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The scholarship support information in Acknowledgement was missing.
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Endothelial dysfunction plays as an important role on mismatch responses that occur during exercise in patients with congestive heart failure (CHF). However, cardiac rehabilitation, a core component of management of CHF patients, can improve endothelial function, contributing to reduce the morbidity and mortality of these patients. The primary aims of this review were to describe the importance of flow-mediated dilatation (FMD) as a non-invasive validation tool to assess endothelial dysfunction and to highlight the relevance of scientific studies that evaluated the effects of exercise interventions on peripheral vascular endothelial function as measured by FMD in patients with CHF with both preserved and reduced ejection fraction.
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Terapia por Ejercicio , Ejercicio Físico , Insuficiencia Cardíaca/rehabilitación , Vasodilatación , Arteria Braquial , Terapia de Resincronización Cardíaca , Endotelio Vascular/fisiopatología , Humanos , Volumen Sistólico , Resultado del Tratamiento , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.
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Sistema de Señalización de MAP Quinasas , Proteína 2 de Unión a Metil-CpG/metabolismo , MicroARNs/metabolismo , Neurogénesis/fisiología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular/genética , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Proteína 2 de Unión a Metil-CpG/genética , Ratones , MicroARNs/genética , Neurogénesis/genética , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/patología , Transducción de SeñalRESUMEN
Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.
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Epigénesis Genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Reprogramación Celular/genética , Metilación de ADN/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Genoma/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Técnicas de Transferencia Nuclear , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mecp2 is an X-linked gene encoding a nuclear protein that binds specifically to methylated DNA (ref. 1) and functions as a general transcriptional repressor by associating with chromatin-remodeling complexes. Mecp2 is expressed at high levels in the postnatal brain, indicating that methylation-dependent regulation of gene expression may have a crucial role in the mammalian central nervous system. Consistent with this notion is the recent demonstration that MECP2 mutations cause Rett syndrome (RTT, MIM 312750), a childhood neurological disorder that represents one of the most common causes of mental retardation in females. Here we show that Mecp2-deficient mice exhibit phenotypes that resemble some of the symptoms of RTT patients. Mecp2-null mice were normal until 5 weeks of age, when they began to develop disease, leading to death between 6 and 12 weeks. Mutant brains showed substantial reduction in both weight and neuronal cell size, but no obvious structural defects or signs of neurodegeneration. Brain-specific deletion of Mecp2 at embryonic day (E) 12 resulted in a phenotype identical to that of the null mutation, indicating that the phenotype is caused by Mecp2 deficiency in the CNS rather than in peripheral tissues. Deletion of Mecp2 in postnatal CNS neurons led to a similar neuronal phenotype, although at a later age. Our results indicate that the role of Mecp2 is not restricted to the immature brain, but becomes critical in mature neurons. Mecp2 deficiency in these neurons is sufficient to cause neuronal dysfunction with symptomatic manifestation similar to Rett syndrome.
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Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas Represoras , Síndrome de Rett/genética , Animales , Diferenciación Celular , Tamaño de la Célula , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Islas de CpG , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Proteína 2 de Unión a Metil-CpG , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Síndrome de Rett/metabolismo , Síndrome de Rett/patologíaRESUMEN
The tissue-specific organization of collagen molecules into tridimensional macroaggregates determines the physiomechanical properties of most connective tissues, but the factors and mechanisms controlling this process are unknown. It has been postulated that quantitatively minor types V and XI collagen regulate the growth of type I and II collagen fibrils, respectively. To test this hypothesis, we created mice that produce a structurally abnormal alpha 2(V) collagen chain. Homozygous mutant mice survive poorly, possibly because of complications from spinal deformities, and exhibit skin and eye abnormalities caused by disorganized type I collagen fibrils. Our results demonstrate that type V collagen is a key determinant in the assembly of tissue-specific matrices, and provide an animal model for human connective tissue disorders.
