| Project/Area Number |
14360187
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied veterinary science
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
INABA Mutsumi Hokkaido University, Graduate School of Veterinary Medicine, Professor, 獣医学研究科, 教授 (00183179)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAKUWA Yuichi Tokyo Women's Medical University, Faculty of Medicine, Professor, 医学部, 教授 (40113740)
SAITO Masayuki Hokkaido University, Graduate School of Veterinary Medicine, Professor, 大学院・獣医学研究科, 教授 (80036441)
MAEDA Yoshimitsu Hokkaido University, Graduate School of Veterinary Medicine, Professor, 大学院・獣医学研究科, 教授 (40002084)
INANAMI Osamu Hokkaido University, Graduate School of Veterinary Medicine, Associate Professor, 大学院・獣医学研究科, 助教授 (10193559)
YAMAMOTO Masayuki Tsukuba University, TARA Center, Professor, 基礎医学系先端学際領域研究センター, 教授 (50166823)
滝口 満喜 北海道大学, 大学院・獣医学研究科, 講師 (70261336)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2003: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2002: ¥9,900,000 (Direct Cost: ¥9,900,000)
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| Keywords | erythroid development / red blood cell / membrane skeleton / band 3 / ankyrin / membrane trafficking / intracellular vesicles / cell culture / 赤芽球系分化 / 赤血球膜 / 膜骨格タンパク質 / スペクトリン / 牛 |
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| Research Abstract |
The "unit-assembly hypothesis" for red cell membrane skeleton during erythroid development was investigated.
Stable transfectants of major constituents of red cell transmembrane proteins were developed in K562 and HEK293 cells using retroviral vectors. Band 3 and glycophorinCweretransportedtotheplasmamembranevia the ER/Golgi pathway. Band 3-binding domain of ankyrin (AnkN90) and protein 4.1 showed membranous localization in the cytoplasm with co-localization with markers for the ER and the Golgi apparatus in band 3-transfeted cells despite of the difference in erythroid differentiation. Endogenous membrane skeletal proteins including ankyrin, protein 4.1, and spectrin also exhibited membranous distribution. These results indicate that the assembly of membrane skeletal proteins occurs at an early stage of erythroidal development on the surface of the ER but not on the inner surface of the plasma membrane.
Stable expression of band 3 caused a reduction in a proliferation rate of K562 cells to approximately 70% that of non-transfected cells. Reduced production of hemoglobin suggestive of erythroid differentiation was also observed. However, no significant changes in intracellular localization of skeletal proteins and cell morphology was detected Synthesis and assembly to the ER of skeletal proteins involving spectrin and subsequent synthesis and localization to the plasma membrane of band 3 were observed in erythroid precursor cells from peripheral blood of cattle in two phase liquid culture system, indicating that the assembly of membrane skeletal proteins to the ER occurs in physiolosical erythropoiesis.
The present study demonstrated that the assembly of the red cell membrane skeleton is an event which takes place at early stages of erythroid development on the ER. The exact mechanism by which a small unit of the ekeleton formed on intracellular vesicles are integrated in the plasma membrane remained to be clarified.
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