RESUMO
Magnetic nanoparticles are of great interest both for fundamental research and emerging applications. In the biomedical field, magnetite (Fe(3)O(4)) has shown promise as a hyperthermia-based tumor therapeutic. However, preparing suitable solubilized magnetite nanoparticles is challenging, primarily due to aggregation and poor biocompatibility. Thus methods for coating Fe(3)O(4) NPs with biocompatible stabilizers are required. We report a new method for preparing Fe(3)O(4) nanoparticles by co-precipitation within the pores of agar gel samples. Permeated agar gels were then dried and ground into a powder, yielding agar-conjugated Fe(3)O(4) nanoparticles. Samples were characterized using XRD, FTIR, TGA, TEM and SQUID. This method for preparing agar-coated Fe(3)O(4) nanoparticles is environmentally friendly, inexpensive and scalable.
Assuntos
Ágar/química , Química Verde/métodos , Nanopartículas de Magnetita/química , Sobrevivência Celular , Células Hep G2 , Humanos , Nanopartículas de Magnetita/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria , Difração de Raios XRESUMO
Ezrin links cortical actin filaments with the cell membrane, and has a critical role in many membrane-initiated events. Fas is directly associated with ezrin, but conflicting results have been reported for the involvement of ezrin in Fas-induced cell death. In this study we show that ezrin was associated with Fas in T cells before stimulation and was released shortly after Fas ligand (FasL) engagement. The knockdown of ezrin moderately increased Fas-triggered or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-triggered cell death in normal T lymphocytes and in H9 cells, but had no effect on death receptor-induced apoptosis in type II cells, such as Jurkat and CEM. Expression of a dominant-negative form of ezrin also led to an increased Fas-induced apoptosis in H9 cells. Ezrin deficiency did not affect the internalization of Fas after Fas ligation. Instead, an enhanced formation of death-inducing signaling complex (DISC) was observed in H9 cells with ezrin knockdown, leading to accelerated caspase-8 activation. Together, our results suggest that ezrin has a negative role in the recruitment of Fas into signaling complexes in type I T cells. Loss of ezrin likely removes the constraint imposed by ezrin and facilitates the assembly of death receptor complex in T cells.
Assuntos
Apoptose/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteína Ligante Fas/imunologia , Receptores de Morte Celular/metabolismo , Transdução de Sinais/fisiologia , Caspase 8/metabolismo , Morte Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteína Ligante Fas/fisiologia , Humanos , Receptores de Morte Celular/efeitos dos fármacos , Receptores de Morte Celular/fisiologia , Linfócitos T , Ligante Indutor de Apoptose Relacionado a TNFRESUMO
Decoy receptor 3 (DcR3/TR6) is a decoy receptor for the Fas ligand (FasL) and can inhibit FasL-induced apoptosis. It has been reported recently that DcR3 can induce T cell activation via co-stimulation of T cells, suggesting that DcR3 may be involved in the pathophysiology of autoimmune diseases. This study aims to analyse the serum DcR3 in patients with systemic lupus erythematosus (SLE) and to investigate the role of DcR3 in the pathogenesis of SLE. Significantly elevated serum DcR3 was observed in SLE patients, and the mean serum DcR3 level was significantly higher for those with active disease [SLE disease activity index (SLEDAI) >/= 10] compared with that in patients with inactive disease (SLEDAI < 10). In addition to reducing activation-induced cell death in activated T cells via neutralization of the FasL, soluble DcR3-Fc enhanced T cell proliferation and increased interleukin-2 and interferon-gamma production via co-stimulation of T cells. Moreover, enhanced T cell reactivity to DcR3-induced co-stimulation was demonstrated in lymphocytes from patients with SLE, suggesting the elevated serum DcR3 may associate with enhanced T cell activation in vivo. These findings are the first to demonstrate that serum DcR3 concentrations are increased in SLE patients, and this may imply a possible role of DcR3 in the pathogenesis of SLE via enhanced T cell hyperreactivity and reduced apoptosis in activated T cells.
