RESUMEN
BACKGROUND: The intake of certain types of resistant starch (RS) has been associated in some studies with increased whole-body insulin sensitivity. This randomised, cross-over pilot trial evaluated the effect of consuming cooked, then chilled potatoes, a source of RS, compared to isoenergetic, carbohydrate (CHO)-containing control foods, on insulin sensitivity and related markers. METHODS: Nineteen adults with body mass index 27.0-39.9 kg m-2 consumed 300 g day-1 RS-enriched potatoes (approximately two potatoes; ~18 g RS) or CHO-based control foods, as part of lunch, evening and snack meals, over a 24-h period. After an overnight fast, insulin sensitivity, CHO metabolism markers, free fatty acids, breath hydrogen levels and appetite were assessed for up to 5 h after the intake of a standard breakfast. The primary endpoint was insulin sensitivity, assessed with the Matsuda index. P < 0.05 (one-sided) was considered statistically significant. RESULTS: Insulin sensitivity was not significantly different between the potato and control conditions. The potato intervention resulted in higher postprandial breath hydrogen (P = 0.037), lower postprandial free fatty acid concentrations (P = 0.039) and lower fasting plasma glucose (P = 0.043) compared to the control condition. Fullness ratings were significantly lower after potato versus control (P = 0.002). No other significant effects were observed; however, there was a trend toward lower fasting insulin (P = 0.077) in the potato versus the control condition. CONCLUSIONS: The results of this pilot study suggest RS-enriched potatoes may have a favourable impact on carbohydrate metabolism and support the view that additional research in a larger study sample is warranted.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina , Almidón Resistente/administración & dosificación , Adulto , Apetito/efectos de los fármacos , Biomarcadores/metabolismo , Glucemia/metabolismo , Estudios Cruzados , Ácidos Grasos no Esterificados/metabolismo , Femenino , Humanos , Masculino , Comidas , Persona de Mediana Edad , Proyectos Piloto , Solanum tuberosum/químicaRESUMEN
Fifteen primiparous crossbred dairy cows that were 114±14d in milk and weighed 533±56kg were used in a replicated 5×5 Latin square to test the efficacy of a calcium montmorillonite clay, NovaSil Plus (NSP; BASF Corp., Ludwigshaven, Germany), for the reduction of aflatoxin (AF) metabolite (AFM1) in milk and the effect of NSP on milk composition. Cows were housed in a freestall barn, fed once a day and milked twice a day. The experiment consisted of five 14-d periods: d 1 through 7 were considered for data collection, and d 8 through 14 were considered a wash-out phase. In each period, cows were randomly assigned to 1 of 5 dietary treatments: (1) control (CON), consisting of a basal total mixed ration (TMR); (2) high-dose NSP diet (NSP-1%), consisting of TMR plus 230 g of NSP; (3) aflatoxin diet (AFD), consisting of the TMR plus AF challenge; (4) low-dose NSP with AF (NSP-0.5%+AFD), composed of TMR plus 115 g of NSP and AF challenge; and (5) high-dose NSP with AF (NSP-1%+AFD), consisting of TMR plus 230 g of NSP and AF challenge. The AF challenge consisted of top dressing a daily dose of 100 µg/kg estimated dry matter intake (DMI); similarly, NSP was fed at 1.0 or 0.5% of estimated DMI. Milk yield and DMI were similar across treatments averaging 21.1±1.33 kg/d and 19.7±0.56 kg/d, respectively. Concentration of milk fat, protein, and lactose were similar across treatments with averages of 4.91±0.20%, 3.85±0.10%, and 4.70±0.06%, respectively. Concentration of vitamin A averaged 0.28±0.03 µg/mL and riboflavin concentration averaged 1.57±0.13 µg/mL across treatments. The concentration of minerals in milk were similar for all treatments. Cows fed CON and NSP-1% yielded the lowest concentration of AFM1 in milk with 0.03 and 0.01±0.06 µg/L. Addition of NSP reduced milk AFM1 from 1.10±0.06 µg/L with the AF diet to 0.58 and 0.32±0.06 µg/L with the NSP-0.5%+AF and NSP-1%+AF diets, respectively. Excretion of AFM1 was reduced by NSP; mean values were 24.38, 11.86, 7.38, 0.64, and 0.23, ± 1.71 µg/d, for AFD, NSP-0.5%+AFD, NSP-1%+AFD, NSP-1%, and CON, respectively. More specifically, 1.07±0.08% of the daily AF intake was transferred to the milk of cows consuming the AFD, whereas the AF transfer rates in milk from cows that consumed the NSP-0.5%+AFD and NSP-1%+AFD were 0.52 and 0.32±0.08%. Results from this research demonstrate that feeding NSP to lactating cows is an effective method to reduce the transfer and excretion of AFM1 in milk with no negative effects on dry matter intake, milk production, and composition.
