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1.
J Lipid Res ; 59(3): 475-487, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29335275

RESUMO

Lipogenesis in liver is highest in the postprandial state; insulin activates SREBP-1c, which transcriptionally activates genes involved in FA synthesis, whereas glucose activates carbohydrate-responsive element-binding protein (ChREBP), which activates both glycolysis and FA synthesis. Whether SREBP-1c and ChREBP act independently of one another is unknown. Here, we characterized mice with liver-specific deletion of ChREBP (L-Chrebp-/- mice). Hepatic ChREBP deficiency resulted in reduced mRNA levels of glycolytic and lipogenic enzymes, particularly in response to sucrose refeeding following fasting, a dietary regimen that elicits maximal lipogenesis. mRNA and protein levels of SREBP-1c, a master transcriptional regulator of lipogenesis, were also reduced in L-Chrebp-/- livers. Adeno-associated virus-mediated restoration of nuclear SREBP-1c in L-Chrebp-/- mice normalized expression of a subset of lipogenic genes, while not affecting glycolytic genes. Conversely, ChREBP overexpression alone failed to support expression of lipogenic genes in the livers of mice lacking active SREBPs as a result of Scap deficiency. Together, these data show that SREBP-1c and ChREBP are both required for coordinated induction of glycolytic and lipogenic mRNAs. Whereas SREBP-1c mediates insulin's induction of lipogenic genes, ChREBP mediates glucose's induction of both glycolytic and lipogenic genes. These overlapping, but distinct, actions ensure that the liver synthesizes FAs only when insulin and carbohydrates are both present.


Assuntos
Glicólise , Lipogênese , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Fatores de Transcrição/deficiência
2.
Microvasc Res ; 83(2): 154-61, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22166857

RESUMO

As the pathogenesis of arterial thrombosis often includes platelet thrombus formation (PTF), antiplatelet agents are commonly used for the prevention of thromboembolic events. Here, using a novel microchip flow-chamber system we developed to quantitatively analyze the PTF process, we evaluated the pharmacological efficacies of antiplatelet agents under different arterial shear rates. Hirudin-anticoagulated whole blood was perfused over a collagen-coated microchip at shear rates of 1000, 1500, and 2000s(-1), and PTF in the absence and presence of various antiplatelet agents was observed microscopically and quantified by measuring flow-pressure changes. The onset of PTF was measured as T(10) (time to reach 10 kPa), and AUC(10) (area under the flow pressure curve for the first 10 min) was calculated to quantify the overall stability of the formed thrombus. Aspirin and AR-C66096 (P2Y(12)-antagonist) at high concentrations (50 µM and 1000 nM, respectively) prolonged T(10) only modestly (AR-C66096>aspirin), but effectively decreased AUC(10), resulting in unstable PTF at all examined shear rates. With dual inhibition using both aspirin (25 µM) and ARC-66096 (250 nM), AUC(10) was drastically reduced. Nearly complete suppression of AUC(10) was also observed with abciximab (2 µg ml(-1)) and beraprost (PGI(2)-analog; 4 nM). Although OS-1 (GPIbα-antagonist; 100 nM) prevented complete capillary occlusion, significant amounts of microscopic thrombi were observed on the collagen surface. In contrast to abciximab and beraprost, OS-1 differentially affected PTF under higher shear conditions. Our novel analytical system is capable of distinguishing the pharmacological effects of various antiplatelet agents under physiological shear rates, suggesting that this system may aid in the determination of the appropriate type and dose of antiplatelet agent in the clinical setting.


Assuntos
Plaquetas/efeitos dos fármacos , Dispositivos Lab-On-A-Chip , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária/instrumentação , Trombose/prevenção & controle , Abciximab , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Adulto , Anticorpos Monoclonais/farmacologia , Área Sob a Curva , Aspirina/farmacologia , Plaquetas/metabolismo , Pressão Sanguínea , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Epoprostenol/análogos & derivados , Epoprostenol/farmacologia , Desenho de Equipamento , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Masculino , Microscopia de Vídeo , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Fluxo Sanguíneo Regional , Estresse Mecânico , Trombose/sangue , Trombose/fisiopatologia , Fatores de Tempo
3.
Biosci Biotechnol Biochem ; 75(1): 165-7, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21228473

RESUMO

ß-Lactoglobulin (BLG), a major allergen of cow's milk, was conjugated with the N-hydroxysuccinimide ester of the amylose-glycylglycine adduct (AG-ONSu) to reduce its immunogenicity, and the biochemical and immunological properties of the resulting conjugate (AG-BLG) were studied. The conjugate was prepared by modifying BLG with AG-ONSu, and was purified in a Sephadex G-100 column. The analytical data for AG-BLG indicated that 10.5 moles of AG-ONSu, with a mean molecular weight of 2,800, was covalently attached to the amino groups of the BLG molecule. Conjugation with AG-ONSu greatly decreased the reactivity of BLG with anti-BLG polyclonal antibodies owing to its shielding action for epitopes on the protein's surface. These findings suggest that AG-ONSu can be used advantageously to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.


