RESUMEN
The enteroendocrine K and L cells are responsible for secretion of glucose-dependent insulinotropic polypeptide (GIP) and glucagon like-peptide 1 (GLP-1), whereas pancreatic α-cells are responsible for secretion of glucagon. In rodents and humans, dysregulation of the secretion of GIP, GLP-1, and glucagon is associated with impaired regulation of metabolism. This study evaluates the consequences of acute removal of Gip- or Gcg-expressing cells on glucose metabolism. Generation of the two diphtheria toxin receptor cellular knockout mice, TgN(GIP.DTR) and TgN(GCG.DTR), allowed us to study effects of acute ablation of K and L cells and α-cells. Diphtheria toxin administration reduced the expression of Gip and content of GIP in the proximal jejunum in TgN(GIP.DTR) and expression of Gcg and content of proglucagon-derived peptides in both proximal jejunum and terminal ileum as well as content of glucagon in pancreas in TgN(GCG.DTR) compared with wild-type mice. GIP response to oral glucose was attenuated following K cell loss, but oral and intraperitoneal glucose tolerances were unaffected. Intraperitoneal glucose tolerance was impaired following combined L cell and α-cell loss and normal following α-cell loss. Oral glucose tolerance was improved following L cell and α-cell loss and supernormal following α-cell loss. We present two mouse models that allow studies of the effects of K cell or L cell and α-cell loss as well as isolated α-cell loss. Our findings show that intraperitoneal glucose tolerance is dependent on an intact L cell mass and underscore the diabetogenic effects of α-cell signaling. Furthermore, the results suggest that K cells are less involved in acute regulation of mouse glucose metabolism than L cells and α-cells.
Asunto(s)
Células Enteroendocrinas/fisiología , Células Secretoras de Glucagón/fisiología , Glucosa/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Toxina Diftérica/genética , Células Enteroendocrinas/clasificación , Células Enteroendocrinas/metabolismo , Femenino , Polipéptido Inhibidor Gástrico/metabolismo , Técnicas de Silenciamiento del Gen , Genes Transgénicos Suicidas/genética , Células Secretoras de Glucagón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Especificidad de Órganos/genéticaRESUMEN
TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial-mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation and EMT, we found TWIST1 to be upregulated during EMT and downregulated early in carcinogenesis. The TWIST1 3'UTR contains putative regulatory elements, including miRNA target sites and two cytoplasmic polyadenylation elements (CPE). We found that miR-580, CPEB1, and CPEB2 act as negative regulators of TWIST1 expression in a sequence-specific and additive/cooperative manner.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/fisiología , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas , Proteína 1 Relacionada con Twist/metabolismo , Regiones no Traducidas 3' , Sitios de Unión , Neoplasias de la Mama/patología , Línea Celular , Movimiento Celular , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Genes Reporteros , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Proteínas Nucleares/genética , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Proteína 1 Relacionada con Twist/genética , Regulación hacia Arriba , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/fisiologíaRESUMEN
Notch signaling is an evolutionarily conserved pathway that controls the developmental choices made by individual cells. Cells communicate via Notch receptors and their ligands, which direct decisions on the fate of stem cells according to the states of their neighbors. In this study we explored Notch signaling after the pulp capping of adult first upper rat molars. The wound was capped with calcium hydroxide. In situ hybridization revealed an increased expression of Notch signaling genes on day 1, which showed a tendency to decrease on day 3. Notch1 increased in the subodontoblast zone and close to the lesion limited to a few cells. Notch2 increased in pulp stroma surrounded by coronal odontoblasts. Notch1 and, especially, Notch3 expression increased, corresponding to perivascular cell groups. A low increase of ligand expression was observed near the injury with Delta1 expression along the dentin wall and Jagged1 in the stroma. Expression of the downstream target, Hes1, was observed along the lesion and adjacent dentin walls. Hes5 expression was not observed. The results indicate that Notch signaling is activated in response to injury and associated with the differentiation of pulp cells into perivascular cells and odontoblasts. The findings are consistent with the concept that the Notch pathway controls stem cell fate during pulp regeneration.
