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1.
HIV Med ; 22(5): 360-371, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33369017

RESUMEN

OBJECTIVES: The aim of this analysis was to characterize transmitted drug resistance (TDR) in Strategic Timing of Antiretroviral Treatment (START) study participants by next-generation sequencing (NGS), a sensitive assay capable of detecting low-frequency variants. METHODS: Stored plasma from participants with entry HIV RNA > 1000 copies/mL were analysed by NGS (Illumina MiSeq). TDR was based on the WHO 2009 surveillance definition with the addition of reverse transcriptase (RT) mutations T215N and E138K, and integrase strand transfer inhibitor (INSTI) surveillance mutations (Stanford HIVdb). Drug resistance mutations (DRMs) detected at three thresholds are reported: > 2%, 5% and 20% of the viral population. RESULTS: Between 2009 and 2013, START enrolled 4684 antiretroviral therapy (ART)-naïve individuals in 35 countries. Baseline NGS data at study entry were available for 2902 participants. Overall prevalence rates of TDR using a detection threshold of 2%/5%/20% were 9.2%/5.6%/3.2% for nucleoside reverse transcriptase inhibitors (NRTIs), 9.2%/6.6%/4.9% for non-NRTIs, 11.4%/5.5%/2.4% for protease inhibitors (PIs) and 3.5%/1.6%/0.1% for INSTI DRMs and varied by geographic region. Using the 2% detection threshold, individual DRMs with the highest prevalence were: PI M46IL (5.5%), RT K103NS (3.5%), RT G190ASE (3.1%), T215ISCDVEN (2.5%), RT M41L (2.2%), RT K219QENR (1.7%) and PI D30N (1.6%). INSTI DRMs were detected almost exclusively below the 20% detection threshold, most commonly Y143H (0.4%), Q148R (0.4%) and T66I (0.4%). CONCLUSIONS: Use of NGS in this study population resulted in the detection of a large proportion of low-level variants which would not have been detected by traditional Sanger sequencing. Global surveillance studies utilizing NGS should provide a more comprehensive assessment of TDR prevalence in different regions of the world.


Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , VIH-1 , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral/genética , Genotipo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación
2.
HIV Med ; 16 Suppl 1: 77-87, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25711326

RESUMEN

OBJECTIVES: HIV-1 transmitted drug resistance (TDR) in treatment-naïve individuals is a well-described phenomenon. Baseline genotypic resistance testing is considered standard of care in most developed areas of the world. The aim of this analysis was to characterize HIV-1 TDR and the use of resistance testing in START trial participants. METHODS: In the Strategic Timing of AntiRetroviral Treatment (START) trial, baseline genotypic resistance testing results were collected at study entry and analysed centrally to determine the prevalence of TDR in the study population. Resistance was based on a modified 2009 World Health Organization definition to reflect newer resistance mutations. RESULTS: Baseline resistance testing was available in 1946 study participants. Higher rates of testing occurred in Europe (86.7%), the USA (81.3%) and Australia (89.9%) as compared with Asia (22.2%), South America (1.8%) and Africa (0.1%). The overall prevalence of TDR was 10.1%, more commonly to nonnucleoside reverse transcriptase inhibitors (4.5%) and nucleoside reverse transcriptase inhibitors (4%) compared with protease inhibitors (2.8%). The most frequent TDR mutations observed were M41L, D67N/G/E, T215F/Y/I/S/C/D/E/V/N, 219Q/E/N/R, K103N/S, and G190A/S/E in reverse transcriptase, and M46I/L and L90M in protease. By country, the prevalence of TDR was highest in Australia (17.5%), France (16.7%), the USA (12.6%) and Spain (12.6%). No participant characteristics were identified as predictors of the presence of TDR. CONCLUSIONS: START participants enrolled in resource-rich areas of the world were more likely to have baseline resistance testing. In Europe, the USA and Australia, TDR prevalence rates varied by country.


