| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
The aim of this st'udy is to observe the shape and distribution of dystrophin molecule in normal human skeletal myofiber by using quick-freeze, deep etch, rotary shadow replica method. Six histochemically normal human quadriceps femoris-muscles undergoing orthopedic operations and six Duchenne biopsied muscles were fixed in chilled 4% paraformaldehyde solution for 2 hours. After washing, the fixed muscles were cut into thin slices by cryostat and the sliced muscles were frozen quickly by liquid helium and then the samples were transferred into freeze fracture apparatus. Fracture was carried out in the vacuum 1, -2 x 10^<-7> Torr at -150ﾟC. The specimens were deep etched at -90ﾟC for 30 minutds, cooled up to -150ﾟC again and rotary shadowed by electron beam gunned platinum and carbon. Electron microscopy of the good quality replicas revealed the orderly arranged myofilaments and, at higher magnification, the myosin cross bridge and about 5nm periodicity of surface contour of actin filam
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ents. There were two modes of fracture and deep-etching faces in myofibers of both Duchenne and normal cbntkol muscles. In one mode, the line of splitting ran frord the basal lamina to the true outer surface bf the muscle plasma membrane, then-enteted the hydrophobic interior of the muscle plasma membrane and jumped into the interior of the myofibers. In this case, the piffotoplasmic fracture face of the muscle plasma membrane and adjacent subsarcolemmal filaments were seen. In the other mode of fracture, the line of splitting ran from the cytoplasm of the myofiberto the cytoplasmic surface of the plasma membrane, then entered the hydrophobic interor of the muscle plasma membrane and jumped into the exterior of the myofibers. In this case, the extracellular fracture face and adjacent cytoplasmic surface of the muscle plasma membrane were noted. At higher magnification, the attachment of individual rod-shaped cytoskeletons of various size to the cytoplasmic surface of the plasma membrane was observed and the elaborate network of cytoskeletal elements was observed in both Duchenne and normal control myofibers. Since th. e marked difference of shape and distribution pattern of individual muscle plasma membrane associated cytoskeletons-were detected between Duchenne and normal control myofibers, dystrophin will be detectecd in the normal muscle specimens labelled by antidystrophin antibodies. Less
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