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1.
Adv Immunol ; 131: 61-99, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27235681

RESUMEN

The generation of antigen-specific neutralizing antibodies and memory B cells is one of the most important immune protections of the host and is the basis for successful vaccination strategies. The protective antibodies, secreted by preexisting long-lived plasma cells and reactivated antigen-experienced memory B cells, constitute the main humoral immune defense. Distinct from the primary antibody response, the humoral memory response is generated much faster and with greater magnitude, and it produces antibodies with higher affinity and variable isotypes. Humoral immunity is critically dependent on the germinal center where high-affinity memory B cells and plasma cells are generated. In this chapter, we focus on recent advances in our understanding of the molecular mechanisms that govern fate decision for memory B cells and plasma cells and the mechanisms that maintain the long-lived plasma-cell pool, with emphasis on how the transcription factor Blimp-1 (B lymphocyte-induced maturation protein-1) helps regulate the above-mentioned immunoregulatory steps to ensure the production and maintenance of antibody-secreting plasma cells as well as how it directs memory cell vs plasma-cell fate. We also discuss the molecular basis of Blimp-1 action and how its expression is regulated.


Asunto(s)
Células Productoras de Anticuerpos/inmunología , Linfocitos B/inmunología , Diferenciación Celular , Inmunidad Humoral , Memoria Inmunológica , Animales , Regulación de la Expresión Génica , Homeostasis , Humanos , Vacunación
2.
Cell Death Differ ; 23(7): 1175-84, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-26823144

RESUMEN

The transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) has crucial roles in the control of plasma cell differentiation and in maintaining survival of plasma cells. However, how Blimp-1 ensures the survival of plasma cell malignancy, multiple myeloma (MM), has remained elusive. Here we identified Aiolos, an anti-apoptotic transcription factor of MM cells, as a Blimp-1-interacting protein by mass spectrometry. ChIP coupled with DNA microarray was used to profile the global binding of Aiolos and Blimp-1 to endogenous targets in MM cells, which revealed their co-binding to a large number of genes, including apoptosis-related genes. Accordingly, Blimp-1 and Aiolos regulate similar transcriptomes in MM cells. Analysis of the binding motifs for Blimp-1 and Aiolos uncovered a partial motif that was similar across sites for both proteins. Aiolos promotes the binding of Blimp-1 to target genes and thereby enhances Blimp-1-dependent transcriptional repression. Furthermore, treatment with an anti-MM agent, lenalidomide, caused ubiquitination and proteasomal degradation of Blimp-1, leading to the de-repression of a new Blimp-1 direct target, CULLIN 4A (CUL4A), and reduced Aiolos levels. Accordingly, lenalidomide-induced cell death was partially rescued by reintroduction of Blimp-1 or knockdown of CUL4A. Thus, we demonstrated the functional impacts and underlying mechanisms of the interaction between Aiolos and Blimp-1 in maintaining MM cell survival. We also showed that interruption of Blimp-1/Aiolos regulatory pathways contributes to lenalidomide-mediated anti-MM activity.


Asunto(s)
Apoptosis , Factor de Transcripción Ikaros/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Inhibidores de la Angiogénesis/farmacología , Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Proteínas Cullin/antagonistas & inhibidores , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Células HEK293 , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/inmunología , Lenalidomida , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/antagonistas & inhibidores , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacología , Ubiquitinación/efectos de los fármacos
3.
Diabetologia ; 56(1): 136-46, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23052053

