RESUMEN
BACKGROUND: In many countries, including South Korea, labour market changes have led to an increase in unstable, temporary jobs. There is evidence that workers in such jobs may experience poorer mental health than those in more stable employment. AIMS: To investigate the association between temporary employment and depressive symptoms in South Korean workers. METHODS: We analysed data from the 2010-2014 Korean Welfare Panel Study (KOWEPS). Employment type was categorized into workers paid per day of labour (day labourers), those on short-term contracts (fixed-term workers) and permanent workers. The association between employment type and depressive symptoms, measured using the Center for Epidemiological Studies Depression scale (CES-D 11), was examined using the generalized estimating equation model. RESULTS: A total of 3756 workers aged 20-59 were included in the 2010 baseline population. Day labourers had the highest mean CES-D 11 score, followed by fixed-term workers and permanent workers. With the day labourer group as reference, fixed-term workers (ß: -1.5027, P < 0.001) and permanent workers (ß: -2.1848, P < 0.001) showed statistically significant decreases in depression scores. CONCLUSIONS: Compared with day labourers, fixed-term workers and permanent workers had progressively lower depression scores. The findings of this study suggest that mental health inequalities based on employment type exist in South Korea.
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Depresión/epidemiología , Empleo/psicología , Adulto , Contratos , Femenino , Humanos , Masculino , Persona de Mediana Edad , República de Corea/epidemiologíaRESUMEN
Cardiac motion and partial volume effects (PVE) are two of the main causes of image degradation in cardiac PET. Motion generates artifacts and blurring while PVE lead to erroneous myocardial activity measurements. Newly available simultaneous PET-MR scanners offer new possibilities in cardiac imaging as MRI can assess wall contractility while collecting PET perfusion data. In this perspective, we develop a list-mode iterative reconstruction framework incorporating both tagged-MR derived non-rigid myocardial wall motion and position dependent detector point spread function (PSF) directly into the PET system matrix. In this manner, our algorithm performs both motion 'deblurring' and PSF deconvolution while reconstructing images with all available PET counts. The proposed methods are evaluated in a beating non-rigid cardiac phantom whose hot myocardial compartment contains small transmural and non-transmural cold defects. In order to accelerate imaging time, we investigate collecting full and half k-space tagged MR data to obtain tagged volumes that are registered using non-rigid B-spline registration to yield wall motion information. Our experimental results show that tagged-MR based motion correction yielded an improvement in defect/myocardium contrast recovery of 34-206% as compared to motion uncorrected studies. Likewise, lesion detectability improved by respectively 115-136% and 62-235% with MR-based motion compensation as compared to gating and no motion correction and made it possible to distinguish non-transmural from transmural defects, which has clinical significance given the inherent limitations of current single modality imaging in identifying the amount of residual ischemia. The incorporation of PSF modeling within the framework of MR-based motion compensation significantly improved defect/myocardium contrast recovery (5.1-8.5%, p < 0.01) and defect detectability (39-56%, p < 0.01). No statistical difference was found in PET contrast and lesion detectability based on motion fields obtained with half and full k-space tagged data.
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Corazón/diagnóstico por imagen , Corazón/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Movimiento , Miocardio/patología , Tomografía de Emisión de Positrones/métodos , Humanos , Fantasmas de Imagen , Dosis de Radiación , Factores de TiempoRESUMEN
PURPOSE: We propose a novel approach for PET respiratory motion correction using tagged-MRI and simultaneous PET-MRI acquisitions. METHODS: We use a tagged-MRI acquisition followed by motion tracking in the phase domain to estimate the nonrigid deformation of biological tissues during breathing. In order to accurately estimate motion even in the presence of noise and susceptibility artifacts, we regularize the traditional HARP tracking strategy using a quadratic roughness penalty on neighboring displacement vectors (R-HARP). We then incorporate the motion fields estimated with R-HARP in the system matrix of an MLEM PET reconstruction algorithm formulated both for sinogram and list-mode data representations. This approach allows reconstruction of all detected coincidences in a single image while modeling the effect of motion both in the emission and the attenuation maps. At present, tagged-MRI does not allow estimation of motion in the lungs and our approach is therefore limited to motion correction in soft tissues. Since it is difficult to assess the accuracy of motion correction approaches in vivo, we evaluated the proposed approach in numerical simulations of simultaneous PET-MRI acquisitions using the NCAT phantom. We also assessed its practical feasibility in PET-MRI acquisitions of a small deformable phantom that mimics the complex deformation pattern of a lung that we imaged on a combined PET-MRI brain scanner. RESULTS: Simulations showed that the R-HARP tracking strategy accurately estimated realistic respiratory motion fields for different levels of noise in the tagged-MRI simulation. In simulations of tumors exhibiting increased uptake, contrast estimation was 20% more accurate with motion correction than without. Signal-to-noise ratio (SNR) was more than 100% greater when performing motion-corrected reconstruction which included all counts, compared to when reconstructing only coincidences detected in the first of eight gated frames. These results were confirmed in our proof-of-principle PET-MRI acquisitions, indicating that our motion correction strategy is accurate, practically feasible, and is therefore ready to be tested in vivo. CONCLUSIONS: This work shows that PET motion correction using motion fields measured with tagged-MRI in simultaneous PET-MRI acquisitions can be made practical for clinical application and that doing so has the potential to remove motion blur in whole-body PET studies of the torso.
