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We studied the effect of E-64, a new thiol protease inhibitor derived from Aspergillus japonicus TPR-64, and of its synthesized analogue, E-64-d, on dystrophic mice ( C57BL 6J -dy ). The locomotor activity of normal mice increased markedly, reaching a plateau at 8 weeks of age. In dystrophic mice, it increased until they were 7 weeks old, thereafter, it decreased gradually. This decrease reflected the degradation of the hind legs. Serum activities of creatine phosphokinase were significantly greater in dystrophic than in normal mice. When 3-week-old dystrophic mice were injected with E-64 (1 mg/kg, i.p.) or E-64-d (40 to 60 mg/kg, p.o.), the decrease in their locomotor activity was retarded and their serum enzyme activities decreased significantly. In addition, the survival time of treated dystrophic mice was prolonged. The locomotor activity of normal mice and their serum enzyme levels were not affected by the administration of E-64-d. We posit that the new thiol protease inhibitors we tested retard the porgression of dystrophy in mice.

We studied the effect of mitochondrial protein synthesis inhibitors on the neurotrophic action of the nerve stump in mice. The sciatic nerve was cut as close to, or as far from the extensor digitorum longus muscles as possible. At 2 and 3 days after denervation, depolarization in the resting membrane potential of muscles with long nerve stumps (14-16 mm) was significantly smaller than in muscles with very short (less than 2 mm) nerve stumps. Chloramphenicol (100 mg/kg, p.o.), erythromycin (4 micrograms/kg, i.p.), ethidium bromide, (10 micrograms/kg, i.p.) or acridine (10 micrograms/kg, i.p.), administered for 2 or 3 days after denervation, increased depolarization in the resting membrane potential of muscles with long nerve stumps; muscles with very short nerve stumps were not affected by these drugs. The administration of chloramphenicol and erythromycin resulted in an earlier increase in the depolarization than ethidium bromide and acridine. Activities of cathepsin B and L of lysosomal proteases in the nerve stumps were enhanced by nerve degeneration; they were not affected by the administration of chloramphenicol or ethidium bromide. We suggest that proteins synthesized in mitochondria of the nerve may participate in trophic actions.

We report that protein synthesis inhibitors exert an inhibitory effect on the trophic action of the nerve stump. The sciatic nerve innervating the extensor digitorum longus muscles of mice was cut either as close to, or as far from, the muscle as possible. Denervation changes in the muscle were evaluated using the resting membrane potential and dose-response curves obtained by plotting acetylcholine-induced contractures. Actinomycin D (2 micrograms/kg, i.p.), ethidium bromide (10 micrograms/kg, i.p.), cycloheximide (1 or 5 mg/kg, i.p.), or chloramphenicol (100 mg/kg, p.o.) administration was immediately after neurotomy and continued daily until the day preceding muscle removal. Although denervation changes occurred significantly later in muscles with a long rather than a short nerve stump, the administrated antibiotics, excluding cycloheximide, accelerated the manifestation of denervation changes in muscles with long nerve stumps without affecting those in muscles with short nerve stumps.

The combined activities of cathepsin B and cathepsin L were studied in the forelimb and hind limb muscles of dystrophic mice. The activities of these proteases in the forelimb and hind limb muscles of young and adult dystrophic mice were significantly higher than those in normal mice. However, clinical involvement of dystrophy appeared in the hind limbs but not in the forelimbs. We therefore suggest that the increase in protease activity begins at a very early age and that the clinical involvement is not linked with the increase in cathepsins B and L.

We studied the effect of chymostatin on dystrophic mice ( C57BL 6J -dy ). The locomotor activity of normal mice increased markedly, attaining a plateau at 8 weeks of age, whereas in dystrophic mice, it increased until the age of 7 weeks, and thereafter decreased gradually. Serum levels of creatine phosphokinase were significantly higher in dystrophic mice compared with normal mice, and dystrophic mice had a reduced muscle protein content. When 3-week-old dystrophic mice received chymostatin (1 mg/kg, i.p.), the decrease in locomotor activity was retarded, serum enzyme levels decreased significantly, and muscle protein content increased significantly. In addition, the survival time of treated dystrophic mice was prolonged. The locomotor activity, serum enzyme levels, and muscle protein content of normal mice were not affected by chymostatin. Therefore, we posit that chymostatin retarded the progression of dystrophy in mice.