948 resultados para Glycogen accumulating organism (gao)


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GP catalyzes the phosphorylation of glycogen to Glc-1-P. Because of its fundamental role in the metabolism of glycogen, GP has been the target for a systematic structure-assisted design of inhibitory compounds, which could be of value in the therapeutic treatment of type 2 diabetes mellitus. The most potent catalytic-site inhibitor of GP identified to date is spirohydantoin of glucopyranose (hydan). In this work, we employ MD free energy simulations to calculate the relative binding affinities for GP of hydan and two spirohydantoin analogues, methyl-hydan and n-hydan, in which a hydrogen atom is replaced by a methyl- or amino group, respectively. The results are compared with the experimental relative affinities of these ligands, estimated by kinetic measurements of the ligand inhibition constants. The calculated binding affinity for methyl-hydan (relative to hydan) is 3.75 +/- 1.4 kcal/mol, in excellent agreement with the experimental value (3.6 +/- 0.2 kcal/mol). For n-hydan, the calculated value is 1.0 +/- 1.1 kcal/mol, somewhat smaller than the experimental result (2.3 +/- 0.1 kcal/mol). A free energy decomposition analysis shows that hydan makes optimum interactions with protein residues and specific water molecules in the catalytic site. In the other two ligands, structural perturbations of the active site by the additional methyl- or amino group reduce the corresponding binding affinities. The computed binding free energies are sensitive to the preference of a specific water molecule for two well-defined positions in the catalytic site. The behavior of this water is analyzed in detail, and the free energy profile for the translocation of the water between the two positions is evaluated. The results provide insights into the role of water molecules in modulating ligand binding affinities. A comparison of the interactions between a set of ligands and their surrounding groups in X-ray structures is often used in the interpretation of binding free energy differences and in guiding the design of new ligands. For the systems in this work, such an approach fails to estimate the order of relative binding strengths, in contrast to the rigorous free energy treatment.

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Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

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Glycogen phosphorylase (GP) is currently exploited as a target for inhibition of hepatic glycogenolysis under high glucose conditions. Spirohydantoin of glucopyranose and N-acetyl-beta-D-glucopyranosylamine have been identified as the most potent inhibitors of GP that bind at the catalytic site. Four spirohydantoin and three beta-D-glucopyranosylamine analogs have been designed, synthesized and tested for inhibition of GP in kinetic experiments. Depending on the functional group introduced, the K(i) values varied from 16.5 microM to 1200 microM. In order to rationalize the kinetic results, we determined the crystal structures of the analogs in complex with GP. All the inhibitors bound at the catalytic site of the enzyme, by making direct and water-mediated hydrogen bonds with the protein and by inducing minor movements of the side chains of Asp283 and Asn284, of the 280s loop that blocks access of the substrate glycogen to the catalytic site, and changes in the water structure in the vicinity of the site. The differences observed in the Ki values of the analogs can be interpreted in terms of variations in hydrogen bonding and van der Waals interactions, desolvation effects, ligand conformational entropy, and displacement of water molecules on ligand binding to the catalytic site.

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In this study we show that both glycogen synthase kinase 3 (GSK3) isoforms, GSK3alpha and GSK3beta, are present in human platelets and are phosphorylated on Ser(21) and Ser(9), respectively, in platelets stimulated with collagen, convulxin and thrombin. Phosphorylation of GSK3alpha/beta was dependent on phosphoinositide 3-kinase (PI3K) activity and independent of platelet aggregation, and correlated with a decrease in GSK3 activity that was preserved by pre-incubating platelets with PI3K inhibitor LY294002. Three structurally distinct GSK3 inhibitors, lithium, SB415286 and TDZD-8, were found to inhibit platelet aggregation. This implicates GSK3 as a potential regulator of platelet function. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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We report the isolation and characterization of a hitherto unknown gram-negative, rod-shaped Neisseria-like organism from an infected wound resulting from a bite from a kinkajou. Based on both phenotypic and phylogenetic evidence, it is proposed that the unknown organism be classified as a new species, Kingella potus sp. nov.

