EFAR - Escola de Farmácia
URI permanente desta comunidadehttp://www.hml.repositorio.ufop.br/handle/123456789/451
Notícias
O curso de Farmácia em Ouro Preto foi criado em 1839, sendo a mais antiga Escola de Farmácia da América Latina.
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6 resultados
Resultados da Pesquisa
Item Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae.(1993) Aelst, Linda Van; Hohmann, Stefan; Bulaya, Botchaka; Koning, Wim de; Sierkstra, Laurens; Neves, Maria José; Luyten, Kattie; Alijo, Rafael; Ramos, José; Coccetti, Paola; Martegani, Enzo; Rocha, Neuza Maria de Magalhaes; Brandão, Rogélio Lopes; Dijck, Patrick Van; Vanhalewyn, Mieke; Durnez, Peter; Jans, Arnold W. H; Thevelein, Johan MariaCells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinosrtot turnover, rapid post-translatronal effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for general Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggsi aHeles (fic/pf, bypi and cifi) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggsi mutants are unable to grow on glucose or Received 25 November, 1992; revised and accepted 15 February, 1993. •For correspondence. Tel. (16) 220931; Fax (16) 204415. tThese two authors contributed equally to this paper. related readily fermentable sugars, apparently owing to unrestricted influx of sugar Into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggsi^ strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggsi mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggsi diploids to sporulate.Item Possible involvement of a phosphatidylinositol-type signaling pathway in glucose-induced activation of plasma membrane ATPase and cellular proton in the yeast Sacchamyces cerevisiae.(1994) Brandão, Rogélio Lopes; Rocha, Neuza Maria de Magalhaes; Alijo, Rafael; Ramos, José; Thevelein, Johan MariaAddition of glucose to cells of the yeast Saccharomyces cerevisiae causes rapid activation of plasma membrane H+-ATPase and a stimulation of cellular H ÷ extrusion. We show that addition of diacylglycerol and other activators of protein kinase C to intact cells also activates the H+-ATPase and causes at the same time a stimulation of H ÷ extrusion from the cells. Both effects are reversed by addition of staurosporine, a protein kinase C inhibitor. Addition of staurosporine or calmidazolium, an inhibitor of Ca2+/calmodulin-dependent protein kinases, separately, causes a partial inhibition of glucose-induced H+-ATPase activation and stimulation of cellular H + extrusion; together they cause a more potent inhibition. Addition of neomycin, which complexes with phosphatidylinositol 4,5-bisphosphate, or addition of compound 48/80, a phospholipase C inhibitor, also causes near complete inhibition. Diacylglycerol and other protein kinase C activators had no effect on the activity of the K+-uptake system and the activity of trehalase and glucose-induced activation of the K+-uptake system and trehalase was not inhibited by neomycin, supporting the specificity of the effects observed on the H+-ATPase. The results support a model in which glucose-induced activation of H+-ATPase is mediated by a phosphatidylinositol-type signaling pathway triggering phosphorylation of the enzyme both by protein kinase C and one or more Ca2+/calmodulin-dependent protein kinases.Item During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1.(1998) Ernandes, José Roberto; Meirsman, Catherine; Rolland, Filip; Winderickx, Joris; Winde, Johannes de; Brandão, Rogélio Lopes; Thevelein, Johan MariaIn the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1 =GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Ä mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total D-glucose measured (intracellular+periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0·5–2 mM in the wild-type strain, &10 mM in a hxk1Ä hxk2Ä glk1Ä and 2–3 mM in a tps1Ä strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity.Item Glucose induced activation of the plasma membrane ATPase in Fusarium oxysporum.