RESUMEN

AIMS/HYPOTHESIS: Recent reports indicate that B lymphocyte-induced maturation protein 1 (BLIMP-1), encoded by the Prdm1 gene, expands its control over T cells and is associated with susceptibility to colitis in mice with T cell-specific BLIMP-1 deficiency. In this study, we aimed to investigate the potential role of BLIMP-1 in regulating autoimmune diabetes and T helper type 17 (Th17) cells. METHODS: We generated T cell-specific Blimp1 (also known as Prdm1) transgenic (Tg) or conditional knockout (CKO) NOD mice, in which Blimp1 is overexpressed or deleted in T cells, respectively. By side-by-side analysing these Tg or CKO mice, we further dissected the potential mechanisms of BLIMP-1-mediated modulation on autoimmune diabetes. RESULTS: Overproduction of BLIMP-1 in T cells significantly attenuated insulitis and the incidence of diabetes in NOD mice. Consistent with these results, the diabetogenic effect of splenocytes was remarkably impaired in Blimp1 Tg mice. Moreover, overproduction of BLIMP-1 repressed the proliferation and activation of lymphocytes and enhanced the function of regulatory T cells (Tregs) in NOD mice. In contrast, mice lacking BLIMP-1 in T cells markedly increased Th1 and Th17 cells, and developed highly proliferative and activated lymphocytes. Strikingly, overexpansion of Th1 and Th17 cells in CKO mice was significantly reduced by introducing a Blimp1 transgene, reinforcing the emerging role of BLIMP-1 in autoimmunity. CONCLUSIONS/INTERPRETATION: We conclude that BLIMP-1 orchestrates a T cell-specific modulation of autoimmunity by affecting lymphocyte proliferation and activation, Th1 and Th17 cell differentiation, and Treg function. Our results provide a theoretical basis for developing BLIMP-1-manipulated therapies for autoimmune diabetes.


Asunto(s)
Autoinmunidad , Diabetes Mellitus Tipo 1/prevención & control , Terapia de Inmunosupresión , Páncreas/inmunología , Células TH1/inmunología , Células Th17/inmunología , Factores de Transcripción/biosíntesis , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Cruzamientos Genéticos , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Femenino , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Páncreas/patología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Organismos Libres de Patógenos Específicos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células TH1/patología , Células Th17/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
Mol Cell Biol ; 20(23): 8684-95, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11073970

RESUMEN

The importance of c-myc as a target of the Blimp-1 repressor has been studied in BCL-1 cells, in which Blimp-1 is sufficient to trigger terminal B-cell differentiation. Our data show that Blimp-1-dependent repression of c-myc is required for BCL-1 differentiation, since constitutive expression of c-Myc blocked differentiation. Furthermore, ectopic expression of cyclin E mimicked the effects of c-Myc on both proliferation and differentiation, indicating that the ability of c-Myc to drive proliferation is responsible for blocking BCL-1 differentiation. However, inhibition of c-Myc by a dominant negative form was not sufficient to drive BCL-1 differentiation. Thus, during Blimp-1-dependent plasma cell differentiation, repression of c-myc is necessary but not sufficient, demonstrating the existence of additional Blimp-1 target genes.


Asunto(s)
Linfocitos B/citología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Ciclina E/metabolismo , Citocinas/farmacología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoglobulina M/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Mutación , Células Plasmáticas/citología , Proteoglicanos/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/biosíntesis , Sindecanos , Factores de Transcripción/genética
5.
J Immunol ; 165(10): 5462-71, 2000 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-11067898

RESUMEN

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor that is sufficient to trigger terminal differentiation in the B cell lymphoma BCL-1. In this study, we have determined the expression pattern of Blimp-1 in vivo in primary and secondary lymphoid organs of humans and immunized mice. Blimp-1 is expressed in plasma cells derived from either a T-independent or T-dependent response in plasma cells that have undergone isotype switching and those resulting from secondary immunization. Blimp-1 is also present in long-lived plasma cells residing in the bone marrow. However, Blimp-1 was not detected in memory B cells. This expression pattern provides further evidence of a critical role for Blimp-1 in plasma cell development, supporting earlier studies in cultured lines. Significantly, Blimp-1 was also found in a fraction (4-15%) of germinal center B cells in murine spleen and human tonsils. Blimp-1 expression in the germinal center is associated with an interesting subset of cells with a phenotype intermediate between germinal center B cells and plasma cells. In the mouse, Blimp-1(+) germinal center B cells peak at day 12 postimmunization and disappear soon thereafter. They are not apoptotic, some are proliferating, they express germinal center markers peanut agglutinin or CD10 but not Bcl-6, and most express CD138 (syndecan-1), IRF4, and cytoplasmic Ig. Together, these data support a model in which B cell fate decisions occur within the germinal center and Blimp-1 expression is critical for commitment to a plasma cell, rather than a memory cell, fate.


Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Ficoll/análogos & derivados , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Proteínas Represoras , Factores de Transcripción/biosíntesis , Animales , Antígenos T-Independientes/inmunología , Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Ficoll/administración & dosificación , Ficoll/inmunología , Centro Germinal/citología , Haptenos/administración & dosificación , Haptenos/inmunología , Hemocianinas/administración & dosificación , Hemocianinas/inmunología , Humanos , Inmunización Secundaria , Memoria Inmunológica , Inmunofenotipificación , Inyecciones Intraperitoneales , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Nitrofenoles/administración & dosificación , Nitrofenoles/inmunología , Fenilacetatos , Células Plasmáticas/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva
6.
Nat Immunol ; 1(6): 526-32, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11101876

RESUMEN

Class II transactivator (CIITA), a coactivator required for class II major histocompatibility complex (MHC) transcription, is expressed in B cells but extinguished in plasma cells. This report identifies B lymphocyte-induced maturation protein I (BLIMP-I), a transcriptional repressor that is capable of triggering plasma cell differentiation, as a developmentally regulated repressor of CIITA transcription. BLIMP-I represses the B cell-specific promoter of the human gene that encodes CIITA (MHC2TA) in a binding site-dependent manner. Decreased CIITA correlates with increased BLIMP-I during plasma cell differentiation in cultured cells. Ectopic expression of BLIMP-I represses endogenous mRNA for CIITA and the CIITA targets, class II MHC, invariant chain and H2-DM (the murine equivalent of HLA-DM) in primary splenic B cells as well as 18-81 pre-B cells. Thus, the BLIMP-I program of B cell differentiation includes loss of antigen presentation via extinction of CIITA expression.


Asunto(s)
Proteínas Nucleares , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Proteínas Represoras , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Presentación de Antígeno , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Secuencia de Bases , Diferenciación Celular , Línea Celular , ADN/genética , Cartilla de ADN/genética , Antígenos HLA-DR/genética , Cadenas alfa de HLA-DR , Humanos , Células Híbridas , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/genética , Factores de Transcripción/genética
7.
J Neurosci ; 19(22): 9821-30, 1999 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-10559391

RESUMEN

Iron chelators are pluripotent neuronal antiapoptotic agents that have been shown to enhance metabolic recovery in cerebral ischemia models. The precise mechanism(s) by which these agents exert their effects remains unclear. Recent studies have demonstrated that iron chelators activate a hypoxia signal transduction pathway in non-neuronal cells that culminates in the stabilization of the transcriptional activator hypoxia-inducible factor-1 (HIF-1) and increased expression of gene products that mediate hypoxic adaptation. We examined the hypothesis that iron chelators prevent oxidative stress-induced death in cortical neuronal cultures by inducing expression of HIF-1 and its target genes. We report that the structurally distinct iron chelators deferoxamine mesylate and mimosine prevent apoptosis induced by glutathione depletion and oxidative stress in embryonic cortical neuronal cultures. The protective effects of iron chelators are correlated with their ability to enhance DNA binding of HIF-1 and activating transcription factor 1(ATF-1)/cAMP response element-binding protein (CREB) to the hypoxia response element in cortical cultures and the H19-7 hippocampal neuronal cell line. We show that mRNA, protein, and/or activity levels for genes whose expression is known to be regulated by HIF-1, including glycolytic enzymes, p21(waf1/cip1), and erythropoietin, are increased in cortical neuronal cultures in response to iron chelator treatment. Finally, we demonstrate that cobalt chloride, which also activates HIF-1 and ATF-1/CREB in cortical cultures, also prevents oxidative stress-induced death in these cells. Altogether, these results suggest that iron chelators exert their neuroprotective effects, in part, by activating a signal transduction pathway leading to increased expression of genes known to compensate for hypoxic or oxidative stress.


