| Summary: | 碩士 === 慈濟大學 === 人類遺傳學研究所 === 92 === Bacillus anthracis is the causative agent of anthrax. The major virulence factor of the bacterium is anthrax exotoxin that composed of three separate proteins, which are protective antigen (PA), lethal factor (LF) and edema factor (EF). PA acts to deliver LF to the cytosol of target cell. LF is a Zn2+-dependent matalloprotease, inactivates members of the mitogen activated protein kinase kinase (MAPKK or MEK) family through proteolysis of their NH2-termini. The mechanism of pathogenesis is not clear. Based on the fact that LT (PA add LF) could kill the macrophages, current model proposed that cytokine-induced shock is responsible for death resulting from anthrax infection. However this model couldn’t provide satisfactory explanation why abnormal bleeding and thrombcytopenia were found in patients and experimental animals. Because abnormally regulated the extracellular signal-regulated kinase (ERK) could block megakaryocyte differentiation, thus we hypothesized that LT could block megakaryocyte differentiation through inactivated MAPK pathway. The human erythro/megakaryocytic (HEL) cell line, which can be induced by TPA (12-O- tetradecanoylphorbol-13-acetate, differentiation inducer) to undergo megakaryocytic differentiation, were treated with recombinant wild-type LT, mutated LT (LF, E687A) or various specific MAPK inhibitors. The changes of the surface markers and phenotype were then analyzed by flow cytometry and microscopy analysis. Specific megakaryocyte differentiation markers (CD41b or CD61) were decreased while specific erythroid differentiation marker (glycophorin A) was increased after various treatments such as lethal dose LT, sub-lethal dose LT and recombinate wild-type LT but not by recombinate mutated LT and MAPK inhibitors. These results suggested that catalytic activative of LF is required to influence megakaryocyte differentiation, and such inhibition is through a MAPK independent pathway.
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