RESUMEN
In this study we have evaluated an alternative 96-well format flow cytometry based (FCtox) method which enable simultaneous detection of cytotoxicity and human leukocyte antigen (HLA) antibody binding. Comparable results were obtained in side-by-side comparisons with conventional complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM) in terms of sensitivity and specificity. There was 91 and 93% agreement between results obtained by FCtox and CDC for T and B cells, respectively. In addition, comparable results were obtained with FCtox IgG and FCXM IgG for both T and B cells. Furthermore, compared with a recently developed and highly sensitive Luminex based C1q assay we obtained close to 90% method agreement with the FCtox assay. Our alternative cytotoxicity and IgG binding assay which exhibit low intra-and inter-assay variation will improve the workflow and speed up the pre-transplant testing and also allow continuous monitoring of assay performance and proper quality assurance.
Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Citotoxicidad Inmunológica , Citometría de Flujo/métodos , Linfocitos B/citología , Linfocitos B/inmunología , Pruebas Inmunológicas de Citotoxicidad/normas , Citometría de Flujo/normas , Humanos , Inmunoglobulina G/metabolismo , Masculino , Variaciones Dependientes del Observador , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Recent studies indicate that chemotherapeutic agents may increase the anti-tumoral immune response. Based on the pivotal role of dendritic cells (DCs) in host tumour-specific immune responses, we investigated the effect of commonly used chemotherapeutic drugs dexamethasone, doxorubicin, cisplatin and irinotecan and glucocorticoids on monocyte-derived DCs (moDCs). Dexamethasone displayed the strongest inhibitory effect on DC differentiation. The effect of cisplatin and irinotecan was moderate, while only weak effects were noticed for doxorubicin. Surprisingly, when the functional consequence of chemotherapy-treated CD14(+) monocytes and their capacity to activate CD4(+) T responders cells were investigated, cisplatin-treated monocytes gave rise to increased T cell proliferation. However, dexamethasone, doxorubicin and irinotecan-pretreated monocytes did not stimulate any increased T cell proliferation. Further investigation of this observation revealed that cisplatin treatment during DC differentiation up-regulated significantly the interferon (IFN)-ß transcript. By contrast, no effect was evident on the expression of interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6 or IFN-α transcripts. Blocking IFN-ß attenuated the cisplatin-enhanced T cell proliferation significantly. In conclusion, cisplatin treatment enhanced the immune stimulatory ability of human monocytes, a mechanism mediated mainly by the increased production of IFN-ß.
Asunto(s)
Presentación de Antígeno/efectos de los fármacos , Antineoplásicos/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Camptotecina/análogos & derivados , Camptotecina/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Cisplatino/farmacología , Citocinas/biosíntesis , Citocinas/genética , Células Dendríticas/citología , Dexametasona/farmacología , Doxorrubicina/farmacología , Humanos , Inmunoterapia , Interferón beta/antagonistas & inhibidores , Interferón beta/biosíntesis , Interferón beta/genética , Irinotecán , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
A 70-year-old man with an uncemented metal-on-polyethylene total hip prosthesis underwent revision arthroplasty 33 months later because of pain, swelling and recurrent dislocation. There appeared to be corrosion and metal release from the prosthetic head, resulting in pseudotumour formation and severe local soft-tissue destruction. The corrosion occurred at the junction between the titanium-molybdenum-zirconium-iron taper and the cobalt-chrome-molybdenum head, but the mechanism was unproven.
