Soluble cholinesterase of muscle from dystrophic and normal mice

The percentage of cholinesterase extractable by isotonic sucrose from dystrophic mouse muscle was greater than that found in normal muscle. Of the total cholinesterase found in normal and dystrophic muscle about 60% was specific AChE and 40% was non-specific cholinesterase. The extract from dystrophic muscle showed, on sucrose sedimentation, one major peak of AChE activity with a sedimentation constant of approximately 4.3 S. This was much higher than that from normal muscle.     

Comparative proteomic analysis of the contractile-protein-depleted fraction from normal versus dystrophic skeletal muscle

In basic and applied myology, gel-based proteomics is routinely used for studying global changes in the protein constellation of contractile fibers during myogenesis, physiological adaptations, neuromuscular degeneration, and the natural aging process. Since the main proteins of the actomyosin apparatus and its auxiliary sarcomeric components often negate weak signals from minor muscle proteins during proteomic investigations, we have here evaluated whether a simple prefractionation step can be employed to eliminate certain aspects of this analytical obstacle. To remove a large portion of highly abundant contractile proteins from skeletal muscle homogenates without the usage of major manipulative steps, differential centrifugation was used to decisively reduce the sample complexity of crude muscle tissue extracts. The resulting protein fraction was separated by two-dimensional gel electrophoresis, and 2D-landmark proteins were identified by mass spectrometry. To evaluate the suitability of the contractile-protein-depleted fraction for comparative proteomics, normal versus dystrophic muscle preparations were examined. The mass spectrometric analysis of differentially expressed proteins, as determined by fluorescence difference in-gel electrophoresis, identified 10 protein species in dystrophic mdx hindlimb muscles. Interesting new biomarker candidates included Hsp70, transferrin, and ferritin, whereby their altered concentration levels in dystrophin-deficient muscle were confirmed by immunoblotting.     

Similarities in protein synthesis and degradation in normal and dystrophic muscle cultures

Protein synthesis and degradation were compared in cultured muscle cells obtained from normal and dystrophic chick embryos under conditions where labeled amino acid reincorporation was not a complicating factor, where fibroblast contamination was minimized, and where the animals compared were as genetically similar as possible. Under these conditions both cell types exhibited a half-time of protein turnover of 34 h. Degradation in both was inhibited 21% by leupeptin (50 μg/ml), and both showed parallel increases in degradation rates under ‘step-down’ conditions.     

Electrophysiological observations on diaphragm muscle from normal and dystrophic hamsters

The cellular electrical activity of diaphragm from F1B normal and BIO 14.6 dystrophic hamsters has been investigated using microelectrodes. Resting membrane potentials and action potentials were recorded from control muscles and from muscles exposed to 2,4-dinitrophenol. The action potentials of normal and dystrophic diaphragms were similar in amplitude and configuration. Treatment with 2,4-dinitrophenol caused the action potential amplitude of both diaphragms to decline by similar amounts. The control resting membrane potential of diaphragm from dystrophic hamsters is not significantly different from that of normal hamsters. Treatment with 2,4-dinitrophenol caused a linear decrease in the resting membrane potentials of both groups of muscles. Dystrophic muscle, however, showed a more rapid decline in excitability when exposed to 2,4-dinitrophenol. This suggests that adenosine triphosphate production in dystrophic muscle is partially inhibited as has been suggested by other workers.     

Satellite cells of normal and dystrophic muscle

The structure, the ultrastructure and the number of myonuclei and satellite cells in Duchenne's muscular dystrophy and in control muscles were compared in order to determine the possible changes in the satellite cells population. The bioptical fragments were obtained from 16 healthy (control) and from 16 dystrophic male children from 12 to 96 months of age. The biopsies were embedded in paraffin and in Durcupan and the sections were stained with ematossilin-eosin, P.A.S. for the light microscope observation and with uranil-acetate and lead-citrate for the electron microscope study. Moreover the semithin sections were stained according to the method of Ontell (1974) that is specific for the satellite cells identification. The morphological aspects of the dystrophic muscles are the same previously reported by other authors. The quantitative analysis of the myonuclei and satellite cells in control and dystrophic muscles was carried out on five random sections of each biopsy. The whole number of nuclei (myonuclei and satellite cell nuclei) and the number of the satellite cells nuclei were evaluated and the mean values in controls and dystrophic muscles were compared with the t Student test. The obtained results show that: 1) in the control muscles the satellite cells number is nearly the same in all ages considered; 2) in the dystrophic muscles the satellite cells number is in a statistically significant way greater than in control muscles and show a moderate trend to increase with aging; 3) in the dystrophic muscles the whole number of nuclei (myonuclei and satellite cells) is greater than in control in a statistically significant way and this increase is due to the number of satellite cells.     

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle

Summary Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9–26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9–20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17–18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.     

Microsomal T system: a stereological analysis of purified microsomes derived from normal and dystrophic skeletal muscle

Heterogeneous populations of microsomes obtained from normal and dystrophic chicken pectoralis muscle were separated into two subfractions by an iterative loading technique. The buoyant density of the sarcoplasmic reticulum (SR) microsomes was increased after loading them with calcium oxalate. Several incubations in the transport medium were necessary to load all of the SR. The fraction that did not form a pellet contained microsomes which displayed freeze-fracture faces that had a low density of particles. A stereological analysis was used on membrane fracture faces of intact muscle to generate reference particle density distributions, which were compared with the distributions measured on the microsomal fracture faces. The concave microsomal fracture faces of purified microsomes which did not load calcium oxalate had particle distributions nearly identical to the distributions of intact P-face T tubules. The morphological data suggest that this subfraction is microsomal T system. Biochemical measurements show negligible amounts of specific Na+, K+-ATPase activity, suggesting that there was little contamination from the surface membrane in this subfraction. Furthermore, an active Ca2+-ATPase is demonstrated in both normal and dystrophic T-tubular membranes.