Assuntos
Amilose/química , Lactoglobulinas/química , Lactoglobulinas/imunologia , Animais , Glicilglicina/química , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoglobulina G/imunologia , Camundongos , Succinimidas/química
4.
Biosci Biotechnol Biochem ; 74(4): 721-6, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20378982

RESUMO

Beta-lactoglobulin (BLG) was conjugated with the N-hydroxysuccinimide ester of the dextran-glycylglycine adduct (DG-ONSu) to reduce the immunogenicity of BLG, a major allergen of cow's milk, and some immunological properties of the conjugate (DG-BLG) were studied. The conjugate was prepared by modifying BLG with DG-ONSu and purified in a Sephadex G-100 column. The analytical data for DG-BLG indicated that 5.2 moles of DG-ONSu with a mean molecular weight of 9,300 were covalently attached to the amino groups of the BLG molecule. Conjugation with DG-ONSu greatly decreased the reactivity of BLG with anti-BLG antibodies and suppressed their production in vivo due to its shielding action for epitope(s) on the protein's molecular surface. It was also found that DG-BLG was resistant to proteolytic enzymes. These findings allow us to suggest that DG-ONSu could be advantageously used to suppress the hypersensitivity mediated by IgG antibodies in milk allergy.


Assuntos
Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Lactoglobulinas/imunologia , Hipersensibilidade a Leite/imunologia , Alérgenos/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Formação de Anticorpos/imunologia , Bovinos , Dextranos/imunologia , Epitopos/imunologia , Feminino , Imunidade/imunologia , Lactoglobulinas/metabolismo , Leite/imunologia , Leite/metabolismo , Succinimidas
5.
Biochem Biophys Res Commun ; 391(2): 1166-9, 2010 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-20025850

RESUMO

Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site ("NES1") of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Glucose/metabolismo , Carioferinas/metabolismo , Sinais de Exportação Nuclear , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Cultivadas , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Ratos , Proteína Exportina 1
6.
J Biol Chem ; 283(36): 24899-908, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18606808

RESUMO

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an alpha-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin alpha. Notably, 14-3-3 appears to compete with importin alpha for ChREBP binding. 14-3-3beta bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 microm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3beta. These results suggest that interactions with importin alpha and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.


Assuntos
Proteínas 14-3-3/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas 14-3-3/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Linhagem Celular , Núcleo Celular/genética , Citosol/metabolismo , Glucose/genética , Glucose/metabolismo , Glicólise/fisiologia , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Lipogênese/fisiologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Peptídeos/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Ratos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/genética , Proteína Exportina 1
8.
Eur J Immunol ; 37(4): 966-77, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17330820

RESUMO

While IL-18 synergizes with IL-12 to induce a Th1 immune response, it also promotes a Th2 response. Here we investigate the modulatory role of IL-18 on the Th1/Th2 cytokine response. The injection of alpha-galactosylceramide (alpha-GalCer), a ligand for NKT cells, elevated mouse serum levels of both IFN-gamma and IL-4. When the mice were treated 2 h before alpha-GalCer challenge with IL-18, IFN-gamma production but not IL-4 production was remarkably up-regulated. In contrast, pretreatment with IL-18 6 h before the challenge enhanced IL-4 production. However, this IL-18-enhanced IL-4 production was not elicited in mice injected with anti-CD3 Ab. Liver mononuclear cells (MNC) produced a similar cytokine production pattern when MNC from mice treated with IL-18 either 2 h or 6 h before challenge were stimulated with alpha-GalCer in vitro. Expression of SOCS1 and SOCS3 was notably up-regulated in the liver MNC from mice pretreated 6 h before with IL-18; in particular, SOCS3 expression was confined to the liver NKT cells. Inhibition of SOCS3 by RNA interference up-regulated the phosphorylation of STAT3 and suppressed in vitro IL-4 production by IL-18-primed liver MNC stimulated with alpha-GalCer, but it did not affect IFN-gamma production. These results suggest that IL-18 time-dependently modulates Th1/Th2 cytokine production in ligand-activated NKT cells by regulating/inducing SOCS3 expression.