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Colágeno/genética , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Mutación , Animales , Secuencia de Bases , Enfermedades del Tejido Conjuntivo/genética , Enfermedades del Tejido Conjuntivo/metabolismo , Enfermedades del Tejido Conjuntivo/patología , ADN/genética , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica , Datos de Secuencia Molecular , Conejos , Eliminación de Secuencia , Piel/metabolismo , Piel/ultraestructuraRESUMEN
Chromosomal deletions ("deficiencies') are powerful tools in the genetic analysis of complex genomes. They have been exploited extensively in Drosophila melanogaster, an organism in which deficiencies can be efficiently induced and selected. Spontaneous deletions in humans have facilitated the dissection of phenotypes in contiguous gene syndromes and led to the positional cloning of critical genes. In mice, deletion complexes created by whole animal irradiation experiments have enabled a systematic characterization of functional units along defined chromosomal regions. However, classical mutagenesis in mice is logistically impractical for generating deletion sets on a genome-wide scale. Here, we report a high-throughput method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. Dozens of deletions of up to several centiMorgans, encompassing a specific locus, can be created in a single experiment and transmitted through the germline. The ability to rapidly create deletion complexes along chromosomes will facilitate systematic functional analyses of the mammalian genome.
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Deleción Cromosómica , Quimera por Radiación , Células Madre/efectos de la radiación , Animales , Supervivencia Celular/efectos de los fármacos , Mapeo Cromosómico , Cruzamientos Genéticos , Relación Dosis-Respuesta en la Radiación , Drosophila melanogaster/genética , Embrión de Mamíferos , Embrión no Mamífero , Femenino , Marcadores Genéticos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Fenotipo , Polimorfismo GenéticoRESUMEN
Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type-specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.
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Apoptosis , Metilación de ADN , Regulación de la Expresión Génica , Genoma , Impresión Genómica , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Virales , Alelos , Animales , Sitios de Ligazón Microbiológica/genética , División Celular , Línea Celular Transformada , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Evolución Molecular , Fibroblastos , Eliminación de Gen , Perfilación de la Expresión Génica , Genes de Partícula A Intracisternal/genética , Integrasas/genética , Integrasas/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , ARN Mensajero/genética , Recombinación Genética/genética , Células Madre/enzimología , Células Madre/metabolismoRESUMEN
Early outgrowth of the vertebrate embryonic limb requires signalling by the apical ectodermal ridge (AER) to the progress zone (PZ), which in response proliferates and lays down the pattern of the presumptive limb in a proximal to distal progression. Signals from the PZ maintain the AER until the anlagen for the distal phalanges have been formed. The semidominant mouse mutant dactylaplasia (Dac) disrupts the maintenance of the AER, leading to truncation of distal structures of the developing footplate, or autopod. Adult Dac homozygotes thus lack hands and feet except for malformed single digits, whereas heterozygotes lack phalanges of the three middle digits. Dac resembles the human autosomal dominant split hand/foot malformation (SHFM) diseases. One of these, SHFM3, maps to chromosome 10q24 (Refs 6,7), which is syntenic to the Dac region on chromosome 19, and may disrupt the orthologue of Dac. We report here the positional cloning of Dac and show that it belongs to the F-box/WD40 gene family, which encodes adapters that target specific proteins for destruction by presenting them to the ubiquitination machinery. In conjuction with recent biochemical studies, this report demonstrates the importance of this gene family in vertebrate embryonic development.
