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Certification of quantum nonlocality plays a central role in practical applications like device-independent quantum cryptography and random number generation protocols. These applications entail the challenging problem of certifying quantum nonlocality, something that is hard to achieve when the target quantum state is only weakly entangled, or when the source of errors is high, e.g. when photons propagate through the atmosphere or a long optical fiber. Here we introduce a technique to find a Bell inequality with the largest possible gap between the quantum prediction and the classical local hidden variable limit for a given set of measurement frequencies. Our method represents an efficient strategy to certify quantum nonlocal correlations from experimental data without requiring extra measurements, in the sense that there is no Bell inequality with a larger gap than the one provided. Furthermore, we also reduce the photodetector efficiency required to close the detection loophole. We illustrate our technique by improving the detection of quantum nonlocality from experimental data obtained with weakly entangled photons.
RESUMEN
OBJECTIVE: To detect doubts and training needs in an urban health and family doctor training centre during the usual practice. MATERIAL AND METHODS: A cross-sectional descriptive study was conducted for one month in an urban health centre in Madrid. Family doctors were interviewed after their daily clinics about the doubts they had identified, choosing two of them. Unresolved questions were grouped by subject and according to the current taxonomies. A teaching program was then developed to tackle them. RESULTS: Out of a total 21 physicians of the centre, 19 attended 10,678 patients during the period. The doubt detection rate was 0.44 doubts for every 10 patients attended. Of the 384 questions chosen, 83.34% were clinical and 16.66% were non-clinical. Just over half (51.2%) of these were still unresolved 15days later the consultation event. The main methods for their resolution were using the scientific bases on the internet (mainly PubMed, UpToDate and Clinical Practice Guidelines; 38%), followed by consultation with other colleagues (34.9%). CONCLUSIONS: Most of the doubts generated during clinics were clinical, although there is a significant burden of bureaucratic questions. More than half of the doubts are not resolved during the consultation or within the following 15days. The scientific databases on the internet are the main sources of information, although consulting other colleagues was often used as well. Additional time for dealing with patients and enhanced access to solve complex questions should be available to improve the success rate.
Asunto(s)
Médicos de Familia/psicología , Atención Primaria de Salud/organización & administración , Servicios Urbanos de Salud/organización & administración , Adulto , Estudios Transversales , Femenino , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Médicos de Familia/educación , EspañaRESUMEN
OBJECTIVE: To assess the association of survival and treatment with colistin and tigecycline in critically ill patients with carbapenem-resistant Acinetobacter baumannii bacteraemia. METHODS: An observational cohort study was carried out. Targeted therapy consisted of monotherapy with colistin (9 million UI/day) or combined therapy with colistin and tigecycline (100 g/day). The primary outcome was 30-day crude mortality. The association between combined targeted therapy and mortality was controlled for empirical therapy with colistin, propensity score of combined therapy and other potential confounding variables in a multivariate Cox regression analysis. RESULTS: A total of 118 cases were analysed. Seventy-six patients (64%) received monotherapy and 42 patients (36%) received combined therapy. The source of bacteraemia was primary in 18% (21/118) of the patients, ventilator-associated pneumonia in 64% (76/118) and other sources in 14% (16/118). The 30-day crude mortality rate was 62% (42/76) for monotherapy and 57% (24/42) for combined therapy. The variables associated with 30-day crude mortality were: Charlson index (hazard ratio (HR) 1.16, 95% CI 1.02-1.32; p 0.028), empirical therapy with colistin (HR 2.25, 95% CI 1.33-3.80; p 0.003) and renal dysfunction before treatment (HR 1.91, 95% CI 1.01-3.61; p 0.045). Combined targeted therapy was not associated with lower adjusted 30-day crude mortality (adjusted HR 1.29, 95% CI 0.64-2.58; p 0.494). CONCLUSIONS: Combined targeted therapy with high-dose colistin and standard dose tigecycline was not associated with lower crude mortality of bacteraemia due to carbapenem-resistant A. baumannii in critically ill patients. TRIAL REGISTRATION: Registered in ClinicalTrials.gov. Identifier: NCT02573064.
