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D Chandrasekaran, O Srivastava, R Pfister; Determination of Molecular Changes in Selected Epithelial Proteins in Human Keratoconus Corneas Compared to NormalCorneas . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1664.
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Purpose: The purpose of the study was to determine molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas. Methods: Two-dimensional (2D) electrophoretic profiles of epithelial cell extracts from normal and keratoconus corneas were compared, and the selected spots were trypsin digested and analyzed by mass spectrometry. Based on the results, α-enolase, ß-actin and keratin 10 were further immunohistochemically analyzed using their respective antibodies. The Western blot analysis of proteins from normal and keratoconus corneas was also performed to determine possible modifications in the three proteins. Results: On comparison of the 2D-gel electrophoretic profiles, two spots were identified in the normal corneas that were absent in the keratoconus corneas. By mass spectrometric analysis, the spots were identified to be α-enolase (42 kDa) and ß-actin (48 kDa). By a similar analysis, only one spot was found to be present in keratoconus corneas and was identified as keratin 10 (59 kDa). Immunohistochemical analysis revealed that in the keratoconus, antibodies to α-enolase, ß-actin or keratin 10 showed weak to almost no immunoreactivity in the epithelial superficial, wing and basal cells when compared to normal corneas. The 2D-gel electrophoresis followed by Western blot analysis showed that in the keratoconus corneas, α-enolase and ß-actin had different molecular weights and charges compared to the normal corneas. Conclusions: Altered expression of three proteins, i.e. α-enolase, ß-actin and keratin 10 was observed in the epithelial cells of keratoconus corneas compared to normal corneas.
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