Assuntos
Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , Membro 6b de Receptores do Fator de Necrose Tumoral/sangue , Linfócitos T/imunologia , Adulto , Apoptose/imunologia , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
The objective of this study was to investigate the molecular mechanisms of gonadotropin-releasing hormone receptor (GnRH-R) involved in the endocrine regulation of reproduction in the orange-spotted grouper, Epinephelus coioides. The full-length cDNA encoding GnRH-R type I was successfully cloned from the pituitary by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods in the grouper. The complete GnRH-R type I cDNA is 1607 bp, which includes an open reading frame of 1092 bp encoding a protein of 364 amino acids, a seven-alpha helix transmembrane domain, a N-terminal extracellular domain, and a C-terminal cytoplasmic domain. The expression of GnRH-R type I was found to be highest in the pituitary. An intramuscular injection of various GnRH types in vivo was attempted. The expression of GnRH-R type I was stimulated by a single injection of salmon GnRH, while in the case of chicken GnRH II treatment, the expression of GnRH-R type I was inhibited. This suggests that the action of chick GnRH II is probably enhanced through the GnRH receptor of different forms. Furthermore, none of them were expressed by an injection of seabream GnRH, and this is likely attributed to the injection dose being below the threshold level, and this remains to be further examined. In conclusion, GnRHs of various types are effective in stimulating the expression of gonadotropins through various forms of the GnRH-R, and multiple forms of the receptor gene likely exist in teleosts.
Assuntos
Expressão Gênica , Perciformes/genética , Filogenia , Receptores LHRH/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Análise por Conglomerados , Biologia Computacional , Primers do DNA , DNA Complementar/genética , Hormônio Liberador de Gonadotropina/metabolismo , Dados de Sequência Molecular , Hipófise/metabolismo , Receptores LHRH/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Recent evidence indicates that the decoy receptor 3 (DcR3) of the TNF receptor superfamily, which initially though prevents cytokine responses of FasL, LIGHT and TL1A by binding and neutralization, can modulate monocyte function through reverse signaling. We show in this work that DcR3 can induce osteoclast formation from human monocytes, murine RAW264.7 macrophages, and bone marrow cells. DcR3-differentiated cells exhibit characteristics unique for osteoclasts, including polynuclear giant morphology, bone resorption, TRAP, CD51/61, and MMP-9 expression. Consistent with the abrogation of osteoclastogenic effect of DcR3 by TNFR-Fc, DcR3 treatment can induce osteoclastogenic cytokine TNF-alpha release through ERK and p38 MAPK signaling pathways. We conclude that DcR3 via coupling reverse signaling of ERK and p38 MAPK and stimulating TNF-alpha synthesis is a critical regulator of osteoclast formation. This action of DcR3 might play an important role in significant osteoclastic activity in osteolytic bone metastases.
Assuntos
Diferenciação Celular/fisiologia , Glicoproteínas de Membrana/farmacologia , Monócitos/fisiologia , Osteoclastos/fisiologia , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Animais , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Reabsorção Óssea/metabolismo , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Citocinas/metabolismo , Citocinas/farmacologia , Dinoprostona/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4 , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/fisiologia , Metaloproteinase 9 da Matriz/genética , Glicoproteínas de Membrana/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ligante RANK , Ratos , Ratos Sprague-Dawley , Receptor Ativador de Fator Nuclear kappa-B , Receptores de Superfície Celular/fisiologia , Receptores do Fator de Necrose Tumoral , Membro 6b de Receptores do Fator de Necrose Tumoral , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores de Vitronectina/metabolismo , Fosfatase Ácida Resistente a Tartarato , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A cDNA sequence of stearoyl-CoA desaturase (SCD) was determined from zebrafish (Danio rerio) and compared to the corresponding genes in several teleosts. Zebrafish SCD cDNA has a size of 1,061 bp, encodes a polypeptide of 325 amino acids, and shares 88, 85, 84, and 83% similarities with tilapia (Oreochromis mossambicus), grass carp (Ctenopharyngodon idella), common carp (Cyprinus carpio), and milkfish (Chanos chanos), respectively. This 1,061 bp sequence specifies a protein that, in common with other fatty acid desaturases, contains three histidine boxes, believed to be involved in catalysis. These observations suggested that SCD genes are highly conserved. In addition, an oligonucleotide probe complementary to zebrafish SCD mRNA was hybridized to mRNA of approximately 396 bases with Northern blot analysis. The Northern blot and RT-PCR analyses showed that the SCD mRNA was expressed predominantly in the liver, intestine, gill, and muscle, while a lower level was found in the brain. Furthermore, we utilized whole-mount in situ hybridization and real-time quantitative RT-PCR to identify expression of the zebrafish SCD gene at five different stages of development. This revealed that very high levels of transcripts were found in zebrafish at all stages during embryogenesis and early development.