Assuntos
Citocinas/biossíntese , Interleucina-18/fisiologia , Células Matadoras Naturais/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Células Cultivadas , Citocinas/classificação , Galactosilceramidas/imunologia , Interferon gama/biossíntese , Interleucina-18/administração & dosagem , Interleucina-4/biossíntese , Células Matadoras Naturais/metabolismo , Ligantes , Fígado/citologia , Fígado/imunologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Células Th1/metabolismo , Células Th2/metabolismo , Fatores de Tempo
9.
J Hepatol ; 43(4): 670-8, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15922476

RESUMO

BACKGROUND/AIMS: The functions of mouse liver NK1.1+ T (NKT) cells stimulated with alpha-galactosylceramide (alpha-GalCer) are enhanced age dependently, and the antitumor and anti-metastatic effect in the liver is dependent on IFN-gamma. However, hepatic injury is independent of IFN-gamma and Fas/Fas-ligand dependent. The aim of this study is to investigate how tumor necrosis factor is involved in the alpha-GalCer-mediated immune phenomena. METHODS: C57BL/6 mice were intraperitoneally treated with anti-TNF antibody 1 h before alpha-GalCer injection, and Fas-ligand expression of NKT cells, the serum ALT levels and histopathological findings of the liver, kidney and lung and mortality after alpha-GalCer injection were evaluated. IFN-gamma production and antitumor immunity in the liver after the intravenous injection of EL-4 cells were also assessed. RESULTS: Serum TNF levels after alpha-GalCer injection increased age dependently in mice. Anti-TNF Ab reduced Fas-ligand (Fas-L) expression of NKT cells while it completely inhibited organ injuries induced by alpha-GalCer and thereby reduced the mortality of old mice, whereas it did not affect the IFN-gamma production from NKT cells, the antitumor immunity in the liver nor the mouse survival after EL-4 injection. CONCLUSIONS: NKT cells activated by alpha-galactosylceramide participated in either antitumor immunity or hepatic injury using IFN-gamma and TNF/Fas-L, respectively.


Assuntos
Galactosilceramidas/toxicidade , Falência Hepática/prevenção & controle , Fator de Necrose Tumoral alfa/imunologia , Envelhecimento , Alanina Transaminase/sangue , Animais , Antineoplásicos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Creatinina/sangue , Citometria de Fluxo , Interferon gama/sangue , L-Lactato Desidrogenase/sangue , Fígado/crescimento & desenvolvimento , Fígado/patologia , Falência Hepática/induzido quimicamente , Linfoma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
10.
J Biochem ; 136(3): 273-7, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15598882

RESUMO

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K, identical to PFKFB3) is expressed in a variety of cells and tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, primary blood mononuclear cells and cancer cells. We observed previously that the enhancer region of the HP2K gene, which has been identified in the 5'-flanking region between -1265 and -1329, could respond to serum stimulation following the transfection of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. The HP2K enhancer region also contains two copies of the hypoxia-inducible factor-1 (HIF-1) binding motif (5'-ACGTG-3'). In this study we performed characterization of the HP2K gene expression in response to hypoxic conditions. Both electrophoretic mobility shift and co-transfection assays of the HP2K promoter-luciferase reporter with HIF-1 expression vectors indicated that HIF-1 binds to the hypoxia-responsive element (HRE) of HP2K, thereby upregulating its gene expression. In addition, we demonstrated using site-directed mutagenesis that a complete tandem repeat of the HIF-1 binding motif with a 4-bp interruption is required for full induction of HP2K expression (up to 22-fold) under hypoxic conditions, and that this response is much stronger than that of the erythropoietin (EPO) gene. These results suggest that the sequence 5'-ACGTGNNNNACGTG-3' in the HP2K enhancer is the authentic HRE consensus motif that mediates increased transcription, under hypoxic conditions, via HIF-1.