(1992) Brandão, Rogélio Lopes; Castro, Ieso de Miranda; Passos, Jomar Becher dos; Nicoli, Jacques Robert; Thevelein, Johan MariaAddition of glucose and other sugars to derepressed cells of the fungus Fusarium oxysporum var. Zini triggered activation of the plasma membrane H+-ATPase within 5 min. Glucose was the best activator while galactose and lactose had a lesser effect. The activation was not prevented by previous addition of cycloheximide and it was fully reversible when the glucose was removed. The activation process in uiuo also caused changes in the kinetic properties of the enzyme. The non-activated enzyme had an apparent K, of about 3.2 mM for ATP whereas the activated enzyme showed an apparent K,,, of 0.26 mM. In addition, the pH optimum of the H+-ATPase changed from 6.0 to 7.5 upon activation. The activated enzyme was more sensitive to inhibition by vanadate. When F. oxysporum was cultivated in media containing glucose as the major carbon source, enhanced M+-ATPase activity was largely confined to the period corresponding to the lag phase, i.e. just before the start of acidification of the medium. This suggests that the activation process might play a role in the onset of extracellular acidification. Addition of glucose to F. oxysporum var. Zini cells also caused an increase in the cAMP level. No reliable increase could be demonstrated for the other sugars. Addition of proton ionophores such as DNP and CCCP at pH 5-0 caused both a large increase in the intracellular level of cAMP and in the activity of the plasma membrane H+- ATPase. Inhibition of the DNP-induced increase in the cAMP level by acridine orange also resulted in inhibition of the activation of plasma membrane H+-ATPase. These results suggest a possible causal relationship between the activity of F. oxysporum var. Zini plasma membrane H+-ATPase and the intracellular level of CAMP.Item Glucose-induced activation of plasma membrane H+-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis.(1992) Passos, Jomar Becher dos; Vanhalewyn, Mieke; Brandão, Rogélio Lopes; Castro, Ieso de Miranda; Nicoli, Jacques Robert; Thevelein, Johan MariaAddition of glucose-related fermentable sugars or pro,tonophores to derepressed cells of the yeast Saccharomyces ceret'isiae causes a 3- to 4-fold activation of the plasma membrane H +-A'fPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP .~jnthesis, incohati~a at the restrictive temperature reduced the extent of H+-ATPase activation, Incubation of nontemperature- sensitive strains, however, at such temperatures also caused reduction of H +-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H+-ATPase aCtivation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H +-A'l'Pase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAlVlP-protein kinase A signaling pathway is not required for glucose activation of the H*-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phnsphorylating enzymes hexokinase Pl and Pll and glucokinase showed that activation of the H+-ATPase with glucose or fructose was completely dependent on the presence cf a kinase able m phnsphorylate the sugar. These and other data concerning the role of init,:al sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H+-ATPase have a common initiation point.Item The PLC1 encoded phospholipase C in the yeast Saccharomyces cerevisiae is essential for glucose-induced phosphatidylinositol turnover and activation of plasma membrane H -ATPase.(1998) Coccetti, Paola; Tisi, Renata; Martegani, Enzo; Teixeira, Leonardo Souza; Brandão, Rogélio Lopes; Castro, Ieso de Miranda; Thevelein, Johan MariaAddition of glucose to glucose-deprived cells of the yeast Saccharomyces cerevisiae triggers rapid turnover of phosphatidylinositol, phosphatidylinositol-phosphate and phosphatidylinositol 4,5-bisphosphate. Glucose stimulation of PI turnover was measured both as an increase in the specific ratio of 32P-labeling and as an increase in the level of diacylglycerol after addition of glucose. Glucose also causes rapid activation of plasma membrane H.-ATPase. We show that in a mutant lacking the PLC1 encoded phospholipase C, both processes were strongly reduced. Compound 48/80, a known inhibitor of mammalian phospholipase C, inhibits both processes. However, activation of the plasma membrane H.- ATPase is only inhibited by concentrations of compound 48/80 that strongly inhibit phospholipid turnover. Growth was inhibited by even lower concentrations. Our data suggest that in yeast cells, glucose triggers through activation of the PLC1 gene product a signaling pathway initiated by phosphatidylinositol turnover and involved in activation of the plasma membrane H.-ATPase.