Assuntos
DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Estearoil-CoA Dessaturase/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Estearoil-CoA Dessaturase/químicaRESUMO
TNF-related apoptosis-inducing ligand (TRAIL, also called Apo2L), a novel member of TNF superfamily, induces apoptosis in transformed cell lines of diverse origin. TRAIL is expressed in most of the cells, and the expression is up-regulated in activated T cells. Four receptors for TRAIL have been identified, and there is complex interplay between TRAIL and TRAIL receptors in vivo. The actual biological function of TRAIL/TRAIL receptor is still not clear. Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. Cross-linking of TRAIL by plate-bound rTRAIL receptor, death receptor 4-Fc fusion protein enhanced T cell proliferation and increased IFN-gamma production in conjunction with immobilized suboptimal anti-CD3 stimulation in mouse splenocytes. The increase of T cell proliferation by death receptor 4-Fc was dose dependent, and this effect could be blocked by soluble rTRAIL proteins, indicating the occurrence of reverse signaling through TRAIL on T cell. The enhanced secretion of IFN-gamma mediated via TRAIL could be blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in inducing apoptosis by binding to the death receptors, TRAIL itself can enhance T cell proliferation after TCR engagement and signal the augmentation of IFN-gamma secretion via a p38-dependent pathway. This provides another example of reverse signaling by a member of TNF superfamily. In conclusion, our data suggest that TRAIL can itself transduce a reverse signal, and this may shed light on the biological function of TRAIL.
Assuntos
Apoptose/imunologia , Interferon gama/biossíntese , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/genética , Proteínas Reguladoras de Apoptose , Complexo CD3/imunologia , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Ligantes , Ativação Linfocitária/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Piridinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Linfócitos T/enzimologia , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Fas (CD95) and Fas ligand (FasL/CD95L) are involved in programmed cell death and the regulation of host immune responses. FasL has been shown to provide immune privilege, thus prolonging the survival of unmatched grafts in a variety of tissues, such as eyes and testis. In murine FasL (mFasL) transgenic mice, FasL provoked granulocyte infiltration and insulitis in the pancreas. We intended to study whether the expression of human FasL, instead of mFasL, on mouse beta islet cells could avoid granulocyte infiltration, and whether islet cells transgenic for FasL could be used in islet transplantation. We produced transgenic mice in which the human FasL transgene was driven by rat insulin promoter and was expressed exclusively in the pancreas islet cells in ICR mice. In contrast to mFasL transgenic mice, histochemical staining showed that the pancreas was intact in human FasL transgenic ICR mice. However, when human FasL transgenic islet cells were transplanted into allogeneic mice with streptozotocin-induced diabetes, human FasL appeared not to prolong graft survival. Intensive granulocyte infiltration into the islet grafts was observed in recipients (Balb/c mice) which received islet grafts from human FasL transgenic mice, but not from nontransgenic, allogeneic ICR mice on day 31. Our observations suggest that FasL alone is insufficient to confer immune protection, and that other environmental factors might contribute to the formation of immune privilege sites in vivo
Assuntos
Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Morte Celular , Diabetes Mellitus Experimental/imunologia , Proteína Ligante Fas , Humanos , Inflamação/patologia , Insulina/genética , Ilhotas Pancreáticas/patologia , Transplante das Ilhotas Pancreáticas/patologia , Células Jurkat , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Infiltração de Neutrófilos , Regiões Promotoras Genéticas/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Transgenes/genéticaRESUMO
We have explored the regulatory roles played by Ca2+-dependent signaling on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6) release in mouse peritoneal macrophages. To elevate intracellular Ca2+, we used thapsigargin (TG) and UTP. Although LPS alone cannot stimulate NO synthesis, co-addition with TG, which sustainably increased [Ca2+]i, resulted in NO release. UTP, via acting on P2Y6 receptors, can stimulate phosphoinositide (PI) turnover and transient [Ca2+]i increase, however, it did not possess the NO priming effect. LPS alone triggered the release of PGE2, TNF-alpha, and IL-6; all of which were potentiated by the presence of TG, but not of UTP. The stimulatory effect of LPS plus TG on NO release was inhibited by the presence of Ro 31-8220, Go6976, KN-93, PD 098059, or SB 203580, and abolished by BAPTA/AM and nuclear factor kappaB (NF-kappaB) inhibitor, PDTC. PGE2, TNF-alpha, and IL-6 release by LPS alone were attenuated by Ro 31-8220, Go6976, PD 098059, SB 203580, and PDTC. Using L-NAME, soluble TNF-alpha receptor, IL-6 antibody, NS-398, and indomethacin, we performed experiments to understand the cross-regulation by the four mediators. The results revealed that TNF-alpha up-regulated NO, PGE2, and IL-6 synthesis; PGE2 up-regulated NO, but down-regulated TNF-alpha synthesis; and PGE2 and IL-6 mutually up-regulated reciprocally. Taken together, murine peritoneal macrophages required a sustained [Ca2+]i increase, which proceeds after TG, but not UTP, stimulation, to enhance LPS-mediated release of inflammatory mediators, particularly for NO induction. Activation of PKC-, ERK-, and p38 MAPK-dependent signaling also are essential for LPS action. The positive regulatory interactions among these mediators might amplify the inflammatory response caused by endotoxin.
Assuntos
Adjuvantes Imunológicos/fisiologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Proteínas Quinases/fisiologia , Tapsigargina/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Cálcio/metabolismo , Cálcio/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Células Cultivadas , Dinoprostona/metabolismo , Interleucina-6/metabolismo , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/fisiologia , NF-kappa B/fisiologia , Óxido Nítrico/metabolismo , Proteína Quinase C/fisiologia , Receptores Purinérgicos P2/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Uridina Trifosfato/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. TNF-related activation-induced cytokine (TRANCE), a member of TNF superfamily, is preferentially expressed on the surface of activated CD4(+) Th1 cells. The soluble receptor activator of NF-kappaB (RANK).Fc fusion protein suppresses IFN-gamma secretion by activated Th1 cells, but does not affect IL-4 secretion by Th2 cells. The suppressive effect on IFN-gamma secretion is observed when Th1 cells are activated by APCs, but not by immobilized anti-TCR beta mAb. In contrast, immobilized RANK.Fc fusion protein augments IFN-gamma secretion by Th1 cells, indicating the occurrence of reverse signaling through TRANCE during T cell/APC interaction. The enhanced secretion of IFN-gamma mediated via TRANCE correlates with the activation of p38 mitogen-activated protein kinase and is blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in activating dendritic cells by binding to the receptor RANK, TRANCE itself can signal the augmentation of IFN-gamma secretion via a p38-dependent pathway, and this provides yet another example of reverse signaling by a member of TNF superfamily.