Asunto(s)
Prótesis de Cadera/efectos adversos , Metales/efectos adversos , Anciano , Artroplastia de Reemplazo de Cadera/métodos , Corrosión , Granuloma de Células Plasmáticas/diagnóstico por imagen , Granuloma de Células Plasmáticas/etiología , Humanos , Masculino , Polietileno , Diseño de Prótesis , Falla de Prótesis , Reoperación/métodos , Tomografía Computarizada por Rayos XRESUMEN
We studied the importance of human leucocyte antigen (HLA)-A, -B and -DRB1 high-resolution matching on the outcome of haematopoietic stem cell transplantation (HSCT) with matched unrelated donors (MUDs) vs single allele-mismatched unrelated donors. Fifty consecutive HSCT patients receiving an HLA-A, -B or -DR allele-level-mismatched unrelated graft (mmUD) were compared with a matched cohort of 100 patients with an HLA-A, -B and -DR-MUD. Rejection occurred in seven patients (14%) in the mmUD group and in four patients (4%) in the MUD group (P = 0.04), but this was mainly an effect of HLA-C mismatch. The cumulative incidence of acute graft vs host disease (GVHD) grades II-IV were 61%, 26% and 33% in the class I mmUD, class II mmUD and MUD groups, respectively. In multivariate analysis, HLA class I mismatch was associated with an increased risk of acute GVHD grades II-IV (2.09, P = 0.007) and transplant-related mortality (TRM) (1.99, P = 0.06). The 5-year overall survival was 81% in patients with a class II allele-mismatched donor compared with 52% (P = 0.025) and 50% (P = 0.017) in patients with a class I mismatch and a MUD. In multivariate analysis, HLA class II allele mismatch was associated with improved survival (3.38, P = 0.019). Relapse-free survival were 53%, 37% and 42% in patients with a class II mmUD, class I mmUD and a MUD, respectively (not significant). An HLA-C or -DQ mismatch had no significant impact on survival, TRM and relapse. In conclusion, compared with MUD, HLA class I allele mmUD had an increased risk of acute GVHD and TRM.
Asunto(s)
Enfermedad Injerto contra Huésped/genética , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-DR/genética , Trasplante de Células Madre Hematopoyéticas , Histocompatibilidad/genética , Adolescente , Adulto , Alelos , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/mortalidad , Antígenos HLA-A/inmunología , Antígenos HLA-B/inmunología , Antígenos HLA-DR/inmunología , Cadenas HLA-DRB1 , Histocompatibilidad/inmunología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Donantes de Tejidos , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Chronic stress has been proposed to aggravate allergic inflammation, whereas acute stress may have functional beneficial effects. The aim of this study was to investigate the influence of timing of single short restraint stress (RST) in a model of eosinophilic airway inflammation. METHODS: The airways of ovalbumin (OVA)-sensitized mice were exposed to an intranasal OVA challenge. RST was applied in two different ways; either 2 h before (pre-stress) or after (post-stress) the OVA challenge, respectively, or as a combination of stress before and after (double-stress) the OVA challenge. One group of mice was also treated with metyrapone (ME) prior to a pre-stress challenge. The inflammatory cell response was evaluated in bronchoalveolar lavage fluid (BALF), lung and nasal tissue, as well as bone marrow. RESULT: RST applied prior to the OVA challenge (pre-stress) inhibited OVA-induced airway inflammation in BALF and lung tissue, and reduced nasal histopathology compared to unstressed mice. Given as post-stress or double-stress, RST did not affect the inflammation in BALF, lungs or nasal tissue. Pre-treatment with ME prevented the pre-challenge stress evoked decrease in inflammation in BALF and lungs. CONCLUSION: Effects of RST on eosinophilic airway inflammation appear to be strongly dependent on timing and, as could be judged from the ME inhibition pattern, also corticosterone dependent. Hypothalamic-pituitary-adrenal axis activation probably influences eosinophilic inflammation through specific sequences of compartmental activation and thereby timing effects are evident on cellular recruitment pattern during the allergic reaction.
