were completely resected for colorectal cancer stages II-IV and adjuvantly...... treated with 5-FU were retrospectively evaluated. Paraffin embedded tumor specimens were assessed for gene copies per nucleus of TYMS, TP and DHFR by fluorescence in situ hybridisation (FISH) using specific peptide nucleic acid probes. Outcome according to gene copies per nucleus above and below...... 1.1-2.2; p = 0.02) and death (HR = 1.6; 95%CI 1.1-2.3; p = 0.01). No significant differences in outcome appeared according to TP and DHFR gene ratios. CONCLUSION: Aberration of TYMS gene is of significance to expression of TYMS, which may influence the biology and 5-FU sensitivity of colorectal...

  19. The correlation between PNP geneit c poyl morphisms and the susceptibility to endemic arseni c poisoning%PNP 基因多态性与地方性砷中毒易感性的关系研究

    Institute of Scientific and Technical Information of China (English)

    侯文胜; 张丽芳; 裴秋玲

    2013-01-01

    目的探讨砷代谢相关酶嘌呤核苷磷酸化酶( PNP)单核苷酸第2外显子多态性与地方性砷中毒患病风险的关系。方法采用聚合酶链反应一单链构象多态性( PCR-SSCP )技术对79名地方性砷中毒患者和110名正常人群PNP基因第2外显子的突变情况进行初筛,并进一步对异常带型进行直接DNA测序,以确定突变位点及类型。对多态性进行单因素分析和多因素非条件Logistic回归分析。结果病例组15位密码子GAA→AAA突变、51位密码子GGT→C/AGT突变和51位密码子GGT→AGT突变的发生率(11.4%)低于对照组(19.1%);病例组20位密码子CAC→CAT 突变的发生率(10.1%)低于对照组(17.3%);病例组57位密码子CCC→CCT突变的发生率(22.8%)低于对照组(38.2%),但五种突变差异均无统计学意义( P >0.05)。在多因素分析中调整年龄、性别等因素后,PNP基因多态性与地方性砷中毒发病仍无统计学意义,与单因素分析的结果相同。结论虽然病例组PNP基因第2外显子上的多态性发生率低于对照组,但是这种差异没有统计学意义,出现这种情况的原因可能与分析的样本量较小,或者是有关的多态性位点在PNP基因其他的外显子或非编码区上有关。%Objetc ive To investigate the correlation between genetic polymorphism of purine nucleoside phosphorylase ( PNP)and susceptibility to endemic arsenic poisoning .Methods Polymerase chain reaction-single strand conformation polymorphism (PCR S-SCP ) technology was used to detect the mutations of PNP gene exon 2 in 79 patients with endemic arsenic poisoning (case group) and 110 healthy subjects (control group),then the PCR products with abnormal band forms were further sequenced in order to find the types and sites of mutation . Chi-square test and multivariate logistic analysis were performed to analyze PNP genetic polymorphisms

  20. Cardiac endothelial transport and metabolism of adenosine and inosine

    Science.gov (United States)

    Schwartz, Lisa M.; Bukowski, Thomas R.; Revkin, James H.; Bassingthwaighte, James B.

    2010-01-01

    The influence of transmembrane flux limitations on cellular metabolism of purine nucleosides was assessed in whole organ studies. Transcapillary transport of the purine nucleosides adenosine (Ado) and inosine (Ino) via paracellular diffusion through interendothelial clefts in parallel with carrier-mediated transendothelial fluxes was studied in isolated, Krebs-Henseleit-perfused rabbit and guinea pig hearts. After injection into coronary inflow, multiple-indicator dilution curves were obtained from coronary outflow for 90 s for 131I-labeled albumin (intravascular reference tracer), [3H]arabinofuranosyl hypoxanthine (AraH; extracellular reference tracer and nonreactive adenosine analog), and either [14C]Ado or [14C]Ino. Ado or Ino was separated from their degradative products, hypoxanthine, xanthine, and uric acid, in each outflow sample by HPLC and radioisotope counting. Ado and Ino, but not AraH, permeate the luminal membrane of endothelial cells via a saturable transporter with permeability-surface area product PSecl and also diffuse passively through interendothelial clefts with the same conductance (PSg) as AraH. These parallel conductances were estimated via fitting with an axially distributed, multi-pathway, four-region blood-tissue exchange model. PSg for AraH were ~4 and 2.5 ml · g−1 · min−1 in rabbits and guinea pigs, respectively. In contrast, transplasmalemmal conductances (endothelial PSecl) were ~0.2 ml · g−1 · min−1 for both Ado and Ino in rabbit hearts but ~2 ml · g−1 · min−1 in guinea pig hearts, an order of magnitude different. Purine nucleoside metabolism also differs between guinea pig and rabbit cardiac endothelium. In guinea pig heart, 50% of the tracer Ado bolus was retained, 35% was washed out as Ado, and 15% was lost as effluent metabolites; 25% of Ino was retained, 50% washed out, and 25% was lost as metabolites. In rabbit heart, 45% of Ado was retained and 5% lost as metabolites, and 7% of Ino was retained and 3% lost as

  1. Ribavirin can be mutagenic for arenaviruses.

    Science.gov (United States)

    Moreno, Héctor; Gallego, Isabel; Sevilla, Noemí; de la Torre, Juan Carlos; Domingo, Esteban; Martín, Verónica

    2011-07-01

    Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under single- and multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed.

  2. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    Energy Technology Data Exchange (ETDEWEB)

    Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  3. A flexible loop as a functional element in the catalytic mechanism of nucleoside hydrolase from Trypanosoma vivax.

    Science.gov (United States)

    Vandemeulebroucke, An; De Vos, Stefan; Van Holsbeke, Els; Steyaert, Jan; Versées, Wim

    2008-08-08

    The nucleoside hydrolase of Trypanosoma vivax hydrolyzes the N-glycosidic bond of purine nucleosides. Structural and kinetic studies on this enzyme have suggested a catalytic role for a flexible loop in the vicinity of the active sites. Here we present the analysis of the role of this flexible loop via the combination of a proline scan of the loop, loop deletion mutagenesis, steady state and pre-steady state analysis, and x-ray crystallography. Our analysis reveals that this loop has an important role in leaving group activation and product release. The catalytic role involves the entire loop and could only be perturbed by deletion of the entire loop and not by single site mutagenesis. We present evidence that the loop closes over the active site during catalysis, thereby ordering a water channel that is involved in leaving group activation. Once chemistry has taken place, the loop dynamics determine the rate of product release.

  4. Pathologic overproduction: the bad side of adenosine.

    Science.gov (United States)

    Borea, Pier Andrea; Gessi, Stefania; Merighi, Stefania; Vincenzi, Fabrizio; Varani, Katia

    2017-03-02

    Adenosine is an endogenous ubiquitous purine nucleoside, increased by hypoxia, ischemia and tissue damage that mediates a number of physiopathological effects by interacting with four G-protein-coupled receptors, identified as A1 , A2A , A2B , and A3 . Physiological and acutely-increased adenosine is associated with beneficial effects mostly including vasodilation and decrease of inflammation. In contrast chronic overproduction of adenosine occurs in important pathological states, where long lasting increases in the nucleoside levels are responsible for the bad side of adenosine associated with chronic inflammation, fibrosis and organ damage. In this review we describe and critically discuss the pathologic overproduction of adenosine analysing when, where and how adenosine exerts its detrimental effects through the body.

  5. Comprehensive identification of 125 multifarious constituents in Shuang-huang-lian powder injection by HPLC-DAD-ESI-IT-TOF-MS.

    Science.gov (United States)

    Sun, Hongyang; Liu, Meixian; Lin, Zongtao; Jiang, Haixiu; Niu, Yanyan; Wang, Hong; Chen, Shizhong

    2015-11-10

    A high-performance liquid chromatography-diode array detector-electrospray ionization-ion trap-time of flight-mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) method was established for excellent separation and structural identification of constituents in Shuang-huang-lian powder injection (SHLPI). The typical ultraviolet absorptions, accurate empirical molecular formula and reasonable fragmentation mechanisms of these ingredients were used for their structural elucidation. In consequence, 125 constituents (33 phenolic acids, 29 flavonoids, 32 phenylethanoid glycosides, 15 iridoid glycosides, 8 lignans, 3 amino acids and 2 purines nucleosides, 2 quinoid glycosides and 1 alkylbenzene glycoside) were either unequivocally identified or tentatively characterized by comparing authentic standards or published data. The result showed that this study could provide valuable information for the quality control and further investigation of SHLPI formula.

  6. Protein Kinase C-Related Kinase (PKN/PRK). Potential Key-Role for PKN1 in Protection of Hypoxic Neurons.

    Science.gov (United States)

    Thauerer, Bettina; Zur Nedden, Stephanie; Baier-Bitterlich, Gabriele

    2014-05-01

    Serine/threonine protein kinase C-related kinase (PKN/PRK) is a family of three isoenzymes (PKN1, PKN2, PKN3), which are widely distributed in eukaryotic organisms and share the same overall domain structure. The Nterminal region encompasses a conserved repeated domain, termed HR1a-c as well as a HR2/C2 domain. The serine/threonine kinase domain is found in the C-terminal region of the protein and shows high sequence homology to other members of the PKC superfamily. In neurons, PKN1 is the most abundant isoform and has been implicated in a variety of functions including cytoskeletal organization and neuronal differentiation and its deregulation may contribute to neuropathological processes such as amyotrophic lateral sclerosis and Alzheimer's disease. We have recently identified a candidate role of PKN1 in the regulation of neuroprotective processes during hypoxic stress. Our key findings were that: 1) the activity of PKN1 was significantly increased by hypoxia (1% O2) and neurotrophins (nerve growth factor and purine nucleosides); 2) Neuronal cells, deficient of PKN1 showed a decrease of cell viability and neurite formation along with a disturbance of the F-actinassociated cytoskeleton; 3) Purine nucleoside-mediated neuroprotection during hypoxia was severely hampered in PKN1 deficient neuronal cells, altogether suggesting a potentially critical role of PKN1 in neuroprotective processes. This review gives an up-to-date overview of the PKN family with a special focus on the neuroprotective role of PKN1 in hypoxia.

  7. Protein Kinase C-Related Kinase (PKN/PRK). Potential Key-Role for PKN1 in Protection of Hypoxic Neurons

    Science.gov (United States)

    Thauerer, Bettina; zur Nedden, Stephanie; Baier-Bitterlich, Gabriele

    2014-01-01

    Serine/threonine protein kinase C-related kinase (PKN/PRK) is a family of three isoenzymes (PKN1, PKN2, PKN3), which are widely distributed in eukaryotic organisms and share the same overall domain structure. The Nterminal region encompasses a conserved repeated domain, termed HR1a-c as well as a HR2/C2 domain. The serine/threonine kinase domain is found in the C-terminal region of the protein and shows high sequence homology to other members of the PKC superfamily. In neurons, PKN1 is the most abundant isoform and has been implicated in a variety of functions including cytoskeletal organization and neuronal differentiation and its deregulation may contribute to neuropathological processes such as amyotrophic lateral sclerosis and Alzheimer’s disease. We have recently identified a candidate role of PKN1 in the regulation of neuroprotective processes during hypoxic stress. Our key findings were that: 1) the activity of PKN1 was significantly increased by hypoxia (1% O2) and neurotrophins (nerve growth factor and purine nucleosides); 2) Neuronal cells, deficient of PKN1 showed a decrease of cell viability and neurite formation along with a disturbance of the F-actinassociated cytoskeleton; 3) Purine nucleoside-mediated neuroprotection during hypoxia was severely hampered in PKN1 deficient neuronal cells, altogether suggesting a potentially critical role of PKN1 in neuroprotective processes. This review gives an up-to-date overview of the PKN family with a special focus on the neuroprotective role of PKN1 in hypoxia. PMID:24851086

  8. DNA damage and radical reactions: Mechanistic aspects, formation in cells and repair studies

    Energy Technology Data Exchange (ETDEWEB)

    Cadet, J.; Ravanat, J.L. [CEA Grenoble, Inst Nanosci and Cryogenie, SCIB-UMR-E 3, Lab Les Acides Nucl, UJF, F-38054 Grenoble 9 (France); Carell, T. [Univ Munich, Dept Chem and Biochem, Ctr Integrat Prot Sci, D-81377 Munich (Germany); Cellai, L. [CNR, Ist Cristalog, Monterotondo Stn, I-00016 Rome (Italy); Chatgilialoglu, Ch. [CNR, ISOF, I-40129 Bologna, (Italy); Gimisis, Th. [Univ Athens, Dept Chem, Organ Chem Lab, Athens 15784, (Greece); Miranda, M. [Univ Politecn Valencia, Inst Technol Quim, Dept Quim, Valencia 46022 (Spain); O' Neill, P. [Univ Oxford, Oxford OX3 7DQ (United Kingdom); Robert, M. [Univ Paris 07, CNRS, UMR 7591, Electrochim Mol Lab, F-75251 Paris 05 (France)

    2008-07-01

    Several examples of oxidative and reductive reactions of DNA components that lead to single and tandem modifications are discussed in this review. These include nucleophilic addition reactions of the one-electron oxidation-mediated guanine radical cation and the one-electron reduced intermediate of 8-bromo-purine 2'-de-oxy-ribo-nucleosides that give rise to either an oxidizing guanine radical or related 5',8-cyclo-purine nucleosides. In addition, mechanistic insights into the reductive pathways involved in the photolyase induced reversal of cyclo-buta-cli-pyrimidine and pyrimidine (6-4) pyrimidone photoproducts are provided. Evidence for the occurrence and validation in cellular DNA of (OH){sup {center_dot}} radical degradation pathways of guanine that have been established in model systems has been gained from the accurate measurement of degradation products. Relevant information on biochemical aspects of the repair of single and clustered oxidatively generated damage to DNA has been gained from detailed investigations that rely on the synthesis of suitable modified probes. Thus the preparation of stable carbocyclic derivatives of purine nucleoside containing defined sequence oligonucleotides has allowed detailed crystallographic studies of the recognition step of the base damage by enzymes implicated in the base excision repair (BER) pathway. Detailed insights are provided on the BER processing of non-double strand break bi-stranded clustered damage that may consist of base lesions, a single strand break or abasic sites and represent one of the main deleterious classes of radiation-induced DNA damage. (authors)

  9. The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Baumann, Martin; Petersen, B.O.

    2009-01-01

    -Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1 -> 4)-Glcp (maltose), alpha-Glcp-(1 -> 4)-GlcNp (maltosamine), alpha-Glcp-(1 -> 4)-GlcNAcp (N...

  10. Efficient one-pot enzymatic synthesis of alpha-(1 -> 4)-glucosidic disaccharides through a coupled reaction catalysed by Lactobacillus acidophilus NCFM maltose phosphorylase

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Dilokpimol, Adiphol; Abou Hachem, Maher

    2010-01-01

    -b-glucopyranosyl( 1 -> 4)-o-glucosaminopyranose I maltosaminej, a-a-glucopyranosyl-(1 -> 4)-b-mannopyranose, alpha-n-glucopyranosyl-(1 -> 4)-t-fucopyranose and alpha-b-glucopyranosyl-(1 -> 4)-D-xylopyranose, respectively, from 0 1 M maltose, 0.5 M N-acetyl glucosamine, 0.1 M glucosamine. 0.1 M mannose, 1 M t-fucose...

  11. The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel a-glucosides through reverse phosphorolysis by maltose phosphorylase

    NARCIS (Netherlands)

    Nakai, H.; Baumann, M.J.; Petersen, B.O.; Westphal, Y.; Schols, H.A.; Dilokpimol, A.; Hachem, M.A.; Lathinen, S.J.; Duus, J.O.; Svensson, B.

    2009-01-01

    A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regula

  12. Plastidial [alpha] -glucan phosphorylase 1 complexes with disproportionating enzyme 1 in Ipomoea batatas storage roots for elevating malto-oligosaccharide metabolism

    National Research Council Canada - National Science Library

    Yi-Chen Lin; Shih-Chung Chang; Rong-Huay Juang

    2017-01-01

    .... The immunohistochemistry analyses also revealed the co-localization of Pho1 and Dpe1 in the amyloplasts, and the protein levels of Pho1 and Dpe1 increased gradually throughout sweet potato storage root development...

  13. Powerful methods to establish chromosomal markers in Lactococcus lactis: an analysis of pyrimidine salvage pathway mutants obtained by positive selections

    DEFF Research Database (Denmark)

    Martinussen, Jan; Hammer, Karin

    1995-01-01

    phosphoribosyltransferase (upp), uridindcytidine kinase (udk), pyrimidine nucleoside phosphorylase (pdp), cytidine/deoxycytidine deaminase (dd), thymidine kinase (tdk) and purine nucleoride phosphorylase (pup). Based on an analysis of the mutants obtained, the pathways by which L. lactis metabolizes uracil...

  14. UPP1 — EDRN Public Portal

    Science.gov (United States)

    UPP1, or uridine phosphorylase 1, is a pyrimidine nucleoside phosphorylase. UPP1, in the presence of orthophosphate, catalyzes the reversible phosphorylytic cleavage of uridine and deoxyuridine to uracil and ribose- or deoxyribose-1-phosphate. Pyrimidine nucleoside phosphorylases can add ribose or deoxyribose to pyrimidine bases to form nucleosides that can be incorporated into RNA or DNA.

  15. Assessment of expression of selected Bcl-2 family proteins in lymphoid infiltration in patients with B-cell chronic lymphocytic leukaemia treated with nucleoside analogues.

    Directory of Open Access Journals (Sweden)

    Janusz Kłoczko

    2008-12-01

    Full Text Available B-cell chronic lymphocytic leukaemia (B-CLL is characterized by clonal growth and accumulation of mature lymphoid cells due to disturbance in genetically regulated form of cell death called apoptosis. The intrinsic mechanism of apoptosis is controlled by Bcl-2 family proteins. Purine nucleoside analogues induce the apoptosis in cells in a state of quiescence. The aim of the study was to assess expression of selected Bcl-2 family proteins in neoplastic infiltration in bone marrow in patients with B-CLL treated with nucleoside analogues. The study comprised examination of bone marrow obtained routinely by trephine biopsy from 18 patients with B-CLL diagnosed before administration of purine nucleoside analogues treatment and after its completion. Expression of Bcl-2, Bcl-x and Bax proteins was examined. Lymphoid cells in bone marrow were present in all patients before administration of treatment. After treatment in two patients bone marrow was infiltrated in diffuse pattern, whereas other patients presented nodular pattern of infiltration. The difference between stage of infiltration before and after treatment was statistically significant (p<0.002. High percentage of infiltration cells with positive anti Bcl-2 reaction from 42.0% in one patient to 85.33+/-3.06% in four patients before treatment was observed. After treatment percentage of infiltration cells with positive anti Bcl-2 antibody reaction was from 33.0+/-18.38% in two patients to 99.0% in one patient. Positive correlation between stage of infiltration and expression of Bcl-2 protein was confirmed before and after treatment. Such correlations were not observed in case of Bax and Bcl-x. Strong staining of immunohistochemical reaction of cells in lymphoid infiltration with Bcl-2 antibody was confirmed. There was a difference between Bcl-/Bax ratio before and after treatment. Immunohistochemical assessment of expression of Bcl-2 family proteins in cells of lymphoid infiltration in bone

  16. Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

    Science.gov (United States)

    Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

    2017-07-01

    Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the absence vs. presence of E. Mechanisms underlying translation of E-contingent A2 neuron responses to FD into regulatory signaling remain to be determined. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Property of glucan phosphorylase from a thermophilic Bacterium(Ⅰ)%一种高温细菌葡聚糖磷酸化酶的性质研究(Ⅰ)

    Institute of Scientific and Technical Information of China (English)

    陈世琼

    2009-01-01

    为了研究极端嗜热细菌腾冲嗜热厌氧杆菌葡聚糖磷酸化酶(Tte-GlgP)的性质,将用大肠杆菌BL21DE3表达的Tte-GlgP进行了纯化,分别用SDS-PAGE电泳法及毛细管电泳法测定了分子量及等电点,结果表明,Tte-GlgP的分子量约为64kDa,等电点约为6.2,与推测的结果一致,说明Tte-GlgP在大肠杆菌体内可能得到了正确的折叠;WESTERN杂交试验的结果表明,Ttc-GlgP在腾冲嗜热厌氧杆菌体内有表达,且终浓度为1.5%的麦芽糖能轻度诱导Tte-GlgP在腾冲嗜热厌氧杆菌体内的表达,而终浓度为1.5%的葡萄糖则轻度抑制Tte-GlgP在腾冲嗜热厌氧杆菌体内的表达,说明Tte-GlgP在腾冲菌体内可能参与了碳水化合物的代谢.本研究为进一步认识和应用Tte-GlgP提供了依据.

  18. 蔗糖磷酸化酶的分离纯化及其催化合成α-熊果苷的研究%Purification of Sucrose Phosphorylase and Enzymatic Synthesis of α-arbutin

    Institute of Scientific and Technical Information of China (English)

    侯顾伟; 马江锋; 隋姗姗; 徐冰; 刘嵘明; 姜岷

    2011-01-01

    考察了肠膜明串珠菌(Leuconostoc mesenteroides)G123厌氧发酵产蔗糖磷酸化酶下游的分离纯化工艺.收集的菌体经超声破碎得到粗酶液,通过硫酸铵沉淀、透析、阴离子交换层析分离后获得了电泳纯的蔗糖磷酸化酶,酶活回收率为31.7%,酶的分子量约为55.7 kD,纯化后的蔗糖磷酸化酶比活为115.3 U/mg.该酶在中性及偏酸性(pH5.5-8.0)情况下,酶稳定性较好,较报道的肠膜明串珠菌(Leuconostoc mesenteroides)B-1149的pH稳定范围宽.同时该酶在37℃保存2 h,酶活几乎没有下降.利用获得的纯酶以氢醌和蔗糖为底物催化合成α-熊果苷,在23 U/mL的酶反应体系中,60%蔗糖、5%氢醌、pH7.5,37℃,反应12 h,氢醌转化率达到16.3%,α-熊果苷的产量为20g/L.

  19. Regulation of phosphorylase kinase by low concentrations of Ca ions upon muscle contraction: the connection between metabolism and muscle contraction and the connection between muscle physiology and Ca-dependent signal transduction

    OpenAIRE

    OZAWA, Eijiro

    2011-01-01

    It had long been one of the crucial questions in muscle physiology how glycogenolysis is regulated in connection with muscle contraction, when we found the answer to this question in the last half of the 1960s. By that time, the two principal currents of muscle physiology, namely, the metabolic flow starting from glycogen and the mechanisms of muscle contraction, had already been clarified at the molecular level thanks to our senior researchers. Thus, the final question we had to answer was h...

  20. 产气肠杆菌EAM-Z1尿苷磷酸化酶的分离纯化及性质研究%Purification and Characterization of a Uridine Phosphorylase from Enterobacter aerogenes EAM-Z1

    Institute of Scientific and Technical Information of China (English)

    阮期平; 周长林; 徐旭东; 吴梧桐

    2003-01-01

    从产气肠杆菌(Enterobacter aerogenes)突变株EAM-Z1中分离出一种具有较高转移酶活性的尿苷磷酸化酶(UPase).经测定这种Upase的分子量为12.8×104,亚基分子量为4.3×104,由3个同型亚基组成.N端氨基酸序列为:MRMVDLIATKRDGGE.等电点为4.46.对尿苷的Km为0.29mmol/L.酶反应的最适pH为7.8,最适温度为50℃.该酶能磷酸化尿苷、胸苷、5-氟尿苷、2′-脱氧-5-氟尿苷及尿嘧啶-β-D-阿拉伯呋喃糖,且具有较高的转移酶活性,能将尿苷和5-氟尿嘧啶转化成5-氟尿苷(一种抗癌药物的中间体),其转化率为47%.该酶的这些特性对于酶法合成核苷类抗肿瘤药物和抗病毒药物是十分有用的.

  1. Influence of the nucleobase on the physicochemical characteristics and biological activities of Sb{sup V}-ribonucleoside complexes

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Claudio S.; Demicheli, Cynthia, E-mail: demichel@netuno.lcc.ufmg.b [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Quimica; Rocha, Iara C.M. da; Melo, Maria N. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia; Monte Neto, Rubens L.; Frezard, Frederic [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Fisiologia e Biofisica

    2010-07-01

    The influence of the nucleobase (uracyl, U; cytosine, C; adenine, A; guanine, G) on the physicochemical characteristics and in vitro biological activities of Sb{sup V}-ribonucleoside complexes has been investigated. The 1:1 Sb-U and Sb-C complexes were characterized by NMR and ESI-MS spectroscopies and elemental analysis. The stability constant and the apparent association and dissociation rate constants of 1:1 Sb{sup V}-U, Sb{sup V}-C and Sb{sup V}-A complexes were determined. Although Sb{sup V} most probably binds via oxygen atoms to the same 2' and 3' positions in the different nucleosides, the ribose conformational changes and the physicochemical characteristics of the complex depend on the nucleobase. The nucleobase had a strong influence on the cytotoxicity against macrophages and the antileishmanial activity of the Sb{sup V}-ribonucleoside complexes. The Sb{sup V}-purine complexes were more cytotoxic and more effective against Leishmania chagasi than the Sb{sup V}-pyrimidine complexes, supporting the model that the interaction of Sb{sup V} with purine nucleosides may mediate the antileishmanial activity of pentavalent antimonial drugs. (author)

  2. T-705 (favipiravir) activity against lethal H5N1 influenza A viruses.

    Science.gov (United States)

    Kiso, Maki; Takahashi, Kazumi; Sakai-Tagawa, Yuko; Shinya, Kyoko; Sakabe, Saori; Le, Quynh Mai; Ozawa, Makoto; Furuta, Yousuke; Kawaoka, Yoshihiro

    2010-01-12

    The neuraminidase inhibitors oseltamivir and zanamivi are used to treat H5N1 influenza. However, oseltamivir-resistant H5N1 viruses have been isolated from oseltamivir-treated patients. Moreover, reassortment between H5N1 viruses and oseltamvir-resistant human H1N1 viruses currently circulating could create oseltamivir-resistant H5N1 viruses, rendering the oseltamivir stockpile obsolete. Therefore, there is a need for unique and effective antivirals to combat H5N1 influenza viruses. The investigational drug T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has antiviral activity against seasonal influenza viruses and a mouse-adapted H5N1 influenza virus derived from a benign duck virus. However, its efficacy against highly pathogenic H5N1 viruses, which are substantially more virulent, remains unclear. Here, we demonstrate that T-705 effectively protects mice from lethal infection with oseltamivir-sensitive or -resistant highly pathogenic H5N1 viruses. Furthermore, our biochemical analysis suggests that T-705 ribofuranosyl triphosphate, an active form of T-705, acts like purines or purine nucleosides in human cells and does not inhibit human DNA synthesis. We conclude that T-705 shows promise as a therapeutic agent for the treatment of highly pathogenic H5N1 influenza patients.

