RESUMO
Tissue engineering of skeletal muscle from cultured cells has been attempted using a variety of synthetic and natural macromolecular scaffolds. Our study describes the application of artificial scaffolds (collagen sponges, CS) consisting of collagen-I with parallel pores (width 20-50 microm) using the permanent myogenic cell line C(2)C(12). CS were infiltrated with a high-density cell suspension, incubated in medium for proliferation of myoblasts prior to further culture in fusion medium to induce differentiation and formation of multinucleated myotubes. This resulted in a parallel arrangement of myotubes within the pore structures. CS with either proliferating cells or with myotubes were grafted into the beds of excised anterior tibial muscles of immunodeficient host mice. The recipient mice were transgenic for enhanced green fluorescent protein (eGFP) to determine a host contribution to the regenerated muscle tissue. Histological analysis 14-50 days after surgery showed that donor muscle fibres had formed in situ with host contributions in the outer portions of the regenerates. The function of the regenerates was assessed by direct electrical stimulation which resulted in the generation of mechanical force. Our study demonstrated that biodegradable CS with parallel pores support the formation of oriented muscle fibres and are compatible with force generation in regenerated muscle.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Colágeno/farmacologia , Colágeno/ultraestrutura , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Porosidade , Próteses e ImplantesRESUMO
1. Searching for the structural requirements improving the potency and the stereoselectivity of Na(+) channel blockers as antimyotonic agents, new derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid alpha-proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. 2. Their ability to block Na(+) currents, elicited from -100 to -20 mV at 0.3 Hz (tonic block) and 2-10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. 3. The alpha-proline derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. 4. Contrarily to tocainide, all proline derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic derivatives markedly decreased the drug potency with respect to tocainide. 5. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. 6. Thus, constraining the chiral centre of tocainide in alpha-proline cycle leads to more potent and stereoselective use-dependent Na(+) channel blockers with improved therapeutic potential.
Assuntos
Antiarrítmicos/farmacologia , Músculo Esquelético/efeitos dos fármacos , Miotonia/tratamento farmacológico , Bloqueadores dos Canais de Sódio , Tocainide/farmacologia , Animais , Relação Dose-Resposta a Droga , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Mutantes , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/fisiologia , Mutação , Miotonia/genética , Miotonia/fisiopatologia , Rana esculenta , Canais de Sódio/fisiologia , Estereoisomerismo , Relação Estrutura-Atividade , Tocainide/químicaRESUMO
The expression of a transgene NI-ROSA LacZ (LacZtg) trapped into the genes for two presumably untranslated, ubiquitously expressed RNAs, was studied in preimplantation mouse embryos with respect to penetrance (fraction of expressing embryos) and to localisation of beta-galactosidase activity. With maternal origin in NMRI mice beta-galactosidase was first detected within one dot in the cytoplasm of zygotes at 30 h post-hCG. The staining pattern progressed to small clusters and to dense, homogeneous staining of the entire cytoplasm during further development. Within the NMRI background, penetrance in utero was delayed by at least 6 h when the transgene was of paternal as compared with maternal origin. Paternal transgene expression increased marginally during culture to 50 h after explantation of embryos at 30-48 h post-hCG and remained low or decreased in the '2-cell block'. Expression of a paternal transgene in preimplantation embryos developing in utero was further delayed in the maternal MF1 as compared with the NMRI background. In contrast to NMRI x NMRI embryos with paternally derived transgene, expression increased with time during the 2-cell block in MF1 x NMRI embryos. Thus, in the earliest phase of mammalian development expression of this LacZtg is influenced by parental origin, maternal genetic background and environment. The spatial distribution of the gene product is developmentally controlled.
