Protective Effect of Heat Shock Protein 70 Against Oxidative Stresses in Human Corneal Fibroblasts

We evaluated DNA protection effect of heat shock protein (HSP) against cytotoxic effects of exogenous nitric oxide (NO) and reactive oxygen intermediate (ROI). Cultured human corneal fibroblasts were divided into 4 groups. Control (Group I) was not exposed to a sub-lethal heat treatment. Other 3 groups were exposed to 43 degrees C for 1 hr, then incubated at 37 degrees C during different duration (1, 6, 24 hr, Group II, III, IV, respectively). Expression pattern of HSP 70 was analyzed by Western blot. Cell viability was measured by MTT assay and the relationship between HSP 70 expression and DNA damage was examined by terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick and labeling (TUNEL) stain and single cell gel electrophoresis. Expression pattern of HSP 70 was dependent on recovery times. Cell viability following heat treatment was significantly increased and the TUNEL positive cell number was decreased at 6 hr. In single cell gel electrophoresis, tail moments were increased in a dose-dependent manner by SNAP and X/XO. Following heat treatment, tail moments showed decreased significantly at 6 hr. These results suggest that induction of HSP 70 by sub-lethal heat treatment is closely related with cytoprotective effects against oxidative stresses in human corneal fibroblasts.

Action of immobilized xanthine oxidase on purines

Xanthine oxidase was covalently immbolized on polyacrylamide gel beads, polyamide- 11 and dacron. Hypoxanthine (15 ml of 200 µM), prepared in 0.1 M phosphate buffer, pH 8.0, was circulated through a column containing 1.0g derivatized enzyme at a flow rate of 1.0 ml/min at 28§C. Specific activities of 0.660, 0.072 and 0.016 Units/mg of protein were demonstrable for the polyacrylamide gel beads, dacron and polyamide-11 derivatives, respectively. The action of these water insoluble enzyme derivatives on 6 mercaptopurine (15 ml of 660 µM) was also investigated, under the same experimental conditions, showing specific activites of 0.063 Units/mg, 0.574 µUnits/mg and 0.118 µUnitis/mg, respectively. The 6-mercaptopurine oxidative pathway catalyzed by immobilized xanthine oxidase on dacron stopped at the intermediate compound 6-mercaptopurine oxidative on dracon stopped at the intermediate compound, 6-mercapto-8-hydroxypurine, so that no 6-thiouric acid was produced, whereas the immobilized preparations using polyacrylamide gel beads and polyamide-11 behaved like the soluble enzyme, namely, 6-thiouric acid was the final product. The behavior of dracon-xanthine oxidase immobilized on these three supports was similar to the soluble enzyme. However, although its oxidation is stoichiometric for polyacrylamide gel beads and polyamide- 11 derivatives, and no xanthine formation is observed (steady-state equilibrium), under the action of the enzymedacron derivative the xanthine formation rate (0.164 µUnits/mg) is higher than the uric acid formation rate (0.017 µUnits/mg) compared to the hypoxanthine consumption (0.072 µUnits/mg). These findings suggest again that xanthine oxidase-dacron derivative is limited to the catalysis of oxidation of hypoxanthine carbon atom number 2 as in 6-mercaptopurine