  3. Leishmania metacyclogenesis is promoted in the absence of purines.

    Directory of Open Access Journals (Sweden)

    Tiago Donatelli Serafim

    Full Text Available Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

  4. A3 Adenosine Receptors Modulate Hypoxia-inducible Factor-1a Expression in Human A375 Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Stefania Merighi

    2005-10-01

    Full Text Available Hypoxia-inducible factor-1 (HIF-1 is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% 02, adenosine upregulates HIF-1α protein expression in a dose-dependent and timedependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2 protein accumulation through the induction of HIF-1α. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1a and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells.

  5. The Role of Adenosine Signaling in Headache: A Review

    Directory of Open Access Journals (Sweden)

    Nathan T. Fried

    2017-03-01

    Full Text Available Migraine is the third most prevalent disease on the planet, yet our understanding of its mechanisms and pathophysiology is surprisingly incomplete. Recent studies have built upon decades of evidence that adenosine, a purine nucleoside that can act as a neuromodulator, is involved in pain transmission and sensitization. Clinical evidence and rodent studies have suggested that adenosine signaling also plays a critical role in migraine headache. This is further supported by the widespread use of caffeine, an adenosine receptor antagonist, in several headache treatments. In this review, we highlight evidence that supports the involvement of adenosine signaling in different forms of headache, headache triggers, and basic headache physiology. This evidence supports adenosine A2A receptors as a critical adenosine receptor subtype involved in headache pain. Adenosine A2A receptor signaling may contribute to headache via the modulation of intracellular Cyclic adenosine monophosphate (cAMP production or 5' AMP-activated protein kinase (AMPK activity in neurons and glia to affect glutamatergic synaptic transmission within the brainstem. This evidence supports the further study of adenosine signaling in headache and potentially illuminates it as a novel therapeutic target for migraine.

  6. Systematic review of the recent evidence for the efficacy and safety of chlorambucil in the treatment of B-cell malignancies.

    Science.gov (United States)

    Lepretre, Stéphane; Dartigeas, C; Feugier, P; Marty, M; Salles, G

    2016-01-01

    Emergence of new agents has deeply modified treatment options and the role of chlorambucil (CLB) in B-cell malignancies. We conducted a systematic review of prospective, randomized, controlled trials (RCTs) investigating the benefits and harms of CLB used alone or in combination with other treatment in patients suffering from chronic lymphocytic leukemia (CLL), low-grade non-Hodgkin lymphoma (NHL) or Waldenström macroglobulinemia (WM). For CLL, review of the nine RCTs showed that the main advantage of CLB is its low toxicity in comparison with purine nucleoside analogs like fludarabine in either CLL or NHL. In CLL, the major disadvantage is the very low rate of complete response, except when combining an anti-CD20 antibody. For B-cell lymphoma and WM, six RCTs were summarized. Results according to the usual criteria are presented and the role of CLB, used mostly in combination with an anti-CD20 antibody, is discussed for each indication, in particular for unfit patients.

  7. Structure and electronic spectra of purine-methyl viologen charge transfer complexes.

    Science.gov (United States)

    Jalilov, Almaz S; Patwardhan, Sameer; Singh, Arunoday; Simeon, Tomekia; Sarjeant, Amy A; Schatz, George C; Lewis, Frederick D

    2014-01-01

    The structure and properties of the electron donor-acceptor complexes formed between methyl viologen and purine nucleosides and nucleotides in water and the solid state have been investigated using a combination of experimental and theoretical methods. Solution studies were performed using UV-vis and (1)H NMR spectroscopy. Theoretical calculations were performed within the framework of density functional theory (DFT). Energy decomposition analysis indicates that dispersion and induction (charge-transfer) interactions dominate the total binding energy, whereas electrostatic interactions are largely repulsive. The appearance of charge transfer bands in the absorption spectra of the complexes are well-described by time-dependent DFT and are further explained in terms of the redox properties of purine monomers and solvation effects. Crystal structures are reported for complexes of methyl viologen with the purines 2'-deoxyguanosine 3'-monophosphate (DAD'DAD' type) and 7-deazaguanosine (DAD'ADAD' type). Comparison of the structures determined in the solid state and by theoretical methods in solution provides valuable insights into the nature of charge-transfer interactions involving purine bases as electron donors.

  8. The A3 adenosine receptor: history and perspectives.

    Science.gov (United States)

    Borea, Pier Andrea; Varani, Katia; Vincenzi, Fabrizio; Baraldi, Pier Giovanni; Tabrizi, Mojgan Aghazadeh; Merighi, Stefania; Gessi, Stefania

    2015-01-01

    By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein-coupled adenosine receptors (ARs): A(1), A(2)A, A(2)B, and A(3). The A(3) adenosine receptor (A(3)AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A(3)AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A(3) agonists or antagonists would be the best choice. Today, the choice has been made and A(3)AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A(3)AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health.

  9. Homeostatic control of brain function – new approaches to understand epileptogenesis

    Directory of Open Access Journals (Sweden)

    Detlev eBoison

    2013-07-01

    Full Text Available Neuronal excitability of the brain and ongoing homeostasis depends not only on intrinsic neuronal properties but also on external environmental factors; together these determine the functionality of neuronal networks. Homeostatic factors become critically important during epileptogenesis, a process which involves complex disruption of self-regulatory mechanisms. Here we focus on the bioenergetic homeostatic network regulator adenosine, a purine nucleoside whose availability is regulated largely by astrocytes. Endogenous adenosine affects complex network function through adenosine receptor-mediated pathways, through mitochondrial bioenergetics, and through adenosine receptor-independent epigenetic functions. Accumulating evidence from our laboratories shows that disruption of adenosine homeostasis plays a major role in epileptogenesis. Conversely, we have found that reconstruction of adenosine’s homeostatic functions provides new hope for the prevention of epileptogenesis. We will discuss how adenosine-based therapeutic approaches may interfere with epileptogenesis on an epigenetic level, and how dietary interventions can be used to restore network homeostasis in the brain. We conclude that reconstruction of homeostatic functions in the brain offers a new conceptual advance for the treatment of neurological conditions that goes far beyond current target-centric treatment approaches.

  10. Adenosine and adenosine receptors: Newer therapeutic perspective

    Directory of Open Access Journals (Sweden)

    Manjunath S

    2009-01-01

    Full Text Available Adenosine, a purine nucleoside has been described as a ′retaliatory metabolite′ by virtue of its ability to function in an autocrine manner and to modify the activity of a range of cell types, following its extracellular accumulation during cell stress or injury. These effects are largely protective and are triggered by binding of adenosine to any of the four adenosine receptor subtypes namely A1, A2a, A2b, A3, which have been cloned in humans, and are expressed in most of the organs. Each is encoded by a separate gene and has different functions, although overlapping. For instance, both A1 and A2a receptors play a role in regulating myocardial oxygen consumption and coronary blood flow. It is a proven fact that adenosine plays pivotal role in different physiological functions, such as induction of sleep, neuroprotection and protection against oxidative stress. Until now adenosine was used for certain conditions like paroxysmal supraventricular tachycardia (PSVT and Wolff Parkinson White (WPW syndrome. Now there is a growing evidence that adenosine receptors could be promising therapeutic targets in a wide range of conditions including cardiac, pulmonary, immunological and inflammatory disorders. After more than three decades of research in medicinal chemistry, a number of selective agonists and antagonists of adenosine receptors have been discovered and some have been clinically evaluated, although none has yet received regulatory approval. So this review focuses mainly on the newer potential role of adenosine and its receptors in different clinical conditions.

  11. Adenosine receptor modulation: potential implications in veterinary medicine.

    Science.gov (United States)

    Dip, Ramiro G

    2009-01-01

    Adenosine is a purine nucleoside whose concentration increases during inflammation and hypoxia and the many roles of this molecule are becoming better understood. Increased reactivity to adenosine of the airways of asthmatic but not of normal subjects underlines the role of adenosine in airway inflammation. The identification and pharmacological characterisation of different adenosine receptors have stimulated the search for subtype-specific ligands able to modulate the effects of this molecule in a directed way. Several compounds of different chemical classes have been identified as having potential drawbacks, including side effects resulting from the broad distribution of the receptors across the organism, have prevented clinical application. In this article, the effects of adenosine's different receptors and the intracellular signalling pathways are reviewed. The potential of adenosine receptor modulation as a therapeutic target for chronic airway inflammation is considered, taking equine recurrent airway disease and feline asthma as examples of naturally occurring airway obstructive diseases. Other potential applications for adenosine receptor modulation are also discussed. As the intrinsic molecular events of adenosine's mechanism of action become uncovered, new concrete therapeutic approaches will become available for the treatment of various conditions in veterinary medicine.

  12. Renal transepithelial transport of nucleosides.

    Science.gov (United States)

    Nelson, J A; Vidale, E; Enigbokan, M

    1988-01-01

    Previous work from this and other laboratories has suggested that the mammalian kidney has unique mechanisms for handling purine nucleosides. For example, in humans and in mice, adenosine undergoes net renal reabsorption whereas deoxyadenosine is secreted [Kuttesch and Nelson: Cancer Chemother. Pharmacol. 8, 221 (1982)]. The relationships between these renal transport systems and classical renal organic cation and anion, carbohydrate, and cell membrane nucleoside transport carriers are not established. To investigate possible relationships between such carriers, we have tested effects of selected classical transport inhibitors on the renal clearances of adenosine, deoxyadenosine, 5'-deoxy-5-fluorouridine (5'-dFUR), and 5-fluorouracil in mice. The secretion of deoxyadenosine and 5'-dFUR, but not the reabsorption of adenosine or 5-fluorouracil, was prevented by the classical nucleoside transport inhibitors, dipyridamole and nitrobenzylthioinosine. Cimetidine, an inhibitor of the organic cation secretory system, also inhibited the secretion of 5'-dFUR, although it did not inhibit deoxyadenosine secretion in earlier studies [Nelson et al.: Biochem. Pharmacol. 32, 2323 (1983)]. The specific inhibitor of glucose renal reabsorption, phloridzin, failed to inhibit the reabsorption of adenosine or the secretion of deoxyadenosine. Failure of the nucleoside transport inhibitors and phloridzin to prevent adenosine reabsorption suggests that adenosine reabsorption may occur via a unique process. On the other hand, inhibition of the net secretion of deoxyadenosine and 5'-dFUR by dipyridamole and nitrobenzylthioinosine implies a role for the carrier that is sensitive to these compounds in the renal secretion (active transport) of these nucleosides.

  13. Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk

    Directory of Open Access Journals (Sweden)

    Kiyoko Kaneko

    2013-11-01

    Full Text Available In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs, i.e., 421C>A (major and 376C>T (minor, in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

  14. Synthesis of RNAs with up to 100 Nucleotides Containing Site-Specific 2-methylseleno Labels for use in X-ray Crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Hobartner,C.; Rieder, R.; Kreutz, C.; Puffer, B.; Lang, K.; Polonskaia, A.; Serganov, A.; Micura, R.

    2005-01-01

    The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1, 4-dimercapto-2, 3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.

  15. Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Hon-Yeung Cheung

    Full Text Available Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.

  16. The role of adenosine receptor agonist and antagonist on Hippocampal MDMA detrimental effects; a structural and behavioral study.

    Science.gov (United States)

    Kermanian, Fatemeh; Mehdizadeh, Mehdi; Soleimani, Mansureh; Ebrahimzadeh Bideskan, Ali Reza; Asadi-Shekaari, Majid; Kheradmand, Hamed; Haghir, Hossein

    2012-12-01

    There is abundant evidence showing that repeated use of MDMA (3, 4-Methylenedioxymethamphetamine, ecstasy) has been associated with depression, anxiety and deficits in learning and memory, suggesting detrimental effects on hippocampus. Adenosine is an endogenous purine nucleoside that has a neuromodulatory role in the central nervous system. In the present study, we investigated the role of A2a adenosine receptors agonist (CGS) and antagonist (SCH) on the body temperature, learning deficits, and hippocampal cell death induced by MDMA administration. In this study, 63 adult, male, Sprague - Dawley rats were subjected to MDMA (10 and 20 mg/kg) followed by intraperitoneal CGS (0.03 mg/kg) or SCH (0.03 mg/kg) injection. The animals were tested for spatial learning in the Morris water maze (MWM) task performance, accompanied by a recording of body temperature, electron microscopy and stereological study. Our results showed that MDMA treatment increased body temperature significantly, and impaired the ability of rats to locate the hidden platform(P mechanism of these interactions requires further studies.

  17. Cladribine Analogues via O6-(Benzotriazolyl Derivatives of Guanine Nucleosides

    Directory of Open Access Journals (Sweden)

    Sakilam Satishkumar

    2015-10-01

    Full Text Available Cladribine, 2-chloro-2′-deoxyadenosine, is a highly efficacious, clinically used nucleoside for the treatment of hairy cell leukemia. It is also being evaluated against other lymphoid malignancies and has been a molecule of interest for well over half a century. In continuation of our interest in the amide bond-activation in purine nucleosides via the use of (benzotriazol-1yl-oxytris(dimethylaminophosphonium hexafluorophosphate, we have evaluated the use of O6-(benzotriazol-1-yl-2′-deoxyguanosine as a potential precursor to cladribine and its analogues. These compounds, after appropriate deprotection, were assessed for their biological activities, and the data are presented herein. Against hairy cell leukemia (HCL, T-cell lymphoma (TCL and chronic lymphocytic leukemia (CLL, cladribine was the most active against all. The bromo analogue of cladribine showed comparable activity to the ribose analogue of cladribine against HCL, but was more active against TCL and CLL. The bromo ribose analogue of cladribine showed activity, but was the least active among the C6-NH2-containing compounds. Substitution with alkyl groups at the exocyclic amino group appears detrimental to activity, and only the C6 piperidinyl cladribine analogue demonstrated any activity. Against adenocarcinoma MDA-MB-231 cells, cladribine and its ribose analogue were most active.

  18. Waldenstrom macroglobulinemia: prognosis and management.

    Science.gov (United States)

    Oza, A; Rajkumar, S V

    2015-03-27

    Waldenstrom macroglobulinemia (WM) is a B-cell lymphoplasmacytic lymphoma characterized by monoclonal immunoglobulin M protein in the serum and infiltration of bone marrow with lymphoplasmacytic cells. Asymptomatic patients can be observed without therapy. First-line therapy should consist of the monoclonal anti-CD20 antibody, rituximab, given typically in combination with other agents. We prefer dexamethasone, rituximab, cyclophosphamide (DRC) as initial therapy for most patients with symptomatic WM. Other reasonable options are bortezomib, rituximab, dexamethasone (BoRD) or bendamustine plus rituximab (BR). All of these regimens are associated with excellent response and tolerability. Initial therapy is usually administered for 6 months, followed by observation. Response to therapy is assessed using the standard response criteria developed by the International Working Group on Waldenstrom macroglobulinemia. Relapse is almost inevitable in WM but may occur years after initial therapy. In symptomatic patients relapsing more than 1-2 years after initial therapy, the original treatment can be repeated. For relapse occurring sooner, an alternative regimen is used. In select patients, high-dose chemotherapy followed by autologous hematopoietic cell transplantation may be an option at relapse. Options for therapy of relapsed WM besides regimens used in the front-line setting include ibrutinib, purine nucleoside analogs (cladribine, fludarabine), carfilzomib and immunomodulatory agents (thalidomide, lenalidomide).

  19. Phytochemical, Therapeutic, and Ethnopharmacological Overview for a Traditionally Important Herb: Boerhavia diffusa Linn.

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    Shikha Mishra

    2014-01-01

    Full Text Available Boerhavia diffusa (BD is a plant of rasayana category as per ayurvedic claims. It is reported to possess antiaging, disease prevention, and life strengthening activities which hold enormous influence in disease burden and affordability/availability of healthcare in the world. Objective. This paper has been compiled to comment on the studies reported for BD to highlight its chemical and therapeutic potential along with its ethnopharmacological considerations. Methods. In the present paper, a detailed account of chemical constituents and pharmacological activities has been presented. All the findings were correlated with modern pharmacological activities to appraise the value of BD. Results. Chemical analysis of BD gives a wide variety of chemical constituents, namely, rotenoids, flavonoids, xanthones, purine nucleoside, lignans, and steroids. Various ethnopharmacological reports emphasize its role in disorders of reproductive system, gastrointestinal system, respiratory system, urinary system, hepatic system/jaundice, cardiovascular system, and cancer. Conclusions. The studies on the therapeutic activities of BD range from studies on crude extracts to isolated compounds; however some of the studies require sophistication and validated results. BD is a plant of enormous importance in the purview of its chemical and therapeutic properties.

  20. Guanosine: a Neuromodulator with Therapeutic Potential in Brain Disorders

    Science.gov (United States)

    Lanznaster, Débora; Dal-Cim, Tharine; Piermartiri, Tetsadê C. B.; Tasca, Carla I.

    2016-01-01

    Guanosine is a purine nucleoside with important functions in cell metabolism and a protective role in response to degenerative diseases or injury. The past decade has seen major advances in identifying the modulatory role of extracellular action of guanosine in the central nervous system (CNS). Evidence from rodent and cell models show a number of neurotrophic and neuroprotective effects of guanosine preventing deleterious consequences of seizures, spinal cord injury, pain, mood disorders and aging-related diseases, such as ischemia, Parkinson’s and Alzheimer’s diseases. The present review describes the findings of in vivo and in vitro studies and offers an update of guanosine effects in the CNS. We address the protein targets for guanosine action and its interaction with glutamatergic and adenosinergic systems and with calcium-activated potassium channels. We also discuss the intracellular mechanisms modulated by guanosine preventing oxidative damage, mitochondrial dysfunction, inflammatory burden and modulation of glutamate transport. New and exciting avenues for future investigation into the protective effects of guanosine include characterization of a selective guanosine receptor. A better understanding of the neuromodulatory action of guanosine will allow the development of therapeutic approach to brain diseases. PMID:27699087

  1. Toxic effect of acyclovir on testicular tissue in rats.

    Science.gov (United States)

    Movahed, Elham; Nejati, Vahid; Sadrkhanlou, Rajabali; Ahmadi, Abbas

    2013-02-01

    Acyclovir (ACV), a synthetic purine nucleoside analogue, is known to be toxic to gonads. The current study evaluated cytotoxicity of ACV on histopathological changes in testis tissue and serum testosterone and lipid peroxidation concentrations of male rats. Animals were divided into five groups. One group served as control and one group served as control sham. In the drug treated groups ACV administered for 15 days. 18 days after the last injection, animals were sacrificed. Histopathological and histomorphometrical analysis of the testis was carried out. Serum levels of testosterone and Lipid Peroxidation and potential fertility of animals was evaluated. Male rats exposed to ACV had significant reduction in serum testosterone concentrations at 16 and 48mg/kg dose-levels (pACV induced histopathological changes in the testis and also increase the mean number of mast cells in peritubular or interstitial tissue in the testis at at 16 and 48mg/kg dose-levels (pACV caused increase of serum level of Lipid Peroxidation at 48mg/kg dose-level (pACV decreased potential fertility in male rats. The present results highly support the idea that ACV has adverse effect on the reproductive system in male rat.

  2. Potential short-term use of oral cladribine in treatment of relapsing-remitting multiple sclerosis

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    Julie A Murphy

    2010-09-01

    Full Text Available Julie A Murphy, Jacklyn A Harris, Andrew J CrannageSt Louis College of Pharmacy, St Louis, MO, USAAbstract: Multiple sclerosis (MS is a chronic, immune-mediated disorder of the central nervous system. The clinical course of MS varies among patients. Currently, interferon (IFN products, including IFN β-1a administered intramuscularly or subcutaneously and IFN β-1b subcutaneously, glatiramer acetate, natalizumab, and mitoxantrone are approved disease-modifying therapies for the treatment of relapsing-remitting MS. Cladribine, also known as 2-chlorodeoxyadenosine, is a synthetic adenosine deaminase-resistant purine nucleoside analog that preferentially depletes lymphocyte subpopulations. This sustained effect on lymphocytes is advantageous for patients with MS. CLARITY (CLAdRIbine Tablets Treating MS OrallY, a Phase III trial, has demonstrated that short-term oral cladribine decreases relapse rates and risk of disability progression in comparison with placebo. Cladribine was well tolerated in the study, with the most common adverse effects being headache, nausea, upper respiratory tract infections, and lymphocytopenia. An ongoing study is evaluating the efficacy and safety of the combination of oral cladribine and IFN-β products. A further ongoing study is examining the use of oral cladribine in clinically isolated syndrome and time to conversion to MS. Although the results of CLARITY are promising, the exact role of oral cladribine may be better defined with the completion of ongoing studies.Keywords: cladribine, multiple sclerosis, relapsing-remitting, oral, disease-modifying therapy

  3. Separation of effects of adenosine on energy metabolism from those on cyclic AMP in rat thymic lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Nordeen, S.K.; Young, D.A.

    1977-08-10

    In rat thymic lymphocytes incubated for 2 h without exogenous energy-providing substrate, adenosine may be substituted for glucose as a means of maximally restoring energy metabolism and those cellular functions whose rates are sensitive to small changes in the energy balance, such as protein synthesis and uridine utilization for RNA synthesis. Since effects of adenosine in thymocytes and other cells have frequently been attributed to changes in cyclic AMP, this report investigates its possible involvement in these glucose-like restorative actions of adenosine. Although the same range of doses of adenosine effective at raising cyclic AMP also elicit roughly parallel stimulations of protein synthesis and uridine utilization, further results dissociate the restorative actions from those on cyclic AMP. (a) Other purine nucleosides mimic the glucose-like actions of adenosine without increasing cyclic AMP; (b) conversely, prostaglandin E/sub 1/ mimics the cyclic AMP response without restoring energy metabolism or energy-dependent functions; and (c) potentiation of the cyclic AMP response, either by inhibiting phosphodiesterase or adenosine deaminase, does not enhance the restorative response to a range of doses of adenosine. Finally, cyclic AMP-mediated glycogenolysis cannot account for the glucose-like effects since addition of adenosine increases, not decreases, levels of glycogen.

  4. The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells.

    Science.gov (United States)

    Wilson, Jeffrey M; Ross, William G; Agbai, Oma N; Frazier, Renea; Figler, Robert A; Rieger, Jayson; Linden, Joel; Ernst, Peter B

    2009-04-15

    The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4(+) T cell responses. While adenosine clearly has direct effects on CD4(+) T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5'-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-alpha and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4(+) T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A(2A) adenosine receptor (A(2A)AR) and A(2B)AR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A(2A)AR(-/-) and A(2B)AR(-/-)mice, it was shown that NECA modulates TNF-alpha, IL-12, IL-10, and CD86 responses predominantly via A(2B)AR. These data indicate that engagement of A(2B)AR modifies murine BMDC maturation and suggest that adenosine regulates CD4(+) T cell responses by selecting for DC with impaired immunogencity.

  5. Ribavirin shows immunomodulatory effects on activated microglia.

    Science.gov (United States)

    Savic, Danijela; Stojiljkovic, Mirjana; Lavrnja, Irena; Parabucki, Ana; Bjelobaba, Ivana; Nedeljkovic, Nadezda; Herdegen, Thomas; Pekovic, Sanja

    2014-12-01

    Abstract Ribavirin (RBV) is synthetic purine nucleoside analogue, licensed as anti-viral drug that displays immunomodulatory actions on various immune cells. Our previous ex vivo studies have demonstrated immunosuppressive effects of RBV on reactive T-lymphocytes in experimental autoimmune encephalomyelitis. Here, we examined the effects of RBV on inflammatory response of microglia. RBV potency to down-regulate microglia inflammatory response was assessed by measuring microglia cell body size, and the production of nitric oxide (NO) and pro- and anti-inflammatory cytokines. RBV exerted cytotoxic effects on LPS-stimulated microglia, leaving non-stimulated microglia unaffected. The exposure of activated microglia to RBV led to: decrease in the level of NO as a result of decreased cell number, lower average cell surface, the reduction of membrane ruffling, the suppression of interleukin-6 release and promoted interleukin-10 production. On the other hand, RBV promoted LPS-induced interleukin-1 beta release. Our results imply that RBV is a complex immunomodulator showing both anti- and pro-inflammatory effects on activated microglia.

  6. Protein (Viridiplantae): 225460885 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available 0:4156 PREDICTED: GDP-L-galactose phosphorylase 1 Vitis vinifera MNTLRIKRVPTVVSNYQKEDSDDGARQVGGCGRNCLKQCCIQG...PTEFRVDKVLQPFDGNKFNFTKVGQEEVLFQFEPSNDEEPEFIPNAPIDVENSTSVVAINVSPIEYGHVLLIPRIFECLPQRIDRESFLLALDMAVEAGNPYFRLGYN...SLGAFATINHLHFQAYYLATPFPIEKAPTRKITTAGNGVKIFELLKYPVRGLVFEGGDTLQDLANTVADSCICLQDNNIPFNVLIADAGKRIFLFAQCYAEKQALGEV...NQELLDTQVNPAVWEVSGHIVLKRKEDYEGASEQNAWRLLAEVSLSEERFQEVNALIFEAIACGDDEKGNLTEDMIEEPDVTPPSHEDAGAINNSSYPAAMVAGKQECLVQQ ...

