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1.
Oncogenesis ; 1: e28, 2012 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-23552841

RESUMO

Transformation by Simian Virus 40 (SV40) large T antigen (LT) is mediated in large part by its interaction with a variety of cellular proteins at distinct binding domains within LT. While the interaction of LT's N-terminus with the tumor suppressor Rb is absolutely required for LT-dependent transformation, the requirement for the interaction of LT's C-terminus with p53 is less clear and cell- and context-dependent. Here, we report a line of transgenic mice expressing a doxycycline-inducible liver-specific viral transcript that produces abundant 17kT, a naturally occurring SV40 early product that is co-linear with LT for the first 131 amino acids and that binds to Rb, but not p53. Comparative analysis of livers of transgenic mice expressing either 17kT or full length LT demonstrates that 17kT stimulates cell proliferation and induces hepatic hyperplasia but is incapable of inducing hepatic dysplasia or promoting hepatocarcinogenesis. Gene expression profiling demonstrates that 17kT and LT invoke a set of shared molecular signatures consistent with the action of LT's N-terminus on Rb-E2F-mediated control of hepatocyte transcription. However, 17kT also induces a unique set of genes, many of which are known transcriptional targets of p53, while LT actively suppresses them. LT also uniquely deregulates the expression of a subset of genes within the imprinted network and rapidly re-programs hepatocyte gene expression to a more fetal-like state. Finally, we provide evidence that the LT/p53 complex provides a gain-of-function for LT-dependent transformation in the liver, and confirm the absolute requirement for LT's C-terminus for liver tumor development by demonstrating that phosphatase and tensin homolog (PTEN)-deficiency readily cooperates with LT, but not 17kT, for tumorigenesis. These results confirm independent and inter-dependent functions for LT's N- and C-terminus and emphasize differences in the requirements for LT's C-terminus in cell-type dependent transformation.

2.
Oncogene ; 27(49): 6334-46, 2008 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-18663356

RESUMO

Viruses of the DNA tumor virus family share the ability to transform vertebrate cells through the action of virus-encoded tumor antigens that interfere with normal cell physiology. They accomplish this very efficiently by inhibiting endogenous tumor suppressor proteins that control cell proliferation and apoptosis. Simian virus 40 (SV40) encodes two oncoproteins, large tumor antigen, which directly inhibits the tumor suppressors p53 and Rb, and small tumor antigen (ST), which interferes with serine/threonine protein phosphatase 2A (PP2A). We have constructed a Drosophila model for SV40 ST expression and show that ST induces supernumerary centrosomes, an activity we also demonstrate in human cells. In early Drosophila embryos, ST also caused increased microtubule stability, chromosome segregation errors, defective assembly of actin into cleavage furrows, cleavage failure, a rise in cyclin E levels and embryonic lethality. Using ST mutants and genetic interaction experiments between ST and PP2A subunit mutations, we show that all of these phenotypes are dependent on ST's interaction with PP2A. These analyses demonstrate the validity and utility of Drosophila as a model for viral oncoprotein function in vivo.


Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Centrossomo/metabolismo , Citoesqueleto/metabolismo , Drosophila/metabolismo , Proteína Fosfatase 2/metabolismo , Vírus 40 dos Símios/imunologia , Animais , Animais Geneticamente Modificados , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Linhagem Celular , Centrossomo/imunologia , Citoesqueleto/genética , Citoesqueleto/imunologia , Drosophila/embriologia , Drosophila/virologia , Embrião não Mamífero , Técnica Indireta de Fluorescência para Anticorpo , Glutationa Transferase/química , Glutationa Transferase/imunologia , Glutationa Transferase/metabolismo , Heterozigoto , Imuno-Histoquímica , Mutação , Proteína Fosfatase 2/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/metabolismo
3.
Proc Natl Acad Sci U S A ; 98(24): 13566-71, 2001 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-11717424

RESUMO

PPT1 and PPT2 encode two lysosomal thioesterases that catalyze the hydrolysis of long chain fatty acyl CoAs. In addition to this function, PPT1 (palmitoyl-protein thioesterase 1) hydrolyzes fatty acids from modified cysteine residues in proteins that are undergoing degradation in the lysosome. PPT1 deficiency in humans causes a neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (also known as infantile Batten disease). In the current work, we engineered disruptions in the PPT1 and PPT2 genes to create "knockout" mice that were deficient in either enzyme. Both lines of mice were viable and fertile. However, both lines developed spasticity (a "clasping" phenotype) at a median age of 21 wk and 29 wk, respectively. Motor abnormalities progressed in the PPT1 knockout mice, leading to death by 10 mo of age. In contrast, the majority of PPT2 mice were alive at 12 mo. Myoclonic jerking and seizures were prominent in the PPT1 mice. Autofluorescent storage material was striking throughout the brains of both strains of mice. Neuronal loss and apoptosis were particularly prominent in PPT1-deficient brains. These studies provide a mouse model for infantile neuronal ceroid lipofuscinosis and further suggest that PPT2 serves a role in the brain that is not carried out by PPT1.