Assuntos
Blastocisto/metabolismo , Embrião de Mamíferos/metabolismo , Óperon Lac , Transgenes , Animais , Citoplasma/metabolismo , Pai , Feminino , Genes Reporter/genética , Impressão Genômica , Masculino , Camundongos , Mães , RNA/metabolismo , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
We analyzed, with respect to heat shock proteins (HSPs), systemically reacting tobacco leaves inoculated with Tobacco mosaic virus (TMV), wild-type vulgare, and temperature-sensitive coat protein (CP) mutants Ni 118 (P20L) and flavum (D19A), kept at 23 or 30 degrees C. HSP18 and HSP70 mRNAs and proteins were induced with temperature-sensitive CP mutants after 1 to 2 days at 30 degrees C. After 4 to 6 days, HSP70 was also induced at 23 degrees C. The induction of HSPs paralleled the amount of insoluble TMV CP in leaf extracts, indicating that denatured TMV CP by itself induces a heat-shock response.
Assuntos
Capsídeo/genética , Nicotiana/virologia , Proteínas de Plantas , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/patogenicidade , Capsídeo/fisiologia , Genes Virais , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Mutação , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Temperatura , Nicotiana/genética , Nicotiana/fisiologiaRESUMO
Myogenesis involves the determination of progenitor cells to myoblasts, their fusion to yield multinuclear myotubes, and the maturation of myotubes to muscle fibres. This development is reflected in a time pattern of gene expression, e.g. of genes coding for desmin, the myogenic factors myogenin and myoD, the acetylcholine receptor alpha-subunit and the muscular chloride channel CIC-1. We attempted to improve yields and myogenic differentiation in culture by using three-dimensional microcarrier systems. Out of a variety of carriers tested in stationary cultures, collagen-coated dextran Cytodex3 beads proved optimal for the proliferation and differentiation of the murine myogenic cell line C2C12. With C2C12 myoblasts in stationary and stirred systems (Spinner- and SuperSpinner flasks), surface adherence, differentiation into myotubes and expression of muscle-specific mRNAs on Cytodex3 beads were the same as in conventional cultures. Other carriers tested (DEAE cellulose, glass, plastic, cellulose, polyester) did not support growth and differentiation of C2C12 cells. The secondary mouse myogenic stem cells M12 and M2.7-MDX proliferated and differentiated well in stationary Cytodex3 cultures, but no differentiation occurred in Spinner flasks. As indicated by light and scanning electron microscopy, C2C12 myotubes formed not only on but also in between Cytodex beads. The secondary cell lines may succumb to shear forces under these conditions.
Assuntos
Técnicas de Cultura de Células/métodos , Músculo Esquelético/citologia , Actinas/genética , Animais , Adesão Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Desmina/genética , Expressão Gênica , Camundongos , Microscopia Eletrônica de Varredura , Microesferas , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.
Assuntos
Distrofina/metabolismo , Mitocôndrias Cardíacas/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Respiração Celular , Creatina Quinase/metabolismo , Distrofina/deficiência , Distrofina/genética , Feminino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Microscopia Confocal , Translocases Mitocondriais de ADP e ATP/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Fosforilação Oxidativa , Especificidade por SubstratoAssuntos
Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 2/genética , Proteínas Nucleares/genética , Mapeamento Físico do Cromossomo , Mapeamento de Híbridos Radioativos , Animais , Cromossomos Artificiais de Levedura/genética , Éxons/genética , Humanos , Internet , Escore Lod , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Homologia de Sequência do Ácido Nucleico , Ubiquitina-Proteína LigasesRESUMO
K(V)3.4 belongs to the shaw subfamily of shaker-type potassium channels. It conducts fast inactivating, high threshold currents in the central nervous system and in fast-twitch skeletal muscle fibers. The corresponding mouse gene, Kcnc4, consists of five exons spanning a region of 20 kb. Approximately 700 bp of regulatory sequence were delineated. It is GC-rich and lacks typical TATA and CAAT motifs. Instead, seven Sp-1 and three E-box elements define putative regulatory sequences. The mouse K(V)3.4 mRNA has a size of 3639 bp, 1120 bp of which are 3' untranslated region. A transcript initiated from an alternative 5'-exon was identified by RACE and verified by genomic analysis. This isoform, designated K(V)3.4d, is predominantly expressed in skeletal muscle and probably results from alternative promoter usage. It encodes a channel protein with a novel N-terminal cytoplasmic domain. It lacks the conserved sequence motifs encoding the shaw-type tetramerization domain and the 'ball' peptide, which confers fast inactivation properties. Another splice variant, K(V)3.4c, is derived by exon skipping in the C-terminal region and is expressed at similar levels in brain and muscle. These data demonstrate that differential splicing and alternative transcription start sites are utilised to generate a set of K(V)3.4 variants in skeletal muscle and brain, presumably involved in the regulation of excitability.
