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1.
PLoS One ; 6(5): e19854, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21603627

RESUMO

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34(+) or CD34(+)CD45(+) hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34(+) precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34(+) hematopoietic precursors generated in vitro versus in vivo.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Hematopoéticas/citologia , Antígenos CD34 , Técnicas de Cultura de Células/métodos , Linhagem Celular , Humanos , Antígenos Comuns de Leucócito , Linfócitos T
2.
Proc Natl Acad Sci U S A ; 107(29): 13034-9, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20615958

RESUMO

Cell death is an important mechanism to limit uncontrolled T-cell expansion during immune responses. Given the role of death-receptor adapter protein Fas-associated death domain (FADD) in apoptosis, it is intriguing that T-cell receptor (TCR)-induced proliferation is blocked in FADD-defective T cells. Necroptosis is an alternate form of death that can be induced by death receptors and is linked to autophagy. It requires the death domain-containing kinase RIP1 and, in certain instances, RIP3. FADD and its apoptotic partner, Caspase-8, have also been implicated in necroptosis. To accurately assess the role of FADD in mature T-cell proliferation and death, we generated a conditional T-cell-specific FADD knockout mouse strain. The T cells of these mice develop normally, but lack FADD at the mature stage. FADD-deficient T cells respond poorly to TCR triggering, exhibit slow cell cycle entry, and fail to expand over time. We find that programmed necrosis occurs during the late stage of normal T-cell proliferation and that this process is greatly amplified in FADD-deficient T cells. Inhibition of necroptosis using an inhibitor of RIP1 kinase activity rescues the FADD knockout proliferative defect. However, TCR-induced necroptosis did not appear to require autophagy or involve RIP3. Consistent with their defective CD8 T-cell response, these mice succumb to Toxoplasma gondii infection more readily than wild-type mice. We conclude that FADD constitutes a mechanism to keep TCR-induced programmed necrotic signaling in check during early phases of T-cell clonal expansion.


Assuntos
Apoptose/imunologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Necrose/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Autofagia , Caspase 8/metabolismo , Ciclo Celular , Proliferação de Células , Suscetibilidade a Doenças , Proteína de Domínio de Morte Associada a Fas/deficiência , Camundongos , Fenótipo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Análise de Sobrevida , Linfócitos T/citologia , Linfócitos T/enzimologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Toxoplasmose/parasitologia
3.
Proc Natl Acad Sci U S A ; 106(2): 522-7, 2009 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-19116268

RESUMO

Allelic exclusion of Ig gene expression is necessary to limit the number of functional receptors to one per B cell. The mechanism underlying allelic exclusion is unknown. Because germline transcription of Ig and TCR loci is tightly correlated with rearrangement, we created two novel knock-in mice that report transcriptional activity of the Jkappa germline promoters in the Igkappa locus. Analysis of these mice revealed that germline transcription is biallelic and occurs in all pre-B cells. Moreover, we found that the two germline promoters in this region are not equivalent but that the distal promoter accounts for the vast majority of observed germline transcript in pre-B cells while the activity of the proximal promoter increases later in development. Allelic exclusion of the Igkappa locus thus occurs at the level of rearrangement, but not germline transcription.


Assuntos
Alelos , Linfócitos B/citologia , Cadeias kappa de Imunoglobulina/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Linfócitos B/imunologia , Técnicas de Introdução de Genes , Rearranjo Gênico , Humanos , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/imunologia
4.
Nat Immunol ; 9(10): 1171-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18724372

RESUMO

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.


Assuntos
Flagelina/imunologia , Macrófagos/imunologia , Complexos Multiproteicos/imunologia , Proteína Inibidora de Apoptose Neuronal/imunologia , Motivos de Aminoácidos/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Citosol , Ensaio de Imunoadsorção Enzimática , Flagelina/química , Immunoblotting , Legionella pneumophila/imunologia , Doença dos Legionários/imunologia , Macrófagos/microbiologia , Camundongos , Proteína Inibidora de Apoptose Neuronal/genética , Receptor 5 Toll-Like/imunologia , Receptor 5 Toll-Like/metabolismo , Transdução Genética
5.
Immunity ; 28(4): 571-80, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18394936

RESUMO

Ligands for the NKG2D stimulatory receptor are frequently upregulated on tumor lines, rendering them sensitive to natural killer (NK) cells, but the role of NKG2D in tumor surveillance has not been addressed in spontaneous cancer models. Here, we provided the first characterization of NKG2D-deficient mice, including evidence that NKG2D was not necessary for NK cell development but was critical for immunosurveillance of epithelial and lymphoid malignancies in two transgenic models of de novo tumorigenesis. In both models, we detected NKG2D ligands on the tumor cell surface ex vivo, providing needed evidence for ligand expression by primary tumors. In a prostate cancer model, aggressive tumors arising in NKG2D-deficient mice expressed higher amounts of NKG2D ligands than did similar tumors in wild-type mice, suggesting an NKG2D-dependent immunoediting of tumors in this model. These findings provide important genetic evidence for surveillance of primary tumors by an NK receptor.