Asunto(s)
Aflatoxinas/toxicidad , Silicatos de Aluminio/química , Bentonita/farmacología , Bovinos/fisiología , Lactancia/efectos de los fármacos , Aflatoxina M1/análisis , Alimentación Animal/análisis , Animales , Calcio/farmacología , Calcio de la Dieta/metabolismo , Arcilla , Dieta/veterinaria , Suplementos Dietéticos , Ingestión de Alimentos/efectos de los fármacos , Femenino , Leche/químicaRESUMEN
We investigated the diversity of endophytic fungi found on grape (Vitis labrusca cv. Niagara Rosada) leaves collected from Salesópolis, SP, Brazil. The fungi were isolated and characterized by amplified ribosomal DNA restriction analysis, followed by sequencing of the ITS1-5.8S-ITS2 rDNA. In addition, the ability of these endophytic fungi to inhibit the grapevine pathogen Fusarium oxysporum f. sp herbemontis was determined in vitro. We also observed that the climatic factors, such as temperature and rainfall, have no effect on the frequency of infection by endophytic fungi. The endophytic fungal community that was identified included Aporospora terricola, Aureobasidium pullulans, Bjerkandera adusta, Colletotrichum boninense, C. gloeosporioides, Diaporthe helianthi, D. phaseolorum, Epicoccum nigrum, Flavodon flavus, Fusarium subglutinans, F. sacchari, Guignardia mangiferae, Lenzites elegans, Paraphaeosphaeria pilleata, Phanerochaete sordida, Phyllosticta sp, Pleurotus nebrodensis, Preussia africana, Tinctoporellus epiniltinus, and Xylaria berteri. Among these isolates, two, C. gloeosporioides and F. flavus, showed potential antagonistic activity against F. oxysporum f. sp herbemontis. We suggest the involvement of the fungal endophyte community of V. labrusca in protecting the host plant against pathogenic Fusarium species. Possibly, some endophytic isolates could be selected for the development of biological control agents for grape fungal disease; alternatively, management strategies could be tailored to increase these beneficial fungi.
Asunto(s)
Ascomicetos/genética , Basidiomycota/genética , Endófitos/genética , Fusarium/fisiología , Hojas de la Planta/microbiología , Vitis/microbiología , Antibiosis , Agentes de Control Biológico , ADN Espaciador Ribosómico/genética , Haplotipos , Tipificación Molecular , Técnicas de Tipificación Micológica , Filogenia , Enfermedades de las Plantas/microbiología , ARN de Hongos/genética , ARN Ribosómico/genética , Análisis de Secuencia de ADNRESUMEN
Wild-type p53 is a stress-responsive tumor suppressor and potent growth inhibitor. Genotoxic stresses (for example, ionizing and ultraviolet radiation or chemotherapeutic drug treatment) can activate p53, but also induce mutations in the P53 gene, and thus select for p53-mutated cells. Nutlin-3a (Nutlin) is pre-clinical drug that activates p53 in a non-genotoxic manner. Nutlin occupies the p53-binding pocket of murine double minute 2 (MDM2), activating p53 by blocking the p53-MDM2 interaction. Because Nutlin neither binds p53 directly nor introduces DNA damage, we hypothesized Nutlin would not induce P53 mutations, and, therefore, not select for p53-mutated cells. To test this, populations of SJSA-1 (p53 wild-type) cancer cells were expanded that survived repeated Nutlin exposures, and individual clones were isolated. Group 1 clones were resistant to Nutlin-induced apoptosis, but still underwent growth arrest. Surprisingly, while some Group 1 clones retained wild-type p53, others acquired a heterozygous p53 mutation. Apoptosis resistance in Group 1 clones was associated with decreased PUMA induction and decreased caspase 3/7 activation. Group 2 clones were resistant to both apoptosis and growth arrest induced by Nutlin. Group 2 clones had acquired mutations in the p53-DNA-binding domain and expressed only mutant p53s that were induced by Nutlin treatment, but were unable to bind the P21 and PUMA gene promoters, and unable to activate transcription. These results demonstrate that non-genotoxic p53 activation (for example, by Nutlin treatment) can lead to the acquisition of somatic mutations in p53 and select for p53-mutated cells. These findings have implications for the potential clinical use of Nutlin and other small molecule MDM2 antagonists.
