June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Redox Proteomic Identification of Molecules in Retinal Endothelial Cells Undergoing Disulfide Reduction as a Result of Radiation
Author Affiliations & Notes
  • Megan Crowe
    Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI
  • Christopher Lieven
    Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI
  • Leonard Levin
    Ophthalmology & Visual Sciences, University of Wisconsin, Madison, WI
    Ophthalmology, McGill University, Montreal, QC, Canada
  • Footnotes
    Commercial Relationships Megan Crowe, None; Christopher Lieven, None; Leonard Levin, Quark (C), Inotek (C), Merz (C), Wisconsin Alumni Research Foundation (P), Cytodefense (I), Teva (C), Allergan (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1783. doi:
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    • Get Citation

      Megan Crowe, Christopher Lieven, Leonard Levin; Redox Proteomic Identification of Molecules in Retinal Endothelial Cells Undergoing Disulfide Reduction as a Result of Radiation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1783.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Radiation retinopathy can be a bystander effect of therapeutic irradiation to adjacent neoplasms. We previously found that redox-active drugs that reduce protein disulfides could be radioprotective. We hypothesized that radiation induced disulfide formation in one or more protein targets, which could be identified with two-dimensional non-reducing/reducing gel electrophoresis.

Methods: Retinal endothelial cells were exposed to either 0 Gy or 30 Gy of 137Cs irradiation. Immediately after, cells were lysed and prepared for gel electrophoresis in the presence of iodoacetamide to preserve sulfhydryls in a reduced state. After being run in first dimension in non-reducing conditions on a polyacrylamide gel, gel lanes were excised and reduced with dithiothreitol. Lanes were then treated with iodoacetamide to alkylate free sulfhydryls. The excised lanes were then rotated so that they were perpendicular to the first dimension and run in the second dimension. Proteins were visualized with SYPRO ruby and spots that differed between the irradiated and control conditions were identified.

Results: Proteins that did not contain disulfide bonds remained in the diagonal formed by running the protein in the second dimension. Homo- or heteromeric proteins containing disulfide bonds appeared as spots below the diagonal. Unexpectedly, one such spot was present in the 0 Gy radiation condition, but not the 30 Gy condition, indicating that in this case irradiation was associated with the reduction of a disulfide bond.

Conclusions: These findings that irradiation can be associated with changes in disulfide-linked proteins that may be independent of direct oxidative.

Keywords: 670 radiation damage: light/UV  
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