RESUMEN
Intellectual disability (ID) is a major health problem in our society. Genetic causes of ID remain unknown because of its vast heterogeneity. Here we report two Finnish families and one Dutch family with affected individuals presenting with mild to moderate ID, neuropsychiatric symptoms and delayed speech development. By utilizing whole exome sequencing (WES), we identified a founder missense variant c.983T>C (p.Leu328Pro) in seven affected individuals from two Finnish consanguineous families and a deletion c.799_1034-429delinsTTATGA (p.Gln267fs) in one affected individual from a consanguineous Dutch family in the C12orf4 gene on chromosome 12. Both the variants co-segregated in the respective families as an autosomal recessive trait. Screening of the p.Leu328Pro variant showed enrichment in the North Eastern sub-isolate of Finland among anonymous local blood donors with a carrier frequency of 1:53, similar to other disease mutations with a founder effect in that region. To date, only one Arab family with a three affected individuals with a frameshift insertion variant in C12orf4 has been reported. In summary, we expand and establish the clinical and mutational spectrum of C12orf4 variants. Our findings implicate C12orf4 as a causative gene for autosomal recessive ID.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Discapacidad Intelectual/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Consanguinidad , Exoma/genética , Salud de la Familia , Femenino , Finlandia , Efecto Fundador , Genes Recesivos , Genotipo , Geografía , Humanos , Masculino , Países Bajos , Linaje , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de AminoácidoRESUMEN
Ductal carcinoma in situ (DCIS) of the breast represents 15-20% of new breast cancer diagnoses in the US annually. However, long-term competing risks of mortality, as well as racial differences in outcomes among US women with DCIS, are unknown. Case data from the years 1978-2010 were obtained using SEER*Stat software available through the National Cancer Institute from the 2010 SEER registries. Included were all women aged 40 and over with newly diagnosed DCIS. There were 67,514 women in the analysis, including 54,518 white women and 6,113 black women. A total of 12,173 deaths were observed over 607,287 person-years of follow-up. The 20-year cumulative incidence of all-cause death among women with DCIS was 39.6% (CI 38.9-40.3). The corresponding 20-year rates for breast cancer death and CVD death were 3.2% (CI 3.0-3.4) and 13.2% (CI 12.8-13.7), respectively. Black women with DCIS had a higher risk of death compared to white women, with these hazard ratios elevated throughout the entire study period. For example, between 1990 and 2010, black women had a higher risk of all-cause death (HR 3.06, CI 2.39-3.91), breast cancer death (HR 5.78, CI 3.16-10.57), and CVD death (HR 6.43, CI 3.61-11.45) compared to white women diagnosed between 50 and 59 years of age. The risk of all-cause and CVD death was greater than breast cancer death among women diagnosed with DCIS over 20 years. Black women had higher risks of dying from all-causes compared to white women. These differences persisted into the modern treatment era.
Asunto(s)
Negro o Afroamericano/estadística & datos numéricos , Neoplasias de la Mama/etnología , Carcinoma Intraductal no Infiltrante/etnología , Enfermedades Cardiovasculares/etnología , Causas de Muerte , Población Blanca/estadística & datos numéricos , Adulto , Anciano , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/mortalidad , Carcinoma Intraductal no Infiltrante/complicaciones , Carcinoma Intraductal no Infiltrante/mortalidad , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/mortalidad , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Medición de Riesgo , Programa de VERF , Tasa de Supervivencia , Estados Unidos/etnologíaRESUMEN
Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.