Assuntos
Proteínas de Transporte/fisiologia , Citocinas/fisiologia , Interferon gama/metabolismo , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Transdução de Sinais/imunologia , Células Th1/metabolismo , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Humanos , Interferon gama/antagonistas & inibidores , Ativação Linfocitária/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/genética , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumor-specific antigen but is only weakly immunogenic. This study demonstrates that the immunogenicity of the tumor Id protein can be dramatically increased by directing it to antigen-presenting cells (APCs). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) present on activated T cells has a strong binding affinity to both B7-1 and B7-2 molecules, which are primarily expressed on APCs. After construction of a fusion protein consisting of Id and CTLA-4 (Id-CTLA4), mice immunized with the fusion protein induced high titers of Id-specific antibody and T-cell proliferative responses without adjuvants and were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was so potent that even low doses (down to 0.1 microg) of the immunogen were able to elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the ability to bind B7 molecules on APCs was shown to be required for the enhanced immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a potential tumor antigen represents an effective approach to prime antitumor immunities in vivo and may be applicable to the design of vaccines for a variety of other diseases. (Blood. 2000;96:3663-3670)
Assuntos
Antígenos de Diferenciação/imunologia , Antígenos de Neoplasias/imunologia , Imunoconjugados , Abatacepte , Animais , Formação de Anticorpos , Células Apresentadoras de Antígenos/imunologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação/química , Antígenos de Diferenciação/metabolismo , Antígenos de Neoplasias/metabolismo , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2 , Antígeno CTLA-4 , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/normas , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunização/métodos , Imunização/normas , Idiótipos de Imunoglobulinas/química , Idiótipos de Imunoglobulinas/imunologia , Idiótipos de Imunoglobulinas/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Ligação Proteica , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Células Tumorais CultivadasRESUMO
LIGHT is a member of the tumor necrosis factor superfamily and is the ligand for LT-betaR, HVEM, and decoy receptor 3. LIGHT has a cytotoxic effect, which is further enhanced by the presence of interferon-gamma (IFN-gamma). Although LIGHT/IFN-gamma can activate caspase activity, neither benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone nor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can completely inhibit LIGHT/IFN-gamma-mediated apoptosis. Moreover, overexpression of Bcl-2 further enhances LIGHT/IFN-gamma-mediated apoptosis. It appears that LIGHT and IFN-gamma act synergistically to activate caspase-3, with the resultant cleavage of Bcl-2, removal of the BH4 domain, leading to conversion of Bcl-2 from an antiapoptotic to a proapoptotic form in p53-deficient hepatocellular carcinoma Hep3BT2 cells. Thus, LIGHT seems to be able to override the protective effect of Bcl-2 and induce cell death. Although benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone can prevent the cleavage of Bcl-2 by LIGHT/IFN-gamma, they only partially inhibit apoptosis in Hep3BT2 cells that are overexpressing Bcl-2. In contrast, both LIGHT/IFN-gamma-mediated apoptosis and Bcl-2 cleavage are inhibited by free radical scavengers, indicating that free radicals may play an essential role in LIGHT/IFN-gamma-mediated apoptosis at a step upstream of caspase-3 activation. These results suggest that LIGHT signaling may diverge into multiple, separate processes.
Assuntos
Apoptose/fisiologia , Genes bcl-2 , Interferon gama/farmacologia , Proteínas de Membrana/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Neoplasias da Mama , Carcinoma Hepatocelular , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Feminino , Humanos , Cinética , Neoplasias Hepáticas , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais Cultivadas , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
LIGHT is a recently identified member of the TNF superfamily and its receptors, herpesvirus entry mediator and lymphotoxin beta receptor, are found in T cells and stromal cells. In this study, we demonstrate that LIGHT is selectively expressed on immature dendritic cells (DCs) generated from human PBMCs. In contrast, LIGHT is not detectable in DCs either freshly isolated from PBMCs or rendered mature in vitro by LPS treatment. Blockade of LIGHT by its soluble receptors, lymphotoxin beta receptor-Ig or HVEM-Ig, inhibits the induction of DC-mediated primary allogeneic T cell response. Furthermore, engagement of LIGHT costimulates human T cell proliferation, amplifies the NF-kappaB signaling pathway, and preferentially induces the production of IFN-gamma, but not IL-4, in the presence of an antigenic signal. Our results suggest that LIGHT is a costimulatory molecule involved in DC-mediated cellular immune responses.
Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Proteínas de Membrana/fisiologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Diferenciação Celular/imunologia , Humanos , Soros Imunes/farmacologia , Imunossupressores/farmacologia , Interferon gama/biossíntese , Interferon gama/metabolismo , Isoantígenos/imunologia , Teste de Cultura Mista de Linfócitos , Receptor beta de Linfotoxina , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Dados de Sequência Molecular , NF-kappa B/metabolismo , Receptores do Fator de Necrose Tumoral/imunologia , Membro 14 de Receptores do Fator de Necrose Tumoral , Receptores Virais/imunologia , Simplexvirus/imunologia , Linfócitos T/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Activation of lymphotoxin-beta receptor (LT-betaR) by conjugation with heterotrimeric lymphotoxin, LT-alpha1/beta2, or by cross-linking with anti-LT-betaR antibodies can trigger apoptosis. We have observed that overexpression of either LT-betaR or the cytoplasmic domain of LT-betaR (LT-betaR(CD)) also induces apoptosis, which may be attributed to the tendency of LT-betaR(CD) to self-associate. The self-association domain of LT-betaR(CD) was mapped to amino acids 324-377, a region of the protein that is also essential for LT-betaR-induced apoptosis. Furthermore, we have shown that LT-betaR(CD)-induced apoptosis could be inhibited by a TRAF3 dominant negative mutant and by the caspase inhibitors Z-VAD-FMK, DEVD-FMK, and CrmA. The ligand-independent apoptosis induced by LT-betaR(CD) will help us to further dissect LT-betaR signaling pathway.
