Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Cell ; 180(6): 1262-1271.e15, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32169219

RESUMO

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

2.
Nurs Manag (Harrow) ; 26(5): 36-41, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31535540

RESUMO

Few nursing staff from band 6 and above receive formal training on how to chair and manage formal business meetings, which can be a challenging experience for novices and when participants fail to engage with the discussion. This article gives an overview of how to lead and manage effective meetings, focusing on process, content, managing conflict and how to engage participants fully. This will give meetings more purpose and ensure participants feel their time is being used efficiently. Learning how to conduct effective meetings will enhance the quality of team working and team effectiveness.

3.
Genome Biol ; 20(1): 171, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31446895

RESUMO

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.


Assuntos
Alelos , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Animais , Blastocisto/metabolismo , Análise Fatorial , Feminino , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos Knockout , Microinjeções , Análise de Regressão , Reprodutibilidade dos Testes
4.
Nature ; 572(7770): 436-437, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31427805
6.
Arterioscler Thromb Vasc Biol ; 38(7): 1576-1593, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29853569

RESUMO

OBJECTIVE: Vascular remodeling is associated with complex molecular changes, including increased Notch2, which promotes quiescence in human smooth muscle cells. We used unbiased protein profiling to understand molecular signatures related to neointimal lesion formation in the presence or absence of Notch2 and to test the hypothesis that loss of Notch2 would increase neointimal lesion formation because of a hyperproliferative injury response. APPROACH AND RESULTS: Murine carotid arteries isolated at 6 or 14 days after ligation injury were analyzed by mass spectrometry using a data-independent acquisition strategy in comparison to uninjured or sham injured arteries. We used a tamoxifen-inducible, cell-specific Cre recombinase strain to delete the Notch2 gene in smooth muscle cells. Vessel morphometric analysis and immunohistochemical staining were used to characterize lesion formation, assess vascular smooth muscle cell proliferation, and validate proteomic findings. Loss of Notch2 in smooth muscle cells leads to protein profile changes in the vessel wall during remodeling but does not alter overall lesion morphology or cell proliferation. Loss of smooth muscle Notch2 also decreases the expression of enhancer of rudimentary homolog, plectin, and annexin A2 in vascular remodeling. CONCLUSIONS: We identified unique protein signatures that represent temporal changes in the vessel wall during neointimal lesion formation in the presence and absence of Notch2. Overall lesion formation was not affected with loss of smooth muscle Notch2, suggesting compensatory pathways. We also validated the regulation of known injury- or Notch-related targets identified in other vascular contexts, providing additional insight into conserved pathways involved in vascular remodeling.


Assuntos
Lesões das Artérias Carótidas/metabolismo , Espectrometria de Massas , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Proteômica/métodos , Receptor Notch2/metabolismo , Remodelação Vascular , Idoso , Idoso de 80 Anos ou mais , Animais , Anexina A2/metabolismo , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Plectina/metabolismo , Receptor Notch2/deficiência , Receptor Notch2/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo
7.
Nature ; 554(7691): 239-243, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29420474

RESUMO

Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.


Assuntos
Elementos Facilitadores Genéticos/genética , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Fenótipo , Animais , Encéfalo/embriologia , Feminino , Genoma , Coração/embriologia , Deformidades Congênitas dos Membros/embriologia , Deformidades Congênitas dos Membros/genética , Masculino , Camundongos , Deleção de Sequência , Análise Espaço-Temporal
8.
Cell ; 172(3): 491-499.e15, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29358049

RESUMO

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.


