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1.
J Appl Physiol (1985) ; 109(3): 901-5, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20558759

RESUMO

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (approximately 15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.


Assuntos
Bioensaio/métodos , Membrana Celular/efeitos dos fármacos , Códon sem Sentido , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/metabolismo , Oxidiazóis/farmacologia , Músculo Quadríceps/metabolismo , Animais , Animais Recém-Nascidos , Membrana Celular/metabolismo , Membrana Celular/patologia , Células Cultivadas , Disferlina , Humanos , Soluções Hipotônicas , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Pressão Osmótica , Músculo Quadríceps/patologia , Transfecção
2.
Nature ; 447(7140): 87-91, 2007 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-17450125

RESUMO

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.


Assuntos
Códon sem Sentido/genética , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Genéticas Inatas/genética , Oxidiazóis/farmacologia , Oxidiazóis/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Alelos , Animais , Disponibilidade Biológica , Distrofina/biossíntese , Distrofina/genética , Doenças Genéticas Inatas/sangue , Humanos , Camundongos , Camundongos Endogâmicos mdx , Oxidiazóis/administração & dosagem , Oxidiazóis/farmacocinética , Fenótipo , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato
3.
J Cell Sci ; 118(Pt 7): 1405-16, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15769854

RESUMO

The functions of gamma-sarcoglycan (gammaSG) in normal myotubes are largely unknown, however gammaSG is known to assemble into a key membrane complex with dystroglycan and its deficiency is one known cause of limb-girdle muscular dystrophy. Previous findings of apoptosis from gammaSG-deficient mice are extended here to cell culture where apoptosis is seen to increase more than tenfold in gammaSG-deficient myotubes compared with normal cells. The deficient myotubes also exhibit an increased contractile prestress that results in greater shortening and widening when the cells are either lightly detached or self-detached. However, micropipette-forced peeling of single myotubes revealed no significant difference in cell adhesion. Consistent with a more contractile phenotype, acto-myosin striations were more prominent in gammaSG-deficient myotubes than in normal cells. An initial phosphoscreen of more than 12 signaling proteins revealed a number of differences between normal and gammaSG(-/-) muscle, both before and after stretching. MAPK-pathway proteins displayed the largest changes in activation, although significant phosphorylation also appeared for other proteins linked to hypertension. We conclude that gammaSG normally moderates contractile prestress in skeletal muscle, and we propose a role for gammaSG in membrane-based signaling of the effects of prestress and sarcomerogenesis.


Assuntos
Apoptose/fisiologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mioblastos Esqueléticos/fisiologia , Sarcoglicanas/fisiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Sarcoglicanas/deficiência , Sarcoglicanas/metabolismo , Transdução de Sinais/fisiologia
4.
Magn Reson Med ; 51(2): 273-7, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14755651

RESUMO

In this study the ability of magnetodendrimers to efficiently label cultured muscle stem cells and allow for subsequent in vivo cell detection was determined. Magnetodendrimer-labeled cells exhibited normal growth rates in culture, and retained their capacity to undergo proliferation and form normal myotubes. Labeled stem cells possessed high in vivo proton relaxivities that enhanced MRI contrast properties and enabled us to noninvasively monitor the stem cells' incorporation into dystrophic muscle. Well defined regions of decreased signal intensity were observed in both T2- and T1-weighted image sequences. MRI was used to longitudinally follow stem cell dynamics in dystrophic muscle with in-plane resolutions on the order of a single muscle fiber (22 x 43 microm2). Regions of decreased signal intensity were well correlated with iron accumulation and other histochemical markers of stem cell incorporation. We concluded that this technique may be useful for continuous noninvasive readouts of stem cell transfer, replacing sequential muscle biopsies and tissue harvesting.


Assuntos
Imageamento por Ressonância Magnética/métodos , Músculo Esquelético/citologia , Células-Tronco/citologia , Animais , Linhagem Celular , Células Cultivadas , Imuno-Histoquímica , Camundongos , Doenças Musculares/terapia , beta-Galactosidase/metabolismo
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