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Extremidades/embriología , Deformidades Congénitas de las Extremidades/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Proteínas F-Box , Heterocigoto , Humanos , Mesodermo/metabolismo , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución TisularRESUMEN
CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Drosophila melanogaster/genética , Genoma , Animales , Animales Modificados Genéticamente , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Genes Letales/genética , Genes Letales/fisiología , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Ratones , Fenotipo , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Transgenes/genética , Transgenes/fisiologíaRESUMEN
Type 2 diabetes mellitus (T2DM) is characterized by endothelial dysfunction that causes micro- and macrovascular complications. Low intensity therapeutic ultrasound (LITUS) may improve endothelial function, but its effects have not been investigated in these patients. The aim of our study was to compare the effects of pulsed (PUT) and continuous (CUT) waveforms of LITUS on the endothelium-dependent vasodilation of T2DM patients. The present randomized crossover trial had a sample of twenty-three patients (7 men) diagnosed with T2DM, 55.6 (±9.1) years old, with a body mass index of 28.6 (±3.3) kg/m2. All patients were randomized and submitted to different waveforms (Placebo, CUT, and PUT) of LITUS and the arterial endothelial function was evaluated. The LITUS of 1 MHz was applied in pulsed (PUT: 20% duty cycle, 0.08 W/cm2 SATA), continuous (CUT: 0.4 W/cm2 SPTA), and Placebo (equipment off) types of waves during 5 min on the brachial artery. Endothelial function was evaluated using the flow-mediated dilation (FMD) technique. PUT (mean difference 2.08%, 95% confidence interval 0.65 to 3.51) and CUT (mean difference 2.32%, 95% confidence interval 0.89 to 3.74) increased the %FMD compared to Placebo. In the effect size analysis, PUT (d=0.65) and CUT (d=0.65) waveforms presented moderate effects in the %FMD compared to Placebo. The vasodilator effect was similar in the different types of waves. Pulsed and continuous waveforms of LITUS of 1 MHz improved the arterial endothelial function in T2DM patients.
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Diabetes Mellitus Tipo 2 , Terapia por Ultrasonido , Masculino , Humanos , Vasodilatación , Terapia por Ultrasonido/métodos , Endotelio Vascular , Vasodilatadores/farmacología , Vasodilatadores/uso terapéutico , Arteria Braquial/diagnóstico por imagenRESUMEN
This study verified the effects of respiratory muscle training (RMT) on hemodynamics, heart rate (HR) variability, and muscle morphology in rats with streptozotocin-induced diabetes mellitus (DM). Thirty-six male Wistar rats were randomized into 4 groups and 34 completed the study: i) sham-sedentary (Sham-ST; n=9); ii) sham-RMT (Sham-RMT; n=9); iii) DM-sedentary (DM-ST; n=8); and iv) DM-RMT (DM-RMT; n=8). Hemodynamics were assessed by central cannulation, and R-R intervals were measured by electrocardiogram. In addition, the effects of RMT on the cross-sectional area of the diaphragm, anterior tibial, and soleus muscles were analyzed. The induction of DM by streptozotocin resulted in weight loss, hyperglycemia, reduced blood pressure, and attenuated left ventricular contraction and relaxation (P<0.05). We also observed a decrease in root mean square of successive differences between adjacent RR intervals (RMSSD) index and in the cross-sectional area of the muscles assessed, specifically the diaphragm, soleus, and anterior tibial muscles in diabetic rats (P<0.05). Interestingly, RMT led to an increase in RMSSD in rats with DM (P<0.05). The induction of DM produced profound deleterious changes in the diaphragmatic and peripheral muscles, as well as impairments in cardiovascular hemodynamics and autonomic control. Nevertheless, RMT may beneficially attenuate autonomic changes and improve parasympathetic modulation.
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Diabetes Mellitus Experimental , Animales , Ejercicios Respiratorios , Frecuencia Cardíaca , Hemodinámica , Masculino , Ratas , Ratas Wistar , Músculos RespiratoriosRESUMEN
Transgenic mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption and normal mice that had been treated with a CD8-specific mAb were infected intranasally with an H3N2 influenza A virus. Both groups of CD8T cell-deficient mice eliminated the virus from the infected respiratory tract. Potent CTL activity was detected in lung lavage populations taken from mice with intact CD8+ T cell function, with minimal levels of cytotoxicity being found for inflammatory cells obtained from the antibody-treated and beta 2-m mutant mice. We therefore conclude that cells infected with an influenza A virus can be cleared from the respiratory tract of mice lacking both functional class I major histocompatibility complex (MHC) glycoproteins and class I MHC-restricted, CD8+ effector T cells.