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Abdomen/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Movimiento , Tomografía de Emisión de Positrones/métodos , Algoritmos , Encéfalo/diagnóstico por imagen , Pulmón/fisiología , Fantasmas de Imagen , Respiración , Dispersión de Radiación , Factores de TiempoRESUMEN
CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. We have reported that CDX2 promotes tumorigenicity in a subset of human colorectal cancer cell lines. Here, we present evidence that CDX2 negatively regulates the well-documented growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3). Specifically, CDX2 binds to the IGFBP-3 gene promoter and can repress IGFBP-3 transcription, protein expression and secretion. Furthermore, inhibition of IGFBP-3 partially rescues the decreased anchorage-independent growth phenotype observed in CDX2 knockout cells. These data demonstrate for the first time that (1) CDX2 can function as a transcriptional repressor, and (2) one mechanism by which CDX2 promotes anchorage-independent growth is by transcriptional repression of IGFBP-3.
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Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas Represoras/metabolismo , Factor de Transcripción CDX2 , Línea Celular Tumoral , Regulación hacia Abajo , Proteínas de Homeodominio/genética , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. CDX2 is a marker of colon cancer, with strong staining in up to 90% of colonic adenocarcinomas. CDX2 heterozygous-null mice develop colonic neoplasms, which have suggested that CDX2 is a tumor suppressor. However, CDX2 has not been reported to affect xenograft growth. Furthermore, CDX2 is rarely mutated in colon cancer, which has led to suggestions that it may play only a minor role as a tumor suppressor in colon cancer. To understand the functional contributions of CDX2 to colon cancer, we disrupted CDX2 in LOVO and SW48 human colon cancer cell lines by targeted homologous recombination. Consistent with the literature, disruption of CDX2 enhanced anchorage-dependent cell proliferation. However, homozygous loss of CDX2 led to significant inhibition of anchorage-independent growth in LOVO cells, and cell lethality in SW48 cells. Further analyses revealed that disruption of CDX2 led to anchorage-independent G1 to S growth arrest and anoikis. In vivo xenograft studies confirmed that disruption of CDX2 inhibited LOVO tumor growth. These data demonstrate that CDX2 mediates anchorage-independent growth and survival. Thus, CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48.
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Neoplasias del Colon/metabolismo , Neoplasias del Colon/prevención & control , Proteínas de Homeodominio/fisiología , Transactivadores/fisiología , Animales , Anoicis , Western Blotting , Factor de Transcripción CDX2 , Adhesión Celular , Proliferación Celular , Neoplasias del Colon/genética , Femenino , Fase G1 , Genes Supresores de Tumor , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Desnudos , Fase S , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Trasplante Heterólogo , Células Tumorales Cultivadas/trasplante , Ensayo de Tumor de Célula MadreRESUMEN
Conversion of cholesterol to biologically active steroids is a multi-step enzymatic process. Along with some important enzymes, like cholesterol side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD), several proteins play key role in steroidogenesis. The role of steroidogenic acute regulatory (StAR) protein is well established. A novel protein, BRE, found mainly in brain, adrenals and gonads, was highly expressed in hyperplastic rat adrenals with impaired steroidogenesis, suggesting its regulation by pituitary hormones. To further elucidate its role in steroidogenic tissues, mouse Leydig tumor cells (mLTC-1) were transfected with BRE antisense probes. Morphologically the BRE antisense cells exhibited large cytoplasmic lipid droplets and failed to shrink in response to human chorionic gonadotropin. Although cAMP production, along with StAR and P450scc mRNA expression, was unaffected in BRE antisense clones, progesterone and testosterone yields were significantly decreased, while pregnenolone was increased in response to human chorionic gonadotropin stimulation or in the presence of 22(R)OH-cholesterol. Furthermore, whereas exogenous progesterone was readily converted to testosterone, pregnenolone was not, suggesting impairment of pregnenolone-to-progesterone conversion, a step metabolized by 3beta-HSD. That steroidogenesis was compromised at the 3beta-HSD step was further confirmed by the reduced expression of 3beta-HSD type I (3ss-HSDI) mRNA in BRE antisense cells compared with controls. Our results suggest that BRE influences steroidogenesis through its effects on 3beta-HSD action, probably affecting its transcription.
