RESUMO
Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in embryonic neurons and to promote retinal ganglion cell (RGC) survival and axonal regrowth in a mouse optic nerve crush model. In order to evaluate the functions of endogenous Armcx1 in vivo, we have created a conditional Armcx1 knockout mouse line in which the entire coding region of the Armcx1 gene is flanked by loxP sites. This Armcx1fl line was crossed with mouse strains in which Cre recombinase expression is driven by the promoters for ß-actin and Six3, in order to achieve deletion of Armcx1 globally and in retinal neurons, respectively. Having confirmed deletion of the gene, we proceeded to characterize the abundance and morphology of RGCs in Armcx1 knockout mice aged to 15 months. Under normal physiological conditions, no evidence of aberrant retinal or optic nerve development or RGC degeneration was observed in these mice. The Armcx1fl mouse should be valuable for future studies investigating mitochondrial morphology and transport in the absence of Armcx1 and in determining the susceptibility of Armcx1-deficient neurons to degeneration in the setting of additional heritable or environmental stressors.
Assuntos
Proteínas do Domínio Armadillo , Células Ganglionares da Retina , Animais , Camundongos , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Camundongos Knockout , Nervo Óptico/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is the most common form of liver tumors. Although HCC is associated with chronic viral infections, alcoholic cirrhosis, and nonalcoholic fatty liver disease, genetic factors that contribute to the HCC risk remain unknown. The BRCA2 DNA repair associated (BRCA2) and cyclin-dependent kinase inhibitor 1A (CDKN1A) interacting protein, known as BCCIP, are essential for cell viability and maintenance of genomic stability. In this study, we established a new genetically engineered mouse model with Bccip deficiency. Mosaic or heterozygous Bccip deletion conferred an increased risk of spontaneous liver tumorigenesis and B-cell lymphoma development at old age. These abnormalities are accompanied with chronic inflammation, histologic features of nonalcoholic steatohepatitis, keratin and ubiquitin aggregates within cytoplasmic Mallory-Denk bodies, and changes of the intracellular distribution of high-mobility group box 1 protein. Our study suggests BCCIP dysregulation as a risk factor for HCC and offers a novel mouse model for future investigations of nonviral or nonalcoholic causes of HCC development.
Assuntos
Proteína BRCA2/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Neoplasias Hepáticas/genética , Linfoma de Células B/genética , Animais , Proteína BRCA2/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Heterozigoto , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Camundongos , Camundongos Knockout , MosaicismoRESUMO
Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.
Assuntos
Envelhecimento/patologia , Quimera/metabolismo , Células-Tronco Embrionárias/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Miocárdio/patologia , Animais , Cálcio/metabolismo , Conexina 43/metabolismo , Distrofina/metabolismo , Feminino , Testes de Função Cardíaca , Humanos , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Miócitos Cardíacos/metabolismo , RegeneraçãoRESUMO
Precise control of lineage-specific gene expression in the neural stem/progenitor cells is crucial for generation of the diversity of neuronal and glial cell types in the central nervous system (CNS). The mechanism underlying such gene regulation, however, is not fully elucidated. Here, we report that a 377 bp evolutionarily conserved DNA fragment (CR5), located approximately 32 kbp upstream of Olig2 transcription start site, acts as a cis-regulator for gene expression in the development of the neonatal forebrain. CR5 is active in a time-specific and brain region-restricted manner. CR5 activity is not detected in the embryonic stage, but it is exclusively in a subset of Sox5+ cells in the neonatal ventral forebrain. Furthermore, we show that Sox5 binding motif in CR5 is important for this cell-specific gene regulatory activity; mutation of Sox5 binding motif in CR5 alters reporter gene expression with different cellular composition. Together, our study provides new insights into the regulation of cell-specific gene expression during CNS development.
Assuntos
Células-Tronco Neurais/metabolismo , Neurogênese/genética , Prosencéfalo/embriologia , Fatores de Transcrição SOXD/genética , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Bovinos , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Fator de Transcrição 2 de Oligodendrócitos , Prosencéfalo/metabolismo , Ligação Proteica , Fatores de Transcrição SOXD/biossíntese , Alinhamento de SequênciaRESUMO
PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.