Assuntos
Adenocarcinoma/imunologia , Fibrossarcoma/imunologia , Síndromes de Imunodeficiência/imunologia , Vigilância Imunológica , Linfoma de Células B/imunologia , Neoplasias da Próstata/imunologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Adenocarcinoma/genética , Animais , Benzo(a)Antracenos/toxicidade , Modelos Animais de Doenças , Feminino , Fibrossarcoma/induzido quimicamente , Fibrossarcoma/genética , Síndromes de Imunodeficiência/genética , Vigilância Imunológica/genética , Linfoma de Células B/genética , Masculino , Metilcolantreno , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Neoplasias da Próstata/genética , Receptores Imunológicos/fisiologia , Receptores de Células Matadoras Naturais
6.
Curr Biol ; 18(2): 129-35, 2008 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-18207746

RESUMO

Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence.


Assuntos
Tolerância a Medicamentos , Endocitose/efeitos dos fármacos , Dependência de Morfina/metabolismo , Morfina/farmacologia , Receptores Opioides mu/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Síndrome de Abstinência a Substâncias/metabolismo
7.
J Exp Med ; 204(13): 3247-56, 2007 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-18056289

RESUMO

Complete IgHC gene rearrangement occurs only in B cells in a stage-specific and ordered manner. We used gene targeting to reposition a distal V(H) gene segment to a region just 5' of the D(H) gene cluster and found its activation to be highly dependent on the chromosomal domain within which it resides. The targeted V(H) gene segment rearranged at a higher frequency than its endogenous counterpart, its rearrangement was no longer ordered, and its ability to be silenced by allelic exclusion was lost. Additionally, the targeted V(H) gene segment lost lineage specificity, as VDJ(H) rearrangement was observed in thymocytes. These data suggest that locus contraction, mimicked by proximal targeting, can override any regulation imposed by DNA sequences immediately surrounding V(H) gene segments.


Assuntos
Rearranjo Gênico , Genes de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , VDJ Recombinases/genética , Alelos , Animais , Linfócitos B/metabolismo , Linhagem da Célula , Mapeamento Cromossômico , Cromossomos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Transdução de Sinais , Timo/metabolismo
8.
Nat Immunol ; 7(1): 103-12, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16299505

RESUMO

Major histocompatibility complex (MHC) class I molecules present thousands of peptides to allow CD8(+) T cells to detect abnormal intracellular proteins. The antigen-processing pathway for generating peptides begins in the cytoplasm, and the MHC molecules are loaded in the endoplasmic reticulum. However, the nature of peptide pool in the endoplasmic reticulum and the proteolytic events that occur in this compartment are unclear. We addressed these issues by generating mice lacking the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP). We found that loss of ERAAP disrupted the generation of naturally processed peptides in the endoplasmic reticulum, decreased the stability of peptide-MHC class I complexes and diminished CD8(+) T cell responses. Thus, trimming of antigenic peptides by ERAAP in the endoplasmic reticulum is essential for the generation of the normal repertoire of processed peptides.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Leucil Aminopeptidase/imunologia , Leucil Aminopeptidase/metabolismo , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Southern Blotting , Linfócitos T CD8-Positivos/imunologia , Cromatografia Líquida de Alta Pressão , Citometria de Fluxo , Leucil Aminopeptidase/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Transfecção
9.
Immunity ; 21(6): 877-89, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15589175

RESUMO

TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R(-/-) mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R(-/-) mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-alpha, and IFN-gamma. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R(-/-) cells. The immediate-early TLR signaling events in TRAIL-R(-/-) macrophages and dendritic cells are normal, but I kappa B-alpha homeostatic regulation and NF-kappa B activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.


