RESUMEN
The existence of two rather than one estrogen receptor, today characterized as estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta), indicates that the mechanism of action of 17beta-estradiol and related synthetic drugs is more complex than previously thought. Because the homology of amino acid residues in the ligand-binding domain (LBD) of ERbeta is high compared with those amino acid residues in ERalpha LBD, previously shown to line the ligand binding cavity or to make direct contacts with ligands, it is not surprising that many ligands have a similar affinity for both receptor subtypes. We report that 17alpha-ethynyl, 17beta-estradiol, for example, has an ERalpha-selective agonist potency and that 16beta,17alpha-epiestriol has an ERbeta-selective agonist potency. We also report that genistein has an ERbeta-selective affinity and potency but an ERalpha-selective efficacy. Furthermore, we show that tamoxifen, 4-OH-tamoxifen, raloxifene, and ICI 164,384 have an ERalpha-selective partial agonist/antagonist function but a pure antagonist effect through ERbeta. In addition, raloxifene displayed an ERalpha-selective antagonist potency, in agreement with its ERalpha-selective affinity. However, although ICI 164,384 showed an ERbeta-selective affinity, it had a similar potency to antagonize the effect of 17beta-estradiol in the ERalpha- and ERbeta-specific reporter cell lines, respectively. In conclusion, our data indicate that the ligand binding cavity of ERbeta is probably more different from that of ERalpha than can be anticipated from the primary sequences of the two ER subtypes and that it will be possible to develop receptor-specific ligands that may form the basis of novel pharmaceuticals with better in vivo efficacy and side effect profile than current available drugs.
Asunto(s)
Antagonistas de Estrógenos/farmacología , Receptores de Estrógenos/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estradiol/análogos & derivados , Estradiol/farmacología , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Humanos , Ligandos , Alcamidas Poliinsaturadas , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacologíaRESUMEN
The estrogenic character of tamoxifen and raloxifene was studied on three different genes, an ERE-reporter construct and two endogenous genes, sex hormone binding globulin (SHBG) and pS2, in two variants of the human liver carcinoma cell line HepG2. On the ERE-reporter construct and the pS2 gene both tamoxifen and raloxifene acted as pure estrogen antagonists, whereas on the SHBG gene they functioned as partial estrogens/antiestrogens at concentrations below 1 microM and as full "agonists" at concentrations higher than 1 microM. The fold stimulatory effect of tamoxifen and raloxifene on SHBG protein expression was similar in the estrogen receptor (ER) expressing HepG2 cells (HepER3) and the parental non-ER expressing HepG2 cells at concentrations above 1 microM. In contrast, the 17beta-estradiol analogue moxestrol stimulated SHBG expression only in the HepER3 cells. Both tamoxifen and raloxifene had an additive effect to estrogen receptor-dependent SHBG gene expression in the HepER3 cells in the presence of saturating concentrations of moxestrol. However, a significant difference was observed in that a much higher concentration of moxestrol was required to see an additive effect of raloxifene compared to tamoxifen. The cytokine IL1-beta completely blocked the tamoxifen-dependent induction of SHBG gene expression in HepER3 cells, but only partly blocked the effect of moxestrol mediated by the ER. In conclusion, our results suggest that the mechanism for the liver-selective "estrogenic" character of tamoxifen and raloxifene is mediated by a non-ER dependent pathway.
Asunto(s)
Congéneres del Estradiol/farmacología , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Piperidinas/farmacología , Biosíntesis de Proteínas , Proteínas , Receptores de Estrógenos/fisiología , Globulina de Unión a Hormona Sexual/biosíntesis , Tamoxifeno/farmacología , Fosfatasa Alcalina/biosíntesis , Carcinoma Hepatocelular , Células Clonales , Etinilestradiol/análogos & derivados , Etinilestradiol/farmacología , Femenino , Genes Reporteros , Humanos , Cinética , Neoplasias Hepáticas , Placenta , Embarazo , Clorhidrato de Raloxifeno , Receptores de Estrógenos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.