Asunto(s)
Asma/inmunología , Bronquios/inmunología , Eosinófilos/inmunología , Neuroinmunomodulación/inmunología , Eosinofilia Pulmonar/inmunología , Estrés Psicológico/inmunología , Animales , Asma/fisiopatología , Bronquios/fisiopatología , Corticosterona/inmunología , Corticosterona/metabolismo , Inhibidores Enzimáticos/farmacología , Sistema Hipotálamo-Hipofisario/inmunología , Sistema Hipotálamo-Hipofisario/metabolismo , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/farmacología , Masculino , Metirapona/farmacología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Sistema Hipófiso-Suprarrenal/inmunología , Sistema Hipófiso-Suprarrenal/metabolismo , Eosinofilia Pulmonar/fisiopatología , Restricción Física , Estrés Psicológico/fisiopatología , Factores de TiempoRESUMEN
BACKGROUND AND AIMS: Glucocorticosteroid treatment (GCS) is effective for attacks of ulcerative colitis (UC). However, 25-30% of patients fails to respond and may be considered steroid resistant. Glucocorticoid receptors (GR) mediate the effects of GCS. Colorectal mucosa levels of GR and NF-κB were analysed before, during and after treatment with GCS-compounds. METHODS: Patients with moderate-severe attacks of ulcerative colitis were included. Patients undergoing colonoscopy with normal finding served as controls. GR and NF-κB levels in colorectal mucosa were analysed by Western Blotting and the DNA-binding activity of NF-κB by EMSA. RESULTS: Twenty-eight patients and seven controls were included. Ten patients were judged clinically steroid resistant. Responders had significantly higher levels of GR in colorectal mucosa after one week of treatment than non-responders (P=0.039) and significantly higher levels of GR were found in responders in remission as compared to before treatment (P=0.013). NF-κB levels did not differ between the groups at first visit. Increasing levels were found only in responders as remission was obtained (P=0.031). EMSA detected 20% lower DNA-binding of NF-κB in responders in remission as compared to first visit (P=0.021). CONCLUSION: GR levels increase in UC-patients responding to GCS-therapy but not in steroid resistant patients and may be the reason for the lack of steroid-efficacy. Increasing NF-κB levels were found in responders attaining remission, possibly reflecting a lower turnover. A decrease in DNA-binding of NF-κB was found in these patients, perhaps because of the increased GR levels counteracting NF-κB activity.
RESUMEN
Glucocorticoids are widely used to treat inflammatory and malignant diseases. However, many individuals show a lack of therapeutic response and unwanted side-effects. Various known and unknown parameters determine glucocorticoid responsiveness, among them glucocorticoid receptor (GR)-interacting proteins. Several of the proteins interacting with GR also participate in other signal transduction pathways such as the AP-1 pathway and the nuclear factor-kappaB pathway. We suggest that a closer study of GR-interacting proteins may shed new light on mechanisms determining glucocorticoid sensitivity. In this commentary, the general mechanisms of GR action will be addressed and a proteomic-based method to study GR-interacting proteins will be described in brief.
Asunto(s)
Antiinflamatorios/uso terapéutico , Inflamación/tratamiento farmacológico , Receptores de Glucocorticoides/metabolismo , Transducción de Señal/fisiología , Proteínas 14-3-3 , Animales , Antiinflamatorios/metabolismo , Citosol/metabolismo , Resistencia a Medicamentos , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Inflamación/metabolismo , Focalización Isoeléctrica , Hígado/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Receptores de Glucocorticoides/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The interplay between the endocrine and immune systems has come into focus in recent years with the insight that endocrine parameters may affect susceptibility to both auto-immune and infectious diseases. Our interest in immunoendocrine regulation led us to investigate the effects of glucocorticoids on Herpes simplex virus type 1 (HSV-1) infections. Glucocorticoids used to treat inflammatory conditions are not yet recommended for HSV-1 therapy, since they have been reported to prolong viral shedding both in vivo and in vitro. Here we report that glucocorticoids did not alter the viral yield in human gingival fibroblast (HGF) cell culture when glucocorticoid treatment and viral infection occured simultaneously, but the viral yield increased when cells were treated with the glucocorticoid dexamethasone (dex) prior to viral infection. We found that viral infection in our primary cell system increased NF-kappaB levels and DNA binding. In addition, the amount of glucocorticoid receptor (GR) increased following viral infection, and HSV-1 infection as such could induce glucocorticoid-driven transcription of a reporter gene in human embryo kidney (HEK) 293 cells stably transfected with GR. Dex treatment did not affect HSV-1-induced binding of p65 to an NF-kappaB element in an electrophoretic mobility shift assay, and acyclovir was still efficient as an anti-viral drug in the presence of dex. Further studies of the observed effects of HSV-1 infection and glucocorticoid treatment on GR and NF-kappaB regulation could give insights into the immunoendocrine mechanisms important for defence and therapy against viral infections.