Assuntos
Proteína BRCA1/metabolismo , Infertilidade Masculina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Proteína BRCA1/química , Dano ao DNA , Reparo do DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi , Recombinação Homóloga , Humanos , Marcação In Situ das Extremidades Cortadas , Infertilidade Masculina/genética , Masculino , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/químicaRESUMO
Limb-girdle muscular dystrophy-2F (LGMD-2F) is an incurable degenerative muscle disorder caused by a mutation in the sarcoglycan-δ (SGδ)-encoding gene (SGCD in humans). The lack of SGδ results in the complete disruption of the sarcoglycan complex (SGC) in the skeletal and cardiac muscle within the larger dystrophin-glycoprotein complex (DGC). The long-term consequences of SG ablation on other members of the DGC are currently unknown. We produced mosaic mice through the injection of wild-type (WT) embryonic stem cells (ESCs) into SGδ-knockout (KO) blastocysts. ESC-derived SGδ was supplied to the sarcolemma of 18-month-old chimeric muscle, which resulted in the restoration of the SGC. Despite SGC rescue, and contrary to previous observations obtained with WT/mdx chimeras (a mouse rescue paradigm for Duchenne muscular dystrophy), low levels of ESC incorporation were insufficient to produce histological corrections in SGδ-KO skeletal muscle or heart. The inefficient process of ESC rescue was more evident in the SGδ-KO diaphragm, which had reduced levels of dystrophin and no compensatory utrophin, and needed almost full WT ESC reconstitution for histological improvement. The results suggest that the SGδ-KO mouse model of LGMD is not amenable to ESC treatment.
Assuntos
Distrofina/metabolismo , Células-Tronco Embrionárias/metabolismo , Sarcoglicanas/metabolismo , Animais , Diafragma/metabolismo , Células-Tronco Embrionárias/citologia , Feminino , Camundongos , Camundongos Knockout , Sarcoglicanas/deficiênciaRESUMO
The mechanisms by which inflammation or autoimmunity causes proteinuric kidney disease remain elusive. Yet proteinuria is a hallmark and a prognostic indicator of kidney disease, and also an independent risk factor for cardiovascular morbidity and mortality. Podocytes are an integral component of the kidney filtration barrier and podocyte injury leads to proteinuria. Here we show that podocytes, which receive signals from the vascular space including circulating antigens, constitutively express TLR16 and TLR8. We find that podocytes can respond to TLR ligands including staphylococcal enterotoxin B (SEB), poly I:C, or lipopolysaccharide (LPS) with pro-inflammatory cytokine release and activation of type I interferon (IFN) signalling. This in turn stimulates podocyte B7-1 expression and actin remodelling in vitro and transient proteinuria in vivo. Importantly, the treatment of mice with a type I IFN receptor-blocking antibody (Ab) prevents LPS-induced proteinuria. These results significantly extend our understanding of podocyte response to immune stimuli and reveal a novel mechanism for infection- or inflammation-induced transient proteinuria. Dysregulation or aberrant activation of this response may result in persistent proteinuria and progressive glomerular disease. In summary, the inhibition of glomerular type I IFN signalling with anti-IFN Abs may be a novel therapy for proteinuric kidney diseases.
Assuntos
Interferon Tipo I/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Transdução de Sinais/fisiologia , Receptores Toll-Like/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Glomérulos Renais/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteinúria/fisiopatologiaRESUMO
BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a â¼20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Instabilidade Cromossômica/genética , Desenvolvimento Embrionário/genética , Animais , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Transgênicos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinação Genética , Troca de Cromátide IrmãRESUMO
The promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha) protein of acute promyelocytic leukemia (APL) is oncogenic in vivo. It has been hypothesized that the ability of PML-RARalpha to inhibit RARalpha function through PML-dependent aberrant recruitment of histone deacetylases (HDACs) and chromatin remodeling is the key initiating event for leukemogenesis. To elucidate the role of HDAC in this process, we have generated HDAC1-RARalpha fusion proteins and tested their activity and oncogenicity in vitro and in vivo in transgenic mice (TM). In parallel, we studied the in vivo leukemogenic potential of dominant negative (DN) and truncated RARalpha mutants, as well as that of PML-RARalpha mutants that are insensitive to retinoic acid. Surprisingly, although HDAC1-RARalpha did act as a bona fide DN RARalpha mutant in cellular in vitro and in cell culture, this fusion protein, as well as other DN RARalpha mutants, did not cause a block in myeloid differentiation in vivo in TM and were not leukemogenic. Comparative analysis of these TM and of TM/PML(-/-) and p53(-/-) compound mutants lends support to a model by which the RARalpha and PML blockade is necessary, but not sufficient, for leukemogenesis and the PML domain of the fusion protein provides unique functions that are required for leukemia initiation.