  7. NCBI nr-aa BLAST: CBRC-PTRO-18-0038 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PTRO-18-0038 ref|ZP_01507184.1| glycogen/starch/alpha-glucan phosphorylases [Burkholderia phytofirm...ans PsJN] gb|EAV08329.1| glycogen/starch/alpha-glucan phosphorylases [Burkholderia phytofirmans PsJN] ZP_01507184.1 4.8 26% ...

  8. Glycogen accumulation and degradation by the trichomonads Trichomonas vaginalis and Trichomonas tenax.

    Science.gov (United States)

    Nielsen, Tyler J; Pradhan, Prajakta; Brittingham, Andrew; Wilson, Wayne A

    2012-01-01

    Several species of trichomonad have been shown to accumulate significant quantities of glycogen during growth, suggesting an important role for this compound in cell physiology. We provide the first analysis of the changes in glycogen content and glycogen phosphorylase activity that occur during in vitro growth of two trichomonad species: Trichomonas vaginalis and Trichomonas tenax. Both species accumulated glycogen following inoculation into fresh medium and utilized this compound during logarithmic growth. Glycogen phosphorylase activity also varied during growth in a species-specific manner. The expression of phosphorylase genes in T. vaginalis remained constant during growth and thus transcriptional control did not explain the observed fluctuations in phosphorylase activity. After cloning, expression, and purification, two recombinant glycogen phosphorylases from T. vaginalis and one recombinant glycogen phosphorylase from T. tenax had robust activity and, in contrast to many other eukaryotic glycogen phosphorylases, did not appear to be regulated by reversible protein phosphorylation. Furthermore, allosteric regulation, if present, was not mediated by compounds known to impact the activity of better characterized phosphorylases. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

  9. Drug: D04537 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available litus [glycogen phosphorylase inhibitor] glycogen phosphorylase inhibitor [HSA:5834...D04537 Drug Ingliforib (USAN/INN) C23H24ClN3O5 457.1404 457.9068 D04537.gif Treatment of type 2 diabetes mel

  10. Constituents from fruits of Cupressus sempervirens.

    Science.gov (United States)

    Rawat, Preeti; Khan, Mohammad F; Kumar, Manmeet; Tamarkar, Akhilesh K; Srivastava, Arvind K; Arya, Kamal R; Maurya, Rakesh

    2010-04-01

    Two new phenolic glycosides (1, 2), along with fourteen known compounds (3-16) have been isolated from the fruit of Cupressus sempervirens. The structures of these compounds were determined by spectroscopic analysis and were evaluated for their inhibitory activity against glycogen phosphorylase and glucose-6-phosphatase enzymes. Compounds 14 showed a moderate inhibition against glucose-6-phosphatase and 15 against glycogen phosphorylase enzymes.

  11. Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Teruya; Kitaguchi, Yuki; Miyazawa, Masayuki [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan); Kamiya, Hiroyuki [Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812 (Japan); Toma, Sachiko; Ikemizu, Shinji [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan); Shirakawa, Masahiro [Graduate School of Engineering, Kyoto University, Kyoto 615-8510 (Japan); Nakabeppu, Yusaku [Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 (Japan); Yamagata, Yuriko, E-mail: yamagata@gpo.kumamoto-u.ac.jp [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan)

    2006-12-01

    The complexes of human MTH1 with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP, were crystallized. The crystals diffracted to 1.95 and 2.22 Å resolution, respectively. Human MutT homologue 1 (hMTH1) hydrolyzes a variety of oxidized purine nucleoside triphosphates, including 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP and 8-oxo-dATP, to their corresponding nucleoside monophosphates, while Esherichia coli MutT possesses prominent substrate specificity for 8-oxoguanine nucleotides. Three types of crystals were obtained corresponding to the following complexes: selenomethionine-labelled hMTH1 with 8-oxo-dGMP (SeMet hMTH1–8-oxo-dGMP), hMTH1 with 8-oxo-dGMP (hMTH1–8-oxo-dGMP) and hMTH1 with 2-oxo-dATP (hMTH1–2-oxo-dATP). Crystals of the SeMet hMTH1–8-oxo-dGMP complex belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 45.8, c = 153.6 Å, and diffracted to 2.90 Å. Crystals of hMTH1–8-oxo-dGMP and hMTH1–2-oxo-dATP belong to space groups P2{sub 1} and P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 34.0, b = 59.0, c = 65.9 Å, β = 90.7° and a = 59.2, b = 67.3, c = 80.0 Å, respectively. Their diffraction data were collected at resolutions of 1.95 and 2.22 Å, respectively.

  12. XANTHINE OXYDASE INHIBITION OF KOMBUCHA TEA IN HYPERURICEMIA INDUCED WISTAR RAT: decrease of uric acid, malondialdehyde, and 8-hydroxy-2'-deoxyguanosine

    Directory of Open Access Journals (Sweden)

    I D. M. Sukrama

    2015-04-01

    Full Text Available Background: Hyperuricemia is a condition of high level of uric acid in the body due to distortion of purine nucleoside metabolism through hipoxanthin, xanthin, and guanin of basic purine. Objective: to find a cure of hyperuricemia base on the utilization of kombucha tea. Methods: This is a true experimental study by applying posttest only control group design to determine whether kombucha tea inhibit xanthine oxidase in hyperuricemic induced rat reveales by decrease of uric acid, malondialdehyde (MDA, and 8-hydroxy-2’-deoxyguanosine (8-OHdG. In this study, hyperuricemia rat was achieved by intake of high purine diet. Rats were fed with a mixture of 4 g/kg BW of Gnetum gnemon with 50 mL/kg BW of chicken liver ad libitum for 9 days. Treatments in this research are combination of fermentation time of Kombucha tea and volume of this tea, i.e fermentation time 4, 8, and 12 days and the volume are 1 mL and 4 mL. Therefore, there would be seven groups of treatment including control group. ANOVA was then applied to determine the treatment effect with p < 0.05 was concidered significant. Results: This study indicates that kombucha tea has an ability to inhibit xanthine oxidase in hyperuricemic induced rat and decrease uric acid, MDA, and 8-OHdG. This ability was achieved with combination treatment of 12 days fermentation and 4 mL of kombucha intake. Xanthine oxidase, uric acid, MDA, and 8-OHdG levels by this treatment were obtained significantly lower compare to control group. Conclusion: This study proved that kombucha tea was potent to cure hyperuricemia of wistar rat via inhibition of xanthine oxidase produced.

  13. The carboxy-terminal domain of Dictyostelium C-module-binding factor is an independent gene regulatory entity.

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    Jörg Lucas

    Full Text Available The C-module-binding factor (CbfA is a multidomain protein that belongs to the family of jumonji-type (JmjC transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD. An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

  14. Inosine Improves Neurogenic Detrusor Overactivity following Spinal Cord Injury.

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    Yeun Goo Chung

    Full Text Available Neurogenic detrusor overactivity and the associated loss of bladder control are among the most challenging complications of spinal cord injury (SCI. Anticholinergic agents are the mainstay for medical treatment of detrusor overactivity. However, their use is limited by significant side effects such that a search for new treatments is warranted. Inosine is a naturally occurring purine nucleoside with neuroprotective, neurotrophic and antioxidant effects that is known to improve motor function in preclinical models of SCI. However, its effect on lower urinary tract function has not been determined. The objectives of this study were to determine the effect of systemic administration of inosine on voiding function following SCI and to delineate potential mechanisms of action. Sprague-Dawley rats underwent complete spinal cord transection, or cord compression by application of an aneurysm clip at T8 for 30 sec. Inosine (225 mg/kg or vehicle was administered daily via intraperitoneal injection either immediately after injury or after a delay of 8 wk. At the end of treatment, voiding behavior was assessed by cystometry. Levels of synaptophysin (SYP, neurofilament 200 (NF200 and TRPV1 in bladder tissues were measured by immunofluorescence imaging. Inosine administration decreased overactivity in both SCI models, with a significant decrease in the frequency of spontaneous non-voiding contractions during filling, compared to vehicle-treated SCI rats (p<0.05, including under conditions of delayed treatment. Immunofluorescence staining demonstrated increased levels of the pan-neuronal marker SYP and the Adelta fiber marker NF200, but decreased staining for the C-fiber marker, TRPV1 in bladder tissues from inosine-treated rats compared to those from vehicle-treated animals, including after delayed treatment. These findings demonstrate that inosine prevents the development of detrusor overactivity and attenuates existing overactivity following SCI, and may

  15. A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.

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    Gabriele De Luca

    2008-11-01

    Full Text Available Several human neurodegenerative disorders are characterized by the accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG in the DNA of affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation of the oxidized nucleotide during replication. Hydrolases that degrade oxidized purine nucleoside triphosphates normally minimize this incorporation. hMTH1 is the major human hydrolase. It degrades both 8-oxodGTP and 8-oxoGTP to the corresponding monophosphates. To investigate whether the incorporation of oxidized nucleic acid precursors contributes to neurodegeneration, we constructed a transgenic mouse in which the human hMTH1 8-oxodGTPase is expressed. hMTH1 expression protected embryonic fibroblasts and mouse tissues against the effects of oxidants. Wild-type mice exposed to 3-nitropropionic acid develop neuropathological and behavioural symptoms that resemble those of Huntington's disease. hMTH1 transgene expression conferred a dramatic protection against these Huntington's disease-like symptoms, including weight loss, dystonia and gait abnormalities, striatal degeneration, and death. In a complementary approach, an in vitro genetic model for Huntington's disease was also used. hMTH1 expression protected progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene from toxicity associated with expression of the mutant huntingtin. The findings implicate oxidized nucleic acid precursors in the neuropathological features of Huntington's disease and identify the utilization of oxidized nucleoside triphosphates by striatal cells as a significant contributor to the pathogenesis of this disorder.

  16. Determination of genes and microRNAs involved in the resistance to fludarabine in vivo in chronic lymphocytic leukemia

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    Muller Arnaud

    2010-05-01

    Full Text Available Abstract Background Chronic lymphocytic leukemia (CLL cells are often affected by genomic aberrations targeting key regulatory genes. Although fludarabine is the standard first line therapy to treat CLL, only few data are available about the resistance of B cells to this purine nucleoside analog in vivo. Here we sought to increase our understanding of fludarabine action and describe the mechanisms leading to resistance in vivo. We performed an analysis of genomic aberrations, gene expression profiles, and microRNAs expression in CLL blood B lymphocytes isolated during the course of patients' treatment with fludarabine. Results In sensitive patients, the differentially expressed genes we identified were mainly involved in p53 signaling, DNA damage response, cell cycle and cell death. In resistant patients, uncommon genomic abnormalities were observed and the resistance toward fludarabine could be characterized based on the expression profiles of genes implicated in lymphocyte proliferation, DNA repair, and cell growth and survival. Of particular interest in some patients was the amplification of MYC (8q observed both at the gene and transcript levels, together with alterations of myc-transcriptional targets, including genes and miRNAs involved in the regulation of cell cycle and proliferation. Differential expression of the sulfatase SULF2 and of miR-29a, -181a, and -221 was also observed between resistant and sensitive patients before treatment. These observations were further confirmed on a validation cohort of CLL patients treated with fludarabine in vitro. Conclusion In the present study we identified genes and miRNAs that may predict clinical resistance of CLL to fludarabine, and describe an interesting oncogenic mechanism in CLL patients resistant to fludarabine by which the complete MYC-specific regulatory network was altered (DNA and RNA levels, and transcriptional targets. These results should prove useful for understanding and

  17. Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.

    Science.gov (United States)

    Worch, Remigiusz; Niedzwiecka, Anna; Stepinski, Janusz; Mazza, Catherine; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Cusack, Stephen; Stolarski, Ryszard

    2005-09-01

    The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

  18. Synthesis and Cellular Uptake of Radioiodine labeled 2{sup '}-deoxyuridine derivatives with HSV1-TK

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Ah; Lee, Kyo Chul; Hong, Su Hee; Kim, Eun Jung; Lee, Jong Chan; An, Gwang Il; Choi, Tae Hyun; Cheon, Gi Jeong; Chun, Kwon Soo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    Several different radiolabeled probes have been developed to image Herpes Simplex Virus Type-1 thyrimidine kinase gene (HSV1-TK) expression. The nucleoside prodrugs under investigation for HSV1-TK imaging generally fall into two main chemical and radioisotope categories: the pyrimidine nucleosides, primarily radioiodinated, and the purin nucleosides, primarily radiofluorinated, and their respective analogues. In non-invasive imaging of the HSV1-TK system, many nucleoside derivatives have been recommended as HSV1-TK substrates. Most of these nucleoside derivatives have been developed as prodrugs for tumor proliferation imaging or as anti-viral drugs. For example, 5-fluorouracil (5-FU) and IUdR have been used as tumor agents and acyclovir (ACV), ganciclovir (GCV) and (E)-5-(2-bromovinyl)-2{sup '}- deoxyuridine (BVDU) as an anti-viral agents for virus infection several 5-substituted uracil nucleoside derivatives have been identified to have high sensitivity and selective accumulation in HSV1-TK expressing cells. Of those, IVDU was shown to be rapidly accumulated in HSV1- TK expressing cells in vitro. Imaging of the HSV1-TK reporter gene along with various reporter probes is of current interest. In contrast to the mammalian kinase, which phosphorylates thymidine preferentially, HSV1-TK is less discriminative and phosphorylates a wide range of nucleoside analogues such as acycloguanosines and 2{sup '}-fluoro-2{sup '}-deoxyuridine derivatives that are not phosphorylated efficiently by the native enzyme. More specifically, 5-substituted 2{sup '}-fluoro-2{sup '}-deoxyarabinofuranosyluracil nucleosides are efficiently phosphorylated by HSV- TK. This property, together with the presence of fluorine in the 2{sup '}-arabino-position, endows the 2{sup '}-fluoro-2{sup '}-deoxyuridines with antiviral activity against HSV.

  19. Blood flows and metabolic components of the cardiome.

    Science.gov (United States)

    Bassingthwaighte, J B; Li, Z; Qian, H

    1998-01-01

    This is a plan for the first stage of The Cardiome Project. The cardiome is the representation, in quantitative, testable form, of the functioning of the normal heart and its responses to intervention. The goal is to integrate the efforts of many years into a comprehensive understandable scheme. Past efforts have spanned the fields of transport within blood vessels, the distributions of regional coronary blood flows, permeation processes through capillary and cell walls, mediated cell membrane transport, extra- and intracellular diffusion, cardiac electrophysiology, the uptake and metabolism of the prime substrates (fatty acid and glucose), the metabolism of the purine nucleosides and nucleotides (mainly adenosine and ATP), the regulation of the ionic currents and of excitation-contraction coupling and finally the regulation of contraction. The central theme is to define the coronary flows and metabolic components of a computer model that will become a part of a three-dimensional heart with appropriate fibre shortening and volume ejection. The components are: (a) coronary flow distributions with appropriate heterogeneity, (b) metabolism of the substrates for energy production, (c) ATP, PCr and energy metabolism and (d) calcium metabolism as it relates to excitation-contraction coupling. The modeling should provide: (1) appropriate responses to regional ischemia induced by constriction of a coronary artery, including tissue contractility loss and aneurysmal dilation of the ischemic region; (2) physiological responses to rate changes such as treppe and changes in metabolic demand and (3) changes in local metabolic needs secondary to changes in the site of pacing stimulation and shortening inactivation or stretch activation of contraction.

  20. Oxidative DNA damage is involved in cigarette smoke-induced lung injury in rats.

    Science.gov (United States)

    Chen, Zhihai; Wang, Dapeng; Liu, Xing; Pei, Weiwei; Li, Jianxiang; Cao, Yi; Zhang, Jie; An, Yan; Nie, Jihua; Tong, Jian

    2015-09-01

    Reactive oxygen species (ROS) induced by exogenous toxicants are suggested to be involved in carcinogenesis by oxidative modification of DNA. 8-Hydroxyl-2-deoxyguanosine (8-OHdG) has been considered as a reliable biomarker for oxidative DNA damage both in vivo and in vitro studies. But the effect of smoking on oxidative damage has not yet been fully elucidated. Wistar rats were exposed to cigarette smoke at concentrations of 20 and 60 % for 30 min, twice/day for 45 weeks. Then the histopathology of lung tissues, levels of ROS, 8-OHdG, and total antioxidant (T-AOC), expression of DNA repair enzymes, e.g. 8-oxyguaine DNA glycosylase (OGG1), and MutThomolog 1 (Oxidized Purine Nucleoside Triphosphatase, MTH1) were determined in urine, peripheral blood lymphocytes, and lung tissue. The results showed that long-term cigarette smoke exposure can cause obvious damages of lung tissue in rats. In addition, a significant and cigarette smoke concentration-dependent increase in ROS and 8-OHdG were observed compared with the non-exposed control rats. In contrast, the expression of OGG1 and MTH1, and T-AOC levels were obviously decreased after long-term exposure to cigarette smoke. These findings indicate that long-term exposure to cigarette smoker increases ROS levels, decreases total antioxidant capacity, and interferes DNA repair capacity that eventually induces oxidative DNA damage, which appears to play an important role in cigarette smoke-induced lung injury in rats, and determination of 8-OHdG levels might be a useful method for monitoring oxidative damage in cigarette smokers.

  1. Leishmania infantum Parasites Subvert the Host Inflammatory Response through the Adenosine A2A Receptor to Promote the Establishment of Infection

    Directory of Open Access Journals (Sweden)

    Mikhael H. F. Lima

    2017-07-01

    Full Text Available Adenosine is an endogenously released purine nucleoside that signals through four widely expressed G protein-coupled receptors: A1, A2A, A2B, and A3. Of these, A2AR is recognized as mediating major adenosine anti-inflammatory activity. During cutaneous leishmaniasis, adenosine induces immunosuppression, which promotes the establishment of infection. Herein, we demonstrated that A2AR signaling is exploited by Leishmania infantum parasites, the etiologic agent that causes Visceral Leishmaniasis, to successfully colonize the vertebrate host. A2AR gene-deleted mice exhibited a well-developed cellular reaction with a strong Th1 immune response in the parasitized organs. An intense infiltration of activated neutrophils into the disease-target organs was observed in A2AR−/− mice. These cells were characterized by high expression of CXCR2 and CD69 on their cell surfaces and increased cxcl1 expression. Interestingly, this phenotype was mediated by IFN-γ on the basis that a neutralizing antibody specific to this cytokine prevented neutrophilic influx into parasitized organs. In evaluating the immunosuppressive effects, we identified a decreased number of CD4+ FOXP3+ T cells and reduced il10 expression in A2AR−/− infected mice. During ex vivo cell culture, A2AR−/− splenocytes produced smaller amounts of IL-10. In conclusion, we demonstrated that the A2AR signaling pathway is detrimental to development of Th1-type adaptive immunity and that this pathway could be associated with the regulatory process. In particular, it promotes parasite surveillance.

  2. Stabilization of RNA stacking by pseudouridine.

    Science.gov (United States)

    Davis, D R

    1995-01-01

    The effect of the modified nucleoside pseudouridine (psi) on RNA structure was compared with uridine. The extent of base stacking in model RNA oligonucleotides was measured by 1H NMR, UV, and CD spectroscopy. The UV and CD results indicate that the model single-stranded oligoribonucleotides AAUA and AA psi A form stacked structures in solution and the CD results for AA psi A are consistent with a general A-form helical conformation. The AA psi A oligomer exhibits a greater degree of UV hypochromicity over the temperature range 5-55 degrees C, consistent with a better stacked, more A-form structure compared with AAUA. The extent of stacking for each nucleotide residue was inferred from the percent 3'-endo sugar conformation as indicated by the H1'-H2' NMR scalar coupling. This indirect indication of stacking was confirmed by sequential NOE experiments. NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides. The N1-H imino proton in AA psi A exchanges slowly with solvent, suggesting a role for the extra imino proton in stabilizing the conformation of pseudouridine. These results show that the conformational stabilization is an intrinsic property of pseudouridine occurring at the nucleotide level. The characteristics of pseudouridine in these models are consistent with earlier studies on intact rRNA, indicating that pseudouridine probably performs the same stabilizing function in most structural contexts. PMID:8559660

  3. Allogeneic haematopoietic stem cell transplantation for mitochondrial neurogastrointestinal encephalomyopathy

    NARCIS (Netherlands)

    Halter, Joerg P.; Schuepbach, W. Michael M.; Mandel, Hanna; Casali, Carlo; Orchard, Kim; Collin, Matthew; Valcarcel, David; Rovelli, Attilio; Filosto, Massimiliano; Dotti, Maria T.; Marotta, Giuseppe; Pintos, Guillem; Barba, Pere; Accarino, Anna; Ferra, Christelle; Illa, Isabel; Beguin, Yves; Bakker, Jaap A.; Boelens, Jaap J.; de Coo, Irenaeus F. M.; Fay, Keith; Sue, Carolyn M.; Nachbaur, David; Zoller, Heinz; Sobreira, Claudia; Simoes, Belinda Pinto; Hammans, Simon R.; Savage, David; Marti, Ramon; Chinnery, Patrick F.; Elhasid, Ronit; Gratwohl, Alois; Hirano, Michio

    2015-01-01

    Haematopoietic stem cell transplantation has been proposed as treatment for mitochondrial neurogastrointestinal encephalomyopathy, a rare fatal autosomal recessive disease due to TYMP mutations that result in thymidine phosphorylase deficiency. We conducted a retrospective analysis of all known pati

  4. Thymidine phosphorylase expressed in monocyte-macrophages enhanced anticancer effect of 5'-deoxy-5-fluorouridine on colorectal carcinoma cells%单核-巨噬细胞表达的胸苷磷酸化酶增强去氧氟尿苷抗结肠癌细胞活性

    Institute of Scientific and Technical Information of China (English)

    张继民; 刘明姬; 溝井賢幸; 椎葉健一; 佐佐木巌; 松野正纪

    2004-01-01

    目的探讨结肠癌组织中胸苷磷酸化酶(dThdPase)的表达,检定单核-巨噬细胞表达的dThdPase对抗癌药物5′-脱氧氟尿苷(5'-DFUR)的转化调控作用.方法对40例结肠癌标本进行免疫组织化学染色,分析其细胞的dThdPas表达,并测定癌组织和周围正常组织的dThdPase活性.应用ELISA法分别检测4株结肠癌细胞LS174T、Clone A、Colo320、MIP101和2株类巨噬细胞THP-1、U937的dThdPase蛋白含量.MTT法测定4株结肠癌细胞对5-Fu和5′-DFUR的半数有效浓度(IC50).把定量5′-DFUR加入培养基中同类巨噬细胞或单核细胞一起培养24 h后,取其上清液二倍稀释加入大肠癌细胞中测定IC50有无改变.并在加入定量5′-DFUR的类巨噬细胞培养液中检测5-Fu的生成.结果结肠癌组织中dThdPase活性明显高于周围正常肠组织,表达dThdPase阳性细胞主要是肿瘤相关巨噬细胞(TAM).4株结肠癌细胞中仅LS174T检出0.5单位/mg的dThdPase蛋白;而THP-1和U937则分别为18.2单位/mg和19.3单位/mg.全部大肠癌细胞对5′-DFUR的IC50均明显高于5-Fu(P<0.0001).但同THP-1、U937或单核细胞一起培养24 h后,其IC50明显下降,仅相当于原来的5.4%~41.8%(P< 0.001).从含定量5′-DFUR的THP-1、U937的培养液中分别检出40.2 μmol/L和29.5 μmol/L的5-Fu.结论结肠癌组织中主要由TAM表达dThdPase活性.在体外结肠癌细胞因缺乏dThdPase活性,对5′-DFUR缺乏敏感性,5′-DFUR在单核-巨噬细胞表达的dThdPase催化下,可转化成5-Fu并释放到培养基中发挥抗癌作用.

  5. Transcription and activity of 5-fluorouracil converting enzymes in fluoropyrimidine resistance in colon cancer in vitro.

    Science.gov (United States)

    Mader, R M; Sieder, A E; Braun, J; Rizovski, B; Kalipciyan, M; Mueller, M W; Jakesz, R; Rainer, H; Steger, G G

    1997-12-01

    Cellular resistance to 5-fluorouracil (5-FU) is not completely understood. Since 5-FU shares the pyrimidine pathway with the physiological pyrimidines, we investigated the relationship between fluoropyrimidine metabolism, nucleic acid uptake and cytotoxicity of 5-FU in eight colon tumour cell lines including 5-FU-resistant subclones. The cytotoxicity of 5-FU was increased up to 423-fold when the anabolites 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5-fluorodeoxyuridine monophosphate (FdUMP) were compared with the parent drug in vitro. The enzymes uridine phosphorylase and thymidine phosphorylase were predictive for the cytotoxicity of 5-FU in 5/7 cell lines. Inhibition of uridine phosphorylase and thymidine phosphorylase by antisense strategies effectively antagonised 5-FU, abolishing 84% and 79% of its toxicity. The importance of thymidine phosphorylase was supported by a highly restricted enzyme activity in 5-FU-resistant cells. In 5-FU naive cells, a stimulating effect of 5-FU on thymidylate synthase mRNA and ribonucleotide reductase mRNA expression was observed. In these cells, antisense oligonucleotides to ribonucleotide reductase significantly reduced cell growth. Downregulation of ribonucleotide reductase mRNA in 5-FU-resistant subclones suggests different mechanisms in primary and secondary resistance to 5-FU. Most of the intracellular 5-FU was selectively incorporated into RNA (range: 45-91%) and generally spared DNA (range: 0.2-11%). In synthesising our data, we conclude that drug resistance could be overwhelmed through bypassing limiting steps in the activation of 5-FU. In the majority of colonic tumours, the activity of uridine phosphorylase and thymidine phosphorylase may have prognostic relevance for the cytotoxicity of 5-FU in vitro.

