Identification and characterization of excretory/secretory products of Giardia duodenalis trophozoites

One of the major questions concerning Giardia is the understanding of pathophysiological processes associated with small intestine abnormalities. There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in the host intestinal epithelium. The present investigation was undertaken to examine the protease activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). E/S products from trophozoites of each strain in conditioned medium were tested with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the protein profiles, and the protease activity was analyzed using substrate-impregnated SDS-PAGE (gelatin and collagen) and hemoglobin assay. The proteases characterization was based on inhibition assays including synthetic inhibitors. Electrophoresis analysis of E/S products revealed a banding pattern composed by few bands (4 to 6 bands) in the migration region of 123 to 28 kDa. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted substrate degradation and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibitor assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases, although the presence of serine proteases was also indicated. Degradation of substrates including collagen and hemoglobin could lead us to speculate different functions of Giardia excreted/secreted proteases in vivo, but to confirm this possibility and to elucidate its implication on host-parasite interactions, further experiments applying protocols for the purification of proteases are necessary. Even so, our observations are relevant and hold the perspective for the understanding about protease activity in Giardia trophozoites of axenic strain isolated in an endemic area.

Characterization of the excretory/secretory products of Dermatobia hominis larvae, the human bot fly

Proteolytic activity in excretory/secretory products (ESP) of first- (L1), second- (L2) and third-instar (L3) larvae of Dermatobia hominis was analyzed through gelatin-gel and colorimetric enzyme assays with the chromogenic substrates azocasein and BApNA. The functional characterization of proteases was based on inhibition assays including synthetic inhibitors. ESP were obtained from new-hatched larvae reared in the laboratory and from second- and third-instar larvae removed from naturally infested cattle. Gelatin-gel analysis evidenced few bands of proteolysis, predominantly of high apparent molecular masses, in ESP of L1, whereas in the gel of L2 and U ESP there was a wide range of proteolytic activity most of them not resolved in a single species. Azocasein assays revealed a progressive increase of protease activity from first- to third-instar larvae. Protease inhibitor assays revealed a predominance of metalloproteases in L1 ESP that could be related to a skin penetration process and to a diversion of host immune response. The predominance of serine proteases in L2 and L3 and the great tryptic activity presented by L3 ESP were attributed to an increasing trophic activity by the growing larvae, since the viability of adult flies strictly depends on larval abilities to assimilate nutrients from the host. Taking together, these results suggest that Dematobia larvae secrete/excrete different proteases that may be related to diverse functions during host penetration and infestation, which reinforces the relevance of the study of such proteolytic enzymes. (C) 2009 Elsevier B.V. All rights reserved.

Production of Crude Xylanase from Thermoascus Aurantiacus CBMAI 756 Aiming the Baking Process

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

Controlled hydrolysis of cheese whey proteins using trypsin and alpha-chymotrypsin

This study examined the production of protein hydrolysates with controlled composition from cheese whey proteins. Cheese whey was characterized and several hydrolysis experiments were made using whey proteins and purified beta -lactoglobulin, as substrates, and trypsin and a-chymotrypsin, as catalysts, at two temperatures and several enzyme concentrations. Maximum degrees of hydrolysis obtained experimentally were compared to the theoretical values and peptide compositions were calculated. For trypsin, 100% of yield was achieved; for alpha -chymotrypsin, hydrolysis seemed to be dependent on the oligopeptide size. The results showed that the two proteases could hydrolyze beta -lactoglobulin. Trypsin and alpha -chymotrypsin were stable at 40 degreesC, but a sharp decrease in the protease activity was observed at 55 degreesC.

ANALISE ANTIGENICA DE PROTEASES DE DIFERENTES CEPAS DE TRICHOMONAS VAGINALIS ISOLADAS DE PACIENTES DA REGIAO DE ARARAQUARA, SP, BRASIL

Trichomonas vaginalis is the flagellate that causes trichomonadiasis, a sexually transmitted disease. Immunological methods have been proposed for the study of antigenic characterization using strains isolated from different patients. This work compares protease profiles from the different strains using gelatin containing polyacrylamide gels to analyse the protease activity. High molecular weight proteases (20 to 100 kDa) were found on gels showing quantitative differences. Human IgG antiproteases were detected by immunoblotting using the same extracts. These proteases could be related with T. vaginalis pathogenesis.