Assuntos
Lipofuscinoses Ceroides Neuronais/enzimologia , Tioléster Hidrolases/fisiologia , Animais , Feminino , Marcação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Lipofuscinoses Ceroides Neuronais/patologia , Fenótipo , Tioléster Hidrolases/genética
4.
Proc Natl Acad Sci U S A ; 98(24): 13607-12, 2001 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-11717426

RESUMO

Site-1 protease (S1P) cleaves membrane-bound sterol regulatory element-binding proteins (SREBPs), allowing their transcription-stimulating domains to translocate to the nucleus where they activate genes governing lipid synthesis. S1P is a potential target for lipid-lowering drugs, but the effect of S1P blockade in animals is unknown. Here, we disrupt the S1P gene in mice. Homozygous germ-line disruptions of S1P were embryonically lethal. To disrupt the gene inducibly in liver, we generated mice homozygous for a floxed S1P allele and heterozygous for a transgene encoding Cre recombinase under control of the IFN-inducible MX1 promoter. When IFN was produced, 70-90% of S1P alleles in liver were inactivated, and S1P mRNA and protein were reduced. Nuclear SREBPs declined, as did mRNAs for SREBP target genes. Cholesterol and fatty acid biosynthesis in hepatocytes declined by 75%. Low density lipoprotein (LDL) receptor mRNA declined by 50%, as did the clearance of (125)I-labeled LDL from plasma, but plasma cholesterol fell, suggesting that LDL production was reduced. These data raise the possibility that S1P inhibitors may be effective lipid-lowering agents, but they suggest that nearly complete inhibition will be required.


Assuntos
Lipídeos/biossíntese , Fígado/metabolismo , Pró-Proteína Convertases , Serina Endopeptidases/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Colesterol/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/biossíntese , Marcação de Genes , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas LDL/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Serina Endopeptidases/genética , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
J Biol Chem ; 276(47): 44018-26, 2001 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-11562365

RESUMO

The 100-base pair ELA1 transcriptional enhancer drives high level transcription to pancreatic acinar cells of transgenic mice and in transfected pancreatic acinar cells in culture. The A element within the enhancer is the sole positively acting element for acinar specificity. We show that the acinar cell-specific bHLH protein PTF1-P48 and the common bHLH cofactor HEB are part of the PTF1 complex that binds the A element and mediates its activity. Acinar-like activity of the enhancer can be reconstituted in HeLa cells by the introduction of P48, HEB, and the PDX1-containing trimeric homeodomain complex that binds the second pancreatic element of the enhancer. The 5' region of the mouse Ptf1-p48 gene from -12.5 to +0.2 kilobase pairs contains the regulatory information to direct expression in transgenic mice to the pancreas and other organs of the gut that express the endogenous Ptf1-p48 gene. The 5'-flanking sequence contains two activating regions, one of which is specific for acinar cells, and a repressing domain active in non-pancreatic cells. Comparison of the 5'-gene flanking regions of the mouse, rat, and human genes identified conserved sequence blocks containing binding sites for known gut transcription factors within the acinar cell-specific control region.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Pâncreas/metabolismo , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Primers do DNA , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Pâncreas/citologia , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/fisiologia
6.
Neuroscience ; 106(2): 263-74, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11566499

RESUMO

Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1. We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.