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Antígenos CD8/inmunología , Genes MHC Clase I , Virus de la Influenza A/inmunología , Pulmón/inmunología , Depleción Linfocítica , Infecciones por Orthomyxoviridae/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/genética , Animales , Citotoxicidad Inmunológica , Femenino , Tamización de Portadores Genéticos , Homocigoto , Inmunofenotipificación , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption lack beta 2-m protein and are deficient for functional major histocompatibility complex class I (MHC-I) molecules. The mutant mice have normal numbers of CD4+8- T helper cells, but lack MHC-I-directed CD4-8+ cytotoxic T lymphocytes (CTLs). In this study we used the beta 2-m mutant mice to study the importance of MHC-I-directed immunity in skin graft rejection. Our results indicate that MHC-I-directed CD8+ CTLs are not essential in the rejection of allografts with whole MHC or multiple minor H differences. However, the absence of MHC-I-guided immunity profoundly reduces the ability of mutant mice to reject H-Y disparate grafts. In addition, we show that natural killer cells which vigorously reject MHC-I-deficient bone marrow grafts, are not effective in the destruction of MHC-I-deficient skin grafts.
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Rechazo de Injerto , Células Asesinas Naturales/inmunología , Trasplante de Piel/inmunología , Linfocitos T/inmunología , Microglobulina beta-2/deficiencia , Animales , Citotoxicidad Inmunológica/inmunología , Fibroblastos/inmunología , Antígeno H-Y/inmunología , Ratones , Ratones Mutantes , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Xist RNA expression, methylation of CpG islands, and hypoacetylation of histone H4 are distinguishing features of inactive X chromatin. Here, we show that these silencing mechanisms act synergistically to maintain the inactive state. Xist RNA has been shown to be essential for initiation of X inactivation, but not required for maintenance. We have developed a system in which the reactivation frequency of individual X-linked genes can be assessed quantitatively. Using a conditional mutant Xist allele, we provide direct evidence for that loss of Xist RNA destabilizes the inactive state in somatic cells, leading to an increased reactivation frequency of an X-linked GFP transgene and of the endogenous hypoxanthine phosphoribosyl transferase (Hprt) gene in mouse embryonic fibroblasts. Demethylation of DNA, using 5-azadC or by introducing a mutation in Dnmt1, and inhibition of histone hypoacetylation using trichostatin A further increases reactivation in Xist mutant fibroblasts, indicating a synergistic interaction of X chromosome silencing mechanisms.
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Metilación de ADN , Compensación de Dosificación (Genética) , Histonas/metabolismo , ARN no Traducido/genética , Factores de Transcripción/genética , Acetilación , Animales , Replicación del ADN/fisiología , Femenino , Feto/citología , Fibroblastos/fisiología , Eliminación de Gen , Silenciador del Gen/fisiología , Genes Reporteros , Proteínas Fluorescentes Verdes , Histona Desacetilasas/metabolismo , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Ratones , Ratones Transgénicos , ARN/fisiología , ARN Largo no CodificanteRESUMEN
Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin-sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.