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Phenotypic and phylogenetic studies were performed on two strains of an unidentified Gram-positive, fastidious, non-spore-forming, coccus-shaped bacterium recovered from human blood. The organism was catalase-negative and grew under strictly anaerobic conditions and in the presence of 2 and 6% O-2. Comparative 16S rRNA gene sequencing demonstrated that the unidentified bacterium was, phylogenetically, far removed from peptostreptococci and related Gram-positive coccus-shaped organisms, but exhibited a phylogenetic association with Clostridium rRNA cluster III [as defined by Collins et al, Int J Syst Bacteriol 44 (1994), 812-826]. Sequence divergence values of 15% or more were observed between the unidentified bacterium and all other recognized species within this and related rRINIA clostridial clusters. Treeing analysis showed that the unknown bacterium formed a deep line branching at the periphery of rRNA cluster III and represents a hitherto unknown genus within this supra-generic grouping. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from blood be classified in a new genus, Fastidiosipila gen. nov., as Fastidiosipila sanguinis sp, nov. The type strain of Fastidiosipila sanguinis is CCUG 47711(T) (= CIP 108292(T)).

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An unknown Gram-positive, catalase-negative, ovoid-shaped bacterium isolated from the submandibular abscess of a rabbit was subjected to a polyphasic taxonomic analysis. Comparative 16S rRNA gene sequencing demonstrated the unknown coccus represents a new subline within the genus Gemella. The unknown isolate was readily distinguished from other recognized members of the genus Gemella, namely Gemella haemolysans, Gemella bergeri, Gemella morbillorum, Gemella palaticanis and Gemella sanguinis, by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium is classified in the genus Gemella as Gemella cuniculi sp. nov. The type strain is CCUG 42726T.

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Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained.

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Glycogen synthase kinase 3 (GSK3, of which there are two isoforms, GSK3alpha and GSK3beta) was originally characterized in the context of regulation of glycogen metabolism, though it is now known to regulate many other cellular processes. Phosphorylation of GSK3alpha(Ser21) and GSK3beta(Ser9) inhibits their activity. In the heart, emphasis has been placed particularly on GSK3beta, rather than GSK3alpha. Importantly, catalytically-active GSK3 generally restrains gene expression and, in the heart, catalytically-active GSK3 has been implicated in anti-hypertrophic signalling. Inhibition of GSK3 results in changes in the activities of transcription and translation factors in the heart and promotes hypertrophic responses, and it is generally assumed that signal transduction from hypertrophic stimuli to GSK3 passes primarily through protein kinase B/Akt (PKB/Akt). However, recent data suggest that the situation is far more complex. We review evidence pertaining to the role of GSK3 in the myocardium and discuss effects of genetic manipulation of GSK3 activity in vivo. We also discuss the signalling pathways potentially regulating GSK3 activity and propose that, depending on the stimulus, phosphorylation of GSK3 is independent of PKB/Akt. Potential GSK3 substrates studied in relation to myocardial hypertrophy include nuclear factors of activated T cells, beta-catenin, GATA4, myocardin, CREB, and eukaryotic initiation factor 2Bvarepsilon. These and other transcription factor substrates putatively important in the heart are considered. We discuss whether cardiac pathologies could be treated by therapeutic intervention at the GSK3 level but conclude that any intervention would be premature without greater understanding of the precise role of GSK3 in cardiac processes.

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OBJECTIVE: Platelet endothelial cell adhesion molecule-1 (PECAM-1) regulates platelet response to multiple agonists. How this immunoreceptor tyrosine-based inhibitory motif-containing receptor inhibits G protein-coupled receptor-mediated thrombin-induced activation of platelets is unknown. APPROACH AND RESULTS: Here, we show that the activation of PECAM-1 inhibits fibrinogen binding to integrin αIIbβ3 and P-selectin surface expression in response to thrombin (0.1-3 U/mL) but not thrombin receptor-activating peptides SFLLRN (3×10(-7)-1×10(-5) mol/L) and GYPGQV (3×10(-6)-1×10(-4) mol/L). We hypothesized a role for PECAM-1 in reducing the tethering of thrombin to glycoprotein Ibα (GPIbα) on the platelet surface. We show that PECAM-1 signaling regulates the binding of fluorescein isothiocyanate-labeled thrombin to the platelet surface and reduces the levels of cell surface GPIbα by promoting its internalization, while concomitantly reducing the binding of platelets to von Willebrand factor under flow in vitro. PECAM-1-mediated internalization of GPIbα was reduced in the presence of both EGTA and cytochalasin D or latrunculin, but not either individually, and was reduced in mice in which tyrosines 747 and 759 of the cytoplasmic tail of β3 integrin were mutated to phenylalanine. Furthermore, PECAM-1 cross-linking led to a significant reduction in the phosphorylation of glycogen synthase kinase-3β Ser(9), but interestingly an increase in glycogen synthase kinase-3α pSer(21). PECAM-1-mediated internalization of GPIbα was reduced by inhibitors of dynamin (Dynasore) and glycogen synthase kinase-3 (CHIR99021), an effect that was enhanced in the presence of EGTA. CONCLUSIONS: PECAM-1 mediates internalization of GPIbα in platelets through dual AKT/protein kinase B/glycogen synthase kinase-3/dynamin-dependent and αIIbβ3-dependent mechanisms. These findings expand our understanding of how PECAM-1 regulates nonimmunoreceptor signaling pathways and helps to explains how PECAM-1 regulates thrombosis.