Assuntos
Genes/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , RNA Mensageiro/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/química , DNA/genética , Éxons , Variação Genética , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Canais de Potássio Shaw , Distribuição Tecidual , Transcrição GênicaRESUMO
We have investigated the physiological role of desmin in skeletal muscle by measuring isometric tension generated in skinned fibres and intact skeletal muscles from desmin knock-out (DES-KO) mice. About 80% of skinned single extensor digitorum longus (EDL) fibres from adult DES-KO mice generated tensions close to that of wild-type (WT) controls. Weights and maximum tensions of intact EDL but not of soleus (SOL) muscles were lowered in DES-KO mice. Repeated contractions with stretch did not affect subsequent isometric tension in EDL muscles of DES-KO mice. Tension during high frequency fatigue (HFF) declined faster and this deficiency was compensated in DES-KO EDL muscles by 5 mM caffeine which had no influence on HFF in WT EDL. Furthermore, caffeine evoked twitch potentiation was higher in DES-KO than in WT muscles. We conclude that desmin is not essential for acute tensile strength but rather for optimal activation of intact myofibres during E-C coupling.
Assuntos
Desmina/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Animais , Cafeína/farmacologia , Desmina/genética , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Tamanho do Órgão , FenótipoRESUMO
ADAM proteases, defined by extracellular disintegrin and metalloprotease domains, are involved in protein processing and cell-cell interactions. Using wobbler (WR) mutant mice, we investigated the role of ADAMs in neurodegeneration and reactive glia activation in the CNS. We found that ADAM8 (CD 156), a suspected leukocyte adhesion molecule, is expressed in the CNS and highly induced in affected CNS areas of WR mice, in brainstem and spinal cord. ADAM8 mRNA and protein are found at low levels throughout the normal mouse CNS, in neurons and oligodendrocytes. In the WR CNS regions in which neurodegeneration occurs, ADAM8 is induced in neurons, reactive astrocytes, and activated microglia. Similarly, the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) is upregulated and shows the same cellular distribution. In primary astrocytes from wild-type and WR mice, in primary cerebellar neurons, and in mouse motoneuron-like NSC19 cells, ADAM8 expression was induced up to 15-fold by mouse TNF-alpha, in a dose-dependent manner. In both cell types, ADAM8 was also induced by human TNF-alpha, indicating that TNF receptor type I (p55) is involved. Induction of ADAM8 mRNA was suppressed by treatment with an interferon-regulating factor 1 (IRF-1) antisense oligonucleotide. We conclude that IRF-1-mediated induction of ADAM8 by TNF-alpha is a signaling pathway relevant for neurodegenerative disorders with glia activation, proposing a role for ADAM8 in cell adhesion during neurodegeneration.
Assuntos
Antígenos CD , Antígenos de Superfície/biossíntese , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Proteínas de Membrana/biossíntese , Neuroglia/metabolismo , Neurônios/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM , Animais , Antígenos de Superfície/análise , Antígenos de Superfície/genética , Comunicação Celular/efeitos dos fármacos , Extratos Celulares/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Citocinas/biossíntese , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Desintegrinas/biossíntese , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Fator Regulador 1 de Interferon , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Metaloendopeptidases/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Neuroglia/citologia , Neuroglia/patologia , Neurônios/citologia , Neurônios/patologia , Oligonucleotídeos Antissenso/farmacologia , Especificidade de Órgãos/genética , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Ativação Transcricional , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.