Asunto(s)
Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1 , Aciclovir/farmacología , Adyuvantes Farmacéuticos/uso terapéutico , Antivirales/farmacología , Células Cultivadas , Células Clonales , ADN/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virología , Herpes Simple/metabolismo , Herpes Simple/virología , Humanos , FN-kappa B/metabolismo , Unión Proteica , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Carga Viral , Esparcimiento de Virus/efectos de los fármacosRESUMEN
Studies of GR-interacting proteins can provide valuable insights into the regulation of GR cellular signalling. The cytoplasmic localization of GR and reports of GR interaction with such a plethora of other cytoplasmic proteins may point to a unique role for GR in modulating and integrating other signalling pathways. A better insight into these interactions could serve as a tool when trying to understand and modify GR signalling.
Asunto(s)
Citosol/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas 14-3-3 , Proteínas del Citoesqueleto/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , FN-kappa B/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-raf/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
OBJECTIVE: Primary glucocorticoid resistance is characterized by decreased sensitivity to cortisol signalling. We have performed genetic analysis of the glucocorticoid receptor (GR) gene in 12 unrelated patients with primary cortisol resistance as defined by a pathological dexamethasone suppression test. METHODS: Exon specific polymerase chain reaction amplification of the GR gene and sequencing of each exon was carried out. The two mutations were characterized in vitro in terms of glucocorticoid driven reporter gene activity in a transient transfection assay and in a ligand binding assay. Molecular modelling of the R477H mutant was performed based on the X-ray structure of the GR-DNA binding domain. RESULTS: Two novel mutations in the GR gene were found: R477H in the DNA-binding domain which is the first reported mutation in that region of the human GR gene and G679S in the ligand binding domain. The R477H mutation showed no transactivating capacity, whereas the G679S mutation had reduced transactivation capacity compared to the wild-type (wt) GR. When tested for ligand binding capacity, the G679S mutation had 50% binding affinity compared to the wt GR. The effect of the point mutation R477H was deduced by a comparison between the wt structure and the model of the mutant. The wt GR has direct and water mediated contact with the phosphate groups of the glucocorticoid responsive element (GRE) whereas, in the model, the mutation R477H has no contact with the GRE. The G679S mutation is located on the surface of the ligand binding domain, at a distance from the steroid-binding site. A previously reported polymorphism, AAT to AAC at amino acid position 766, was found in four of the patients. CONCLUSIONS: In two of 12 patients with clinical glucocorticoid resistance, mutant forms of GR could be found. The glucocorticoid resistance in vivo in these two patients corresponds to impaired function of the two mutated GR forms in two in vitro assays. The relevance of the conservative polymorphism for the glucocorticoid insensitivity noted in these patients remains to be clarified.
