| Project/Area Number |
03670007
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Yamanashi Medical University (1992-1993)
Shinshu University (1991) |
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Principal Investigator |
OHNO Shinichi Yamanashi Medical University, Professor, 医学部, 教授 (50109170)
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| Project Period (FY) |
1991 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1992: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
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| Keywords | quick-freezing / deep-etching / normal erythrocyte / abnormal erythrocyte / membrane skeleton / chicken / frog / spectrin / 赤血球 / ディ-プエッチング法 |
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| Research Abstract |
(1) A novel method for prepare exposed cytoplasmic aspects of erythrocyte membranes is described, which improves the resolution in direct electron microscopic images. Normal human erythrocytes were briefly fixed with paraformaldehyde. A drop containing the erythrocyte pellets was sandwiched between 2 coverslips. The attached erythrocytes were split open and postfixed with glutaraldehyde. All specimens were quickly frozen in an isopentane-propane mixture, deeply etched and rotary shadowed with platinum and carbon. Filamentous structures were seen to form networks on the cytoplasmic sides of the erythrocyte. This method will be useful in the analysis of the in situ ultrastructure of cytoplasmic sides of normal erythrocyte membranes.
(2) Spherocytic and elliptocytic erythrocytes with hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) were split open mechanically and were immunostained with anti-spectrin antibody. Replica membranes were prepared by a quick-freezing and deep-etching method and were checked by electron microscopy. The in situ membrane skeletons of normal erythrocytes consisted mainly of reticular patterns of spectrin filaments, which formed networks on the cytoplasmic sides of membranes. In contrast, the membrane skeletons of abnormally shaped erythrocytes (HS and HE) were much less filamentous and more granular than those of normal erythrocytes. This abnormal organization may be one of the factors that induce abnormally shaped erythrocytes in HS and HE patients.
(3) Ultrastructures of membrane skeletons in frog or chicken erythrocytes were investigated in the same procedure. Microtubules and actin filaments were also localized on the cytoplasmic sides of erythrocyte membranes in addition to spectrin networks. It is suggested that primitive membrane skeletons contain not only spectrin, but also cytoskeletal proteins.
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