1992 Fiscal Year Annual Research Report

単一ニューロンと微小脳領野の特異的蛋白質の解析:疾患脳への応用

Research Project

Project/Area Number	04454305
Research Institution	Yokohama City University
Principal Investigator	加藤尚彦横浜市立大学, 医学部, 教授 (80010023)
Co-Investigator(Kenkyū-buntansha)	礒部俊明東京都立大学, 理学部, 助教授 (70106607)
Keywords	ミクロウエスタンブロット法 / ミクロ電気泳動法 / S-アンティゲン / 脳特異蛋白質
Research Abstract	初年度は分析技術の開発を目指した.都立大学の奥山・磯部の微量二次元蛋白泳動分析装置のスラブゲル(40×40×1mm)を利用し,0.3〜1×0.3〜1×7mmのsilicon棒(computerのwaferを切り出す技術を応用して作製)を用いて,この大きさのmicrowellをゲルの上端に作った.0.4〜3.9μg乾燥重量の網膜とその層構造を眼球の凍結乾燥切片から切り出し,パラフィン油を満たした小さな井戸(油井)の底に取った0.4μ1のSDS-DTT-緩衝液の小滴の中に加えて溶解して,顕微鏡下で上述のmicrowellに注入した.そして10mm電気泳動した後,Enprotech社のsemidry systemを用いてPVDF紙に泳動した蛋白質をtransferした.網膜の特異蛋白であるS-antigenに対する抗体でPVDF紙を処理し,^<125>I-protein Aで抗体をラベルし,Fujix Bas 2000 Bio-lmage Analyzerを用いて,0.6〜10mm^2のS-antigenのspotを定量した(Micro Western Blot法:加藤).現在この新しい超微量定量法を脳特異蛋白であるV1に応用し,小脳の層構造やPurkinje細胞を分析している.また1〜2μgの脊髄前角や1〜5個の脊髄前角細胞体(10〜30ng)試料を泳動し,銀染色により特有の泳動パタンを得ることが出来た.一方 computerを用いて小型電磁弁の開閉をcontrolし,20×20×0.5〜1mmのスラブゲルに4〜17%のゲル密度匂配を作製する装置を組み立てた(組立に時間を要したので,未だ泳動は行っていない).またラットの生後3〜7日の細胞分裂を続ける小脳の水溶性蛋白質を抽出し,独自に組み立てた二次元高度液体クロマトグラフィーを用いて,形態形成に関与するアミノ酸配列を持つ蛋白質V1を単離して一次構造を決定した (礒部)

Research Products
(6 results)

All Other

All Publications (6 results)

[Publications] Akavayashi,A. and Kato,T.: "Microdetermination of 2-deoxyglucose and 2-deoxyglucose 6-phosphate to determine glucose utilization rates in single neurons and small CNS regions after injecting nontracer amounts of 2-deoxyglucose." Jouranal of Neurochemistry. 59. 888-896 (1992)
[Publications] Tsutsumi,O.,Satoh,K.,Taketani,Y.and Kato,T.: "Determination of enzyme activities of energy metabolism in maturing rat oocytes." Molecular Reproduction and Development. 33. 333-337 (1992)
[Publications] Morita,Y.,Tsutsumi,O.,Hosoya,I.,Taketani,Y.,Oka,O.and Kato,T.: "Expression and possible function of glucose transporter protein Glutl during preimplantation mouse development from oocytes to blastocysts." Biochemical and Biophysical Research Communication. 188. 8-15 (1992)
[Publications] Akabayashi,A. and Kato,T.: "Glucose utilization rates in single neurons and neuropil determined by injecting non-tracer of 2-deoxyglucose." Journal of Neurochemistry. (1992)
[Publications] Aitken,A.,Colline,D.B.,van Heusen,B.P.H.,Isobe,T,.Rosenboom,P.and Soil,J.: "14-3-3 proteins;a highly conserved,widespread family of eukaryotic proteins." TIBS (Trends in Biochemistry). 17. 498-501 (1992)
[Publications] Kato,T.(Eds.Satake,M.,Obata,K.,Hatanaka,H.,Miyamoto,E.and Okuyama,T.): "Energy metabolism in the single neurons and small CNS regions from rats,In Molecular Basis of Neural Connectivity" Hohko-Do,Niigata, 194-196(199) (1992)

1992 Fiscal Year Annual Research Report

単一ニューロンと微小脳領野の特異的蛋白質の解析:疾患脳への応用

Principal Investigator

加藤 尚彦 横浜市立大学, 医学部, 教授 (80010023)

Research Products

[Publications] Akavayashi,A. and Kato,T.: "Microdetermination of 2-deoxyglucose and 2-deoxyglucose 6-phosphate to determine glucose utilization rates in single neurons and small CNS regions after injecting nontracer amounts of 2-deoxyglucose." Jouranal of Neurochemistry. 59. 888-896 (1992)

[Publications] Tsutsumi,O.,Satoh,K.,Taketani,Y.and Kato,T.: "Determination of enzyme activities of energy metabolism in maturing rat oocytes." Molecular Reproduction and Development. 33. 333-337 (1992)

[Publications] Morita,Y.,Tsutsumi,O.,Hosoya,I.,Taketani,Y.,Oka,O.and Kato,T.: "Expression and possible function of glucose transporter protein Glutl during preimplantation mouse development from oocytes to blastocysts." Biochemical and Biophysical Research Communication. 188. 8-15 (1992)

[Publications] Akabayashi,A. and Kato,T.: "Glucose utilization rates in single neurons and neuropil determined by injecting non-tracer of 2-deoxyglucose." Journal of Neurochemistry. (1992)

[Publications] Aitken,A.,Colline,D.B.,van Heusen,B.P.H.,Isobe,T,.Rosenboom,P.and Soil,J.: "14-3-3 proteins;a highly conserved,widespread family of eukaryotic proteins." TIBS (Trends in Biochemistry). 17. 498-501 (1992)

[Publications] Kato,T.(Eds.Satake,M.,Obata,K.,Hatanaka,H.,Miyamoto,E.and Okuyama,T.): "Energy metabolism in the single neurons and small CNS regions from rats,In Molecular Basis of Neural Connectivity" Hohko-Do,Niigata, 194-196(199) (1992)

加藤尚彦横浜市立大学, 医学部, 教授 (80010023)