Asunto(s)
Apoptosis/fisiología , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/farmacología , Ciclinas/genética , Proteínas de Unión al ADN/metabolismo , Deferoxamina/farmacología , Eritropoyetina/genética , Regulación de la Expresión Génica , Neuronas/citología , Neuronas/fisiología , Proteínas Nucleares/metabolismo , Estrés Oxidativo/fisiología , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 1 , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Quelantes/farmacología , Cobalto/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inducción Enzimática/efectos de los fármacos , Feto , Fructosa-Bifosfato Aldolasa/biosíntesis , Fructosa-Bifosfato Aldolasa/genética , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Glucólisis , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , L-Lactato Deshidrogenasa/biosíntesis , L-Lactato Deshidrogenasa/genética , Mimosina/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores , Ratas , Ratas Sprague-Dawley
8.
J Biol Chem ; 274(19): 13650-5, 1999 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-10224137

RESUMEN

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O-2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O-2 levels appears to activate or modulate cell death, as a recombinant SV expressing the O-2 scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O-2 rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O-2 by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.


Asunto(s)
Apoptosis/fisiología , Virus Sindbis/fisiología , Superóxidos/metabolismo , Células 3T3 , Animales , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Paraquat/farmacología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Virus Sindbis/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Células Tumorales Cultivadas
10.
Biochem Biophys Res Commun ; 249(2): 325-31, 1998 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-9712695

RESUMEN

The calcium-sensing receptor (CaR) is a membrane-bound, G-protein-coupled receptor present on parathyroid cells which monitors the level of extracellular calcium (Ca2+o) and transduces signals involved in serum calcium regulation. Expression of CaR protein in tissues with functions unrelated to systemic calcium homeostasis, including the brain, suggests that extracellular calcium (Ca2+o) may act as a first messenger to regulate diverse cellular functions. To test this hypothesis, we examined the effect of increasing Ca2+o on apoptosis induced by Sindbis Virus in AT-3 prostate carcinoma cells. We found a steep increase in cell survival with between 5 and 7 mM added Ca2+o (EC50 = 6.1 mM). Magnesium, a less potent agonist of the calcium sensing receptor, was also protective (EC50 = 23.4 mM). Northern and immunocytochemical analyses confirmed the presence of the CaR message and protein in AT-3 prostate carcinoma cells. Enforced expression of CaR protein by stable transfection in human embryonic kidney (HEK)-293 cells, which normally don't express the receptor, resulted in resistance to SV-induced apoptosis in the presence of elevated Ca2+o. In addition to preventing SV-induced death, elevated Ca2+o also abrogated apoptosis induced by c-Myc overexpression/serum deprivation in rat 1A fibroblasts, and these fibroblasts were shown to express CaR message and protein. Altogether, these observations suggest that Ca2+o can act with the CaR to prevent apoptosis and define a novel mechanism by which calcium ions can regulate cell survival.


Asunto(s)
Apoptosis/efectos de los fármacos , Calcio/farmacología , Receptores de Superficie Celular/fisiología , Animales , Northern Blotting , Calcio/administración & dosificación , Línea Celular , Embrión de Mamíferos , Expresión Génica , Humanos , Inmunohistoquímica , Riñón , Masculino , Neoplasias de la Próstata , Ratas , Receptores Sensibles al Calcio , Receptores de Superficie Celular/genética , Virus Sindbis/fisiología , Transfección , Células Tumorales Cultivadas
11.
J Cell Biol ; 141(7): 1479-87, 1998 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-9647642

RESUMEN

Recent studies have established cell type- specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-kappaB. In each of these studies, inhibitors of NF-kappaB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-kappaB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-kappaB activation and apoptosis in cultured cell lines. To address whether Sindbis virus- induced NF-kappaB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-kappaB activity. Complete suppression of virus-induced NF-kappaB activity neither prevents nor potentiates Sindbis virus-induced apoptosis. In contrast, inhibition of NF-kappaB activity before infection inhibits Sindbis virus-induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-kappaB activity, regulates expression of gene products required for Sindbis virus-induced death. Furthermore, we show that in the same cell line, NF-kappaB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-kappaB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-kappaB activity relative to the death stimulus.


