Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) CeU 51, 919-928], is expressed in striated and smooth muscles as well as in nonmuscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 ± 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel a-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function.
Palyelanal (DMD) patients and rndjc mice through mutations which disrupt the reading frame of the transcript preventing dystrophin expression in the corresponding skeletal muscles [g-14]. I-Iigh concentrations of dystrophin (or a dyscrophin-like protein) have been observed at the neuromuscular junctions (NMJ) in normal rats, rabbits or mice as well as in the electric organ of Torpedo marmwata recently reported a positive immunofluorescence response at the NMJ of DMD patients and rndx mice, using antibodies raised against the central and distal parts of chicken skeletal muscle dystrophin [20,21].We now observe a protein of Mr 400000 which is present in extracts of mdx muscle from regions which contain NMJs and is absent from those which do not. This NMJ-associated homologue of dystrophin has at least 2 epitopes which are different to the usual Xp21 form of dystrophin expressed along the sarcolcmma of muscle fibres in normal muscles. This protein is also expressed at the NMJ of a DMD patient who lacks the first 52 exons of the Xp21 dystrophin gene. MATERIALS AND METI-IODS Biopsy tnarerials and diagnosesThe patient was a g-year-old male who has followed a severe, Duchcnne course and is also mentally retarded. No hybridization
The differentiation of skeletal muscle cells from mdx mice which lack dystrophin expression was examined after glucocorticoid treatment, namely alpha-methylprednisolone (PDN). Primary skeletal muscle cell cultures were established from newborn mdx, congenic C57BL/10, and allogenic BALB/C mice. We show that PDN promotes the myogenesis of both mdx- and control mice-derived cultures as determined by 1) the number of myotubes, 2) acetylcholine receptors, and 3) dystrophin and dystrophin-related protein levels. These results support the hypothesis that PDN could enhance the myogenesis of satellite cells and increase dystrophin-related protein expression in DMD treated patients.
Abstract. Two mAbs, one specific for cardiac a-myosin heavy chains (MHC) and the other specific for cardiac 13-MHC, were used to investigate the heavychain dimeric organization of rat cardiac ventricular myosin. Epitopes of the two mAbs were mapped on the myosin molecule by electron microscopy of rotary shadowed mAb-myosin complexes, mAbs were clearly identifiable by the different locations of their binding sites on the myosin rod. Thus, myosin molecules could be directly discriminated according to their r or I3-MHC content. 0m-MHC and [313-MHC homodimers were visualizeA in complexes consisting of two molecules of the same mAb bound to one myosin molecule. By simultaneously using the ot-MHC-specific mAb and the 13-MHC-specific mAb, r heterodimers were visualized in complexes formed by one molecule of each of the two mAbs bound to one myosin molecule. Proportions of rand I]I3-MHC homodimers and r heterodimers were estimated from quantifications of mAb-myosin complexes and compared with the proportions given by electrophoreses under nondenaturing conditions. This visualization of cardiac myosin molecules clearly demonstrates the arrangement of ~t-and 13-MHC in 0t0t-MHC homodimers, 13~MHC homodimers, and r heterodimers, as initially proposed by Hoh, J. E Y., G. P. S. Yeoh, M. A. W. Thomas, and L. Higginbottom (1979).
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