Assuntos
Histona Desacetilases/metabolismo , Leucemia Promielocítica Aguda/enzimologia , Leucemia Promielocítica Aguda/etiologia , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/fisiologia , Animais , Linhagem Celular , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteína da Leucemia Promielocítica , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
BACKGROUND: Topors is a nuclear protein that co-localizes with promyelocytic leukemia bodies and has both ubiquitin and SUMO E3 ligase activity. Expression studies implicated Topors as a tumor suppressor in various malignancies. To gain insight into the function of Topors, we generated a Topors-deficient mouse strain. RESULTS: Mice homozygous for a mutant Topors allele exhibited a high rate of perinatal mortality and decreased lifespan. In addition, heterozygotes were found to have an increased incidence of malignancy, involving a variety of tissues. Consistent with this finding, primary embryonic fibroblasts lacking Topors exhibited an increased rate of malignant transformation, associated with aneuploidy and defective chromosomal segregation. While loss of Topors did not alter sensitivity to DNA-damaging or microtubule-targeting agents, cells lacking Topors exhibited altered pericentric heterochromatin, manifested by mislocalization of HP1alpha and an increase in transcription from pericentric major satellite DNA. Topors-deficient cells exhibited a transcriptional profile similar to that of cells treated with histone deacetylase inhibitors, and were resistant to the anti-proliferative effects of the histone deacetylase inhibitor trichostatin A. CONCLUSION: These results indicate a unique role for Topors in the maintenance of genomic stability and pericentric heterochromatin, as well as in cellular sensitivity to histone deacetylase inhibitors.
Assuntos
Neoplasias/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Homólogo 5 da Proteína Cromobox , Fibroblastos , Instabilidade Genômica , Heterozigoto , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos , Camundongos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, we generated mice deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals.
Assuntos
Núcleo Celular/metabolismo , Segregação de Cromossomos , Embrião de Mamíferos/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/fisiologia , Animais , Apoptose , Blastocisto/citologia , Blastocisto/metabolismo , Núcleo Celular/genética , Perda do Embrião/genética , Embrião de Mamíferos/citologia , Feminino , Imunofluorescência , Proteínas Ativadoras de GTPase/metabolismo , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Adaptations to chronic hypoxia (CH) could reflect cellular changes within the cardiorespiratory regions of the rostral ventrolateral medulla (RVLM), the C1 region, and the pre-Bötzinger complex (pre-BötC). Previous studies have shown that the hypoxic chemosensitivity of these regions are heme oxygenase (HO) dependent and that CH induces HO-1. To determine the time course of HO-1 induction within these regions and explore its relevance to the respiratory and sympathetic responses during CH, the expression of HO-1 mRNA and protein in the RVLM and measures of respiration, sigh frequency, and sympathetic activity (spectral analysis of heart rate) were examined during 10 days of CH. Respiratory and sympathetic responses to acute hypoxia were obtained in chronically instrumented awake wild-type (WT) and HO-1 null mice. After 4 days of CH, there was a significant induction of HO-1 within the C1 region and pre-BötC. WT mice acclimated to CH by increasing peak diaphragm EMG after 10 days of CH but had no change in the respiratory response to acute hypoxia. There were no significant differences between WT and HO-1 null mice. In WT mice, hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity initially declined before returning toward baseline after 5 days of CH, correlating with the induction of HO-1. In contrast, HO-1 null mice had a persistent decline in hypoxic sigh frequency and hypoxic sensitivity of sympathetic activity. We conclude that induction of HO-1 in these RVLM cardiorespiratory regions may be important for the hypoxic sensitivity of sighs and sympathetic activity during CH.
Assuntos
Adaptação Fisiológica/fisiologia , Sistema Cardiovascular/fisiopatologia , Heme Oxigenase-1/metabolismo , Hipóxia/fisiopatologia , Sistema Respiratório/fisiopatologia , Animais , Diafragma/fisiopatologia , Eletromiografia , Expressão Gênica/genética , Frequência Cardíaca/fisiologia , Heme Oxigenase-1/genética , Hipercapnia/fisiopatologia , Hipóxia/metabolismo , Bulbo/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neurônios/metabolismo , Sistema Nervoso Parassimpático/fisiopatologia , Receptores da Neurocinina-1/metabolismo , Mecânica Respiratória/fisiologia , Sistema Nervoso Simpático/fisiopatologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.
Assuntos
Adenosina Desaminase/metabolismo , Evolução Molecular Direcionada , Edição de Genes , Adenosina Desaminase/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Humanos , Mutação INDEL , CamundongosRESUMO
Chronic intermittent hypoxia (CIH) increases sympathetic tone and respiratory instability. Our previous work showed that chronic hypoxia induces the oxygen-sensing enzyme heme oxygenase-1 (HO-1) within the C1 sympathoexcitatory region and the pre-Bötzinger complex (pre-BötC). We therefore examined the effect of CIH on time course of induced expression of HO-1 within these regions and determined whether the induction of HO-1 correlated with changes in respiratory, sigh frequency, and sympathetic responses (spectral analysis of heart rate) to acute hypoxia (10% O2) during 10 days of exposure to CIH in chronically instrumented awake wild-type (WT) and HO-1 null mice (HO-1-/-). HO-1 was induced within the C1 and pre-BötC regions after 1 day of CIH. There were no significant differences in the baseline respiratory parameters between WT and HO-1-/- Prior to CIH, acute hypoxia increased respiratory frequency in both WT and HO-1-/-; however, minute diaphragm electromyogram activity increased in WT but not HO-1-/- The hypoxic respiratory response after 1 and 10 days of CIH was restored in HO-1-/- CIH resulted in an initial significant decline in 1) the hypoxic sigh frequency response, which was restored in WT but not HO-1-/-, and 2) the baseline sympathetic activity in WT and HO-1-/-, which remained stable subsequently in WT but not in HO-1-/- We conclude that 1) CIH induces expression of HO-1 in the C1 and pre-BötC regions within 1 day and 2) HO-1 is necessary for hypoxia respiratory response and contributes to the maintenance of the hypoxic sigh responses and baseline sympathetic activity during CIH.
