Long-term treatment with a beta-blocker timolol attenuates renal-damage in diabetic rats via enhancing kidney antioxidant-defense system |
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Authors: | Hilal Gokturk N Nuray Ulusu Muslum Gok Erkan Tuncay Belgin Can Belma Turan |
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Institution: | 1. Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37232, USA 3. Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY, 40536, USA 4. Markey Cancer Center, University of Kentucky, Lexington, KY, 40536, USA 5. Kentucky Center for Structural Biology, University of Kentucky, Lexington, KY, 40536, USA 6. Department of Chemistry, University of Kentucky, Lexington, KY, 40536, USA 2. Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37232, USA
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Abstract: | Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 85, 15, and 1 % of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the translocation of RBD Raf1 -GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility, and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems. |
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