Assuntos
Apoptose/genética , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso , Sistema Nervoso/embriologia , Neurônios/metabolismo , Neurotransmissores/metabolismo , Proteínas/metabolismo , Células-Tronco/metabolismo , Proteínas de Transporte Vesicular , Animais , Fator Apoptótico 1 Ativador de Proteases , Caspases/metabolismo , Ciclo Celular/genética , Diferenciação Celular/genética , Grupo dos Citocromos c/metabolismo , Gânglios Sensitivos/citologia , Gânglios Sensitivos/embriologia , Gânglios Sensitivos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Munc18 , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Neurônios/citologia , Proteínas/genética , Receptor trkA/deficiência , Receptor trkA/genética , Transdução de Sinais/genética , Células-Tronco/citologia
7.
Science ; 293(5537): 2084-7, 2001 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-11557891

RESUMO

Transcription factor TFIID, composed of TBP and TAFII subunits, is a central component of the RNA polymerase II machinery. Here, we report that the tissue-selective TAFII105 subunit of TFIID is essential for proper development and function of the mouse ovary. Female mice lacking TAFII105 are viable but infertile because of a defect in folliculogenesis correlating with restricted expression of TAFII105 in the granulosa cells of the ovarian follicle. Gene expression profiling has uncovered a defective inhibin-activin signaling pathway in TAFII105-deficient ovaries. Together, these studies suggest that TAFII105 mediates the transcription of a subset of genes required for proper folliculogenesis in the ovary and establishes TAFII105 as a cell type-specific component of the mammalian transcriptional machinery.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células da Granulosa/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Ovário/fisiologia , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células da Granulosa/metabolismo , Hibridização In Situ , Infertilidade Feminina , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Tamanho do Órgão , Especificidade de Órgãos , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Ovulação , Subunidades Proteicas , Transdução de Sinais , Fator de Transcrição TFIID , Fatores de Transcrição/genética , Fatores de Transcrição TFII/metabolismo
8.
Cancer Res ; 61(14): 5552-7, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11454706

RESUMO

Apurinic/apyrimidinic endonuclease is a key enzyme in the process of base excision repair, required for the repair of spontaneous base damage that arises as a result of oxidative damage to DNA. In mice, this endonuclease is coded by the Apex gene, disruption of which is incompatible with embryonic life. Here we confirm the embryonic lethality of Apex-null mice and report the phenotypic characterization of mice that are heterozygous mutants for the Apex gene (Apex+/-). We show that Apex heterozygous mutant cells and animals are abnormally sensitive to increased oxidative stress. Additionally, such animals manifest elevated levels of oxidative stress markers in serum, and we show that dietary supplementation with antioxidants restores these to normal levels. Apex+/- embryos and pups manifest reduced survival that can also be partially rescued by dietary supplementation with antioxidants. These results are consistent with a proposed role for this enzyme in protection against the deleterious effects of oxidative stress and raise the possibility that humans with heterozygous mutations in the homologous HAP1 gene may be at increased risk for the phenotypic consequences of oxidative stress in cells.


Assuntos
Carbono-Oxigênio Liases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Heterozigoto , Estresse Oxidativo/genética , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/patologia , Animais , Ácido Ascórbico/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Suplementos Nutricionais , Dinoprosta/sangue , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Genótipo , Peróxidos Lipídicos/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linfoma/genética , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Paraquat/farmacologia , Fenótipo , Vitamina E/administração & dosagem , Vitamina K/farmacologia
9.
Genes Dev ; 15(10): 1206-16, 2001 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-11358865

RESUMO

In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting-refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Colesterol/deficiência , Proteínas de Ligação a DNA/metabolismo , Insulina/metabolismo , Lipídeos/biossíntese , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição , Proteínas Virais , Animais , Northern Blotting , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Immunoblotting , Integrases/genética , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Recombinação Genética , Proteína de Ligação a Elemento Regulador de Esterol 1
10.
Dev Biol ; 230(2): 230-42, 2001 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11161575

RESUMO

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.


Assuntos
Desenvolvimento Embrionário e Fetal , Endotélio Vascular/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Integrases/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas Virais , Animais , Endocárdio/embriologia , Elementos Facilitadores Genéticos , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Regiões Promotoras Genéticas , Receptor TIE-2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Galactosidase/análise , beta-Galactosidase/genética
11.
Mol Cell ; 6(1): 77-86, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10949029