Assuntos
Hipóxia , Fosfofrutoquinase-2/química , Placenta/enzimologia , Fatores de Transcrição/genética , Motivos de Aminoácidos , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Elementos Facilitadores Genéticos , Eritropoetina/genética , Eritropoetina/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Luciferases/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos/química , Fosfofrutoquinase-2/fisiologia , Regiões Promotoras Genéticas , Isoformas de Proteínas , Estrutura Terciária de Proteína , Fatores de Tempo , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Regulação para Cima
11.
Biochem Biophys Res Commun ; 325(4): 1367-75, 2004 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-15555578

RESUMO

We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [(35)S]procaspase-9, but not [(35)S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Blástula/fisiologia , Caspases/metabolismo , Oócitos/fisiologia , Xenopus laevis/embriologia , Xenopus laevis/fisiologia , Adenosilmetionina Descarboxilase/genética , Animais , Apoptose/fisiologia , Blástula/citologia , Caspase 1/genética , Caspase 1/metabolismo , Caspase 9 , Caspases/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Oócitos/citologia , Proteínas Recombinantes/metabolismo
12.
Immunology ; 113(1): 35-43, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15312134

RESUMO

We previously reported that mouse NK1.1 Ag+ T (NKT) cells activated by interleukin-12 (IL-12) act as anti-tumour/anti-metastatic effectors. However, IL-12 reportedly induces a rapid disappearance of liver NKT cells by activation-induced apoptosis. In the present study, however, we show that injection of IL-12 into mice merely down-regulates the NK1.1 expression of liver NKT cells and Vbeta8+ intermediate T-cell receptor cells and CD1d/alpha-galactosylceramide (alpha-GalCer)-tetramer reactive cells in the liver remained and did not decrease. Furthermore, when IL-12-pretreated (24 hr before) mice were injected with alpha-GalCer, not only serum interferon-gamma but also serum IL-4 concentrations increased several-fold in comparison to the control alpha-GalCer-injected mice. However, IL-12 pretreatment markedly up-regulated serum ALT levels and Fas-ligand expression on NKT cells after alpha-GalCer injection in middle-aged mice only. Consistently, the liver mononuclear cells (MNC) from IL-12-pretreated mice stimulated with alpha-GalCer in vitro produced much greater amounts of interferon-gamma and IL-4, and also showed a more potent cytotoxicity against tumour targets than those from mice pretreated with phosphate-buffered saline. Liver MNC from middle-aged mice, but not from young mice pretreated with IL-12, also showed increased cytotoxicity following in vitro alpha-GalCer stimulation against cultured hepatocytes. Furthermore, IL-12 treatment of middle-aged mice enhanced tumour necrosis factor receptor 1 mRNA expression in liver Vbeta8+ T cells, and in vitro experiments also revealed that IL-12 pretreatment of liver MNC from middle-aged mice enhanced their tumour necrosis factor-alpha production after alpha-GalCer stimulation. Synthetic ligand-mediated functions of NKT cells, including IL-4 production, are thus enhanced by IL-12 pretreatment.


Assuntos
Antígenos/análise , Interleucina-12/imunologia , Células Matadoras Naturais/imunologia , Fígado/imunologia , Proteínas/análise , Alanina Transaminase/sangue , Animais , Antígenos Ly , Antígenos de Superfície , Células Cultivadas , Meios de Cultura , Citocinas/sangue , Citotoxicidade Imunológica , Proteína Ligante Fas , Galactosilceramidas/imunologia , Expressão Gênica , Hepatócitos/imunologia , Lectinas Tipo C , Ligantes , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK , RNA Mensageiro/genética , Receptores de Interleucina/biossíntese , Receptores de Interleucina/genética , Receptores de Interleucina-12 , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismo
13.
Immunology ; 108(2): 211-9, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12562330

RESUMO

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK-type T cell); CD56 single positive (CD56)-T cells, CD56/CD57 double positive (DP)-T cells and CD57 single positive (CD57)-T cells in the peripheral blood. All NK-type T-cell populations expressed CD122 and intermediate levels of T-cell receptor (TCR; regular CD8+ T cells are CD122- and express high levels of TCR). The number of both DP-T cells and CD57-T cells, but not CD56-T cells, gradually increased with age. All NK-type T-cell populations produced larger amounts of interferon-gamma than did regular CD8+ T cells after stimulation with interleukin (IL)-2, IL-12 and IL-15. However, CD56-T cells and CD57-T cells but not DP-T cells showed a potent antitumour cytotoxity to NK-sensitive K562 cells, whereas only CD56-T cells showed a potent cytotoxity to NK-resistant Raji cells. Furthermore, although NK-type T cells produced large amounts of soluble Fas-ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VbetaT cells were found in each NK-type T-cell population. The non-variant CDR3 region(s) for the TCRbeta chain(s) showed CD57-T cells and CD56-T cells to be derived from distinct origins, while the DP-T cell population consisted of a mixture of the clones seen in both CD56-T cells and CD57-T cells. Our results suggest that CD57-T cells and CD56-T cells are functionally and ontogenically different populations while DP-T cells appear to originate from both CD56-T cells and CD57-T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/sangue , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Envelhecimento/imunologia , Sequência de Bases , Antígeno CD56/sangue , Antígenos CD57/sangue , Citotoxicidade Imunológica , Proteína Ligante Fas , Humanos , Interferon gama/biossíntese , Glicoproteínas de Membrana/biossíntese , Pessoa de Meia-Idade , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Interleucina-2/sangue
14.
J Immunol ; 169(11): 6127-32, 2002 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-12444115