  6. Long-Term Survival on Capecitabine in Two Gemcitabine Refractory Pancreatic Cancer Patients. Is there a Pharmacogenetic Explanation?

    Directory of Open Access Journals (Sweden)

    Muhammad Wasif Saif

    2007-11-01

    Full Text Available Context Capecitabine has shown efficacy in treatment of metastatic pancreatic cancer. Several researchers have identified thymidine phosphorylase, dihydropyrimidine dehydrogenase, or their ratio as indicators of response to capecitabine in various cancers. Case report We report two patients with metastatic pancreatic carcinoma who had long-term survivals on capecitabine after gemcitabine failure. These two cases prompted us to measure thymidine phosphorylase and dihydropyrimidine dehydrogenase levels to facilitate discourses regarding their relationship with efficacy of capecitabine. We also describe a novel method of measuring thymidine phosphorylase level from serum without an invasive tissue biopsy. One patient is alive as of today, with improved performance status, 50 months after capecitabine was started. CA 19-9 and CT scans remained stable during 57 cycles. Her thymidine phosphorylase level was 1.77 compared to a control level of 1.00. Dihydropyrimidine dehydrogenase level was 4.14 compared to a control level of 1.00. Their ratio was 0.43. The other patient was alive on capecitabine for 24 months. His performance status, bilirubin, AST, and ALT improved on capecitabine. CT scans and CA 19-9 remained stable during this period. He had thymidine phosphorylase level of 5.56, dihydropyrimidine dehydrogenase level of 2.74, and their ratio of 2.03. Conclusion Capecitabine resulted in long term survivals in two patients with metastatic pancreatic cancer after gemcitabine failure. The use of capecitabine as second-line treatment in metastatic pancreatic cancer should be further explored along with the role of thymidine phosphorylase and dihydropyrimidine dehydrogenase levels in its activity. A non-invasive method of thymidine phosphorylase measurement we described should be validated in larger trials.

  7. Treatment of experimental colitis in mice with LMP-420, an inhibitor of TNF transcription

    Directory of Open Access Journals (Sweden)

    Cianciolo George

    2008-03-01

    Full Text Available Abstract Background LMP-420 is a boronic acid-containing purine nucleoside analogue that transcriptionally inhibits TNF production but is non-cytotoxic to TNF-producing cells. Methods This study investigated the efficacy of LMP-420 as an anti-inflammatory agent in acute and chronic colitis induced by oral administration of dextran sulfate sodium (DSS to mice and in chronic colitis following piroxicam administration to IL-10-deficient mice. The severity of colon inflammation was assessed histologically. TNF levels were measured by enzyme immunoassay. Results Administration of DSS for 7 days resulted in severe acute colitis that was associated with a marked increase in stool and colon tissue TNF levels. Initiation of therapy with intraperitoneal (i.p. LMP-420 on day 4 of DSS exposure decreased colonic TNF to near normal levels on day 7. However, neither i.p. nor oral treatment with LMP-420 affected the development or severity of acute DSS colitis. Initiation of LMP-420 therapy after 3 cycles of DSS administration to establish chronic colitis also had no effect on the severity of chronic colitis. Analysis of colonic TNF combined with longitudinal analysis of TNF and TNF receptor (TNF-RII levels in stool during the development of chronic DSS colitis demonstrated that the initially elevated colonic TNF levels returned to normal despite intense on-going inflammation in mice with chronic colitis. RAG-2-/- mice deficient in T and B cells also developed severe ongoing colitis in response to 3 cycles of DSS, but showed marked differences vs. wild type mice in stool TNF and TNF-RII in response to DSS exposure. Systemic and oral LMP-420 treatment for 16 days decreased colonic TNF levels in IL-10-deficient mice with chronic colitis, with a trend to decreased histologic inflammation for oral LMP-420. Conclusion These studies demonstrate that short-term treatment with a transcriptional inhibitor of TNF production can decrease systemic and local colonic levels

  8. Cytotoxic Evaluation of Melia Azedarach in Comparison with, Azadirachta Indica and its Phytochemical Investigation

    Directory of Open Access Journals (Sweden)

    Samineh Jafari

    2013-05-01

    Full Text Available Melia azedarach L. is an important medicinal plant that is used for variety of ailments in Iranian traditional medicine. Azadirachta indica A. Juss is its allied species and possesses similar properties and effects. The present study was undertaken to investigate anticancer activity of these M. azedarach in comparison with A. indica on cancer cell lines and also to evaluate their safety in humans by testing them on normal cell line. The study also aimed to determine the active components that are responsible for medicinal effects of M. azedarach in traditional usages.Methods:In this study, the cytotoxic activity of crude extracts from M. azedarach and A. indica leaves, pulps and seeds as well as three main fractions of their leaf extracts were assayed against HT-29, A-549, MCF-7 and HepG-2 and MDBK cell lines. MTT assay was used to evaluate their cytotoxic activities. Methanol leaf fraction of M. azedarach as the safest leaf fraction in terms of cytotoxicity was subjected for phytochemical study.Results:Results of the present study indicated that seed kernel extract of M. azedarach had the highest cytotoxic activity and selectivity to cancer cell lines (IC50 range of 8.18- 60.10 μg mL-1. In contrast to crude seed extract of A. indica, crude pulp and crude leaf extracts of this plant showed remarkably stronger anti-prolifrative activity (IC50 ranges of 83.45 - 212.16 μg mL-1 and 34.11- 95.51 μg mL-1 respectively than those of M. azedarach (all IC50 values of both plants > 650 μg mL-1. The phytochemical analysis led to the isolation of four flavonol 3-O-glycosides including rutin, kaempferol-3-O-robinobioside, kaempferol-3-O-rutinoside and isoquercetin along with a purin nucleoside, β-adenosine.Conclusions:The anti-prolifrative potentials of extracts from different parts of M. azedarach and A. indica were determined. By comparison, methanol leaf fraction of M. azedarach seems to be safer in terms of cytotoxicity. Our study shows

  9. Enzymic degradiation of starch granules in the cotyledons of germinating peas.

    Science.gov (United States)

    Juliano, B O; Varner, J E

    1969-06-01

    Starch, total alpha- and beta-amylase, and phosphorylase levels and the zymogram patterns of these 3 starch-degrading enzymes were determined in the cotyledons of smooth pea (Pisum sativum L.) during the first 15 days of germination. Starch is degraded slowly in the first 6 days; during this time, alpha-amylase is very low, beta-amylase is present at a constant level while phosphorylase gradually increases and reaches a peak on the fifth day. Beginning on the sixth day there is a more rapid degradation of starch which coincides with alpha-amylase production. One phosphorylase band and 2 beta-amylase bands are present in the zymogram of the imbibed cotyledon. An additional phosphorylase band and 1 alpha-amylase band appear during germination. Seeds imbibed in benzyladenine, chloramphenicol, and in cycloheximide show retarded growth and slower starch degradation and enzyme production than the controls. We conclude that alpha-amylase is the major enzyme involved in the initial degradation of starch into more soluble forms while phosphorylase and beta-amylase assist in the further conversion to free sugars.

  10. Clofarabine.

    Science.gov (United States)

    2004-01-01

    Clofarabine [Clofarex] is a purine nucleoside in development with Bio-envision, the Southern Research Institute and ILEX Oncology as an anticancer agent. Clofarabine's nucleoside structure is such that both the purine and ribose rings are halogenated, which allows it to inhibit DNA synthesis at two critical junctures: DNA polymerase I and RNA reductase. An intravenous infusion and an oral formulation are undergoing clinical development. Clofarabine was originated by the Southern Research Institute. In August 1998 Bioenvision signed a co-development agreement with the Southern Research Institute, under which it obtained the right to manufacture, market and distribute clofarabine worldwide, except Japan and Southeast Asia. In addition, the company appears to have licensed rights from the Institute that cover the development and marketing of other purine nucleoside analogues that have relevance in the treatment of leukaemia and lymphoma. Bioenvision will pay royalties to the Southern Research Institute for sales of clofarabine. Bioenvision extended its option in May 2004 to manufacture, market and distribute clofarabine in Japan and Southeast Asia, and is seeking a co-marketing partner to convert the option into a license agreement following the terms agreed upon between Bioenvision and the Southern Research Institute. Bioenvision and ILEX Products (a wholly owned subsidiary of ILEX Oncology) signed an agreement in February 2004 that converted ILEX's option (agreed in March 2001) to market and distribute clofarabine in the US and Canada. As part of the deal, Bioenvision received a $US3.5 million payment from ILEX in December 2003. In March 2004, Genzyme Corporation announced that it had signed a merger agreement with ILEX Oncology under which ILEX shareholders will receive shares of Genzyme common stock valued at approximately $US1 billion in equity value. Genzyme's business combination with ILEX is expected to be completed by the middle of 2004, Genzyme will

  11. Facile preparation of highly crystalline lamellae of (1 → 3)-β-D-glucan using an extract of Euglena gracilis.

    Science.gov (United States)

    Ogawa, Yu; Noda, Kazuhiro; Kimura, Satoshi; Kitaoka, Motomitsu; Wada, Masahisa

    2014-03-01

    In vitro synthesis of (1 → 3)-β-D-glucan was performed using laminaribiose phosphorylase obtained by an extraction of Euglena gracilis with sucrose phosphorylase. The synthetic product was a linear (1 → 3)-β-D-glucan with a narrow distribution of degree of polymerization (DP) centered on DP=30. X-ray diffraction and electron microscopy revealed that the glucan molecules obtained were self-organized as highly crystalline hexagonal lamellae. This synthetic product has quite high structural homogeneity at every level from primary to higher-order structure, which is a great advantage for the detailed analyses of physiological functions of (1 → 3)-β-D-glucan.

  12. Temporal changes in glycogenolytic enzyme mRNAs during myogenesis of primary porcine satellite cells

    DEFF Research Database (Denmark)

    Henckel, Poul; Theil, Peter Kappel; Sørensen, Inge Lise

    2007-01-01

    , phosphorylase kinase, phosphorylase and glycogen debranching enzyme, and no alterations of the transporter molecule GLUT4, clearly indicate that glycogenolytic enzymes of potential importance to meat quality development are regulated at the gene level during myogenesis, and are heavily involved in muscle cell...... and muscle fibre development. The genes, however, are not influenced by insulin, and the lack of response to insulin of expression of gene-encoding enzymes involved in the formation and degradation of glycogen may question the applicability of porcine cell culture systems, like the one applied, as a model...

  13. Genetic engineering of cyanobacteria

    DEFF Research Database (Denmark)

    Jacobsen, Jacob Hedemand

    , including genetic tools that allow metabolic engineering. The cyanobacterial phylum represents a diverse group of aerobic photosynthetic bacteria that are widespread in nature. Cyanobacteria shaped our atmosphere by oxygen evolution through the splitting of water using energy from sunlight. The sole carbon...... and characterized for growth phenotype and glycogen content. While no difference in growth rate or glycogen content was detected between the phosphorylase double mutant and wild type strain, we found that both glycogen phophyrylases must be genetically inactivated to eliminate glycogen phosphorylase activity...

  14. Engineering Neurospora crassa for cellobionate production directly from cellulose without any enzyme addition

    Science.gov (United States)

    A previously engineered strain of N. crassa F5'ace-1'cre-1'ndvB) with six out of seven ß-glucosidase (bgl) genes, two transcription factors (cre1 and ace-1) and cellobionate phosphorylase (ndvB) deleted was able to produce cellobiose and cellobionate directly from cellulose without the addition of e...

  15. Regulation of skeletal muscle glycogenolysis during exercise

    DEFF Research Database (Denmark)

    Hargreaves, M; Richter, Erik

    1988-01-01

    Muscle-glycogen breakdown during exercise is influenced by both local and systemic factors. Contractions per se increase glycogenolysis via a calcium-induced, transient increase in the activity of phosphorylase a, and probably also via increased concentrations of Pi. In fast-twitch muscle, increa...

  16. Glycogenin activity and mRNA expression in response to volitional exhaustion in human skeletal muscle

    DEFF Research Database (Denmark)

    Shearer, Jane; Graham, Terry E.; Battram, Danielle S.

    2005-01-01

    Glycogenolysis results in the selective catabolism of individual glycogen granules by glycogen phosphorylase. However, once the carbohydrate portion of the granule is metabolized, the fate of glycogenin, the protein primer of granule formation, is not known. To examine this, male subjects (n = 6)...

  17. Transferases for alkylation, glycosylation and phosphorylation

    NARCIS (Netherlands)

    Auriol, D.; ter Halle, R.; Lefèvre, F.; Visser, D.F.; Gordon, G.E.R.; Bode, M.L.; Mathiba, K.; Brady, D.; De Winter, K.; Desmet, T.; Cerdobbel, A.; Soetaert, W.; van Herk, T.; Hartog, A.F.; Wever, R.; Brzezińska-rodak, M.; Klimek-Ochab, M.; Żymańczyk-Duda, E.; Mukherjee, J.; Gupta, M.N.; Yin, W.B.; Li, S.M.; Gruber-Khadjawi, M.; Whittall, J.; Sutton, P.W.

    2012-01-01

    This chapter contains sections titled: Industrial Production of Caffeic Acid-α-D-O-Glucoside Enzymatic Synthesis of 5-Methyluridine by Transglycosylation of Guanosine and Thymine Preparation and Use of Sucrose Phosphorylase as Cross-Linked Enzyme Aggregate (CLEA) Enzymatic Synthesis of Phosphorylate

  18. Regulation of skeletal muscle glycogenolysis during exercise

    DEFF Research Database (Denmark)

    Hargreaves, M; Richter, Erik

    1988-01-01

    Muscle-glycogen breakdown during exercise is influenced by both local and systemic factors. Contractions per se increase glycogenolysis via a calcium-induced, transient increase in the activity of phosphorylase a, and probably also via increased concentrations of Pi. In fast-twitch muscle, increa...

  19. Hormone-sensitive lipase as mediator of lipolysis in contracting skeletal muscle

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia;

    2005-01-01

    The authors propose that the enzyme hormone-sensitive lipase (HSL), which is the rate-limiting enzyme for hydrolysis of triacylglycerol in adipocytes, also regulates the intramyocellular triacylglycerol mobilization and is controlled by mechanisms similar to those regulating glycogen phosphorylase...

  20. Physical properties and structure of enzymatically synthesized amylopectin analogs

    NARCIS (Netherlands)

    Ciric, Jelena; Woortman, Albert J. J.; Gordiichuk, Pavlo; Stuart, Marc C. A.; Loos, Katja

    2013-01-01

    The mechanism of the enzymatic polymerization of amylopectin analogs with phosphorylase b and glycogen branching enzyme is very intriguing. Recently, size exclusion chromatography with multi-detection of enzymatically synthesized amylopectin analogs in combination with MALDI-ToF MS analysis of enzym

  1. DPD screening to prevent toxicity in fluoropyrimidine treated patients

    NARCIS (Netherlands)

    Staveren, van M.C.

    2016-01-01

    Fluoropyrimidines are being used in the treatment of different types of cancer. The most common fluoropyrimidine is 5-flourouracil (5-FU), which is administered intravenously as a bolus or as prolonged infusion. Many tissues throughout the body express thymidine phosphorylase. Dihydropyrimidine Dehy

  2. Physical training for McArdle disease

    DEFF Research Database (Denmark)

    Quinlivan, Rosaline; Vissing, John; Hilton-Jones, David

    2011-01-01

    McArdle disease is a rare metabolic myopathy caused by a complete absence of the enzyme muscle glycogen phosphorylase. Affected people experience symptoms of fatigue and cramping within minutes of exercise and are at risk for acute muscle injury (rhabdomyolysis) and acute renal failure. If the fi...

  3. GenBank blastn search result: AK063766 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK063766 001-121-B07 BD056942.1 Transgenic potatoes having reduced levels of alpha ...glucan L- or H-type tuber phosphorylase activity with reduced cold-sweetening.|PAT PAT 7e-87 Plus Plus ...

  4. GenBank blastn search result: AK105410 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105410 001-123-F09 BD056943.1 Transgenic potatoes having reduced levels of alpha ...glucan L- or H-type tuber phosphorylase activity with reduced cold-sweetening.|PAT PAT 3e-49 Plus Plus ...

  5. GenBank blastn search result: AK063766 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK063766 001-121-B07 BD056944.1 Transgenic potatoes having reduced levels of alpha ...glucan L- or H-type tuber phosphorylase activity with reduced cold-sweetening.|PAT PAT 2e-53 Plus Plus ...

  6. Saccharide modified silica particles by enzymatic grafting

    NARCIS (Netherlands)

    Loos, Katja; Braunmühl, Volker von; Stadler, Reimund; Landfester, K; Spiess, HW

    1997-01-01

    The surface of silica particles has been chemically modified with oligo- or poly-(α,1→4)-D-glucopyranose (amylose) chains of various length by covalently attaching maltoheptaose derivatives to the solid support and enzymatic polymerization of glucose-1-phosphate with a potato phosphorylase as cataly

  7. Regulation of glycogenolysis in the locust fat body during flight

    NARCIS (Netherlands)

    Marrewijk, W.J.A. van; Broek, A.Th.M. van den; Beenakkers, A.M.Th.

    1980-01-01

    Glycogen reserves in the fat body of Locusta migratoria decrease dramatically during the first two hours of flight. In fat body of rested locusts only 10% of glycogen phosphorylase occurs in the active form. The enzyme is activated significantly during flight, when up to one-third of the total phosp

  8. The Dynamics of Complex Formation between Amy lose Brushes on Gold and Fatty Acids by QCM-D

    NARCIS (Netherlands)

    Cao, Zheng; Tsoufis, Theodoros; Svaldo-Lanero, Tiziana; Duwez, Anne-Sophie; Rudolf, Petra; Loos, Katja

    2013-01-01

    Amy lose brushes were synthesized by enzymatic polymerization with glucose-l-phosphate as monomer and rabbit muscle phosphorylase b as catalyst on gold-covered surfaces of a quartz crystal microbalance. Fourier transform infrared (FT-IR) spectra confirmed the presence of the characteristic absorptio

  9. Characterization of Components of the Staphylococcus aureus mRNA Degradosome Holoenzyme-Like Complex▿†

    OpenAIRE

    Roux, Christelle M.; DeMuth, Jonathon P.; Dunman, Paul M.

    2011-01-01

    Bacterial two-hybrid analysis identified the Staphylococcus aureus RNA degradosome-like complex to include RNase J1, RNase J2, RNase Y, polynucleotide phosphorylase (PNPase), enolase, phosphofructokinase, and a DEAD box RNA helicase. Results also revealed that the recently recognized RNase RnpA interacts with the S. aureus degradosome and that this interaction is conserved in other Gram-positive organisms.

  10. Dysregulation of multiple facets of glycogen metabolism in a murine model of Pompe disease.

    Directory of Open Access Journals (Sweden)

    Kristin M Taylor

    Full Text Available Pompe disease, also known as glycogen storage disease (GSD type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA. The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA(-/- mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA(-/- mouse strains were found to have elevated glycogen synthase (GS, glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS. Treating GAA(-/- mice with recombinant human GAA (rhGAA led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA(-/- mice, phosphorylase activity is suppressed in the GAA(-/- mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity.

  11. Anemia hemolítica auto-imune e outras manifestações imunes da leucemia linfocítica crônica Autoimmune hemolytic anemia and other autoimmune diseases related to chronic lymphocytic leukemia

    Directory of Open Access Journals (Sweden)

    José O. Bordin

    2005-12-01

    PRCA in about 1% of all CLL patients. Prednisone is the first-line treatment for immune cytopenia related to CLL. About 60% of patients relapse when treatment is stopped therefore splenectomy, intravenous immunoglobulin, or cyclosporine are reasonable second-line treatments. Although the data on pathophysiology are very limited, it appears that the autoimmune mechanisms are related to the activity of the leukemic B lymphocytes that act as aberrant antigen-presenting cells, and are effective in processing and presenting proteins derived from red cells or platelets to auto-reactive TH cells. TH cells specific for certain auto-antigens escape from control mechanisms and when activated may initiate auto-immune disease. Patients with AIHA secondary to CLL should not receive fludarabine because there is an increased frequency of severe and fatal AIHA in patients treated with purine nucleoside analogues. Patients who are more immunosuppressed due to several previous treatments are at higher risk for developing this auto-immune complication.

  12. [Use of properties and regulation peculiarities of enzymes of glycogenolysis in fish skeletal muscle depending on peculiarities of motor activity of species].

    Science.gov (United States)

    Serebrenikova, T P; Nesterov, V P

    2008-01-01

    Levels of activity, properties, and peculiarities of activation of glycogen phosphorylase (GP; EC 2.4.1.1) and glycogen phosphorylase kinase (GPK; EC 2.7.1.38) were studied in the white skeletal muscle of fish differing in motor behavior. No differences in the GP and GPK activity levels were revealed in laskir Diplodus annularis (L.), horse mackerel Trachurus mediterraneus ponticus, salmon Salmo trutta morphario, scorpena Scorpaena porcus, Scophtalnus maeoticus, and carp Cyprinus carpio; however, properties of the isolated enzymes and peculiarities of formation of their activated forms during swimming in a hydrodynamic tube are determined by functional peculiarities of the muscle tissue and are associated with the motor activity character of the species. In fish capable for the spurt type of swimming (scorpena, salmon) the more rapid ion regulation plays the predominant role. In other species, the glycogenolysis hormonal regulation leading to a change of the GPK activity index has been found.

  13. Seasonal Variation of the Glycogen Enzyme Activity in Diploid and Triploid Pacific Oyster Gonad During Sexual Maturation

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The glycogen content and the activities of two key enzymes in glycogen metabolism, glycogen phosphorylase and glycogen synthetase, in the gonad of diploid and triploid Pacific oysters (Crassostrea gigas) were compared during maturation. The glycogen content in the gonad of diploids decreased with gametogenesis (by 85.7%), but the glycogen content in the gonad of triploids did not vary significantly. Activity of glycogen phosphorylase (GP) in the gonad of diploids decreased with gametogenesis (by 55.5%), while GP activity of triploids did not vary significantly during maturation. Activity of glycogen synthetase (GS) in the gonad of diploids increased slightly with gametogenesis, reaching a peak in June. Activity of GS declined sharply from June to July, which might be due to gonad spawning. GS activity of triploid oysters in spawning time (July and August) was significantly higher than that

  14. Effects of electrostimulation on glycogenolysis in cultured rat myotubes

    DEFF Research Database (Denmark)

    Elsner, Peter; Grunnet, Niels; Quistorff, Bjørn

    2003-01-01

    A model for electrostimulation causing contractions of primary cultures of rat myotubes was established. The kinetics of glycogen degradation was investigated for a 2-h period to elucidate the coupling between contraction and glycogenolytic flux. Electrostimulation caused contraction and increased...... glycogenolytic flux, but had no effect on glycogen phosphorylase-a activity. Forskolin increased glycogenolytic flux more than electrostimulation, and caused a fast activation of glycogen phosphorylase, while it did not elicit contraction. The effects of electrostimulation and forskolin on glycogenolytic flux...... were partly additive. The metabolism of glucose and glycogen was almost equally anaerobic and aerobic. The ATP content remained constant during glycogenolysis, but phosphocreatine decreased with the largest decrease in electrostimulated cells. The calculated ATP turnover rate increased about 3 times...

  15. Hormone-sensitive lipase (HSL) expression and regulation in skeletal muscle

    DEFF Research Database (Denmark)

    Langfort, J; Ploug, T; Ihlemann, J;

    1998-01-01

    Because the enzymatic regulation of muscle triglyceride metabolism is poorly understood we explored the character and activation of neutral lipase in muscle. Western blotting of isolated rat muscle fibers demonstrated expression of hormone-sensitive lipase (HSL). In incubated soleus muscle...... epinephrine increased neutral lipase activity by beta-adrenergic mechanisms involving cyclic AMP-dependent protein kinase (PKA). The increase was paralleled by an increase in glycogen phosphorylase activity and could be abolished by antiserum against HSL. Electrical stimulation caused a transient increase...... in activity of both neutral lipase and glycogen phosphorylase. The increase in lipase activity during contractions was not influenced by sympathectomy or propranolol. Training diminished the epinephrine induced lipase activation in muscle but enhanced the activation as well as the overall concentration...

  16. Biological effects of synthetic AKH in Manduca sexta and estimates of the amount of AKH in corpora cardiaca.

    Science.gov (United States)

    Ziegler, R

    1990-01-01

    Dose-response curves were measured with synthetic Manduca adipokinetic hormone (AKH) for glycogen phosphorylase activation in larvae and for lipid mobilization in adults. Both responses are known hormonal functions in Manduca sexta. In ligated larvae, full activation of glycogen phosphorylase was achieved with 0.1 pmol and half-maximal activation with 0.03-0.04 pmol. Maximal lipid mobilization in adults required 10 pmol and half-maximal mobilization 0.15 to 0.2 pmol, respectively. An estimate of AKH content of corpora cardiaca from M. sexta was gained by comparing the dose-response curves for synthetic Manduca AKH with curves from gland extracts. Corpora cardiaca extracts were also quantitated by high performance liquid chromatography. According to both estimates corpora cardiaca of adults contain 10-20 pmol AKH per pair, while a pair of larval corpora cardiaca contains 0.7-2 pmol.

  17. Developmental changes in the response of the fat body of Manduca sexta to injections of Corpora cardiaca extracts.

    Science.gov (United States)

    Ziegler, R

    1984-04-01

    The Corpora cardiaca of larval and adult Manduca sexta contain glycogen phosphorylase-activating and adipokinetic activity. Depending on the age of the injected animal, injection of Corpora cardiaca extracts induces different responses. In larvae of the 1st and 2nd day of the Vth instar, phosphorylase is activated strongly by injections of Corpora cardiaca extracts, whereas in older animals it is only slightly activated. Mobilization of lipids in response to Corpora cardiaca extract injections is found only in animals injected after the imaginal molt; in younger animals this response is absent. It is argued that this difference in responsiveness may be due to a change in the properties of the target organ fat body.

