RESUMO
Although peritoneal resident macrophages (PRM) or peritoneal exudate macrophages (PEM) were activated by lipopolysaccharide (LPS) to kill tumor cells in vitro, lung macrophages (LM) obtained by mincing lung tissues or by harvesting bronchial lavage were not activated by LPS under any experimental conditions, i.e., different LPS concentrations, incubation times and cytotoxicity assay methods. The unresponsiveness of LM to LPS was seen in all of the mouse strains tested. Treatment of LM with indomethacin did not affect the unresponsiveness, although it greatly augmented the cytotoxicity of PRM stimulated with LPS. LM treated in vitro with crude lymphokines (LK) did not show cytotoxicity, but became sensitive to LPS and cytotoxic for tumor cells. LM treated first with crude LK and then with LPS were cytotoxic, but LM treated first with LPS and then with crude LK were not. The ability of crude LK to render LM responsive to LPS was neutralized by rabbit anti-mouse gamma interferon (IFN-gamma) antiserum but not by anti-mouse IFN-(alpha + beta) antiserum. LM treated with recombinant murine IFN-gamma became responsive to LPS and showed cytotoxicity. LM were resistant to direct toxicity of LPS under conditions in which significant populations of PRM and PEM died. However, LM became sensitive to direct toxicity of LPS by treatment with crude LK or recombinant murine IFN-gamma. Fluorescence microscopy showed that almost all PRM and PEM were stained with fluorescein isothiocyanate (FITC)-LPS, while less than 5% of the LM were stained. Instead, approximately 60% of the LM treated with the crude LK or recombinant IFN-gamma for 20 h were stained with FITC-LPS. Fluorescence-activated cell sorter (FACS) analysis confirmed this result. The staining of IFN-gamma treated LM with FITC-LPS was inhibited by polymyxin B or unlabeled LPS. These results suggest that the defective responsiveness of LM to LPS is due to the lack or very low expression of LPS-binding sites on the cell surface and that in vitro treatment with IFN-gamma brings about the expression of them and renders LM responsive to LPS.
Assuntos
Interferon gama/farmacologia , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/farmacologia , Animais , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Leucemia Experimental/imunologia , Receptores de Lipopolissacarídeos , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos ICR , Receptores Imunológicos/efeitos dos fármacosRESUMO
The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.
Assuntos
Antígenos de Superfície/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Sistema Respiratório/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/metabolismo , Humanos , Transporte de Íons , Camundongos , Sintaxina 1 , XenopusRESUMO
It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.
Assuntos
Antígenos de Superfície/farmacologia , Produtos do Gene tat/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neurotransmissores/metabolismo , Células PC12/efeitos dos fármacos , Proteínas de Transporte Vesicular , Animais , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Células PC12/metabolismo , Ratos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas SNARE , Sintaxina 1RESUMO
We reported previously that a reorganization of microfilaments can be observed when macrophages are stimulated with lipopolysaccharide (LPS). This reorganization is not detected with current methods in macrophages of an LPS-nonresponsive C3H/HeJ mouse. These results suggest that the observed microfilament response might be involved in a macrophage-activating process induced by LPS. To investigate this, we studied the effect of cytochalasin D (CD), which inhibits reorganization of microfilaments, on LPS-induced tumor necrosis factor (TNF) production. A concentration of CD incapable of affecting filamentous actin by itself was used in these experiments. When this concentration of CD was added after LPS stimulation, microfilament reorganization and TNF production were inhibited. The suppressive effect of CD on TNF production was confirmed by the observation that TNF-alpha mRNA expression was also inhibited by CD. This inhibitory effect of CD was not specific to TNF, because the production of interleukin-1 and prostaglandin E2 were also inhibited. These effects of CD were observed only when CD was added within the first 20 min after LPS stimulation. These results suggest that the CD-sensitive microfilament response is essential in the signaling pathway for the production of certain monokines in LPS-stimulated macrophages.
