Identificação e caracterização dos produtos de excreção/secreção de trofozóitos de Giardia duodenalis
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Data
2008-04-28
Autores
Carvalho, Thaís Batista de [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente estudo foi desenvolvido com o objetivo de identificar e caracterizar os produtos de excreção/ secreção (PE/S) de trofozoítos de Giardia duodenalis de uma cepa axênica isolada no Brasil (BTU-11), tendo por referência a cepa padrão Portland 1 (P-1) isolada nos Estados Unidos. Os PE/S foram obtidos a partir dos sobrenadantes de cultura de trofozoítos mantidos em meio RPMI por 6 horas a 37oC. Os PE/S de cada cepa foram analisados quanto ao perfil eletroforético em géis de poliacrilamida (SDSPAGE) e a atividade proteolítica foi avaliada em géis contendo gelatina e colágeno tipo I a 0,2% e em ensaios empregando hemoglobina a 0,1%. A caracterização das proteases dos PE/S foi realizada em ensaios para avaliar o efeito de inibidores sintéticos de cisteína, serina, metalo e aspartil proteases sobre a degradação de cada substrato testado. A análise dos perfis protéicos revelou padrões simples, porém distintos quanto ao número de bandas de proteínas, visualizando nos PE/S das cepas P-1 e BTU-11, respectivamente, seis e quatro bandas com massas moleculares distribuídas na faixa de 123 a 28 kDa. Quanto à atividade proteolítica, todos os substratos foram degradados por enzimas presentes nos PE/S. A atividade gelatinolítica foi observada na faixa de 77 a 18 kDa, destacando-se seis bandas evidentes de aproximadamente 77, 66, 54, 52, 21 e 18 kDa. A degradação do colágeno foi detectada na faixa de 145 a 18 kDa, sendo que as zonas de proteólise de maior evidência foram observada nas faixas de 145 a 82 kDa e de 56 a 34 kDa, onde destacam-se as bandas de 145, 96, 82 e 34 kDa. A análise dos padrões de hidrólise da hemoglobina demonstrou similaridade entre as cepas e revelou degradação total da cadeia dimérica e lise parcial da cadeia monomérica. A atividade gelatinolítica e...
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 in conditioned medium by trophozoites of each strain were tested in SDS-polyacrylamide gel electrophoresis for the protein profiles, and the proteases activity was analyzed using substrate gelatin and collagen impregnated SDS-PAGE and haemoglobin assay. The proteases characterization was based on inhibition assays including synthetic inhibitors for cysteine, serine, metallo and aspartic proteases. Electrophoresis analysis of E/S products revealed a banding pattern composed by few bands (4-6 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 degradation of both substrates 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 haemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibiton assays showed that the main proteolytic activity in E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of haemoglobin. These observations are relevants, especially ...(Complete abstract click electronic access below)
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 in conditioned medium by trophozoites of each strain were tested in SDS-polyacrylamide gel electrophoresis for the protein profiles, and the proteases activity was analyzed using substrate gelatin and collagen impregnated SDS-PAGE and haemoglobin assay. The proteases characterization was based on inhibition assays including synthetic inhibitors for cysteine, serine, metallo and aspartic proteases. Electrophoresis analysis of E/S products revealed a banding pattern composed by few bands (4-6 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 degradation of both substrates 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 haemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibiton assays showed that the main proteolytic activity in E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of haemoglobin. These observations are relevants, especially ...(Complete abstract click electronic access below)
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Palavras-chave
Giardia, Trofozoítos
Como citar
CARVALHO, Thaís Batista de. Identificação e caracterização dos produtos de excreção/secreção de trofozóitos de Giardia duodenalis. 2008. 77 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, 2008.