Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Targeted Nucleotide Exchange in Bovine Myostatin Gene

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Morphological and histochemical study of the myotomal muscle in pacu (Piaractus mesopotamicus Holmberg, 1887) during the initial growth phases

Morphological and histochemical methods were used to evaluate the myotomal muscle characteristics of pacu (Piaractus mesopotamicus) from hatching to 40 days old. During the larval period, the musculature consisted predominantly of white muscle. White and red muscle mass increased at 10, 20, 30 and 40 days after hatching. The larvae had round muscle fibers with a moderate degree of maturation and central nuclei. In subsequent phases, small and immature fibers were visible near larger and more differentiated fibers. Undifferentiated cells or presumptive myoblasts located in embryogenic zones were visible in the dorsal and ventral regions, and were more evident at 30 and 40 days. The red muscle fibers located in the subdermal region, had oxidative metabolism and slow contraction, whereas the more predominant white muscle fibers had glycolytic metabolism and fast contraction. Our findings indicate that during the initial phases, myotomal muscle growth in pacu occurs by both, muscle fiber hypertrophy and hyperplasia. The analysis of frequency of red and white muscle fibers shows that hyperplastic growth is intense in this period. As the growth rate in adult fish is related to the number of muscle fibers in young fish, extrinsic factors could change the muscle fiber phenotype and influence their ultimate size.

The Effects of a 785-nm AlGaInP Laser on the Regeneration of Rat Anterior Tibialis Muscle After Surgically-Induced Injury

Objective: This study aims to investigate the effects of low-level laser therapy (LLLT) on muscle regeneration. For this purpose, the anterior tibialis muscle of 48 male Wistar rats received AlGaInP laser treatment (785 nm) after surgically-induced injury.Background Data: Few studies have been conducted on the effects of LLLT on muscle regeneration at different irradiation doses.Materials and Methods: The animals were randomized into four groups: uninjured rats (UN); uninjured and laser-irradiated rats (ULI); injured rats (IN); and injured and laser-irradiated rats (ILI). The direct contact laser treatment was started 24 h after surgery. An AlGaInP diode laser emitting 75 mW of continuous power at 785 nm was used for irradiation. The laser probe was placed at three treatment points to deliver 0.9 J per point, for a total dose of 2.7 J per treatment session. The animals were euthanized after treatment sessions 1, 2, and 4. Mounted sections were stained with hematoxylin and eosin and used for quantitative morphological analysis, in which the number of leukocytes and fibroblasts were counted over an area of 4480 mu m(2). The data were statistically analyzed by analysis of variance (ANOVA) and the Bonferroni t-test.Results: Quantitative data showed that the number of both polymorphonuclear and mononuclear leukocytes in the inflammatory infiltrate at the injury site was smaller in the ILI(1), ILI(2), and ILI(4) subgroups compared with their respective control subgroups (IN(1), IN(2), and IN(4)) for sessions 1, 2, and 4, respectively (p < 0.05). on the other hand, the number of fibroblasts increased after the fourth treatment session (p < 0.05). With regard to the regeneration of muscle fibers following injury, only after the fourth treatment session was it possible to find muscle precursor cells such as myoblasts and some myotubes in the ILI(4) subgroup.Conclusion: During the acute inflammatory phase, the AlGaInP laser treatment was found to have anti-inflammatory effects, reducing the number of leukocytes at the injury site and accelerating the regeneration of connective tissue.

Mecanismos celulares e moleculares que controlam o desenvolvimento e o crescimento muscular

O músculo estriado esquelético é formado pela associação de fibras musculares com a matriz extracelular. Esse tecido possui alta plasticidade e o conhecimento das características morfológicas, da miogênese, e da dinâmica do crescimento é importante para o entendimento da morfofisiologia bem como para a seleção de animais visando a melhoria na produção de carne. A maioria dos músculos estriados originam-se de células precursoras do mesoderma a partir dos somitos do embrião e o controle da diferenciação ocorre pela ação de fatores indutores ou inibidores. Um grupo de fatores transcricionais, pertencentes à família MyoD tem um papel central na diferenciação muscular. Coletivamente chamados de Fatores de Regulação Miogênica (MRFs), são conhecidos quatro tipos: MyoD, myf-5, miogenina e MRF4. Esses fatores ligam-se à seqüências de DNA conhecidas como Ebox (CANNTG) na região promotora de vários genes músculo-específicos, levando à expressão dos mesmos. As células embrionárias com potencial para diferenciação em células musculares (células precursoras miogênicas) expressam MyoD e Myf-5 e são denominadas de mioblastos. Essas células proliferam, saem do ciclo celular, expressam miogenina e MRF4, que regulam a fusão e a diferenciação da fibra muscular. Uma população de mioblastos que se diferencia mais tardiamente, as células miossatélites, são responsáveis pelo crescimento muscular no período pós natal, que pode ocorrer por hiperplasia e hipertrofia das fibras. As células satélites quiescentes não expressam os MRFs, porém, sob a ação de estímulos como fatores de crescimento ou citocinas, ocorre a ativação desse tipo celular que prolifera e expressa os MRFs de maneira similar ao que ocorre com as células precursoras miogênicas durante a miogênese. Os mecanismos de crescimento muscular são regulados pela expressão temporal dos (MRFs), que controlam a expressão dos genes relacionados com o crescimento muscular.