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Infecciones por Acinetobacter/tratamiento farmacológico , Bacteriemia/tratamiento farmacológico , Colistina/administración & dosificación , Minociclina/análogos & derivados , Infecciones por Acinetobacter/mortalidad , Acinetobacter baumannii/efectos de los fármacos , Adulto , Bacteriemia/mortalidad , Carbapenémicos/farmacología , Estudios de Cohortes , Colistina/farmacología , Enfermedad Crítica , Quimioterapia Combinada , Femenino , Humanos , Masculino , Persona de Mediana Edad , Minociclina/administración & dosificación , Minociclina/farmacología , Puntaje de Propensión , Análisis de Supervivencia , Tigeciclina , Resultado del TratamientoRESUMEN
Diagnosis of acute hepatitis E virus (HEV) infection is established by detection of anti-HEV IgM antibodies by ELISA or by amplification of serum viral RNA. Here, we evaluate the diagnostic value of testing HEV RNA in saliva to identify patients with acute HEV infection. Prospective proof-of-concept study including patients with acute hepatitis. Whole blood and neat saliva samples were obtained from all patients. Saliva samples were processed and analysed for HEV RNA by RT-PCR within 2 hr after collection. A total of 34 patients with acute hepatitis and 12 healthy donors were included in the study. HEV RNA in serum was confirmed by RT-PCR in eight of these patients (23.5%; 95% CI: 12.2%-40.2%). HEV was isolated in the saliva of eight of 34 patients (23.5%; 95% CI: 12.2%-40.2%). All patients with HEV RNA amplified in saliva had detectable HEV RNA in serum. HEV was isolated neither in the saliva of any of the 26 patients without detectable HEV RNA in serum nor in healthy donors. Our study suggests that acute HEV infection could be diagnosed by assessing viral load in saliva.
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Virus de la Hepatitis E , Hepatitis E/diagnóstico , ARN Viral/aislamiento & purificación , Saliva/virología , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , ARN Viral/sangre , ARN Viral/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Pruebas Serológicas , Adulto JovenRESUMEN
Transcription of both Xenopus thyroid hormone receptor (TR) genes, xTR alpha and -beta, is strongly up-regulated by their own ligand T3 during natural or T3-induced metamorphosis of tadpoles and in some Xenopus cell lines. To explain this autoinduction, we analyzed the sequence of 1.6 kilobases of xTR beta promoter for putative T3-responsive elements. Two direct repeat +4 AGGTCA hexamer motifs (DR+4), an imperfect distal (-793/-778) and a perfect proximal (-5/11) site, a DR+1 site, and some possible half-sites were located in the 1.6-kilobase promoter. Transfection of Xenopus XTC-2 cells (which express xTR alpha and -beta) and XL-2 cells (which predominantly express TR alpha) with chloramphenicol acetyltransferase reporter constructs of deletion mutants and promoter fragments showed that the distal and proximal DR+4 sites responded to T3, although other flanking sequences may also play a role. The thyroid hormone-responsive element half-site present as DR+1 in the up-stream sequence at -1260/-950, when cloned in front of a heterologous promoter, functions independently. T3 enhanced transcription from the two DR+4-containing fragments when present together by only 2- to 3-fold due to a high basal activity. Overexpression of unliganded xTR alpha and xTR beta in XTC-2 cells repressed basal activity, which was then enhanced 7- to 4-fold by T3, respectively; with XL-2 cells cotransfected with xTR beta, T3 inducibility increased to 16-fold. Electrophoretic mobility shift assays with recombinant Xenopus TR alpha, TR beta, retinoid-X receptor-alpha (RXR alpha) and RXR gamma proteins showed that TR-RXR heterodimers, but not TR or RXR monomers or homodimers, strongly bound the natural and synthetic distal and proximal DR+4 elements in a ligand-independent manner. TR/RXR heterodimers exhibited the highest binding affinity for a 28-mer oligonucleotide probe for the -5/11 proximal DR+4 site, with only slight binding to DR+1 (retinoid-X-responsive element-like) site. The xTR beta promoter binding to XTC-2 cell nuclear extract suggested the in vivo relevance of the findings with recombinant TR/RXR heterodimers. It is concluded that xTR alpha and -beta proteins are capable of regulating the expression of xTR beta gene, which can explain its autoinduction seen during T3-induced metamorphosis.
