| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Oxidative stress is involved in the mechanisms of degenerative diseases and ageing. It causes damages of subcellular structures and bio-molecules. An age-related pigment, lipofuscin, accumulated in lysosomes is one of products of the oxidative damage in the cytoplasm. Previously we found that dystrophin-deficient muscles of 2 to 7-year-old patients with Duchenne-type muscular dystrophy (DMD) and 10-week-old mdx mice, model animals of DMD, accumulate an ageing pigment lipofuscin, which is a product of oxidative stress, earlier than age-matched normal controls (Nakae et al., Histochemistry and cell Biology, 2001, 115, 205-214 ; Nakae et al., Journal of Molecular Histology, 2005, 35, 489-499). Further, most apoptotic myofibres in mdx muscles contain lipofuscin granules (Nakae et al., Histochemistry and Cell Biology, 2001, 115, 205-214). One of the four bases in DNA, guanine, which has the lowest oxidation potential, is preferentially attacked by low levels of oxidative stress and oxidised
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to 8-oxoguanine. Therefore, 8-oxoguanine is a sensitive marker of oxidative stress in nuclei. In the present study, we developed a new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress and apply it to DMD and mdx muscles (Nakae et al., Histochemistry and Cell Biology, 2005, 124, 335-345). The oxidative indices independent of DNA contents in cell nuclei were determined in situ using the technique with Neutral Red for DNA staining, a monoclonal antibody specific for 8-oxoguanine and a antibody for a cell marker, laminin. We found that the mean index for the myonuclei in bicepts brachii muscles of 2- to 7-year-old DMD patients was 14% higher than that in age-matched normal controls. The mean index for the myonuclei in diaphragm muscles of 8-week-old mdx mice was 30% higher than that in age-matched normal controls. However, the mean indices for the myonuclei in lingual muscles of mdx and normal controls were similarly low (Nakae et al., Acta Anatomica Nipponica, 2006, 81(Suppl), 214). Lipofuscin granules were abundant in mdx diaphragm muscle, rare in mdx lingual muscle and absent in normal diaphragm and lingual muscles. Focal degeneration and regeneration were observed in mdx diaphragm muscles but not in mdx lingual muscles. These results suggest that oxidative stress is related to the pathology of dystrofin-deficient muscular dystrophy. Our technique for the quantitative assessment of oxidative damage in nuclear DNA in situ was confirmed to be applicable in biomedical research.
In the present study we also discovered a compound "A" effective to ameliorate muscular dystrophy of mdx mice. This compound may be useful for medical therapy of DMD. The molecular mechanism of the action of the compound in dystrophic muscles is being investigated. Less
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