RESUMO

We recently reported that the direct antitumor effectors in the liver induced by alpha-galactosylceramide (alpha-GalCer) are NK cells that are activated by the IFN-gamma produced from NK1.1 Ag(+) T cells (NKT cells) specifically stimulated with alpha-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the alpha-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-gamma and IL-4 concentrations as well as alanine aminotransferase levels after the alpha-GalCer injection increased in an age-dependent manner. An alpha-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of alpha-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-gamma and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1(+) cells. Furthermore, liver mononuclear cells from old mice stimulated with alpha-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most alpha-GalCer-injected old mice, but no young mice, died, while anti-IFN-gamma Ab pretreatment completely inhibited mouse mortality. However, alpha-GalCer-induced hepatic injury did not improve at all by anti-IFN-gamma Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-gamma is responsible for alpha-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-gamma.


Assuntos
Envelhecimento/imunologia , Antígenos/metabolismo , Células Matadoras Naturais/imunologia , Fígado/imunologia , Proteínas/metabolismo , Subpopulações de Linfócitos T/imunologia , Alanina Transaminase/sangue , Animais , Antígenos Ly , Antígenos de Superfície , Divisão Celular , Citocinas/biossíntese , Citotoxicidade Imunológica , Proteína Ligante Fas , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Hepatócitos/imunologia , Técnicas In Vitro , Interferon gama/biossíntese , Interferon gama/sangue , Células Matadoras Naturais/citologia , Lectinas Tipo C , Ligantes , Fígado/lesões , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Subpopulações de Linfócitos T/citologia , Receptor fas/metabolismo
15.
Exp Cell Res ; 273(2): 147-56, 2002 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11822870

RESUMO

Although there have been many reports on the relationship between activation of telomerase and carcinogenesis, the role of telomerase in normal cellular growth is still unclear. In this study, we analyzed the relationship between upregulation of telomerase activity and cell cycle progression during the liver regeneration process by using an in vivo mouse two-thirds partial hepatectomy (PH) model as well as by using in vitro hepatocyte culture systems. Furthermore, we also investigated the effects of growth factors on telomerase activity during liver regeneration and the influence of MAPK pathway inhibitors (MEK inhibitors PD98059 and U0126; p38 MAPK inhibitor SB203580) on the telomerase activity of regenerating hepatocytes in vitro. An upregulation of the telomerase activity was found at 24 h after PH, and thereafter an increase in the S-phase fraction was observed at 36-48 h. There was no remarkable change in the telomere length after PH. Preoperative treatment with EGF and HGF increased the in vivo telomerase activity. In a hepatocyte primary culture, the upregulation of the telomerase activity required the presence of EGF, and this upregulation was accelerated by the addition of HGF. A remarkable activation of p44/42 MAPK was seen but no such activation of p38 MAPK was observed at 48 h after PH. Although SB203580 had no effect on the telomerase activity of regenerating hepatocytes, treatment with MEK inhibitors (PD 98059, U0126) significantly repressed the telomerase activity. In conclusion, the telomerase activity is upregulated before hepatocytes enter the S phase, and both EGF and HGF play important roles in this step. In addition, the activation of the p44/42 MAPK pathway seems to play an essential role in telomerase upregulation during the liver regeneration process.


Assuntos
Regeneração Hepática/fisiologia , Transdução de Sinais , Telomerase/metabolismo , Animais , Ciclo Celular , Divisão Celular , Células Cultivadas , Proteínas de Ligação a DNA , Ativação Enzimática , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Telomerase/genética , Telômero , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno
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