Asunto(s)
Recubrimiento de la Pulpa Dental , Pulpa Dental/metabolismo , Receptor Notch2/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Pulpa Dental/citología , Pulpa Dental/lesiones , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Masculino , Maxilar , Proteínas de la Membrana/metabolismo , Diente Molar , Ratas , Ratas Wistar , Receptor Notch1/metabolismo , Receptor Notch3 , Traumatismos de los Dientes/metabolismo , Factor de Transcripción HES-1 , Regulación hacia ArribaRESUMEN
Basic helix-loop-helix (bHLH) transcription factors have been shown to be important regulatory proteins for tissue determination and differentiation. We cloned the chicken homologue of the gene of the murine Twist-related bHLH protein Dermo-1, which we named cDermo-1, and analyzed its sequence and embryonic expression. Our sequence data suggest a decisive role of Dermo-1 proteins in the evolution of amniote skin. We present a detailed analysis of cDermo-1 expression during avian embryonic development. cDermo-1 is first expressed in a variety of mesodermal tissues of the chick embryo including the limb buds, but later becomes restricted to the subectodermal mesenchyme of the integument and the developing feather buds, indicating a role of cDermo-1 during avian skin and feather development.
Asunto(s)
Piel/embriología , Piel/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Embrión de Pollo , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Piel/citología , Factores de Transcripción/fisiologíaAsunto(s)
Regulación del Desarrollo de la Expresión Génica , Músculos/embriología , Músculos/metabolismo , Enfermedades Neuromusculares/genética , Animales , Tipificación del Cuerpo/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Proteínas de Unión al ADN/metabolismo , Inducción Embrionaria , Extremidades/embriología , Corazón/embriología , Factores de Transcripción MEF2 , Músculos/patología , Músculos/fisiología , Miocardio/citología , Miocardio/metabolismo , Miocardio/patología , Factores Reguladores Miogénicos , Enfermedades Neuromusculares/metabolismo , Enfermedades Neuromusculares/patología , Regeneración/genética , Transducción de Señal , Somitos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: We studied the effects of angiotensin II (Ang II) and diastolic overstretch on the induction of cardiac growth in isometrically contracting muscle preparations from human right atria and left ventricles. We used the gene expression of brain natriuretic peptide (BNP) as a molecular marker of cardiac hypertrophy. METHODS AND RESULTS: Northern blot analysis was performed in human atrial muscle preparations, which were either incubated in 10(-6) mol/L Ang II for 45 minutes or diastolically stretched to 120% of optimum muscle length. Similar experiments were performed with human left ventricular muscle preparations. Results were as follows: (1) BNP gene expression increased in human atrial myocardium 4-fold when stimulated by Ang II (n=7, P<0.001). (2) Diastolic overstretch increased BNP expression in a time-dependent manner. The linear regression equations for the BNP/GAPDH ratio as a function of time (hours) were y=1.21+0.62x (P:<0.001) for overstretched preparations and y=1.07-0.01x (P:=NS) for atrial preparations kept at physiological muscle length. (3) In left ventricular human muscle preparations, diastolic overstretch and Ang II increased BNP gene expression as well. (4) In addition, the Ang II subtype 1 receptor blocker losartan was able to block the effects of Ang II and diastolic overstretch. CONCLUSIONS: Cardiac hypertrophy can be induced in isolated human atrial and left ventricular intact myocardium by Ang II and diastolic overstretch but not by isometric afterload. The fact that the induction of cardiac growth is inhibited by the blockade of Ang II subtype 1 receptors is of scientific and clinical importance.
Asunto(s)
Angiotensina II/fisiología , Cardiomegalia/patología , Fibras Musculares Esqueléticas/patología , Miocardio/metabolismo , Miocardio/patología , Péptido Natriurético Encefálico/metabolismo , Angiotensina II/farmacología , Northern Blotting , Cardiomegalia/fisiopatología , Diástole , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Humanos , Técnicas In Vitro , Contracción Isométrica , Contracción Miocárdica , Miocardio/ultraestructura , ARN Mensajero/metabolismo , Estrés MecánicoRESUMEN
The vascular wall is built up of a heterogeneous population of smooth muscle cells, which exhibit not only morphological distinctions but also important differences in the composition of their structural and contractile proteins. "Epithelioid" smooth muscle cells correspond to an intimal-like type and display features associated with immaturity, whereas "spindle-shaped" cells closely resemble the more typical medial smooth muscle population. We have investigated the integration of these two cell types into the vascular architecture of an in vivo wound-healing model. Stably transfected with the beta-galactosidase gene, intima- and media-like cells were injected intravenously into the chicken chorioallantoic membrane, within which superficial foci of granulation tissue had been created by thermal or chemical injury. At 24 to 72 h after injection, cells had honed in on the lesion sites and were observed in juxtaposition to the endothelial lining of the capillaries. They began to deposit laminin, thereby indicating an impending role in the formation of the vascular wall. Intima- and media-like smooth muscle cells did not differ in their capacity to associate with capillaries, and, in so doing, their biochemical lineage characteristics became indistinguishable from one another. However, intima-like cells also penetrated the adventitial and medial layers of arteries. These findings reveal vascular smooth muscle cells to possess an extraordinary degree of plasticity, being able to adapt flexibly to changes in functional demands.