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Colágeno/metabolismo , Matriz Extracelular/ultraestructura , Fibronectinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Unión Proteica , TransfecciónRESUMEN
Histone variant macroH2A1 (macroH2A1) contains an NH(2)-terminal domain that is highly similar to core histone H2A and a larger COOH-terminal domain of unknown function. MacroH2A1 is expressed at similar levels in male and female embryonic stem (ES) cells and adult tissues, but a portion of total macroH2A1 protein localizes to the inactive X chromosomes (Xi) of differentiated female cells in concentrations called macrochromatin bodies. Here, we show that centrosomes of undifferentiated male and female ES cells harbor a substantial store of macroH2A1 as a nonchromatin-associated pool. Greater than 95% of centrosomes from undifferentiated ES cells contain macroH2A1. Cell fractionation experiments confirmed that macroH2A1 resides at a pericentrosomal location in close proximity to the known centrosomal proteins gamma-tubulin and Skp1. Retention of macroH2A1 at centrosomes was partially labile in the presence of nocodazole suggesting that intact microtubules are necessary for accumulation of macroH2A1 at centrosomes. Upon differentiation of female ES cells, Xist RNA expression became upregulated and monoallelic as judged by fluorescent in situ hybridization, but early Xist signals lacked associated macroH2A1. Xi acquired macroH2A1 soon thereafter as indicated by the colocalization of Xist RNA and macroH2A1. Accumulation of macroH2A1 on X chromosomes occurred with a corresponding loss of centrosomal macroH2A1. Our results define a sequence for the loading of macroH2A1 on the Xi and place this event in the context of differentiation and Xist expression. Furthermore, these results suggest a role for the centrosome in the X inactivation process.
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Centrosoma/fisiología , Histonas/metabolismo , Células Madre/fisiología , Cromosoma X/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Centrosoma/efectos de los fármacos , Centrosoma/ultraestructura , Citocalasina B/farmacología , Femenino , Histonas/análisis , Histonas/genética , Hibridación in Situ , Masculino , Ratones , Nocodazol/farmacología , Células Madre/citología , Tubulina (Proteína)/análisisRESUMEN
Degradation of type I collagen, the most abundant collagen, is initiated by collagenase cleavage at a highly conserved site between Gly775 and Ile776 of the alpha 1 (I) chain. Mutations at or around this site render type I collagen resistant to collagenase digestion in vitro. We show here that mice carrying a collagenase-resistant mutant Col1a-1 transgene die late in embryo-genesis, ascribable to overexpression of the transgene, since the same mutation introduced into the endogenous Col1a-1 gene by gene targeting permitted normal development of mutant mice to young adulthood. With increasing age, animals carrying the targeted mutation developed marked fibrosis of the dermis similar to that in human scleroderma. Postpartum involution of the uterus in the mutant mice was also impaired, with persistence of collagenous nodules in the uterine wall. Although type I collagen from the homozygous mutant mice was resistant to cleavage by human or rat fibroblast collagenases at the helical site, only the rat collagenase cleaved collagen trimers at an additional, novel site in the nonhelical N-telopeptide domain. Our results suggest that cleavage by murine collagenase at the N-telopeptide site could account for resorption of type I collagen during embryonic and early adult life. During intense collagen resorption, however, such as in the immediate postpartum uterus and in the dermis later in life, cleavage at the helical site is essential for normal collagen turnover. Thus, type I collagen is degraded by at least two differentially controlled mechanisms involving collagenases with distinct, but overlapping, substrate specificities.
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Colágeno/fisiología , Colagenasas/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Genes Letales , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Enfermedades de la Piel/genética , Enfermedades de la Piel/patología , Relación Estructura-ActividadRESUMEN
Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.
Asunto(s)
Membrana Basal/ultraestructura , Moléculas de Adhesión Celular/metabolismo , Epidermis/crecimiento & desarrollo , Integrinas/fisiología , Queratinocitos/fisiología , Animales , Animales Recién Nacidos , Antígenos de Superficie/metabolismo , Vesícula/genética , Adhesión Celular , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Integrina alfa3beta1 , Integrina alfa6beta4 , Integrinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , KalininaRESUMEN
The ability to introduce foreign genes into the germ line and the successful expression of the inserted gene in the organism have allowed the genetic manipulation of animals on an unprecedented scale. The information gained from the use of the transgenic technology is relevant to almost any aspect of modern biology including developmental gene regulation, the action of oncogenes, the immune system, and mammalian development. Because specific mutations can be introduced into transgenic mice, it becomes feasible to generate precise animal models for human genetic diseases and to begin a systematic genetic dissection of the mammalian genome.