Assuntos
Apoptose/fisiologia , Citoplasma/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Apoptose/efeitos dos fármacos , Sequência de Bases , Inibidores de Cisteína Proteinase/farmacologia , Primers do DNA , Células HeLa , Humanos , Receptor beta de Linfotoxina , Microscopia de Fluorescência , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transdução de SinaisRESUMO
Protein kinases are involved in signal transduction pathways and play fundamental roles in the regulation of cell functions. Here we report that the gene G11 located in the human major histocompatibility complex encodes a novel Ser/Thr protein kinase. The G11 gene products of 41.5 and 30 kDa were expressed in insect cells using the baculovirus system and transiently in the mammalian cell line COS-7. It was found that after immunoprecipitation of the G11 polypeptides from recombinant baculovirus-infected insect cell lysates or transfected COS-7 cell lysates the immunoprecipitates contained a Mn2+-dependent protein kinase activity that phosphorylated alpha-casein at Ser/Thr residues and histone at Ser residues. Furthermore, mutation of the ATP-binding site by converting the invariant lysine in the catalytic domain (amino acid 317) to a proline resulted in the complete ablation of the enzyme activity. This was consistent with the observation that the G11 polypeptide can be covalently modified by the reactive ATP analogue 5'-p-fluorosulfonylbenzoyladenosine in the absence of ATP, and that this modification is prevented in the presence of 1 mM ATP, indicating that the kinase domain of the G11 polypeptide is capable of binding ATP. Immunofluorescence staining of transfected COS-7 cells transiently expressing G11 revealed that this novel Ser/Thr protein kinase is localized predominantly in the nucleus.
Assuntos
Complexo Principal de Histocompatibilidade , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Adenosina/análogos & derivados , Marcadores de Afinidade , Animais , Células COS , Caseínas/metabolismo , Compartimento Celular , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Recombinantes , SpodopteraRESUMO
LIGHT is a new member of tumor necrosis factor (TNF) cytokine family derived from an activated T cell cDNA library. LIGHT mRNA is highly expressed in splenocytes, activated PBL, CD8(+) tumor infiltrating lymphocytes, granulocytes, and monocytes but not in the thymus and the tumor cells examined. Introduction of LIGHT cDNA into MDA-MB-231 human breast carcinoma caused complete tumor suppression in vivo. Histological examination showed marked neutrophil infiltration and necrosis in LIGHT expressing but not in the parental or the Neo-transfected MDA-MB-231 tumors. Interferon gamma (IFNgamma) dramatically enhances LIGHT-mediated apoptosis. LIGHT protein triggers apoptosis of various tumor cells expressing both lymphotoxin beta receptor (LTbetaR) and TR2/HVEM receptors, and its cytotoxicity can be blocked specifically by addition of a LTbetaR-Fc or a TR2/HVEM-Fc fusion protein. However, LIGHT was not cytolytic to the tumor cells that express only the LTbetaR or the TR2/HVEM or hematopoietic cells examined that express only the TR2/HVEM, such as PBL, Jurkat cells, or CD8(+) TIL cells. In contrast, treatment of the activated PBL with LIGHT resulted in release of IFNgamma. Our data suggest that LIGHT triggers distinct biological responses based on the expression patterns of its receptors on the target cells. Thus, LIGHT may play a role in the immune modulation and have a potential value in cancer therapy.