Assuntos
Citocalasina D/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Actinas/biossíntese , Animais , Northern Blotting , Células Cultivadas , DNA/metabolismo , Dinoprostona/metabolismo , Interleucina-1/metabolismo , Cinética , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Tioglicolatos/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Numerous factors are known to bind human immunodeficiency virus (HIV) long terminal repeat (LTR) and activate viral transcription, but little is known as to how they function in naturally activated T cells and to what extent their binding is relevant to HIV replication in vivo. To characterize the HIV LTR-binding factors responsible for antigen-dependent activation of HIV, we examined replication of LTR mutant viruses in CD4+ T cells activated by different stimuli. NF-kappaB or Sp1 mutant virus replicated well in CD4+ T cells activated by phorbol ester and calcium ionophore. When they were activated by antigen-pulsed dendritic cells, the replication of the Sp1-deleted virus was severely impaired in CD45RA+, but not in CD45RO+ T cell subsets that dominantly produce interleukin-2 (IL-2). Stimulation via CD3/CD28 induced a high level of IL-2 production in both T cell subsets, but Sp1-deleted virus poorly replicated in CD45RA+ subset. The level of NF-kappaB and Sp1-binding factors did not differ between these subsets. Our results suggest that additional cofactors distinct from IL-2-inducing signaling molecules are important for LTR activation during antigen-dependent T cell activation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Ativação Linfocitária/imunologia , Ativação Transcricional , Tuberculina/imunologia , Apresentação de Antígeno/imunologia , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Produtos do Gene tat/genética , Produtos do Gene tat/fisiologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Interleucina-2/biossíntese , Ionomicina/farmacologia , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/efeitos dos fármacos , Mutação/genética , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Elementos de Resposta/genética , Fator de Transcrição Sp1/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Acetato de Tetradecanoilforbol/farmacologia , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The effects of a monosaccharide precursor of Escherichia coli lipid A (lipid X) on murine macrophages were studied. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3. Lipid X, as well as lipopolysaccharide and lipid A, enhanced O2- generation in mouse peritoneal macrophages and a macrophage-like cell line, J774.1, and further induced the tumor-cytotoxic activity of peritoneal macrophages. Elimination of a 1-phosphate or 3-O-beta-hydroxymyristoyl groups are essential for the elevated O2- generation and induction of tumoricidal activity due to lipid X.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Escherichia coli/imunologia , Glicolipídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Superóxidos/metabolismo , Animais , Linhagem Celular , Leucemia Experimental/imunologia , Lipídeo A/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos ICRRESUMO
Susceptibility to HIV infection was examined in macrophages differentiated from human monocytes by macrophage colony-stimulating factor (M-CSF) or granulocyte/macrophage colony-stimulating factor (GM-CSF). The replication of macrophage-tropic human immunodeficiency virus type-1 (HIV-1), which was determined by reverse transcriptase (RT) activity, was significantly suppressed in macrophages induced by GM-CSF (GM-type macrophages) but not in those induced by M-CSF (M-type macrophages). Multinucleated giant cells were formed only in M-type macrophages after HIV infection. However, the expression of CD4 molecules on the surface of both types of macrophages was similar and the proviral DNA was detectable in cell lysates of both macrophages, although the amount of proviral DNA in M-type macrophages was higher than that in GM-type macrophages. Many steps have been defined in HIV infection and replication, such as adsorption of HIV to the cell surface, internalization of the viral core into the cytoplasm, uncoating of viral RNA, reverse transcription and integration of proviral DNA into cellular DNA, transcription and translation of proviral DNA, assembly of viral components, and budding of virus particles. Our findings suggested that the suppression of HIV-1 replication in macrophages induced by GM-CSF is mainly due to a disturbance at certain steps of replication after synthesis of the proviral DNA. Thus, the suppression of HIV replication in GM-type macrophages may provide a model of the latency of HIV infection in vivo.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Replicação Viral/efeitos dos fármacos , Antígenos de Superfície/metabolismo , Sequência de Bases , Antígenos CD4/metabolismo , Diferenciação Celular , Células Cultivadas , Primers do DNA/genética , DNA Viral/genética , DNA Viral/metabolismo , Genes gag , Infecções por HIV/patologia , HIV-1/genética , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Microscopia Eletrônica , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/efeitos dos fármacos , Provírus/efeitos dos fármacos , Provírus/genética , Provírus/fisiologiaRESUMO
We raised polyclonal and monoclonal antibodies against rat recombinant HPC-1/syntaxin 1A lacking a transmembrane domain. The polyclonal antibody recognized two major bands at 35 and 40 kDa from rat brain membranes. A hybridoma clone designated 14D8, however, recognized only one band at 35 kDa. A polyclonal antibody detected recombinant syntaxin 1B, as well as HPC-1/syntaxin 1A on an immunoblot, whereas 14D8 recognized recombinant HPC-1/ syntaxin 1A, but not syntaxin 1B. Therefore, 14D8 is specific for HPC-1/syntaxin 1A. Using this monoclonal antibody, we investigated the expression of HPC-1/syntaxin 1A in the rat hippocampal membranes. HPC-1/syntaxin 1A was present even in the embryonic d 19 (E19) hippocampal membranes, and it increased during the next two postnatal wk. Pyramidal cell axons were intensely stained with the 14D8 monoclonal antibody, suggesting that HPC-1/syntaxin 1A was not restricted to the presynaptic terminal. Furthermore, we investigated the phosphorylation of HPC-1/syntaxin 1A in the rat brain membranes. HPC-1/syntaxin 1A affinity-purified on a 14D8 IgG-coupled column was recognized by antiphosphoserine antibody, but not by antiphosphotyrosine and phosphothreonine antibodies.