Asunto(s)
Empalme Alternativo , Resistencia a la Insulina/genética , Distrofia Miotónica/genética , Receptor de Insulina/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Humanos , Músculo Esquelético/metabolismo , Distrofia Miotónica/fisiopatología , Isoformas de Proteínas/genética , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Repeticiones de TrinucleótidosRESUMEN
AIMS: To evaluate the variance in current UK clinical practice and clinical outcomes for direct percutaneous radiologically inserted gastrostomy (RIG). MATERIALS AND METHODS: A prospective UK multicentre survey of RIG performed between October 2008 and August 2010 was performed through the British Society of Gastrointestinal and Abdominal Radiology (BSGAR). RESULTS: Data from 684 patients were provided by 45 radiologists working at 17 UK centres. Two hundred and sixty-three cases (40%) were performed with loop-retained catheters, and 346 (53%) with balloon-retained devices. Sixty percent of all patients experienced pain in the first 24 h, but settled in the majority thereafter. Early complications, defined as occurring in the first 24 h, included minor bleeding (1%), wound infection (3%), peritonism (2%), and tube misplacement (1%). Late complications, defined as occurring between day 2 and day 30 post-procedure, included mild pain (30%), persisting peritonism (2%), and 30 day mortality of 1% (5/665). Pre-procedural antibiotics or anti-methicillin-resistant Staphylococcus aureus (MRSA) prophylaxis did not affect the rate of wound infection, peritonitis, post-procedural pain, or mortality. Ninety-three percent of cases were performed using gastropexy. Gastropexy decreased post-procedural pain (p < 0.001), but gastropexy-related complications occurred in 5% of patients. However, post-procedure pain increased with the number of gastropexy sutures used (p < 0.001). The use of gastropexy did not affect the overall complication rate or mortality. Post-procedure pain increased significantly as tube size increased (p < 0.001). The use of balloon-retention feeding tubes was associated with more pain than the deployment of loop-retention devices (p < 0.001). CONCLUSION: RIG is a relatively safe procedure with a mortality of 1%, with or without gastropexy. Pain is the commonest complication. The use of gastropexy, fixation dressing or skin sutures, smaller tube sizes, and loop-retention catheters significantly reduced the incidence of pain. There was a gastropexy-related complication rate in 5% of patients. Neither pre-procedural antibiotics nor anti-MRSA prophylaxis affected the rate of wound infection.
Asunto(s)
Gastrostomía/métodos , Intubación Gastrointestinal/métodos , Radiografía Intervencional/métodos , Estómago/diagnóstico por imagen , Estómago/cirugía , Cirugía Asistida por Computador/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Profilaxis Antibiótica/métodos , Femenino , Estudios de Seguimiento , Gastropexia/métodos , Gastrostomía/efectos adversos , Gastrostomía/instrumentación , Humanos , Intubación Gastrointestinal/efectos adversos , Intubación Gastrointestinal/instrumentación , Masculino , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Persona de Mediana Edad , Aptitud Física , Complicaciones Posoperatorias/etiología , Estudios Prospectivos , Análisis de Supervivencia , Resultado del Tratamiento , Reino Unido , Adulto JovenRESUMEN
We report the formation of a three-dimensionally ordered array of air bubbles of monodisperse pore size in a polymer film through a templating mechanism based on thermocapillary convection. Dilute solutions of a simple, coil-like polymer in a volatile solvent are cast on a glass slide in the presence of moist air flowing across the surface. Evaporative cooling and the generation of an ordered array of breath figures leads to the formation of multilayers of hexagonally packed water droplets that are preserved in the final, solid polymer film as spherical air bubbles. The dimensions of these bubbles can be controlled simply by changing the velocity of the airflow across the surface. When these three-dimensionally ordered macroporous materials have pore dimensions comparable to the wavelength of visible light, they are of interest as photonic band gaps and optical stop-bands.
Asunto(s)
Movimientos del Aire , Modelos Químicos , Poliestirenos/química , Procesamiento de Imagen Asistido por Computador , Microscopía Confocal , Porosidad , Agua/químicaRESUMEN
Myotonic dystrophy (DM) is caused by a CTG expansion in the 3' untranslated region of the DM gene. One model of DM pathogenesis suggests that RNAs from the expanded allele create a gain-of-function mutation by the inappropriate binding of proteins to the CUG repeats. Data presented here indicate that the conserved heterogeneous nuclear ribonucleoprotein, CUG-binding protein (CUG-BP), may mediate the trans-dominant effect of the RNA. CUG-BP was found to bind to the human cardiac troponin T (cTNT) pre-messenger RNA and regulate its alternative splicing. Splicing of cTNT was disrupted in DM striated muscle and in normal cells expressing transcripts that contain CUG repeats. Altered expression of genes regulated posttranscriptionally by CUG-BP therefore may contribute to DM pathogenesis.