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Background and Aims Ptilotus polystachyus (green mulla mulla; ptilotus) is a short-lived perennial herb that occurs widely in Australia in arid and semi-arid regions with nutrient poor soils. As this species shows potential for domestication, its response to addition of phosphorus (P) and nitrogen (N) was compared to a variety of the domesticated exotic perennial pasture herb Cichorium intybus (chicory), ‘Puna’. Methods Pots were filled with 3 kg of an extremely nutrient-deficient sterilized field soil that contained 3 mg kg−1 mineral N and 2 mg kg−1 bicarbonate-extractable P. The growth and P and N accumulation of ptilotus and chicory in response to seven rates of readily available phosphorus (0–300 mg P pot−1) and nitrogen (N) (0–270 mg N pot−1) was examined. Key Results Ptilotus grew extremely well under low P conditions: shoot dry weights were 23, 6 and 1·7 times greater than for chicory at the three lowest levels of P addition, 0, 15 and 30 mg P pot−1, respectively. Ptilotus could not downregulate P uptake. Concentrations of P in shoots approached 4 % of dry weight and cryo-scanning electron microscopy and X-ray microanalysis showed 35–196 mm of P in cell vacuoles in a range of tissues from young leaves. Ptilotus had a remarkable tolerance of high P concentrations in shoots. While chicory exhibited symptoms of P toxicity at the highest rate of P addition (300 mg P pot−1), no symptoms were present for ptilotus. The two species responded in a similar manner to addition of N. Conclusions In comparison to chicory, ptilotus demonstrated an impressive ability to grow well under conditions of low and high P availability. Further study of the mechanisms of P uptake and tolerance in ptilotus is warranted.

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The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was their infused with glucagon (1 nM), isoproterenot (2 mu M), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase. Copyright (c) 2008 John Wiley & Sons, Ltd.

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IL-1 beta, TNF-alpha, cytokine-induced neutrophil chemoattractant-2 alpha/beta, and IL-10 measurements were performed in elicited peritoneal cells from control, diabetic, and insulin-treated diabetic rats. Production/liberation of these cytokines was decreased in elicited peritoneal cells from diabetic rats. These changes were abolished by insulin treatment of diabetic rats. The alterations observed might be involved in the impaired inflammatory response and high occurrence of apoptosis observed in neutrophils under diabetic states.

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Glycogen content of white and red skeletal muscles, cardiac muscle, and liver was investigated in conditions where changes in plasma levels of non-esterified fatty acids (NEFA) occur. The experiments were performed in fed and 12 and 48 h-fasted rats. The animals were also submitted to swimming for 10 and 30 min. Glycogen content was also investigated in both pharmacologically induced low plasma NEFA levels fasted rats and pharmacologically induced high plasma NEFA levels fed rats. The participation of Akt and glycogen synthase kinase-3 (GSK-3) in the changes observed was investigated. Plasma levels of NEFA, glucose, and insulin were determined in all conditions. Fasting increased plasma NEFA levels and reduced glycogen content in the liver and skeletal muscles. However, an increase of glycogen content was observed in the heart under this condition. Akt and GSK-3 phosphorylation was reduced during fasting in the liver and skeletal muscles but it remained unchanged in the heart. Our results suggest that in conditions of increased plasma NEFA levels, changes in insulin-stimulated phosphorylation of Akt and GSK-3 and glycogen content vary differently in liver, skeletal muscles, and heart. Akt and GSK-3 phosphorylation and glycogen content are decreased in liver and skeletal Muscles, but in the heart it remain unchanged (Akt and GSK-3 phosphorylation) or increased (glycogen content) due to consistent increase of plasma NEFA levels. Copyright (C) 2009 John Wiley & Sons, Ltd.

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Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.