|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1997 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1996 : ¥1,200,000 (Direct Cost : ¥1,200,000)
1. Intracellular antioxidant enzymes such as CuZn-superoxide dismutase (SOD), MnSOD and cellular glutathione peroxidase (GPX) were semi-quantitated by immunofluorescent histochemical staining-image analysis. The concentrations of the 3 enzymes increased cooperatively in epithelial tissues of various organ systems during perinatal development in rat.
2. Rocket immunoelectrophoresis of plasma GPX was developed, using specific polyclonal antibodies against the rat enzyme. This method enabled to determine the specific activity of plasma GPX in biological fluid, and was useful for studying post-translational modification of this enzyme.
3. Semi-quantitative analysis after immunoelectron microscopy (i.e., immunogold labeling) revealed that the alveolar macrophages and type 2 pneumocytes in rat lungs were rich in cellular GPX.The intracellular localization of cellular GPX in those tissues was similar to that observed previously in rat hepatocytes.
4. By transient transfection of cellular GPX to
Cos-7 cells, the expression was enhanced 3 times, with translocation of the enzyme to both mitochondria and nucleus.
5. By permanent expression of GPX in a human tumor cell line, the enzyme level was enhanced 30 times, with relative richness in mitochondria, nucleus and cytoplasmic matirx.
6. In selenium deficient rat kidneys, both cellular and plasma GPX were decreased in a similar fashion. The expression was inhibited at multiple steps : i.e., pre-translational, translational and posttranslational levels. The study also revealed that plasma GPX was expressed in the proximal tubules of rat kidneys and was almost completely secreted to the plasma.
7. By collaboraion with various institutions, the pathognomonic structures for familial amyotrophic lateral sclerosis in anterior horn neurons of the autopsied sampled were found to be rich in immunoreactive CuZnSOD,as stained by our polyclonal antibodies. Contribution of proteolytic mechanism mediated by ubiquitin to pathogenesis was suggested. The findings supported the gain-of-function theory for the mutant CuZnSOD to cause degenerative changes. Less