Assuntos
Sequência Conservada , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos , Animais , Encéfalo/anormalidades , Encéfalo/embriologia , Encéfalo/metabolismo , Feminino , Deleção de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
J Pediatr ; 194: 22-27.e5, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29217101

RESUMO

OBJECTIVE: To describe the successful implementation of an in situ simulation program to diagnose and correct latent safety threats in a level 4 neonatal intensive care unit (NICU) to mitigate a methicillin-resistant Staphylococcus aureus (MRSA) outbreak. STUDY DESIGN: An investigational report describes a simulation intervention that occurred during a 4-month MRSA outbreak in a single-center, 46-bed, newly renovated level 4 NICU. The simulation program was developed for all NICU providers in which they were exposed to a 30-minute in situ human simulation intervention that included education, evaluation, and debriefing to resolve perceived or observed latent safety threats. The primary study outcome was improved hand hygiene compliance and an enhanced estimate of the culture of safety during a 6-month period. RESULTS: A total of 99 healthcare providers including physicians, nurses, respiratory therapists, and environmental service workers completed the course. Before the simulation intervention, there were 18 patients colonized or infected with a single MRSA clone; after the intervention, there were no new episodes of colonization or infection. CONCLUSIONS: An in situ, simulation-based intervention can counter threats to patient safety related to workflow and lapses in infection control practices and improve patient outcomes.


Assuntos
Surtos de Doenças/prevenção & controle , Controle de Infecções , Unidades de Terapia Intensiva Neonatal , Staphylococcus aureus Resistente à Meticilina , Treinamento por Simulação , Infecções Estafilocócicas/prevenção & controle , Humanos , Recém-Nascido , Infecções Estafilocócicas/epidemiologia
10.
G3 (Bethesda) ; 6(7): 2051-61, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27175020

RESUMO

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Marcação de Genes/métodos , Fatores de Troca do Nucleotídeo Guanina/genética , RNA Guia/genética , Alelos , Animais , Sequência de Bases , Reparo do DNA por Junção de Extremidades , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endonucleases/metabolismo , Éxons , Feminino , Edição de Genes , Fatores de Troca do Nucleotídeo Guanina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microinjeções , Mutagênese Insercional , RNA Guia/metabolismo , Zigoto/citologia , Zigoto/metabolismo
11.
Osiris ; 31: 94-115, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-30125077

RESUMO

Most scholarship on the medicalization of emotions has focused on projects that locate emotions, one way or another, within individual brains and minds. The story of mother love and mental illness, in contrast, is a medicalization story that frames the problem of pathological emotions as a relational issue. Bad mother love was seen as both a pathology (for the mother) and a pathogen (for her vulnerable child).Moreover, different forms of pathological mother love­smothering love, ambivalent love, love that masked an actual desire to dominate and control­were supposed to have different effects on children, ranging from lack of fitness for military service to homosexuality to juvenile delinquency to outright psychosis, especially schizophrenia. Understanding why mother love came to be associated with mental illness­and, equally, what led to this viewpoint's rapid decline into disrepute­requires us to go beyond simply invoking the trope of "mother blaming" and leaving things at that. This essay is a first effort at a richer narrative, one that blends perspectives from the history of emotions and the history of science and medicine.


Assuntos
Emoções , Medicalização , Transtornos Mentais/psicologia , Relações Mãe-Filho , Mães , Criança , Feminino , Humanos , Amor
12.
Am Psychol ; 70(7): 621-31, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26436312

RESUMO

In the past 20 years, mindfulness therapeutic programs have moved firmly into the mainstream of clinical practice and beyond. As they have, we have also seen the development of an increasingly vocal critique. At issue is often less whether or not these mindfulness practices "work," and more whether there is a danger in dissociating them from the ethical frameworks for which they were originally developed. Mindfulness, the argument goes, was never supposed to be about weight loss, better sex, helping children perform better in school, helping employees be more productive in the workplace, or even improving the functioning of anxious, depressed people. It was never supposed to be a merchandized commodity to be bought and sold. The larger clinical and religious community, however, has not always been troubled by the idea that (PsycINFO Database Record