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Autorreceptores/genética , Regulación de la Expresión Génica , Genes , Receptores de Hormona Tiroidea/genética , Triyodotironina/farmacología , Xenopus laevis/genética , Animales , Autorreceptores/biosíntesis , Secuencia de Bases , Núcleo Celular/química , Regulación de la Expresión Génica/efectos de los fármacos , Larva , Metamorfosis Biológica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/biosíntesis , Proteínas Recombinantes de Fusión/farmacología , Receptor alfa de Ácido Retinoico , Eliminación de Secuencia , Transcripción Genética , Transfección , Xenopus laevis/crecimiento & desarrolloRESUMEN
To determine if the autoinduction of thyroid hormone receptor (TR) alpha and beta mRNAs during metamorphosis in Xenopus tadpoles can be reproduced in cultured cells, we have screened four Xenopus cell lines (XTC-2, XL-177, XL2 and Kr) for receptor transcripts and their response to thyroid hormone. Exposure of XTC-2 cells to 10(-9) M triiodothyronine (T3) for 24 h upregulated TR alpha and beta mRNAs by 2-4- and 10-40-fold, respectively. In view of the marked similarity of the differential distribution of the two transcripts and their upregulation by T3 to the pattern of autoinduction seen in whole tadpoles, the process was studied in greater detail in XTC-2 cells. The time-course of autoinduction of TR alpha and beta mRNAs in these cells also resembled that in vivo, the two transcripts being significantly induced by 3-6 h after T3. Dose-response to T3, and the relative responses to its active and inactive analogs, confirmed that the process of autoinduction was initiated by thyroid hormone receptor with the same functional characteristics as that found in all amphibian and mammalian tissues. Experiments performed with cycloheximide suggested that intermediary protein(s) were involved in autoinduction, so that TR genes cannot be considered as 'immediate early' genes for this process. The possible advantages of studying thyroid hormone action in metamorphosis in XTC-2 cells are briefly discussed.
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Metamorfosis Biológica/genética , Receptores de Hormona Tiroidea/biosíntesis , Tiroxina/farmacología , Triyodotironina/farmacología , Xenopus/crecimiento & desarrollo , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , ARN Mensajero/biosíntesis , Receptores de Hormona Tiroidea/genética , Tiroxina/análogos & derivados , Triyodotironina/análogos & derivados , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Although downregulation of receptors by their respective hormonal ligands is a well-studied phenomenon, relatively less is known about autoupregulation of receptors. However, an increasing number of observations of the latter process are now being reported. Here, we discuss the phenomenon of autoinduction of nuclear receptors of the steroid/thyroid hormone gene family, and its significance in the context of the developmental and gene regulatory function of the ligands. Much of this review is illustrated by recent work from our laboratory on the autoregulation of Xenopus estrogen (ER) and thyroid hormone (TR) receptors and their transcripts, accompanying or anticipating vitellogenesis and metamorphosis, respectively. The activation by estrogen (E2) of the silent vitellogenin genes and the induction of FOSP-1 genes in primary cultures of hepatocytes from male Xenopus and oviduct cells, respectively, are tightly coupled to a substantial upregulation of ER protein and its transcript. The developmental competence to activate vitellogenin in response to E2 was found to be acquired during late metamorphosis. Since the latter process is obligatorily controlled by thyroid hormones (TH), we extended our studies to the developmental and hormonal regulation of Xenopus TR genes. Although very low levels of TR alpha and beta mRNAs are detectable in embryos and early larvae, there is a large increase in the accumulation of both transcripts before the onset of metamorphosis (stage 54 tadpoles), by which time the larval thyroid gland has first begun to secrete TH. Filter and in situ hybridization revealed that the two transcripts were differentially regulated and were not equally distributed in all regions or tissues of the tadpole. Their concentration peaks at metamorphic climax and drops to low levels in froglets and adult Xenopus. Exogenous TH given to pre-metamorphic tadpoles is known to induce metamorphosis precociously. Administration of triiodothyronine (T3) to early tadpoles (stages 50/52) caused a rapid upregulation of TR alpha and beta mRNAs which was particularly marked for the beta transcript (20- to 50-fold increase in steady-state levels). This autoinduction, which is the earliest response to T3, is mimicked to variable degrees in some Xenopus cell lines. In XTC-2 cells, in which the in vivo process is fully reproduced, it was possible to show with cycloheximide that the increase in TR mRNA requires protein synthesis. It was also possible to show by transfection of XTC-2 cells with a reporter-promoter construct of Xenopus albumin gene, which is a target for T3, that the extra TR mRNA increases functional receptor in the cell. Although the role of TH is well-known in metamorphosis, we found that TR is also autoinduced in primary culture of adult male Xenopus hepatocytes. The significance of this finding lies in the fact that T3 potentiates the autoinduction of ER and the de novo activation of vitellogenin genes by E2. Prolactin (PRL) is known to exert a "juvenilizing" action by preventing the induction of amphibian metamorphosis by TH. It is therefore highly significant that PRL prevented both the autoinduction of TR alpha and beta mRNAs in whole tadpoles and organ cultures and the activation of TR target genes, such as those encoding albumin and 63 kDa adult-type keratin. Although how PRL exerts its antimetamorphic effect is not known, these findings lead us to propose a dual threshold model for the autoinduction of TR, whereby the autoinduction of TR genes requires a very low level of TR and TH to rapidly augment the amount of functional TR. This higher amount of receptor would be required to achieve a higher threshold to activate "downstream" or target genes which specify the adult phenotype at the end of metamorphosis. Finally, a survey of recent literature shows that the phenomenon of autoinduction is not restricted to Xenopus ER and TR but is more widespread among members of the nuclear receptor family.
