RESUMO
Autosomal recessive limb-girdle muscular dystrophy type 2G (LGMD2G) is an adult-onset myopathy characterized by distal lower limb weakness, calf hypertrophy and progressive decline in ambulation. The disease is caused by mutations in Tcap, a z-disc protein of skeletal muscle, although the precise mechanisms resulting in clinical symptoms are unknown. To provide a model for preclinical trials and for mechanistic studies, we generated knockout (KO) mice carrying a null mutation in the Tcap gene. Here we present the first report of a Tcap KO mouse model for LGMD2G and the results of an investigation into the effects of Tcap deficiency on skeletal muscle function in 4- and 12-month-old mice. Muscle histology of Tcap-null mice revealed abnormal myofiber size variation with central nucleation, similar to findings in the muscles of LGMD2G patients. An analysis of a Tcap binding protein, myostatin, showed that deletion of Tcap was accompanied by increased protein levels of myostatin. Our Tcap-null mice exhibited a decline in the ability to maintain balance on a rotating rod, relative to wild-type controls. No differences were detected in force or fatigue assays of isolated extensor digitorum longus (EDL) and soleus (SOL) muscles. Finally, a mechanical investigation of EDL and SOL indicated an increase in muscle stiffness in KO animals. We are the first to establish a viable KO mouse model of Tcap deficiency and our model mice demonstrate a dystrophic phenotype comparable to humans with LGMD2G.
Assuntos
Modelos Animais de Doenças , Proteínas Musculares/genética , Músculo Esquelético/fisiopatologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/fisiopatologia , Fenótipo , Fatores Etários , Análise de Variância , Animais , Conectina , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Marcação de Genes/métodos , Vetores Genéticos/genética , Immunoblotting , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Proteínas Musculares/fisiologia , Músculo Esquelético/ultraestrutura , Miostatina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Teste de Desempenho do Rota-RodRESUMO
Co2+, Fe2+, Cu2+ and Zn2+ were separated and detected following derivatization with 4-(2-pyridylazo)resorcinol by capillary electrophoresis with UV-Vis detection. Parameters which were examined include both on-column and pre-column complexation, limit of detection, capillary loadability, linear dynamic range and reproducibility. A sample stacking technique was investigated in order to obtain better detection limits and greater sensitivity. Detection limits of 1 x 10(-8) M were achieved for Co2+, Fe2+ and Zn2+ and 4 x 10(-7) M for Cu2+. Mass detection limits were 0.2 fmol for Co2+, Fe2+ and Zn2+ and 7.0 fmol for Cu2+. The use of this method for the determination of metals in vitamin tablets and a pond water sample is presented.