Asunto(s)
Empalme Alternativo , Distrofia Miotónica/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Repeticiones de Trinucleótidos , Proteínas CELF1 , Línea Celular , Núcleo Celular/metabolismo , Exones , Humanos , Intrones , Músculo Esquelético/citología , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Mutación , Distrofia Miotónica/metabolismo , Proteína Quinasa de Distrofia Miotónica , Fosforilación , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Transcripción Genética , Transfección , Troponina/genética , Troponina TRESUMEN
We have recently shown that the orphan nuclear receptor Nur77 (NGFI-B) is most active in transcription when it is interacting with a cognate DNA sequence as a homodimer. Further, we have shown that the target for Nur77 dimers, the Nur response element (NurRE), is responsive to physiological stimuli in both endocrine and lymphoid cells, whereas other DNA targets of Nur77 action are not. The Nur77 subfamily also includes two related receptors, Nur-related factor 1 (Nurr1) and neuron-derived orphan receptor 1 (NOR-1). Often, more than one member of this subfamily is induced in response to extracellular signals. We now show that Nur77 and Nurr1 form heterodimers in vitro in the presence or absence of NurRE, and we have documented interactions between these proteins in vivo by using a two-hybrid system in mammalian cells. These heterodimers synergistically enhance transcription from NurRE reporters in comparison to that seen with homodimers. The naturally occurring NurRE from the pro-opiomelanocortin gene preferentially binds and activates transcription in the presence of Nur77 homo- or heterodimers, while a consensus NurRE sequence does not show this preference. Taken together, the data indicate that members of the Nur77 subfamily are most potent as heterodimers and that different dimers exhibit target sequence preference. Thus, we propose that a combinatorial code relying on specific NurRE sequences might be responsible for the activation of subsets of target genes by one of the members of the Nur77 subfamily of transcription factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proopiomelanocortina/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Elementos de Respuesta , Factores de Transcripción/metabolismo , Activación Transcripcional , Hormona Liberadora de Corticotropina/farmacología , Dimerización , Genes Reporteros , Modelos Genéticos , Proteínas del Tejido Nervioso/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Unión Proteica , Receptores de Esteroides , Receptores de Hormona Tiroidea , Técnicas del Sistema de Dos HíbridosRESUMEN
Within the nuclear receptor family, Nur77 (also known as NGFI-B) distinguishes itself by its ability to bind a target sequence (the NBRE) as a monomer and by its role in T-cell receptor (TCR)-induced apoptosis in T cells. We now report on a novel mechanism of Nur77 action that is mediated by homodimers. These dimers bind a Nur77 response element (NurRE), which has been identified as a target of CRH-induced Nur77 in the pro-opiomelanocortin (POMC) gene promoter. Both halves of the palindromic NurRE are required for responsiveness to physiological signals, like CRH in pituitary-derived AtT-20 cells. Similarly, in T-cell hybridomas, TCR activation induced NurRE but not NBRE reporters. The in vivo signaling function of Nur77 thus appears to be mediated by dimers acting on a palindromic response element of unusual spacing between its half-sites. This mechanism may represent the biologically relevant paradigm of action for this subfamily of orphan nuclear receptors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Adenohipófisis/fisiología , Proopiomelanocortina/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/fisiología , Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Animales , Colforsina/farmacología , Hormona Liberadora de Corticotropina/farmacología , AMP Cíclico/fisiología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Dimerización , Regulación de la Expresión Génica/fisiología , Hibridomas , Ratones , Mutación , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Adenohipófisis/citología , Regiones Promotoras Genéticas/genética , Receptores de Antígenos de Linfocitos T/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Esteroides , Linfocitos T/citología , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Two important functions of glucocorticoids (Gc), namely, suppression of immune system function and feedback repression of the hypothalamo-pituitary-adrenal (HPA) axis, are mediated through repression of gene transcription. Previous studies have indicated that this repression is exerted in part through antagonism between the glucocorticoid receptors (GR) and the AP-1 family of transcription factors. However, this mechanism could not account for repression of the pro-opiomelanocortin (POMC) gene, an important regulator of the HPA axis. Our recent identification of the orphan nuclear receptor Nur77 as a mediator of CRH induction of POMC transcription led us, in the present work, to show that Gc antagonize this positive signal at two levels. First, Gc partly blunt the CRH induction of Nur77 mRNA, and second, they antagonize Nur77-dependent transcription. GR repression is exerted by antagonism of Nur77 action on the NurRE element of the POMC gene. Gc antagonism of NurRE activity was observed in response to physiological stimuli in both endocrine (CRH induction of POMC) and lymphoid (T-cell receptor activation) cells. In transfection experiments, transcriptional activation by Nur77 and the repressor activity of liganded GR titrated each other on their cognate DNA target. In vitro binding experiments as well as mutation analysis of GR suggest that the mechanism of GR antagonism of Nur77 is very similar to that of the antagonism between GR and AP-1. The convergence of positive signals mediated by Nur77 (and also probably by related family members) and negative signals exerted by GR appears to be a general mechanism for control of transcription, since it is active in both endocrine and lymphoid cells.
