Aspects of normal and dystrophic chicken muscle grown in vitro.

Pectoral muscle from normal and dystrophic New Hampshire chicken embryos was dissociated and grown in vitro. Marked differences between the two types of cell cultures were observed with the light and electron microscopes during early myogenic stages. The diseased myoblasts assumed a polarized affect and fused into smaller and fewer myotubes. Pseudostraps rather than true muscle straps were often seen in diseased cultures. There was also a delay in the appearance of myosin containing thick myofilaments in differentiating dystrophic muscle cells.     

Leucine pools in normal and dystrophic chicken skeletal muscle cells in culture

The specific radioactivity of [3H]Leu in the extracellular, intracellular, and Leu-tRNA pools of normal (white leghorn) and dystrophic (line 307) embryonic chick breast muscle cultures was analyzed as a function of equilibration time and extracellular Leu concentration (0.05-5 mM). The primary results were the following 1) [3H]Leu equilibrated to a constant specific radioactivity in the intracellular and Leu-tRNA pools within 2 min after addition to both normal and dystrophic cultures. 2) After equilibration, the extracellular [3H] Leu specific radioactivity in dystrophic cell culture medium was lower than that of medium exposed to normal cells (especially at low Leu concentrations), probably because of increased release of unlabeled Leu from the dystrophic cells as a result of faster protein breakdown. Accordingly, the specific radioactivities in the intracellular and the Leu-tRNA pools were also lower in dystrophic cells. 3) At 5 mM extracellular Leu, the specific radioactivity in the Leu-tRNA pool was approximately 40% lower than the specific radioactivity in the intracellular pool in both normal and dystrophic cells. Thus, high concentrations of extracellular Leu cannot be used to "flood out" reutilization of unlabeled Leu (released by protein degradation) during protein synthesis. 4) At 5.0 mM extracellular Leu, the specific radioactivity of [3H]Leu in the intracellular pool was comparable to that in the extracellular pool in normal and dystrophic cells; however, the specific radioactivity of Leu-tRNA (i.e. the immediate precursor to protein synthesis) was only 55-65% of the extracellular specific radioactivity in normal and dystrophic cells. In conclusion, reutilization of Leu from protein degradation is higher in dystrophic muscle cell cultures than in normal muscle cell cultures, and accurate rates of protein synthesis in cell cultures can only be obtained if specific radioactivity of amino acid in tRNA is measured.     

Myoglobin in control and dystrophic chicken muscle

This study shows no differences in the character of myoglobin in muscles from control and genetically dystrophic chickens. Our data on cellulose chromatography, acrylamide gel electrophoresis, sedimentation velocities, spectra, isoelectric points, and amino acid analyses provide useful information on the first avian myoglobin to be added to the list of mammalian and fish myoglobins which have already been characterized.     

Retarded decline in poly-ADPR content and poly-ADPR synthetase activity in chicken dystrophic muscle

The activity of poly-ADPR synthetase declines just after hatching in normal chick muscle nuclei. However, in dystrophic chick muscle nuclei it decreases 5 weeks after hatching. A delayed decrease in the amount of poly-ADPR is also observed in dystrophic chick muscle nuclei. These observations suggest that dystrophic muscle follows an abnormal developmental program.     

Arylamidase and cathepsin-A activity of normal and dystrophic human muscle.

Human skeletal muscle homogenate has been shown to contain enzymes that catalyze the hydrolysis of L-leucyl p-nitroanilide and carbobenzoxyglutamyl-L-tyrosine, known substrates, respectively, for arylamidase and cathepsin A. The muscle arylamidase was found to be inhibited by p-chloromercuribenzoate. Addition of Co2+ resulted in slight stimulation of its activity. Neither ethylenediamine tetraacetate nor thiol compounds had any appreciable effect on the enzyme. When compared to controls, no significant differences in muscle arylamidase levels were observed in patients with muscular dystrophies and certain selected neuromuscular diseases. Cathepsin A was, however, increased in muscles moderately affected by muscular dystrophy and denervating diseases.     

Effect of leupeptin of protein turnover in normal and dystrophic chicken skeletal muscle cells in culture

The effect of leupeptin on turnover of the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain was evaluated in muscle cell cultures. Cultures were prepared from the breast muscle of 12-day normal (white leghorn) and dystrophic (line 307) chick embryos. After 7 days in culture, cells were labeled for 16 hr with 1 μCi/ml of [³H]Leu and then “chased” for an additional 24 hr in culture medium containing no [³H]Leu and 0–75 μg/ml leupeptin. Cultures were analyzed for radioactivity in the soluble protein fraction, the myofibrillar protein fraction, and myosin heavy chain. Leupeptin (75 μg/ml) virtually eliminated loss of radioactivity from the soluble protein fraction, but only minimally affected loss of radioactivity from the myofibrillar fraction or myosin heavy chain. Normal and dystrophic muscle cells responded identically to leipeptin treatment. Thus, the muscle proteases that are specifically inhibited by leupeptin seem to have no major role in initiating myofibrillar protein turnover.