  18. Genetic engineering of cyanobacteria

    DEFF Research Database (Denmark)

    Jacobsen, Jacob Hedemand

    , including genetic tools that allow metabolic engineering. The cyanobacterial phylum represents a diverse group of aerobic photosynthetic bacteria that are widespread in nature. Cyanobacteria shaped our atmosphere by oxygen evolution through the splitting of water using energy from sunlight. The sole carbon...... and its natural ability to take up and stably integrate heterologous DNA make Synechococcus sp. PCC 7002 a good candidate for metabolic engineering. For targeted gene inactivation, a suite of vectors were made by adaptation of a system previously used in plants and fungi. The vectors include a cassette...... and characterized for growth phenotype and glycogen content. While no difference in growth rate or glycogen content was detected between the phosphorylase double mutant and wild type strain, we found that both glycogen phophyrylases must be genetically inactivated to eliminate glycogen phosphorylase activity...

  19. TAS-102: a novel antimetabolite for the 21st century.

    Science.gov (United States)

    Uboha, Nataliya; Hochster, Howard S

    2016-01-01

    TAS-102, a novel antimetabolite combination chemotherapy agent, consists of a rediscovered antimetabolite agent, trifluorothymidine (trifluridine) combined with the metabolic inhibitor of thymidine phosphorylase, tipiracil, in a 1:0.5 molar ratio. Mechanism of action studies suggest that this agent works by incorporation into DNA. Both preclinical and clinical studies demonstrate that this agent is noncross-resistant with 5-fluorouracil. Tipiracil may also have antiangiogenic effects through inhibition of thymidine phosphorylase. Recent randomized Phase II and III trials demonstrate clinical activity (improved progression-free survival, time to decrease in performance status, prolonged overall survival) in metastatic colorectal cancer refractory to all standard agents. Monotherapy with TAS-102 has now been approved for this indication in Japan and in the USA.

  20. Enzymatic regulation of glycogenolysis in a subarctic population of the wood frog: implications for extreme freeze tolerance.

    Directory of Open Access Journals (Sweden)

    M Clara F do Amaral

    Full Text Available The wood frog, Rana sylvatica, from Interior Alaska survives freezing at -16°C, a temperature 10-13°C below that tolerated by its southern conspecifics. We investigated the hepatic freezing response in this northern phenotype to determine if its profound freeze tolerance is associated with an enhanced glucosic cryoprotectant system. Alaskan frogs had a larger liver glycogen reserve that was mobilized faster during early freezing as compared to conspecifics from a cool-temperate region (southern Ohio, USA. In Alaskan frogs the rapid glucose production in the first hours of freezing was associated with a 7-fold increase in glycogen phosphorylase activity above unfrozen frog levels, and the activity of this enzyme was higher than that of frozen Ohioan frogs. Freezing of Ohioan frogs induced a more modest (4-fold increase in glycogen phosphorylase activity above unfrozen frog values. Relative to the Ohioan frogs, Alaskan frogs maintained a higher total protein kinase A activity throughout an experimental freezing/thawing time course, and this may have potentiated glycogenolysis during early freezing. We found populational variation in the activity and protein level of protein kinase A which suggested that the Alaskan population had a more efficient form of this enzyme. Alaskan frogs modulated their glycogenolytic response by decreasing the activity of glycogen phosphorylase after cryoprotectant mobilization was well under way, thereby conserving their hepatic glycogen reserve. Ohioan frogs, however, sustained high glycogen phosphorylase activity until early thawing and consumed nearly all their liver glycogen. These unique hepatic responses of Alaskan R. sylvatica likely contribute to this phenotype's exceptional freeze tolerance, which is necessary for their survival in a subarctic climate.

  1. [Regularities of organ-specific expression of enzyme systems in cattle].

    Science.gov (United States)

    Tatarenko, O F; Glazko, V I

    1992-01-01

    The organ specificity of creatine kinase, esterase, isocitrate dehydrogenase lactate dehydrogenase, nucleoside phosphorylase, adenylate kinase, hexokinase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase of black-white cattle has been studied. Esterases, creatine kinase, adenylate kinase, hexokinase and glucose-6-phosphate dehydrogenase have a very wide spectrum of the organ variabilities. Liver and heart have the largest specificity of enzymes activity. Some peculiarities of isozyme spectrum are found in ovaries and spleen.

  2. Fat metabolism during exercise in patients with McArdle disease

    DEFF Research Database (Denmark)

    Ørngreen, M C; Jeppesen, T D; Andersen, S Tvede;

    2009-01-01

    OBJECTIVE: It is known that muscle phosphorylase deficiency restricts carbohydrate utilization, but the implications for muscle fat metabolism have not been studied. We questioned whether patients with McArdle disease can compensate for the blocked muscle glycogen breakdown by enhancing fat oxida...... not increase fatty acid oxidation suggests that blocked glycogenolysis may limit the capacity of fat oxidation to compensate for the energy deficit in McArdle disease....

  3. The influence of simulated microgravity on the formation of storage polysaccharides of potato minitubers

    Science.gov (United States)

    Shniukova, Ye. I.; Nedukha, O. M.

    A simulated microgravity influenced essentially the metabolism of storage polysaccharides of the potato minitubers. An increase of synthesis and accumulation of starch and an enhancement of phosphorylase activity were observed in the minitubers during a long-term clinorotation. At the same time the mono- and disaccharides content decreased, which pointed to the intensification of their utilization for starch synthesis. The correlation between the enhancement of enzyme activity and the increase of starch content is revealed.

  4. Genetic Mapping in Xenopus Laevis: Eight Linkage Groups Established

    OpenAIRE

    Graf, J. D.

    1989-01-01

    Inheritance of alleles at 29 electrophoretically detected protein loci and one pigment locus (albinism) was analyzed in Xenopus laevis by backcrossing multiply heterozygous individuals generated by intersubspecies hybridization. Pairwise linkage tests revealed eight classical linkage groups. These groups have been provisionally numbered from 1 to 8 in an arbitrarily chosen order. Linkage group 1 includes ALB-2 (albumin), ADH-1 (alcohol dehydrogenase), NP (nucleoside phosphorylase), and a(p) (...

  5. Physiological function of the maltose operon regulator, MalR, in Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Rådström Peter

    2002-09-01

    Full Text Available Abstract Background Maltose metabolism is initiated by an ATP-dependent permease system in Lactococcus lactis. The subsequent degradation of intracellular maltose is performed by the concerted action of Pi-dependent maltose phosphorylase and β-phosphoglucomutase. In some Gram-positive bacteria, maltose metabolism is regulated by a maltose operon regulator (MalR, belonging to the LacI-GalR family of transcriptional regulators. A gene presumed to encode MalR has been found directly downstream the maltose phosphorylase-encoding gene, malP in L. lactis. The purpose of this study was to investigate the physiological role of the MalR protein in maltose metabolism in L. lactis. Results A L. lactis ssp. lactis mutant, TMB5004, deficient in the putative MalR protein, was physiologically characterised. The mutant was not able to ferment maltose, while its capability to grow on glucose as well as trehalose was not affected. The activity of maltose phosphorylase and β-phosphoglucomutase was not affected in the mutant. However, the specific maltose uptake rate in the wild type was, at its lowest, five times higher than in the mutant. This difference in maltose uptake increased as the maltose concentration in the assay was increased. Conclusion According to amino acid sequence similarities, the presumed MalR is a member of the LacI-GalR family of transcriptional regulators. Due to the suggested activating effect on maltose transport and absence of effect on the activities of maltose phosphorylase and β-phosphoglucomutase, MalR of L. lactis is considered rather as an activator than a repressor.

  6. Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties

    OpenAIRE

    Rubio-Villena, Carla; Sanz, Pascual; Garcia-Gimeno, Maria Adelaida

    2015-01-01

    Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyz...

  7. Drug discovery for type 2 diabetes mellitus and metabolic syndrome: ten novel biological targets

    Directory of Open Access Journals (Sweden)

    Ivan Nikolaevich Tyurenkov

    2015-03-01

    Full Text Available In this review we discuss ten promising biological targets of interest for treating type 2 diabetes mellitus, obesity and metabolic syndrome. Namely, we address current experimental and clinical data on several new compounds that affect SGLT2, 11b-HSD1, PTP1B, SCD1, Il-1β, fructose-1,6-bisphosphatase, glycogen phosphorylase, SIRT1, DGAT-1 and GPR119. The body of data shows potential of these substances to become effective antidiabetic agents.

  8. MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

    Science.gov (United States)

    The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

  9. DPD screening to prevent toxicity in fluoropyrimidine treated patients

    OpenAIRE

    Staveren, van, W.A.

    2016-01-01

    Fluoropyrimidines are being used in the treatment of different types of cancer. The most common fluoropyrimidine is 5-flourouracil (5-FU), which is administered intravenously as a bolus or as prolonged infusion. Many tissues throughout the body express thymidine phosphorylase. Dihydropyrimidine Dehydrogenase (DPD) is involved in the degradation of endogenous pyrimidine nucleosides, but also in the degradation of fluoropyrimidines. More than 80% of the amount of 5-FU administered is catabolize...

  10. Molecular Toxicology of Chromatin.

    Science.gov (United States)

    1987-12-31

    Vig ) + polymerase (0.2 jig): lane 5: Form Ir (1 jig) + NAD + (0.05 mM) + BA (0.05 mM) + histones (0.2 gg) + polymerase (0.2 jig); lane 6: Form Ir...Phosphorylase B, 66.2 - Bovine serum albumin, 42.6 , Ovalbumin; 31.0 - Carbonic anhydrase; 21.5 - Soybean trypsin inhibitor, 14.4 - lysozyme. Protein

  11. Mechanism by which glucose and insulin inhibit net hepatic glycogenolysis in humans.

    OpenAIRE

    Petersen, K F; Laurent, D.; Rothman, D L; Cline, G W; Shulman, G I

    1998-01-01

    13C NMR spectroscopy was used to assess flux rates of hepatic glycogen synthase and phosphorylase in overnight-fasted subjects under one of four hypoglucagonemic conditions: protocol I, hyperglycemic (approximately 10 mM) -hypoinsulinemia (approximately 40 pM); protocol II, euglycemic (approximately 5 mM) -hyperinsulinemia (approximately 400 pM); protocol III, hyperglycemic (approximately 10 mM) -hyperinsulinemia (approximately 400 pM); and protocol IV; euglycemic (approximately 5 mM) -hypoin...

  12. Fructose effect to enhance liver glycogen deposition is due to inhibition of glycogenolysis

    Energy Technology Data Exchange (ETDEWEB)

    Youn, J.; Kaslow, H.; Bergman, R.

    1987-05-01

    The effect of fructose on glycogen degradation was examined by measuring flux of (/sup 14/C) from prelabeled glycogen in perfused rat livers. During 2 h refeeding of fasted rats hepatic glycogen was labeled by injection of (U /sup 14/C) galactose (0.1 mg and 0.02 ..mu..Ci/g of body weight). Refed livers were perfused for 30 min with glucose only (10 mM) and for 60 min with glucose (10 mM) without (n=5) or with fructose (1, 2, 10 mM; n=5 for each). With fructose, label production immediately declined and remained suppressed through the end of perfusion (P < 0.05). Suppression was dose-dependent: steady state label production was suppressed 45, 64, and 72% by 1, 2, and 10 mM fructose (P < 0.0001), without significant changes in glycogen synthase or phosphorylase. These results suggest the existence of allosteric inhibition of phosphorylase in the presence of fructose. Fructose 1-phosphate (F1P) accumulated in proportion to fructose (0.11 +/- 0.01 without fructose, 0.86 +/- 0.03, 1.81 +/- 0.18, and 8.23 +/- 0.6 ..mu..moles/g of liver with 1, 2, and 10 mM fructose. Maximum inhibition of phosphorylase was 82%; FIP concentration for half inhibition was 0.57 ..mu..moles/g of liver, well within the concentration of F1P attained in refeeding. Fructose enhances net glycogen synthesis in liver by suppressing glycogenolysis and the suppression is presumably caused by allosteric inhibition of phosphorylase by F1P.

  13. Enzymatic regulation of glycogenolysis in a subarctic population of the wood frog: implications for extreme freeze tolerance.

    Science.gov (United States)

    do Amaral, M Clara F; Lee, Richard E; Costanzo, Jon P

    2013-01-01

    The wood frog, Rana sylvatica, from Interior Alaska survives freezing at -16°C, a temperature 10-13°C below that tolerated by its southern conspecifics. We investigated the hepatic freezing response in this northern phenotype to determine if its profound freeze tolerance is associated with an enhanced glucosic cryoprotectant system. Alaskan frogs had a larger liver glycogen reserve that was mobilized faster during early freezing as compared to conspecifics from a cool-temperate region (southern Ohio, USA). In Alaskan frogs the rapid glucose production in the first hours of freezing was associated with a 7-fold increase in glycogen phosphorylase activity above unfrozen frog levels, and the activity of this enzyme was higher than that of frozen Ohioan frogs. Freezing of Ohioan frogs induced a more modest (4-fold) increase in glycogen phosphorylase activity above unfrozen frog values. Relative to the Ohioan frogs, Alaskan frogs maintained a higher total protein kinase A activity throughout an experimental freezing/thawing time course, and this may have potentiated glycogenolysis during early freezing. We found populational variation in the activity and protein level of protein kinase A which suggested that the Alaskan population had a more efficient form of this enzyme. Alaskan frogs modulated their glycogenolytic response by decreasing the activity of glycogen phosphorylase after cryoprotectant mobilization was well under way, thereby conserving their hepatic glycogen reserve. Ohioan frogs, however, sustained high glycogen phosphorylase activity until early thawing and consumed nearly all their liver glycogen. These unique hepatic responses of Alaskan R. sylvatica likely contribute to this phenotype's exceptional freeze tolerance, which is necessary for their survival in a subarctic climate.

  14. Toxicity and Kinetics of (3H)Microcystin-LR in Isolated Perfused Rat Livers

    Science.gov (United States)

    1990-03-20

    constant flow of 10 ml/m±:n in~ a recirculating system. During the 60-mmn exposure, toxcin caused stimulation of glycogenolysis . liver engorgemient...ml/min in a recirculating system. During the 60-min exposure, toxin caused stimulation of glycogenolysis , liver engorgement, and cessation of bile...with the observation that MCYST activates phosphorylase a (Runnegar et al., 1987), the rate-limiting enzyme for glycogenolysis . MCYST stlmulation of

  15. Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis.

    Science.gov (United States)

    Kosar-Hashemi, Behjat; Irwin, Jennifer A; Higgins, Jody; Rahman, Sadequr; Morell, Matthew K

    2006-05-01

    A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.

  16. Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy.

    Science.gov (United States)

    Garcia-Diaz, Beatriz; Garone, Caterina; Barca, Emanuele; Mojahed, Hamed; Gutierrez, Purification; Pizzorno, Giuseppe; Tanji, Kurenai; Arias-Mendoza, Fernando; Quinzii, Caterina M; Hirano, Michio

    2014-05-01

    Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that

  17. Efficacy of trifluridine and tipiracil (TAS-102) versus placebo, with supportive care, in a randomized, controlled trial of patients with metastatic colorectal cancer from Spain: results of a subgroup analysis of the phase 3 RECOURSE trial

    OpenAIRE

    Longo-Muñoz, F.; Argiles, G.; Tabernero, J.; Cervantes, A.; Gravalos, C.; Pericay, C.; Gil-Calle, S.; Mizuguchi, H; Carrato-Mena, A.; Limón, M. L.; Garcia-Carbonero, R.

    2016-01-01

    Purpose TAS-102 is a combination of the thymidine-based nucleoside analog trifluridine and the thymidine phosphorylase inhibitor tipiracil. Efficacy and safety of TAS-102 in patients with metastatic colorectal cancer (mCRC) refractory or intolerant to standard therapies were evaluated in the phase 3 RECOURSE trial. Results of RECOURSE demonstrated significant improvement in overall survival (OS) and progression-free survival (PFS) with TAS-102 versus placebo [hazard ratio (HR) = 0.68 and 0.48...

  18. Phase 1 study of cardiac safety of TAS-102 in patients with advanced solid tumors

    OpenAIRE

    Bendell, Johanna C.; Patel, Manish R.; Yoshida, Kenichiro; Seraj, Jabed; Weaver, Racquel; Lemech, Charlotte; Todaro, Thomas G.; Pant, Shubham; Arkenau, Hendrik-Tobias

    2016-01-01

    Purpose TAS-102 is a novel oral agent combining the antineoplastic thymidine-based nucleoside analogue, trifluridine, and the thymidine phosphorylase inhibitor, tipiracil (molar ratio 1:0.5). TAS-102 has shown good activity in refractory metastatic colorectal cancer with acceptable safety. No QT prolongation was seen in clinical studies. This study aimed to investigate TAS-102 cardiac safety for regulatory requirements. Methods This was a phase 1, non-randomized study in adults with advanced ...

  19. TAS-102: a novel antimetabolite for the 21st century

    OpenAIRE

    Uboha, Nataliya; Hochster, Howard S.

    2015-01-01

    TAS-102, a novel antimetabolite combination chemotherapy agent, consists of a rediscovered antimetabolite agent, trifluorothymidine (trifluridine) combined with the metabolic inhibitor of thymidine phosphorylase, tipiracil, in a 1:0.5 molar ratio. Mechanism of action studies suggest that this agent works by incorporation into DNA. Both preclinical and clinical studies demonstrate that this agent is noncross-resistant with 5-fluorouracil. Tipiracil may also have antiangiogenic effects through ...

  20. Novel PHKG2 mutation causing GSD IX with prominent liver disease: report of three cases and review of literature.

    Science.gov (United States)

    Albash, Buthainah; Imtiaz, Faiqa; Al-Zaidan, Hamad; Al-Manea, Hadeel; Banemai, Mohammed; Allam, R; Al-Suheel, Ali; Al-Owain, Mohammed

    2014-05-01

    Glycogen storage disease type IX (GSD IX) is a common form of glycogenosis due to mutations in PHKA1, PHKA2, or PHKB and PHKG2 genes resulting in the deficiency of phosphorylase kinase. The first two genes are X-linked while the latter two follow an autosomal recessive inheritance. The majority of cases of GSD IX are attributed to defects in PHKA2 which usually cause a mild disease. We report three patients with PHKG2-related GSD IX presenting with significant hepatic involvement, fibrosis, and cirrhosis. Interestingly, the homozygosity mapping resolved a dilemma about an erroneously normal phosphorylase kinase activity in patient 1. The novel mutation found in all the three patients (p.G220E) affects the catalytic subunit of the phosphorylase kinase. Increasing evidence shows that patients with PHKG2 mutations have a severe hepatic phenotype within the heterogeneous GSD IX disorder. Therefore, defect in PHKG2 should be considered in patients with suspected glycogenosis associated with significant liver fibrosis and cirrhosis.

  1. Study of bioenergetics of mouse pregnant uterine muscle by magnetic resonance spectroscopy (MRS)

    Energy Technology Data Exchange (ETDEWEB)

    Negami, Akira; Tominaga, Toshiro

    1989-06-01

    To investigate the bioenergetics of uterine muscles in vivo, we examined the energy state of mouse preterm uterus by means of magnetic resonance spectroscopy. Full-term mouse uterus contained ATP, PCr, phospho-di and mono ester (PDE and PME) and inorganic phosphate (Pi). The oxytocin-induced uterine muscle contraction peaks level and positions changed. Multiple peak analysis indicated a muscle contraction induced increase in the Pi concentration and decrease in the PCr concentration. The peak position of Pi was shifted in the contractive state also, indicating that the intracellular pH was lower than in the non-contractive state and this low pH level was recovered within several minutes. There was no change in the AMP peak neight in the contractive and non-contractive states. These data indicated that the energetics of mouse uterine muscle was maintained by the ATP-PCr system and acidosis of muscle was recovered within several minutes at rest. The constant AMP peak levels may indicate that phosphorylase is not regulated by AMP, but the phosphorylated phosphorylase kinase and pH levels in the contractive and non-contractive states also may indicate that phosphorylase kinase is not regulated by proteolysis or by the intracellular pH level but by the elevated intracellular calcium ion and calmodulin system. (author).

  2. Glucose 1-phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules.

    Science.gov (United States)

    Fettke, Joerg; Albrecht, Tanja; Hejazi, Mahdi; Mahlow, Sebastian; Nakamura, Yasunori; Steup, Martin

    2010-02-01

    Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [(14)C]glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

  3. Mode of action of dopamine in inducing hyperglycemia in the fresh water edible crab, Oziothelphusa senex senex.

    Science.gov (United States)

    Swetha, Ch; Sainath, S B; Reddy, P Sreenivasula

    2014-11-01

    The objective of this study was to investigate the mode of action of dopamine in regulating hemolymph sugar level in the fresh water edible crab, Oziothelphusa senex senex. Injection of dopamine produced hyperglycemia in a dose-dependent manner in intact crabs but not in eyestalkless crabs. Administration of dopamine resulted in a significant decrease in total carbohydrates and glycogen levels with a significant increase in glycogen phosphorylase activity levels in hepatopancreas and muscle of intact crabs, indicating dopamine-induced glycogenolysis resulting in hyperglycemia. Bilateral eyestalk ablation resulted in significant increase in the total carbohydrates and glycogen levels with a significant decrease in the activity levels of phosphorylase in the hepatopancreas and muscle of the crabs. Eyestalk ablation resulted in significant decrease in hemolymph hyperglycemic hormone levels. The levels of hyperglycemic hormone in the hemolymph of dopamine injected crabs were significantly higher than in control crabs. However, no significant changes in the levels of hemolymph hyperglycemic hormone and sugar and tissue carbohydrate and phosphorylase activity were observed in dopamine injected eyestalk ablated crabs when compared with eyestalk ablated crabs. These results support an earlier hypothesis in crustaceans that dopamine acts as a neurotransmitter and induces hyperglycemia by triggering the release of hyperglycemic hormone in the crab, O. senex senex. © 2014 Wiley Periodicals, Inc.

  4. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

    Energy Technology Data Exchange (ETDEWEB)

    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-11-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating (/sup 3/H)thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase.

  5. Contribution of hepatic glycogenolysis and gluconeogenesis in the defense against short-term insulin induced hypoglycemia in rats.

    Science.gov (United States)

    Nascimento, Kátia F; Garcia, Rosângela F; Gazola, Vilma A F G; de Souza, Helenir Medri; Obici, Simoni; Bazotte, Roberto B

    2008-05-07

    In this study, the contribution of liver glycogenolysis and gluconeogenesis in the defense against short-term insulin induced hypoglycemia (IIH) was investigated. For this purpose, we used an experimental model in which IIH was obtained by administering an IP injection of a pharmacological dose (1 U/kg) of regular insulin to rats that had been deprived of food for a period of six hours. This experimental model is suitable to study the simultaneous participation of glycogen breakdown and gluconeogenesis in the defense against IIH. The livers of IIH rats showed insignificant changes in the glycogen concentration, total phosphorylase, active phosphorylase, and percent of active phosphorylase. Our results also indicated that the livers of IIH rats that received the concentration of L-alanine, L-glutamine, L-lactate, or glycerol found in the blood during IIH (basal values) showed negligible glucose production. Nonetheless, glucose, urea, and pyruvate production increased (P<0.05) if the livers were perfused with a saturating concentration of gluconeogenic precursors. In agreement with these results, IIH rats that received intragastric L-alanine, L-glutamine, or L-lactate showed increased (P<0.05) glycemia 30 min after the administration of these substances. However, when using glycerol, higher glycemia (P<0.05) was observed at 2 and 5 min, but not 30 min after the administration of this hepatic gluconeogenic precursor. Thus, we can conclude that the oral availability of gluconeogenic precursors could allow for their use as important antidote in the defense against IIH.

  6. Astrocytic glycogenolysis: mechanisms and functions.

    Science.gov (United States)

    Hertz, Leif; Xu, Junnan; Song, Dan; Du, Ting; Li, Baoman; Yan, Enzhi; Peng, Liang

    2015-02-01

    Until the demonstration little more than 20 years ago that glycogenolysis occurs during normal whisker stimulation glycogenolysis was regarded as a relatively uninteresting emergency procedure. Since then, a series of important astrocytic functions has been shown to be critically dependent on glycogenolytic activity to support the signaling mechanisms necessary for these functions to operate. This applies to glutamate formation and uptake and to release of ATP as a transmitter, stimulated by other transmitters or elevated K(+) concentrations and affecting not only other astrocytes but also most other brain cells. It is also relevant for astrocytic K(+) uptake both during the period when the extracellular K(+) concentration is still elevated after neuronal excitation, and capable of stimulating glycogenolytic activity, and during the subsequent undershoot after intense neuronal activity, when glycogenolysis may be stimulated by noradrenaline. Both elevated K(+) concentrations and several transmitters, including the β-adrenergic agonist isoproterenol and vasopressin increase free cytosolic Ca(2+) concentration in astrocytes, which stimulates phosphorylase kinase so that it activates the transformation of the inactive glycogen phosphorylase a to the active phosphorylase b. Contrary to common belief cyclic AMP plays at most a facilitatory role, and only when free cytosolic Ca(2+) concentration is also increased. Cyclic AMP is not increased during activation of glycogenolysis by either elevated K(+) concentrations or the stimulation of the serotonergic 5-HT(2B) receptor. Not all agents that stimulate glycogenolysis do so by directly activating phophorylase kinase--some do so by activating processes requiring glycogenolysis, e.g. for synthesis of glutamate.