Assuntos
Astrócitos/enzimologia , Sistema Nervoso Central/enzimologia , Metaloendopeptidases/biossíntese , Doenças Neurodegenerativas/enzimologia , Animais , Astrócitos/imunologia , Astrócitos/patologia , Linhagem Celular , Células Cultivadas , Sistema Nervoso Central/patologia , Indução Enzimática/genética , Indução Enzimática/imunologia , Regulação da Expressão Gênica/imunologia , Metaloproteinase 14 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , RNA Mensageiro/biossíntese , Proteínas Recombinantes/farmacologia , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidores Teciduais de Metaloproteinases/biossíntese , Inibidores Teciduais de Metaloproteinases/genética , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
The YPT1/RAB1 protein, a key regulator of the intracellular vesicle transport in eukaryotes, is highly conserved in function and amino acid sequence. Here we report that the most highly conserved nucleotide sequence of the Rab1a gene of amniote vertebrates corresponds to the 3'-untranslated region (3'-UTR) of the mRNA. Sequences of 27 species ranging from mammals to sauropsida are >91% identical in this region. Secondary structure prediction procedures applied to the 3'-UTR sequences between positions 750 and 984 and 1428 (mouse cDNA: Y00094), respectively, of the RAB1a mRNAs revealed families of alternative structures around nucleotide position 800 as recurrent features. The two hairpin loops are also predicted for marsupials, despite of their exceptional extension of the A-rich sequence in between. Yet, sequence conservation is much higher than required to conserve secondary structure. Implications for posttranscriptional regulation and protein binding are discussed.
Assuntos
Regiões 3' não Traduzidas/genética , Sequência Conservada/genética , Marsupiais/genética , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas rab1 de Ligação ao GTP/genética , Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Regulação da Expressão Gênica/genética , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/fisiologia , Alinhamento de SequênciaRESUMO
We identified a novel gene family in vertebrates which is preferentially expressed in developing and adult striated muscle. Three genes of the Popeye (POP) family were detected in human and mouse and two in chicken. Chromosomal mapping indicates that Pop1 and Pop3 genes are clustered on mouse chromosome 10, whereas Pop2 maps to mouse chromosome 16. We found evidence that POP1 and POP3 in chicken may also be linked and multiple transcript isoforms are generated from this locus. The POP genes encode proteins with three potential transmembrane domains that are conserved in all family members. Individual POP genes exhibit specific expression patterns during development and postnatally. Chicken POP3 and mouse Pop1 are first preferentially expressed in atrium and later also in the subepicardial compact layer of the ventricles. Chicken POP1 and mouse Pop2 are expressed in the entire heart except the outflow tract. All three Pop genes are expressed in heart and skeletal muscle of the adult mouse and lower in lung. Pop1 and Pop2 expression is upregulated in uterus of pregnant mice. Like the mouse genes, human POP genes are predominantly expressed in skeletal and cardiac muscle. The strong conservation of POP genes during evolution and their preferential expression in heart and skeletal muscle suggest that these novel proteins may have an important function in these tissues in vertebrates.
