| Budget Amount *help |
¥26,000,000 (Direct Cost: ¥26,000,000)
Fiscal Year 1993: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1992: ¥22,000,000 (Direct Cost: ¥22,000,000)
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| Research Abstract |
The gene cloning, sequencing and expression localization of several proteins initimately related to the intracellular Ca-signal mechanism were undertaken in the present study. These proteins included calbindin calreticulin, Ca/calmodulin dependent protein kinase(CaMK) II and IV, diacylglycerol kinase(DGK), calpain, calpastatin, 14-3-3 protein (presumptive regulator for Ca-dependent protein kinase C) ; and further protein phosphatases (PP-2A, 2B, 2C), -adrenergic receptor kinase and neuronal and non-neuronal enolases. By in situ hybridization histochemistry using specific cDNA probes for each protein which were obtained by PCR amplification, spatial and temporal heterogeneity in the expression intensity was remarkable in developing and mature rat brain. For example, cerebellar granule cells expressed intensely CaMKII and IV and PP subunits and subtypes, while no significant expression for calbindin and calreticulin was detected in these cells. This heterogeneity suggests the functional heterogeneity of these Ca-signal-related proteins in different neuronal populations, and further suggests the existence of multiple isoforms for some of these proteins. The latter possibility was pursued by using DGK and CaMKIV as targets. As a result, 80 kDa- and 90 kDa-DGKs were identified by the gene cloning. They showed 58% identity to each other and contained zinc finger-like sequences, E-F hand motifs and ATP-binding sites. The gene expression for 80 kDa-DGK was confined to oligodendrocytes, suggesting the involvement of this isoform in Ca-signals related to the myelin formation. The gene of 90 kDa-DGK was expressed predominantly in neurons of the caudate putamen, suggesting its involvement in Ca-signals related the dopaminergic transmission. In addition, a cDNA encoding CaMKIV -subtype was isolated and sequenced from rat brain. Although addition of 28 amino acids in its amino terminal region was the only difference of the -subtype from the *, the diffirential gene expression fo
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