  7. Regulation of glycogen breakdown and its consequences for skeletal muscle function after training.

    Science.gov (United States)

    Katz, Abram; Westerblad, Håkan

    2014-10-01

    Repeated bouts of physical exercise, i.e., training, induce mitochondrial biogenesis and result in improved physical performance and attenuation of glycogen breakdown during submaximal exercise. It has been suggested that as a consequence of the increased mitochondrial volume, a smaller degree of metabolic stress (e.g., smaller increases in ADP and Pi) is required to maintain mitochondrial respiration in the trained state during exercise at the same absolute intensity. The lower degree of Pi accumulation is believed to account for the diminished glycogen breakdown, since Pi is a substrate for glycogen phosphorylase, the rate-limiting enzyme for glycogenolysis. However, in this review, we present an alternative explanation for the diminished glycogen breakdown. Thus, the lower degree of metabolic stress after training is also associated with smaller increases in AMP (free concentration during contraction at specific intracellular sites) and this results in less activation of phosphorylase b (the non-phosphorylated form of phosphorylase), resulting in diminished glycogen breakdown. Concomitantly, the smaller accumulation of Pi, which interferes with cross-bridge function and intracellular Ca(2+) handling, contributes to the increased fatigue resistance. The delay in glycogen depletion also contributes to enhanced performance during prolonged exercise by functioning as an energy reserve.

  8. Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature.

    Science.gov (United States)

    Fettke, Joerg; Leifels, Lydia; Brust, Henrike; Herbst, Karoline; Steup, Martin

    2012-05-01

    Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹⁴C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹⁴-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹⁴C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹⁴C]glucose 1-phosphate or adenosine-[U-¹⁴C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹⁴C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional

  9. Sexual dimorphism in adrenergic regulation of hepatic glycogenolysis

    Energy Technology Data Exchange (ETDEWEB)

    Studer, R.K.

    1987-04-01

    The total phosphorylase a plus b of hepatocytes isolated from females and incubated in the absence or presence of estradiol and progesterone at concentrations found in vivo does not vary during the estrous cycle. However, there is a slight but significant influence of the estrous cycle on basal and epinephrine-stimulated phosphorylase a activity, with a nadir being seen on diestrus. The relative contributions of the ..cap alpha..- and ..beta..-mediated pathways to phosphorylase a activation do not vary with the estrous cycle but are constant at 75 and 56%, respectively, of the response to 5 x 10/sup -8/ M epinephrine. When the epinephrine-stimulated glucose release from glycogen stores in cells from females and males is compared, the release from the female is greater than that from the male, while the ..cap alpha..-receptor-mediated stimulation in the female is comparable with that in the male. The epinephrine-stimulated increase in cytostolic free calcium (Ca/sub i/) is greater in the male than the female at 10/sup -6/ M but greater in the female than the male at 5 x 10/sup -9/ M. The changes in Ca/sub i/ are equivalent at intermediate epinephrine concentrations. When considered with the prior analysis of /sup 45/Ca efflux after adrenergic stimulation, this suggests there may be a sexual dimorphism in hepatocyte calcium transport systems. The glucose release for a given increase in Ca/sub i/ is greater in the female than the male probably due to the concomitant action of the ..beta..-mediated increase in cAMP and the ..cap alpha..-mediated increase in Ca/sub i/. This supports the conclusion that the ..beta..-mediated component does make a significant contribution to the catecholamine regulation of glycogenolysis in hepatocytes from adult female rats.

  10. The regulation of ascorbate biosynthesis.

    Science.gov (United States)

    Bulley, Sean; Laing, William

    2016-10-01

    We review the regulation of ascorbate (vitamin C) biosynthesis, focusing on the l-galactose pathway. We discuss the regulation of ascorbate biosynthesis at the level of gene transcription (both repression and enhancement) and translation (feedback inhibition of translation by ascorbate concentration) and discuss the eight proteins that have been demonstrated to date to affect ascorbate concentration in plant tissues. GDP-galactose phosphorylase (GGP) and GDP-mannose epimerase are critical steps that regulate ascorbate biosynthesis. These and other biosynthetic genes are controlled at the transcriptional level, while GGP is also controlled at the translational level. Ascorbate feedback on enzyme activity has not been observed unequivocally.

  11. Dicty_cDB: CHH787 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ytica glycogen phosphorylase-2 mRNA, partial cds. 70 2e-08 1 AZ550934 |AZ550934.1 ENTEY06TR Entamoeba histolytica Shear....1 ENTKO14TR Entamoeba histolytica Sheared DNA Entamoeba histolytica genomic, DNA sequence. 68 9e-08 1 BH149...199 |BH149199.1 ENTQG01TR Entamoeba histolytica Sheared DNA Entamoeba histolytica... genomic, DNA sequence. 68 9e-08 1 AZ668559 |AZ668559.1 ENTLD89TR Entamoeba histolytica Sheared DNA Entamoeb...a histolytica genomic, DNA sequence. 64 1e-06 1 AZ674540 |AZ674540.1 ENTHI71TFB Entamoeba histolytica Sheare

  12. Epilepsy and McArdle Disease in A Child

    Directory of Open Access Journals (Sweden)

    Faruk incecik

    2015-03-01

    Full Text Available McArdle's disease, defined by the lack of functional glycogen phosphorylase in striated muscle, is inherited as an autosomal recessive trait. Patients typically suffer from reduced exercise tolerance, with muscle cramps and pain provoked by exercise, along with easy fatigability and weakness after exercise. Following prolonged exertion, contractures, rhabdomyolysis, and myoglobinuria may occur. Central nervous system symptoms have rarely been reported in McArdle disease. In this case report, a 13-year-old boy with epilepsy and McArdle's disease is presented. [Cukurova Med J 2015; 40(Suppl 1: 5-7

  13. Affi-gel blue treatment simplifies the protein composition of sarcoplasmic reticulum vesicles.

    Science.gov (United States)

    Papp, S; Dux, L; Martonosi, A

    1986-04-01

    Sarcoplasmic reticulum vesicles isolated by conventional techniques usually contain, in addition to the recognized sarcoplasmic reticulum components, several other proteins (phosphorylase, myosin, glyceraldehyde-3-phosphate dehydrogenase, etc.) in variable amounts; these proteins complicate the interpretation of chemical modification data. Incubation of sarcoplasmic reticulum vesicles with Affi-Gel blue particles for 1-4 h at 2 degrees C, followed by sedimentation of the Affi-Gel in a clinical centrifuge, simplifies the protein composition by selective adsorption of the accessory proteins, and improves the consistency of the preparations. The Affi-Gel blue treatment is recommended as part of the standard procedure for the isolation of sarcoplasmic reticulum vesicles.

  14. Anesthetic Management of a Child with Mitochondrial Neurogastrointestinal Encephalopathy

    Science.gov (United States)

    Casarez, Vianey Q.; Zavala, Acsa M.; Owusu-Agyemang, Pascal; Hagan, Katherine

    2015-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with deficiency of thymidine phosphorylase (TP). Associated manifestations include visual and hearing impairments, peripheral neuropathies, leukoencephalopathy, and malnutrition from concomitant gastrointestinal dysmotility and pseudoobstruction. Given the altered metabolic state in these patients, specific consideration of medication selection is advised. This case report will describe the anesthetic management used in a 10-year-old girl with MNGIE. She had multiple anesthetics while undergoing allogeneic hematopoietic stem cell transplantation. This case report will discuss the successful repeated use of the same anesthetic in this pediatric patient, with the avoidance of volatile anesthetic agents, propofol, and muscle relaxant. PMID:26124966

  15. Assignment of the protein kinase C [delta] polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14

    Energy Technology Data Exchange (ETDEWEB)

    Huppi, K.; Siwarski, D.; Goodnight, J.; Mischak, H. (Molecular Genetics Section Lab. of Genetics, Bethesda, MD (United States))

    1994-01-01

    The protein kinase C (pkc) enzymes are a family of serine-threonine protein kinases, each encoded by a distinct and separate gene. The chromosomal locations of human PRKCA, PRKCB, and PRKCG have previously been established. The authors now report that PRKCD, a novel member of the pkc gene family, maps to human chromosome 3. The chromosomal location of Pkcd has also been determined in the mouse by analysis of recombination frequency in an interspecific panel of back-cross mice. They find that the locus encoding pkcd resides proximal to nucleoside phosphorylase (Np-2) and Tcra on mouse chromosome 14 in a region syntenic with human 3p. 9 refs., 2 tabs.

  16. Assignment of the protein kinase C delta polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14.

    Science.gov (United States)

    Huppi, K; Siwarski, D; Goodnight, J; Mischak, H

    1994-01-01

    The protein kinase C (pkc) enzymes are a family of serine-threonine protein kinases, each encoded by a distinct and separate gene. The chromosomal locations of human PRKCA, PRKCB, and PRKCG have previously been established. We now report that PRKCD, a novel member of the pkc gene family, maps to human chromosome 3. The chromosomal location of Pkcd has also been determined in the mouse by analysis of recombination frequency in an interspecific panel of backcross mice. We find that the locus encoding pkcd resides proximal to nucleoside phosphorylase (Np-2) and Tcra on mouse chromosome 14 in a region syntenic with human 3p.

  17. [Clonage of the "malA" region of "Escherichia coli" K12: nucleotide sequence of the regulatory region and the promoters, identification and purification of the MalT-activator protein (author's transl)].

    Science.gov (United States)

    Raibaud, O; Débarbouillé, M; Cossart, P

    1982-01-01

    A 5,800-bp (base pair) HindIII-EcoRI DNA fragment containing malT, the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase, was cloned into pBR322. A sequence of 802 bp was established in a DNA segment containing the promotor for malPQ and the promoter for malT. A total of 611 bp separates the initiation codons for these two genes, which are transcribed in opposite directions. The malT product was identified as a 94,000 dalton polypeptide.

  18. sup 13 C and sup 31 P NMR studies of myocardial metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Laughlin, M.R.

    1988-01-01

    The fluxes through two enzyme systems have been measured in perfused or in in vivo heart using NMR: phosphocreatine kinase, and glycogen synthase and phosphorylase. The rates of synthesis and degradation of glycogen were monitored in vivo in fed, fasted, and diabetic rat heart during infusions of {sup 13}C-1-glucose and insulin using proton-decoupled {sup 13}C-NMR at 1.9 and 4.7 tesla. The enzyme activities of glycogen synthase and glycogen phosphorylase were also measured in this tissue which had been freeze clamped at the end of the experiment, for comparison with the synthetic rates. For normal fed, fasted, and diabetic animals, synthesis rates were 0.28, 0.16, and 0.15 {mu}mol/min.gww respectively. Glycogen synthase i activity was 0.23, 0.14, and 0.14 {mu}mol/min.gww in these hearts at the end of the experiment, when measured at appropriate substrate and activator concentrations, and follow activation time courses that are consistent with being the main rate determinant for net synthesis in all cases. Turnover of glycogen was studied by observing the preformed {sup 13}C-1-glycogen signal during infusion of {sup 12}C-glucose and insulin, and was found to be close to zero. Extracted phosphorylase a activity was approximately ten times that of synthase i under these circumstances. In order to fully interpret the turnover studies, glycogenolysis of preformed {sup 13}C-glycogen was observed after a bolus of glucagon. The glycogen had either been synthesized from {sup 13}C-1-glucose for a single hour, or during an hour of {sup 13}C-glucose and a subsequent hour of {sup 12}C-glucose infusion. The author observed that breakdown follows an exponential time course related to the phosphorylase a activation state and that the last synthesized glycogen breaks down at the rate of 2.5 {mu}mol/min.gww, five times faster than that synthesized an hour earlier.

  19. Anesthetic Management of a Child with Mitochondrial Neurogastrointestinal Encephalopathy

    Directory of Open Access Journals (Sweden)

    Vianey Q. Casarez

    2015-01-01

    Full Text Available Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE is an autosomal recessive disorder associated with deficiency of thymidine phosphorylase (TP. Associated manifestations include visual and hearing impairments, peripheral neuropathies, leukoencephalopathy, and malnutrition from concomitant gastrointestinal dysmotility and pseudoobstruction. Given the altered metabolic state in these patients, specific consideration of medication selection is advised. This case report will describe the anesthetic management used in a 10-year-old girl with MNGIE. She had multiple anesthetics while undergoing allogeneic hematopoietic stem cell transplantation. This case report will discuss the successful repeated use of the same anesthetic in this pediatric patient, with the avoidance of volatile anesthetic agents, propofol, and muscle relaxant.

  20. Fat and Sugar Metabolism During Exercise in Patients With Metabolic Myopathy

    Science.gov (United States)

    2017-08-31

    Metabolism, Inborn Errors; Lipid Metabolism, Inborn Errors; Carbohydrate Metabolism, Inborn Errors; Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency; Glycogenin-1 Deficiency (Glycogen Storage Disease Type XV); Carnitine Palmitoyl Transferase 2 Deficiency; VLCAD Deficiency; Medium-chain Acyl-CoA Dehydrogenase Deficiency; Multiple Acyl-CoA Dehydrogenase Deficiency; Carnitine Transporter Deficiency; Neutral Lipid Storage Disease; Glycogen Storage Disease Type II; Glycogen Storage Disease Type III; Glycogen Storage Disease Type IV; Glycogen Storage Disease Type V; Muscle Phosphofructokinase Deficiency; Phosphoglucomutase 1 Deficiency; Phosphoglycerate Mutase Deficiency; Phosphoglycerate Kinase Deficiency; Phosphorylase Kinase Deficiency; Beta Enolase Deficiency; Lactate Dehydrogenase Deficiency; Glycogen Synthase Deficiency

  1. Physical training for McArdle disease

    DEFF Research Database (Denmark)

    Quinlivan, Rosaline; Vissing, John; Hilton-Jones, David;

    2011-01-01

    McArdle disease is a rare metabolic myopathy caused by a complete absence of the enzyme muscle glycogen phosphorylase. Affected people experience symptoms of fatigue and cramping within minutes of exercise and are at risk for acute muscle injury (rhabdomyolysis) and acute renal failure. If the fi....... If the first few minutes of exercise are paced, a 'second wind' will occur enabling exercise to continue. This is due to mobilisation and utilisation of alternative fuel substrates. Aerobic training appears to improve work capacity by increasing cardiovascular fitness....

  2. Physiology of sexual reproduction. I. A histochemical study of the embryo sac development in Zephyranthes rosea and Lagenaria vulgaris.

    Science.gov (United States)

    Malik, C P; Vermani, S

    1975-01-01

    Using histochemical techniques, changes in the localization of different reserve substances (e.g. pectic compounds, starch, polysaccharides, proteins, nucleic acids, ascorbic acid) and enzymes (Acid phosphatase, alkaline phosphatase, ATP-ase, 5-nucleotidase, esterase, phosphorylase, succinate dehydrogenase, cytochrome oxidase and lipase) have been studied in the young and fertilized ovules of Zephyranthes sp. and Lagenaria sp. etc. Extensive changes in the enzyme activity and reserve substances are demonstrated during megasporogenesis and megagametogenesis and most of the substances exhibited interesting distributional pattern. Similarly, all the enzymes investigated have specific locale of distribution in the tissues which displayed differentiation of embryo sac. The earlier changes observed are in the megaspore which contained many reserve substances (starch; nucleic acids; ascorbic acid; proteins) and enzymes (peroxidase, succinate dehydrogenase, acid phosphatase, alkaline phosphatase and ATP-ase). In the matured embryo sac different cells have differential localization of the substances. Based on histochemical studies, distinct differences are made out between egg and synergids; egg and central cell. In general antipodals have maximum accumulation of physiologically active substances and intense activity of different enzymes. Nucellus cells also stored diverse substances and enzymes especially towards the chalazal end. Pollination stimulated accumulation of several reserve substances and enzymes in the tip of nucellus beak, micropylar zone and these included starch, peroxidase, phosphorylase succinate dehydrogenase, cytochrome oxidase etc.

  3. Eco-physiological studies on Indian arid zone plants. III. Effect of sodium chloride and gibberellin on the activity of the enzymes of carbohydrate metabolism in leaves of Pennisetum typhoides

    Energy Technology Data Exchange (ETDEWEB)

    Huber, W.; Rustagi, P.N.; Sankhla, N.

    1974-01-01

    Seedlings of Pennisetum typhoides were grown in sodium chloride (NaCl) and gibberellic acid (GA/sub 3/) separately and in combination, and the effects on the activity of amylase, phosphorylase, aldolase, invertase, hexose-phosphateisomerase, sucrose-synthetase and sucrose-6-phosphate-synthetase were studied. Treatment of the seedlings with NaCl caused an inhibition of the activity of amylase and invertase in the leaf homogenate, but enhanced that of phosphorylase, aldolase, sucrose-synthetase and sucrose-6-phosphate-synthetase. GA/sub 3/ alone, as observed earlier, promoted the activity of invertase but indicated no significant influence on the other enzymes tested. In combination with salt, however, GA/sub 3/ tended to counteract, partially or wholly, the effect of NaCl on the activity of severe enzymes tested. The possible significance of the similarities between the action of abscisic acid (ABA) and salinity in influencing growth and metabolism of plants during stress is discussed. 34 references, 3 figures.

  4. Effect of diabetes on glycogen metabolism in rat retina.

    Science.gov (United States)

    Sánchez-Chávez, Gustavo; Hernández-Berrones, Jethro; Luna-Ulloa, Luis Bernardo; Coffe, Víctor; Salceda, Rocío

    2008-07-01

    Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.

  5. A whole-body model for glycogen regulation reveals a critical role for substrate cycling in maintaining blood glucose homeostasis.

    Directory of Open Access Journals (Sweden)

    Ke Xu

    2011-12-01

    Full Text Available Timely, and sometimes rapid, metabolic adaptation to changes in food supply is critical for survival as an organism moves from the fasted to the fed state, and vice versa. These transitions necessitate major metabolic changes to maintain energy homeostasis as the source of blood glucose moves away from ingested carbohydrates, through hepatic glycogen stores, towards gluconeogenesis. The integration of hepatic glycogen regulation with extra-hepatic energetics is a key aspect of these adaptive mechanisms. Here we use computational modeling to explore hepatic glycogen regulation under fed and fasting conditions in the context of a whole-body model. The model was validated against previous experimental results concerning glycogen phosphorylase a (active and glycogen synthase a dynamics. The model qualitatively reproduced physiological changes that occur during transition from the fed to the fasted state. Analysis of the model reveals a critical role for the inhibition of glycogen synthase phosphatase by glycogen phosphorylase a. This negative regulation leads to high levels of glycogen synthase activity during fasting conditions, which in turn increases substrate (futile cycling, priming the system for a rapid response once an external source of glucose is restored. This work demonstrates that a mechanistic understanding of the design principles used by metabolic control circuits to maintain homeostasis can benefit from the incorporation of mathematical descriptions of these networks into "whole-body" contextual models that mimic in vivo conditions.

  6. Dietary Management of the Ketogenic Glycogen Storage Diseases

    Directory of Open Access Journals (Sweden)

    Kaustuv Bhattacharya MBBS, MRCPCH, FRACP, MD

    2016-08-01

    Full Text Available The glycogen storage diseases (GSDs comprise a group of rare inherited disorders of glycogen metabolism. The hepatic glycogenolytic forms of these disorders are typically associated with hypoglycemia and hepatomegaly. For GSD I, secondary metabolic disturbances include fasting hyperlactatemia, hyperuricemia, and hyperlipidemia. Glycogen storage disease III is caused by reduced activity of the debrancher enzyme, GSD VI by phosphorylase, and GSD IX by phosphorylase kinase. It has often been reported that the non-GSD I group of disorders have a benign course. However, myopathy, cardiomyopathy, and cirrhosis have been reported significant clinical morbidities associated with GSD III and IX in particular. There have been a range of reports indicating high-protein diets, high-fat diets, medium chain triglyceride (MCT, modified Atkins diet, and therapeutic ketones as rescuing severe phenotypes of GSD III in particular. The etiology of these severe phenotypes has not been defined. Cases presented in this report indicate potential harm from excessive simple sugar use in GSD IX C. Review of the literature indicates that most interventions have reduced the glycemic load and provide alternate substrates for energy in rescue situations. Prevention of complications is most likely to occur with a mixed balanced low glycemic index diet potentially with relative increases in protein.

  7. Dietary Management of the Ketogenic Glycogen Storage Diseases

    Directory of Open Access Journals (Sweden)

    Kaustuv Bhattacharya MBBS, MRCPCH, FRACP, MD

    2016-08-01

    Full Text Available The glycogen storage diseases (GSDs comprise a group of rare inherited disorders of glycogen metabolism. The hepatic glycogenolytic forms of these disorders are typically associated with hypoglycemia and hepatomegaly. For GSD I, secondary metabolic disturbances include fasting hyperlactatemia, hyperuricemia, and hyperlipidemia. Glycogen storage disease III is caused by reduced activity of the debrancher enzyme, GSD VI by phosphorylase, and GSD IX by phosphorylase kinase. It has often been reported that the non-GSD I group of disorders have a benign course. However, myopathy, cardiomyopathy, and cirrhosis have been reported significant clinical morbidities associated with GSD III and IX in particular. There have been a range of reports indicating high-protein diets, high-fat diets, medium chain triglyceride (MCT, modified Atkins diet, and therapeutic ketones as rescuing severe phenotypes of GSD III in particular. The etiology of these severe phenotypes has not been defined. Cases presented in this report indicate potential harm from excessive simple sugar use in GSD IX C. Review of the literature indicates that most interventions have reduced the glycemic load and provide alternate substrates for energy in rescue situations. Prevention of complications is most likely to occur with a mixed balanced low glycemic index diet potentially with relative increases in protein.

  8. The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5-fluoro-2'-deoxyuridine, is independent of activation by thymidine kinase and insensitive to degradation by phosphorolytic enzymes.

    Science.gov (United States)

    Vande Voorde, Johan; Liekens, Sandra; McGuigan, Christopher; Murziani, Paola G S; Slusarczyk, Magdalena; Balzarini, Jan

    2011-09-01

    A novel phosphoramidate nucleotide prodrug of the anticancer nucleoside analogue 5-fluoro-2'-deoxyuridine (5-FdUrd) was synthesized and evaluated for its cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, NUC-3073 markedly kept its antiproliferative activity in TK-deficient tumour cells, and thus is largely independent of intracellular TK activity to exert its cytostatic action. NUC-3073 was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. NUC-3073 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with our observations that 5-FdUrd, but not NUC-3073, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, we propose NUC-3073 as a novel 5-FdUrd phosphoramidate prodrug that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP which is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue.

  9. Glycogen serves as an energy source that maintains astrocyte cell proliferation in the neonatal telencephalon.

    Science.gov (United States)

    Gotoh, Hitoshi; Nomura, Tadashi; Ono, Katsuhiko

    2017-06-01

    Large amounts of energy are required when cells undergo cell proliferation and differentiation for mammalian neuronal development. Early neonatal mice face transient starvation and use stored energy for survival or to support development. Glycogen is a branched polysaccharide that is formed by glucose, and serves as an astrocytic energy store for rapid energy requirements. Although it is present in radial glial cells and astrocytes, the role of glycogen during development remains unclear. In the present study, we demonstrated that glycogen accumulated in glutamate aspartate transporter (GLAST)+ astrocytes in the subventricular zone and rostral migratory stream. Glycogen levels markedly decreased after birth due to the increase of glycogen phosphorylase, an essential enzyme for glycogen metabolism. In primary cultures and in vivo, the inhibition of glycogen phosphorylase decreased the proliferation of astrocytic cells. The number of cells in the G1 phase increased in combination with the up-regulation of cyclin-dependent kinase inhibitors or down-regulation of the phosphorylation of retinoblastoma protein (pRB), a determinant for cell cycle progression. These results suggest that glycogen accumulates in astrocytes located in specific areas during the prenatal stage and is used as an energy source to maintain normal development in the early postnatal stage.

  10. Glycogen storage diseases: New perspectives

    Institute of Scientific and Technical Information of China (English)

    Hasan (O)zen

    2007-01-01

    Glycogen storage diseases (GSD) are inherited metabolic disorders of glycogen metabolism. Different hormones,including insulin, glucagon, and cortisol regulate the relationship of glycolysis, gluconeogenesis and glycogen synthesis. The overall GSD incidence is estimated 1 case per 20 000-43 000 live births. There are over 12 types and they are classified based on the enzyme deficiency and the affected tissue. Disorders of glycogen degradation may affect primarily the liver, the muscle,or both. Type Ⅰ a involves the liver, kidney and intestine (and Ⅰ b also leukocytes), and the clinical manifestations are hepatomegaly, failure to thrive, hypoglycemia,hyperlactatemia, hyperuricemia and hyperlipidemia. Type Ⅲa involves both the liver and muscle, and Ⅲb solely the liver. The liver symptoms generally improve with age.Type Ⅳ usually presents in the first year of life, with hepatomegaly and growth retardation. The disease in general is progressive to cirrhosis. Type Ⅵ and Ⅸ are a heterogeneous group of diseases caused by a deficiency of the liver phosphorylase and phosphorylase kinase system. There is no hyperuricemia or hyperlactatemia.Type Ⅺ is characterized by hepatic glycogenosis and renal Fanconi syndrome. Type Ⅱ is a prototype of inborn lysosomal storage diseases and involves many organs but primarily the muscle. Types Ⅴ and Ⅶ involve only the muscle.

  11. Short-term Influence of Herbicide Quinclorac on Enzyme Activities in Flooded Paddy Soils

    Institute of Scientific and Technical Information of China (English)

    L(U) Zhen-Mei; MIN Hang; YE Yang-Fang

    2004-01-01

    The influence of quinclorac (3,7-dichloroquinoline-8-carboxylic acid) on enzyme activities in flooded paddy soils was assessed under laboratory conditions. The enzymes differed markedly in their response to quinclorac. Quinclorac inhibited proteinase, hydrogen peroxidase, phosphorylase, and urease activities.The higher the concentration of quinclorac applied, the more significant the inhibition to these observed activities with a longer time required to recover to the level of the control. However, soils supplemented with quinclorac were nonpersistent for proteinase, phosphorylase and urease as opposed to soils without quinclorac. Dehydrogenase activity was also sensitive to quinclorac. Three soil samples with concentrations of quinclorac higher than 1 μg g-1 soil declined to less than 20% of that in the control. However, the highest dehydrogenase activity (up to 3.28-fold) was detected in soils with 2 μg g-1 soil quinclorac on the 25th day after treatment. Quinclorac had a relatively mild effect on saccharase activity at the concentrations used in this experiment and a stimulatory one on soil respiration when added to soil at normal field concentrations.Nonetheless it was inhibited at higher concentrations in paddy soils. Quinclorac is still relatively safe to the soil ecosystem when applied at a normal concentration (0.67 μg g-1 dried soil) but may have some effects on soil enzymes at higher concentrations.