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Regulación del Desarrollo de la Expresión Génica , Receptores de Estrógenos/genética , Receptores de Hormona Tiroidea/genética , Animales , Técnicas de Cultivo , Regulación del Desarrollo de la Expresión Génica/fisiología , Metamorfosis Biológica , Prolactina/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Xenopus/embriologíaRESUMEN
Analytical goals for rectilinearity based on within-subject biological variation have not yet been advocated. On the other hand, the statistical tests to evaluate rectilinearity may be too restrictive for clinical purposes. If rectilinearity of the least-squares regression is rejected by the statistical test used, we propose to compare the systematic error introduced using such a regression line as the calibration function, with the allowable total error. Since total error ideally should be less than the within-subject biological coefficient of variation (C.V.Bw) the goal for rectilinearity we propose is that the maximum allowable systematic relative error produced by the calibration function (LoRi) when a lack of rectilinearity really occurs is: LoRi < C.V.Bw -1.96 C.V.M, where C.V.M is the between-run metrological coefficient of variation of the measurement procedure, corresponding to the value of concentration under study.
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Calibración/normas , Química Clínica/normas , Algoritmos , Química Clínica/estadística & datos numéricos , Humanos , Análisis de los Mínimos Cuadrados , Análisis de RegresiónRESUMEN
BACKGROUND: The usefulness of the concentration of salivary 17-hydroxyprogesterone (17-OHPRG) in the diagnosis of congenital adrenal hyperplasia by partial deficiency of 21-hydroxylase was studied. As a biologic medium, saliva has important advantages such as facility in sample collection and the avoidance of the stress of venous puncture. METHODS: Salivary 17-OHPRG was measured by a direct solid phase radioimmunoassay. A control group made up of 28 males and 26 females was studied. The group of patients included 30 women, 10 of them with a previous diagnosis of partial deficit of 21-hydroxylase. Basal values were established in the control group and were compared with those found in the patients in whom a stimulation test with adrenocorticotropin (ACTH) was performed collecting blood and saliva samples. RESULTS: The levels of 17-OHPRG observed in the patients with partial deficiency of 21-hydroxylase were significantly higher than those found in the control group and in the group with hirsutism, including both basal levels and those following stimulation. The correlation between the values found in blood and saliva was very significant. CONCLUSIONS: The measure of 17-hydroxyprogesterone in saliva by a method of direct radioimmunoassay is a valid alternative test to serum measure in both basal conditions and following a stimulation test.
