Estudo da expressão dos genes de choque térmico hsp90, hsp60 e hsp10 do fungo aquático Blastocladiella emersonii / Study of expression of heat shock genes Hsp90, hsp60, hsp10 of aquatic fungi Blastocladiella emersonii

A proteína de choque térmico Hsp90 é uma chaperone molecular encontrada no citosol. O cDNA imcompleto desta proteína foi isolado de uma biblioteca construída a partir de mRNA de células de esporulação de B. emersonii submitidas a choque térmico. Um clone genômico contendo a seqüência completa do gene hsp90 também foi isolado, seqüênciado e caracterizado. A região codificadora do gene hsp90 é interrompida por um único íntron de 184 nucleotídeos. A seqüência de aminoácidos deduzida indicou uma proteína de 710 resíduos, com massa molecular calculada de 80.792 Da e um pl médio de 4,85. Experimentos de extensão de oligonucleotídeo e RACE-PCR demonstraram um sítio único de início de transcrição localizado...

Dose-dependent differential effect of hemin on protein synthesis and cell proliferation in Leishmania donovani promastigotes cultured in vitro.

Leishmania donovani requires an exogenous source of heme for growth and transformation. In in vitro culture of the free-living promastigotes, exogenously added hemin enhances cell proliferation. In this investigation, the question of the function of heme with particular reference to protein synthesis and cell proliferation has been addressed. The results of in vitro cell culture experiments demonstrated that hemin (10 microM) alone is suitable for supporting optimum level of protein synthesis, and thereby cell proliferation of promastigotes to an extent that it can replace fetal bovine serum. However, in situ labelling experiments along with Western blots revealed that high concentration of hemin (50 microM) reduced the level of protein synthesis in general and of beta-tubulin in particular with a concomitant induction of hsp90, and induced consequent morphological changes that are observed during in situ transformation of promastigotes in mammalian macrophages. These results therefore suggest that sudden exposure to high concentration of heme in mammalian macrophages may be one of the key factors that trigger promastigote to amastigote transformation in L. donovani. Furthermore, hemin with its dual characteristic could be used as a tool to understand molecular mechanism of cell proliferation and transformation in these parasites.