Asunto(s)
Transmisión de Enfermedad Infecciosa , Farmacorresistencia Viral , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Adolescente , Adulto , Anciano , Femenino , Genotipo , Técnicas de Genotipaje , VIH-1/clasificación , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Adulto Joven
3.
Science ; 209(4457): 695-7, 1980 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-7394526

RESUMEN

Messenger RNA coding for acetylcholine receptor peptides has been identified. This polyadenylate [poly(A)+]RNA from Torpedo californica directs, in a cell-free system, the synthesis of peptides 60,000, 51,000, 49,000 41,000, and 35,000 daltons which account for approximately 2 percent of the total synthesized proteins. The results suggest that several different messenger RNA's code for the receptor subunits. These proteins react specifically to antiserum to native acteylcholine receptor, suggesting that the primary translational product has conformational features similar to the native receptor. Further, the results support the idea that there is post-translational modification of receptor subunits as the molecular weights of the cell-free synthesized proteins differ from those of purified receptor subunits.


Asunto(s)
Acetilcolina/metabolismo , Metabolismo , Biosíntesis de Péptidos , Biosíntesis de Proteínas , Receptores Colinérgicos/metabolismo , Animales , Bungarotoxinas/metabolismo , Sistema Libre de Células , Peces , Poli A/metabolismo , ARN Mensajero
4.
Science ; 241(4863): 350-3, 1988 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-2838908

RESUMEN

Although steroid hormone receptors are known to activate gene expression by binding to specific hormone-dependent enhancers, the mechanisms by which steroids inhibit the transcription of specific genes are unknown. It is shown here by gene transfer studies that the same glucocorticoid receptor that activates gene expression can negatively regulate expression of the human glycoprotein hormone alpha-subunit gene. Glucocorticoid inhibition was conferred by a 52-nucleotide region that also contains elements crucial both for adenosine 3',5'-monophosphate (cAMP) responsiveness and for placental-specific expression of this gene and was observed only under conditions in which these elements were functioning as enhancers. Purified glucocorticoid receptor was found to bind to DNA that overlap the cAMP responsive elements sites in this region. It is hypothesized that steroid receptors negatively regulate gene expression by interfering with the activity or binding of other important transcription factors.


Asunto(s)
Gonadotropina Coriónica/genética , AMP Cíclico/fisiología , Dexametasona/farmacología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Receptores de Esteroides/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Línea Celular , Proteínas de Unión al ADN/fisiología , Humanos , Técnicas In Vitro , Factores de Transcripción/fisiología
5.
Science ; 205(4406): 602-7, 1979 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-377496

RESUMEN

The nucleotide sequence of a DNA complementary to human growth hormone messenger RNA was cloned; it contains 29 nucleotides in its 5' untranslated region, the 651 nucleotides coding for the prehormone, and the entire 3' untranslated region (108 nucleotides). The data reported predict the previously unknown sequence of the signal peptide of human growth hormone and, by comparison with the previously determined sequences of rat growth hormone and human chorionic somatomammotropin, strengthens the hypothesis that these genes evolved by gene duplication from a common ancestral sequence. The human growth hormone gene sequences have been linked in phase to a fragment of the trp D gene of Escherichia coli in a plasmid vehicle, and a fusion protein is synthesized at high level (approximately 3 percent of bacterial protein) under the control of the regulatory region of the trp operon. This fusion protein (70 percent of whose amino acids are coded for by the human growth hormone gene) reacts specifically with antibodies to human growth hormone and is stable in E. coli.


Asunto(s)
ADN Recombinante/metabolismo , Escherichia coli/metabolismo , Hormona del Crecimiento/biosíntesis , Plásmidos , Biosíntesis de Proteínas , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Humanos , Hipófisis/metabolismo , Neoplasias Hipofisarias/metabolismo , Poli A/metabolismo , Prolactina/biosíntesis , ARN Mensajero/metabolismo
6.
Science ; 171(3967): 189-91, 1971 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-4395230

RESUMEN

Mouse lymphoma cells in culture which are killed by adrenal steroids contain specific cortisol receptors that may be involved in the initial events of hormone action. The similarity of these receptors to those in hepatoma tissue culture cells, where adrenal steroids induce tyrosine aminotransferase, suggests that certain aspects of steroid action are similar in the two systems. In three steroid-resistant lymphoma cell populations specific binding was less than in the parent lines, suggesting that conversion to steroid resistance may be associated with changes in specific steroid binding.


