| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1992: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
1) Stimulation of fura-2-loaded or [^3H]arachidonic acid-labeled human megakaryoblastic leukemia CMK cells with thrombin or an analogue of thromboxane A_2 (STA_2) caused a transient increase in intracellular Ca^<2+> concentration and a marked arachidonic acid liberation. The latter response was synergistically argumented by pretreatment with a phorbol ester (PMA), though PMA itself did not cause significant arachidonate release.
2) Biphasic production of diacylglycerol (DG), peaking at 10sec and 5min, was observed in thrombin-triggered CMK cells. The generation of phosphatidylethanol in the presence of ethanol suggested that the second DG peak derives via a phospholipase D pathway in [^3H]myristic acid-labeled cells. It was hardly possible to obtain reproducible data in differentiating CMK cells.
3) Screening of rat megakryocyte cDNA library with chicken v-src as a probe enabled to obtain 8 positive clones. Rat hck cDNA was cloned, and its complete sequence was determined. Rat hck, consi
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sting 1911bp,encodes 503 amino acid with an apparent molecular weight of 57,126.
4) Molecular mechanisms of phospholipase Cgamma(PLCgamma)activation were examined in both differentiated rat primary cultured hepatocytes and undifferentiated human hepato-carcinoma, HepG2 cells, stimulated with hepatocyte growth factor (HGF). i) HGF caused a significant inositol 1,4,5-trisphosphate (InsP_3) formation in rat normal hepatocytes, whereas not in transformed HepG2 cells. Pretreatment of the cells with a tyrosine kinase inhibitor, genistein, prevented HGF-induced InsP_3 formation. Essentially the same results were obtained with [^3H]inositol-labeled cells. Since the HGF-receptor is the proto-oncogene product of c-met, the results suggested agonist-induced InsP_3 formation by the activation of PLC through receptor tyrosine kinase. ii) Immunoprecipitation of PLCgamma revealed tyrosine phosphorylation of PLCgamma in HGF-stimulated normal hepatocytes, which was inhibited by the genistein pretreatment, indicating the phosphorylation through the activation of receptor tyrosine kinase. No significant changes in tyrosiene phosphorylation was observed in transformed HepG2 cells, where HGF-receptor is expressed to nearly the same extent as normal hepatocytes, suggesting abnormal coupling between HGF-receptor and PLCgamma in transformed cells. Less
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