Asunto(s)
Fosfatasa Ácida/química , Huesos/enzimología , Glicoproteínas/química , Isoenzimas/química , Proteínas de Plantas/química , Proteínas Recombinantes/química , Fosfatasa Ácida/genética , Fosfatasa Ácida/aislamiento & purificación , Animales , Baculoviridae/genética , Conformación de Carbohidratos , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Glicoproteínas/genética , Glicoproteínas/aislamiento & purificación , Hierro/análisis , Oligosacáridos/química , Fosfoproteínas Fosfatasas , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Espectrofotometría , Spodoptera/genética , Relación Estructura-Actividad , Fosfatasa Ácida TartratorresistenteRESUMEN
Eight donor hearts and six explanted hearts due to dilated cardiomyopathy, normal skeletal muscle and liver were analysed. Glucocorticoid receptor (GR) and thyroid hormone receptor (T3R) isoforms beta 1, beta 2, alpha 1 and alpha 2 mRNA abundance were determined by solution hybridization. Both GR and T3R receptor mRNA isoforms were lower in the myocardium as compared to skeletal muscle and in particular to liver. GR mRNA abundance was higher than that of any T3R isoform while the sum of ligand-binding isoforms (beta 1, beta 2 and alpha 1) were similar in the myocardium and in skeletal muscle as opposed to the liver where GR mRNA was higher. GR mRNA abundance was similar in right and left ventricles from donor hearts and in cardiomyopathy. T3R beta 1 showed higher levels in the right ventricle with higher levels in cardiomyopathy as compared to donor heart. T3R isoform alpha 1 and especially the alpha 1/alpha 2 ratio were lower in left ventricle in cardiomyopathy compared to donor hearts. In conclusion, GR and T3R isoforms mRNA abundance are low in the human myocardium. In the failing myocardium GR and T3R beta 2 and alpha 2 show no signs of reactivity while T3R beta 1 and alpha 1 mRNA adapt.
Asunto(s)
Cardiomiopatía Dilatada/genética , Miocardio/metabolismo , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Hormona Tiroidea/genética , Adolescente , Adulto , Humanos , ARN Mensajero/genéticaRESUMEN
Amiodarone, a powerful antiarrhythmic drug, likely exerts its major effect by antagonism of thyroid hormone (T3), probably at the receptor level. T3 is known to regulate beta-adrenergic receptor density in the heart but the effects of sympathomimetic drugs on thyroid hormone receptors (T3R) is not known. The aim of this study was to investigate how amiodarone and isoproterenol affect T3R-mRNA in cultured cardiomyocytes. Confluent, isoproterenol pretreated, AT-1 cardiomyocytes were treated with isoproterenol free medium, amiodarone, T3 and amiodarone together with T3 for 48 hours. Solution hybridization for the determination of mRNA for T3R alpha 1, alpha 2, beta 1 and beta 2 were performed. In itself isoproterenol upregulated T3R alpha 1, T3R beta 1, T3R beta 2 (p < 0.05), but did not affect the levels of T3R alpha 2. Amiodarone and T3, respectively, downregulated T3R alpha 1, T3R beta 1, T3R beta 2 (p < 0.05), but did not affect the levels of T3R alpha 2. Amiodarone and T3, added together, upregulated T3R alpha 2 and T3R beta 1 (p < 0.05) as compared to amiodarone or T3 alone. There was an antagonistic effect between amiodarone and T3 for the regulation T3R beta 1. This is the first evidence showing that amiodarone regulates T3R-mRNA concentrations during cathecholamine stress. Isoproterenol regulation of T3R-mRNA levels provides further evidence for the close interaction between the thyroid hormone and the beta-adrenergic systems.