Assuntos
Diafragma/fisiopatologia , Frequência Cardíaca , Heme Oxigenase-1/metabolismo , Hipóxia/fisiopatologia , Bulbo/metabolismo , Proteínas de Membrana/metabolismo , Oxigênio/metabolismo , Sistema Nervoso Simpático/fisiopatologia , Adaptação Fisiológica , Animais , Doença Crônica , Diafragma/inervação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mecânica RespiratóriaRESUMO
Acute promyelocytic leukemia (APL) is characterized by the accumulation of abnormal promyelocytes in the bone marrow (BM), and by the presence of a reciprocal chromosomal translocation involving retinoic acid receptor alpha (RARalpha). To date, five RARalpha partner genes have been identified in APL. NuMA-RARalpha was identified in a pediatric case of APL carrying a translocation t(11;17)(q13;q21). Using a construct containing the NuMA-RARalpha fusion gene driven by the human cathepsin G promoter (hCG-NuMA-RARalpha), two transgenic mouse lines were generated. Transgenic mice were observed to have a genetic myeloproliferation (increased granulopoiesis in BM) at an early age, and rapidly developed a myeloproliferative disease-like myeloid leukemia. This leukemia was morphologically and immunophenotypically indistinguishable from human APL, with a penetrance of 100%. The phenotype of transgenic mice was consistent with a blockade of neutrophil differentiation. NuMA-RARalpha is therefore sufficient for disease development in this APL model.
Assuntos
Catepsinas/genética , Leucemia Mieloide/genética , Proteínas Nucleares/genética , Receptores do Ácido Retinoico/genética , Animais , Antígenos Nucleares , Sequência de Bases , Catepsina G , Proteínas de Ciclo Celular , Primers do DNA , Genótipo , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Leucemia Mieloide/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas à Matriz Nuclear , Receptor alfa de Ácido Retinoico , Serina EndopeptidasesRESUMO
PDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse.
RESUMO
Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdxâ¶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdxâ¶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdxâ¶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdxâ¶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdxâ¶utrophin chimeras, despite the fact that utrophin was compromised in the mdxâ¶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle.
Assuntos
Células-Tronco Pluripotentes Induzidas/transplante , Distrofia Muscular Animal/terapia , Transplante de Células-Tronco/métodos , Utrofina/farmacologia , Tecido Adiposo , Animais , Blastocisto , Composição Corporal , Peso Corporal , Camundongos , Músculo Esquelético , Utrofina/administração & dosagemRESUMO
Embryonic stem cells have the capacity to differentiate into a wide range of cell types. We previously described that blastocyst injection of wild type (WT) embryonic stem cells (ESCs) into various knockout (KO) mouse models of human disease prevents disease from occurring. In this study we ask if the blastocyst approach can also correct defects in a mouse model of transgenic (Tg) overexpression of a pro-apoptotic factor. We injected ROSA26 (LacZ-marked) WT ESCs into human mammalian sterile 20 like-kinase 1 (Mst1) Tg blastocysts. Mst1 Tg mice overexpress Mst1, a pro-apoptotic factor, in a cardiac-specific manner. As a result, Mst1 Tg mice develop adult dilated cardiomyopathy driven by apoptosis, reduction in cell density and no hypertrophic compensation. Incorporation of WT ESCs generated WT/Mst1 chimeric mice with normal hearts at histological and functional levels. Accordingly, apoptosis and cell density parameters were normalized. The experiments suggest that an adult-onset cardiac myopathy induced by overexpression of the pro-apoptotic Mst1 can be reversed by developmental incorporation of WT ESCs. The findings also suggest that since forced expression of the Mst1 transgene is not abolished in the rescued chimeras, the WT ES-derived cells normalize pathways that lie downstream of Mst1. The results expand the therapeutic capability of the ESCs to mouse models that overproduce detrimental proteins.
Assuntos
Blastocisto/citologia , Cardiomiopatias/prevenção & controle , Células-Tronco Embrionárias/transplante , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cardiomiopatias/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Serina-Treonina Quinases/genética , Regulação para CimaRESUMO
Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.