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Hiperplasia Suprarrenal Congénita/diagnóstico , Hirsutismo/metabolismo , Hidroxiprogesteronas/análisis , Saliva/química , 17-alfa-Hidroxiprogesterona , Hiperplasia Suprarrenal Congénita/metabolismo , Adulto , Femenino , Humanos , Masculino , RadioinmunoensayoRESUMEN
BACKGROUND: We studied pS2 protein in breast tumors and its relation with estrogen and progesterone receptors and with anatomopathological characteristics of the tumors. MATERIAL AND METHODS: We measured the pS2 content (by IRMA) and steroid receptors content (by EIA) in 151 breast tumors. Results were compared and correlated with tumoral characteristics. RESULTS: 53% of tumors were pS2+. Among them, 91% were estrogen receptors +.86% of estrogen receptors negative tumors were pS2-. We observed correlation between pS2 and estrogen receptors values (r = 0.56; p < 0.0001) and between pS2 and progesterone receptors values (r = 0.53; p < 0.0001). Distributing the tumors in pS2+ and pS2-, we observed association between pS2+ and estrogen receptors + (chi 2 = 45.6; p < 0.0001) and pS2+ and progesterone receptors + (chi 2 = 43.1; p < 0.0001). However, we found a 18.5% of estrogen receptors + pS2- tumors. We observed a significant difference between GII and GIII tumoral grades (chi 2 = 5.51; p < 0.019), with a majority of pS2+ tumors in GII and pS2- tumors in GIII. CONCLUSIONS: The estrogen receptors in estrogen receptors + ps2- tumors may be non functional. The presence of pS2 protein alternative to that of progesterone receptors may indicate a functional heterogeneity of the estrogen receptors system, which is of interest in evaluating prognosis and response to the hormonal therapy.
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Neoplasias de la Mama/metabolismo , Estrógenos/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Proteínas , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Femenino , Humanos , Persona de Mediana Edad , Factor Trefoil-1 , Proteínas Supresoras de TumorRESUMEN
OBJETIVO: Detectar las dudas y las necesidades formativas de los médicos de familia de un centro de salud urbano y docente durante la práctica clínica habitual. MATERIAL Y MÉTODOS: Estudio descriptivo transversal de un mes de duración en un centro de salud urbano de Madrid. Los médicos de familia del equipo fueron entrevistados tras la consulta sobre las dudas que habían tenido, eligiendo dos de ellas. Las dudas no resueltas en las dos semanas siguientes se agruparon por materias y según las taxonomías vigentes. Posteriormente se elaboró un programa docente para resolverlas. RESULTADOS: Participaron 19 de los 21 médicos del centro, y en el periodo de estudio atendieron a 10.678 pacientes. Se detectaron 0,44 dudas por cada 10 pacientes atendidos. De las 384 dudas elegidas, el 83,34% fueron clínicas y el 16,66%, no clínicas. El 51,2% de estas quedaron sin resolver en los 15días tras la consulta. Los métodos para su resolución fueron las bases científicas de internet (PubMed, UpToDate y guías de práctica clínica, principalmente; 38%), seguido de la consulta a otros colegas (34,9%). CONCLUSIONES: La mayoría de las dudas generadas durante la consulta son clínicas, aunque existe una carga importante de preguntas burocráticas. Más de la mitad de las dudas no se resuelven durante la consulta ni en los 15días posteriores. Las bases de datos científicas en internet son la principal fuente de información, seguida de la consulta a otros colegas. Deberían mejorar los tiempos por paciente y el acceso a fuentes de información fiables y ágiles, para mejorar estos aspectos
OBJECTIVE: To detect doubts and training needs in an urban health and family doctor training centre during the usual practice. MATERIAL AND METHODS: A cross-sectional descriptive study was conducted for one month in an urban health centre in Madrid. Family doctors were interviewed after their daily clinics about the doubts they had identified, choosing two of them. Unresolved questions were grouped by subject and according to the current taxonomies. A teaching program was then developed to tackle them. RESULTS: Out of a total 21 physicians of the centre, 19 attended 10,678 patients during the period. The doubt detection rate was 0.44 doubts for every 10 patients attended. Of the 384 questions chosen, 83.34% were clinical and 16.66% were non-clinical. Just over half (51.2%) of these were still unresolved 15days later the consultation event. The main methods for their resolution were using the scientific bases on the internet (mainly PubMed, UpToDate and Clinical Practice Guidelines; 38%), followed by consultation with other colleagues (34.9%). CONCLUSIONS: Most of the doubts generated during clinics were clinical, although there is a significant burden of bureaucratic questions. More than half of the doubts are not resolved during the consultation or within the following 15days. The scientific databases on the internet are the main sources of information, although consulting other colleagues was often used as well. Additional time for dealing with patients and enhanced access to solve complex questions should be available to improve the success rate
