| Co-Investigator(Kenkyū-buntansha) |
KASAHARA Jiro Kumamoto University School of Medicine, Pharmacology, Instructor, 医学部, 助手 (10295131)
YAMAMOTO Hideyuki Kumamoto University School of Medicine, Pharmacology, Assistant Professor, 医学部, 講師 (60191433)
FUKUNAGA Kohji Kumamoto University School of Medicine, Pharmacology, Associate Professor, 医学部, 助教授 (90136721)
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| Research Abstract |
H.A.Lester reported that the development of cellular models to express important proteins such as receptors, enzymes, ion channels and so on by transfecting known cell lines with the cDNAs serves for the establishment of useful systems to evaluate drug efficacy and create new drugs. In other words, the establishment of stable cell lines in which functional proteins are not endogenously present, but overexpressed be useful and important.
Recently, we have studied on activation and autophosphorylation of CaM kinase II in primary cultures and established cell lines in response to extracellular stimuli such as neurotransmitters, cell growth factors and so on. We reported the activation of CaM kinase II in the CA1 area of the hippocampus during LTP induction.
The present study were carried out as follows. 1)We prepared the cDNAs of CaM kinase IIalpha, beta, gamma, delta subunits by the PCR method with synthesized primers. The cDNAs were inserted into the expression vector pCAGGSneo. PC12 cells or NG108-15 cells were transfected with the cDNAs by electropolation or the lipofectamine method. Mock cells into which only the vector is inserted without the cDNAs of CaM kinase II subunit isoforms were used. 2)Stable PC12 cells transfected with the cDNAs of CaM kinase IIalpha were prepared. 3)The expression of CaM kinase IIalpha in stable cell lines inhibited neurite outgrowth induced by dibutyryl cAMP.4)The specific antibody to CaM kinase IIdelta was prepared by immunizing rabbits. 5)The cDNAs of CaM kinase IIdelta subunit isoforms were prepared by the PCR method and sequenced. Three isoforms of CaM kinase IIdelta had nuclear localization signal. 6)The immunohistochemistry of the sagittal sections of rat cerebellum demonstrated that CaM kinase IIdelta is localized in the nuclei of granule cells, but not in the nuclei of Purkinje cells.
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