DEFAR - Departamento de Farmácia
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Resultados da Pesquisa
Item Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lin.(2002) Barata, Ricardo Andrade; Andrade, Milton Hércules Guerra de; Rodrigues, Roberta Dias; Castro, Ieso de MirandaAn alkaline serineprotease, capable of hydrolyzing Nu-benzoyl-DL arginine p-nitroanilide, was secreted by Fusurium oxysporum var. hi grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, aMnity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45OC and showed a rapid decrease of activity at 48OC. The optimum pH was around 8.0. Characterization of the protease showed that Ca*+ and MgZ+ cations increased the activity, which was not inhibited by EDTA or l,lO-phenanthroline. The enzyme activity on Nubenzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride,p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and IVtosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5x lo-4 M. The protease had a Michaelis-Menten constant of 0.16 mM and a V,, of 0.60 pm01 released product .min-‘.mg’ enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nu-benzoyl-DL arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A Ki value of 0.04 mM was obtained.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 Yeast genes YOL002C and Sul1 are involved in neomycine resistance.(2001) Castro, Ieso de Miranda; Cabral, Daniela Bastos; Trópia, Maria José Magalhães; Barnabé, Fabiana Maria de Almeida; Brandão, Rogélio LopesIn previous studies we suggested the importance of the control of plasma membrane H+-ATPase by a phosphatidylinositol-like pathway for cellular proton extrusion in Saccharomyces cerevisiae (Brandão et al. 1994; Coccetti et al. 1998). The observations that provided the model above include the inhibition of the glucose-induced activation of the plasma membrane H+-ATPase as well as the inhibition of the glucose-induced external acidification by neomycin, a known inhibitor of the phosphatidylinositol turnover in eukaryotic cells. In this work, using two libraries, we isolated two yeast clones that were able to prevent the inhibition of glucose-induced activation of the H+-ATPase by neomycin. We show that the YOL002C gene, which encodes a protein of unknown function, and the SUL1 gene, which is a sulphate transporter belonging to the major facilitator superfamily, suppress growth inhibition by neomycin. However, they are not required for glucose-induced activation of the plasma membrane H+-ATPase. The resistance of the clones to neomycin is probably related to the level and/or activity of proteins functioning as drug extrusion pumps.Item Relationship between Protein kinase C and derepression of different enzymes.(2002) Salgado, Ana Paula Carneiro; Schuller, Dorit; Casal, Margarida; Leão, Cecília; Leiper, F. C.; Carling, D.; Gomes, Luciano; Trópia, Maria José Magalhães; Castro, Ieso de Miranda; Brandão, Rogélio LopesThe PKC1 gene in the yeast Saccharomyces cerevisiae encodes for protein kinase C which is known to control a MAP kinase cascade consisting of di¡erent kinases: Bck1, Mkk1 and Mkk2, and Mpk1. This cascade a¡ects the cell wall integrity but the phenotype of pkc1v mutants suggests additional targets that have not yet been identi¢ed [Heinisch et al., Mol. Microbiol. 32 (1999) 671^680]. The pkc1v mutant, as opposed to other mutants in the MAP kinase cascade, displays defects in the control of carbon metabolism. One of them occurs in the derepression of SUC2 gene after exhaustion of glucose from the medium, suggesting an involvement of Pkc1p in the derepression process that is not shared by the downstream MAP kinase cascade. In this work, we demonstrate that Pkc1p is required for the increase of the activity of enzymatic systems during the derepression process. We observed that Pkc1p is involved in the derepression of invertase and alcohol dehydrogenase activities. On the other hand, it seems not to be necessary for the derepression of the enzymes of the GAL system. Our results suggest that Pkc1p is acting through the main glucose repression pathway, since introduction of an additional mutation in the PKC1 gene in yeast strains already presenting mutations in the HXKII or MIG1 genes does not interfere with the typical derepressed phenotype observed in these single mutants. Moreover, our data indicate that Pkc1p participates in this process through the control of the cellular localization of the Mig1 transcriptional factor.Item Deficiency of Pkc1 activity affects glycerol metabolism in Saccharomices cerevisiae.(2005) Gomes, Katia das Neves; Freitas, Suzy Magaly Alves Cabral de; Pais, Thiago Martins; Fietto, Juliana Lopes Rangel; Totola, Antonio Helvecio; Arantes, Rosa Maria Esteves; Martins, António; Lucas, Cândida Manuel Ribeiro Simões; Schuller, Dorit; Casal, Margarida; Castro, Ieso de Miranda; Fietto, Luciano Gomes; Rogelio, Lopes BrandãoProtein kinase C is apparently involved in the control of many cellular systems: the cell wall integrity pathway, the synthesis of ribosomes, the appropriated reallocation of transcription factors under specific stress conditions and also the regulation of N-glycosylation activity. All these observations suggest the existence of additional targets not yet identified. In the context of the control of carbon metabolism, previous data had demonstrated that Pkc1p might play a central role in the control of cellular growth and metabolism in yeast. In particular, it has been suggested that it might be involved in the derepression of genes under glucose-repression by driving an appropriated subcellular localization of transcriptional factors, such as Mig1p. In this work, we show that a pkc1D mutant is unable to grow on glycerol because it cannot perform the derepression of the GUT1 gene that encodes glycerol kinase. Additionally, active transport is also partially affected. Using this phenotype, we were able to isolate a new pkc1D revertant. We also isolated two transformants identified as the nuclear exportin Msn5 and the histone deacetylase Hos2 extragenic suppressors of this mutation. Based on these results, we postulate that Pkc1p may be involved in the control of the cellular localization and/or regulation of the activity of nuclear proteins implicated in gene expression.Item Biochemical and molecular characterization of Saccharomyces cerevisiae strains obtained from sugar-cane juice fermentations and their impact in cachaça production.(2008) Oliveira, Valdinéia Aparecida de; Vicente, Maristela de Araújo; Fietto, Luciano Gomes; Castro, Ieso de Miranda; Coutrim, Maurício Xavier; Schüller, Dorit; Alves, Henrique; Casal, Margarida; Santos, Juliana de Oliveira; Araújo, Leandro Dias; Silva, Paulo Henrique Alves da; Brandão, Rogélio LopesSaccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachac¸a, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachac¸a producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5 ,5 -trifluor-D,L-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachac¸a producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachac¸a with a higher quality standard.