| Budget Amount *help |
¥3,680,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥180,000)
Fiscal Year 2007: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Cytotoxic protein MM29kD, which is derived from a crystal protein produced by Bacillus thuringiensis, is highly toxic to Jurkat cell, leading to cell death. MM29kD recognizes Jurkat cell and bind to the surface of the target cell. It is supposed that a receptor on the cell surface is involved in this process. In this study, I search for functional receptors for MM29kD, and analyse the intracellular events induced by MM29kD.
When Jurkat cells were treated with phosphatidyl inositol-specific phospholipase C (PI-PLC), the susceptibility of Jurkat toward MM29kD decreased more than 10-fold. PI-PLC cleaves the phosphate linkage of the phosphatidyl inositol to release GPI-anchored proteins from the cell surface. It was, therefore, suggested that GPI-anchored molecules on the surface of Jurkat cells were, as functional receptors, involved in determining the susceptibility toward MM29kD. Moreover, total cell membrane proteins from Jurkat were separated with 2-D gel electrophoresis followed by LC
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/MS analyses and MM29kD-binding assay. The results suggested that CD7 was one of the MM29kD-binding protein on the cell surface of Jurkat. It is known that CD7 is highly expressed in cancer cells derived from T cell. Thus, CD7 may be one of the most probable candidates for MM29kD receptors. Based on these results, it is suggested that, at least, both GPI-anchored proteins and CD7 are involved in determining the susceptibility of Jurkat cells toward MM29kD.
Furthermore, I investigate intracellular events in Jurkat induced by MM29kD. The total proteins of the Jurkat cell that had been treated with MM29kD were separated by 2-D gel electrophoresis. As judged by the positions and intensities of the protein spots, low molecular weight protein tyrosine phosphatase (LMW-PTP) was found to alter remarkably upon administration of MM29kD. In addition, it was strongly suggested that the LMW-PTP was inactivated by reactive oxygen species (ROS), production of which was demonstrated to be induced by MM29kD. According to the results thus far obtained, MM29kD induces apoptosis in Jurkat cells, leading to cell death. Thus, it is supposed that ROS, which is produced in Jurkat cells upon administration of MM29kD, damages the mitochondrial membrane to make the cytochrome c released into the cytoplasm, leading to induction of apoptosis. Less
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