Asunto(s)
Hirsutismo/genética , Hidrocortisona/fisiología , Mutación Puntual , Receptores de Glucocorticoides/genética , Adulto , Aldosterona/orina , Dexametasona , Resistencia a Medicamentos/genética , Femenino , Glucocorticoides , Hirsutismo/orina , Humanos , Hidrocortisona/orina , Masculino , Persona de Mediana Edad , Modelos Moleculares , Polimorfismo GenéticoRESUMEN
BACKGROUND AND AIMS: Up to 30% of patients with severe-to-moderate attacks of ulcerative colitis (UC) respond poorly to glucocorticosteroid (GCS) treatment. The reason for this unresponsiveness is unknown. AIM: Our aim was to evaluate possible differences in glucocorticoid receptor (GR) density in peripheral leukocytes and effects of low-dose GCS treatment on GR density and on the hypothalamic-pituitary-adrenal axis in UC patients who had received high-dose GCS treatment due to a moderate or severe attack. Eleven UC patients in remission who were responders (Rs) to previous GCS treatment were compared with 10 patients who failed GCS therapy and had a colectomy (nonresponders. NRs). Ten healthy individuals served as controls. METHODS: Quantitation of GR mRNA by a solution hybridization assay in peripheral leukocytes and a low-dose adrenocorticotropin hormone stimulation test was performed before and after low-dose dexamethasone (DEX) treatment for 14 days. The glucocorticoid-responsive gene for metallothionein IIa (MTIIa) was also analyzed by a solution hybridization assay in peripheral leukocytes. RESULTS: Overall, basal GR mRNA levels were higher in patients than in controls (p < 0.0001). There were no significant differences between NRs and Rs. None of the groups down-regulated their GR mRNA levels in response to DEX treatment. Basal and stimulated cortisol levels decreased significantly only among NRs after DEX (p = 0.012 and 0.0093). MTIIa levels were lower in NRs as compared with Rs, both in mononuclear (p = 0.0059) and in polynuclear leukocytes (p = 0.030). CONCLUSION: Patients with UC in remission exhibit higher levels of GR mRNA in peripheral leukocytes. We speculate that this may be secondary to an underlying up-regulation of proinflammatory factors also present in patients in clinical remission. Differences in GR mRNA levels per se thus may not be important for the ability of patients with UC to respond to GCS treatment. The hypothalamic pituitary adrenal axis was suppressed by low-dose DEX treatment only in NRs, possibly indicating that steroid-resistance is not a generalized phenomenon. Lower levels of MTIIa in NRs may indicate a diminished efficiency of GR regulation in steroid-refractory patients.
Asunto(s)
Antiinflamatorios/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Dexametasona/uso terapéutico , Leucocitos/metabolismo , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/metabolismo , Adulto , Antiinflamatorios/farmacología , Estudios de Casos y Controles , Colectomía , Colitis Ulcerosa/sangre , Colitis Ulcerosa/cirugía , Dexametasona/farmacología , Femenino , Glucocorticoides/uso terapéutico , Humanos , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Masculino , Metalotioneína/sangre , Persona de Mediana Edad , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Receptores de Glucocorticoides/genética , Resultado del TratamientoRESUMEN
The glucocorticoid receptor (GR) functions as a ligand-dependent transcription factor. In the present study we describe a specific immunoaffinity chromatography purification of GR from liver cytosol from adrenalectomized rats that may be used to identify hitherto unknown cytosolic GR interacting proteins. We have identified the ubiquitously expressed 14-3-3 as well as Raf-1, a downstream effector of Ras, as GR co-purifying proteins. In our semi-quantitative analysis liganded/activated GR showed the strongest interaction with 14-3-3 and Raf-1, but 14-3-3 was also found to co-purify with GR in a nonliganded/nonactivated state. By extensive salt washes we were also able to demonstrate that the glucocorticoid induced interaction between GR, 14-3-3, and Raf-1, respectively, is remarkably stable and withstood 2.4 m salt. The interaction between GR and 14-3-3 was also verified by 14-3-3 co-immunoprecipitation studies. Our observations that GR and Raf-1 are found within the same protein complex ("receptosome") in the cytoplasm of rat liver cells could provide a mechanistic explanation for glucocorticoid effects on the Raf-1-Ras signaling pathway.