Asunto(s)
Hormona Liberadora de Corticotropina/farmacología , Proteínas de Unión al ADN/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Glucocorticoides/genética , Factores de Transcripción/genética , Activación Transcripcional/fisiología , Animales , Línea Celular , ADN/metabolismo , ADN Recombinante , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Hibridomas , Ratones , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Hipófisis/citología , Hipófisis/fisiología , Proopiomelanocortina/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Receptores de Antígenos de Linfocitos T , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides , Transducción de Señal/genética , Linfocitos T/fisiología , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Apoptosis is a normal physiological process which eliminates cells that do not receive adequate extracellular signals. One of the pathways signalling apoptosis is controlled by the small GTPases of the Rho family, also involved in cell proliferation, differentiation and motility. Another major apoptosis signalling pathway involves the p53 tumour suppressor which is activated by a variety of stress and mediates growth arrest or apoptosis in normal cells. We show here that upon detachment from the extracellular matrix, fibroblasts undergo rapid apoptosis that can be rescued by constitutive activation of Rac1 and Cdc42Hs GTPases. Conversely, inhibition of Rac1 and Cdc42Hs efficiently triggers apoptosis in adherent cells. Interestingly, apoptosis is not observed in p53-/- cells either cultured in suspension or inhibited for Rac1 and Cdc42Hs activity. Moreover, Rac1 and Cdc42Hs extinction in normal cells activates endogenous p53. Using specific inhibitors of MAPK pathways, we demonstrate that, in our experimental system, p38 signals survival, while ERK activity is required for apoptosis. Our data constitute the first demonstration that Rac1 and Cdc42Hs control pathways that require simultaneous signalling through MAPK ERK and p53 to induce apoptosis.
Asunto(s)
Apoptosis , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Células 3T3 , Animales , Adhesión Celular , Supervivencia Celular , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismoRESUMEN
Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5' element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3' element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.
Asunto(s)
Ciclo Celular/genética , Ciclina A/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Linfocitos T/metabolismo , Factores de Transcripción/aislamiento & purificación , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Humanos , Hidroxiurea/farmacología , Sustancias Macromoleculares , Ratones , Microinyecciones , Datos de Secuencia Molecular , Peso Molecular , Fase de Descanso del Ciclo Celular , Fase S/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
Cyclin A transcription is cell cycle regulated and induced by cell proliferative signals. To understand the mechanisms underlined in this regulation in normal human cells, we have analysed in vivo protein-DNA interactions at the Cyclin A locus in primary T lymphocytes. Stimulation of purified T lymphocytes by a combination of monoclonal antibodies directed at CD2 and CD28 adhesion molecules gives rise to a long lasting proliferation in the absence of accessory cells. Cyclin A was observed after 4 days of costimulation with anti CD2 + CD28 whereas stimulation by anti CD2 or anti CD28 alone was not effective. In vivo genomic DMS footprinting revealed upstream of the major transcription initiation sites, the presence of at least three protein binding sites, two of which were constitutively occupied. They bind in vitro respectively ATF-1 and NF-Y proteins. The third site was occupied in quiescent cells or in cells stimulated by anti CD2 or anti CD28 alone. The mitogenic combination of anti CD2 + anti CD28 released the footprint as cells were committed to proliferation. Consistent with theses results, nuclear extracts prepared from quiescent cells formed a specific complex with this element, whereas extracts prepared from cells treated with anti CD2 + anti CD28 failed to do so after cells entered a proliferative state.