Asunto(s)
Técnicas de Cultivo , Hidrocortisona/farmacología , Linfoma , Receptores de Droga , 17-Cetosteroides/farmacología , Androstanos/farmacología , Animales , Carcinoma Hepatocelular/enzimología , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Inducción Enzimática , Hidroxiprogesteronas/farmacología , Neoplasias Hepáticas , Ratones , Testosterona/farmacología , Tritio , Tirosina Transaminasa/biosíntesis
7.
Science ; 175(4018): 189-90, 1972 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-4332760

RESUMEN

The cellular site of binding of dexamethasone by specific glucocorticoid receptors in cultured hepatoma cells was investigated with the use of certain mercurials. p-Chloromercuribenzene sulfonate and p-chloromercuribenzoate inhibit the binding of steroid by receptors in cell-free extracts, but they allow the steroid-receptor complex to form in whole cells. In contrast, HgCl(2) inhibits binding both in extracts and cells. Since both organic mercury compounds, unlike HgCl(2), do not readily enter intact cells, it appears that the specific steroid binding occurs inside the cell rather than at the cell membrane.


Asunto(s)
Sitios de Unión , Dexametasona/metabolismo , Receptores de Droga , Animales , Autorradiografía , Sitios de Unión/efectos de los fármacos , Carcinoma Hepatocelular , Sistema Libre de Células , Células Cultivadas , Cloruros/farmacología , Cloromercuribenzoatos/farmacología , Neoplasias Hepáticas , Mercurio/farmacología , Ratas , Ácidos Sulfónicos/farmacología , Tritio
8.
Science ; 209(4453): 289-92, 1980 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-7384802

RESUMEN

The human genes for growth hormone (GH), chorionic somatomammotropin (CSH), and a third growth hormone-like gene (GHL) have been located on chromosome 17 in humans. DNA fragments of 2.6, 2.8, and 9.5 kilobase pairs containing GH, CSH, and GHL, respectively, were identified in human genomic DNA, and a 7.5-kilobase DNA fragment related to growth hormone DNA sequences was found in mouse cells. In somatic hybrids of human and mouse cells containing reduced numbers of human chromosomes, but a normal complement of mouse chromosomes, the mouse, 7.5-kolobase DNA fragment was always present, whereas the 2.6-, 2.8-, and 9.5-kilobase human fragments were present only when human chromosome 17 was also present.


Asunto(s)
Cromosomas Humanos 16-18 , ADN , Genes , Hormona del Crecimiento/biosíntesis , Lactógeno Placentario/biosíntesis , Animales , Secuencia de Bases , Línea Celular , ADN/metabolismo , Humanos , Células Híbridas/metabolismo , Ratones , Translocación Genética
9.
Science ; 243(4896): 1346-51, 1989 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-2493678

RESUMEN

The x-ray crystal structure of recombinant human renin has been determined. Molecular dynamics techniques that included crystallographic data as a restraint were used to improve an initial model based on porcine pepsinogen. The present agreement factor for data from 8.0 to 2.5 angstroms (A) is 0.236. Some of the surface loops are poorly determined, and these disordered regions border a 30 A wide solvent channel. Comparison of renin with other aspartyl proteinases shows that, although the structural cores and active sites are highly conserved, surface residues, some of which are critical for specificity, vary greatly (up to 10A). Knowledge of the actual structure, as opposed to the use of models based on related enzymes, should facilitate the design of renin inhibitors.


Asunto(s)
Proteínas Recombinantes , Renina , Ácido Aspártico Endopeptidasas , Fármacos Cardiovasculares/farmacología , Endopeptidasas/metabolismo , Humanos , Modelos Moleculares , Pepsina A/metabolismo , Conformación Proteica , Proteínas Recombinantes/metabolismo , Renina/metabolismo
10.
Science ; 280(5370): 1747-9, 1998 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-9624051

RESUMEN

The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define the site that binds the coactivators, glucocorticoid receptor-interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription involves formation of this surface by folding the carboxyl-terminal alpha helix against a scaffold of three other helices. These features may represent general ones for nuclear receptors.


Asunto(s)
Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Triyodotironina/metabolismo , Células HeLa , Histona Acetiltransferasas , Humanos , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Coactivador 1 de Receptor Nuclear , Coactivador 2 del Receptor Nuclear , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/genética , Proteínas Recombinantes de Fusión/metabolismo , Receptores X Retinoide , Triyodotironina/farmacología
11.
J Clin Invest ; 60(6): 1230-9, 1977 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-199617