Asunto(s)
Imidazoles/farmacología , Mutación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Células Clonales , Humanos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Osteosarcoma/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismoAsunto(s)
Carcinoma Papilar/secundario , Cistadenocarcinoma Seroso/secundario , Neoplasias Ováricas/patología , Neoplasias del Cuello Uterino/secundario , Adenocarcinoma Papilar/tratamiento farmacológico , Adenocarcinoma Papilar/secundario , Adenocarcinoma Papilar/cirugía , Carcinoma Papilar/tratamiento farmacológico , Carcinoma Papilar/cirugía , Quimioterapia Adyuvante , Cistadenocarcinoma Seroso/tratamiento farmacológico , Cistadenocarcinoma Seroso/cirugía , Endometrio/patología , Trompas Uterinas/patología , Femenino , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/cirugía , Neoplasias del Cuello Uterino/cirugíaRESUMEN
P53 wild-type and p53-null or mutant cells undergo a G(2)-phase cell-cycle arrest in response to ionizing radiation (IR). In this study we examined the effect of heat-shock protein 90 (HSP90) inhibitor, geldanamycin (GA), on IR-induced G(2) arrest in human colon adenocarcinoma cells with different p53 status. We show that GA treatment abrogates IR-induced G(2)-phase arrest in cells null or mutant for p53. Specifically, GA treatment pushed irradiated p53 signaling-defective cells into a premature mitosis characterized by aberrant mitotic figures, increased gammaH2AX expression and formation of micronucleated cells. Cells expressing wild-type p53 were resistant to GA-induced G(2) checkpoint abrogation. Notably, GA treatment decreased levels of G(2) regulatory proteins Wee1 and Chk1, and inhibitory phosphorylation of Cdc2, independent of p53 status. Further investigation identified p21 as the potential downstream effector of p53 that mediates resistance to G(2) checkpoint abrogation. Clonogenic survival studies demonstrated higher sensitivity to GA alone or combination IR plus GA treatment in p53 and p21-null cells. Collectively, these data demonstrate potential mechanisms through which HSP90 inhibition can enhance the effects of ionizing radiation in p53-compromised cancer cells. Combination IR plus HSP90 inhibitor therapies may be particularly useful in treating cancers that lack wild-type p53.
Asunto(s)
Benzoquinonas/farmacología , Neoplasias del Colon/radioterapia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Mitosis/efectos de los fármacos , Proteína p53 Supresora de Tumor/fisiología , División Celular , Neoplasias del Colon/patología , Fase G2 , Células HCT116 , Células HT29 , HumanosRESUMEN
Mesenchymal stem cells are an easily obtainable stem cell source from bone marrow. Presently, they are the most widely used cell type for cellular replacement strategies in humans as a result of extensive research that has demonstrated that these cells are capable of self-renewal, able to undergo multi-lineage differentiation, engraft, and ameliorate symptoms in numerous animal models. In this study, we comprehensively characterize human second trimester mesenchymal stem cells (STMSCs). We demonstrate that STMSCs are easily expandable to clinical relevance and express pluripotent markers such as Oct-4, Nanog, Sox-2, and SSEA-4 at the cellular and molecular level. Moreover, we directionally differentiate STMSCs into osteogenic, chondrogenic, adipogenic, neurogenic, and cardiogenic cell lineages. These studies demonstrate the plasticity of STMSCs and the potential for their use in cellular replacement therapy.