Asunto(s)
Antígenos CD2/fisiología , Antígenos CD28/fisiología , Ciclinas/genética , Regulación de la Expresión Génica , Linfocitos T/fisiología , Factor de Transcripción Activador 1 , Anticuerpos Monoclonales , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Ciclo Celular/fisiología , División Celular/fisiología , Células Cultivadas , Ciclinas/metabolismo , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Activación de Linfocitos , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Linfocitos T/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
Cyclin A is a positive regulatory component of kinases required for the progression through S phase and for the transition between the G2 and M phases of the cell division cycle. Previous studies have demonstrated that the promoter of its gene is under transcriptional repression in quiescent cells. Whereas the DNA sequences mediating this effect have been clearly delineated, the nature of the proteins acting in trans is still debated. Indirect observations suggest the involvement of proteins related to the retinoblastoma tumor suppressor protein (pRb). However, the precise role of these proteins has been difficult to assess, since most experiments designed to analyse their function have been carried out in transformed cell lines. Nevertheless, a current model has emerged whereby the role of the p130 protein would be restricted to resting and early G1 cells and p107, absent in quiescent cells, would be involved later in the control of the G1/S transition, whilst pRb would be effective throughout the cell cycle. We show here that cyclin A transcriptional inhibition is relieved in primary fibroblasts from pRb(-/-) embryos and not in fibroblasts from p13O(-/-), p107(-/-) or even p130(-/-)/p107(-/-) double mutant embryos. This suggests a unique role for pRb in controlling the extinction of specific genes in G0, providing thus the first example of non-overlapping functions achieved by the different pocket proteins.
Asunto(s)
Ciclina A/genética , Regulación hacia Abajo , Proteínas , Proteína de Retinoblastoma/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , ADN , Ratones , Datos de Secuencia Molecular , Mutagénesis , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Transcripción GenéticaRESUMEN
Many cells, when cultured in suspension, fail to express cyclin A, a regulatory component of cell cycle kinases cdc2 and cdk2 and as a consequence, do not enter S phase. However, many cell type-specific differences are disclosed between not only normal and transformed cells, but also between cell lines whose proliferation is strictly anchorage-dependent. These apparent discrepancies are seen in established cell lines most probably because of adaptative events that have occurred during cell culture. We have therefore used primary cells to understand how cyclin A transcription is controlled by cell anchorage properties. To this aim, we have used embryonic fibroblasts from either wild type, Rb(-/-) or p107(-/-)/p130(-/-) mice and tested the effect of an ectopic expression of Rb mutants. In the experiments reported here, we show that anchorage-dependent expression of cyclin A (i) is reflected by the in vivo occupancy of a negative DNA regulatory element previously shown to be instrumental in the down regulation of cyclin A transcription in quiescent cells (Cell Cycle Responsive Element: CCRE) (ii) requires a functional Rb but neither p107 nor p130 (iii) mutation of the CCRE abolishes both adhesion-dependent regulation and response to Rb.
Asunto(s)
Ciclina A/genética , Regulación de la Expresión Génica , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Proteínas , Proteína de Retinoblastoma/fisiología , Animales , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Proteínas Nucleares/genética , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/genética , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del RetinoblastomaRESUMEN
We compared the effect of estradiol on activator protein-1 (AP-1) activity in estrogen receptor positive (ER alpha+) and estrogen receptor negative (ER alpha-) human breast cancer cell lines transiently transfected with the AP-1-responsive reporter plasmid AP-1-TK-CAT and an ER alpha expression vector. While estradiol increased AP-1 activity in the ER alpha+ cell lines MCF7, ZR75.1, and T47D, it decreased (MDA-MB231 and BT20 cells) or had no significant effect (MDA-MB435 cells) on AP-1-mediated transcription in ER alpha- cells. Estradiol also inhibited AP-1 activity in ER alpha-MDA-MB231 cells stably transfected with ER alpha and in which ER alpha levels are close to those found in MCF7. Use of ER alpha mutant expression vectors demonstrated that the DNA-binding domain of ER alpha was needed for stimulation or inhibition of AP-1 activity by estradiol but suggested that ER alpha binding to estrogen-responsive elements was not required for these effects. Changes in regulation paralleled quantitative and qualitative changes in protein binding to AP-1 sites, as demonstrated by gel shift assay: protein binding was greater and DNA/protein complexes migrated faster for ER alpha--than for ER alpha+ cells. In fact, by Northern blot, a high level of Fra-1 mRNA was found in BT20 and MDA-MB231 cells as compared with ER alpha+ cells, and MDA-MB435 cells showed an intermediary level of expression. The differential expression of Fra-1 in MCF7 and MDA-MB231 cells was confirmed at the protein level by supershift experiments. In addition, overexpression of Fra-1 in MCF7 cells decreased the positive effect of estradiol while inhibition of Fra-1 expression in MDA-MB231 cells, by transient transfection of the Fra-1 antisense expression vector, abolished the negative effect of the hormone. In conclusion, we demonstrated that ER alpha- breast cancer cell lines differ from ER+ cells by a high level of AP-1 DNA-binding activity due, at least in part, to high Fra-1 constitutive expression. High Fra-1 concentration is crucial for the negative regulation of AP-1 activity by estradiol and thus may take part in estradiol-induced inhibition of cell proliferation in ER alpha- breast cancer cells transfected with ER alpha expression construct.
