DEFAR - Departamento de Farmácia

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Xanthones and Phenylcoumarins from Kielmeyera pumila.
(1988) Nagem, Tanus Jorge; Silva, Maurício de Abreu e
In continuation of our studies on extracts of the Guttiferae family we have examined the stems and fruit of Kielmeyera pumila Pohl, collected in the region of Ouro Preto, State of Minas Gerais, from a specimen identified by the botanist Jo& Badini (Herb. Prof. JosC Badini, Universidade Federal de Ouro Preto, Minas Gerais, Brazil). Several species of this genus has been shown to contain xanthones [l]. Investigation of the stems of the K. pumih has yielded, besides sitosterol, two previously known xanthones (osajaxanthone [Z] and 6-deoxyjacareubin [3]) and two known phenylcoumarins, the 1,2-dihydro-5- hydroxy-2-(l-hydroxy-l-methylethyl)-4-(3-methylbutyryl)- 6-phenyl-furo [2,3-h] [l] benzopyran-8-one [4] and mammeigin (1) [S]. The fruit of the same plant yielded friedelin, a-amyrin, shikimic acid, mammeigin and an isomer of mammeigin to which we have given the trivial name isomammeigin (2).
Regulation of sugar transport systems in Fusarium oxysporum var. lini.
(1990) Brandão, Rogélio Lopes; Dias, Maria da Conceição Loureiro
Fusarium oxysporum var. Iini (ATCC 10960) formed a facilitated diffusion system for glucose (Ks, about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (K5, about 40 ,iM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (Ks, about 30 mM).
Humoral immune response in dogs experimentally infected with Trypanosoma cruzi.
(1991) Lana, Marta de; Vieira, Lauro Mello; Coelho, George Luiz Lins Machado; Chiari, Egler; Veloso, Vanja Maria; Tafuri, Washington Luiz
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 Lopes
The 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.
Glycerol utilization in Fusarium oxysporum var lini : regulation of transport and metabolism.
(1991) Castro, Ieso de Miranda; Dias, Maria da Conceição Loureiro
Glycerol 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.
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 Maria
Addition 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.
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 Maria
Addition 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.
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 Maria
Cells 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.
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 Maria
Addition 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.
Utilization of lactic acid by Fusarium oxysporum var. lini : regulation of transport and metabolism.
(1994) Castro, Ieso de Miranda; Dias, Maria da Conceição Loureiro
Lactic 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.
Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human schistosomiasis. IV. A role for prostaglandin-induced inhibition of in vitro granuloma formation.
(1994) Goes, Alfredo Miranda de; Rezende, Simone Aparecida; Gazzinelli, Giovanni; Doughty, Barbara L.
The prostaglandins (PG) are known to regulate immune cell function (s) and participate in the progression of both acute and chronic inflammatory reactions. Using an in vitro model of Schistosoma mansoni egg-induced hypersensitivity granulomas, we have delineated the role of immune complexes (IC) in the induction andrelease of PG and their inhibitory effects on granuloma development. The hypersensitivity- type granuloma reaction to soluble egg antigen (SEA) was examined using a model of in vitro granuloma ,formation. Our results show that granuloma formation was dramatically suppressed by the addition to the granuloma cultures of IC, PGE,, PGE2, while PGF, alpha had no significant effect. The inhibition of the PG function was achieved by the introduction of anti-PG antibodies that blocked suppression of granuloma,formation. It appears in this model system that IC may inhibit the activity of granuloma formation by stimulating the monocyte-macrophage lineage to release inhibitory mediators. Our results suggest that the prostaglandins E series may be important in the generation and maintenance of suppression of the granulomatous inflammatory response to S. mansoni egg antigens .
Maillard reaction during the processing of "Doce de Leite".