Assuntos
Moléculas de Adesão Celular , Coração/embriologia , Família Multigênica , Proteínas Musculares/isolamento & purificação , Músculo Esquelético/embriologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Proteínas Aviárias , Embrião de Galinha , Indução Embrionária , Feminino , Biblioteca Gênica , Átrios do Coração/embriologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/genética , Pericárdio/embriologia , Gravidez , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , ÚteroRESUMO
Titin, also called connectin, is a giant muscle protein that spans the distance from the sarcomeric Z-disc to the M-band. Titin is thought to direct the assembly of sarcomeres and to maintain sarcomeric integrity by interacting with numerous sarcomeric proteins and providing a mechanical linkage. Since severe defects of such an important molecule are likely to result in embryonic lethality, a cell culture model should offer the best practicable tool to probe the cellular functions of titin. The myofibroblast cell line BHK-21/C13 was described to assemble myofibrils in culture. We have now characterized the sub-line BHK-21-Bi, which bears a small deletion within the titin gene. RNA analysis revealed that in this mutant cell line only a small internal portion of the titin mRNA is deleted. However, western blots, immunofluorescence microscopy and immunoprecipitation experiments showed that only the N-terminal, approx. 100 kDa central Z-disc portion of the 3 MDa titin protein is expressed, due to the homozygous deletion in the gene. Most importantly, in BHK-21-Bi cells the formation of thick myosin filaments and the assembly of myofibrils are impaired, although sarcomeric proteins are expressed. Lack of thick filament formation and of ordered actin-myosin arrays was confirmed by electron microscopy. Myogenisation induced by transfection with MyoD yielded myofibrils only in myotubes formed from wild type and not from mutant cells, ruling out that a principal failure in myogenic commitment of the BHK-21-Bi cells might cause the observed effects. These experiments provide the first direct evidence for the crucial role of titin in both thick filament formation as a molecular ruler and in the coordination of myofibrillogenesis.
Assuntos
Proteínas Musculares/fisiologia , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Proteínas Quinases/fisiologia , Animais , Proteínas de Ligação a Calmodulina/fisiologia , Linhagem Celular , Conectina , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , RNA Mensageiro/análise , Deleção de SequênciaRESUMO
For reconstruction or repair of damaged tissues, an artificially regulated switch from proliferation to differentiation would be of great advantage. To achieve conditional myogenesis, we expressed MyoD in mouse C3H 10T1/2 fibroblastic cells, using a gene regulation system based on the synthetic steroid RU 486. By stable co-transfection of a plasmid construct with the RU 486 dependent activator and an integrating inducible MyoD construct, a cell clone, designated 10T-RM, was obtained in which MyoD expression was stringently controlled by RU 486. 12 h after addition of 10 nM RU 486 to 10T-RM cells, saturation levels of MyoD mRNA were observed and >/=2 days later, mRNA for embryonal myosin heavy chain (MyHC(emb)) was abundant and mononucleated cells fused into myotubes.
Assuntos
Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Mifepristona/farmacologia , Músculos/citologia , Músculos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular/efeitos dos fármacos , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Genes Reporter/genética , Camundongos , Camundongos Endogâmicos C3H , Desenvolvimento Muscular , Músculos/embriologia , Proteína MyoD/genética , Cadeias Pesadas de Miosina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacos , TransfecçãoRESUMO
The wobbler mouse (phenotype WR; genotype wr/wr) has been investigated as a model for neurodegenerative diseases like SMA and ALS. A new diagnostic marker based on a polymorphism in the closely linked chaperonine gene Cct4 enabled us to diagnose the allelic status at the wr locus within the original background strain C57BL/6. Using this marker, we investigated the spatiotemporal progression of neuropathology in WR mice from postnatal day (d.p.n.) 10 to 60. Neurodegeneration starts at 13 d.p.n. in the thalamus (N. ventralis), in deep cerebellar nuclei, brain stem (N. vestibularis) and spinal cord interneurons. The motor nuclei of spinal nerves and motoneurons degenerate from 15 d.p.n. onward. Reactive astrocytes are observed around 17 d.p.n. in the white and grey matter of the spinal cord. Microgliosis occurs only from 23 d.p.n. onward. Our data demonstrate that in the WR disease, neurodegeneration in thalamus, cerebellum, and brain stem precedes motoneuron degeneration, astrogliosis and microgliosis.
Assuntos
Degeneração Neural/patologia , Neuroglia/fisiologia , Doenças Neuromusculares/patologia , Alelos , Animais , Astrócitos/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Microglia/fisiologia , Degeneração Neural/genética , Doenças Neuromusculares/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético/genética , Desempenho Psicomotor/fisiologiaRESUMO
Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of alpha-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.