Asunto(s)
Alantoides/citología , Corion/citología , Músculo Liso Vascular/citología , Cicatrización de Heridas/fisiología , Alantoides/lesiones , Alantoides/metabolismo , Animales , Capilares/ultraestructura , Carbocianinas/farmacocinética , Diferenciación Celular/fisiología , Embrión de Pollo , Pollos , Corion/lesiones , Corion/metabolismo , Cicatriz/patología , Molde por Corrosión , Colorantes Fluorescentes/farmacocinética , Ratones , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Músculo Liso Vascular/metabolismo , Neovascularización Fisiológica/fisiología , Túnica Íntima/metabolismo , Túnica Íntima/ultraestructura , beta-Galactosidasa/farmacocinéticaRESUMEN
During a gene trap screen, an insertion of the gene trap vector into the dystrophin gene, creating a new allele for the Dmd gene, has been discovered. Because the ROSA beta geo vector was used, the new allele is called Dmd(mdx-beta geo). The insertion occurred 3' of exon 63 of the dystrophin gene, resulting in a mutation that affects all presently known dystrophin isoforms. In contrast to spontaneous or ENU-induced alleles, Dmd(mdx-beta geo) can be used to follow dystrophin expression by staining for beta-galactosidase activity. The high sensitivity of this method revealed additional and earlier expression of dystrophin during embryogenesis than that seen previously with other methods. Dystrophin promoters are active predominantly in the dermamyotome, limb buds, telencephalon, floor plate, eye, liver, pancreas anlagen, and cardiovascular system. Adult Dmd(mdx-beta geo) mice show reporter gene expression in brain, eye, liver, pancreas, and lung. In skeletal and heart muscle, beta-galactosidase activity is not detectable, confirming Western blot data that indicate the absence of the mutant full-length protein in these tissues. Hemizygous Dmd(mdx-beta geo) mice show muscular dystrophy with degenerating muscle fibers, cellular infiltration, and regenerated muscle fibers that have centrally located nuclei. Some mutant animals develop a dilated esophagus, probably due to constriction by the hypertrophic crura of the diaphragm.
Asunto(s)
Alelos , Distrofina/genética , Mutación , Animales , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Desarrollo Embrionario y Fetal/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Técnicas Genéticas , Vectores Genéticos , Hibridación in Situ , Operón Lac , Masculino , Ratones , Ratones Endogámicos mdx , Ratones Transgénicos , Datos de Secuencia Molecular , Distrofia Muscular Animal/embriología , Distrofia Muscular Animal/genética , Fenotipo , Embarazo , Distribución TisularRESUMEN
Saethre-Chotzen syndrome, a common autosomal dominant craniosynostosis in humans, is characterized by brachydactyly, soft tissue syndactyly and facial dysmorphism including ptosis, facial asymmetry, and prominent ear crura. Previously, we identified a yeast artificial chromosome that encompassed the breakpoint of an apparently balanced t(6;7) (q16.2;p15.3) translocation associated with a mild form of Saethre-Chotzen syndrome. We now describe, at the DNA sequence level, the region on chromosome 7 affected by this translocation event. The rearrangement occurred approximately 5 kb 3' of the human TWIST locus and deleted 518 bp of chromosome 7. The TWIST gene codes for a transcription factor containing a basic helix-loop-helix (b-HLH) motif and has recently been described as a candidate gene for Saethre-Chotzen syndrome, based on the detection of mutations within the coding region. Potential exon sequences flanking the chromosome 7 translocation breakpoint did not hit known genes in database searches. The chromosome rearrangement downstream of TWIST is compatible with the notion that this is a Saethre-Chotzen syndrome gene and implies loss of function of one allele by a positional effect as a possible mechanism of mutation to evoke the syndrome.