Asunto(s)
Diferenciación Celular/genética , Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Células Madre Multipotentes/metabolismo , Actinas/genética , Actinas/metabolismo , Biomarcadores/análisis , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Citometría de Flujo , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Cariotipificación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Miocardio/citología , Miocardio/metabolismo , Proteína Homeótica Nanog , Sistema Nervioso/citología , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Embarazo , Segundo Trimestre del Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Osteopetrosis means a low function of osteoclasts, that induces bone resorption with a decrease in bone density. It has two genetic forms: a benign form with autosomal dominant inheritance (AD) and a malignant form with autosomal recessive inheritance (AR). The most successful therapy is bone marrow transplantation (rate of success 80%). We will present a case of malignant osteopetrosis of a five years old child. The late diagnosis (at 18 month) and the malignant evolution has made possible only palliative treatment, without surgical or ophthalmologic-neurosurgical implication. The prognosis is unfavorable with 70% survival to six years and 30% to ten years.
Asunto(s)
Exoftalmia/etiología , Órbita , Osteopetrosis/complicaciones , Osteopetrosis/diagnóstico , Cráneo , Ceguera/etiología , Médula Ósea/patología , Preescolar , Diagnóstico Diferencial , Femenino , Genes Recesivos , Humanos , Órbita/diagnóstico por imagen , Osteopetrosis/genética , Osteopetrosis/terapia , Cuidados Paliativos , Pronóstico , Radiografía , Cráneo/diagnóstico por imagenRESUMEN
Activation of dendritic cells (DCs) and macrophages by infectious agents leads to secretion of interleukin 12 (IL-12), which subsequently induces interferon-gamma (IFN-gamma) production by multiple cell types that include DCs and macrophages. In turn, IFN-gamma acts on macrophages to augment IL-12 secretion and to produce nitric oxide (NO), which eradicates infected microbes. We show here that in cytokine common gamma subunit-deficient and/or IL-2 receptor beta-deficient mice, production of IL-12, IFN-gamma and NO by DCs and macrophages was severely impaired, as was up-regulation of major histocompatibility complex class II and CD40. Similar phenotypes were observed in DCs and macrophages from IL-15-deficient mice but not in those from IL-2-deficient mice. This shows that the IL-15-IL-15R interaction is critical in early activation of antigen-presenting cells and plays an important role in the innate immune system.
Asunto(s)
Células Dendríticas/inmunología , Interleucina-15/fisiología , Macrófagos/inmunología , Receptores de Interleucina-2/fisiología , Animales , Antígenos CD40/biosíntesis , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Antígenos de Histocompatibilidad Clase II/biosíntesis , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Interleucina-12/farmacología , Interleucina-15/genética , Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2 , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/genética , Receptores de Interleucina-15 , Regulación hacia ArribaRESUMEN
MDM2 can bind the N terminus of p53 and promote its ubiquitination and export from the nucleus to the cytoplasm, where p53 can then be degraded by cytoplasmic proteasomes. Several studies have reported that an intact MDM2 binding domain is necessary for p53 to be targeted for ubiquitination, nuclear export, and degradation by MDM2. In the current study, we examined whether the MDM2 binding domain of p53 could be provided in trans through oligomerization between two p53 molecules. p53 proteins mutated in their MDM2 binding domains were unable to bind MDM2 directly and were resistant to MDM2-mediated ubiquitination, nuclear export, and degradation when expressed with MDM2 alone. However, these same p53 mutants formed a complex with MDM2 and were efficiently ubiquitinated, exported from the nucleus, and degraded when co-expressed with MDM2 and wild-type p53. Moreover, this effect required MDM2 binding by wild-type p53 as well as oligomerization between wild-type p53 and the MDM2 binding-deficient p53 mutants. Taken together, these results support a model whereby MDM2 binding-deficient forms of p53 can bind MDM2 indirectly through oligomerization with wild-type p53 and are subsequently targeted for ubiquitination, nuclear export, and degradation. These findings may have important implications regarding the DNA damage response of p53.
Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Daño del ADN , Epítopos , Genes p53/genética , Humanos , Immunoblotting , Microscopía Fluorescente , Mutación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-mdm2 , Estrés Fisiológico , Transfección , Células Tumorales CultivadasRESUMEN
Mice lacking IFN-regulatory factor (IRF)-1 have reduced numbers of mature CD8+ T cells within the thymus and peripheral lymphoid organs, suggesting a critical role of IRF-1 in CD8(+) T cell differentiation. Here we show that endogenous Bcl-2 expression is substantially reduced in IRF-1(-/-)CD8+ thymocytes and that introduction of a human Bcl-2 transgene driven by Emu or lck promoter in IRF-1(-/-) mice restores the CD8(+) T cell development. Restored CD8+ T cells are functionally mature in terms of allogeneic MLR and cytokine production. In contrast to thymus-derived CD8+ T cells, other lymphocyte subsets including NK, NK T, and TCR-gammadelta(+) intestinal intraepithelial lymphocytes, which are also impaired in IRF-1(-/-) mice, are not rescued by expressing human Bcl-2. Our results indicate that IRF-1 differentially regulates the development of these lymphocyte subsets and that survival signals involving Bcl-2 are critical for the development of thymus-dependent CD8+ T cells.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Mucosa Intestinal/metabolismo , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Subgrupos de Linfocitos T/metabolismo , Timo/citología , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/inmunología , Técnicas de Transferencia de Gen , Humanos , Factor 1 Regulador del Interferón , Interferón gamma/biosíntesis , Interferón gamma/fisiología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/genética , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfoproteínas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/administración & dosificación , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timo/inmunología , Transgenes/inmunologíaRESUMEN
Mimecan is a small leucine-rich proteoglycan that can occur as either keratan sulfate proteoglycan in the cornea or as glycoprotein in many connective tissues. As yet, there is no information on its transcriptional regulation. Recently we demonstrated the presence of eight mimecan mRNA transcripts generated by alternative transcription initiation, alternative polyadenylation, and differential splicing, all of which encode an identical protein. Here we report a conserved consensus p53-binding DNA sequence in the first intron of bovine and human mimecan genes and show that wild-type p53 binds to this sequence in vitro. Co-transfections of Saos-2, HeLa, NIH 3T3, and primary bovine corneal keratocytes with bovine mimecan promoter/luciferase reporter constructs in combination with p53 expression vectors activate the second mimecan promoter through the p53-binding sequence. In addition, we show absence of mimecan expression in different tumors and cancer cell lines, where p53 frequently is inactivated/mutated. Thus, this work provides novel information that links mimecan to the p53 network.
Asunto(s)
ADN/genética , ADN/metabolismo , Glicoproteínas/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Bovinos , Células Cultivadas , Sondas de ADN/genética , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Intrones , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional , Transfección , Células Tumorales CultivadasRESUMEN
p53 is stabilized in response to DNA damaging stress. This stabilization is thought to result from phosphorylation in the N-terminus of p53, which inhibits p53:MDM2 binding, and prevents MDM2 from promoting p53 ubiquitination. In this report, the DNA alkylating agents mitomycin C (MMC) and methylmethane sulfonate (MMS), as well as UV radiation, stabilized p53 in a manner independent of phosphorylation in p53 N-terminus. This stabilization coincided with decreased levels of MDM2 mRNA and protein, and a corresponding decrease in p53 ubiquitination. Importantly, MDM2 overexpression inhibited the stabilization of p53 and decrease in ubiquitination following MMC, MMS, and UV treatment. This indicates that downregulation of MDM2 contributes to the stabilization of p53 in response to these agents.