(1994) Pavlovic, Suzana; Santos, Rinaldo Cardoso dos; Glória, Maria Beatriz Abreu
‘Doce de leite’, a dairy product widely consumed in Brazil as a dessert or cake filling, is obtained from the heat treatment of milk and sucrose. On heating, the Maillard reaction occurs with the formation of desirable browncoloured products with a characteristic and pleasant flavour. However, the reaction can also lead to changes in nutritive value. In order to follow chemically the extension of the Maillard reaction and its effect on the nutritive value of ‘doce de leite’ its commercial processing was followed. Increases in the level of free 5-hydroxymethylfurfural and in absorption at 420 nm by pigments attached to the protein were observed. The amino acid analysis showed a significant decrease in lysine levels (33%) as well as in arginine (11%) and histidine (10%). There was also a reduction in available lysine levels, as measured by the fluorodinitrobenzene (50.6%) and by the 2,4,6-trinitrobenzenesulphonic acid (23.5%) methods. These results suggest a deleterious effect of the processing of ‘doce de leite’ on the nutritive value of the milk.
Cell surface analysis of trypanosomes of the subgenus Schizotrypanum isolate from bats.
(1996) Pinto, Artur da Silveira; Teixeira, Luiz Fernando de Medeiros; Padron, Thais Cristina Baeta Soares Souto; Andrade, Arnaldo Feitosa Braga de
Two stocks (M5, M29) of trypanosomes of the subgenus Schizotrypanum were isolated from the bat Phylloslomus hastatus and analyzed for cell electrophoretic mobility (EPM) and lectin binding surface sites. Epimastigotes from the M5 and M29 stocks presented a mean EPM of around - 0.57 and -0.56 μm, s-1.V-1.cm, respectively. Differences in the agglutination profiles were detected between epimastigotes or trypomastigotes from the two parasite populations using lectins with specificity for D-GlcNAc, D-GalNAc, D-Gal and D-Man as probe. Major variation was observed between epimastigote forms. Additionally, the D-GlcNAc binding lectins WGA and BS II strongly interacted with the trypomastigote from both M5 and M29 stocks; this fact is evidence that these trypanosomes are distinct from Trypanosoma (Schizotrypanum) cruzi.
A possible tsunami deposit at the Cretaceous-Tertiary boundary in Pemambuco, northeastern Brazil.
(1996) Albertão, Gilberto Athayde; Martins Júnior, Paulo Pereira
Interdisciplinary and integrated investigations of a stratigraphic succession spanning the Cretaceous-Tertiary (K-T) boundary in Pemambuco (the Poty Quarry section, near Recife), northeastern Brazil, provides direct evidence for the hypothesis of an extraterrestrial bolide impact event. Discussions on the exact position of the K-T boundary point to na impact event in the earliest Danian. One particular bed at the base of the Maria Farinha Formation shows sedimentary characteristics and exotic (probably impact-derived) material which suggest the action of a tsunami wave. The distribution of iridium concentrations throughout the studied succession records a major peak of iridium (up to 69 times the background levels) at about 15-20 cm above the main tsunami bed.
Characterization of two isolates of Trypanosoma cruzi obtained from the patient Berenice, the first human case of Chagas disease.
(1996) Lana, Marta de; Chiari, Cléa de Andrade; Chiari, Egler; Morel, Carlos Medicis; Gonçalves, Antônio M.; Romanha, Alvaro José
Two isolates of Trypanosoma cruzi were obtained from the patient Berenice, the first human case of Chagas’ disease (Chagas 1909), when she was 55 and 71 years old, respectively. The isolates were characterized on the basis of their epimastigote-trypomastigote differentiation in liquid media and of the electrophoretic pattern of EcoR1 digestion products of kinetoplast DNA (k- DNA) minicircles (schizodeme) and isoenzyme patterns (zymodeme). Clear differences were found between the isolates, suggesting the occurrence of a heterogeneous population of T. cruzi in the infection of this patient.
IL-10 plays a role in modulation of human granulomatous hypersensitivity against Schistosoma mansoni eggs induced by immune complexes.