RESUMO

In mice with too little fat (lipodystrophy) or too much fat (ob/ob), leptin deficiency leads to hyperglycemia, hyperinsulinemia, and insulin resistance. In both disorders, the liver overproduces glucose as a result of resistance to the normal action of insulin in repressing mRNAs for gluconeogenic enzymes. Here we show that chronic hyperinsulinemia downregulates the mRNA for IRS-2, an essential component of the insulin-signaling pathway in liver, thereby producing insulin resistance. Despite IRS-2 deficiency, insulin continues to stimulate production of SREBP-1c, a transcription factor that activates fatty acid synthesis. The combination of insulin resistance (inappropriate gluconeogenesis) and insulin sensitivity (elevated lipogenesis) establishes a vicious cycle that aggravates hyperinsulinemia and insulin resistance in lipodystrophic and ob/ob mice.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/metabolismo , Resistência à Insulina/fisiologia , Lipodistrofia/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Obesidade/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição , Animais , Sequência de Bases , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/efeitos dos fármacos , Glucagon/farmacologia , Humanos , Técnicas In Vitro , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Lipodistrofia/genética , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Modelos Biológicos , Proteínas Nucleares/genética , Obesidade/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína de Ligação a Elemento Regulador de Esterol 1
12.
J Clin Invest ; 105(10): 1373-82, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10811845

RESUMO

Endothelin-converting enzyme-1 and -2 (ECE-1 and -2) are membrane-bound metalloproteases that can cleave biologically the inactive endothelin-1 (ET-1) precursor to form active ET-1 in vitro. We previously reported developmental defects in specific subsets of neural crest-derived tissues, including branchial arch-derived craniofacial structures, aortic arch arteries, and the cardiac outflow tract in ECE-1 knockout mice. To examine the role of ECE-2 in cardiovascular development, we have now generated a null mutation in ECE-2 by homologous recombination. ECE-2 null mice develop normally, are healthy into adulthood, are fertile in both sexes, and live a normal life span. However, when they are bred into an ECE-1-null background, defects in cardiac outflow structures become more severe than those in ECE-1 single knockout embryos. In addition, ECE-1(-/-); ECE-2(-/-) double null embryos exhibited abnormal atrioventricular valve formation, a phenotype never seen in ECE-1 single knockout embryos. In the developing mouse heart, ECE-2 mRNA is expressed in the endocardial cushion mesenchyme from embyronic day (E) 12.5, in contrast to the endocardial expression of ECE-1. Levels of mature ET-1 and ET-2 in whole ECE-1(-/-); ECE-2(-/-) embryos at E12.5 do not differ appreciably from those of ECE-1(-/-) embryos. The significant residual ET-1/ET-2 in the ECE-1(-/-); ECE-2(-/-) embryos indicates that proteases distinct from ECE-1 and ECE-2 can carry out ET-1 activation in vivo.


Assuntos
Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/fisiologia , Coração Fetal/embriologia , Coração Fetal/enzimologia , Metaloendopeptidases/genética , Metaloendopeptidases/fisiologia , Animais , Sequência de Bases , Primers do DNA/genética , Endotelina-1/metabolismo , Endotelina-2/metabolismo , Enzimas Conversoras de Endotelina , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Cardiopatias Congênitas/enzimologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Hibridização In Situ , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual
13.
Science ; 287(5454): 864-9, 2000 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-10657302

RESUMO

Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.


Assuntos
Encéfalo/embriologia , Encéfalo/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurotransmissores/metabolismo , Sinapses/fisiologia , Proteínas de Transporte Vesicular , Animais , Apoptose , Encéfalo/citologia , Diferenciação Celular , Divisão Celular , Deleção de Genes , Cones de Crescimento/fisiologia , Camundongos , Camundongos Knockout , Proteínas Munc18 , Degeneração Neural , Proteínas do Tecido Nervoso/genética , Vias Neurais , Junção Neuromuscular/embriologia , Junção Neuromuscular/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Sinapses/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura
14.
15.
Dev Biol ; 218(2): 248-58, 2000 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-10656767

RESUMO

Release of cytochrome c from the mitochondria, and subsequent binding to apoptotic protease-activating factor-1 (Apaf-1), is a key trigger of apoptotic events. A complex composed of Apaf-1, dATP, and cytochrome c activates a series of cytoplasmic proteases called caspases, leading to apoptotic cell death. We have disrupted the Apaf-1 gene in the mouse. Like previous reports on this knockout model, we find that most Apaf-1 mutants die perinatally and frequently exhibit exencephaly and cranioschesis. We additionally find that the neural lesions that develop in the knockout are due to an excess of neural progenitor cells that manifests as early as embryonic day 9.5 in development. In contrast to previous reports on the Apaf-1 knockout mice, we find that 5% of the mutants successfully survive to adulthood. In these survivors, the brain develops normally, but in males, there is degeneration of spermatogonia resulting in the virtual absence of sperm. Thus, cytochrome c-mediated apoptosis is not absolutely required for normal neural development, but is essential for spermatogenesis. These findings strongly suggest that alternative apoptotic pathways work in conjunction with and parallel to Apaf-1 and can modify its effect on programmed cell death.