Asunto(s)
Neoplasias de la Mama/metabolismo , Estradiol/farmacología , Expresión Génica , Proteínas Proto-Oncogénicas c-fos/genética , Factor de Transcripción AP-1/metabolismo , Northern Blotting , Neoplasias de la Mama/química , ADN/metabolismo , Genes fos/genética , Humanos , Proteínas Proto-Oncogénicas c-fos/farmacología , ARN Mensajero/metabolismo , Receptores de Estrógenos/análisis , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes de Fusión , Factor de Transcripción AP-1/genética , Transfección , Células Tumorales CultivadasRESUMEN
Although the incidence of bovine spongiform encephalopathy in cattle continues to decline in the United Kingdom, it remains important to maintain vigilance of all potential routes of transmission of infection to humans. Initial studies have demonstrated a potential risk of carcass contamination with brain tissue following the use of captive bolt gun stunning in cattle. The objective of this study was to further explore these initial findings particularly in regard to captive bolt guns currently in use in the United Kingdom. Brain tissue fragments or elevated levels of a marker protein for brain tissue were detected in venous blood samples from 4% (95% confidence interval, 1.6 to 9.8%) of cattle stunned by penetrating captive bolt gun and from 2% (95% confidence interval, 0.6 to 7%) of those stunned by nonpenetrating captive bolt gun.
Asunto(s)
Mataderos , Sistema Nervioso Central/lesiones , Encefalopatía Espongiforme Bovina/transmisión , Contaminación de Alimentos/prevención & control , Animales , Bovinos , Contaminación de Equipos , Contaminación de Alimentos/análisis , Proteínas del Tejido Nervioso/aislamiento & purificaciónRESUMEN
In MCF7 human breast cancer cells, the antiestrogens 4-hydroxy-tamoxifen and ICI 164,384 inhibit the mitogenic activity of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). These growth factors also stimulate the expression of cathepsin-D and pS2 genes. Therefore, we studied the effects of antiestrogens on growth factor induction of pS2 and cathepsin-D mRNA. The two antiestrogens strongly inhibited the transcriptional induction of pS2 by growth factors. On the contrary, estradiol and IGF-I or EGF had an additive effect on pS2 mRNA accumulation. Growth factor induction of cathepsin-D was also inhibited by ICI 164,384. By contrast, 4-hydroxytamoxifen had an agonist effect on cathepsin-D and an additive effect on IGF-I-induced mRNA. When 12-O-tetradecanoylphorbol-13-acetate or 8-bromo-cAMP (8-Br-cAMP) was used instead of growth factors, similar effects of 4-hydroxytamoxifen and ICI 164,384 were obtained on pS2 (12-O-tetradecanoylphorbol-13-acetate and 8-Br-cAMP) and cathepsin-D (8-Br-cAMP) induction. A mechanism based on the classical competitive inhibition by antiestrogens of estrogen binding and action on the estrogen receptor was very unlikely, as 1) no antigrowth factor activity was obtained with R5020, which was a potent inhibitor of estrogen induction of pS2 and cathepsin-D mRNA; 2) in the Ishikawa endometrial cancer cell line, the cathepsin-D gene is unresponsive to estrogen, but was inhibited by antiestrogen after its induction by EGF or 8-Br-cAMP; and 3) the residual estrogen concentration in cells was too low to induce the expression of estrogen-specific genes. However, antiestrogens did not inhibit the expression of all genes induced by growth factors, as they were without effect on IGF-I induction of glyceraldehyde-3-phosphate dehydrogenase mRNA. These results demonstrate that antiestrogens can modulate the transcription of some growth factor-induced genes and strongly suggest that this effect is not due to interference with residual estrogens.
