Protein profiles of sarcoplasmic reticulum from normal and dystrophic mouse muscle.

Sarcoplasmic reticulum (SR) was isolated from skeletal muscle of dystrophic (C57BL/6J dy2J/dy2J) mice and the protein composition analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Densitometric analysis of dystrophic SR preparations indicated a decrease in the Ca2+-ATPase and calsequestrin, and the appearance of a protein with molecular weight 72 000. These differences in the protein profiles between normal and dystrophic SR became more apparent as the disease progressed. The observations are discussed in relation to secondary changes in the dystrophic process such as changes in fibre type and the presence of immature fibres.

A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing.

The application of silver staining methods to the detection of proteins on ultrathin isoelectric focusing gel systems requires the optimization of many steps in the procedure in order to obtain reproducible staining of proteins with acceptable levels of background. Three different methods which have been reported for detecting proteins by silver staining in sodium dodecyl sulfate-polyacrylamide gel systems were investigated. A major problem with staining ultrathin isoelectric focusing gels was found to be surface staining that was associated with gels cast on support films. A modification of the method of Poehling and Neuhoff (H.-M. Poehling and V. Neuhoff, 1981, Electrophoresis 2, 141-147) was found to give the best results.

Deformability and cell membrane properties of erythrocytes in Duchenne muscular dystrophy.

In many investigations of the hypothesis of a membrane defect in Duchenne muscular dystrophy, the erythrocyte has been the cell of choice, but reported abnormalities in the Duchenne erythrocyte are disputed. Techniques of microsieving, cell surface staining and measurement of cell water diffusion turnover rate have been used in this investigation to examine erythrocyte properties. Measurements of pressure drop-velocity relations were carried out in a capillary pore rheometer using pure erythrocyte suspensions. The study showed that Duchenne erythrocytes do not behave differently from normal, results which are in substantial agreement with similar microsieving studies. A nuclear magnetic resonance method was used to measure cell water diffusion efflux which, using our cell surface data, could be related to cell membrane permeability. Under normal conditions and conditions of chemical interference with the membrane protein, the results revealed that the membrane pathways involved in water diffusion in Duchenne erythrocytes are unaltered.

In vitro skeletal muscle protein and RNA incorporation in neuromuscular disease.

Skeletal muscle of Duchenne muscular dystrophy (DMD) patients, DMD carriers and of patients with other neuromuscular diseases was sampled at diagnostic muscle biopsy. The incorporation of isotopic precursors into protein and RNA in a 1-hour in vitro incubation of these samples was then determined. Individual DMD results overlap with these obtained with normal muscle, and an inverse relationship is observed between DMD patient age and incorporation into skeletal muscle protein. The protein and RNA incorporation of DMD carrier skeletal muscle was normal. Limb-girdle dystrophy shows an increase in protein incorporation, while nemaline myopathy, dystrophia myotonica and polymyositis show increases in both protein and RNA incorporation over normal skeletal muscle. The significance of the incorporation observed in the neuromuscular disorders studied in discussed.

In vitro radioisotope precursor incorporation into ribonucleic acid and protein in dystrophic mouse gastrocnemius muscle (C57 BL/6J dy2J/dy2J).

An in vitro tissue slice technique has been developed and used to compare radioisotope precurosr incorporation into RNA and protein in normal and dystrophic mouse gastrophic muscle. Significant differences are observed with both RNA and protein when incorporation is measured on a fresh weight of muscle basis. Specific activity comparisons show significantly increased incorporation with protein but not with RNA. A comparison of the results with in vivo studies has been made. The in vitro system developed is applicable to the study of macromolecular metabolism in normal and diseased human muscle tissue.

Studies in vivo of ribonucleic acid turnover in mice (strain C57 BL/6J) homozygous and heterozygous for the dystrophic gene, dy(2J).

In vivo incorporation of [5-(3)H]uridine into RNA of 40-day-old mice, homozygous and heterozygous for the dy(2J) dystrophic allele, has been examined. Increased incorporation is seen in leg skeletal muscle but no increase is liver tissue. Determination of DNA and RNA content of the muscle tissue examined showed that the increased incorporation was not due to muscle volume changes that are associated with wasting in dystrophic muscle. The results obtained with the dy(2J) allele are discussed with respect to related investigation with the dy dystrophic allele.