Assuntos
Infertilidade Masculina/genética , Proteínas/genética , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Fator Apoptótico 1 Ativador de Proteases , Sequência de Bases , Caspase 3 , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Primers do DNA , Ativação Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Proteínas/metabolismo , Espermatozoides/citologia , Espermatozoides/enzimologia , Espermatozoides/metabolismo
16.
J Immunol ; 164(2): 812-24, 2000 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-10623827

RESUMO

The mouse Ig kappa L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon V kappa-J kappa joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream V kappa 2 gene family including their entire 5' and 3' flanking sequences, along with one member of a downstream V kappa 21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Ig kappa miniloci that contained differing lengths of sequences 5' of the most distal V kappa 2 gene family member. In transgenic mice, Ig kappa miniloci exhibited position-independent and copy number-dependent germline transcription. Ig kappa miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal V kappa gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5' region exhibited significantly greater recombination of the upstream V kappa 2 genes but not the downstream V kappa 21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Ig kappa gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.


Assuntos
Cromossomos Artificiais de Levedura/imunologia , Mapeamento de Sequências Contíguas , Rearranjo Gênico de Cadeia Leve de Linfócito B/genética , Cadeias kappa de Imunoglobulina/genética , Transcrição Gênica/imunologia , Transgenes/imunologia , Alelos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cromossomos Artificiais de Levedura/genética , Cruzamentos Genéticos , Dosagem de Genes , Genes de Imunoglobulinas/genética , Marcadores Genéticos/imunologia , Células Germinativas/imunologia , Células Germinativas/metabolismo , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/química , Camundongos , Camundongos Transgênicos , Família Multigênica/imunologia , Reprodutibilidade dos Testes
17.
Neuron ; 24(3): 687-700, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10595519

RESUMO

We have generated mice lacking synaptogyrin I and synaptophysin I to explore the functions of these abundant tyrosine-phosphorylated proteins of synaptic vesicles. Single and double knockout mice were alive and fertile without significant morphological or biochemical changes. Electrophysiological recordings in the hippocampal CA1 region revealed that short-term and long-term synaptic plasticity were severely reduced in the synaptophysin/synaptogyrin double knockout mice. LTP was decreased independent of the induction protocol, suggesting that the defect in LTP was not caused by insufficient induction. Our data show that synaptogyrin I and synaptophysin I perform redundant and essential functions in synaptic plasticity without being required for neurotransmitter release itself.


Assuntos
Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Sinaptofisina/fisiologia , Animais , Encéfalo/patologia , Estimulação Elétrica , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout/genética , Camundongos Knockout/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurotransmissores/metabolismo , Linhagem , Sinaptogirinas , Sinaptofisina/deficiência , Sinaptofisina/genética , Fatores de Tempo
18.
J Biol Chem ; 274(46): 32551-4, 1999 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-10551807

RESUMO

Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTPgammaS in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores.


Assuntos
Proteínas de Transporte/genética , Grânulos Citoplasmáticos/metabolismo , Exocitose/genética , Marcação de Genes , Proteínas de Membrana/genética , Animais , Proteínas de Transporte/metabolismo , Clonagem Molecular , Condutividade Elétrica , Endocitose/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Mastócitos , Fusão de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Proteínas de Transporte Vesicular
20.
Neuron ; 24(2): 377-87, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10571231

RESUMO

Synapsins constitute a family of synaptic vesicle proteins essential for regulating neurotransmitter release. Only two domains are conserved in all synapsins: a short N-terminal A domain with a single phosphorylation site for cAMP-dependent protein kinase (PKA) and CaM Kinase I, and a large central C domain that binds ATP and may be enzymatic. We now demonstrate that synapsin phosphorylation in the A domain, at the only phosphorylation site shared by all synapsins, dissociates synapsins from synaptic vesicles. Furthermore, we show that the A domain binds phospholipids and is inhibited by phosphorylation. Our results suggest a novel mechanism by which proteins reversibly bind to membranes using a phosphorylation-dependent phospholipid-binding domain. The dynamic association of synapsins with synaptic vesicles correlates with their role in activity-dependent plasticity.


Assuntos
Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Sequência de Aminoácidos/genética , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/fisiologia , Exocitose/fisiologia , Camundongos , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Fosforilação , Ratos , Especificidade por Substrato , Sinapsinas/genética
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