Assuntos
Anticorpos Monoclonais , Antígenos de Superfície/imunologia , Imunoglobulina G , Proteínas do Tecido Nervoso/imunologia , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Córtex Cerebral/química , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/química , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células PC12/química , Fosforilação , Coelhos , Ratos , Ratos Endogâmicos , Retina/química , Coloração pela Prata , Sintaxina 1RESUMO
To clarify the pathogenesis of excessive proliferation of extrinsic nerve fibers in the aganglionic colon of patients with Hirschsprung's disease (HD), we immunohistochemically determined the role that exocytosis-related proteins play in the regulation of exocytosis using the antibody to HPC-1/syntaxin 1A, an exocytosis-related protein. Localization of exocytosis-related proteins (HPC-1/syntaxin 1A, N-ethylmalemide-sensitive fusion protein (NSF), soluble NSF attachment protein (SNAP), synaptotagmin, synaptobrevin, and synaptosome-associated protein 25 (SNAP-25)) was determined in surgical specimens obtained from normal proximal and aganglionic distal segments of the colon of 7 infant patients with HD. In the normal ganglionic colon, Auerbach's plexus, Meisner's plexus, nerve fibers in the muscle layer, and ganglion cells were immunopositive for all six kinds of antisera. In the aganglionic segments, numerous proliferated nerve fibers and hypertrophied nerve bundles were detected in the submucosal layer and myenteric layer by NSF, SNAP, synaptotagmin, synaptobrevin, and SNAP-25. However, HPC-1/syntaxin 1A was not recognized in the proliferated nerve fibers of the submucosal layer or the hypertrophied nerve bundles of the aganglionic segment. These findings show that immunoreactivity of HPC-1/syntaxin 1A was decreased in the affected bowel segments of patients with HD and may be related to the pathogenesis of extrinsic nerve-fiber proliferation in the aganglionic colon of HD.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas de Ligação ao Cálcio , Colo/patologia , Sistema Nervoso Entérico/patologia , Doença de Hirschsprung/patologia , Hipertrofia/patologia , Fibras Nervosas/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Vesicular , Antígenos de Superfície/imunologia , Proteínas de Transporte/metabolismo , Colo/inervação , Colo/metabolismo , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/fisiopatologia , Doença de Hirschsprung/metabolismo , Doença de Hirschsprung/fisiopatologia , Humanos , Hipertrofia/metabolismo , Hipertrofia/fisiopatologia , Imuno-Histoquímica , Lactente , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso/inervação , Músculo Liso/metabolismo , Músculo Liso/patologia , Plexo Mientérico/metabolismo , Plexo Mientérico/patologia , Plexo Mientérico/fisiopatologia , Proteínas Sensíveis a N-Etilmaleimida , Fibras Nervosas/metabolismo , Proteínas do Tecido Nervoso/imunologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas R-SNARE , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Plexo Submucoso/metabolismo , Plexo Submucoso/patologia , Plexo Submucoso/fisiopatologia , Sinaptotagminas , Sintaxina 1RESUMO
Cultivation of human peripheral blood monocytes with granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-4 facilitates generation of strongly antigen-presenting dendritic cells (DC). These monocyte-derived DC (mdDC) were used here to further delineate differentiation pathways in the myeloid lineage. Incubation of mdDC with TNF or soluble CD40L led to enhanced MHC and accessory surface antigen expression with significantly elevated T cell stimulatory activity, indicative of DC maturation. In contrast, after cytokine withdrawal or incubation with M-CSF, mdDC differentiated to macrophages. Cells became adherent, monocyte/macrophage surface markers were upregulated, and MHC and accessory surface proteins were downregulated. Furthermore, the multilaminar MHC class II compartments (MIIC) were lost and the T cell stimulating capacity largely diminished. Thus, mdDC show a high developmental plasticity by retaining their ability to become macrophages or to continue their differentiation towards mature DC.
