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doi:10.1016/S0891-5849(01)00752-3 
Copyright © 2001 Elsevier Science Inc. All rights reserved.
Original contribution
Reduced levels of oxidized and glycoxidized proteins in human fibroblasts exposed to repeated mild heat shock during serial passaging in vitro
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Philippe Verbekea, Brian F. C. Clarka and Suresh I. S. Rattana, 
, 
Received 7 August 2001;
Abstract
Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, N
-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.
Author Keywords: Replicative senescence; Hormesis; Oxidation; Glycoxidation; Heat shock response; Stress; Anti-aging; Fibroblast; Free radicals
Abbreviations: AGE, advanced glycation endproduct; AP, Amadori product; CML, N-carboxymethyl-lysine; CPDL, cumulative population doubling level; DNPH, 2,4-dinitrophenylhydrazine; ELISA, enzyme-linked immunosorbent assay; GSH, reduced glutathione; GSSG, oxidized glutathione; HPLC, high performance liquid chromatography; HS, heat shock; HSF, human skin fibroblast; HSP, heat shock protein; PAGE, polyacrylamide gel electrophoresis; RMHS, repeated mild heat shock; ROS, radical oxygen species
Article Outline
- • Introduction
- • Materials and methods
- • Cell culture and repeated mild heat shock
- • Determination of HSP and oxidized carbonylated protein levels by western blot analysis
- • Determination of Furosine level
- • Determination of total AGE products and CML-rich proteins levels
- • Determination of cellular glutathione content
- • Statistical analysis
- • Results
- • Cell growth and replicative senescence
- • Effect of RMHS on accumulation of carbonylated proteins
- • Effect of RMHS on accumulation of glycation products
- • Effect of RMHS on the basal glutathione concentration
- • Effect of RMHS on oxidative-related HSP level
- • Discussion
- • Acknowledgements
- • References
Address correspondence to: Dr. Suresh I. S. Rattan, Department of Molecular and Structural Biology (IMSB), University of Aarhus, Gustav Wieds Vej 10-C, DK-8000 Aarhus -C, Denmark; Tel: +45 89 42 50 34; Fax: +45 86 12 31 78; email: rattan@imsb.au.dk
