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Structural analysis of oxidatively modified protein as a oxidative stress probe

Research Project

Project/Area Number 13660122
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 食品科学・栄養科学
Research InstitutionNagoya University

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)

Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
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)
KeywordsOxidized 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
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 … More 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

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Ichihashi et al.: "Endogenous formation of protein adducts with carcinogenic aldehydes"J. Biol. Chem.. 276. 23903-23913 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Itakura et al.: "Evidence that malondialdehyde-derived conjugated Schiff base is not a fluorescent age pigment"Chem. Res. Toxicol.. 14. 473-475 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Yamada et al.: "Lipofuscin-like fluorescent pigments derived from malondialdehyde"J. Lipid Res.. 42. 1187-1196 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Furuhata et al.: "Thiolation of protein-bound carcinogenic aldehydes"J. Biol. Chem.. 277. 27919-27926 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Noiri et al.: "Serum protein acrolein adducts"Free Radic. Biol. Med.. 33. 1651-1656 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Ichihashi,K., Osawa,T., Toyokuni S., and Uchida,K.: "Endogenous formation of protein adducts with carcinogenic aldehydes : implications for oxidative stress"J.Biol.Chem.. 276. 23903-23913 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Itakura,K. and Uchida,K.: "Evidence that malondialdehyde-derived conjugated Schiff base is not a fluorescent age pigment"Chem.Res.Toxicol. 14. 473-475 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Yamada et al.: "Lipofuscin-like fluorescent pigments derived from malondialdehyde"J.Lipid Res.. 42. 1187-1196 (2001)

    • Related Report
      2002 Annual Research Report
  • [Publications] Furuhata et al.: "Thiolation of protein-bound carninogenid aldehyde"J.Biol.Chem.. 277. 27919-27926 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Noiri et al.: "Serum protein acrolein adducts"Free Radic.Biol.Med.. 33. 1651-1656 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ichihashi et al.: "Enclogenous formation of protin adducts with carcinogenic aldehydes"J. Biol. Chem.. 276. 23903-23913 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Itakura, Uchida: "Evidence that malondialdehyde-derived conjugateil Schitt base is not a fluorescent age pigment"Chem. Res. Toxical.. 14. 473-475 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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