Identificação e caracterização dos produtos de excreção/secreção de trofozóitos de Giardia duodenalis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mycelial biomass and biochemical properties of proteases produced by Lentinus citrinus DPUA 1535 (higher basidiomycetes) in submerged cultivation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Uso de complexo enzimático em dietas para tilápia do Nilo: digestibilidade, atividade enzimática, desempenho produtivo e parâmetros fisiológicos

Pós-graduação em Zootecnia - FCAV

Partial characterization of protease from a thermophilic fungus, Thermoascus aurantiacus, and its hydrolytic activity on bovine casein

Proteases are one of the most important groups of industrial enzymes, with considerable application in the food industry. The aim of this work was to study a novel protease produced by the thermophilic fungus, Thermoascus aurantiacus, through solid-state fermentation (SSF). The enzyme acted optimally at pH 5.5 and 60 degrees C it was stable up to 60 degrees C for 1 h and in the pH range 3.0-9.5. To elucidate the enzyme's proteolytic activity, its hydrolytic profile on bovine casein, an important protein in the food industry, was studied by enzymatic hydrolysis on skim milk, analyzed by gel electrophoresis (UREA-PAGE), which clearly showed that the protease does not have the same specificity as bovine chymosin. (c) 2006 Elsevier Ltd. All rights reserved.

Protease inhibition activity of extracts from Salicaceae species from Brazilian Cerrado and Atlantic Rain Forest and of an enriched fraction of clerodane diterpenes (casearins)

Considerando o uso popular de Casearia sylvestris Sw., Salicaceae, para o tratamento de problemas gástricos e resultados pré-clínicos que mostraram potencial atividade anti-ulcerogênica, foi realizado um screening farmacológico para avaliar a atividade biológica de outras espécies de Salicaceae. Para isso, foi utilizado um ensaio de inibição de proteases como um modelo farmacológico molecular para screening de extratos com atividade anti-ulcerogênica. Os extratos etanólico e aquoso dos galhos e folhas de C. gossypiosperma, C. decandra e C. rupestris mostraram inibição da atividade da pepsina em aproximadamente 50% com a concentração de 1 μg/mL. Curiosamente, C. obliquoa e Flacourtia ramontchi não apresentaram atividade sobre a pepsina, mas seus extratos mais apolares mostraram atividade inibitória sobre a subtilisina. A fração enriquecida de diterpenos clerodânicos mostrou atividade inibitória (42,75%) sobre a pepsina com a concentração de 1 μg/mL, mas não sobre a subtilisina (23,76%). Os resultados obtidos com os extratos e folhas das espécies testadas mostraram um padrão de atividade diferente sobre os dois tipos de proteases, a pepsina e a subtilisina, as quais estão relacionadas com diferentes tipos de atividades biológicas. Ainda mais, os resultados com a fração enriquecida de diterpenos clerodânicos sugerem que estas substâncias podem estar relacionadas com a atividade do extrato bruto de C. sylvestris.

In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

As cisteína-proteases estão entre os alvos mais promissores para o desenvolvimento de novos agentes terapêuticos, visto que participam de eventos fundamentais do ciclo de vida de muitos microorganismos, inclusive Giardia. Como a atividade das proteases pode ser controlada por inibidores específicos, essas substâncias têm sido avaliadas quanto ao potencial antiparasitário. Diante disso, o presente estudo teve por objetivo avaliar o efeito in vitro do inibidor de cisteína-proteases E-64 sobre o crescimento, a aderência e a viabilidade de trofozoítos de cepa de Giardia isolada em Botucatu. Nos ensaios de crescimento e aderência, o número de trofozoítos foi estimado microscopicamente em hemocitômetro, enquanto que a viabilidade celular foi avaliada pelo método do MTT. No presente estudo, embora o metronidazol tenha se apresentado bastante efetivo, o E-64 mostrou ser capaz de inibir o crescimento, a aderência e a viabilidade em taxas superiores a 50%, especialmente nos cultivos expostos à concentração de 100 µM. A despeito de preliminares, esses resultados demonstram que o inibidor E-64 pode interferir em processos primordiais para a sobrevivência do parasita, além do que, abrem novas perspectivas para investigações futuras a fim de se avaliar o real potencial giardicida dos inibidores de proteases.

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 Proteolytic activity is catalytically related to the HslVU protease?

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P 1, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli Hs1VU protease suggested two putative threonine catalytic groups, one in the N-domain (T 136, K 168, and Y 264) and the other in the C-domain (T 375, K 409, and S 502). Mutagenesis studies showed that the replacement of K 409 by A caused a complete loss of the proteolytic activity, whereas the mutation of K 168 to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T 375, K 409, and S 502 at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.

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.