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Stefan Löfgren, M. Rohan Fernando, Kui-Yi Xing, Yin Wang, Charles A. Kuszynski, Ye-Shih Ho, Marjorie F. Lou; Effect of Thioltransferase (Glutaredoxin) Deletion on Cellular Sensitivity to Oxidative Stress and Cell Proliferation in Lens Epithelial Cells of Thioltransferase Knockout Mouse. Invest. Ophthalmol. Vis. Sci. 2008;49(10):4497-4505. doi: 10.1167/iovs.07-1404.
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purpose. To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase −/−) lens epithelial cells (LECs) as a model.
methods. Primary LEC cultures were obtained from wild-type (TTase +/+) and TTase −/− mice. Characterization and validation of the cells were determined by immunoblotting for TTase and α-crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H2O2 stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase −/− cells was achieved with reagent.
results. Primary LEC cultures obtained from wild-type (TTase +/+) and TTase −/− mice were characterized and found to contain lens-specific α-crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase −/− cells and its presence in the wild-type cells. TTase −/− LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase +/+ cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H2O2 after challenge with low levels of H2O2. Reloading of purified TTase into the TTase −/− cells restored the antioxidant function in TTase −/− cells to a near normal state.
conclusions. These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.
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