Assuntos
Imunidade Inata/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Citocinas/biossíntese , Citocinas/metabolismo , Deleção de Genes , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Muromegalovirus/imunologia , Mycobacterium bovis/imunologia , Receptores de Superfície Celular/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Ligante Indutor de Apoptose Relacionado a TNF , Fatores de Tempo , Receptor 2 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/metabolismo
10.
Cell ; 118(1): 19-29, 2004 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-15242641

RESUMO

Regulated gene rearrangement is thought to underlie allelic exclusion, the observation that an individual B cell expresses only a single immunoglobulin molecule. Previous data has implicated transcriptional activation of rearranging loci in the regulation of their accessibility to the V(D)J recombinase. Using homologous recombination in ES cells, we have generated "knockin" mice which express a GFP cDNA from an unrearranged immunoglobulin kappa light-chain allele. Surprisingly, we find that only a small fraction of kappa alleles are highly transcribed in a population of pre-B cells, that such transcription is monoallelic, and that these highly transcribed alleles account for the vast majority of kappa light-chain gene rearrangement. These data lead us to suggest that probabilistic enhancer activation and allelic competition are part of the mechanism of kappa locus allelic exclusion and may be a general mechanism contributing to cellular differentiation during development.


Assuntos
Alelos , Linfócitos B/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/genética , Células-Tronco/imunologia , Ativação Transcricional , Animais , Linfócitos B/citologia , Biomarcadores , Células da Medula Óssea/imunologia , Linhagem da Célula , Células Cultivadas , Elementos Facilitadores Genéticos , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Frequência do Gene , Rearranjo Gênico de Cadeia Leve de Linfócito B/imunologia , Genes de Imunoglobulinas , Genes Reporter , Proteínas de Fluorescência Verde , Heterozigoto , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Mutantes , Regiões Promotoras Genéticas , Recombinação Genética , Baço/citologia
11.
Proc Natl Acad Sci U S A ; 101(7): 2052-7, 2004 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-14769931

RESUMO

Mutations in the human DDB2 gene give rise to xeroderma pigmentosum group E, a disease characterized by increased skin tumorigenesis in response to UV-irradiation. Cell strains derived from xeroderma pigmentosum group E individuals also have enhanced resistance to UV-irradiation due to decreased p53-mediated apoptosis. To further address the precise function(s) of DDB2 and the consequence of non-naturally occurring DDB2 mutations, we generated mice with a disruption of the gene. The mice exhibited significantly enhanced skin carcinogenesis in response to UV-irradiation, and cells from the DDB2(-/-) mice were abnormally resistant to killing by the radiation and had diminished UV-induced, p53-mediated apoptosis. Notably, the cancer-prone phenotype and the resistance to cellular killing were not observed after exposure to the chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), to which mice carrying defective nucleotide excision repair genes respond with enhanced tumors and cell killing. Although cells from heterozygous DDB2(+/-) mice appeared normal, these mice had enhanced skin carcinogenesis after UV-irradiation, so that XP-E heterozygotes might be at risk for carcinogenesis. In sum, these results demonstrate that DDB2 is well conserved between humans and mice and functions as a tumor suppressor, at least in part, by controlling p53-mediated apoptosis after UV-irradiation.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinógenos/farmacologia , Proteínas de Ligação a DNA/deficiência , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Raios Ultravioleta , 9,10-Dimetil-1,2-benzantraceno/farmacologia , Animais , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/efeitos da radiação , Proteínas de Ligação a DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Deleção de Genes , Camundongos , Proteína Supressora de Tumor p53/metabolismo
12.
J Immunol ; 172(2): 1074-82, 2004 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-14707081

RESUMO

Several gene families are known in which member genes are expressed in variegated patterns in differentiated cell types. Mechanisms responsible for imposition of a variegated pattern of gene expression are unknown. Members of the closely linked Ly49 inhibitory receptor gene family are expressed in a variegated fashion by NK cells. Variegated expression of these genes results in subsets of NK cells that differ in specificity for MHC class I molecules. To address the mechanisms underlying variegation, a 30-kb genomic fragment containing a single Ly49 gene was used to generate a panel of murine transgenic lines. The results demonstrated that, in almost all of the lines, the isolated Ly49A gene was expressed in a variegated pattern, remarkably similar in nearly all respects to the expression pattern of the endogenous Ly49A gene. Furthermore, the developmental timing of gene expression and regulation by host MHC molecules closely mirrored that of the endogenous Ly49A gene. Therefore, Ly49 variegation does not require competition in cis between different Ly49 genes, and the sequences imposing variegation are located proximally to Ly49 genes. Efforts to define regulatory elements of the Ly49A gene led to the identification of a DNase I hypersensitive site 4.5 kb upstream of the Ly49A gene transcription initiation site, which was shown to be essential for transgene expression. Highly related sequence elements were found upstream of other Ly49 genes, suggesting that a similar regulatory element controls each Ly49 gene.