Asunto(s)
Humanos , Masculino , Femenino , Persona de Mediana Edad , Médicos de Familia/psicología , Atención Primaria de Salud/organización & administración , Servicios Urbanos de Salud/organización & administración , Estudios Transversales , Entrevistas como Asunto , Médicos de Familia/educación , EspañaRESUMEN
We have prepared and analyzed 40 HSV-1 intratypic recombinants with regard to plaque morphology and glycoprotein C(gC) phenotypes. Vero cells have been cotransfected with the intact genome of HSV-1(F) and cloned or uncloned DNA fragments from HSV-1(MP) and recombinants inducing the fusion of Vero cells [syncytial (Syn) recombinants] have been selected and purified. Marker transfer of the Syn phenotype has been observed with the cloned BamHI L and B fragments (0.706-0.745 and 0.745-0.810 map units, respectively) as well as with the uncloned HpaI TXO fragment (0.710-0.761) from MP DNA. No marker transfer has been observed with F DNA alone or with the cloned BamHI N fragment (0.863-0.898 map units). When viruses expressing the Syn phenotype in Vero cells were tested in HEp-2 cells, three kinds of recombinants were observed. Members of the first class expressed a wild type, cytoaggregating (Syn+), plaque morphology in these cells. Members of the second class induced the complete fusion (Syn phenotype) of the cells. Members of the third class induced an intermediate plaque morphology, characterized by the formation of groups of polykaryocytes (fused cells) but without formation of a complete syncytium. All recombinants expressing the Syn+ phenotype in HEp-2 cells were also gC+, whereas recombinants expressing the Syn phenotype in these cells were gC- with one exception, in which low levels of gC could be detected (but clearly less than with HSV-1(F]. Concerning polykaryocytic class of recombinants, some of them were gC+ while others expressed only low amounts of gC; no gC- virus was observed within this class of recombinants. The three classes of recombinants were observed with each of the cloned BamHI L and B fragments and also with the HpaI TXO fragment, suggesting the existence of multiple adjacent or overlapping loci affecting plaque morphology and the control of the accumulation or the synthesis of gC at both sides of 0.745 map units.
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Simplexvirus/genética , Proteínas del Envoltorio Viral/genética , Proteínas Virales/genética , Animales , Línea Celular , Mapeo Cromosómico , ADN Viral/genética , Genes Reguladores , Genes Virales , Humanos , Fenotipo , Recombinación Genética , Proteínas Virales de Fusión , Ensayo de Placa ViralRESUMEN
Osteoclasts are postmitotic, multinucleated giant cells generated by the fusion of hematopoietic mononuclear precursors from the monocyte-macrophage lineage. In culture, adherent macrophages from blood-derived monocytes grow, gather, and fuse together to form multinucleated osteoclast-like cells. These events are controlled by 1,25(OH)(2)D(3). To sort out new 1,25(OH)(2)D(3) target genes involved in osteoclast differentiation, we have performed an RT-PCR differential display using mRNA from macrophages induced for 10 h by 1,25(OH)(2)D(3) compared to nontreated cells. We have identified a new target gene, a chick ATP-dependent Ca(2+) pump, ChkSERCA3. Although the level of the corresponding transcript increases during the differentiation process from macrophages to osteoclast-like cells, its steady-state level is downregulated by hormone treatment. The action of 1,25(OH)(2)D(3) on the Ca(2+)-ATPase gene expression is independent of de novo protein synthesis and is hormone dose dependent. This expression in adult chick was restricted to the hematopoietic cell lineage, spleen, lung, intestine, and brain, whereas no expression was detected in embryos. The function of the protein can be predicted from its high homology with the other members of the SR ATP-dependent Ca(2+) pump family, i.e., storage and control of cytosolic Ca(2+) directly regulated by 1, 25(OH)(2)D(3), further supporting the critical role for intracellular calcium in highly specialized cells such as osteoclasts.