RESUMEN

l-Thyroxine is converted to 3,5,3'-l-triiodothyronine (T(3)) as well as to 3,3',5'-l-triiodothyronine (reverse T(3)). One product of further deiodination is 3,3'-diiodothyronine (3,3'T(2)). The serum levels of reverse T(3) and 3,3'T(2) change considerably in various physiological and disease states. We previously found that reverse T(3) and 3,3'T(2) bind to the solubilized hepatic nuclear "receptors" for thyroid hormones. This led us to study binding and actions of these metabolites in cultured rat pituitary cells in which glucose consumption and growth hormone production are regulated by T(3) and l-thyroxine. Reverse T(3) and 3,3'T(2) stimulated growth hormone production and glucose consumption and inhibited nuclear binding of radioactive T(3). Either metabolite produced maximal effects that equaled those of T(3), and neither inhibited the T(3) response. Further, additive effects were observed when reverse T(3) was combined with submaximal concentrations of T(3). In serum-free and serum-containing media, concentrations of 3,3'T(2) 50- to 70- and 10- to 100-fold greater, respectively, than those of T(3) were required for equivalent stimulations and for inhibition of nuclear binding by T(3). The relative activity differences under the two conditions can be attributed to weaker serum protein binding of 3,3'T(2) than T(3). With cells in serum-free media, reverse T(3) was a less avid competitor than 3,3'T(2) for T(3) binding by the nuclear receptors, and was less potent than 3,3'T(2) (0.001 the potency of T(3)) in inducing growth hormone production or glucose oxidation. In incubations with serum-containing media, reverse T(3) was an ineffective competitor for T(3) binding, and had only 0.1 the inducing potency of 3,3'T(2) (0.001 the potency of T(3)). The weaker activity of reverse T(3) relative to 3,3'T(2) in serum-containing media could be explained by stronger serum binding of reverse T(3) than 3,3'T(2). In addition, after long-term incubation of cells with radioactive reverse T(3), much of the cell-associated radioactivity was recovered as 3,3'T(2). These studies suggest that reverse T(3) and 3,3'T(2) can stimulate thyroid hormone-regulated functions as weak agonists by acting via the same receptors that mediate T(3) actions. Moreover, some of the effects of reverse T(3) may be due to 3,3'T(2) produced by deiodination of reverse T(3).


Asunto(s)
Glucosa/metabolismo , Hormona del Crecimiento/biosíntesis , Hipófisis/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Tironinas/análogos & derivados , Triyodotironina/farmacología , Animales , Línea Celular , Técnicas In Vitro , Isomerismo , Hipófisis/metabolismo , Neoplasias Hipofisarias , Ratas , Tironinas/farmacología , Tritio
12.
J Clin Invest ; 58(3): 579-89, 1976 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-182723

RESUMEN

Aldosterone receptors from rat kidney slices were utilized in a competitive binding technique to analyze the contribution of various steroids to plasma "mineralocorticoid" activity and to assess their possible role in hypertension. To consider simultaneously the plasma binding, steroids were incubated with slices in undiluted plasma; competitor activities for [3H]aldosterone binding were aldosterone, 100%; deoxycorticosterone, 16.2%; cortisol, 0.4%; and 18-hydroxy-deoxy-corticosterone and d18-hydroxy-corticosterone, 0.1%. These steroids were more active in buffer than plasma, suggesting that they bind to plasma and that this reduces their receptor binding. Analysis of the competition data suggests that at normal plasma concentrations, aldosterone occupies the receptors to a major extent, cortisol occupies some of the receptors, and deoxycorticosterone and 8-hydroxydeoxycorticosterone contribute little to receptor occupancy. Two steroids implicated in low-renin essential hypertension, 16beta-hydroxy-dehydro-epiandrosterone and 16-oxoandrostenediol, did not have significant competitor activity. Competitor activity in plasmas from normal subjects taken at 12 noon (upright) was greater than that in those taken at 8 a.m. (supine). Since the 12 noon samples had higher aldosterone and lower cortisol levels than the 8 a.m. samples, the competitor activity under these physiological circumstances reflects aldosterone more than cortisol. The competitor activities of plasmas from patients relative to normal subjects (100+/-12.1%; mean+/-SEM) were: normal renin "essential" hypertension, 117+/-14%; low-renin essential hypertension, 101+/-6.6%; and primary aldosteronism, 176+/-14.3%. Thus a significant increase in activity of steroids that interact with mineralocorticoid receptors was detected in primary aldosteronism (P LESS THAN 0.01) BUT WAS NOT DETECTED IN LOW-RENIN OR NORMAL-RENIN ESSENTIAL HYPERTENSION.