Asunto(s)
Acrocefalosindactilia/genética , Proteínas Nucleares , Factores de Transcripción/genética , Translocación Genética , Secuencia de Bases , Rotura Cromosómica , Mapeo Cromosómico , Cromosomas Humanos Par 7 , Cósmidos/genética , ADN Complementario , Genoma Humano , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia de ADN , Eliminación de Secuencia , Transcripción Genética , Proteína 1 Relacionada con TwistRESUMEN
We isolated an abundant novel cDNA SSEC-8 from a subtraction cDNA library enriched for maternal transcripts that are still present in the mouse 2 cell stage embryo. This gene is evolutionarily conserved and maps to the distal region of mouse chromosome 2. The deduced polypeptide sequence of the encoded protein contains a conserved helix-loop-helix (HLH) motif without a basic DNA binding domain, suggesting that it functions as a negative regulator of basic (b) HLH transcription factors. Gel mobility shift assays show that in vitro translated protein prevents the E12/MyoD bHLH dimer from binding to DNA. Also, transient overexpression of this protein in C2C12 cells reduced the transcription of a CAT-reporter regulated by an E12/MyoD driven enhancer. The 3'-UTR contains consensus sequences of cytoplasmic polyadenylation elements (CPE's), and the length of its poly (A) tail changes during oocyte maturation, indicating that its expression is controlled by timely activation of translation. This new gene, Maid, models the translational and transcriptional regulation of gene expression during the transition from gamete to embryo.
Asunto(s)
Secuencias Hélice-Asa-Hélice , Óvulo/metabolismo , Cigoto/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , Femenino , Genes Homeobox , Ligamiento Genético , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Poli A/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
Inhibition of myogenic differentiation can be achieved by various mechanisms. The murine bHLH protein Twist has been shown to inhibit muscle differentiation in mammalian cells. Here we demonstrate that this inhibition is cell autonomous and does not alter cell proliferation. By overexpression of E12, we can distinguish the inhibitory mechanisms of Twist and the dominant negative HLH factor Id. A difference is seen both for the native muscle-specific enhancers of myogenin and myosin light chain 1/3 and for an enhancer consisting only of four E-boxes. Mutagenesis experiments revealed that both the basic region and an evolutionarily conserved carboxy-terminal domain are required for the Twist-specific type of inhibition. Loss of either of these regions renders Twist less efficient and more similar to Id. Gel mobility shift assays demonstrate that Twist can bind to the muscle creatine kinase E-box and inhibit DNA binding of heterodimers of E12 with myogenic bHLH transcription factors like MyoD. However, a fourfold excess of Twist compared to MyoD is required for both effects. Our results suggest that Twist inhibits muscle-specific gene activation by formation of actively inhibitory complexes rather than by sequestering E-proteins.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Musculares/genética , Músculos/embriología , Proteínas Nucleares/fisiología , Proteínas Represoras , Animales , Diferenciación Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Secuencias Hélice-Asa-Hélice/genética , Secuencias Hélice-Asa-Hélice/fisiología , Proteína 1 Inhibidora de la Diferenciación , Sustancias Macromoleculares , Ratones , Proteínas Musculares/biosíntesis , Músculos/citología , Mutagénesis Sitio-Dirigida , Proteína MioD/antagonistas & inhibidores , Proteína MioD/fisiología , Proteínas Nucleares/genética , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/fisiología , Activación Transcripcional , Transfección , Proteína 1 Relacionada con TwistRESUMEN
The murine homologue of the Drosophila twist gene has been shown to be essential for head mesenchyme formation and to act as an inhibitor of muscle differentiation. This paper presents a detailed analysis of M-twist expression patterns from day 7 post coitum (p.c.) to day 18 p.c., indicating a more general function of the M-twist gene. At day 7 p.c., M-twist is expressed in the mesoderm outside the primitive streak. Later M-twist message is predominantly found in the somites, the head mesenchyme, the branchial arches, the limbs, and in the mesenchyme underneath the epidermis. Beginning at day 8 p.c., M-twist is mainly expressed in undifferentiated cells committed to muscle and cartilage development: this expression is consistent with a suggested role of M-twist in inhibiting overt muscle and cartilage differentiation. However, during organogenesis, M-twist is expressed in several areas of mesenchyme-epithelia interactions, suggesting additional tissue specific functions.