Asunto(s)
Apoptosis , Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , FN-kappa B/metabolismo , Virus Sindbis/fisiología , Células 3T3 , Acetilcisteína/farmacología , Animales , Antivirales/farmacología , Proteínas de Unión al ADN/genética , Vectores Genéticos , Peróxido de Hidrógeno/toxicidad , Ratones , Inhibidor NF-kappaB alfa , Subunidad p50 de NF-kappa B , Ratas , Estaurosporina/toxicidad , Factores de Tiempo , Factor de Transcripción ReIA , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/toxicidad
12.
J Neurosci ; 18(11): 4083-95, 1998 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-9592089

RESUMEN

Catalase is an antioxidant enzyme that has been shown to inhibit apoptotic or necrotic neuronal death induced by hydrogen peroxide. We report the purification of a contaminating antiapoptotic activity from a commercial bovine liver catalase preparation by following its ability to inhibit apoptosis when applied extracellularly in multiple death paradigms. The antiapoptotic activity was identified by protein microsequencing as arginase, a urea cycle and nitric oxide synthase-regulating enzyme, and confirmed by demonstrating the presence of antiapoptotic activity in a >97% pure preparation of recombinant arginase. The pluripotency of recombinant arginase was demonstrated by its ability to inhibit apoptosis in multiple paradigms including rat cortical neurons induced to die by glutathione depletion and oxidative stress, by 100 nM staurosporine treatment, or by Sindbis virus infection. The protective effects of arginase in these apoptotic paradigms, in contrast to previous studies on excitotoxic neuronal necrosis, are independent of nitric oxide synthase inhibition. Rather, arginase-induced depletion of arginine leads to inhibition of protein synthesis, resulting in cell survival. Because inhibitors of nitric oxide synthesis and of protein synthesis have been shown to decrease necrotic and apoptotic death, respectively, in animal models of stroke and spinal cord injury, arginine-depleting enzymes, capable of simultaneously inhibiting protein synthesis and nitric oxide generation, may be propitious therapeutic agents for acute neurological diseases. Furthermore, our results suggest caution in attributing the cytoprotective effects of some catalase preparations to catalase.


Asunto(s)
Apoptosis/fisiología , Arginasa/metabolismo , Hígado/enzimología , Neuronas/citología , Óxido Nítrico/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Arginasa/genética , Arginasa/farmacología , Catalasa/genética , Catalasa/metabolismo , Catalasa/farmacología , Bovinos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Inhibidores Enzimáticos/farmacología , Feto/citología , Glutatión/metabolismo , Datos de Secuencia Molecular , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/fisiología , Ratas , Ratas Sprague-Dawley , Estaurosporina/farmacología
13.
Cell Death Differ ; 5(7): 577-83, 1998 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10200512

RESUMEN

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.


Asunto(s)
Apoptosis , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Leupeptinas/farmacología , Complejos Multienzimáticos/metabolismo , Relación Dosis-Respuesta a Droga , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal , Virus Sindbis/fisiología
14.
J Immunol ; 158(5): 2318-26, 1997 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-9036980

RESUMEN

The unique immunologic environment of the central nervous system (CNS) regulates most local inflammatory responses. In some circumstances, however, immune-mediated injury to the brain can occur. To understand how lymphocytes are regulated within the CNS during an inflammatory response that does not produce immunopathology, we have studied T cells isolated from the brains of mice with Sindbis virus (SV) encephalitis. Even though they express activation markers, these T cells are arrested in the cell cycle and do not proliferate in vitro. Altered phosphorylation of the retinoblastoma gene product, a critical cell cycle regulator, appears to mediate this effect. Furthermore, while brain-derived T cells generate IFN-gamma, IL-4, and IL-10, these T cells are deficient in IL-2 production compared with peripheral T cells. This pattern of cytokine production occurs in cells that do not activate NF-kappaB normally. When T cells producing both IL-2 and IFN-gamma are adoptively transferred into SV-infected mice, some of these cells traffic into the brain. Those that enter the brain selectively down-regulate IL-2 production over time. Since normal brain lipids can inhibit IL-2 production and T cell proliferation in vitro, these substances may mediate these same effects in vivo. Collectively, these data show that the local environment of the CNS during SV encephalitis exerts a complex regulatory effect on T cells that are recruited into the brain. We speculate that this effect serves to prevent excessive local T cell reactivity. Whether and how this regulation might fail in the setting of autoimmune neurologic disease remains to be explored.