Asunto(s)
Neoplasias de la Mama/metabolismo , Catepsina D/genética , Antagonistas de Estrógenos/farmacología , Sustancias de Crecimiento/farmacología , Proteínas de Neoplasias/genética , Proteínas , ARN Mensajero/biosíntesis , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Factor de Crecimiento Epidérmico/farmacología , Estradiol/análogos & derivados , Estradiol/farmacología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Alcamidas Poliinsaturadas , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The bipartite repressor elements, termed cell cycle-dependent element (CDE)/cell cycle regulatory element (CCRE)-cell cycle homology region (CHR) control the growth-dependent transcription of the cyclin A, cdc25C, cdc2 genes. Here, we have identified a functional element displaying the signature of the CDE-CHR in the promoter of the mouse RB2 (p130) gene, encoding the retinoblastoma protein family (pRB)-related protein p130. This element locates close to the major transcription start site where it makes major groove contacts with proteins that can be detected in a cellular context using in vivo genomic footprinting techniques. Inactivation of either the CDE or CHR sequence strongly up-regulates the p130 promoter activity in exponentially growing cells, a situation where endogenous p130 gene expression is almost undetectable. Electrophoretic mobility shift assays suggest that two different protein complexes bind independently to the p130 CDE and CHR elements, and that the protein(s) bound to the CDE might be related to those bound on cyclin A and cdc2 promoters.
Asunto(s)
Regulación de la Expresión Génica , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Proteínas , Animales , Secuencia de Bases , Clonación Molecular , ADN , Proteínas de Unión al ADN/metabolismo , Genes cdc , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Proteína p130 Similar a la del Retinoblastoma , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Use of a beta-lactamase stable antibiotic is called for in cases of acute otitis media (AOM) likely to be caused by beta-lactamase-producing Haemophilus influenzae or Moraxella catarrhalis. Two beta-lactamase-stable agents commonly used for empirical treatment of AOM are amoxicillin/clavulanate and cefixime. METHODS: A multicenter, randomized clinical trial compared cefixime (CFX; 8 mg/kg once daily) with amoxicillin/clavulanate (A/C; 40 mg/kg/day in three divided doses) for the treatment of children with AOM. Three hundred thirteen children were randomly assigned to a 10-day course of either CFX (n = 158) or A/C (n = 155). Based on history, physical examinations and otoscopic and tympanometric assessments, clinical responses were evaluated as cure, improvement, failure, relapse or nonevaluable. Compliance and patient/parent acceptability were also analyzed. RESULTS: Overall favorable clinical responses (cure plus improvement) were comparable post-therapy for the two treatments (CFX = 76%; A/C = 77%). Significant differences in response rates for both treatments were noted among different geographic regions, with the highest response rates observed in the Northeast and South. Acceptability of CFX was significantly better than that of A/C (P = 0.0001), and the adverse experience rate was lower (P = 0.001). The most frequently reported adverse experiences were diarrhea (CFX 15.2%, A/C 29.7%) and vomiting (CFX 3.2%, A/C 10.32%). Relapse rates were 26% for CFX and 29% for A/C. CONCLUSION: This study demonstrated that CFX has comparable clinical efficacy and a better adverse events profile than A/C when used to treat AOM of childhood.
Asunto(s)
Cefotaxima/análogos & derivados , Cefalosporinas/uso terapéutico , Quimioterapia Combinada/uso terapéutico , Otitis Media/tratamiento farmacológico , Enfermedad Aguda , Amoxicilina/uso terapéutico , Combinación Amoxicilina-Clavulanato de Potasio , Cefixima , Cefotaxima/uso terapéutico , Niño , Preescolar , Ácidos Clavulánicos/uso terapéutico , Femenino , Humanos , Lactante , Masculino , Estudios Prospectivos , Resultado del TratamientoRESUMEN
Whereas orphan nuclear receptors of the Nur77 (NGFI-B) subfamily were previously known to act on transcription as monomers or as heterodimers with RXR, we have recently shown that Nur77 homodimers potently activate transcription upon interaction with a novel palindromic response element, the NurRE. In fact, reporter plasmids containing the NurRE respond to physiological stimuli in conditions where the NBRE, a binding site for Nur77 monomers, does not. Nur77 and its related receptors were shown to be important mediators for control of apoptosis induced by the T-cell receptor, and they also mediate the effect of the hypothalamic hormone CRH on transcription of the pituitary pro-opiomelanocotin (POMC) gene. In both systems, glucocorticoids antagonize the stimulatory effects of Nur77 on transcription by a mechanism that involves protein:protein interactions. Thus, the Nur77 signalling pathway appears to be a point of convergence for stimulatory signals and glucocorticoid repression in both endocrine and lymphoid systems.