Assuntos
Células Dendríticas/citologia , Monócitos/efeitos dos fármacos , Apresentação de Antígeno , Antígenos CD/biossíntese , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40 , Adesão Celular , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Antígenos HLA/biossíntese , Humanos , Células de Langerhans/imunologia , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Glicoproteínas de Membrana/farmacologia , Monócitos/citologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The monoclonal antibody HPC-1 recognizes a protein antigen in the hippocampus, and its specific reactivity to the plasma membrane of the amacrine cell somas and the inner plexiform layer in rat retina has been reported. Sequencing the cDNA indicated in our previous study that the HPC-1 antigen was a membrane protein. By means of immunoblotting, an antiserum against the fusion protein of Escherichia coli beta-galactosidase and the HPC-1 antigen detected several proteins of about 35 kDa in the nervous tissues including retina, cerebral cortex, hippocampus, cerebellum and spinal cord, but no signal was obtained in the non-neuronal tissues. Immunofluorescent histochemistry of the various rat tissues revealed that the HPC-1 antigen was confined to the nervous system, including the matrices of the cerebral cortex and hippocampus, the molecular layer, membranes of granular cell somas and glomeruli in the cerebellum and gray matter of spinal cord. However, little staining was seen in the white matter of the central nervous tissues. Thus, the HPC-1 antigen was accumulated in the synapse-rich regions of neuronal cells. In situ hybridization revealed that the HPC-1 mRNA was present in most, if not all, neurons in the central and peripheral nervous systems except for the retina. In the retina, mRNA signals were detected in amacrine and ganglion cells in which HPC-1 immunoreactivity was absent in their soma, suggesting polarized localization of the HPC-1 mRNA on the ganglion cell axon terminal.
Assuntos
Antígenos de Superfície/biossíntese , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , RNA Mensageiro/biossíntese , Retina/metabolismo , Medula Espinal/metabolismo , Animais , Anticorpos Monoclonais , Antígenos de Superfície/análise , Elementos Antissenso (Genética) , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Digoxigenina , Escherichia coli/enzimologia , Expressão Gênica , Hipocampo/metabolismo , Soros Imunes , Imuno-Histoquímica , Hibridização In Situ , Proteínas do Tecido Nervoso/análise , Especificidade de Órgãos , Sondas RNA , RNA Mensageiro/análise , Ratos , Proteínas Recombinantes de Fusão/análise , Sintaxina 1 , beta-Galactosidase/análiseRESUMO
HPC-1/syntaxin 1A (HPC-1), which plays an important role in vesicular transport to the plasma membrane, possesses a hydrophobic sequence at its C terminus. When expressed from cDNA in COS cells, wild-type HPC-1 was localized in the Golgi complex and the plasma membrane. Truncation of the hydrophobic domain resulted in the cytoplasmic localization of the mutant, thus indicating that the domain indeed functions as a membrane anchor. A fusion protein with the C-terminal glycosylation sites was glycosylated in transfected cells, providing evidence that HPC-1 has a transmembrane structure, and that the protein is first inserted into the endoplasmic reticulum and then transported to the plasma membrane. A chimeric protein consisting of Escherichia coli maltose-binding protein with the last 24 amino acids of HPC-1 was inserted into the endoplasmic reticulum in a transmembrane topology and localized along the exocytic pathway of transfected cells similar to HPC-1. These results indicate that the portion is important for intracellular localization of HPC-1.
Assuntos
Antígenos de Superfície/metabolismo , Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Superfície/química , Antígenos de Superfície/fisiologia , Transporte Biológico , Células COS , Retículo Endoplasmático/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Sintaxina 1RESUMO
Human peripheral monocytes cultured with GM-CSF and IL-4 differentiated to dendritic cells (DCs) and with GM-CSF alone to macrophages. Superoxide-generating ability in such DCs was found to be suppressed whereas that in macrophages remained constant. To examine the reason for the suppression in DCs, we evaluated by immunoblotting the levels of essential components of the superoxide generating system in the cells during the differentiation. In contrast to the levels of cytosolic 47- and 65-kDa components and Rac-p21, which remained constant throughout cultivation, those of the large and the small subunits of cytochrome b558 were found to decrease quickly by day 2 during cultivation of monocytes with GM-CSF and IL-4. DCs obtained after 7 days of cultivation had lost the large subunit almost completely and most of the small subunit. A cell surface epitope of the cytochrome detected by a monoclonal antibody also decreased during the differentiation. On the other hand, these components, including both subunits of cytochrome b558, were maintained in the cells during differentiation of monocytes to macrophages. These results indicate that the decreased levels of cytochrome b558, especially that of the large subunit, is responsible for the low level of superoxide-generating ability of DCs and that the suppression is caused by IL-4.