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Calcitriol/farmacología , ATPasas Transportadoras de Calcio/genética , Pollos/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Monocitos/metabolismo , Retículo Sarcoplasmático/enzimología , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Diferenciación Celular/efectos de los fármacos , Clonación Molecular , Células Gigantes/citología , Células Gigantes/efectos de los fármacos , Células Gigantes/metabolismo , Humanos , Isoenzimas/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Filogenia , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismoRESUMEN
The carbonic anhydrase II gene, whose transcription is enhanced by 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3), encodes an important enzyme in bone-resorbing cells derived from the fusion of monocytic progenitors. We analyzed the 1,25-(OH)2D3-mediated activation of the avian gene by transient transfection assays with promoter/reporter constructs into HD11 chicken macrophages and by DNA mobility shift assays. Deletion and mobility shift analyses indicated that the -62/-29 region confers 1,25-(OH)2D3 responsiveness and forms DNA-protein complexes. The addition of an anti-vitamin D receptor (VDR) antibody inhibited binding to this sequence, whereas anti-retinoid X receptor (RXR) antibody generated a lower mobility complex. Therefore, we concluded that this element binds a VDR.RXR heterodimer, but the addition of extra 1,25-(OH)2D3 had no effect on the formation of this complex. Moreover, the use of nuclear extracts from 1,25-(OH)2D3-treated macrophages led to the formation of an additional high mobility complex also composed of VDR.RXR heterodimer. Mutations provided evidence that the 1, 25-(OH)2D3-mediated activation of the carbonic anhydrase II gene is mediated by VDR.RXR heterodimers bound to a DR3-type vitamin D response element with sequence AGGGCAtggAGTTCG. This vitamin D response element is also functional in the ROS 17/2.8 osteoblasts.
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Anhidrasas Carbónicas/genética , Regiones Promotoras Genéticas , Proteína de Unión a Vitamina D/química , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Pollos , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Dimerización , Mutagénesis , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Células Tumorales Cultivadas , Proteína de Unión a Vitamina D/metabolismoRESUMEN
We have tested a number of herpes simplex virus type 1 (HSV-1) populations for their ability to enter and express virus polypeptides in non-permissive rat XC cells. The viruses tested included 40 intratypic F(MP) recombinants and different batches of virus, belonging to the same strains, that had been produced in two different permissive systems (HEp-2 or Vero cells). Our results indicated that the ability to infect XC cells was determined in part by genetic elements of the virus genome and in part by phenotypic characteristics conferred by the permissive cell that had produced the virus: a virus strain like HSV-1 MP which, when produced in HEp-2 cells, was able to infect XC cells, lost this ability when produced in Vero cells. Working only with viruses produced in HEp-2 cells we showed that the ability to enter XC cells could be transferred from the MP strain to the F strain (which does not normally infect XC cells efficiently) by transfer of the cloned BamHI B (map units 0.745 to 0.81) or BamHI L (map units 0.706 to 0.745) fragments isolated from MP DNA. The implicated locus or loci seemed to segregate, however, from loci controlling gC synthesis and cell fusion, which have been described as mapping in the same region.
Asunto(s)
Transformación Celular Viral , Simplexvirus/fisiología , Animales , Línea Celular , Humanos , Ratas , Simplexvirus/genética , Especificidad de la Especie , Células VeroRESUMEN
The ability to induce the synthesis of virus polypeptides in nonpermissive XC cells by several strains, variants, and intratypic recombinants of HSV-1 has been investigated. Our results show that (i) whereas HSV-1 strains mP and MP, and recombinants F(MP)A through D (Ruyechan et al., J. Virol. 29, 677-697, 1979) and HFEMtsB5/MP3 (Manservigi et al., Proc. Nat. Acad. Sci. USA 74, 3913-3917, 1977) induced in XC cells the synthesis of several virus polypeptides (the "positive" viruses), in the case of HSV-1 strain F, variants HFEMtsB5 and mPtsHA1 (both tested at 33 degrees), and recombinants F(MP)E and F(MP)F, no virus specific polypeptides could be detected in these cells (the "negative" viruses). (ii) Failure of the "negative" viruses to synthesize polypeptides in XC cells could be explained by failure of the virions to penetrate the cells since polyethylene glycol, a fusion promotor agent, enabled these viruses to overcome the blockage of their expression. (iii) The ability to modulate penetration into XC cells is genetically determined. A locus affecting this function maps between coordinates 0.70 to 0.82 of HSV-1 DNA and is closely linked to Cr, a locus controlling the synthesis or accumulation of virus glycoprotein C (gC) (Ruyechan et al., 1979). (iv) A decrease in the amount of gC, relative to gB, is associated with an enhanced ability to enter XC cells, suggesting that gC may control penetration into these restrictive cells by negatively modulating the gB promoted fusion between host cell and virion membranes.
