Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
General anatomy (including Histology/Embryology)
|Research Institution||Tohoku University|
KONDO Hisatake Tohoku University, Histology, Professor, 医学部, 教授 (20004723)
SPENER F. Univ.Muenster, Biochem., Professor, 生化学, 教授
CANTLEY L.C. Harvard Univ.Cell Biology, Professor, 医学部, 教授
GLOMSET J.A. Univ.Washington, Biochem., Professor, 医学部, 教授
HUNT D.M. Univ.Coll.London, Eye Inst.Professor, 眼研究所, 教授
GOTO Kaoru Yamagata University, Anatomy, Professor, 医学部, 教授 (30234975)
CANFLEY L.C. ハーバード大学, 医学部, 教授
野沢 義則 岐阜大学, 医学部, 教授 (10021362)
GLOMSET J.A ワシントン大学, 医学部, 教授
大和田 祐二 東北大学, 医学部, 助手 (20292211)
阪上 洋行 東北大学, 医学部, 助手 (90261528)
|Project Period (FY)
1997 – 1998
Completed(Fiscal Year 1998)
|Budget Amount *help
¥15,800,000 (Direct Cost : ¥15,800,000)
Fiscal Year 1998 : ¥7,500,000 (Direct Cost : ¥7,500,000)
Fiscal Year 1997 : ¥8,300,000 (Direct Cost : ¥8,300,000)
|Keywords||lipid kinase / PI-signaling / FABP / CDP-DG synthase / gene knockout / in situ hybridization / brain / PI3-キナーゼ / PI4-キナーゼ / PLD / 脳内発現局在 / ジアシルグリセロールキナーゼ / PI3キナーゼ / 遺伝子クローニング / ゲノム解析 / 脂肪酸結合蛋白|
A cDNA encoded a 462-amino acid protein, which showed CDP-diacylglycerol synthase (CDS) activity was cloned for the first time as the vertebrate enzyme molecule from rat brain cDNA library. The deduced molecular mass of this CDS was 53 kDa, and putative primary structure included several possible membrane-spanning regions. This enzyme molecule preferred 1-stearoyl- 2-arachidonoyl phosphatidate as a substrate and its activity was strongly inhibited by phosphatidylinositol (PtdIns) 4,5-bisphosphate. The molecule was mainly localized in close association with the membrane of the endoplasmic reticulum of over-expressed in vitro cells. The intense mRNA expression of CDS was localized in the cerebellar Purkinje cells, pineal body, and the inner segment of photoreceptor cells, and postmitotic spermatocytes and spermatids of testis of mature rats. This finding is closely similar to that of arachidonoyl-diacylglycerol kinase (DGK) and in this regard we discussed with Dr.J.A.Glomset who first id
entified the DGK and attempted to examine the colocalization of the two molecules by immunohistochemistry by Drs.K.Goto.
A novel DGK was identified by cDNA cloning from human brain and retina cDNA library and termed DGKtheta in co-operation with Dr.W.J.van Blitterswijk. DGKtheta was composed of 951 amino acids and contained three cysteine-rich domain, a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. In adult rat brain high expression was detected in the cerebellar cortex and hippocampus.
The cloning and characterization of a novel class II phosphoinositide 3-kinase was succeeded from the cDNA library of regenerating rat liver. This enzyme molecule composed of1505 amino acids contained a C2 domain and displayed a restricted substrate specificity for PtdIns and PtdIns 4-P and, thus termed PI3K-IIgamma. This molecule was localized in the juxtanuclear Golgi region in the over-expressed in vitro cells. The mRNA expression was confined to the liver throughout the development and its expression increased during liver regeneration after partial hepatectomy. Further molecular characterization is under way in co-operation with Dr.L.C.Cantley.
By in situ hybridization histochemistry a dramatic increase in expression of Akt was detected in affected hypoglossal nucleus after 2-7 days following axotomy of the hypoglossal nerve. Such an increase in the gene expression after axotomy was observed in a similar time course for PI3 kinase and skin type-fatty acid binding protein (FABP), suggesting a functional relation between PI-signal and FABP.Thus the gene knockout for s-FABP is under way in co-operation with Dr.F.Spener.
The gene expression localization of phospholipase D in the brain is also under way in cooperation with Dr.Y.Nozawa. The chromosome mapping of the genome for PtdIns 4-kinase was attempted in cooperation with Dr.D.M.Hunt and started. However, in the course of experiment we unfortunately found that it has already done by another foreign research group and thus gave up this attempt. Less