DEFAR - Artigos publicados em periódicos
URI permanente para esta coleçãohttp://www.hml.repositorio.ufop.br/handle/123456789/531
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Item Effect of substrate and pH on the activity of proteases from Fusarium oxysporum var. lini.(1991) Castro, Ieso de Miranda; Lima, Angélica Alves; Paula, Carmem Aparecida de; Nicoli, Jacques Robert; Brandão, Rogélio LopesThe results obtained in this work suggest that both the pH (through selective inhibition) and the carbon source (through repression and acidification or alkalinization of the medium) may play an important role in the distribution of extracellular proteases in Fusarium oxysporum var. lini.Item Glycerol utilization in Fusarium oxysporum var lini : regulation of transport and metabolism.(1991) Castro, Ieso de Miranda; Dias, Maria da Conceição LoureiroGlycerol was transported in the fungus Fusarium oxysporum var. lini by a facilitated diffusion transport system with a half-saturation constant, Ks, of 0.5 mM and a maximum velocity, Vmax, of 0.9 mmol (g dry wt)-l h-l at pH 5 and 25OC. 1,2-Propanediol was a competitive inhibitor of glycerol transport, but the cells did not actively accumulate 1,2-propanediol. The transport system was partially constitutive. In cells grown in the presence of glucose, glycerol was not transported, indicating that the synthesis of the system was under glucose repression. Glycerol base and NADP+-dependent glycerol dehydrogenase activities were present under all physiological conditions tested. A flavin-dependent glycerol phosphate dehydrogenase was induced only when glycerol was the sole energy source in the medium. This enzyme, together with the transport system, constitute the regulated steps in the glycerol metabolic pathway.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 Utilization of lactic acid by Fusarium oxysporum var. lini : regulation of transport and metabolism.(1994) Castro, Ieso de Miranda; Dias, Maria da Conceição LoureiroLactic acid was transported in Fusarium oxysporum var. lini ATCC 10960 by a saturable transport system that had a half-saturation constant of 56.6 7.5 FLM and a maximum velocity of 0.61 0.10 mmol h-1 g-1 (dry weight) at 26°C and pH 5.0. This transport system was inducible and was not expressed in the presence of a repressing substrate. Evidence is presented that the anionic form lactate- was taken up by the cells. Propionic, acetic, pyruvic, and bromoacetic acids but not succinic acid competitively inhibited the transport of lactic acid. Bromoacetic acid, which was not metabolized, was taken up to a steady-state level when intracellular and extracellular concentrations were identical, indicating that the transport system was not accumulative. The enzymatic activity that was physiologically more relevant in the metabolism of lactic acid was lactate: ferricytochrome c oxidase. This enzyme did not exhibit stereospecifity and was induced by lactic acid.Item Intracellular signal triggered by cholera toxin in Saccharomyces boulardii and Saccharomyces cerevisiae.(1998) Brandão, Rogélio Lopes; Castro, Ieso de Miranda; Bambirra, Eduardo Alves; Amaral, Sheila Coutinho; Fietto, Luciano Gomes; Trópia, Maria José Magalhães; Neves, Maria José; Santos, Raquel Gouvêa dos; Gomes, Newton Carlos Marcial; Nicoli, Jacques RobertAs is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.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.