Asunto(s)
Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Tirosina 3-Monooxigenasa/metabolismo , Proteínas 14-3-3 , Animales , Western Blotting , Cromatografía de Afinidad , Citoplasma/metabolismo , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Ligandos , Hígado/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Proto-Oncogénicas c-raf/aislamiento & purificación , Ratas , Receptores de Glucocorticoides/aislamiento & purificación , Cloruro de Sodio/farmacología , Tirosina 3-Monooxigenasa/aislamiento & purificaciónRESUMEN
Sine the autumn of 1995, Benecol, a proprietary brand of cholesterol-lowering margarine, has been available in ordinary grocery shops in Finland. The active ingredient is a sitostanol ester. Several studies in humans have shown use of the margarine to result in an approximately 10 per cent reduction in total serum cholesterol, and a 13-15 per cent reduction of LDL-cholesterol. However, further studies are required of its phyto-oestrogenic and endocrine effects, and its effects on growing children, particularly regarding subsequent fertility in boys. Although the margarine is classed as a 'functional food' in Finland, the question arises where the line is to be drawn between medicines and food-stuffs.
Asunto(s)
Colesterol/sangre , Margarina , Sitoesteroles/administración & dosificación , Anticolesterolemiantes/administración & dosificación , Finlandia , Alimentos Formulados , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Sitoesteroles/químicaRESUMEN
Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript generates two receptor isoforms, hGRalpha and hGRbeta, with different carboxyl termini diverging at amino acid 727. By reverse transcriptase-polymerase chain reactions it was previously demonstrated that the hGRbeta message had a widespread tissue distribution. To demonstrate the presence of hGRbeta as protein we produced specific rabbit antisera to hGRbeta, as well as a hGRbeta-specific mouse monoclonal IgM antibody, by peptide immunizations. By SDS-polyacrylamide gel electrophoresis and Western immunoblotting we showed that hGRbeta is endogenously expressed at the protein level in HeLa cells and human lymphatic leukemia cells. Using an antibody directed against an epitope shared by both isoforms we showed a relatively lower expression of the hGRbeta form. We also showed that hGRbeta bound to hsp90 by immunoprecipitation of in vitro translated hGRbeta in reticulocyte lysate with hsp90-specific antibodies, a coprecipitation occurring also in the presence of dexamethasone. We could not demonstrate that hGRbeta inhibited the effects of dexamethasone-activated hGRalpha on a glucocorticoid-responsive reporter gene. In conclusion, low hGRbeta expression levels and hGRbeta-hsp90 interaction maintained in the presence of ligand and lack of inhibition of hormone-activated hGRalpha effects challenge the concept of the hGRbeta isoform as a proposed dominant negative inhibitor of hGRalpha activity.
Asunto(s)
Receptores de Glucocorticoides/fisiología , Proteínas Represoras/fisiología , Empalme Alternativo , Animales , Células COS , Dexametasona/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/genéticaRESUMEN
The effect of glucocorticoid hormones, purified glucocorticoid receptor (GR) and purified heat shock protein M(r) 90,000 (hsp90) on microtubule (MT) assembly in vitro was tested by a spectrophotometric MT assembly assay and electron microscopy. GR significantly prolonged the nucleation phase, slowed down the assembly rate and reduced the maximal amplitude of MT assembly compared with control. The effects were partially reversed by the addition of glucocorticoid hormone. GR associated with MTs. These results indicate that GR affects MT assembly in vitro, which may be a functional correlate to the structural association of GR with MTs. This implies that factors affecting GR may affect MT assembly in vivo.