Assuntos
Núcleo Celular/metabolismo , Proteínas Contráteis , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Peptídeos/química , Motivos de Aminoácidos , Animais , Bovinos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/química , Profilinas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas do Complexo SMN , Medula Espinal/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Técnicas do Sistema de Duplo-HíbridoRESUMO
To detect cation channels, the expression of which is dependent on the physiological state of muscle, single-channel activities of dissociated fibres of the mouse interosseus muscle were recorded using the patch-clamp technique in the cell-attached mode. Fibres were prepared from juvenile and adult wild-type (WT), from chloride channel-deficient myotonic and from denervated adult WT muscles. In all cases delayed-rectifier K+ channels (KDR) with a unitary conductance of 11 pS were recorded in more than 95% of sarcolemmal patches, but with a low, steady-state open probability. Inwards-rectifying K+ channels (KIR) with a conductance of 31 pS in 140 mM [K+]o were active in about 50% of the membrane patches from WT and in more than 90% of those from myotonic fibres. A hitherto undescribed, inwards-rectifying, cation channel, provisionally termed CIR, with fast kinetics and a unitary conductance of 36 pS, was active in nearly every membrane patch from juvenile mice, both WT and myotonic. The abundance of CIR decreased during development, but was not changed 7 days after denervation of adult WT muscle. Ca(2+)-dependent K+ channels were seen sporadically. Channels with the characteristics of adenosine 5'-triphosphate (ATP)-sensitive K+ channels were recorded frequently upon excision of membrane patches, but remained inactive in most cell-attached recordings. In conclusion, of the investigated ion channels, only KIR was responsive to the activity pattern of adult muscle, whereas CIR was down-regulated during muscle maturation.
Assuntos
Envelhecimento/metabolismo , Músculo Esquelético/metabolismo , Miotonia/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Animais , Canais de Cloreto/metabolismo , Canais de Potássio de Retificação Tardia , Condutividade Elétrica , Feminino , Membro Posterior , Técnicas In Vitro , Masculino , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/fisiologiaRESUMO
Chloride currents (I(Cl)) were investigated with the two-electrode voltage-clamp technique in enzymatically isolated fibers from interosseus muscles of wild-type (WT), denervated WT, and myotonic (ADR, ClC-1-deficient) mice. Characteristics of I(Cl) were consistent with previous observations on rat muscle fibers and cultured nonmuscle cells transfected with hClC-1 cDNA. In the presence of 0.1 mM anthracene-9-carboxylic acid and in ADR fibers, I(Cl) was reduced by >90%. WT interosseus fibers denervated 6-7 days prior to isolation showed approximately 50% I(Cl) compared to control fibers. Addition of 3.3 microM staurosporine, a nonspecific inhibitor of protein kinases, increased I(Cl) in WT interosseus fibers by a factor of approximately two and altered its kinetic characteristics. We conclude that in dissociated fibers cultured for 1-2 days, in contrast to freshly isolated muscles, chloride conductance is downregulated by a mechanism involving protein phosphorylation. In situ, this short-term regulation may complement transcriptional long-term regulation of ClC-1.
Assuntos
Canais de Cloreto/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Canais de Cloreto/efeitos dos fármacos , Denervação , Técnicas In Vitro , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/ultraestrutura , Técnicas de Patch-Clamp , Fosforilação , Ratos , Estaurosporina/farmacologiaRESUMO
Embryos from certain mouse strains are arrested at the 2-cell stage in cell culture ('2-cell block'), whereas those from other strains develop to the blastocyst stage under the same conditions. It was previously shown that blocking embryos can be rescued in culture by aggregation with an excess of 2-cell stages of a non-blocking strain such as CBA x C57BL/6 F2. Here we have employed a LacZ transgene in a blocking strain (NMRI) to follow the fate of rescued blastomeres up to the blastocyst stage. We found that rescued blastomeres can participate in both inner cell mass and trophoblast formation, thus completely overcoming the 2-cell block.