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3-Hidroxiesteroide Deshidrogenasas/metabolismo , Células Intersticiales del Testículo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Testosterona/biosíntesis , Animales , Elementos sin Sentido (Genética)/farmacología , Western Blotting/métodos , Línea Celular Tumoral , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Gonadotropina Coriónica/farmacología , AMP Cíclico/biosíntesis , Depresión Química , Glutatión Transferasa/genética , Humanos , Masculino , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares , Fosfoproteínas/genética , Pregnenolona/biosíntesis , Progesterona/biosíntesis , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: Our previous work on linkage analysis showed that histamine release from basophils to anti-IgE stimuli was linked to the gene marker of chromosome 11q13, where the beta chain of the high-affinity receptor for IgE (FcepsilonRI-beta) is located. OBJECTIVE: To evaluate the association between FcepsilonRI-mediated histamine release from basophils and four bi-allelic single nucleotide polymorphisms of the FcepsilonRI-beta gene. METHODS: Phenotypes of asthma, such as maximal histamine release from basophils and atopy, were measured from 80 randomly recruited asthmatic children. Polymorphisms of the FcepsilonRI-beta gene were determined by PCR-based methods. RESULTS: The polymorphism in exon 7, resulting in Glu to Gly substitution, was significantly associated with histamine release from basophils to anti-IgE stimuli, but not with total IgE levels and skin test responses to aeroallergens. CONCLUSION: This study supports a role for the FcepsilonRI-beta gene in the expression of high affinity IgE receptor-mediated histamine release from basophils.
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Asma/genética , Basófilos/inmunología , Receptores de IgE/genética , Receptores de IgE/fisiología , Adolescente , Alelos , Basófilos/metabolismo , Calcimicina/farmacología , Niño , Femenino , Liberación de Histamina/efectos de los fármacos , Liberación de Histamina/fisiología , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Distribución AleatoriaRESUMEN
An MN9D dopaminergic neuronal cell line overexpressing calbindin-D28K (MN9D/Calbindin) was established in order to investigate directly the potential role of calcium-binding protein in neuronal differentiation. Overexpression of calbindin-D28K in MN9D cells resulted in significant increases in the number of neurites, the length of primary neurites, and the total extent of neurites. This robust neurite outgrowth occurred without cessation of cell division. Analysis of immunoblots revealed that this morphological differentiation was accompanied by increased expression of such markers of maturation as the synaptosomal protein SNAP-25. During calbindin-D28K-evoked neurite outgrowth in MN9D cells, phosphorylation of p38 mitogen-activated protein kinase (MAPK) dramatically increased while the levels and extent of phosphorylation of such other MAPKs as c-Jun N-terminal kinase (JNK) or extracellular response kinase (ERK) were not altered. Consequently, calbindin-D28K-induced neurite outgrowth was largely abolished by treatment with a p38 inhibitor, PD 169316, while the level of SNAP-25 in MN9D/Calbindin cells was not altered by this treatment. These data support an idea that calbindin-D28K and its associated p38 signaling pathway play a role in dopaminergic neuronal differentiation.