Asunto(s)
Alquilantes/farmacología , Regulación hacia Abajo/efectos de los fármacos , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinas/antagonistas & inhibidores , Ubiquitinas/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Regulación hacia Abajo/efectos de la radiación , Humanos , Metilmetanosulfonato/farmacología , Mitomicina/farmacología , Mutación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Fosfoserina/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Rayos UltravioletaRESUMEN
Wild-type p53 is stabilized and accumulates in the nucleus of DNA damaged cells. The effect of stabilizing p53 is to inhibit cell growth, either through a G1 cell cycle arrest or apoptotic cell death. MDM2 can inhibit p53 activity, in part, by promoting its rapid degradation through the ubiquitin proteolysis pathway. In the current study, MDM2-mediated degradation of p53 was partially inhibited in cells treated with leptomycin B (LMB), a specific inhibitor of nuclear export. In contrast, levels of ubiquitinated p53 increased in LMB-treated cells, indicating that nuclear export is not required for p53 ubiquitination. To investigate this further, p53 mutants were generated which localize to either the nucleus or cytoplasm, and their susceptibility to MDM2-mediated ubiquitination was assessed. p53 mutants that localized to either the nucleus or the cytoplasm were efficiently ubiquitinated, and their steady-state levels decreased, when coexpressed with MDM2. In addition, an MDM2-mutant that localized to the cytoplasm was able to ubiquitinate and degrade a p53 mutant which was similarly localized in the cytoplasm. Our results indicate that nuclear export is not required for p53 ubiquitination, and that p53 proteins that localize to either the nucleus or cytoplasm can be ubiquitinated and degraded by MDM2.
Asunto(s)
Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinas/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN/genética , Ácidos Grasos Insaturados/farmacología , Humanos , Masculino , Proteínas Proto-Oncogénicas c-mdm2 , Salmón , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ubiquitinas/genéticaRESUMEN
MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that nuclear export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 nuclear export has not been clarified. Here we show that MDM2 can promote the nuclear export of p53 in transiently transfected cells. This activity requires the nuclear-export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 nuclear export. Finally, inhibition of nuclear export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the export of p53 to the cytoplasm through the activity of the p53 NES.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Dedos de Zinc , Transporte Activo de Núcleo Celular , Humanos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ubiquitinas/metabolismoRESUMEN
p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.
Asunto(s)
Fase G1/efectos de la radiación , Proteína p53 Supresora de Tumor/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Animales , Técnicas de Cultivo de Célula , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Humanos , Factores de TiempoRESUMEN
We investigated the role of antigen-presenting cells in early interferon (IFN)-gamma production in normal and recombinase activating gene 2-deficient (Rag-2(-/-)) mice in response to Listeria monocytogenes (LM) infection and interleukin (IL)-12 administration. Levels of serum IFN-gamma in Rag-2(-/-) mice were comparable to those of normal mice upon either LM infection or IL-12 injection. Depletion of natural killer (NK) cells by administration of anti-asialoGM1 antibodies had little effect on IFN-gamma levels in the sera of Rag-2(-/-) mice after LM infection or IL-12 injection. Incubation of splenocytes from NK cell-depleted Rag-2(-/-) mice with LM resulted in the production of IFN-gamma that was completely blocked by addition of anti-IL-12 antibodies. Both dendritic cells (DCs) and monocytes purified from splenocytes were capable of producing IFN-gamma when cultured in the presence of IL-12. Intracellular immunofluorescence analysis confirmed the IFN-gamma production from DCs. It was further shown that IFN-gamma was produced predominantly by CD8alpha+ lymphoid DCs rather than CD8alpha- myeloid DCs. Collectively, our data indicated that DCs are potent in producing IFN-gamma in response to IL-12 produced by bacterial infection and play an important role in innate immunity and subsequent T helper cell type 1 development in vivo.