Asunto(s)
Amiodarona/farmacología , Regulación hacia Abajo , Isoproterenol/farmacología , Miocardio/metabolismo , Receptores de Hormona Tiroidea/biosíntesis , Animales , Secuencia de Bases , Células Cultivadas , Corazón/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Miocardio/citología , ARN Mensajero/análisis , Receptores de Hormona Tiroidea/genéticaRESUMEN
Full length human glucocorticoid receptor and truncated receptor derivatives lacking the major amino-terminal trans-activating domain were expressed in stably transfected Chinese hamster ovary (CHO) cells. The receptors were co-expressed together with human metallothionein IIa, and the expression levels were amplified in the presence of increasing concentrations of metal. In amplified cells, both synthesized receptor forms showed the expected molecular weights, as assayed by affinity labeling and immunoblotting. They were expressed at concentrations of about 350,000-520,000 molecules/cell which corresponds to a 10-fold increase in receptor levels as compared to rat liver cells. The hormone (agonist or antagonist) binding properties of the expressed proteins were very similar to those characteristic of authentic glucocorticoid receptors in tissues or cultured cells. Moreover, the expressed proteins specifically recognized a glucocorticoid-response element sequence motif in in vitro protein-DNA binding experiments. The activation of a glucocorticoid-responsive reporter gene by the expressed full length receptor was dramatic (about 75-fold) and strictly ligand-dependent. In contrast, the expressed amino-terminal deletion mutant exhibited considerably weaker functional activity but showed normal hormone-binding properties. Upon exposure to dexamethasone in vivo, the expressed receptor mRNAs and proteins were down-regulated about 2- to 6-fold, indicating that regulatory signals important for autoregulation may be contained within structures corresponding to the ligand and DNA-binding domains. Transcription from the expression vector was not negatively regulated from the hormone, strongly arguing that receptor down-regulation was due to a post-transcriptional mechanism. In conclusion, this expression system should be a useful tool for further structural and functional studies of the receptor, including the biochemistry of its activation from a cryptic to a functional species, and its ligand-dependent autoregulation.
Asunto(s)
Regulación hacia Abajo , ARN Mensajero/genética , Receptores de Glucocorticoides/metabolismo , Marcadores de Afinidad , Animales , Autorradiografía , Secuencia de Bases , Células Cultivadas , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Humanos , Ligandos , Datos de Secuencia Molecular , Plásmidos , Esteroides/metabolismo , TransfecciónRESUMEN
We have cloned the human thyroid hormone receptor beta 1 (hThR beta) from the human breast cancer cell line T47D using the PCR technique. A recombinant baculovirus transfer vector pVL1392/hThR beta was constructed and the full length receptor was expressed in the insect cell line Spodoptera frugiperda (Sf9). Approx. 10-15 x 10(6) receptors are expressed/cell which implies a production level of 2.5-4.0 mg hThR beta/l of cell culture. The expressed hThR beta displayed a single class of binding sites for T3 with high affinity. Western blot analysis using a polyclonal antibody indicated that the molecular weight of the baculovirus expressed receptor is approx. 50 kDa. Crude nuclear extract of hThR beta labeled with [125I]T3 sedimented as a 4 S peak on a glycerol gradient. No receptor could be detected in the cytoplasm indicating its proper translocation to the nuclear compartment. An oligonucleotide containing a palindromic thyroid hormone response element is specifically recognized and retarded in a gel-mobility-shift assay in the presence of nuclear extract of Sf9 cells expressing hThR beta. These data suggest that hThR beta expressed in Sf9 cells is functional and displays characteristics virtually indistinguishable from those of the thyroid hormone receptor (ThR) extracted from mammalian cells. Furthermore, the data indicate that the baculovirus expression system is adequate for large-scale production of receptor for detailed structural and functional studies.
Asunto(s)
Baculoviridae/genética , Clonación Molecular , Expresión Génica , Vectores Genéticos , Mariposas Nocturnas/metabolismo , Receptores de Hormona Tiroidea/genética , Animales , Secuencia de Bases , Western Blotting , Neoplasias de la Mama , Línea Celular , Núcleo Celular/metabolismo , Centrifugación por Gradiente de Densidad , ADN/genética , ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa , Receptores de Hormona Tiroidea/metabolismo , Triyodotironina/metabolismo , Células Tumorales CultivadasRESUMEN
Replication of human immunodeficiency virus type 1 (HIV-1) isolates in peripheral blood mononuclear cells (PBMC) has been studied by in situ hybridization using the riboprobe BH10-R3 from HTLV-IIIB. Two series of isolates were tested: (a) 20 isolates from individuals with varying severity of HIV-1 infection and (b) sequential isolates from 5 subjects showing signs of clinical progression over a 45 month observation period. The results show that HIV-1 isolates with distinct replicative capacity can be distinguished by the intensity of radioactive labeling over single infected cells after in situ hybridization. Sequential isolates from patients with clinically progressive HIV-1 infection show a gradual increase in replicative capacity over time. In PBMC cultures infected with such sequential isolates, intensity of radioactive label over single infected cells increases and is strongest with isolates obtained at the time of low CD4 counts in blood. The results suggest that the restriction of virus replication that operates in the early stages of HIV-1 infection is gradually lost with progression of the disease.
