Structural analysis of oxidatively modified protein as a oxidative stress probe
| Project/Area Number |
13660122
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
食品科学・栄養科学
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| Research Institution | Nagoya University |
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Principal Investigator |
UCHIDA Koji Nagoya University, Graduate School of Bioagricultural Sciences, Associate Professor, 大学院・生命農学研究科, 助教授 (40203533)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Oxidized cholesterol esters / Low density lipoprotein / Lipid peroxidation / Oxidative modification of protein / Reactive aldehydes / Monoclonal antibody / Lysine adduct / Oxiative stress / 免疫化学 / NMR解析 |
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| Research Abstract |
It has been proposed that plasma low-density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apo B). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apo B. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu^<2+> produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol (5-OVC) as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-OVC peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde leve
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ls was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester-core aldehydes, and (ii) the increase in the amounts of apo B-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester-core aldehydes were further converted to their 7-ketocholesterol and apo B-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody (mAb2A81) directed against 9-ONC-modified protein and found that it extensively recognized protein-bound cholesteryl ester-core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester-core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins. Less
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Report
(3 results)
Research Products
(14 results)
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[Publications] Yamada,S., Kumazawa,S., Ishii,T., Nakayama,T., Itakura,K., Shibata,N., Kobayashi,M., Sakai,K., Osawa,T., and Uchida,K.: "Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine"J.Lipid Res.. 42. 1187-1196 (2001)
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[Publications] Kawai,Y., Saito,A., Shibata,N., Kobayashi,M., Yamada,S., Osawa,T., and Uchida,K.: "Covalent binding of oxidized cholesteryl esters to protein : Implications for oxidative modification of low density lipoprotein and atherosclerosis"J.Biol.Chem. in press. (2003)
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Related Report
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