Asunto(s)
Microtúbulos/ultraestructura , Receptores de Glucocorticoides/fisiología , Animales , Western Blotting , Bovinos , Proteínas HSP90 de Choque Térmico/fisiología , Cinética , Microscopía Electrónica , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Ratas , EspectrofotometríaRESUMEN
We have cloned a member of the nuclear receptor superfamily. The cDNA was isolated from a rat liver library and encodes a protein of 446 aa with a predicted mass of 50 kDa. This clone (OR-1) shows no striking homology to any known member of the steroid/thyroid hormone receptor superfamily. The most related receptor is the ecdysone receptor and the highest homologies represent < 10% in the amino-terminal domain, between 15-37% in the carboxyl-terminal domain and 50-62% in the DNA binding domain. The expression of OR-1 appears to be widespread in both fetal and adult rat tissues. Potential DNA response elements composed of a direct repeat of the hexameric motif AGGTCA spaced by 0-6 nt were tested in gel shift experiments. OR-1 was shown to interact with the 9-cis-retinoic acid receptor (retinoid X receptor, RXR) and the OR-1/RXR complex to bind to a direct repeat spaced by 4 nt (DR4). In transfection experiments, OR-1 appears to activate RXR-mediated function through the DR4. Therefore OR-1 might modulate 9-cis-retinoic acid signaling by interacting with RXR.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Hígado/metabolismo , Receptores de Ácido Retinoico/biosíntesis , Receptores de Ácido Retinoico/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Clonación Molecular , Cricetinae , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Feto , Biblioteca de Genes , Hibridación in Situ , Isomerismo , Receptores X del Hígado , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Especificidad de Órganos , Receptores Nucleares Huérfanos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Transfección , Tretinoina/metabolismoRESUMEN
The cellular distribution of the glucocorticoid receptor (GR) has not yet been firmly established. The extensive literature indicates that GR is present both in the cytoplasm and the cell nucleus, however, some studies have failed to detect cytoplasmic GR. It is still controversial as to whether GR is randomly diffusing in the cytoplasm and nucleus, or if the GR-distribution is organized or controlled in some way, which may be of importance for the transduction of glucocorticoid effects to cells. There is evidence that both non-activated and activated GR is associated with the plasma membrane, a number of cytoplasmic organelles and the nucleus. Both morphological and biochemical evidence show that GR is associated with microtubules during different stages of the cell cycle, i.e. GR co-localizes, co-purifies and co-polymerizes with tubulin. This indicates that GR is structurally linked to the intracellular MT-network which may be of importance in the mechanism of action of glucocorticoid hormones. The literature in this field is reviewed including the reported data on subcellular GR-localization.
Asunto(s)
Compartimento Celular , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Receptores de Glucocorticoides/fisiología , Ciclo Celular/fisiología , Fibroblastos/ultraestructura , Humanos , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/ultraestructura , Receptores de Glucocorticoides/aislamiento & purificación , Receptores de Glucocorticoides/ultraestructuraRESUMEN
This paper describes the localization of the alpha-type peroxisome proliferator-activated receptor (PPAR alpha) in the rat brain using immunocytochemistry and in situ hybridization. Expression of PPAR alpha mRNA was highest in the granular cells of the cerebellar cortex and in the dentate gyrus, with a somewhat lower expression in areas CA1-CA4 of the hippocampus. PPAR alpha mRNA was also found in some neurones of the cerebral cortex (layers II-IV) and the molecular layer of the cerebellar cortex, and in the olfactory tubercle. Immunocytochemistry revealed nuclear PPAR alpha-immunoreactivity (-IR) in the same areas as seen with the in situ hybridization. Furthermore, PPAR alpha-IR was also localized in oligodendrocytes, whereas the other glial cell types appeared to lack PPAR alpha. These results suggest that peroxisome proliferators and chemicals acting similarly have effects on discrete populations of neurones. The presence of PPAR alpha in oligodendrocytes lends further support to the suggestion that peroxisomes are important in the assembly and degradation of myelin.