Assuntos
Células Dendríticas/citologia , Monócitos/citologia , NADPH Oxidases , Superóxidos/metabolismo , Acridinas/química , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Grupo dos Citocromos b/química , Grupo dos Citocromos b/imunologia , Grupo dos Citocromos b/metabolismo , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Immunoblotting , Interleucina-4/farmacologia , Medições Luminescentes , Monócitos/efeitos dos fármacos , CoelhosRESUMO
Monoclonal antibody 5E5 recognized an intranuclear antigen of neurons in the rat. We isolated 5E5cDNA and determined its nucleotide and deduced amino acid sequences. The 5E5cDNA had an open reading frame of 825 amino acids and its amino acid sequence showed no significant homology to any protein or to any DNA binding motif so far known. 5E5 protein had an abundance of basic amino acids, especially arginine, and included a glycine-rich region and a proline cluster. Monoclonal antibody 12H raised against 5E5cDNA fusion protein recognized an intranuclear substance in rat brain sections and a single protein band of about 98 kDa in the brain nuclear extract fraction on immunoblotting. DNA-cellulose column chromatography indicated that 5E5 protein might have DNA-binding ability. Transfection studies indicated that 5E5 protein expressed in COS-1 is localized in cell nuclei. These results suggest that 5E5 protein is a possible DNA-binding protein which is expressed especially in neurons.
Assuntos
Antígenos , Química Encefálica/fisiologia , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Peso Molecular , Ratos , Sirtuína 2 , SirtuínasRESUMO
The membrane protein syntaxin (originally named HPC-1) is involved in vesicle trafficking and required for neurotransmitter release at nerve terminals. The presence of syntaxin on target membranes is hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins. To elucidate the function of syntaxin 1A in exocytosis, HPC-1/syntaxin 1A-reduced PC12h cells (PC12h/Deltasyx) that were stably transfected with a plasmid for antisense syntaxin 1A expression were constructed. Depolarizing stimulation of PC12h/Deltasyx enhanced dopamine release, compared with PC12h. There was a strong inverse correlation between syntaxin 1A protein expression and enhancement of dopamine release. Reduction of syntaxin 1A had no effect on increase of the cytoplasmic free Ca2+ concentration by depolarized stimulation. Moreover, PC12h/Deltasyx clones similarly enhanced of exocytosis by native secretagogues. These results indicate that syntaxin 1A has more than one function in exocytosis.
Assuntos
Antígenos de Superfície/fisiologia , Exocitose/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Transporte Vesicular , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/farmacologia , Cálcio/metabolismo , Dopamina/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Células PC12 , RNA Antissenso/genética , Ratos , Proteínas SNARE , Sintaxina 1 , TransfecçãoRESUMO
We have previously isolated a lipopolysaccharide (LPS)-resistant mutant (named LR-9) of a cultured macrophage-like cell line, J774.1. This mutant had defective LPS binding [Hara-Kuge, S., Amano, F., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 6606-6610]. In this study, we found that: (1) LPS-binding to parental J774.1 cells was dependent on a serum factor with a molecular weight of about 60 kDa, probably LPS binding protein (LBP); (2) LPS-binding to J774.1 cells was markedly reduced by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC); (3) mutant LR-9 cells were defective in LPS-binding even in the presence of serum; (4) LR-9 cells lacked CD14 protein on flow cytometric and immunoblot analyses, but retained normal CD14 mRNA levels on RNA blot analysis; (5) small amounts of LPS (1 to 10 ng/ml) activated J774.1, but not LR-9 cells, to secrete tumor necrosis factor-alpha and to release arachidonate metabolites, whereas both J774.1 and LR-9 were activated by large concentrations of LPS (100 to 1,000 ng/ml). These results provide genetic evidence that CD14 molecules in J774.1 cells play a crucial role in LPS-binding and in LPS-triggered signal transduction, and indicate that large amounts of LPS can activate J774.1 cells without the participation of CD14 molecules.
