Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/fisiologia , Serina Endopeptidases/química , Serina Endopeptidases/fisiologia , Sequência de Aminoácidos , Animais , Ânions , Catálise , Cisteína Endopeptidases/classificação , Humanos , Peptídeos/metabolismo , Conformação Proteica , Serina Endopeptidases/classificação , Relação Estrutura-AtividadeRESUMO
Coculture of mouse bone marrow-derived immature mast cells (BMMC) with Swiss 3T3 fibroblasts in the presence of stem cell factor (SCF) promotes morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype, which is accompanied by increased expression of several unique genes. Here we report the molecular identification of one of them, mast cell maturation-associated inducible gene (MMIG)-1. The MMIG-1 cDNA encodes a 117-kDa cytosolic protein that comprises an N-terminal PYRIN domain, a central nucleotide-binding domain, and nine C-terminal leucine-rich repeats. MMIG-1 shows >85% sequence similarity to human cryopyrin/PYPAF1, a causal gene for familial cold urticaria and Muckle-Wells syndrome. MMIG-1 was distributed in the cytosol of CTMC-like differentiated BMMC. MMIG-1 underwent alternative splicing in the leucine-rich repeats and each variant was induced differently in BMMC during coculture. Moreover, its expression was increased in the ears of mice with experimental atopic dermatitis. Thus, MMIG-1, a likely mouse PYPAF1 ortholog, may play a role in mast cell-directed inflammatory diseases.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Diferenciação Celular , Regulação da Expressão Gênica , Mastócitos/citologia , Mastócitos/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Técnicas de Cocultura , DNA Complementar/genética , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/genética , Dinitrofluorbenzeno/farmacologia , Fibroblastos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Coculture of mouse bone marrow-derived mast cells (BMMC) with fibroblasts in the presence of stem cell factor (SCF) facilitates morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. By means of cDNA subtraction, we identified several inducible genes during this mast cell maturation process. Of approximately 100 sequenced clones induced, nearly 50% were chromosome 14-associated serine proteases. Approximately 14% encoded NDRG1, a 43-kDa cytosolic protein that has been implicated in cell differentiation. NDRG1 was distributed in the cytosol of cultured mast cells and CTMC in rat skin. Overexpression of NDRG1 in RBL-2H3 cells resulted in enhanced degranulation in response to various stimuli. Thus, NDRG1 may be a mast cell maturation-associated inducible protein that allows the cells to be susceptible to extracellular stimuli leading to degranulation. Additionally, several unique maturation-associated inducible genes were identified, molecular and functional characterization of which will provide new insights into mast cell biology.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Mastócitos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Citosol/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Imuno-Histoquímica , Inflamação , Insetos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Ratos , TransfecçãoRESUMO
We prepared sigma(1)-receptor selective 1-([4-methoxy-(11)C]-3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine ([(11)C]SA4503) and its fluorinated analog 1-([4-methoxy-(11)C]3,4-dimethoxyphenethyl)-4-[3-(4-fluorophenyl)propyl]piperazine ([(11)C]SA5845), and their [(11)C]ethoxy and [(18)F]fluoroethoxy analogs, and evaluated their potential for positron emission tomography studies. [(11)C]SA4503 is most selective for sigma(1) receptors, and the other five showed affinities for sigma(1) and sigma(2) receptors with a different extent. All radioligands showed the receptor-specific binding in the brain, and visualized similar regional brain distributions by ex vivo autoradiography. The [(11)C]ethoxy analogs were relatively labile for metabolism.
Assuntos
Nootrópicos/síntese química , Piperazinas/síntese química , Animais , Cobaias , Masculino , Camundongos , Nootrópicos/metabolismo , Nootrópicos/farmacocinética , Piperazinas/metabolismo , Piperazinas/farmacocinética , Ratos , Ratos Wistar , Receptores sigma/agonistas , Receptores sigma/metabolismo , Relação Estrutura-Atividade , Distribuição Tecidual , Tomografia Computadorizada de EmissãoRESUMO
Elevation of the vitelline coat of eggs of the ascidian, Halocynthia roretzi, was induced by 12-O-tetradecanoylphorbol-13-acetate or phorbol-12, 13-didecanoate, but not by their 4-epimers. After elevation of the vitelline coat in this way, eggs were unable to undergo cleavage on addition of sperm. This elevation of the vitelline coat by phorbol esters was inhibited by specific inhibitors of trypsin-like enzyme, calmodulin, phospholipase A2 , and protein kinase C. In association with elevation of the vitelline coat, a trypsin-like enzyme was released from the eggs. The properties of this enzyme were similar to those of the enzyme released by calcium ionophore, which also induced elevation of the vitelline coat, suggesting that similar exocytosis of intracellular granules occurred in both cases. Calmodulin, possibly involved in the elevation process, was isolated from eggs and characterized.
