Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, the optimal hydrolysis temperature for sucrose, raffinose and inulin was 55 degrees C and the optimal pH for sucrose was 4.75, the apparent K-m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides, the results obtained suggest the hypothesis that enzyme production was constitutive.

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80 kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECNI components. (c) 2006 Elsevier SAS. All rights reserved.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Inserções de aminoácidos na protease têm sido raramente descritas em pacientes infectados pelo HIV. Uma destas inserções foi, recentemente, descrita no codon 35, embora seu impacto na resistência mantém-se pouco conhecido. Este trabalho apresenta um caso de uma variante viral com inserção no codon 35 da protease, descrita pela primeira vez em Bauru, São Paulo, Brasil, circulante em um homem, caucasiano, com 38 anos, o qual apresenta infecção assintomática pelo HIV desde 1997. A variante isolada mostrou uma inserção no codon 35 da protease de dois aminoácidos: uma treonina e um ácido aspártico, resultando na sequência de aminoácidos E35E_TD.

Experimental poisoning by Senecio brasiliensis in calves: quantitative and semi-quantitative study on changes in the hepatic extracellular matrix and sinusoidal cells

A matriz extracelular (MEC) desempenha um papel importante em lesões hepáticas crônicas e tem sido estudada em modelos de intoxicação experimental. em bovinos, no entanto, não há estudos específicos sobre a MEC hepática normal ou com lesões crônicas. Por isso, foi desenvolvido um modelo de intoxicação experimental hepático usando Senecio brasilliensis, uma planta que contém alcalóides pirrolizidínicos e causa lesão hepática dependente da dose. Cinco bezerros receberam por via oral, 0.38g/kg de folhas secas por 24 dias. Biópsias hepáticas foram obtidas a cada 15 dias durante 60 dias. Sinais clínicos de complicações digestivas surgiram da terceira semana do experimento. Um bezerro morreu aos 45 dias e os outros quatro foram avaliados até os 60 dias. As biópsias hepáticas foram processadas para microscopia óptica, imuno-histoquímica e microscopia eletrônica de transmissão. No trigésimo dia, as lesões hepáticas eram progessivas caracterizadas por vacuolização hepatocelular, necrose, apoptose, megalocitose, e fibrose centrolobular, pericelular e portal. Foram realizadas avaliações quantitativas e semi-quantitativas de componentes da MEC hepática antes e após o aparecimento das lesões. Foi realizada morfometria do colágeno total e do sistema de fibras elásticas. Colágeno total e colágenos tipos I e III aumentaram progressivamente em todos os locais do fígado. Mudanças na localização, quantidade e disposição do sistema de fibras elásticas foram também observadas. Houve um aumento significativo de células de Kupffer aos 30 dias e de células sinusoidais totais aos 45 e 60 dias. As lesões hepáticas neste experimento foram progressivas mesmo após a remoção da planta. Lesões de fibrose severa foram localizadas principalmente nos espaços porta, seguido por fibrose veno-oclusiva e pericelular. Os colágenos tipo I e tipo III foram observados no fígado normal e no fígado dos bezerros afetados, com predomínio do tipo I. Nos bezerros afetados o aumento do colágeno total e do sistema de fibras elásticas foi paralelo ao aumento no número das células sinusoidais.

Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

Effect of green Coffea arabica L. seed oil on extracellular matrix components and water-channel expression in in vitro and ex vivo human skin models

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystallization, preliminary X-ray analysis and Patterson search of a new aspartic protease isolated from human urine

Aspartic protease (EC 3.4.23) make up a widely distributed class of enzymes in animals, plants, microbes and, viruses. In animals these enzymes perform diverse functions, which range from digestion of food proteins to very specific regulatory roles. In contrast the information about the well-characterized aspartic proteases, very little is known about the corresponding enzyme in urine. A new aspartic protease isolated from human urine has been crystallized and X-ray diffraction data collected to 2.45 Angstrom resolution using a synchrotron radiation source. Crystals belong to the space group P2(1)2(1)2(1) the cell parameters obtained were a=50.99, b=75.56 and c=89.90 Angstrom. Preliminary analysis revealed the presence of one molecule in the asymmetric unit. The structure was determined using the molecular replacement technique and is currently being refined using simulated annealing and conjugate gradient protocols.

Mucosa-associated but not luminal Escherichia coli is augmented in Crohn's disease and ulcerative colitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prostate carcinogenesis induced by N-methyl-N-nitrosourea (mnu) in gerbils: Histopathological diagnosis and potential invasiveness mediated by extracellular matrix components

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protease activity in Giardia duodenalis trophozoites of axenic strains isolated from symptomatic and asymptomatic patients

We have examined by gelatin-SDS-PAGE the protease activity in cell lysates of Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic patient (BTU-11) and an asymptomatic carrier (BTU-10), and the reference strain Portland 1 (P1). The proteolysis band patterns showed differences among strains isolated from asymptomatic and symptomatic individuals. The lysate of the strain BTU-10, showed only five hydrolysis bands, while a greater number of bands (10-11 bands) was seen in strains BTU-11 and P1. The protease activity in all lysates was inhibited by cysteine (E-64 and iodoacetamide) and serine proteases (TPCK and TLCK) inhibitors, but not by PMSF and EDTA. In general, the results revealed protease activities in G. duodenalis trophozoites of Brazilian axenic strains and the predominance of cysteine proteinases. It should be stressed the inter-strain difference in hydrolysis band patterns observed between strains isolated from symptomatic patients and the strain obtained from an asymptomatic carrier.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

Purification and Characterization of Two Extracellular Xylanases from Penicillium sclerotiorum: A Novel Acidophilic Xylanase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)