Assuntos
Transtornos Mentais/terapia , Atenção Plena/ética , Atenção Plena/história , Estresse Psicológico/terapia , História do Século XX , História do Século XXI , Humanos , Transtornos Mentais/psicologia , Atenção Plena/métodos , Estresse Psicológico/psicologia
13.
Circ Res ; 113(8): 975-85, 2013 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-23965337

RESUMO

RATIONALE: Deregulated vascular smooth muscle cell (VSMC) proliferation contributes to multiple vascular pathologies, and Notch signaling regulates VSMC phenotype. OBJECTIVE: Previous work focused on Notch1 and Notch3 in VSMC during vascular disease; however, the role of Notch2 is unknown. Because injured murine carotid arteries display increased Notch2 in VSMC as compared with uninjured arteries, we sought to understand the impact of Notch2 signaling in VSMCs. METHODS AND RESULTS: In human primary VSMCs, Jagged-1 (Jag-1) significantly reduced proliferation through specific activation of Notch2. Increased levels of p27(kip1) were observed downstream of Jag-1/Notch2 signaling and were required for cell cycle exit. Jag-1 activation of Notch resulted in increased phosphorylation on serine 10, decreased ubiquitination, and prolonged half-life of p27(kip1). Jag-1/Notch2 signaling robustly decreased S-phase kinase-associated protein, an F-box protein that degrades p27(kip1) during G1. Overexpression of S-phase kinase-associated protein before Notch activation by Jag-1 suppressed the induction of p27(kip1). Additionally, increased Notch2 and p27(kip1) expression was colocalized to the nonproliferative zone of injured arteries as indicated by co-staining with proliferating cell nuclear antigen, whereas Notch3 was expressed throughout normal and injured arteries, suggesting Notch2 may negatively regulate lesion formation. CONCLUSIONS: We propose a receptor-specific function for Notch2 in regulating Jag-1-induced p27(kip1) expression and growth arrest in VSMCs. During vascular remodeling, colocalization of Notch2 and p27(kip1) to the nonproliferating region supports a model where Notch2 activation may negatively regulate VSMC proliferation to lessen the severity of the lesion. Thus, Notch2 is a potential target for control of VSMC hyperplasia.


Assuntos
Lesões das Artérias Carótidas/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Notch2/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Ciclo Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/genética , Modelos Animais de Doenças , Humanos , Hiperplasia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Interferência de RNA , Receptor Notch2/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais , Fatores de Tempo , Transfecção
15.
Genetics ; 186(2): 451-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20628038

RESUMO

Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Técnicas de Inativação de Genes/métodos , Marcação de Genes/métodos , Mutagênese Sítio-Dirigida , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Notch3 , Receptores Notch/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Proteínas Serrate-Jagged , Dedos de Zinco
16.
Genesis ; 48(9): 563-7, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20645309

RESUMO

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2-expressing populations using Tie2-Cre;Rosa26R-EYFP mice. In Tie2-Cre;Rosa26R-EYFP mice, analysis of bone marrow cells showed Cre-mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ∼ 84% of each lineage was EYFP(+), and nearly all cells that come from Tie2-expressing lineages are CD45(+), confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2(+) origin. Our findings of high levels of Tie2-Cre recombination in the hematopoietic lineage have implications for the use of the Tie2-Cre mouse as a lineage-restricted driver strain.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Recombinação Genética/genética , Animais , Proteínas de Bactérias/metabolismo , Células da Medula Óssea/metabolismo , Primers do DNA/genética , Citometria de Fluxo , Integrases/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Receptor TIE-2 , Baço/metabolismo
17.
Circ Res ; 103(4): 423-31, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18617694