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Dopamina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Proteína G de Unión al Calcio S100/biosíntesis , Animales , Calbindina 1 , Calbindinas , Diferenciación Celular , División Celular , Pollos , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Immunoblotting , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Neuroblastoma/metabolismo , Neuronas/citología , Neuronas/enzimología , Fosforilación , Unión Proteica , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The effects of neurotoxins on levels of mitochondrially encoded gene transcripts in a dopaminergic neuronal cell line, MN9D, were examined following treatment with 200 microM N-methyl-4-phenylpyridinium (MPP(+)) or 6-hydroxydopamine (6-OHDA). As confirmed by a Northern blot analysis, levels of cytochrome c oxidase subunit 3 (COX III) and ATPase subunit 6 (ATPase 6) transcript were decreased in a time-dependent manner following treatment with MPP(+) but not with 6-OHDA. Accordingly, enzymatic activity of cytochrome c oxidase (COX) and the intracellular ATP content were also decreased in MPP(+)-treated cells while these remained unaltered in 6-OHDA-treated cells. In the cell death paradigm induced by MPP(+), overexpression of Bcl-2 in MN9D cells (MN9D/Bcl-2) significantly blocked MPP(+)-induced downregulation of COX III and ATPase 6 transcripts. In MN9D/Bcl-2 cells, MPP(+)-induced downregulation of COX activity and the intracellular level of ATP was also blocked. Treatment with a pan-caspase inhibitor, however, neither prevented MPP(+)-induced downregulation of COX activity nor affected intracellular level of ATP in MN9D cells. Taken together, our present data suggest that Bcl-2 may play a regulatory role in energy metabolism by preventing downregulation of mitochondrially encoded gene(s) at a point distinct from its known anticaspase activity in MPP(+)-induced dopaminergic neuronal death.
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1-Metil-4-fenilpiridinio/farmacología , Dopaminérgicos/farmacología , Mitocondrias/enzimología , Mitocondrias/genética , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Animales , Muerte Celular , Línea Celular , ADN Mitocondrial/genética , Regulación hacia Abajo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , ATPasas de Translocación de Protón Mitocondriales , Neuronas/efectos de los fármacos , Oxidopamina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
The present study was designed to test whether GnRH regulates pituitary adenylate cyclase-activating polypeptide mRNA levels in a stage-dependent manner during follicle development in the rat ovary. The granulosa cells of preovulatory and immature follicles obtained from PMSG- and estrogen-treated rats, respectively, were cultured in serum-free conditions in the presence of various hormones. GnRH receptor mRNA expression was detected in both preovulatory and immature granulosa cells and was down-regulated by gonadotropins. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner. In situ hybridization analysis of cultured preovulatory follicles revealed that GnRH-induced pituitary adenylate cyclase- activating polypeptide signals were detected in granulosa cells, but not thecal cells. In immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. Furthermore, treatment with GnRH antagonist inhibited LH-induced pituitary adenylate cyclase-activating polypeptide gene expression in preovulatory granulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase-activating polypeptide gene expression in immature granulosa cells. Interestingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltri fluoromethyl ketone, an inhibitor of phospholipase A(2), but not by an inhibitor of protein kinase A or C. Lastly, treatment of preovulatory follicles with pituitary adenylate cyclase-activating polypeptide antagonist suppressed GnRH-stimulated progesterone production during 6--9 h of culture. Taken together, these results demonstrate the stage-dependent regulation of pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the stimulatory and inhibitory effect in granulosa cells of preovulatory and immature follicles, respectively.
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Fase Folicular , Hormona Liberadora de Gonadotropina/fisiología , Células de la Granulosa/metabolismo , Neuropéptidos/genética , Ovario/metabolismo , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Activación Enzimática/fisiología , Femenino , Hormona Folículo Estimulante/farmacología , Hormona Liberadora de Gonadotropina/agonistas , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hormona Luteinizante/farmacología , Neuropéptidos/antagonistas & inhibidores , Folículo Ovárico/metabolismo , Fosfolipasas A/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Progesterona/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
NGFI-B is an immediate-early gene that encodes an orphan nuclear receptor. The present study was designed to examine the localization and gonadotropin regulation of NGFI-B expression in the rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of NGFI-B during prepubertal development. Treatment of immature rats with PMSG, however, decreased ovarian NGFI-B expression. The major cell types expressing NGFI-B messenger RNA were thecal cells of follicles in different sizes. In contrast, treatment of PMSG-primed rats with human (h) CG resulted in the rapid and transient stimulation of ovarian NGFI-B messenger RNA, reaching a peak within 1 h. In situ hybridization analysis revealed that hCG treatment induced the expression of NGFI-B in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of NGFI-B messenger RNA and protein. LH-stimulated NGFI-B expression in preovulatory follicles was abolished by alpha-amanitin, but was superinduced by cycloheximide. Furthermore, treatment of adult cycling rats with pentobarbital abolished NGFI-B expression on proestrus, and exogenous administration of hCG restored it, indicating the role of the preovulatory surge of LH in the stimulation of NGFI-B expression. These results demonstrate the cell type-specific expression and gonadotropin induction of NGFI-B in granulosa cells of preovulatory follicles and suggest a role for NGFI-B in the ovulatory process.
