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1.
Mol Cell Endocrinol ; 173(1-2): 193-202, 2001 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-11223190

RESUMO

The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of -/- mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30-60% those in wild-type mice, with heterozygotes (+/-) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3' side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5' side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.


Assuntos
Dexametasona/metabolismo , Resistência a Medicamentos/genética , Deleção de Genes , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Adrenalectomia , Animais , Western Blotting , Morte Celular/efeitos dos fármacos , Extratos Celulares , Dexametasona/farmacologia , Indução Enzimática/efeitos dos fármacos , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Glucose-6-Fosfatase/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ensaios de Proteção de Nucleases , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Timo/citologia , Timo/efeitos dos fármacos
2.
Immunity ; 13(2): 179-86, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10981961

RESUMO

Glucocorticoids are believed to play a role in T cell development and selection, although their precise function is controversial. Glucocorticoid receptor (GR)-deficient mice were used to directly investigate this problem. GR-deficient thymocytes were resistant to dexamethasone-mediated apoptosis, confirming the absence of glucocorticoid responsiveness. An absence of GR signaling had no impact on thymocyte development either in vivo or in vitro. T cell differentiation, including positive selection, was normal as assessed by normal development of CD4+CD8+, alphabetaTCR+CD4+, and alphabetaTCR+CD8+ thymocytes. Negative selection, mediated by the superantigen staphylococcal enterotoxin B (SEB), or anti-CD3/CD28, was also normal in the absence of GR signaling. In contrast to earlier reports, these data demonstrate that GR signaling is not essential for intrathymic T cell development or selection.


Assuntos
Receptores de Glucocorticoides/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia , Timo/fisiologia , Animais , Diferenciação Celular/fisiologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Marcação de Genes , Glucocorticoides/fisiologia , Camundongos , Técnicas de Cultura de Órgãos , Linfócitos T/citologia , Timo/citologia , Timo/embriologia
3.
Immunol Today ; 21(12): 606-11, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11114419

RESUMO

A role for glucocorticoids in thymopoiesis has been suggested by studies using glucocorticoid receptor (GR) anti-sense transgenic mice, glucocorticoid synthesis inhibitors and GR antagonists. Unfortunately, no consensus has been reached on exactly how glucocorticoids influence T-cell development. The most recent approach, using GR knockout (GR(-/-)) mice, indicates that GR signaling is, in fact, dispensable in this entire process.


Assuntos
Glucocorticoides/fisiologia , Estresse Fisiológico/imunologia , Subpopulações de Linfócitos T/imunologia , Aminoglutetimida/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Deleção Clonal/efeitos dos fármacos , DNA Antissenso/genética , Glucocorticoides/antagonistas & inibidores , Glucocorticoides/deficiência , Glucocorticoides/genética , Hematopoese/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Ativação Linfocitária/efeitos dos fármacos , Metirapona/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mifepristona/farmacologia , Modelos Biológicos , Receptores de Glucocorticoides/deficiência , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Estresse Fisiológico/fisiopatologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Timo/embriologia , Timo/imunologia
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