RESUMEN
At the end of an immune response, apoptosis drastically reduces the numbers of activated T cells. It has been a matter of intense research how this form of apoptosis is regulated and initiated, and a number of proteins have been identified that contribute to this process. The present, widely accepted model assumes that the interplay of pro- and anti -apoptotic Bcl-2 family members determines the onset of activated T cell death, with the BH3-only protein Bim activating pro-apoptotic Bax/Bak. In the search for up-stream signals, factors from other immune cells have been shown to play a role, and the NFkappaB family member Bcl-3 has been implicated as a signalling-intermediate in T cells. Recent work has tested the interrelation of these factors and has suggested that Bcl-3 acts as a regulator of Bim activation, that the induction of apoptosis through Bim can be complemented by its relative Puma, and that the presence of certain cytokines during T cell activation delays the activation of Bim and Puma. Here we discuss these recent insights and provide a view on how the regulation of activated T cell death is achieved and how extrinsic signals may translate into the activation of the apoptotic pathway.
Asunto(s)
Apoptosis , Transducción de Señal , Linfocitos T/patología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Antígenos CD28/metabolismo , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Humanos , Sistema Inmunológico , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/metabolismo , Transcripción GenéticaRESUMEN
Apoptosis of activated T cells is critical for the termination of immune responses. Here we show that adjuvant-stimulated dendritic cells secrete cytokines that prime activated T cells for survival and analyze the roles of the NF-kappaB regulator Bcl-3 and the proapoptotic Bcl-2 family members Bim and Puma. Bcl-3 overexpression increased survival, and activated bcl-3-/- T cells died abnormally rapidly. Cytokines from adjuvant-stimulated dendritic cells induced Bcl-3, but survival through cytokine priming was Bcl-3-independent. Apoptosis inhibition by Bcl-3 involved blockade of Bim activation, because Bim was overactivated in Bcl-3-deficient cells, and Bcl-3 failed to increase survival of bim-/- T cells. However, adjuvants increased survival also in Bim-deficient T cells. This Bim-independent death pathway is at least in part regulated by Puma, as shown by analysis of puma-/- and noxa-/- T cells. IL-1, IL-7, and IL-15 primed T cells for survival even in the absence of Bim or Puma. Our data define interrelations and a Bim-independent pathway to activated T cell death.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adyuvantes Inmunológicos , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas del Linfoma 3 de Células B , Proteína 11 Similar a Bcl2 , Células Cultivadas , Células Dendríticas/metabolismo , Interleucinas/farmacología , Activación de Linfocitos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Solubilidad , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Factores de Transcripción , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Vibrio parahaemolyticus, V. cholerae, and V. vulnificus were isolated from 10.3%, 1.0%, and 0.1% of 885 blue mussel samples, respectively. Four of the samples contained trh(+) V. parahaemolyticus, while no tdh-positive isolates were detected. The V. cholerae isolates were non-O:1/non-O:139 serotypes and were ctxA negative.
Asunto(s)
Mytilus edulis/microbiología , Vibrio cholerae/aislamiento & purificación , Vibrio parahaemolyticus/aislamiento & purificación , Vibrio vulnificus/aislamiento & purificación , Animales , Proteínas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Noruega , Vibrio cholerae/clasificación , Vibrio cholerae/genética , Vibrio parahaemolyticus/genética , Vibrio vulnificus/genéticaRESUMEN
Toll-like receptors (TLR) are activated by microbial components and transmit signals that induce cell activation and differentiation. A number of recent reports further indicate that TLR also have the potential to induce apoptosis upon ligand binding. Here we investigate the apoptosis-inducing capacity of TLR9, the receptor for microbial CpG-DNA. Unlike ligands for TLR2 and TLR4, CpG-DNA failed to induce apoptosis in RAW264.7 mouse macrophages. In human embryonic kidney fibroblasts transfected stably to express TLR9, CpG-DNA weakly induced apoptosis in one clone but not others without an obvious allocation to differences in TLR-signaling events. Analysis of the apoptotic signaling showed that the mitochondrial pathway of apoptosis was triggered by TLR9, as mitochondrial Bax was activated upstream of caspase-cleavage. CpG-DNA-induced apoptosis was reduced by cycloheximide suggesting that de novo protein synthesis was required. Strikingly, stimulation with CpG-DNA resulted in a strongly increased sensitivity of TLR9-expressing fibroblasts to apoptosis induced by staurosporine and UV-irradiation. These results identify a mitochondrial pathway to apoptosis that can be triggered by TLR9 and that may serve to sensitize cells from the innate immune system to apoptosis in the course of an immune response.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/fisiología , Fibroblastos/fisiología , Receptores de Superficie Celular/fisiología , Animales , Apoptosis/genética , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Receptor Toll-Like 9RESUMEN
The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of the CN ligands of the active site iron of the NiFe-hydrogenases using carbamoylphosphate as a substrate. HypE protein from Escherichia coli was purified from a transformant overexpressing the hypE gene from a plasmid. Purified HypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statistically significant amounts of metals or of cofactors absorbing in the UV and visible light range. The protein displays low intrinsic ATPase activity with ADP and phosphate as the products, the apparent K(m) being 25 micro m and the k(cat) 1.7 x 10(-3) s(-1). Removal of the C-terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the K(m) (47 micro m) but not significantly the k(cat) (2.1 x 10(-3) s(-1)). During the carbamoyltransfer reaction, HypE and HypF enter a complex which is rather tight at stoichiometric ratios of the two proteins. A mutant HypE variant was generated by amino acid replacements in the nucleoside triphosphate binding region, which showed no intrinsic ATPase activity. The variant was active as an acceptor in the transcarbamoylation reaction but did not dehydrate the thiocarboxamide to the thiocyanate. The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Hidrogenasas/química , Hidrogenasas/metabolismo , Agua/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Catálisis , Mutación/genéticaRESUMEN
HypF has been characterized as an auxiliary protein whose function is required for the synthesis of active [NiFe] hydrogenases in Escherichia coli and other bacteria. To approach the functional analysis, in particular the involvement in CO/CN ligand synthesis, HypF was purified from an overproducing strain to apparent homogeneity. The purified protein behaves as a monomer on size exclusion chromatography, and it is devoid of nickel or other cofactors. As indicated by the existence of a sequence motif also present in several O-carbamoyltransferases, HypF interacts with carbamoyl phosphate as a substrate and releases inorganic phosphate. In addition, HypF also possesses ATP cleavage activity that gives rise to AMP and pyrophosphate as products and that is dependent on the presence of carbamoyl phosphate. This and the fact that HypF catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP exchange reaction suggest that the protein catalyzes activation of carbamoyl phosphate. Extensive mutagenesis of the putative functional motifs deduced from the derived amino acid sequence showed a full correlation of the resulting variants between their activity in hydrogenase maturation and the in vitro reactivity with carbamoyl phosphate. The results are discussed in terms of the involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Carbamoil Fosfato/metabolismo , Catálisis , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Hidrogenasas/metabolismo , Hidrólisis , Cinética , Ligandos , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Unión Proteica , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
In contrast to other countries as USA, Australia and some European countries there are no German studies concerning psychiatric disorders in runaway and homeless children and adolescents. Although the presence of street youths faces a big problem, there is a deficit of empirical data among this group for the planning of more efficient differential programming. In this pilot study we investigated male and female street adolescents among ICD-10 respectively DSM-IV criteria. The goal of this study was to build gender-specific hypotheses and to investigate the applicability and practicability of the standardised diagnostic instruments assessing psychiatric and substance-related disorders.