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Stephen Barnes, Kyle Floyd, Landon Wilson, David Anderson, Kevin Schey; Genistein in various dietary forms does not alter C-terminal truncation of alphaA-crystallin in cataracts in ICR/f rats. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2981. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that the diet used in an ICR/f rat model of lens cataractogenesis influences the development of lens opacity. In particular, diets containing the antioxidant genistein in various forms (its aglycone GEN, as soybean beta-glucosides BGs, and complex isoflavone glucosides CGs) accelerated the early development of opacity compared to a casein-based and isoflavone-free diet.
In the present study we applied imaging mass spectrometry on thin sections of lens from ICR/f rats at the end of the GEN diet studies. Quantitative LC-MS protein analysis was also performed on successive lens sections to determine the effect of GENon the apparent amounts of truncated forms of alphaA-crystallin.
Full length (yellow, aa1-173) and C-terminal truncated forms (blue, aa1-156 and red, aa1-53?) of alphaA-crystallin were discretely and differentially distributed in the lens. The images obtained were unaffected by the depth of the section taken from the lens. Analysis of IMS images of lenses from ICR/f rats obtained at the time of necropsy at 110 days of age (full cataract formation) revealed that there were no systematic differences between rats fed the GEN-containing and GEN-free diets. Quantitative, multiple reaction monitoring LC-MS analysis of peptides specific for the full-length and the C-terminal truncations of alphaA-crystallin was performed using chymotryptic digests of lens section homogenates collected at the time of imaging. Data were normalized to an added non-lens internal standard (digested BSA) and alpha-tubulin as a housekeeping protein. Statistical analysis revealed that full length alphaA-crystallin was unaffected by GEN diets. In general, there were small, GEN-associated increases in certain C-truncated alphaA-crystallin species (aa1-156, aa1-157 and aa1-168) compared with the casein control diet; however, there were no differences observed for the aa1-151 and aa1-163 fragments.
In conclusion, the effects of dietary supplemented forms of genistein have only minor changes in alphaA-crystallin C-terminal truncations when analyzed after lens cataract formation.
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