Asunto(s)
Desarrollo Embrionario/fisiología , Ratones/embriología , Proteínas Nucleares/genética , Animales , Cartílago/embriología , Desarrollo Embrionario y Fetal/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Válvulas Cardíacas/embriología , Secuencias Hélice-Asa-Hélice/fisiología , Hibridación in Situ , Esbozos de los Miembros/fisiología , Mesodermo/citología , Mesodermo/fisiología , Músculos/embriología , Factores Reguladores Miogénicos/genética , Cresta Neural/citología , Cresta Neural/fisiología , Embarazo , ARN Mensajero/análisis , Proteína 1 Relacionada con TwistRESUMEN
The mouse M-twist gene codes for a basic helix-loop-helix protein which was shown to be inhibitory for differentiation of myogenic cells in culture. Mouse embryonic stem (ES) cells of line BLC6 efficiently differentiating into skeletal muscle cells when cultivated as embryo-like aggregates (embryoid bodies) were stably transfected with the plasmid pME18s-twist containing the M-twist gene under the control of the modified SV40 early promoter SR alpha. Two pME18s-twist-expressing clones showed delayed and reduced skeletal muscle cell differentiation depending on the level of exogenous M-twist expression compared to control cells. By morphological analysis using phase contrast microscopy and hematoxylin-eosin staining, the development of first myocytes and formation of myotubes in embryoid body outgrowths of these clones were found to be delayed for about 3 days in comparison to control cells. Immunofluorescence studies with a monoclonal antibody against sarcomeric myosin heavy chain revealed that myogenic cells appeared in so-called myogenic centers showing a reduced number of myocytes and myotubes in the M-twist-expressing clones. Using RT-PCR analysis the expression of the skeletal muscle determination genes myf5, myogenin, and MyoD as well as muscle-specific genes coding for the gamma-subunit of the nicotinic acetylcholine receptor and the cell adhesion molecule M-cadherin were found to appear with a delay of at least 1 to 4 days in the pME18s-twist-transfected cells during the development of embryoid bodies. We conclude that the constitutive expression of the mouse M-twist gene during ES-cell-derived differentiation has an inhibitory effect on skeletal muscle cell development depending on the level of exogenous M-twist expression.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/embriología , Factores Reguladores Miogénicos/biosíntesis , Proteínas Nucleares/biosíntesis , Células Madre/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Células Clonales , Técnica del Anticuerpo Fluorescente , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Factores Reguladores Miogénicos/genética , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , Células Madre/citología , Transcripción Genética , Transfección , Proteína 1 Relacionada con TwistRESUMEN
The twist gene encodes a transcription factor containing a conserved basic helix-loop-helix domain. During development, transcription factors of this type are normally associated with the induction of differentiation. Yet the expression pattern of the murine M-twist suggests an inhibitory role during muscle differentiation. Following stable transfection of myogenic mouse cells with an M-twist expression vector, 75% of M-twist-expressing clones were impaired in their ability to differentiate. In contrast, only 15% of control clones were unable to differentiate. Treatment with antisense oligonucleotides restored differentiation capacity in a concentration-dependent manner. Control oligonucleotides had no effect. These experiments show that the mouse twist gene can act as an inhibitor of muscle differentiation and that this inhibition is reversible.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Musculares/genética , Músculos/embriología , Factores Reguladores Miogénicos , Proteínas Nucleares/fisiología , Transactivadores , Animales , Diferenciación Celular , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Hibridación in Situ , Ratones , Músculos/citología , Proteína MioD/genética , Factor 5 Regulador Miogénico , Miogenina/genética , ARN Mensajero/genética , Proteína 1 Relacionada con TwistRESUMEN
The differential expression of genes triggering myogenesis might cause or reflect differences among myoblasts. Little is known about the presence of MyoD1 and myogenin during the process of regeneration. We therefore examined the expression of MyoD1 and myogenin in muscle regeneration after grafting. Immunostaining of regenerating skeletal muscle of the mouse revealed the presence of both MyoD1 and myogenin. In mononucleated cells the proteins were not detected until shortly before fusion into myotubes. They persisted in the nuclei of regenerated muscle fibers for at least 2 weeks. MyoD1 and myogenin were not detected in nonregenerating control muscle.