RESUMO

Notch signaling is critical for the development and maintenance of the cardiovasculature, with loss-of-function studies defining roles of Notch1 in the endothelial/hematopoietic lineages. No in vivo studies have addressed complementary gain-of-function strategies within these tissues to define consequences of Notch activation. We developed a transgenic model of Cre recombinase-mediated activation of a constitutively active mouse Notch1 allele (N1ICD(+)) and studied transgene activation in Tie2-expressing lineages. The in vivo phenotype was compared to effects of Notch1 activation on endothelial tubulogenesis, paracrine regulation of smooth muscle cell proliferation, and hematopoiesis. N1ICD(+) embryos showed midgestation lethality with defects in angiogenic remodeling of embryonic and yolk sac vasculature, cardiac development, smooth muscle cell investment of vessels, and hematopoietic differentiation. Angiogenic defects corresponded with impaired endothelial tubulogenesis in vitro following Notch1 activation and paracrine inhibition of smooth muscle cells when grown with Notch1-activated endothelial cells. Flow cytometric analysis of hematopoietic and endothelial precursor populations demonstrated a significant loss of CD71(+)/Ter119(+) populations with an active N1ICD(+) allele and a corresponding increase in c-Kit(+)/CD71 and Flk1(+) populations, suggesting a developmental block during the transition between c-Kit- and Ter119-expressing erythroblasts. Cardiovascular lineages are sensitive to an imbalance in Notch signaling, with aberrant activation reflecting a vascular phenotype comparable to a loss-of-function Notch1 mutation.


Assuntos
Sistema Cardiovascular/embriologia , Endotélio Vascular/metabolismo , Sistema Hematopoético/embriologia , Músculo Liso Vascular/metabolismo , Receptor Notch1/metabolismo , Receptor TIE-2/metabolismo , Animais , Sistema Cardiovascular/metabolismo , Células Cultivadas , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Sistema Hematopoético/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Mutação , Receptor Notch1/genética , Receptor TIE-2/genética , Transdução de Sinais/fisiologia , Saco Vitelino/irrigação sanguínea , Saco Vitelino/metabolismo
18.
Dev Biol ; 321(1): 64-76, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18582454

RESUMO

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.


Assuntos
Condrócitos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Diferenciação Celular , Proliferação de Células , Condrócitos/citologia , Camundongos , Camundongos Transgênicos , Osteogênese , Fator de Transcrição STAT1/metabolismo , Ubiquitinação , Regulação para Cima
19.
BMJ ; 336(7651): 967-8, 2008 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-18390494
20.
J Vasc Res ; 45(1): 1-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-17898542

RESUMO

BACKGROUND: Our study characterizes Delta-like 1 (Dll1) in the adult mouse, particularly in normal versus injured vasculature, with the aid of the transgenic Dll1(LacZ) line. METHODS: Normal mouse adult tissues or those from the Dll1(LacZ) reporter line were analyzed for Dll1 expression and promoter activity. Vascular tissue was analyzed before and after carotid artery ligation. RESULTS: In wild-type mice, Dll1 transcript expression was widespread. Similarly, the Dll1(LacZ) reporter had beta-galactosidase activity detectable in the cerebellum, cerebrum, spinal cord, liver, lung and cornea, although the normal adult vasculature had no reporter expression. Following arterial ligation, there was acute induction of Dll1(LacZ) reporter expression, both in the ligated left carotid artery, and the uninjured right contralateral artery. Expression returned to low/undetectable levels 4-10 days after arterial ligation. CONCLUSION: The expression of Dll1 in the adult mouse is more widespread than previously realized, although not in resting large arteries in the adult mouse. Following arterial injury, Dll1 promoter activity is induced selectively in the endothelial cells of both the injured artery and the contralateral uninjured artery. Our results show that while overall expression in the adult mouse is widespread, Dll1 may be selectively expressed in the endothelium of injured vasculature, similar to the endothelial-restricted expression of Dll4.


Assuntos
Artérias Carótidas/metabolismo , Endotélio Vascular/metabolismo , Genes Reporter , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Óperon Lac , Regiões Promotoras Genéticas , Animais , Proteínas de Ligação ao Cálcio , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio Vascular/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Fatores de Tempo , Distribuição Tecidual , Transcrição Genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA