| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Besides fundamental neuronal functions including cellular excitability, axonal transport, and neurotransmitter release, the signal nutrient calcium plays crucial roles in higher functions in the central nervous system (CNS) such as integration of synaptic inputs, construction of neural network, and synaptic plasticity. Calcium is also involved in pathogenesis of degenerative brain diseases and ischemic brain vessel damage. Ca signaling mechanisms in CNS are thus far more diverse as compared to those in peripheral tissues, implying diverse species of Ca-binding proteins which mediates/regulates Ca functions in CNS.In this context, our attentions have been focused on molecular diversity of cephalocalcin, tentatively named to a Ca-binding protein exclusively localized or especially enriched in CNS.Using Ca-induced selective elution from anion exchangers (DEAE-Sephacel/Resource Q), we attained a large scale purification of cephalocalcin from bovine brain stem to give a single 30-kDa entity
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as visualized by SDS PAGE.2-D native PAGE of the purified sample, howerve, showed a cluster of plural spots. Such a unique cluster of cephalocalcin was ubiquitous in vertebrate classes V-IX.Despite reactive thiols detected in cephalocalcin, interconversion of thiol/disulfide did not account for the diversity of cephalocalcin. Peptide maps of two major forms of cephalocalcin, which were subjected to reduced-alkylation in the presence of denaturing agents in advance, were almost identical but displayd distinct fragments, indicating differences in peptide structures. When tissue extracts obtained from rat CNS by micropunch were analyzed, cephalocalcin cluster was dense in cerebellum, brainstem, olfactory bulb, retina, and spinal cord, but sparse in cerebral cortex and basal ganglia. Molecular forms of cephalocalcin were differentially distributed in loci of rat CNS.During development of chick embryo, two major forms appeared in cerebellum and retina with different time courses. We also obtained preliminary evidence that nutritional disturbance by deficiency of zinc, which bound to cephalocalcin in a non-competitive manner with Ca, affected cephalocalcin levels and distribution pattern in CNS.Overall evidence indicates that the molecular diversity of cephalocalcin is of biological significance and it enriches repertoire of Ca-binding proteins in CNS. Less
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