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Proteínas de Unión al ADN/genética , Gonadotropinas/fisiología , Folículo Ovárico/fisiología , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Animales , Gonadotropina Coriónica/farmacología , Técnicas de Cultivo , Estro/fisiología , Femenino , Fase Folicular/fisiología , Gonadotropinas Equinas/farmacología , Hormona Luteinizante/farmacología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Folículo Ovárico/efectos de los fármacos , Ovario/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , OvinosRESUMEN
P11, a member of the S100 family of calcium-binding proteins, has been shown to interact with BAD (Bcl-xL/Bcl-2-associated death promoter) in the yeast two-hybrid protein-protein interaction assay. Because overexpression of P11 dampens the proapoptotic activity of BAD in transfected cells, we tested the possibility that the expression of this antiapoptotic protein may be regulated by gonadotropins and other survival factors in the ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of P11 messenger RNA (mRNA) during prepubertal development in the theca cells of preantral and early antral follicles. Treatment of immature rats with PMSG did not affect P11 expression, whereas treatment of PMSG-primed rats with an ovulatory dose of human (h)CG stimulated ovarian P11 mRNA within 6-9 h in the granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time-dependent stimulation of P11 by gonadotropins. In addition, treatment of cultured preovulatory follicles with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-stimulated P11 mRNA, whereas treatment with forskolin, an adenylate cyclase activator, but not the protein kinase C activator, 2-O-tetradecanol-phorbal-13-acetate, mimicked the LH action, suggesting the role of adenylate cyclase activation in P11 expression. Treatment with other follicle survival factors, including the epidermal growth factor, the basic fibroblast growth factor, and interleukin-1beta, could also stimulate P11 expression in cultured preovulatory follicles. These results demonstrate the expression of P11 mRNA in theca cells of different-sized follicles and in granulosa cells of preovulatory follicles following gonadotropin stimulation, and suggest that P11 may mediate, at least partially, the survival action of gonadotropins during the ovulatory process.
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Anexina A2 , Proteínas de Unión al Calcio/genética , Expresión Génica/efectos de los fármacos , Gonadotropinas/farmacología , Células de la Granulosa/metabolismo , Folículo Ovárico/metabolismo , Ovario/metabolismo , Proteínas S100 , Inhibidores de Adenilato Ciclasa , Animales , Apoptosis , Northern Blotting , Gonadotropina Coriónica/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Gonadotropinas Equinas/farmacología , Iminas/farmacología , Hormona Luteinizante/farmacología , Ovario/química , Ovulación , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Maduración Sexual , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
It has been proposed recently that two types of GnRH receptors (GnRHR) exist in a particular species. Here we present data demonstrating that at least three types of GnRHR are expressed in a single diploid species, the bullfrog. Three different cDNAs, encoding distinct types of bullfrog GnRHR (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3), were isolated from pituitary and hindbrain of the bullfrog. BfGnRHR-1 mRNA was expressed predominantly in pituitary, whereas bfGnRHR-2 and -3 mRNAs were expressed in brain. The bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 proteins have an amino acid identity of approximately 30% to approximately 35% with mammalian GnRHRs and approximately 40% to approximately 50% with nonmammalian GnRHRs. Interestingly, bfGnRHR-2 has an 85% amino acid homology with Xenopus GnRHR. Less than 53% amino acid identity was observed among the three bfGnRHRs. All isolated cDNAs encode functional receptors because their transient expression in COS-7 cells resulted in a ligand-dependent increase in inositol phosphate production. Notably, all three receptors exhibited a differential ligand selectivity. For all receptors, cGnRH-II has a higher potency than mGnRH. In addition, salmon GnRH also has a strikingly high potency to stimulate all three receptors. In conclusion, we demonstrated the presence of three GnRHRs in the bullfrog. Their expression in pituitary and brain suggests that bfGnRHRs play an important role in the regulation of reproductive functions in the bullfrog.