Asunto(s)
Infecciones por VIH/microbiología , VIH-1/fisiología , Replicación Viral , Infecciones por VIH/sangre , Infecciones por VIH/etiología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Leucocitos Mononucleares/microbiología , Masculino , Hibridación de Ácido Nucleico , Sondas ARN , Factores de TiempoRESUMEN
The presence of HIV gag and env proteins (HIV Ag) and virus replicating cells was studied by immunohistochemistry and in situ hybridization, respectively, in brain specimens from five HIV infected patients. HIV antigens were detected in 3 of 5 brains in micronodular areas characterized by increased cellularity and the presence of multinuclear giant cells. By double immunostaining, HIV Ag positive cells were shown to express markers common to macrophages and microglia i.e. Leu M5+, My4+, HLA-Dr+, RCA-1+, and to a lesser extent CD4+ (Leu3+). Another macrophage specific marker, KiM6, was found only on HIV+ cells in HIV infected specimens and not in uninfected, control brains. Medium-sized, virus replicating cells were found exclusively in micronodular areas, but in much smaller quantities than HIV Ag+ cells. Our observations provide further evidence to support the hypothesis that macrophages play an important role in CNS infection by HIV and additionally support the concept that reactive microglial originate from activated macrophages infiltrating the brain. Both direct effects of viral components and cell mediated reactions can be implicated from our findings as mechanisms involved in the pathogenesis of the CNS lesions.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/microbiología , Encefalopatías/microbiología , Encéfalo/microbiología , Demencia/microbiología , VIH/análisis , Síndrome de Inmunodeficiencia Adquirida/patología , Anticuerpos Monoclonales , Antígenos de Diferenciación/análisis , Antígenos Virales/análisis , Encéfalo/patología , Encefalopatías/patología , ADN Viral/análisis , Demencia/patología , Antígenos HLA/análisis , HumanosRESUMEN
Serologic studies were done to estimate the antibody prevalence against human herpesvirus 6 (HHV-6) in patients with malignant lymphomas, Sjögren's syndrome and sarcoidosis. Serologic studies showed IgG antibody titers against HHV-6 in up to 41% of patients with sarcoidosis, 50-70% with malignant lymphomas and in 36% with Sjögren's syndrome. In situ hybridization on lymph node biopsies was positive for HHV-6 genome in 1 out of 5 sarcoidosis lymph nodes.
Asunto(s)
Anticuerpos Antivirales/análisis , Herpesviridae/inmunología , Linfoma/inmunología , Sarcoidosis/inmunología , Síndrome de Sjögren/inmunología , Técnica del Anticuerpo Fluorescente , Herpesviridae/ultraestructura , Humanos , Inmunoglobulina G/análisis , Inmunohistoquímica , Linfoma/microbiología , Microscopía Electrónica , Hibridación de Ácido Nucleico , Sarcoidosis/microbiología , Síndrome de Sjögren/microbiologíaRESUMEN
The gene encoding the human vasoactive intestinal peptide (VIP) and the histidine-methionine amide (PHM-27) peptide hormone was isolated from lambda phage libraries. The human gene was found to be composed of seven exons spanning approximately 9 kilobase pairs. The first exon codes for an untranslated leader sequence, and the second exon codes for a putative signal peptide. DNA sequences coding for the VIP and PHM-27 hormones are located in two different exons. Southern blot analysis with genomic DNA suggested that a single copy of the VIP/PHM-27 gene is present in the human haploid genome. The expression of VIP/PHM-27 precursor mRNA in various tissues in the rat was analyzed by RNA gel blot hybridization. In the organs examined, expression was only detected in the brain and duodenum. RNA isolated from various regions of the rat brain--including the cortex, hypothalamus, and hippocampus--hybridized to both VIP- and PHM-27-specific probes. The same pattern of hybridization was found when VIP- and PHM-27-specific probes were used, suggesting that possible differences in the localization of VIP and PHM-27 peptides between different brain regions cannot be accounted for by differential RNA processing.