Cytoplasmic superoxide causes bone fragility owing to low-turnover osteoporosis and impaired collagen cross-linking.

The aging process correlates with the accumulation of cellular and tissue damage caused by oxidative stress. Although previous studies have suggested that oxidative stress plays a pathologic role in the development of bone fragility, little direct evidence has been found. In order to investigate the pathologic significance of oxidative stress in bones, we analyzed the bone tissue of mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, encoded by the Sod1 gene; Sod1(-/-)). In this study, we showed for the first time that in vivo cytoplasmic superoxide caused a distinct weakness in bone stiffness and decreased BMD, aging-like changes in collagen cross-linking, and transcriptional alterations in the genes associated with osteogenesis. We also showed that the surface areas of osteoblasts and osteoclasts were decreased significantly in the lumbar vertebrae of Sod1(-/-) mice, indicating the occurrence of low-turnover osteopenia. In vitro experiments demonstrated that intracellular oxidative stress induced cell death and reduced the proliferation in primary osteoblasts but not in osteoclasts, indicating that impaired osteoblast viability caused the decrease in osteoblast number and suppressed RANKL/M-CSF osteoclastogenic signaling in bone. Furthermore, treatment with an antioxidant, vitamin C, effectively improved bone fragility and osteoblastic survival. These results imply that intracellular redox imbalance caused by SOD1 deficiency plays a pivotal role in the development and progression of bone fragility both in vivo and in vitro. We herein present a valuable model for investigating the effects of oxidative stress on bone fragility in order to develop suitable therapeutic interventions.

Oxidative stress in skeletal muscle causes severe disturbance of exercise activity without muscle atrophy.

The increase in reactive oxygen species (ROS) levels that occurs during intense exercise has been proposed to be one of the major causes of muscle fatigue. In addition, the accumulation of cellular damage due to ROS is widely regarded to be one of the factors triggering age-related pathological conditions in skeletal muscle. To investigate the pathological significance of oxidative stress in skeletal muscle, we generated skeletal muscle-specific manganese superoxide dismutase-deficient (muscle-Sod2(-/-)) mice. The mutant mice showed severe disturbances in exercise activity, but no atrophic changes in their skeletal muscles. In histological and histochemical analyses, the mutant mice showed centralized nuclei in their muscle fibers and selective loss of enzymatic activity in mitochondrial respiratory chain complexes. In addition, the mutant mice displayed increased oxidative damage and reduced ATP content in their muscle tissue. Furthermore, a single administration of the antioxidant EUK-8 significantly improved exercise activity and increased the cellular ATP level in skeletal muscle. These results imply that the superoxide anions generated in mitochondria play a pivotal role in the progression of exercise intolerance.

Skin atrophy in cytoplasmic SOD-deficient mice and its complete recovery using a vitamin C derivative.

Intrinsic skin ageing is characterized by atrophy and loss of elasticity. Although the skin hypertrophy induced by photoageing has been studied, the molecular mechanisms of skin atrophy during ageing remain unclear. Here, we report that copper/zinc superoxide dismutase (CuZn-SOD)-deficient mice show atrophic morphology in their skin. This atrophy is accompanied by the degeneration of collagen and elastic fibers, and skin hydroxyproline is also significantly reduced in deficient mice. These imply that the dysfunction of collagen and elastin biosynthesis are involved in the progression of skin thinning. Furthermore, transdermal administration of a vitamin C derivative which can permeate through the membrane, completely reversed the skin thinning and deterioration of collagen and elastin in the mutant mice. These indicate that the vitamin C derivative is a powerful agent for alleviating skin ageing through regeneration of collagen and elastin. The CuZn-SOD-deficient mice might be applicable to evaluation of therapeutic medicines against skin ageing.

High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system.

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins.

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.