| Project/Area Number |
11670643
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurology
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| Research Institution | Showa University |
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Principal Investigator |
WAKAYAMA Yoshihiro School of Medicine, Showa University professor, 医学部, 教授 (40138467)
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| Co-Investigator(Kenkyū-buntansha) |
JIMI Takahiro School of Medicine, Showa University Assistant professor, 医学部, 助教授 (30196654)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Duchenne dystrophy / Fukuyama dystrophy / aquaporin 4 / protein / genomic DNA / mRNA / depletion / mechanism / Duchenne筋ジストロフィー症 / 福山型筋ジストロフィー / 骨格筋細胞膜 / feeze fracture replica / orthogonal arrays / PCR / RT-PCR |
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| Research Abstract |
Previous our freeze fracture electron microscopic studies demonstrated that the muscle plasma membrance of boys with Duchenne muscular dystrophy (DMD) and children with Fukuyama congenital muscular dystrophy (FCMD) contained the markedly reduced number of orthogonal arrays (OAs). Recent investigations revealed that the major protein component of OAs was aquaporin4 (AQP4) which was a member of water channel protein family. Present investigations were undertaken to analyze the mechanism of the depletion of AQP4 in the muscle plasma membranes with DMD and FCMD at protein genomic DNA and mRNA levels. The blood and muscle samples were taken under informed consent from 6 boys with DMD 4 children with FCMD 5 patients with myotonic dystrophy 1 patient with limb-girdle muscular dystrophy 1 patient with myasthenia gravis and 6 normal controls. At protein level immunohistochemical staining and immunoblot analyses were done using rabbit anti AQP4 and anti β-spectrin antibodies and monoclonal anti
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dystrophin antibody (Novocastra DYS-1). The immunohistochemistry of DMD and FCMD muscles using anti AQP4 antibody showed that the DMD and FCMD muscles were composed of the negatively stained myofibers mixed with the positively stained sporadic myofibers. The immunostainability of DMD and FCMD muscles using anti dystrophin antibody was negative and positive respectively. The anti β-spectrin antibody stained basically all of the DMD and FCMD myofibers. The immunostaining of normal and disease control muscles by using these three antibodies revealed the positive staining at their cell periphery in all myofibers. Immunoblot analyses showed that the staining intensity of DMD and FCMD muscles with anit AQP4 antibody was markedly decreased : while the immunostaining reactions of DMD and FCMD muscles with anti dystrophin antibody were negative and positive, respectively and those of DMD and FCMD muscles with anit β-spectrin antibody were positive similar to those of normal and disease control muscles. The genomic DNA of AQP4 molecule was detected by PCR in all blood samples including DMD and FCMD samples. Further the AQP4 mRNA of DMD and FCMD muscles was analyzed by RT-PCR and was markedly decreased in both dystrophies. The results of this study suggested that the reduced expression of AQP4 in DMD and FCMD muscles was due to the decreased level of AQP4 mRNA in these muscles. Less
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