Asunto(s)
Antígenos CD8/inmunología , Células Dendríticas/inmunología , Interferón gamma/metabolismo , Interleucina-12/farmacología , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Células Asesinas Naturales/inmunología , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Ratones , Ratones Noqueados , Bazo/inmunología , Transposasas/genéticaRESUMEN
Wild-type p53 is degraded in part through the ubiquitin proteolysis pathway. Recent studies indicate that MDM2 can bind p53 and promote its rapid degradation although the molecular basis for this degradation has not been clarified. This report demonstrates that MDM2 can promote the ubiquitination of wild-type p53 and cancer-derived p53 mutants in transiently transfected cells. Deletion mutants that disrupted the oligomerization domain of p53 displayed low binding affinity for MDM2 and were poor substrates for ubiquitination. However, efficient MDM2 binding and ubiquitination were restored when an oligomerization-deficient p53 mutant was fused to the dimerization domain from another protein. These results indicate that oligomerization is required for p53 to efficiently bind and be ubiquitinated by MDM2. p53 ubiquitination was inhibited in cells exposed to UV radiation, and this inhibition coincided with a decrease in MDM2 protein levels and p53.MDM2 complex formation. In contrast, p53 dimerization was unaffected following UV treatment. These results suggest that UV radiation may stabilize p53 by blocking the ubiquitination and degradation of p53 mediated by MDM2.
Asunto(s)
Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Dedos de Zinc , Humanos , Mutación Puntual , Conformación Proteica , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/efectos de la radiación , Ubiquitinas/metabolismo , Rayos UltravioletaRESUMEN
Using mice deficient for LFA-1, CD44, and ICAM-1, we examined the role of these adhesion molecules in NK1.1+TCR alpha beta+ (NKT) cell development. Although no defect in NKT cell development was observed in CD44-/- and ICAM-1-/- mice, a dramatic reduction of liver NKT cells was observed in LFA-1-/- mice. Normal numbers of NKT cells were present in other lymphoid organs in LFA-1-/- mice. When LFA-1-/- splenocytes were injected i.v. into wild-type mice, the frequency of NKT cells among donor-derived cells in the recipient liver was normal. In contrast, when LFA-1-/- bone marrow (BM) cells were injected i.v. into irradiated wild-type mice, the frequency of liver NKT cells was significantly lower than that of mice injected with wild-type BM cells. Collectively, these data indicate that LFA-1 is required for the development of liver NKT cells, rather than the migration to and/or subsequent establishment of mature NKT cells in the liver.
Asunto(s)
Antígenos Ly , Antígenos/metabolismo , Hígado/citología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Proteínas/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/citología , Animales , Antígenos de Neoplasias/metabolismo , Antígenos de Superficie , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Receptores de Hialuranos/genética , Receptores de Hialuranos/fisiología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/fisiología , Lectinas Tipo C , Hígado/inmunología , Hígado/metabolismo , Antígeno-1 Asociado a Función de Linfocito/genética , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Mutantes , Subfamilia B de Receptores Similares a Lectina de Células NK , Receptores Similares a Lectina de Células NK , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Hypoxia, a result of DNA-damaging agents such as ionizing radiation, induces the nuclear accumulation of the p53 tumor suppressor protein. However, unlike the effect in ionizing radiation, hypoxia readily induces the nuclear accumulation of p53 in HPV E6-infected cells. In HPV-infected cells, a key regulator of p53 protein levels is the E6 oncoprotein. In association with the endogenous cellular protein E6-associated protein (E6AP), E6 can accelerate the degradation of p53 under aerobic conditions. To better define the mechanism of p53 induction in E6-infected cells by hypoxia, we studied the expression and association of E6 and E6AP with p53 in vivo. We found that hypoxia did not alter the protein levels of E6 or E6AP as compared with those found under aerobic growth conditions, indicating that protein inhibition of E6 or E6AP alone is not sufficient to explain the increased accumulation of p53 under hypoxic conditions. However, p53 did fail to coprecipitate with E6AP under hypoxia, indicating that hypoxia uncouples the interaction of p53 with E6 and E6AP. We also present evidence to indicate that hypoxia decreases the expression of the endogenous cellular regulator of p53 protein, the human MDM2 protein, resulting in an inhibition of p53 export from the nucleus to the cytoplasm for degradation. Taken together, these results suggest that the hypoxic induction of p53 is attributable to the down-regulation of MDM2 protein levels and uncoupling of p53 from its interaction with the E6/E6AP complex.