  12. Mitochondrial Neurogastrointestinal Encephalomyopathy Treated with Stem Cell Transplantation: A Case Report and Review of Literature.

    Science.gov (United States)

    Peedikayil, Musthafa Chalikandy; Kagevi, Eje Ingvar; Abufarhaneh, Ehab; Alsayed, Moeenaldeen Dia; Alzahrani, Hazzaa Abdulla

    2015-06-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder. The mutation in the ECGF1 gene causes severe deficiency of thymidine phosphorylase (TP), which in turn increases thymidine and deoxyuridine in the blood, serum, and tissue. The toxic levels of these products cause malfunction of the mitochondrial respiratory chain and mitochondrial DNA. Commonly, patients become symptomatic between 15 and 20 years of age (range 5 months to 35 years). The most commonly affected systems are gastrointestinal, followed by ocular, and nervous system. The disease is often fatal; high mortality rate is reported between 20 and 40 years of age. Treatment modalities that can increase thymidine phosphorylase activity and decrease thymidine and deoxy-uridine have shown symptomatic improvements in patients with MNGIE. Platelet transfusion, hemodialysis, peritoneal dialysis or allogeneic hematopoietic stem cell transplantation (HSCT) have been tried. The survival and long-term benefits of these measures are still not clear. Engrafted patients after stem cell transplantation have showed improvements in serum thymidine and deoxyuridine. We are reporting a case of MNGIE from Saudi Arabia, who underwent allogeneic hematopoietic stem cell transplantation. No MNGIE case has been previously reported from Saudi Arabia or the Gulf Arab countries. From the available literature, so far only 11 patients with MNGIE have undergone stem cell transplantation.

  13. Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction.

    Science.gov (United States)

    Hyzewicz, Janek; Tanihata, Jun; Kuraoka, Mutsuki; Ito, Naoki; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

    2015-05-01

    High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein-protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs.

  14. Effect of molecular crowding on the enzymes of glycogenolysis.

    Science.gov (United States)

    Chebotareva, N A

    2007-12-01

    Cell cytoplasm contains high concentrations of macromolecules occupying a significant part of the cell volume (crowding conditions). According to modern concepts, crowding has a pronounced effect on the rate and equilibrium of biochemical reactions and stimulates the formation of more compact structures. This review considers different aspects of the crowding effect in vivo and in vitro, its role in regulation of cell volume, the effect of crowding on various interactions, such as protein-ligand and protein-protein interactions, as well as on protein denaturation, conformation transitions of macromolecules, and supramolecular structure formation. The influence of crowding arising from the presence of high concentrations of osmolytes on the interactions of the enzymes of glycogenolysis has been demonstrated. It has been established that, in accordance with predictions of crowding theory, trimethylamine N-oxide (TMAO) and betaine highly stimulate the association of phosphorylase kinase (PhK) and its interaction with glycogen. However, high concentrations of proline, betaine, and TMAO completely suppress the formation of PhK complex with phosphorylase b (Phb). The protective effect of osmolyte-induced molecular crowding on Phb denaturation by guanidine hydrochloride is shown. The influence of crowding on the interaction of Phb with allosteric inhibitor FAD has been revealed. The results show that, under crowding conditions, the equilibrium of the isomerization of Phb shifts towards a more compact dimeric state with decreased affinity for FAD.

  15. Kinetic analysis of glycogen turnover: relevance to human brain 13C-NMR spectroscopy.

    Science.gov (United States)

    DiNuzzo, Mauro

    2013-10-01

    A biophysical model of the glycogen molecule is developed, which takes into account the points of attack of synthase and phosphorylase at the level of the individual glucose chain. Under the sole assumption of steric effects governing enzyme accessibility to glucosyl residues, the model reproduces the known equilibrium structure of cellular glycogen at steady state. In particular, experimental data are reproduced assuming that synthase (1) operates preferentially on inner chains of the molecule and (2) exhibits a faster mobility than phosphorylase in translocating from an attacked chain to another. The model is then used to examine the turnover of outer versus inner tiers during the labeling process of isotopic enrichment (IE) experiments. Simulated data are fitted to in vivo (13)C nuclear magnetic resonance spectroscopy measurements obtained in the human brain under resting conditions. Within this experimental set-up, analysis of simulated label incorporation and retention shows that 7% to 35% of labeled glucose is lost from the rapidly turning-over surface of the glycogen molecule when stimulation onset is delayed by 7 to 11.5 hours after the end of [1-(13)C]glucose infusion as done in actual procedures. The substantial label washout before stimulation suggests that much of the subsequent activation-induced glycogenolysis could remain undetected. Overall, these results show that the molecular structure significantly affects the patterns of synthesis and degradation of glycogen, which is relevant for appropriate design of labeling experiments aiming at investigating the functional roles of this glucose reserve.

  16. Effect of exposure to sublethal concentrations of sodium cyanide on the carbohydrate metabolism of the Indian Major Carp Labeo rohita (Hamilton, 1822

    Directory of Open Access Journals (Sweden)

    Praveen N. Dube

    2013-07-01

    Full Text Available Experiments were designed to study in-vivo effects of sodium cyanide on biochemical endpoints in the freshwater fish Labeo rohita. Fish were exposed to two sublethal concentrations (0.106 and 0.064mg/L for a period of 15 days. Levels of glycogen, pyruvate, lactate and the enzymatic activities of lactate dehydrogenase (LDH, succinate dehydrogenase (SDH, glucose-6-phosphate dehydrogenase (G6PDH, phosphorylase, alkaline phosphatase (ALP, acid phosphatase (AcP were assessed in different tissues (liver, muscle and gills. Result indicated a steady decrease in glycogen, pyruvate, SDH, ALP and AcP activity with a concomitant increase in the lactate, phosphorylase, LDH and G6PD activity in all selected tissues. The alterations in all the above biochemical parameters were significantly (p<0.05 time and dose dependent. In all the above parameters, liver pointing out the intensity of cyanide intoxication compare to muscle and gills. Study revealed change in the metabolic energy by means of altered metabolic profile of the fish. Further, these observations indicated that even sublethal concentrations of sodium cyanide might not be fully devoid of deleterious influence on metabolism in L. rohita.

  17. Review on TAS-102 development and its use for metastatic colorectal cancer.

    Science.gov (United States)

    Mota, Jose Mauricio; Fonseca, Leonardo G; Braghiroli, Maria Ignez; Hoff, Paulo M

    2016-08-01

    TAS-102 is the combination of trifluridine (TFT) with tipiracil (TPI) in a 1:0.5 molar ratio. TFT is a fluoropyrimidine that retains cytotoxic activity in 5-fluorouracil resistant cell lines. Due to TFT short half-life, early clinical development was discouraging. Thereafter, TFT was shown to be promptly degraded by thymidine phosphorylase, also known as platelet-derived endothelial cell growth factor, a pro-angiogenic protein and a poor prognosis marker in colorectal cancer. TPI is a specific antagonist of thymidine phosphorylase and led to an increase in TFT serum levels when both agents are combined. Moreover, TPI is a potential anti-angiogenic molecule and could exert antitumor actions per se. TAS-102 was tested in several Phase I studies published in the early 21st century. The best regimen was settled as 70mg/m(2)/day, q12h, orally given at days 1-5 and days 8-13, each 28days. Recently, the first Phase III trial evaluating TAS-102 in refractory colorectal cancer patients was published. The RECOURSE trial demonstrated a survival advantage of the agent over supportive care, and definitely established TAS-102 as a novel strategy in the current armamentarium against colorectal cancer. Here we review the preclinical data regarding TFT and TPI that led to the development of TAS-102, and the set of clinical data that ultimately proved that TAS-102 improved outcomes in colorectal cancer patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. TAS-102 an Emerging Oral Fluoropyrimidine.

    Science.gov (United States)

    Chen, Jiezhong; Han, Mei; Saif, Muhammad Wasif

    2016-01-01

    Colon cancer is a common type of cancer with high mortality. The standard therapy for colon cancer is 5-FU-based regimen, although the current response rate to 5-FU is only 10-15%. Various approaches have been used to improve the efficacy of 5-FU including inhibition of its degradation enzyme dihydropyrimidine dehydrogenase (DPD) such as S1, UTF, use of 5-FU pro-drug capecitabine to exploit thymidine phosphorylase (TP) and supplementation of reduced folate acid to increase cytotoxicity. TAS-102 is a newly-developed anti-folate drug containing the 5-FU analogue trifluridine (TFD) and tipiracil hydrochloride (TPI). TPI is an inhibitor of TFD degradation enzyme thymidine phosphorylase and thus increases the bioavailability of TFD. In the present review, we summarize recent progress with regard to TAS-102, including pre-clinical tests and clinical trials. We further propose several approaches to further improve the efficacy of TAS-102 including combination with targeted therapy and immune therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  19. The malQ gene is essential for starch metabolism in Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Yutaka Sato

    2013-08-01

    Full Text Available Background: The malQ and glgP genes, respectively, annotated as putative 4-α-glucanotransferase and putative glycogen phosphorylase are located with a 29 nucleotide overlap on the Streptococcus mutans genome. We found that the glgP gene of this organism was induced with maltose, and the gene likely constituted an operon with the upstream gene malQ. This putative operon was negatively regulated with the malR gene located upstream from the malQ gene and a MalR-binding consensus sequence was found upstream of the malQ gene. S. mutans is not able to catabolize starch. However, this organism utilizes maltose degraded from starch in the presence of saliva amylase. Therefore, we hypothesized that the MalQ/GlgP system may participate in the metabolism of starch-degradation products. Methods: A DNA fragment amplified from the malQ or glgP gene overexpressed His-tagged proteins with the plasmid pBAD/HisA. S. mutans malQ and/or glgP mutants were also constructed. Purified proteins were assayed for glucose-releasing and phosphorylase activities with appropriate buffers containing maltose, maltotriose, maltodextrin, or amylodextrin as a substrate, and were photometrically assayed with a glucose-6-phosphate dehydrogenase–NADP system. Results: Purified MalQ protein released glucose from maltose and maltotriose but did not from either maltodextrin or amylodextrin. The purified GlgP protein did not exhibit a phosphorylase reaction with maltose or maltotriose but generated glucose-1-phosphate from maltodextrin and amylodextrin. However, the GlgP protein released glucose-1-phosphate from maltose and maltotriose in the presence of the MalQ protein. In addition, the MalQ enzyme activity with maltose released not only glucose but also produced maltooligosaccharides as substrates for the GlgP protein. Conclusion: These results suggest that the malQ gene encodes 4-α-glucanotransferase but not α-1,4-glucosidase activity. The malQ mutant could not grow in the

  20. Effect of partial drying and partial baking on starch degradation, on related enzymatic activities and on chemical composition of upper flue-cured tobacco leaves%半晾半烤对上部烟叶淀粉降解和相关酶活性及品质的影响

    Institute of Scientific and Technical Information of China (English)

    王文超; 贺帆; 徐成龙; 王涛; 石盼盼; 宫长荣

    2012-01-01

    The upper leaves of flue-cured tobacco of tobacco cultivars Zhongyan 100 and Yunyan 87 were used to study the effect of different drying periods (48, 60, 72 h) on the activities of amylase and starch phosphorylase. Compared to the routine baking process, amylase activity of the leaves pretreated with air drying reached its first peak 12 h earlier during the baking process, which extended the life time of amylase; the starch phosphorylase activity in the early period of baking process decreased, but in the late period the decrease was not obvious. The leaves pretreated with 60 h of air drying had high activities of amylase and starch phosphorylase, high amylase degradation, and the amylase content was low while the contents of water-soluble total sugar and reducing sugar were high in these leaves, these cured upper leaves with balanced chemical components are considered highly applicable.%以烤烟品种中烟100和云烟87的上部叶为材料,研究不同晾黄时间(48、60、72 h)对烟叶烘烤过程中淀粉降解及淀粉酶和淀粉磷酸化酶活性的影响.结果表明:与对照(常规烘烤)相比,晾制处理的烟叶在烘烤过程中淀粉酶活性提前12h达到第1次高峰,适当延长了淀粉酶的有效活性时间;与对照相比,烘烤前期淀粉磷酸化酶活性有所降低,在烘烤后期下降不明显,其中,晾黄60 h后烘烤烟叶的淀粉酶和淀粉磷酸化酶活性、淀粉降解量均较高,烤后烟叶淀粉含量较低,水溶性总糖、还原糖含量较高,各种化学成分较为协调,上部烟叶的可用性较高.

  1. Chronic primary intestinal pseudo-obstruction from visceral myopathy

    Directory of Open Access Journals (Sweden)

    M. T. Muñoz-Yagüe

    Full Text Available Chronic intestinal pseudo-obstruction is an uncommon syndrome characterized by relapsing episodes suggesting intestinal obstruction during which no mechanical causes are identified to account for symptoms. Etiologic factors may be manifold. Among them a number of neurologic conditions, gastrointestinal smooth muscle myopathies, endocrino-metabolic and autoimmune diseases, and the use of selected drugs stand out. We report a case of chronic intestinal pseudo-obstruction originating in a sporadic, primary intestinal myopathy that corresponds to no type thus far described. A histological study of the intestinal wall showed disrupted muscle bundles and the presence of interstitial edema. Myocytes had severe degenerative changes, and no alterations were seen in submucosal and myenteric plexus neurons. The activity of enzyme complexes in the mitochondrial respiratory chain, and of thymidine phosphorylase was normal. No mitochondrial DNA changes were seen.

  2. Chronic primary intestinal pseudo-obstruction from visceral myopathy.

    Science.gov (United States)

    Muñoz-Yagüe, M T; Marín, J C; Colina, F; Ibarrola, C; López-Alonso, G; Martín, M A; Solís-Herruzo, J A

    2006-04-01

    Chronic intestinal pseudo-obstruction is an uncommon syndrome characterized by relapsing episodes suggesting intestinal obstruction during which no mechanical causes are identified to account for symptoms. Etiologic factors may be manifold. Among them a number of neurologic conditions, gastrointestinal smooth muscle myopathies, endocrino-metabolic and autoimmune diseases, and the use of selected drugs stand out. We report a case of chronic intestinal pseudo-obstruction originating in a sporadic, primary intestinal myopathy that corresponds to no type thus far described. A histological study of the intestinal wall showed disrupted muscle bundles and the presence of interstitial edema. Myocytes had severe degenerative changes, and no alterations were seen in submucosal and myenteric plexus neurons. The activity of enzyme complexes in the mitochondrial respiratory chain, and of thymidine phosphorylase was normal. No mitochondrial DNA changes were seen.

  3. Differential Muscle Involvement in Mice and Humans Affected by McArdle Disease

    DEFF Research Database (Denmark)

    Krag, Thomas O; Pinós, Tomàs; Nielsen, Tue L;

    2016-01-01

    , variations in fiber size, vacuoles, and some internal nuclei associated with cytosolic glycogen accumulation and ongoing regeneration; structural damage was seen only in a minority of human patients. Neither liver nor brain isoforms of glycogen phosphorylase were upregulated in muscles, thus providing...... no substitution for the missing muscle isoform. In the mice, the tibialis anterior (TA) muscles were invariably more damaged than the quadriceps muscles. This may relate to a 7-fold higher level of myophosphorylase in TA compared to quadriceps in wild-type mice and suggests higher glucose turnover in the TA. Thus......McArdle disease (muscle glycogenosis type V) is caused by myophosphorylase deficiency, which leads to impaired glycogen breakdown. We investigated how myophosphorylase deficiency affects muscle physiology, morphology, and glucose metabolism in 20-week-old McArdle mice and compared the findings...

  4. P-Ser-HPr-a link between carbon metabolism and the virulence of some pathogenic bacteria

    DEFF Research Database (Denmark)

    Mijakovic, Ivan

    2005-01-01

    HPr kinase/phosphorylase phosphorylates HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system, at serine-46. P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria, but also plays a role in virulence development of certain...... pathogens. In Listeria monocytogenes, several virulence genes, which depend on the transcription activator PrfA, are repressed by glucose, fructose, etc., in a catabolite repressor (CcpA)-independent mechanism. However, the catabolite co-repressor P-Ser-HPr was found to inhibit the activity of Prf...... is probably also mediated via P-Ser-HPr, since ccpA disruption leads to elevated amounts of P-Ser-HPr. Indeed, a ccpA ptsH1 double mutant exhibited normal PrfA activity. In S. pyogenes, the expression of several virulence genes depends on the transcription activator Mga. Interestingly, the mga promoter...

  5. Thermal expansion behavior of A- and B-type amylose crystals in the low-temperature region.

    Science.gov (United States)

    Kobayashi, Kayoko; Kimura, Satoshi; Naito, Philip-Kunio; Togawa, Eiji; Wada, Masahisa

    2015-10-20

    The thermal expansion behaviors of A-type and B-type amylose crystals, which were prepared by recrystallization of short amylose chains synthesized by phosphorylase, were investigated using synchrotron X-ray powder diffraction between 100 and 300K. For both types of crystals, the room-temperature phase (RT phase), which is the usually observed phase, transitioned to a low-temperature phase (LT phase), on cooling. The phase transitions took place reversibly with rapid changes in the unit-cell parameters around 200-270K. The differences between the RT and LT phase were investigated using solid-state (13)C NMR spectroscopy, which revealed there were changes in molecular chain conformations. These results suggest that the phase transition of water molecules on the crystalline surfaces affects the thermal behavior and structure of polysaccharide crystals.

  6. Insights into fluorometabolite biosynthesis in Streptomyces cattleya DSM46488 through genome sequence and knockout mutants.

    Science.gov (United States)

    Zhao, Chunhua; Li, Peng; Deng, Zixin; Ou, Hong-Yu; McGlinchey, Ryan P; O'Hagan, David

    2012-10-01

    Streptomyces cattleya DSM 46488 is unusual in its ability to biosynthesise fluorine containing natural products, where it can produce fluoroacetate and 4-fluorothreonine. The individual enzymes involved in fluorometabolite biosynthesis have already been demonstrated in in vitro investigations. Candidate genes for the individual biosynthetic steps were located from recent genome sequences. In vivo inactivation of individual genes including those encoding the S-adenosyl-l-methionine:fluoride adenosyltransferase (fluorinase, SCATT_41540), 5'-fluoro-5'-deoxyadenosine phosphorylase (SCATT_41550), fluoroacetyl-CoA thioesterase (SCATT_41470), 5-fluoro-5-deoxyribose-1-phosphate isomerase (SCATT_20080) and a 4-fluorothreonine acetaldehyde transaldolase (SCATT_p11780) confirm that they are essential for fluorometabolite production. Notably gene disruption of the transaldolase (SCATT_p11780) resulted in a mutant which could produce fluoroacetate but was blocked in its ability to biosynthesise 4-fluorothreonine, revealing a branchpoint role for the PLP-transaldolase.

  7. MITOCHONDRIAL NEUROGASTROINTESTINAL ENCEPHALOMYOPATHY (MNGIE

    Directory of Open Access Journals (Sweden)

    P. Ayatollahi

    2006-06-01

    Full Text Available Mitochondrial neurogastrointestinal encephalo-myopathy (MNGIE is a rare autosomal recessive disease caused by thymidine phosphorylase (TP gene mutation. Here we report a patient with MNGIE in whom sensorimotor polyneuropathy was the first presenting symptom and had a fluctuating course. This 26-year-old female patient developed acute-onset demyelinating polyneuropathy from the age of 6 with two relapses later on. In addition, she had gastrointestinal symptoms (diarrhea, recurrent abdominal pain, progressive weight loss and ophthalmoparesis. Brain magnetic resonance imaging showed white matter abnormalities, and muscle biopsy showed ragged red fibers. This constellation of clinical and laboratory findings raised the diagnosis of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE. This report highlights the uncommon clinical characteristics of this rare disease.

  8. Formation of a covalent Nepsilon2-guanylylhistidyl reaction intermediate by the GTP:GTP guanylyltransferase from the brine shrimp Artemia.

    Science.gov (United States)

    Cartwright, J L; McLennan, A G

    1999-01-01

    The chemical nature of the enzyme-nucleotide phosphoramidate reaction intermediate employed by the unique GTP:GTP guanylyltransferase from yolk platelets of Artemia franciscana cysts to synthesize diguanosine tetraphosphate (Gp4G) has been investigated. Labeling of the enzyme with [alpha-32P]GTP followed by isolation of the labeled phosphoamino acid by periodate treatment and alkaline hydrolysis and comparison of the product with phosphoamino acid standards by thin-layer and ion-exchange chromatography showed that the linkage involves the Nepsilon2 ring nitrogen of an enzyme histidyl residue. Thus, this enzyme is distinct from the mRNA capping enzymes which can also synthesize Gp4G but which employ a lysyl-nucleotide intermediate. Based on its reaction mechanism and substrate specificity, GTP:GTP guanylyltransferase may belong to the GAFH superfamily which includes the histidine triad proteins, Ap4A phosphorylases, and galactose-1-phosphate uridylyltransferase.

  9. Converting bulk sugars into prebiotics: semi-rational design of a transglucosylase with controlled selectivity.

    Science.gov (United States)

    Verhaeghe, Tom; De Winter, Karel; Berland, Magali; De Vreese, Rob; D'hooghe, Matthias; Offmann, Bernard; Desmet, Tom

    2016-03-01

    Despite the growing importance of prebiotics in nutrition and gastroenterology, their structural variety is currently still very limited. The lack of straightforward procedures to gain new products in sufficient amounts often hampers application testing and further development. Although the enzyme sucrose phosphorylase can be used to produce the rare disaccharide kojibiose (α-1,2-glucobiose) from the bulk sugars sucrose and glucose, the target compound is only a side product that is difficult to isolate. Accordingly, for this biocatalyst to become economically attractive, the formation of other glucobioses should be avoided and therefore we applied semi-rational mutagenesis and low-throughput screening, which resulted in a double mutant (L341I_Q345S) with a selectivity of 95% for kojibiose. That way, an efficient and scalable production process with a yield of 74% could be established, and with a simple yeast treatment and crystallization step over a hundred grams of highly pure kojibiose (>99.5%) was obtained.

  10. Dietary starch types affect liver nutrient metabolism of finishing pigs.

    Science.gov (United States)

    Xie, Chen; Li, Yanjiao; Li, Jiaolong; Zhang, Lin; Zhou, Guanghong; Gao, Feng

    2017-09-01

    This study aimed to evaluate the effect of different starch types on liver nutrient metabolism of finishing pigs. In all ninety barrows were randomly allocated to three diets with five replicates of six pigs, containing purified waxy maize starch (WMS), non-waxy maize starch (NMS) and pea starch (PS) (the amylose to amylopectin ratios were 0·07, 0·19 and 0·28, respectively). After 28 d of treatments, two per pen (close to the average body weight of the pen) were weighed individually, slaughtered and liver samples were collected. Compared with the WMS diet, the PS diet decreased the activities of glycogen phosphorylase, phosphoenolpyruvate carboxykinase and the expression of phosphoenolpyruvate carboxykinase 1 in liver (P0·05). Compared with the WMS diet, the PS diet reduced the expressions of glutamate dehydrogenase and carbamoyl phosphate synthetase 1 in liver (Pliver compared with the WMS diet (Pliver of finishing pigs.

  11. Discovery of new nanomolar inhibitors of GPa: Extension of 2-oxo-1,2-dihydropyridinyl-3-yl amide-based GPa inhibitors.

    Science.gov (United States)

    Loughlin, Wendy A; Jenkins, Ian D; Karis, N David; Healy, Peter C

    2017-02-15

    Glycogen Phosphorylase (GP) is a functionally active dimeric enzyme, which is a target for inhibition of the conversion of glycogen to glucose-1-phosphate. In this study we report the design and synthesis of 14 new pyridone derivatives, and seek to extend the SAR analysis of these compounds. The SAR revealed the minor influence of the amide group, importance of the pyridone ring both spatially around the pyridine ring and for possible π-stacking, and confirmed a preference for inclusion of 3,4-dichlorobenzyl moieties, as bookends to the pyridone scaffold. Upon exploring a dimer strategy as part of the SAR analysis, the first extended 2-oxo-dihydropyridinyl-3-yl amide nanomolar based inhibitors of GPa (IC50 = 230 and 260 nM) were identified. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. 2-Oxo-1,2-dihydropyridinyl-3-yl amide-based GPa inhibitors: Design, synthesis and structure-activity relationship study.