Asunto(s)
Encéfalo/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Autorradiografía , Inmunohistoquímica , Hibridación in Situ , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/genética , Distribución Tisular , Factores de Transcripción/genéticaRESUMEN
We have used a panel of monoclonal antibodies and a cell-free transcription assay to study the function of the tau 1 transactivation domain of the human glucocorticoid receptor. Three antibodies (monoclonal antibodies 250, 275, and 286) specifically inhibited tau 1-dependent transcription, but had little or no effect on either basal transcription or the activity of an unrelated yeast transcription factor. This inhibition was not due to interference of DNA binding activity, as all three antibodies super shifted tau 1-containing protein-DNA complexes. Epitopes for all three antibodies were localized to a region between amino acids 190 and 200, which lies within the recently defined 41-amino acid core region of tau 1 that is required for transactivation (Dahlman-Wright, K., Almlöf, T., McEwan, I.J., Gustafsson, J-A., and Wright, A. P.H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1619-1623). In contrast to the effect on tau 1-dependent transcription none of the antibodies tested antagonized the squelching ability of the tau 1 domain, suggesting that tau 1-mediated transactivation involves interactions in addition to those identified by the squelching assay. Consistent with this, a comparison of the kinetics of tau 1 squelching and inhibition of transactivation by monoclonal antibodies suggested a role for tau 1 mediated transcriptional induction at two or more steps during transcription initiation by RNA polymerase II.
Asunto(s)
ARN Polimerasa II/metabolismo , Receptores de Glucocorticoides/metabolismo , Transcripción Genética , Animales , Anticuerpos Monoclonales , Sistema Libre de Células , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Mapeo Epitopo , Epítopos , Humanos , Modelos Genéticos , Ratas , Receptores de Glucocorticoides/inmunología , Relación Estructura-Actividad , Activación TranscripcionalRESUMEN
By means of a monoclonal mouse immunoglobulin G2a antibody against the rat liver glucocorticoid receptor and the indirect immunoperoxidase technique, the distribution of glucocorticoid receptors in neuronal and glial cell populations was mapped in the central nervous system of the male rat. The mapping was complemented by computer-assisted morphometric and microdensitometric evaluation of glucocorticoid receptor immunoreactivity in many brain regions. The quantitative analysis allowed us to achieve for the first time an objective characterization of glucocorticoid receptor distribution in the CNS, thus avoiding the ambiguities of previous mapping studies based on subjective evaluations. In addition, a taxonomic analysis of central nervous system regions containing glucocorticoid receptor immunoreactivity was carried out utilizing the quantitative parameters obtained in the morphometric evaluation. Nuclei of neuronal and glial cells containing glucocorticoid receptor immunoreactivity were detected in a widespread, but still highly heterogeneous, fashion in the central nervous system, underlining the view that glucocorticoids can control a large number of central nervous system target cells via effects on gene expression. Many nerve cell populations have been shown to contain substantial amounts of nuclear glucocorticoid receptor immunoreactivity, whereas only a low density of glial cells, in both gray and white matter, show nuclear glucocorticoid receptor immunoreactivity. Thus, in most brain areas, the major target for glucocorticoids appears to be the nerve cells. Interestingly, an inverse correlation was found in the regional density of glucocorticoid receptor-immunoreactive nerve and glial cells, suggesting that glucocorticoids may influence a brain area either via glial cells or, more frequently, via nerve cells. The results on mapping highlight the impact of glucocorticoids in areas both traditionally and not traditionally involved in stress responses. The distribution of glucocorticoid receptor immunoreactivity also emphasizes a role of glucocorticoids in the regulation of the afferent regions of the basal ganglia and the cerebellar cortex, and of both afferent and efferent layers of the cerebral cortex. Glucocorticoid receptor immunoreactivity is widely distributed over the thalamus, probably leading to modulation of activity in the various thalamocortical pathways transmitting inter alia specific sensory information to the cerebral cortex. Many unspecific afferents to the cerebral cortex are potentially regulated by glucocorticoid receptors such as the noradrenaline and 5-hydroxytryptamine afferents, since their nerve cells of origin contain strong glucocorticoid receptor immunoreactivity. Eight brain regions involving sensory, motor and limbic areas were shown to have a similarity with regard to glucocorticoid receptor-immunoreactive parameters at the level of 95%. The density of glucocorticoid receptor-immunoreactive nerve cells appeared to be the main factor in determining such a very high level of similarity. Overall, our results emphasize that glucocorticoids may appropriately tune networks of different areas to obtain optimal integration and in this way improve survival of the animal under challenging conditions.