Asunto(s)
Infecciones por Alphavirus/inmunología , Encéfalo/inmunología , Encefalitis Viral/inmunología , Linfocitos T/inmunología , Enfermedad Aguda , Traslado Adoptivo , Infecciones por Alphavirus/metabolismo , Infecciones por Alphavirus/patología , Animales , Encéfalo/metabolismo , Química Encefálica/inmunología , Células Cultivadas , Citocinas/biosíntesis , Citocinas/metabolismo , Encefalitis Viral/metabolismo , Encefalitis Viral/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , FN-kappa B/deficiencia , FN-kappa B/metabolismo , Fosforilación , Proteína de Retinoblastoma/metabolismo , Virus Sindbis/inmunología , Linfocitos T/metabolismo , Linfocitos T/trasplante
15.
J Immunol ; 157(10): 4333-40, 1996 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-8906807

RESUMEN

Gangliosides may regulate the activity of the immune system in vivo, particularly within tissues such as neoplasms or the central nervous system, where they are most abundant. However, the specific mechanisms by which gangliosides modulate immune function remain incompletely understood. We have characterized the effects that brain-derived gangliosides have on specific steps of the T cell activation process in vitro. Gangliosides inhibit T cell proliferation downstream from the early activation events that are bypassed pharmacologically using the combination of a phorbol ester plus a calcium ionophore. These lipids block IL-2 and IFN-gamma gene transcription without inhibiting the production of IL-4 and IL-10 mRNA. This may be accounted for by the ability of gangliosides to prevent the activation of NF-kappaB in mitogen-stimulated T cells. Despite inhibiting IL-2 production, the antiproliferative effects of gangliosides are not reversed by adding supplemental IL-2 to the culture media. This defect persists because gangliosides also block the entry of activated T cells into the cell cycle. In this setting, phosphorylation of the retinoblastoma gene product, a protein whose phosphorylation state is an important regulator of normal cell cycle progression, is prevented. These studies help to define how gangliosides modulate T cell effector function in vitro. They also highlight the fact that certain T cell responses, namely the production of Th2-associated cytokines, are not inhibited by their actions.


Asunto(s)
Encéfalo/metabolismo , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Gangliósidos/biosíntesis , Gangliósidos/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Interleucina-2/farmacología , Ionóforos/farmacología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/antagonistas & inhibidores , FN-kappa B/biosíntesis , Fosforilación/efectos de los fármacos , Proteína de Retinoblastoma/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología
16.
J Cell Biol ; 131(5): 1149-61, 1995 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8522579

RESUMEN

Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SV-induced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 microM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Over-expression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SV-induced NF-kappa B activation. To determine if NF-kappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.


Asunto(s)
Apoptosis , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Virus Sindbis/fisiología , Reactivos de Sulfhidrilo/farmacología , Acetilcisteína/farmacología , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Quelantes/farmacología , ADN , Ditiotreitol/farmacología , Hierro , Peroxidación de Lípido/efectos de los fármacos , Masculino , Mercaptoetanol/farmacología , Ratones , Datos de Secuencia Molecular , FN-kappa B/antagonistas & inhibidores , Neuroblastoma/patología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Pirrolidinas/farmacología , Ratas , Tiocarbamatos/farmacología , Células Tumorales Cultivadas
17.
Biotechniques ; 14(5): 795-8, 1993 May.
Artículo en Inglés | MEDLINE | ID: mdl-8512705

RESUMEN

An alternative dimethoxytrityl-on (dmt-on) method is described to purify hydrophobic oligodeoxyribonucleoside methyl-phosphonates (OM) with a phosphodiester linkage at the 5' end, instead of the conventional dmt-off method using a DEAE ion-exchange column. This method is modified from the reverse-phase method for purification of normal oligonucleotides.


Asunto(s)
Oligodesoxirribonucleótidos/aislamiento & purificación , Secuencia de Bases , Biotecnología , Cromatografía DEAE-Celulosa , ADN sin Sentido/química , ADN sin Sentido/aislamiento & purificación , Etilenodiaminas , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Organofosfonatos/aislamiento & purificación
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