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Rana catesbeiana/genética , Receptores LHRH/clasificación , Receptores LHRH/metabolismo , Secuencia de Aminoácidos , Animales , Southern Blotting , Células COS , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Regulación de la Expresión Génica , Humanos , Fosfatos de Inositol/metabolismo , Ligandos , Datos de Secuencia Molecular , Hipófisis/química , Isoformas de Proteínas/química , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores LHRH/química , Receptores LHRH/genética , Rombencéfalo/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TransfecciónRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates pituitary hormone biosynthesis and secretion through its cognate receptors. PACAP also plays an important role in the regulation of ovarian steroid biosynthesis. If so, there might be a feedback regulation of hypothalamic PACAP synthesis by the pituitary and by ovarian steroids. In the present study, we used RNase protection assays to determine changes in mRNA levels of PACAP and type I PACAP receptor (PAC(1)) under the conditions of ovariectomy and replacement with ovarian steroids. Progesterone (P) alone or in combination with estradiol (E) induced significant increases in PACAP mRNA level in the medial basal hypothalamus (MBH) and PAC(1) mRNA levels in MBH and the preoptic area (POA). This finding suggests that feedback regulation takes place between the ovary and hypothalamic PACAP neurons. P is known to be a major regulatory feedback factor for hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, but P receptor is not present in these neurons. Therefore, we examined a possible involvement of PACAP in the feedback regulatory pathway of P to LHRH neurons. After an antisense PAC(1) oligodeoxynucleotide (ODN) was i.c.v.-injected into the third ventricle of E and P-treated rats, LHRH mRNA levels were determined. The ODN markedly decreased the P-induced increase in the LHRH mRNA level. Taken together, the present data suggest that PACAP may play a role as a mediator in the regulation of LHRH synthetic machinery by stimulatory feedback of P.
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Hipotálamo Medio/fisiología , Neuropéptidos/genética , Adenohipófisis/fisiología , Progesterona/farmacología , Receptores de la Hormona Hipofisaria/genética , Animales , Elementos sin Sentido (Genética) , Química Encefálica/efectos de los fármacos , Química Encefálica/genética , Retroalimentación/fisiología , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Hormona Liberadora de Gonadotropina/genética , Hipotálamo Medio/citología , Inyecciones Intraventriculares , Neuronas/química , Neuronas/fisiología , Ovariectomía , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Adenohipófisis/citología , Área Preóptica/citología , Área Preóptica/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa HipofisariaRESUMEN
Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and known to stimulate the production of cAMP in anterior pituitary cells. In the recent report, the expression of PACAP was detected in preovulatory follicles, and treatment with PACAP stimulated the production of progesterone and prostaglandin E(2) through the action of AC and PLC pathways in the ovary. PACAP binds to three type receptors. Type I A receptor is coupled to adenylate cyclase (AC) and phospholipase C (PLC) pathways, while type I B and type II receptors are only coupled to AC. Thus, the present study aimed to evaluate the temporal expression of PACAP and its type I A receptor mRNAs in the rat ovary after treatment with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Northern blot analysis showed that PACAP transcripts were transiently expressed from 3-9 hr after hCG treatment, reaching a maximum at 6 hr. During these time points, PACAP mRNAs were specifically and strongly expressed in granulosa cells and cumulus cells of large preovulatory follicles and interstitial glandular cells. Type I A receptor mRNAs were also transiently expressed in granulosa cells of large preovulatory follicles from 3-9 hr after hCG treatment. PACAP and its type I A receptor mRNAs were expressed in the same preovulatory follicles. These results demonstrate that PACAP acts as an autoregulator or pararegulator through type I A receptor in granulosa cells and cumulus cells of large preovulatory follicles. Thus, we suggest that PACAP may have a critical role in granulosa cells of preovulatory follicles for the preparation of ovulation.