Assuntos
Proteínas de Fase Aguda , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Transporte/metabolismo , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana , Diester Fosfórico Hidrolases/metabolismo , Ácido Araquidônico/metabolismo , Benzoquinonas , Linhagem Celular , Lactamas Macrocíclicas , Receptores de Lipopolissacarídeos , Mutação , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Quinonas/farmacologia , Rifabutina/análogos & derivados , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Injection of ibotenic acid into the caudate nucleus caused an increase in the uptake of 2-deoxyglucose (2DG) in the ipsilateral substantia nigra pars reticulata (SNr). Increased 2DG uptake was completely suppressed by chronic infusion of muscimol, the gamma-aminobutyric acid (GABA) agonist. While delayed shorter infusion of muscimol from the 3rd to the 7th day, when 2DG accumulation was the most prominent, partially prevented this increase. These data suggest that GABA-mediated transneuronal processes play an important role in the delayed elevation in 2DG uptake in SNR but hyperexcitation due to disinhibition by the loss of GABAergic inputs may play only a partial role in this increase.
Assuntos
Núcleo Caudado/fisiologia , Glucose/metabolismo , Muscimol/farmacologia , Substância Negra/efeitos dos fármacos , Animais , Morte Celular/fisiologia , Desoxiglucose/metabolismo , Ácido Ibotênico , Infusões Parenterais , Masculino , Ratos , Ratos Endogâmicos , Substância Negra/metabolismoRESUMO
A monoclonal antibody, 2F7, raised against a nuclear protein subfraction recognized the nuclear membrane of nervous and muscular tissues of guinea pig, rat and rabbit, but no other tissue was stained. In the nervous system, both neurons and glial cells were labelled. An electron microscopic immunohistochemical study demonstrated that 2F7 antibody labelled the inner surface of the nuclear membrane. Western blot analysis on the nuclear envelope fraction containing nuclear lamina revealed that this antibody reacted to two minor component proteins of 80 and 82KDa. These 2F7 antigens expressed preferentially in the nervous and muscular tissues were distinct from the major nuclear envelope proteins reported so far and might be related to neuronal or muscular tissue-specific functions.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Proteínas de Membrana/imunologia , Proteínas Musculares/imunologia , Proteínas do Tecido Nervoso/imunologia , Membrana Nuclear/imunologia , Animais , Feminino , Cobaias , Masculino , Microscopia Eletrônica , Coelhos , Ratos , Ratos EndogâmicosRESUMO
To clarify the function of HPC-1/syntaxin 1A in the mammalian central synapse, the effects of intracellularly applied antibody on the synaptic transmission were examined at the autapse of the cultured rat hippocampal neuron. Intracellularly applied antibody against HPC-1/syntaxin 1A (IgG, 0.3 mg ml(-1)) during whole-cell recording enhanced the autaptic excitatory postsynaptic current (EPSC). Pre-immune IgG (0.3 mg ml(-1)) showed no effect. The amplitude-distribution of an asynchronous EPSC was not affected by administration of this antibody, indicating that the increase in the amplitude of the evoked EPSC was attributable to an increase in transmitter release from the presynaptic terminal HPC-1/syntaxin 1A could be involved in suppressing as well as facilitating process of the exocytosis at the mammalian central synapse.
Assuntos
Antígenos de Superfície/fisiologia , Hipocampo/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Antígenos de Superfície/imunologia , Células Cultivadas , Potenciais Evocados/fisiologia , Hipocampo/citologia , Imunoglobulina G/farmacologia , Proteínas do Tecido Nervoso/imunologia , Neurônios/citologia , Ratos , Sintaxina 1RESUMO
An ornithine-containing lipid (Orn-L) or a serine-containing lipid (Ser-L) from Flavobacterium meningosepticum exhibited strong mitogenicity for the splenocytes from both LPS-responder C3H/HeSlc and LPS-low-responder C3H/HeJ mice. The potency of the lipoamino acids was the same as that of LPS for responder mice. The lipoamino acids were B-lymphocyte mitogens. Furthermore, Orn-L or Ser-L exhibited strong adjuvanticity. Compared with the adjuvanticity of LPS, the activity of Orn-L was rather high. Based on these data, together with the previously reported data of macrophage activation, we propose that the lipoamino acids are non-toxic, potent immunoactivators.