Assuntos
Antígenos Ly/biossíntese , Antígenos Ly/genética , Regulação da Expressão Gênica/imunologia , Sequências Reguladoras de Ácido Nucleico/imunologia , Transgenes/imunologia , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/metabolismo , Animais , Animais Recém-Nascidos , Sequência Conservada/imunologia , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Variação Genética/imunologia , Imunidade Celular/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Família Multigênica/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Regiões Promotoras Genéticas/imunologia , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores Semelhantes a Lectina de Células NK
13.
J Biol Chem ; 278(47): 47104-9, 2003 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-13129926

RESUMO

Nur77 and its family members, Nor-1 and Nurr1, are orphan steroid receptors implicated in a wide variety of biological processes, including apoptosis and dopamine neuron agenesis. Expression of these family members can be detected at low levels in many tissues but they are expressed at very high levels when cells are stimulated by outside signals, including serum, nerve growth factor, and receptor engagement. Introduction of a dominant negative Nur77 protein that blocks the activities of all family members led to inhibition of apoptosis in T cells. Nur77-deficient mice, however, exhibit no phenotype, and a line of Nor-1 mutant mice was reported to exhibit a mild ear development phenotype but no other gross abnormalities. Here, we report the generation of Nor-1-deficient mice with a block in early embryonic development. Nor-1 is expressed early during embryogenesis, and its loss leads to embryonic lethality around embryonic day 8.5 of gestation. The mutant embryos fail to complete gastrulation and display distinct morphological abnormalities, including a decrease in overall size, developmental delay and an accumulation of mesoderm in the primitive streak during gastrulation. Abnormal expression of a number of early developmental markers and defects in growth or distribution of emerging mesoderm cells were also detected. These data suggest that Nor-1 plays a crucial role in gastrulation.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas do Tecido Nervoso , Proteínas Nucleares/fisiologia , Animais , Biomarcadores/análise , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Perda do Embrião , Embrião de Mamíferos , Desenvolvimento Embrionário e Fetal , Gástrula , Hibridização In Situ , Mesoderma , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Fenótipo , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides/fisiologia , Receptores dos Hormônios Tireóideos , Fatores de Transcrição/fisiologia
14.
Immunity ; 19(1): 105-17, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12871643

RESUMO

Although expression of the RAG1 and RAG2 genes is essential for lymphocyte development, the mechanisms responsible for the lymphoid- and developmental stage-specific regulation of these genes are poorly understood. We have identified a novel, evolutionarily conserved transcriptional enhancer in the RAG locus, called Erag, which was essential for the expression of a chromosomal reporter gene driven by either RAG promoter. Targeted deletion of Erag in the mouse germline results in a partial block in B cell development associated with deficient V(D)J recombination, whereas T cell development appears unaffected. We found that E2A transcription factors bind to Erag in vivo and can transactivate Erag-dependent reporter constructs in cotransfected cell lines. These findings lead us to conclude that RAG transcription is regulated by distinct elements in developing B and T cells and that Erag is required for optimal levels of RAG expression in early B cell precursors but not in T cells.


Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica , Genes RAG-1 , Animais , Linfócitos B/citologia , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Linhagem Celular , DNA Nucleotidiltransferases/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos B/fisiologia , Recombinases , Fatores de Transcrição/metabolismo , Transfecção
15.
Immunity ; 18(4): 513-21, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12705854

RESUMO

FADD is an adaptor known to transmit apoptotic signals from members of the tumor necrosis factor receptor family. We show here that FADD has a domain implicated in cell proliferation. Mice bearing the Asp mutation in the serine 191 phosphorylation site are runted and anemic and display splenomegaly. Apoptosis is unimpaired in these mice, but they exhibit many immune developmental problems indicative of proliferative defects. Mutant FADD T cells are defective in cell cycle progression, suggesting that regulation of phosphorylation at serine 191 is essential for growth/proliferation. Remarkably, serine 191 is conserved among mammalian FADD proteins, but this C-terminal region is absent in lower organisms, suggesting that FADD acquired a domain during evolution, rendering it a "proliferation-apoptosis coupler" that balances cell proliferation and apoptosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/fisiologia , Sequência de Aminoácidos , Anemia/etiologia , Animais , Apoptose , Proteínas de Transporte/química , Ciclo Celular , Divisão Celular , Proteína de Domínio de Morte Associada a Fas , Camundongos , Dados de Sequência Molecular , Esplenomegalia/etiologia , Relação Estrutura-Atividade , Linfócitos T/fisiologia
16.
Immunity ; 17(5): 639-51, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12433370