Assuntos
Apoptose , Genes Supressores de Tumor , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Mapeamento Cromossômico , Meios de Cultura Livres de Soro , Feminino , Técnicas de Transferência de Genes , Humanos , Hibridização in Situ Fluorescente , Interferon gama/metabolismo , Ligantes , Ativação Linfocitária , Linfócitos do Interstício Tumoral , Receptor beta de Linfotoxina , Masculino , Proteínas de Membrana/genética , Membro 14 de Receptores do Fator de Necrose Tumoral , Células Tumorais Cultivadas , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/genéticaRESUMO
The lymphotoxin-beta receptor (LT-betaR) has been shown to be the receptor for the membrane-bound lymphotoxin heterotrimers LTalpha1/beta2 and LTalpha2/beta1. The extracellular domain of LT-betaR shows extensive similarity with members of the tumor necrosis factor receptor family, while its cytoplasmic domain is distinct and lacks any inherent enzymatic activity. This suggests that the interaction of LT-betaR with other molecules might be important for signal transduction. Here we demonstrate the association of a fusion protein, comprising glutathione S-transferase and the cytoplasmic domain of LT-betaR (GST-LT-betaR(CD)), with several proteins in the size range 29-80 kDa from HepG2 cell lysates. We present evidence that two of these proteins are serine/threonine kinases, which associate with amino acids 324-377 of the cytoplasmic domain of LT-betaR and phosphorylate this receptor. The characteristics of these novel kinases indicate that they are distinct from the previously described tumor necrosis factor receptor-associated kinases. This suggests the presence of novel signal transduction pathway(s) for LT-betaR.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Aminoácidos/metabolismo , Animais , Antígenos CD/metabolismo , Sítios de Ligação , Citoplasma/metabolismo , Glutationa/metabolismo , Humanos , Receptor beta de Linfotoxina , Mapeamento de Peptídeos , Fosforilação , Proteínas Quinases/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Twelve transcriptional units have now been located in a 160 kb segment of DNA that includes the genes encoding members of the serum complement system C2, Factor B (Bf) and C4 within the class III region of the human major histocompatibility complex (MHC). The common arrangement of these genes is tel-C2-Bf-RD-G11-C4A-[P450c21A-YA-XA]-C4B-[P450c21B-YB ]-+ ++TNX-cen. Characterisation of cDNA and genomic clones corresponding to the novel gene G11 has revealed that the gene spans approximately 9.1 kb of DNA and is split into 7 exons. The 5' end of the gene is associated with a CpG-island while the 3' end of the gene lies 611 bp from the transcriptional start site of the C4A gene. The approximately 1.4 kb G11 mRNA, which is expressed in a number of different cell types including monocytes, hepatocytes, epithelial cells, T and B lymphocytes, encodes protein products of 254 or 258 amino acids due to differential use of two splice sites lying 12 bp apart at the end of exon 3. These polypeptides share homology with a limited number of proteins including human cytochrome P450XIB1 and the tyrosine kinase transforming protein from fujinami virus. Duplication of the C4/P450c21 transcriptional unit occurred by a nonhomologous recombination event. Sequence analysis of a 1.5 kb segment of DNA flanking the C4B gene has revealed that 914 bp of the 3' end of the G11 gene also lies 611 bp from the transcriptional start site of the C4B gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Complemento C4a/genética , Complexo Principal de Histocompatibilidade/genética , Proteínas Nucleares , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA , Humanos , Dados de Sequência Molecular , Família Multigênica , Proteínas Serina-Treonina Quinases , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
We have previously described multiallelic restriction fragment length polymorphisms of the C2 gene, suggesting the presence of a variable number of tandem repeats (VNTR) locus. We report here the cloning and sequencing of the polymorphic fragments from the two most common alleles of the gene, a and b. The results confirm the presence of a VNTR locus consisting of a nucleotide sequence, 41 bp in average length, repeated tandemly 23 and 17 times in alleles a and b, respectively. The difference in the number of repeats between the two alleles is due to the deletion/insertion of two noncontiguous segments, 143 and 118 bp long, of allele a, and of a 40-bp segment of allele b. The VNTR region is associated with a SINE (short interspersed sequence)-type retroposon, SINE-R.C2, located within the third intron of the C2 gene. SINE-R.C2 is a member of a previously described large retroposon family of the human genome, apparently derived from the human endogenous retrovirus, (HERV) K10, which is homologous to the mouse mammary tumor virus.