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom

Myotoxin-I (MjTX-I) was purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. Its molecular weight, estimated by SDS-PAGE, was 13,400 (reduced) or 26,000 (unreduced). The extinction coefficient (E-1.0 cm(1.0 mg/ml)) of MjTX-I was 1.145 at lambda = 278 nm, pH 7.0, and its isoelectric point was 8.2 at ionic strength mu = 0.1. When lyophilized and stored at 4 degrees C, dimeric, trimeric, and pentameric forms of the protein were identified by SDS-PAGE. This heterogeneous sample could be separated into three fractions by gel filtration on Sephadex 6-50. The fractions were analyzed by isoelectric focusing, immunoelectrophoresis, and amino acid composition, which indicated that heterogeneity was the result of different levels of self-association. Protein sequencing indicated that MjTX-I is a Lys49 myotoxin and consists of 121 amino acids (M-r = 13,669), containing a high proportion of basic and hydrophobic residues. It shares a high degree of sequence identity with other Lys49 PLA(2)-like myotoxins, but shows a significantly lower identity with catalytically active Asp49 PLA(2)s. The three-dimensional structure of MjTX-I was modeled based on the crystal structures of three highly homologous Lys49 PLA(2)-like myotoxins. This model showed that the amino acid substitutions are conservative, and mainly the beta-wing region, and the C-terminal extended random coil. MjTX-I displays local myotoxic and edema-inducing activities in mice, and is lethal by intraperitoneal injection, with an LD50 value of 8.5 +/- 0.8 mg/kg, In addition, it is cytotoxic to myoblasts/ myotubes in culture, and disrupts negatively charged liposomes. In comparison with the freshly prepared dimeric sample, the more aggregated forms showed significantly reduced myotoxic activity. However, the edema-inducing activity of MjTX-I was independent of molecular association. Phospholipase A(2) activity on egg yolk, as well as anticoagulant activity, were undetectable both in the native and in the more associated forms. His, Tyr, and Trp residues of the toxin were chemically modified by specific reagents. Although the myotoxic and lethal activities of the modified toxins were reduced by these treatments, neither its edema-inducing or Liposome-disrupting activities were significantly altered. Rabbit antibodies to native MjTX-I cross-reacted with the chemically modified forms, and both the native and modified MjTX-I preparations were recognized by antibodies against the C-terminal region 115-129 of myotoxin II from B. asper, a highly Lys49 PLA(2)-homologue with high sequencial similarity. (C) 2000 Academic Press.

The role of myofibroblasts and interstitial fibrosis in the progression of membranous nephropathy

Renal interstitial fibrosis has been observed in a large number of nephropathies and contributes to the progressive deterioration of renal function. Myofibroblasts have been implicated in the reparative process of tissue injury, including renal scarring secondary to glomerular diseases. We performed a retrospective study on 28 patients with biopsy-proven primary membranous nephropathy, to determine whether interstitial myofibroblasts and tubulointerstitial lesions correlated with renal function at follow-up. Tubulointerstitial pathology was evaluated by morphometric and semiquantitative methods. Interstitial myofibroblasts were counted; 24-hour urinary protein and serum creatinine at the time of diagnosis and at the end of follow-up were available for all the patients. There were 20 males and 8 females, age 2-67 years (mean 42.3±153), most of them with nephrotic syndrome (78.6%). The final renal function had deteriorated in 16 patients (57.1%) and in 5 patients (17.8%) reached end-stage. The renal outcome was correlated with histological changes. We found a positive correlation between the severity of tubulointerstitial damage and the deterioration of the final serum creatinine (r 2=0.185; p=0.016). Myofibroblasts did not predict impaired renal function at the final follow-up. The current data do not support previous suggestions that myofibroblasts are a useful a predictor of end-stage renal disease.

Transmissão de produtos farmacêuticos tópicos através de energia ultra-sônica na regeneração de lesões da musculatura esquelética - enfoque morfológico

Pós-graduação em Ciências da Motricidade - IBRC

Crioconservação de embriões de peixes neotropicais

Pós-graduação em Biologia Animal - IBILCE

Loss of nuclear poly(A)-binding protein 1 (PABPN1) causes defects in myogenesis and mRNA biogenesis

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Expressão de microRNAs envolvidos com o crescimento muscular do pacu (Piaractus mesopotamicus): análises in vivo e in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)