Asunto(s)
Proteínas Musculares/análisis , Músculos/metabolismo , Proteína MioD , Proteínas Nucleares/análisis , Fosfoproteínas/análisis , Regeneración/fisiología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Músculos/citología , Músculos/fisiología , MiogeninaRESUMEN
Parvalbumin (PV) is a soluble Ca++ binding protein which is particularly concentrated in fast muscles of rodents. We have developed a new protocol to fix frozen sections of muscle by formaldehyde vapor, which enabled us to immunochemically stain serial frozen sections for PV. Fiber types were defined on the basis of myosin ATPase stability, and of isomyosins identified by a variety of antibodies because ATPase stability alone yielded ambiguous results in the mouse. Slow Type I fibers in mouse and rat were devoid of PV and had intermediate to high SDH levels. Fast fiber subtypes IIA, IIB, and IIX-like were defined in the mouse on the basis of the similarity of their myosin heavy chain immunoreactivity to these types in the rat. The soleus muscle was usually PV negative, but a small population of strongly PV-positive IIX-like fibers was present in the mouse. In mouse fast muscle, small diameter IIA fibers were PV negative with high SDH activity. In both mouse and rat, PV reactivities of IIB and IIX fibers were higher than those of IIA and I, whereas SDH levels of IIA, IIX, and I fibers were higher than those of IIB. Thus, PV content correlated with the type of myosin ATPase but not with SDH levels. The method described for immunocytochemistry of PV may be applicable to other highly soluble proteins.
Asunto(s)
Secciones por Congelación , Músculos/metabolismo , Miosinas/metabolismo , Parvalbúminas/metabolismo , Succinato Deshidrogenasa/metabolismo , Animales , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas EndogámicasRESUMEN
Developmental aspects of an animal model of myotonia, the mouse mutant called "arrested development of righting response" (ADR phenotype), were studied. Adult ADR muscle is characterized by a low chloride conductance of the membrane, leading to hyperexcitability, and by a low parvalbumin content. The myotonic hyperexcitability (as measured by the extent of "aftercontractions") of ADR muscle increased steeply between postnatal days 9 and 18, by which time it had approached the adult level. To study the tissue autonomy of the myotonic phenotype, muscle grafts were performed in all four combinations between ADR and wildtype (WT phenotype) donors and hosts. In most experiments, the relative contributions of donor and host to the regenerated muscles were determined by an allelic marker (glucose phosphate isomerase). In WT and ADR hosts, ADR grafts showed myotonic responses that in WT nude mouse hosts were incomplete and similar to those of juvenile ADR muscle. In no case did grafts from WT donors show any myotonia. This shows that the myotonic ADR phenotype is based on an intrinsic muscle property most likely related to the plasma membrane. The parvalbumin contents of grafted muscles, when compared with those of untransplanted muscles, indicated graft-host interaction in the expression of this secondary phenotypic property.
Asunto(s)
Músculos/fisiopatología , Miotonía/fisiopatología , Factores de Edad , Animales , Glucosa-6-Fosfato Isomerasa/metabolismo , Ratones , Ratones Mutantes , Contracción Muscular , Desarrollo de Músculos , Músculos/trasplante , Succinato Deshidrogenasa/metabolismoRESUMEN
The muscle physiology and histochemistry of a hereditary neuromuscular syndrome of the mouse, "arrested development of righting response" (ADR), was studied. The speed of single twitches of fast ADR limb muscles was normal up to an age of about 60 days but decreased at later ages. At any age between 10 and 120 days postnatal, fast and slow muscles of the mutant displayed after-contractions of 1-3 (5) seconds duration. These coincided with electrical after-activity of muscle, as demonstrated by electromyography. After-contractions and EMG signals were suppressed by the membrane-stabilizing drug tocainide. These physiological data suggest that ADR is a myotonia. With a few exceptions, limb and trunk muscles of ADR animals showed a uniform oxidative phenotype with a lack of large diameter glycolytic fibers. The histochemical muscle phenotype of the ADR mouse was partially reversed by a long-term treatment with tocainide.