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Células de la Granulosa/metabolismo , Neuropéptidos/metabolismo , Folículo Ovárico/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Animales , Northern Blotting , Gonadotropina Coriónica/farmacología , Femenino , Gonadotropinas Equinas/farmacología , Humanos , Hibridación in Situ , Inyecciones Intraperitoneales , Ovulación/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Embarazo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa HipofisariaRESUMEN
The present study was designed to determine whether progesterone might have a role in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide (Pacap) gene expression in rat ovary. Northern blot analysis revealed that treatment of pregnant mare's serum gonadotrophin (PMSG)-primed immature rats with the progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane, 1 h before HCG, resulted in a dose-dependent inhibition of the HCG-induced Pacap gene expression. In-situ hybridization demonstrated that the number of pre-ovulatory follicles expressing Pacap mRNA in their granulosa cells was greatly reduced in ovaries treated with RU486. Moreover, the suppressive effect of RU486 or epostane on the LH-induced Pacap gene expression in cultured pre-ovulatory follicles was reversed by co-treatment with the synthetic progestin R5020. We further cloned the 5'-flanking region of the rat Pacap gene and identified the presence of a consensus progesterone receptor element. When luciferase fusion genes containing Pacap gene promoter were transiently transfected into granulosa cells of pre-ovulatory follicles, luciferase activity was markedly stimulated by LH. Treatment with RU486 or epostane resulted in partial suppression of LH-stimulated PACAP promoter activity. Taken together, these results indicate that progesterone, acting through progesterone receptors, plays a role in gonadotrophin induction of Pacap gene expression in granulosa cells of pre-ovulatory follicles, and thereby may be involved in the process of ovulation.
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Regulación de la Expresión Génica/efectos de los fármacos , Gonadotropinas/farmacología , Neuropéptidos/genética , Folículo Ovárico/efectos de los fármacos , Progesterona/fisiología , Androstenoles/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Secuencia de Consenso , Técnicas de Cultivo , Femenino , Células de la Granulosa/citología , Ratones , Mifepristona/farmacología , Datos de Secuencia Molecular , Ovulación/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Progestinas/farmacología , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
Expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with considerable homology to vasoactive intestinal peptide, has been shown to be stimulated by gonadotropins in the ovary. The present studies further evaluated the cell-type specific expression and gonadotropin regulation of PACAP type I receptor (PACAPR) messenger RNA in immature rat ovaries and in cultured preovulatory follicles. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of PACAPR during prepubertal development. The major cell types expressing PACAPR messenger RNA were granulosa cells of large preantral follicles. Treatment of immature rats with PMSG caused a decrease in ovarian PACAPR expression. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3-6 h in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of PACAPR by gonadotropins in granulosa cells of preovulatory follicles. Moreover, RNase protection assay revealed that the short variant of ovarian PACAPR was the predominant form stimulated during prepubertal development and by gonadotropins. These results demonstrate the expression of PACAPR messenger RNA in granulosa cells of growing follicles and of preovulatory follicles stimulated by gonadotropins, and suggest that PACAP may play a role in the growth of developing follicles and in ovulation as an autocrine/paracrine factor.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Folículo Ovárico/fisiología , Ovario/fisiología , Receptores de la Hormona Hipofisaria/genética , Transcripción Genética , Envejecimiento , Animales , Células Cultivadas , Gonadotropina Coriónica/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Gonadotropinas Equinas/farmacología , Humanos , Hormona Luteinizante/farmacología , Folículo Ovárico/citología , Folículo Ovárico/efectos de los fármacos , Ovario/efectos de los fármacos , Ovario/crecimiento & desarrollo , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Maduración SexualRESUMEN
CD44 is a cell adhesion molecule with numerous isoforms created by mRNA alternative splicing. Expression of CD44 variants has been suggested to play a potential role in tumor progression and metastasis. We designed primers CD44V, CD44V6/7, CD44R1 and CD44V6-10 to analyze and compare the roles of each CD44 variants. Expressions of CD44 variants were investigated in normal colonic mucosa, the lymph nodes which was histopathologically free of cancer cell, and cancer tissues of 44 human colorectal cancer patients by RT-PCR method. The expression of CD44V was observed in 28 out of 39 (71.8%) tumors and 7 out of 11 (63.6%) N1 normal regional lymph nodes, and CD44V6/7 was observed in 28 out of 39 (71.8%) tumors and 9 out of 11 (81.8%) N1 normal regional lymph nodes. The expressions of CD44V and CD44V6/7 were most frequently observed compared with any other CD44 variants. In normal colonic mucosa, the expression of CD44 variants are low but in cancer tissue and its regional lymph node, the expression of CD44V and CD44V6/7 were significantly higher and more frequent than any other CD44 variants (p<0.05). These results suggest that CD44V and CD44V6/7 can be a molecular marker for colorectal cancer and its micrometastasis to the regional normal lymph node.