    Science.gov (United States)

    Loughlin, Wendy A; Jenkins, Ian D; Karis, N David; Schweiker, Stephanie S; Healy, Peter C

    2016-03-23

    Glycogen phosphorylase (GP), which plays a crucial role in the conversion of glycogen to glucose-1-phosphate, is a target for therapeutic intervention in diabetes. In this study, we report the design and synthesis of 29 new derivatives of 2-oxo-1,2-dihydro pyridin-3-yl amides, as potential inhibitors of GP. The hit rate (45%) was high with 13 compounds inhibiting GPa (between 33% at 4.40 mM and an IC50 of 1.92 μM). Two lead compounds were identified as compounds exhibiting good GPa inhibition (IC50 = 2.1 and 1.92 μM). SAR analysis of these compounds revealed sensitivity of GPa to the length of the 2-oxo-1,2-dihydro pyridin-3-yl amide derivative and a preference for inclusion of a 3,4-dichlorobenzyl moiety. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  13. Dicty_cDB: CHR451 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available CH (Link to library) CHR451 (Link to dictyBase) - - - Contig-U16403-1 - (Link to Original site) - - CHR...451Z 602 - - - - Show CHR451 Library CH (Link to library) Clone ID CHR451 (Link to dicty...iol.tsukuba.ac.jp/CSM/CH/CHR4-C/CHR451Q.Seq.d/ Representative seq. ID - (Link to ...Original site) Representative DNA sequence >CHR451 (CHR451Q) /CSM/CH/CHR4-C/CHR451Q.Seq.d/ XXXXXXXXXXTACTGAA...42 |X62142.1 D. discoideum glpV gene for glycogen phosphorylase 1. 94 1e-17 3 BX538351 |BX538351.1 Cryptosporidium parvum chr

  14. High glycogen levels in the hippocampus of patients with epilepsy

    DEFF Research Database (Denmark)

    Dalsgaard, Mads K; Madsen, Flemming F; Secher, Niels H

    2006-01-01

    biopsies were obtained from pathologic hippocampus (n=19) and from apparently 'normal' cortical grey and white matter. We determined the in vivo brain glycogen level and the activity of glycogen phosphorylase and synthase. Regional differences in glycogen concentration were examined similarly in healthy...... pigs (n=5). In the patients, the glycogen concentration in 'normal' grey and white matter was 5 to 6 mmol/L, but much higher in the hippocampus, 13.1+/-4.3 mmol/L (mean+/-s.d.; P..., glycogen was similarly higher than in grey and white matter. Consequently, in human grey and white matter and, particularly, in the hippocampus of patients with temporal lope epilepsy, glycogen constitutes a large, active energy reserve, which may be of importance for energy provision during sustained...

  15. Identification of a glycogenolysis-inhibiting peptide from the corpora cardiaca of locusts.

    Science.gov (United States)

    Clynen, Elke; Huybrechts, Jurgen; Baggerman, Geert; Van Doorn, Jan; Van Der Horst, Dick; De Loof, Arnold; Schoofs, Liliane

    2003-08-01

    A mass spectrometric study of the peptidome of the neurohemal part of the corpora cardiaca of Locusta migratoria and Schistocerca gregaria shows that it contains several unknown peptides. We were able to identify the sequence of one of these peptides as pQSDLFLLSPK. This sequence is identical to the part of the Locusta insulin-related peptide (IRP) precursor that is situated between the signal peptide and the B-chain. We designated this peptide as IRP copeptide. This IRP copeptide is also present in the pars intercerebralis, which is likely to be the site of synthesis. It is identical in both L. migratoria and S. gregaria. It shows no effect on the hemolymph lipid concentration in vivo or muscle contraction in vitro. The IRP copeptide is able to cause a decreased phosphorylase activity in locust fat body in vitro, opposite to the effect of the adipokinetic hormones and therefore possibly represents a glycogenolysis-inhibiting peptide.

  16. A Bacterial Glucanotransferase Can Replace the Complex Maltose Metabolism Required for Starch to Sucrose Conversion in Leaves at Night

    DEFF Research Database (Denmark)

    Ruzanski, Christian; Smirnova, Julia; Rejzek, Martin

    2013-01-01

    Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl...... moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan...... approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed...

  17. [Several features of the metabolism of the fast and slow muscles of Emys orbicularis tortoises].

    Science.gov (United States)

    Lebedinskaia, I I; Ogorodnikova, L G

    1978-01-01

    In skeletal muscles of the tortoise E. orbicularis, studies have been made on the content of glycogen, lactic acid, on the activity of glucose-6-phosphatase and phosphorylase. Histochemical studies were made on the lipid content. Experiments were performed on fast and slow bundles from mm. iliofibularis, testo cervicalis and retractor capitis. For comparison, the same indices of carbohydrate metabolism were investigated in fast m. plantaris and slow m. soleus of rats. In rats, slow muscles exhibit aerobic metabolism, in fast muscles--anaerobic one. In tortoises, slow muscles exhibit intermediate type of metabolism. Fast muscles show an anaerobic type or metabolism which is less intensive than anaerobic metabolism in slow muscles. Significant differences in some of the indices of carbohydrate metabolism were found in muscles which perform different functions in the organism.

  18. Network analysis of biochemical logic for noise reduction and stability: a system of three coupled enzymatic and gates.

    Science.gov (United States)

    Privman, Vladimir; Arugula, Mary A; Halámek, Jan; Pita, Marcos; Katz, Evgeny

    2009-04-16

    We develop an approach aimed at optimizing the parameters of a network of biochemical logic gates for reduction of the "analog" noise buildup. Experiments for three coupled enzymatic AND gates are reported, illustrating our procedure. Specifically, starch, one of the controlled network inputs, is converted to maltose by beta-amylase. With the use of phosphate (another controlled input), maltose phosphorylase then produces glucose. Finally, nicotinamide adenine dinucleotide (NAD(+)), the third controlled input, is reduced under the action of glucose dehydrogenase to yield the optically detected signal. Network functioning is analyzed by varying selective inputs and fitting standardized few-parameters "response-surface" functions assumed for each gate. This allows a certain probe of the individual gate quality, but primarily yields information on the relative contribution of the gates to noise amplification. The derived information is then used to modify our experimental system to put it in a regime of a less noisy operation.

  19. Enzymatic Synthesis of 5-Fluorouridine with Enterobacter Aero-genes%酶法合成5-氟尿苷

    Institute of Scientific and Technical Information of China (English)

    雷霆; 周长林; 窦洁; 吴梧桐

    2000-01-01

    采用产气肠杆菌(Enterobacter aerogenes) CPU 7856变异株的游离细胞,利用其中的嘧啶核苷磷酸化酶(Uridine Phosphorylase)通过碱基交换进行酶法合成目的物.试验以30mmol/L尿苷(UR),30mmol/L 5-氟尿嘧啶( FU),30mmol/L磷酸盐缓冲液(pH7.8)的混合物加入10%的产气肠杆菌湿菌体反应3h,底物UR的转化率可达50%.

  20. Glycogen shortage during fasting triggers liver–brain–adipose neurocircuitry to facilitate fat utilization

    Science.gov (United States)

    Izumida, Yoshihiko; Yahagi, Naoya; Takeuchi, Yoshinori; Nishi, Makiko; Shikama, Akito; Takarada, Ayako; Masuda, Yukari; Kubota, Midori; Matsuzaka, Takashi; Nakagawa, Yoshimi; Iizuka, Yoko; Itaka, Keiji; Kataoka, Kazunori; Shioda, Seiji; Niijima, Akira; Yamada, Tetsuya; Katagiri, Hideki; Nagai, Ryozo; Yamada, Nobuhiro; Kadowaki, Takashi; Shimano, Hitoshi

    2013-01-01

    During fasting, animals maintain their energy balance by shifting their energy source from carbohydrates to triglycerides. However, the trigger for this switch has not yet been entirely elucidated. Here we show that a selective hepatic vagotomy slows the speed of fat consumption by attenuating sympathetic nerve-mediated lipolysis in adipose tissue. Hepatic glycogen pre-loading by the adenoviral overexpression of glycogen synthase or the transcription factor TFE3 abolished this liver–brain–adipose axis activation. Moreover, the blockade of glycolysis through the knockdown of the glycogen phosphorylase gene and the resulting elevation in the glycogen content abolished the lipolytic signal from the liver, indicating that glycogen is the key to triggering this neurocircuitry. These results demonstrate that liver glycogen shortage activates a liver–brain–adipose neural axis that has an important role in switching the fuel source from glycogen to triglycerides under prolonged fasting conditions. PMID:23939267

  1. The activity of carbohydrate-degrading enzymes in the development of brood and newly emerged workers and drones of the Carniolan honeybee, Apis mellifera carnica.

    Science.gov (United States)

    Żółtowska, Krystyna; Lipiński, Zbigniew; Łopieńska-Biernat, Elżbieta; Farjan, Marek; Dmitryjuk, Małgorzata

    2012-01-01

    The activity of glycogen Phosphorylase and carbohydrate hydrolyzing enzymes α-amylase, glucoamylase, trehalase, and sucrase was studied in the development of the Carniolan honey bee, Apis mellifera carnica Pollman (Hymenoptera: Apidae), from newly hatched larva to freshly emerged imago of worker and drone. Phosphorolytic degradation of glycogen was significantly stronger than hydrolytic degradation in all developmental stages. Developmental profiles of hydrolase activity were similar in both sexes of brood; high activity was found in unsealed larvae, the lowest in prepupae followed by an increase in enzymatic activity. Especially intensive increases in activity occurred in the last stage of pupae and newly emerged imago. Besides α-amylase, the activities of other enzymes were higher in drone than in worker broods. Among drones, activity of glucoamylase was particularly high, ranging from around three times higher in the youngest larvae to 13 times higher in the oldest pupae. This confirms earlier suggestions about higher rates of metabolism in drone broods than in worker broods.

  2. High-resolution melting facilitates mutation screening of PYGM in patients with McArdle disease

    DEFF Research Database (Denmark)

    Duno, M.; Quinlivan, R.; Vissing, J.

    2009-01-01

    variations. Thirteen of these are pathogenic, and three were classified as polymorphisms. Nine variations had not previously been described. One of the novel mutations, c.2430C > T, was initially predicted to result in a silent G810G change, but cDNA analysis demonstrated that the mutation led to abnormal m......Mutations in PYGM, encoding the muscle-specific glycogen phosphorylase (myophosphorylase), are responsible for McArdle disease. Among Caucasians, a large proportion of patients are homozygous for the R50X mutation, but other mutations can affect all the 20 exons of PYGM, making mutation detection...... laborious. We have developed a high-resolution melting (HRM) assay for mutation detection in PYGM. Twelve McArdle patients were investigated, in whom pre-screening had ruled out homozygosity or compound heterozygosity for the two common G205S and R50X mutations. In total, we identified 16 different...

  3. Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae, larvae

    Directory of Open Access Journals (Sweden)

    Jordie D. Fraser

    2017-06-01

    Full Text Available Winter mortality is a major factor regulating population size of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae. Glycerol is the major cryoprotectant in this freeze intolerant insect. We report findings from a gene expression study on an overwintering mountain pine beetle population over the course of 35 weeks. mRNA transcript levels suggest glycerol production in the mountain pine beetle occurs through glycogenolytic, gluconeogenic and potentially glyceroneogenic pathways, but not from metabolism of lipids. A two-week lag period between fall glycogen phosphorylase transcript and phosphoenolpyruvate carboxykinase transcript up-regulation suggests that gluconeogenesis serves as a secondary glycerol-production process, subsequent to exhaustion of the primary glycogenolytic source. These results provide a first look at the details of seasonal gene expression related to the production of glycerol in the mountain pine beetle.

  4. Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), larvae.

    Science.gov (United States)

    Fraser, Jordie D; Bonnett, Tiffany R; Keeling, Christopher I; Huber, Dezene P W

    2017-01-01

    Winter mortality is a major factor regulating population size of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Glycerol is the major cryoprotectant in this freeze intolerant insect. We report findings from a gene expression study on an overwintering mountain pine beetle population over the course of 35 weeks. mRNA transcript levels suggest glycerol production in the mountain pine beetle occurs through glycogenolytic, gluconeogenic and potentially glyceroneogenic pathways, but not from metabolism of lipids. A two-week lag period between fall glycogen phosphorylase transcript and phosphoenolpyruvate carboxykinase transcript up-regulation suggests that gluconeogenesis serves as a secondary glycerol-production process, subsequent to exhaustion of the primary glycogenolytic source. These results provide a first look at the details of seasonal gene expression related to the production of glycerol in the mountain pine beetle.

  5. A reference proteomic database of Lactobacillus plantarum CMCC-P0002.

    Directory of Open Access Journals (Sweden)

    Li Zhu

    Full Text Available Lactobacillus plantarum is a widespread probiotic bacteria found in many fermented food products. In this study, the whole-cell proteins and secretory proteins of L. plantarum were separated by two-dimensional electrophoresis method. A total of 434 proteins were identified by tandem mass spectrometry, including a plasmid-encoded hypothetical protein pLP9000_05. The information of first 20 highest abundance proteins was listed for the further genetic manipulation of L. plantarum, such as construction of high-level expressions system. Furthermore, the first interaction map of L. plantarum was established by Blue-Native/SDS-PAGE technique. A heterodimeric complex composed of maltose phosphorylase Map3 and Map2, and two homodimeric complexes composed of Map3 and Map2 respectively, were identified at the same time, indicating the important roles of these proteins. These findings provided valuable information for the further proteomic researches of L. plantarum.

  6. McArdle Disease and Exercise Physiology

    Directory of Open Access Journals (Sweden)

    Yu Kitaoka

    2014-02-01

    Full Text Available McArdle disease (glycogen storage disease Type V; MD is a metabolic myopathy caused by a deficiency in muscle glycogen phosphorylase. Since muscle glycogen is an important fuel for muscle during exercise, this inborn error of metabolism provides a model for understanding the role of glycogen in muscle function and the compensatory adaptations that occur in response to impaired glycogenolysis. Patients with MD have exercise intolerance with symptoms including premature fatigue, myalgia, and/or muscle cramps. Despite this, MD patients are able to perform prolonged exercise as a result of the “second wind” phenomenon, owing to the improved delivery of extra-muscular fuels during exercise. The present review will cover what this disease can teach us about exercise physiology, and particularly focuses on the compensatory pathways for energy delivery to muscle in the absence of glycogenolysis.

  7. A case of McArdle disease: efficacy of vitamin B6 on fatigability and impaired glycogenolysis.

    Science.gov (United States)

    Izumi, Rumiko; Suzuki, Naoki; Kato, Kazuhiro; Warita, Hitoshi; Tateyama, Maki; Nakashima, Ichiro; Itoyama, Yasuto

    2010-01-01

    McArdle disease is a glycogenetic myopathy caused by a deficit of myophosphorylase inherited in an autosomal recessive pattern. Here, we report a case of McArdle disease in which fatigability was the only subjective complaint. Objective neurological findings were normal except for very mild muscle weakness in limbs and an elevated serum creatine kinase level. Ischemic forearm exercise test showed deficient glycogenolysis. In the muscle biopsy specimen, periodic acid Schiff (PAS) stained subsarcolemmal glycogen was increased and the muscle phosphorylase A activity was decreased. After administration of vitamin B6, fatigability was diminished and ischemic forearm exercise test showed improved glycogenolysis. Vitamin B6 may be beneficial for McArdle disease, especially for its easy fatigability.

  8. Basal glycogenolysis in mouse skeletal muscle: in vitro model predicts in vivo fluxes

    Science.gov (United States)

    Lambeth, Melissa J.; Kushmerick, Martin J.; Marcinek, David J.; Conley, Kevin E.

    2002-01-01

    A previously published mammalian kinetic model of skeletal muscle glycogenolysis, consisting of literature in vitro parameters, was modified by substituting mouse specific Vmax values. The model demonstrates that glycogen breakdown to lactate is under ATPase control. Our criteria to test whether in vitro parameters could reproduce in vivo dynamics was the ability of the model to fit phosphocreatine (PCr) and inorganic phosphate (Pi) dynamic NMR data from ischemic basal mouse hindlimbs and predict biochemically-assayed lactate concentrations. Fitting was accomplished by optimizing four parameters--the ATPase rate coefficient, fraction of activated glycogen phosphorylase, and the equilibrium constants of creatine kinase and adenylate kinase (due to the absence of pH in the model). The optimized parameter values were physiologically reasonable, the resultant model fit the [PCr] and [Pi] timecourses well, and the model predicted the final measured lactate concentration. This result demonstrates that additional features of in vivo enzyme binding are not necessary for quantitative description of glycogenolytic dynamics.

  9. Influence of supplemental maslinic acid (olive-derived triterpene) on the post-mortem muscle properties and quality traits of gilthead seabream

    DEFF Research Database (Denmark)

    Matos, E.; Silva, Tomé Santos; Wulff, Tune

    2013-01-01

    , enzymatic activities and protein expression in the muscle were assessed. Supplementing gilthead seabream diets with maslinic acid mainly resulted in hypertrophy of muscle fibres and inhibition of cathepsin B activity, with no observed differences in terms of glycogen and ATP content of the muscle, as well...... as glycogen phosphorylase activity. Proteomic analysis showed a low impact of maslinic acid supplementation on muscle metabolism, with most changes reflecting increased stress coping capacity and muscle hypertrophy in maslinic acid-fed fish. As a finishing strategy to improve the muscle's energetic status......Maslinic acid, a natural triterpene, was evaluated as a dietary supplement to modulate glycogen post-mortem mobilization in gilthead seabream muscle. For this purpose, a multidisciplinary trial was undertaken, where flesh quality criteria, as well as biochemical and histological parameters...

  10. Immunological and biochemical responses in Mya arenaria (Mollusca Bivalvia) exposed in vivo to estradiol-17beta.

    Science.gov (United States)

    Gauthier-Clerc, S; Pellerin, J; Fournier, M; Amiard, J-C

    2006-11-01

    Soft-shell clams Mya arenaria were injected with 10, 20 or 40 nmol of estradiol 17beta (E2). We observed a significant inhibiting effect of E2 on phagocytic activity of hemocytes from clams exposed to 10 and 20 nmol. A dose-response increase of the glycogen phosphorylase in the gonad tended to show a remobilisation of glycogen reserves involved in vitellogenesis although the exposure time must have been too short to observe a decrease in glycogen reserves or an increase in RNA concentration. Both results corroborate those of other studies about estrogen involvement in controlling immune capacity and energy metabolism related to vitellogenesis in bivalves. We can assume that immune parameters should now be taken into consideration in assessing endocrine disruption in bivalves. Nevertheless further studies are needed to understand the controlling pathways of E2 with a special regard on its interactions with other effectors involved in bivalve immunity and reproduction as well.

  11. Lactate production and clearance in exercise. Effects of training. A mini-review

    DEFF Research Database (Denmark)

    Stallknecht, B; Vissing, J; Galbo, H

    1998-01-01

    -induced increase in pyruvate dehydrogenase activity and is relatively impaired by low oxygen availability and low mitochondrial oxidative capacity. During exercise lactate is eliminated in liver, heart, and resting and working muscle. In muscle, elimination depends on plasma concentration, fiber type, and fiber......Lactate accumulates if pyruvate formation exceeds pyruvate oxidation. Accelerated glycogenolysis is essential for lactate production. Glycogen and epinephrine enhance glycogen phosphorylase activity and this is higher in type II b than in type I fibers. Pyruvate oxidation is enhanced by exercise...... conditions. Due to influence on hormonal response, mitochondrial oxidative capacity and fiber recruitment, training diminishes glycogenolysis and lactate production. Training also increases lactate clearance. This reflects increased hepatic capacity for gluconeogenesis as well as increased lactate transport...

  12. Contribution of cathepsins B, L and D to muscle protein profiles correlated with texture in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Godiksen, Helene; Morzel, M.; Hyldig, Grethe;

    2009-01-01

    Post-mortem softening of fish tissue often results in low yield and decreased product quality. In this study, proteolytic profiles of trout stored 5 days oil ice were obtained by SDS-PAGE. The link between protein hand intensities and firmness of trout fillets was examined through a correlation...... Study. In parallel, trout extracts were incubated with cathepsin B, cathepsin L and cathepsin D, alone or in combination, in order to evaluate the effect of each cathepsin on the texture-related proteins. Proteins from both myofibrillar (alpha-actinin, actin, MLC1, MLC2. and N-terminal 70 kDa MHC...... fragment) and sarcoplastic (glycogen phosphorylase, creatine kinase, and TPI) fractions correlated closely with firmness. Cathepsins D, B and L affected, respectively, 10, 9 and 4 out of the 17 protein bands correlating with firmness, and most changes induced by cathepsin D were unfavourable to firmness...

  13. [Glycogen storage disease type IX presenting as abdominal distention, hepatomegaly and hypoglycemia during infancy].

    Science.gov (United States)

    Soler Palacín, P; Tomasa Wörner, N; Sánchez de Toledo Sancho, J; Yeste Fernández, D; Gussinyé Canadell, M; Carrascosa Lezcano, A

    2004-11-01

    Glycogen storage diseases are a rare group of disorders in daily pediatric practice but must be taken into account when a patient presents with poor physical growth, hepatomegaly, hypoglycemia, hypotonia and/or other metabolic disturbances. Early diagnosis allows treatment that might improve the patient's outcome to be started or, at the very least, genetic counseling to be given to the parents. We present a 10-month-old boy who presented with growth retardation, abdominal distention and hepatomegaly and who was finally diagnosed with glycogenosis type IX. Definitive diagnosis was obtained by demonstrating the enzyme defect (phosphorylase beta-kinase) in affected tissues. Enteral nutrition was started using a diurnal high-carbohydrate diet with frequent feedings and nocturnal nasogastric continuous feeding, achieving optimal growth parameters and clinical response.

  14. One-pot microbial synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase.

    Science.gov (United States)

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-06-01

    A one-pot enzymatic synthesis of 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase was established. Glycolysis by baker's yeast (Saccharomyces cerevisiae) generated ATP which was used to produce D: -glyceraldehyde 3-phosphate production from glucose via fructose 1,6-diphosphate. The D: -glyceraldehyde 3-phosphate produced was transformed to 2'-deoxyribonucleoside via 2-deoxyribose 5-phosphate and then 2-deoxyribose 1-phosphate in the presence of acetaldehyde and a nucleobase by deoxyriboaldolase, phosphopentomutase expressed in Escherichia coli, and a commercial nucleoside phosphorylase. About 33 mM 2'-deoxyinosine was produced from 600 mM glucose, 333 mM acetaldehyde and 100 mM adenine in 24 h. 2'-Deoxyinosine was produced from adenine due to the adenosine deaminase activity of E. coli transformants.

  15. Mitochondrial neurogastrointestinal encephalomyopathy treated with peritoneal dialysis and bone marrow transplantation.

    Science.gov (United States)

    Ariaudo, Claudia; Daidola, Germana; Ferrero, Bruno; Guarena, Cesare; Burdese, Manuel; Segoloni, Giuseppe Paolo; Biancone, Luigi

    2015-02-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease caused by thymidine phosphorylase deficiency which leads to toxic accumulations of thymidine (dThd) and deoxyuridine (dUrd). It lacks an established treatment and the prognosis is traditionally poor. We report a case of a young female patient with normal renal function and MNGIE treated by peritoneal dialysis (PD) and allogeneic bone marrow transplantation (BMT). PD was effective in reducing dThd and dUrd plasma levels and in improving clinical symptoms. To our knowledge, this is the first report on the beneficial effects of PD regarding MNGIE neurological symptoms. PD, therefore, should be considered especially in medically compromised patients as a supportive treatment to improve clinical conditions before BMT.

  16. A novel antimetabolite: TAS-102 for metastatic colorectal cancer.

    Science.gov (United States)

    Miyamoto, Yuji; Lenz, Heinz-Josef; Baba, Hideo

    2016-01-01

    TAS-102 is a new oral anti-tumor drug, composed of a thymidine-based nucleoside analog (trifluridine: FTD) and a thymidine phosphorylase inhibitor (tipiracil hydrochloride: TPI). TAS-102 has been shown to significantly improve overall survival and progression-free survival in patients with refractory metastatic colorectal cancer (mCRC) in placebo-controlled randomized phase II and III trials. The current review summarizes mechanisms of action, pharmacokinetics/dynamics and preclinical and clinical data of TAS-102 in colorectal cancer. TAS-102 is a new salvage-line treatment option for patients with mCRC. TAS-102 is well tolerated and has great potential in future clinical drug combination therapies.

  17. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    Science.gov (United States)

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin-cadmium induced diabetic nephrotoxic rats.

    Science.gov (United States)

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-09-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)-cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ-Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ-Cd induced diabetic nephrotoxic rats.

  19. Identity of the Growth-Limiting Nutrient Strongly Affects Storage Carbohydrate Accumulation in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae▿ † ‡

    Science.gov (United States)

    Hazelwood, Lucie A.; Walsh, Michael C.; Luttik, Marijke A. H.; Daran-Lapujade, Pascale; Pronk, Jack T.; Daran, Jean-Marc

    2009-01-01

    Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negatively correlated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (dilution rate, 0.10 h−1) of S. cerevisiae, the identity of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate, or zinc) strongly affected storage carbohydrate accumulation. The glycogen contents of the biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of the biomass from cultures grown under the other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of S. cerevisiae is not a generic response to excess glucose but instead is strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and msn2Δ msn4Δ strains confirmed that transcriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supported involvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higher than the ratio in the other glucose-excess cultures. PMID:19734328

  20. SNPs in genes functional in starch-sugar interconversion associate with natural variation of tuber starch and sugar content of potato (Solanum tuberosum L.).

    Science.gov (United States)

    Schreiber, Lena; Nader-Nieto, Anna Camila; Schönhals, Elske Maria; Walkemeier, Birgit; Gebhardt, Christiane

    2014-07-31

    Starch accumulation and breakdown are vital processes in plant storage organs such as seeds, roots, and tubers. In tubers of potato (Solanum tuberosum L.) a small fraction of starch is converted into the reducing sugars glucose and fructose. Reducing sugars accumulate in response to cold temperatures. Even small quantities of reducing sugars affect negatively the quality of processed products such as chips and French fries. Tuber starch and sugar content are inversely correlated complex traits that are controlled by multiple genetic and environmental factors. Based on in silico annotation of the potato genome sequence, 123 loci are involved in starch-sugar interconversion, approximately half of which have been previously cloned and characterized. By means of candidate gene association mapping, we identified single-nucleotide polymorphisms (SNPs) in eight genes known to have key functions in starch-sugar interconversion, which were diagnostic for increased tuber starch and/or decreased sugar content and vice versa. Most positive or negative effects of SNPs on tuber-reducing sugar content were reproducible in two different collections of potato cultivars. The diagnostic SNP markers are useful for breeding applications. An allele of the plastidic starch phosphorylase PHO1a associated with increased tuber starch content was cloned as full-length cDNA and characterized. The PHO1a-HA allele has several amino acid changes, one of which is unique among all known starch/glycogen phosphorylases. This mutation might cause reduced enzyme activity due to impaired formation of the active dimers, thereby limiting starch breakdown.