(1997) Rezende, Simone Aparecida; Teixeira, David Nascimento Silva; Drummond, Sandra Costa; Goes, Alfredo Miranda de
It has been demonstrated that the chronic intestinal form of schistosomiasis is associated with the establishment and maintenance of a variety of immunoregulatory mechanisms that lead to a diminished granulomatous reaction to parasite eggs. Using an in vitro model of granuloma reaction we showed that immune complexes (IC) isolated from the sera of chronic intestinal schistosomiasis patients were able to reduce the granulomatous hypersensitivity (developed by peripheral blood mononuclear cells (PBMC) from schistosomiasis patients) to soluble egg antigen (SEA)-conjugated polyacrylamide beads (PB–SEA). This inhibitory activity, mediated by IC, was also observed in the proliferative response of PBMC stimulated with SEA and soluble worm antigen preparation (SWAP). Furthermore, we observed a decrease in TNF-α and an increase in IL-10 production by PBMC treated with IC in an in vitro granuloma reaction. This phenomenon was also seen in a cell proliferation assay when PBMC were treated with IC and stimulated with S. mansoni antigens. These results demonstrate that circulating IC may down-regulate PBMC reactivity to S. mansoni antigens by changing the cytokine pattern produced by these cells.
Purification and substrate specificity of an angiotensin converting elastase-2 from the rat mesenteric arterial bed perfusate.
(1998) Paula, Carmem Aparecida de; Sousa, Marcelo Valle de; Salgado, Maria Cristina de Oliveira; Oliveira, Eduardo Brandt de
A soluble angiotensin (Ang) II-generating enzyme has been purified to homogeneity from the rat mesenteric arterial bed(MAB) perfusate by a combination of gel filtration and affinity chromatographies. The enzyme is a glycoprotein of 28.5 kDa(SDS-PAGE), whose N-terminal sequence is identical with that of the rat pancreatic elastase-2 ; therefore the enzyme willhenceforth be referred to as rat MAB elastase-2. When Ang I was used as the substrate, the enzyme specifically released AngII and the dipeptide His-Leu (Km=36WM;Kcat= 1530 min31). The catalytic efficiency (Kcat/Km= 42.5 min31WM31) of thisreaction was comparable to those of other known Ang I-converting enzymes. The proteolytic specificity of the purifiedenzyme toward mellitin, oxidized insulin B chain, somatostatin-14 and renin substrate tetradecapeptide suggested that theenzyme-substrate interaction was defined by an extended substrate binding site, typical of elastases-2 of pancreatic origin. According to the sensitivity of the rat MAB elastase-2 to various inhibitors this enzyme could be described as a member ofthe chymostatin-sensitive group of Ang II-forming serine proteases. The localization and biochemical properties of thisenzyme suggest that it might play a role in the regional control of vascular tonus.
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 Maria
Addition 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.
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 Robert
As 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.
Compared vectorial transmissibility of pure and mixed clonal genotypes of Trypanosoma cruzi in Triatoma infestans.
(1998) Pinto, Artur da Silveira; Lana, Marta de; Bastrenta, Brigitte; Barnabé, Christian; Quesney, Virginie; Noel, Sébastien; Tibayrenc, Michel
A total of 15 mixtures involving 9 di erent stocks attributed to the 19/20, 32 and 39 major clonal genotypes of Trypanosoma cruzi were used to infect third-instar nymphs of Triatoma infestans via an arti®- cial feeding device. Three biological parameters were considered: (1) the percentage of infected insects (%II), (2) the number of ¯agellates per insect (NFI), and (3) the percentage of trypomastigotes per insect (%DIF). Ge- netic characterization by both multilocus enzyme elec- trophoresis (MLEE) and random ampli®cation of polymorphic DNA (RAPD) indicated that in almost all cases (87%), mixtures remained present after completion of the whole cycle in the insect vector. Two lines of comparison were performed: (1) pure clonal genotypes versus corresponding mixed clonal genotypes and (2) the actual behavior of mixed clonal genotypes versus the expected behavior of the theoretical mixture (i.e. the arithmetic mean of the results observed for each of the two clonal genotypes taken separately). Statistical analyses of the variables were made di cult because of the presence of large standard deviations. Nevertheless, in several cases, mixtures di ered signi®cantly from pure clonal genotypes, and in one case the actual mixture di ered signi®cantly from the theoretical mixture. In some cases, interaction (either potentialization or re- ciprocal inhibition) could be suspected.