Asunto(s)
Recombinación Genética , Simplexvirus/patogenicidad , Proteínas del Envoltorio Viral , Proteínas Virales/análisis , Animales , Línea Celular , Chlorocebus aethiops , Glicoproteínas/biosíntesis , Humanos , Polietilenglicoles/farmacología , Simplexvirus/genética , Proteínas Virales/biosíntesis , VirulenciaRESUMEN
The expression of immediate-early (IE) genes of herpes simplex virus type 1 (HSV-1, MP strain) in non-permissive rat XC cells was analysed and compared with the expression of IE genes in permissive HEp-2 cells by the following three methods: analysis of virus polypeptide synthesis in infected cells, Northern blot hybridization between poly(A) nuclear or cytoplasmic RNA and in vitro labelled virus DNA or plasmid-cloned fragments corresponding to IE genes, and ability of poly(A) cytoplasmic RNAs to direct synthesis of virus polypeptides in vitro. ICP4 (175K), ICP0 (110K) and ICP27 (62K) were synthesized in XC cells although in smaller amounts than in HEp-2 cells; ICP4 is functional since early and late polypeptides could be observed. Their corresponding mRNAs were present at low levels in nuclei and in cytoplasm and are functional since the polypeptides were synthesized in a rabbit reticulocyte system. ICP22 (68K) was not detectable in infected XC cells; its mRNA was present in nuclei and in cytoplasm, but it is not functional since the corresponding polypeptide was not synthesized in a rabbit reticulocyte system. This suggests some structural differences in the ICP22 mRNA molecules in infected XC and HEp-2 cells and implicates cellular determinants in the control of the expression of HSV-1 IE genes.
Asunto(s)
Herpes Simple/genética , Simplexvirus/genética , Proteínas Virales/genética , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , ADN Viral/genética , Regulación de la Expresión Génica , Genes Virales , Humanos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Transcripción Genética , Replicación ViralRESUMEN
We have investigated the effect of Epstein-Barr virus nuclear antigen 1 (EBNA-1), a nuclear protein encoded by EBV, on herpes simplex virus type 1 (HSV-1) infection either in cells constitutively expressing EBNA-1 or in transient expression assays. Rat-1 cells and rat embryo fibroblasts (REF) immortalized by c-myc or E1A were transfected with a specific EBV DNA fragment coding for EBNA-1. Cloned cell lines which constitutively expressed this antigen were infected with HSV-1. Our results indicate that in EBNA-1-expressing cells, virus growth was higher than in control cells for different virus strains or rodent cell lines. This increase was maximal when cells were infected at low multiplicity, as determined by virus growth, and correlated with the stimulation of viral DNA synthesis. REF + c-myc and Vero cells were cotransfected by an EBNA-1 expression vector driven by Moloney murine leukaemia virus LTR and HSV-1 immediate-early (alpha 0) or early thymidine kinase upstream promoter regulatory regions linked to chloramphenicol acetyltransferase (CAT) coding sequences as effectors. In both cell lines, stimulation of CAT expression by EBNA-1 was observed only with the immediate-early promoter. These results suggest that EBNA-1 can transactivate immediate-early HSV-1 expression.
Asunto(s)
Antígenos Virales/biosíntesis , Simplexvirus/crecimiento & desarrollo , Animales , Antígenos Virales/genética , Línea Celular Transformada , Núcleo Celular/inmunología , Replicación del ADN , ADN Viral/biosíntesis , Antígenos Nucleares del Virus de Epstein-Barr , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Humanos , Simplexvirus/genética , Simplexvirus/fisiología , Activación Transcripcional , TransfecciónRESUMEN
Impaired synthesis of adrenal steroid hormones because of steroid 21-hydroxylase deficiency is one of the most common inborn errors of metabolism. To expedite molecular diagnosis in families with 21-hydroxylase deficiency, we have designed a rapid strategy to determine nine of the most common mutations in the 21-hydroxylase gene. According to the mutation to be detected, we apply either of two simple strategies: digestion with adequate restriction enzyme or use of the amplification-created restriction site (ACRS) approach and subsequent restriction analysis. Both procedures are rapid and, being nonradioactive, are safer to perform; moreover determination of zygosity in the analyzed mutations requires only one tube per mutation.