Asunto(s)
Aldosterona/metabolismo , Hipertensión/sangre , Mineralocorticoides/sangre , Receptores de Superficie Celular , Androstenodioles/sangre , Androstenodioles/farmacología , Animales , Unión Competitiva , Corticosterona/análogos & derivados , Corticosterona/sangre , Corticosterona/farmacología , Depresión Química , Desoxicorticosterona/sangre , Desoxicorticosterona/farmacología , Hidrocortisona/sangre , Hidrocortisona/farmacología , Postura , Ratas , Factores de Tiempo
13.
Mol Cell Biol ; 5(11): 2984-92, 1985 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-3018491

RESUMEN

In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.


Asunto(s)
Genes Reguladores , Genes , Hormona del Crecimiento/genética , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Animales , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , Humanos , Cinética , Datos de Secuencia Molecular , Unión Proteica , Ratas , Receptores de Glucocorticoides/genética , Simplexvirus/enzimología , Simplexvirus/genética , Timidina Quinasa/genética , Transcripción Genética , Transfección
14.
Mol Cell Biol ; 12(3): 1352-6, 1992 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-1545816

RESUMEN

Activators of protein kinase C, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), are known to regulate the expression of many genes, including the tumor necrosis factor alpha (TNF) gene, by affecting the level or activity of upstream transcription factors. To investigate the mechanism whereby TPA activates the TNF promoter, a series of 5'-deletion mutants of the human TNF promoter linked to chloramphenicol acetyltransferase was transfected into U937 human promonocytic cells. TPA produced a 7- to 11-fold activation of all TNF promoters tested, even those promoters truncated to contain only the core promoter with no upstream enhancer elements. The proximal TNF promoter containing only 28 nucleotides upstream and 10 nucleotides downstream of the RNA start site confers TPA activation to a variety of unrelated upstream enhancer elements and transcription factors, including Sp1, CTF/NF1, cyclic AMP-response element, GAL-E1a, and GAL-VP16. The level of activation by TPA depends on the TATA box structure, since the TPA response is greater in promoters containing the sequence TATAAA than in those containing TATTAA or TATTTA. These findings suggest that the core promoter region is a target for gene regulation by second-messenger pathways.


Asunto(s)
Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/genética , Secuencia de Bases , Línea Celular , ADN , Humanos , Datos de Secuencia Molecular , Mutación , Plásmidos , Proteína Quinasa C/metabolismo
15.
Mol Cell Biol ; 13(5): 3042-9, 1993 May.
Artículo en Inglés | MEDLINE | ID: mdl-8474460

RESUMEN

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes fos , Genes jun , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Regulación de la Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Timidina Quinasa/genética , Transfección
16.
Mol Cell Biol ; 7(3): 1193-7, 1987 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-3561414

RESUMEN

Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.


Asunto(s)
Regulación de la Expresión Génica , Hormona del Crecimiento/genética , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Animales , Secuencia de Bases , Células Cultivadas , Deleción Cromosómica , ADN/genética , Especificidad de Órganos , Adenohipófisis/metabolismo , Ratas , Transfección
17.
Mol Cell Biol ; 15(3): 1554-63, 1995 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-7862148

RESUMEN

We investigated how overexpression of human TATA-box-binding protein (TBP) affects the action of estrogen receptor (ER) and compared the response with that of other activators. When ER activates a simple promoter, consisting of a response element and either the collagenase or tk TATA box, TBP overexpression potentiates transcription. TBP potentiates only estrogen-induced and not basal transcription and does so independent of spacing between response element and TATA box. TBP overexpression also reduces autoinhibition by overexpressed ER, suggesting that one target of the autoinhibition may be TBP itself. Both AF-1 and AF-2 domains of ER are potentiated by TBP, and each domain binds TBP in vitro. Like ER, chimeric GAL4/VP16 and GAL4/Tat activators are also potentiated by TBP, as is the synergistic activation by ER and GAL4/VP16 on a complex promoter. Unlike ER, GAL4/Sp1 and GAL4/NF-I become less potent when TBP is overexpressed. Furthermore, synergy between ER and Sp1 or between ER and NF-I, whether these are supplied by transfected GAL4 fusions or by the endogenous genes, is inhibited by TBP overexpression. Thus, ER resembles VP16 in response to TBP overexpression and is different from Sp1 and NF-I, which predominate over ER in setting the response on complex promoters.


Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Estradiol/farmacología , Receptores de Estrógenos/metabolismo , TATA Box , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , Cricetinae , Proteínas de Unión al ADN/metabolismo , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Receptores de Estrógenos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteína de Unión a TATA-Box
18.
Mol Cell Biol ; 17(8): 4687-95, 1997 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9234725

RESUMEN

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha). We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity.


Asunto(s)
Mutación , Receptores de Hormona Tiroidea/metabolismo , Proteínas de Saccharomyces cerevisiae , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional/genética , Triyodotironina/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Secuencia Conservada/genética , Proteínas de Unión al ADN , Células HeLa , Humanos , Ligandos , Modelos Moleculares , Coactivador 2 del Receptor Nuclear , Conformación Proteica , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/genética , Proteínas Recombinantes de Fusión , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
19.
Circ Res ; 89(7): 591-8, 2001 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-11577024

RESUMEN

Physiological and pathological cardiac hypertrophy have directionally opposite changes in transcription of thyroid hormone (TH)-responsive genes, including alpha- and beta-myosin heavy chain (MyHC) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), and TH treatment can reverse molecular and functional abnormalities in pathological hypertrophy, such as pressure overload. These findings suggest relative hypothyroidism in pathological hypertrophy, but serum levels of TH are usually normal. We studied the regulation of TH receptors (TRs) beta1, alpha1, and alpha2 in pathological and physiological rat cardiac hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH target genes, alpha- and beta-MyHC and SERCA. All 3 TR subtypes in myocytes were downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype, phenylephrine in culture and pressure overload in vivo. Myocyte TRbeta1 was upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine, T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and promoters. In addition, TR cotransfection and treatment with the TRbeta1-selective agonist GC-1 suggested different functional coupling of the TR isoforms, TRbeta1 to transcription of beta-MyHC, SERCA, and TRbeta1, and TRalpha1 to alpha-MyHC transcription and increased myocyte size. We conclude that TR isoforms have distinct regulation and function in rat cardiac myocytes. Changes in myocyte TR levels can explain in part the characteristic molecular phenotypes in physiological and pathological cardiac hypertrophy.


Asunto(s)
Cardiomegalia/fisiopatología , Regulación de la Expresión Génica , Miocardio/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Animales , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Actividad Motora , Miocardio/citología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Fenilefrina/farmacología , Condicionamiento Físico Animal , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Hormona Tiroidea/agonistas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Transfección , Triyodotironina/farmacología
20.
Circulation ; 103(8): 1089-94, 2001 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-11222471

RESUMEN

BACKGROUND: We have previously demonstrated that changes in myosin heavy chain (MHC) isoforms that occur in failing human hearts resemble the pattern produced in rodent myocardium in response to hypothyroidism. Because thyroid hormone status is usually within normal limits in these patients, we hypothesized that failing/hypertrophied human myocardium might have a defect in thyroid hormone signaling due to alterations in expression of thyroid hormone receptors (TRs). METHODS AND RESULTS: To examine this hypothesis, we used RNase protection assay to measure mRNA levels of TRs in failing left ventricles that exhibited a fetal pattern of gene expression, ie, decreased expression of alpha-MHC with increased beta-MHC expression compared with left ventricles from age-matched controls. We detected expression of TR-alpha(1), -alpha(2), and -beta(1) isoforms in human left ventricles. In failing left ventricles, TR-alpha(1) was downregulated, whereas TR-alpha(2), a splice variant that does not bind thyroid hormone but inhibits responses to liganded TRs, was increased. Expression levels of TR-beta(1) did not differ significantly between the 2 groups. According to linear regression analysis, expression levels of TR-alpha(1) and -alpha(2) were positively and negatively correlated with those of alpha-MHC, respectively. CONCLUSIONS: We conclude that decreases in TR-alpha(1) and increases in TR-alpha(2) may lead to local attenuation of thyroid hormone signaling in the failing human heart and that the resulting tissue-specific hypothyroidism is a candidate for the molecular mechanism that induces fetal gene expression in the failing human ventricle.


Asunto(s)
Expresión Génica , Cardiopatías/genética , Receptores de Hormona Tiroidea/genética , Transducción de Señal/genética , Adulto , Factor Natriurético Atrial/genética , Femenino , Proteínas Fetales/genética , Humanos , Complejo Mayor de Histocompatibilidad/genética , Masculino , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Receptores de Hormona Tiroidea/biosíntesis
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