RESUMO

Previous in vitro studies defined the minimal regions of RAG1 and RAG2 essential for V(D)J recombination. In order to characterize the role of the C-terminal "dispensable" portion of RAG2, we generated core-RAG2 knock-in mice. We found that the core-RAG2-containing recombinase complex is selectively defective in catalyzing V-to-DJ rearrangement at the IgH and TCRbeta loci, resulting in partial developmental blocks in B and T lymphopoiesis. Analysis of recombination intermediates showed defects at the cleavage phase of the reaction. We also observed a reduction in overall recombinase activity in core-RAG2-expressing thymocytes, leading us to suggest that the interaction of a defective recombinase with RSS sequences unique to VH and Vbeta gene segments may underlie the specific V-to-DJ rearrangement defect in core-RAG2 mice.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/genética , Recombinação Genética/imunologia , Linfócitos T/imunologia , Animais , Diferenciação Celular/genética , Linhagem Celular , DNA Nucleotidiltransferases/imunologia , Proteínas de Ligação a DNA/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/imunologia , Camundongos , VDJ Recombinases
17.
J Biol Chem ; 277(45): 43344-51, 2002 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-12221099

RESUMO

During angiogenesis, endothelial cells undergo proliferation, reorganization, and stabilization to establish a mature vascular network. This process is critical for establishing a functional circulatory system during development and contributes to the pathological process of tumor growth. Here we report that embryos deficient for the ERK5 MAPK die between embryonic days 10.5 and 11.5 with angiogenic failure and cardiovascular defects. We show that ERK5 deficiency leads to an increased expression of the vascular endothelial growth factor (VEGF), dysregulation of which has been shown to impede angiogenic remodeling and vascular stabilization. Our data also reveal that ERK5 negatively regulates transcription from the vegf locus during hypoxic responses. Importantly, ERK5 is required at an earlier developmental stage than p38alpha, and p38alpha does not compensate for ERK5 deficiency. These results demonstrate that ERK5 plays a specific role in the regulation of early angiogenesis.


Assuntos
Fatores de Crescimento Endotelial/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linfocinas/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas Repressoras/metabolismo , Animais , Hipóxia Celular , Fatores de Crescimento Endotelial/antagonistas & inibidores , Éxons , Feminino , Morte Fetal , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Íntrons , Linfocinas/antagonistas & inibidores , Camundongos , Camundongos Knockout , Proteína Quinase 7 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/deficiência , Proteínas Quinases Ativadas por Mitógeno/genética , Gravidez , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Saco Vitelino/irrigação sanguínea , Saco Vitelino/patologia
18.
Proc Natl Acad Sci U S A ; 99(2): 868-73, 2002 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-11782535

RESUMO

Natural killer (NK) cells are believed to achieve self-tolerance through the expression of self-MHC-specific inhibitory receptors, such as members of the Ly49 and CD94/NKG2 families. Individual Ly49 genes are stochastically expressed by NK subsets and are expressed in a monoallelic fashion, but little is known about the mechanisms underlying CD94/NKG2A expression. We show here that, like Ly49 genes, mouse Nkg2a is stochastically and monoallelically expressed. Thus, a single general mechanism controls expression of all known MHC-specific receptors by mouse NK cells. In addition, we find that DBA/2J mice are naturally CD94-deficient and do not express cell-surface CD94/NKG2A receptors, even on neonatal NK cells. Thus, self-tolerance of neonatal NK cells cannot be attributed to CD94/NKG2A expression. Taken together, the results lead to a reconsideration of current models of NK cell development and self-tolerance.


Assuntos
Antígenos Ly , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Glicoproteínas de Membrana/deficiência , Receptores Imunológicos/genética , Alelos , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos CD/genética , Diferenciação Celular , Expressão Gênica , Células Matadoras Naturais/citologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Modelos Imunológicos , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Receptores Semelhantes a Lectina de Células NK , Receptores de Células Matadoras Naturais , Tolerância a Antígenos Próprios , Subpopulações de Linfócitos T/imunologia , Timo/imunologia
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