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Neoplasias Colorrectales/inmunología , Receptores de Hialuranos/genética , Ganglios Linfáticos/inmunología , Empalme Alternativo , Neoplasias Colorrectales/patología , Expresión Génica , Humanos , Ganglios Linfáticos/patología , Isoformas de Proteínas/genéticaRESUMEN
Genomic DNA amplification in tumors is frequently associated with an increased gene copy number of oncogenes or other cancer-related genes. We have used a two-dimensional whole-genome scanning technique to identify gene amplification events in esophageal adenocarcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification encompassing this fragment was confirmed by Southern blot analysis. The corresponding DNA sequence was matched by BLAST to a BAC contig, which allowed the use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our laboratory (Lin, L. et al., Cancer Res., 60: 1341-1347,2000), was used to characterize the amplicon. Genomic DNA from 65 esophageal and 11 gastric cardia adenocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequence tagged site markers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0.8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Fourteen expressed sequence tags (ESTs) covering the minimal region were then assayed for potential gene overexpression using quantitative reverse transcription-PCR. Seven of the selected ESTs were found to be both amplified and overexpressed. Among these seven ESTs, cyclin E showed the highest frequency of gene amplification and overexpression in the tumors examined, which allowed us to finalize the core-amplified region to <300 kb. These results indicate that cyclin E is the likely target gene selected by the amplification event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplification underlies the cyclin E gene overexpression. Our study represents the first extensive analysis of the 19q12 amplicon, and is the first to physically map the core-amplified domain to a region of <300 kb that includes cyclin E. Amplification of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esophageal and 9.1% of gastric cardia adenocarcinomas.
Asunto(s)
Adenocarcinoma/genética , Cromosomas Humanos Par 19 , Ciclina E/genética , Neoplasias Esofágicas/genética , Southern Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Mapeo Cromosómico , Clonación Molecular , Mapeo Contig , Ciclina E/biosíntesis , Electroforesis en Gel Bidimensional , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Esófago/metabolismo , Esófago/patología , Etiquetas de Secuencia Expresada , Humanos , Neoplasias Pulmonares/genética , Modelos Genéticos , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
The majority of ovarian follicles undergo atresia mediated by apoptosis. Bcl-2-related proteins act as regulators of apoptosis via the formation of dimers with proteins inside and outside the Bcl-2 family. Previous studies have identified BAD as a proapoptotic Bcl-2 family member expressed in the ovary. It is known that BAD phosphorylation induced by survival factors leads to its preferential binding to 14-3-3 and suppression of the death-inducing function of BAD. To identify ovarian binding partners for hypophosphorylated BAD, we performed a yeast two-hybrid screening of a rat ovary complementary DNA library using as bait a mutant BAD incapable of binding to 14-3-3. Screening of yeast transformants yielded positive clones encoding the rat ortholog of Mcl-1 (myeloid cell leukemia-1), an antiapoptotic Bcl-2 protein. Amino acid sequence analysis revealed that rat and human Mcl-1 showed a complete conservation of the Bcl-2 homology domains BH1, BH2, and BH3. In the yeast two-hybrid system, Mcl-1 binds to the hypophosphorylated mutant of BAD and interacts preferentially with different proapoptotic (Bax, Bak, Bok, Bik, and BOD) compared with antiapoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, CED-9, and BHRF-1). Northern blot hybridization demonstrated expression of Mcl-1 transcripts of 2.3 and 3.7 kb in the ovary and diverse other rat tissues. In immature rats, PMSG treatment led to a transient increase in the 2.3-kb Mcl-1 transcript, peaking at 6 h after injection and returning to baseline levels after 24 h. Moreover, the same transcript was induced in the PMSG-primed preovulatory rat ovary 6 h after the administration of ovulatory doses of either hCG or FSH. In situ hybridization studies revealed that the gonadotropin stimulation of ovarian Mcl-1 message occurs in both granulosa and thecal cells. In conclusion, rat Mcl-1 was identified as an ovarian BAD-interacting protein and the message for the antiapoptotic Mcl-1 protein was induced after treatment with gonadotropins in granulosa and thecal cells of growing follicles.