RESUMEN
The prevalence of hepatitis B virus (HBV) is a global healthcare threat, particularly chronic hepatitis B (CHB) that might lead to hepatocellular carcinoma (HCC) should not be neglected. Although many types of HBV diagnosis detection methods are available, some technical challenges, such as the high cost or lack of practical feasibility, need to be overcome. In this study, the polycrystalline silicon nanowire field-effect transistors (pSiNWFETs) were fabricated through commercial process technology and then chemically functionalized for sensing hepatitis B virus surface antigen (HBsAg) and hepatitis B virus X protein (HBx) at the femto-molar level. These two proteins have been suggested to be related to the HCC development, while the former is also the hallmark for HBV diagnosis, and the latter is an RNA-binding protein. Interestingly, these two proteins carried opposite net charges, which could serve as complementary candidates for evaluating the charge-based sensing mechanism in the pSiNWFET. The measurements on the threshold voltage shifts of pSiNWFETs showed a consistent correspondence to the polarity of the charges on the proteins studied. We believe that this report can pave the way towards developing an approachable tool for biomedical applications.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/análisis , Hepatitis B/diagnóstico , Nanocables , Transactivadores/análisis , Proteínas Reguladoras y Accesorias Virales/análisis , Carcinoma Hepatocelular , Atención a la Salud , Virus de la Hepatitis B , Humanos , Neoplasias Hepáticas , SilicioRESUMEN
The aim of this study was to establish the effect of a 70% ethanol extract of Elaeocarpus sylvestris (ESE) on varicella-zoster virus (VZV) replication and identify the specific bioactive component(s) underlying its activity. ESE induced a significant reduction in replication of the clinical strain of VZV. Activity-guided fractionation indicated that the ethyl acetate (EtOAc) fraction of ESE contains the active compound(s) inhibiting VZV replication. High-Performance Liquid Chromatography coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (HPLC-Q-TOF-MS/MS) analysis of the EtOAc fraction of ESE facilitated the identification of 13 chemical components. Among these, 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG) markedly suppressed VZV-induced c-Jun N-terminal kinase (JNK) activation, expression of viral immediate-early 62 (IE62) protein and VZV replication. Our results collectively support the utility of PGG as a potential candidate anti-viral drug to treat VZV-associated diseases.
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Elaeocarpaceae/química , Herpesvirus Humano 3/efectos de los fármacos , Taninos Hidrolizables/farmacología , Extractos Vegetales/química , Replicación Viral/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Herpesvirus Humano 3/fisiología , Humanos , Taninos Hidrolizables/aislamiento & purificación , Proteínas Inmediatas-Precoces/análisis , Proteínas Quinasas JNK Activadas por Mitógenos/análisis , Espectrometría de Masa por Ionización de Electrospray , Transactivadores/análisis , Proteínas del Envoltorio Viral/análisisRESUMEN
BACKGROUND: Pseudomyxoma peritonei (PMP) is a rare locoregional disease characterized by disseminated intraperitoneal mucinous tumors. However, little is known about PMP from urachal neoplasm as a result of its rarity. METHODS: A total of 9 patients with PMP of urachal origin were treated by cytoreductive surgery (CRS) plus hyperthermic intraperitoneal chemotherapy (HIPEC) in our institution. All specimens of surgeries were submitted for pathologic examination. Representative slides of tumors and normal urachus were submitted for immunohistochemical staining. RESULTS: Four patients were men; the median age was 48 years (range 27-65 years). Initial radiologic examination of all patients showed a cystic tumor located between posterior aspect of umbilicus and the dome of urinary bladder, with or without leaking mucin. Complete CRS and HIPEC were performed in all patients. Until the latest follow-up, local recurrence occurred in 1 patient. Other 8 patients had a median disease-free survival of 27.5 months. Primary urachal tumors of 9 cases were all mucinous adenocarcinoma. Six patients had low-grade mucinous carcinoma peritonei, and 3 patients had high-grade mucinous carcinoma peritonei. Signet ring cells were noted in 4 patients. All tumor specimens of 9 patients were diffuse positive for CK-20, CDX-2, MUC-2, and MUC-5AC, and were variant positive for CK-7. CONCLUSIONS: PMP arising from urachus comes from neoplastic cells with development of intestinal-type mucinous neoplasm. It shares a similar pathophysiology as PMP from appendix. CRS including total urethrectomy, partial cystectomy, and peritonectomy plus HIPEC can be considered as a new option of treatment for PMP originating from urachus.
Asunto(s)
Adenocarcinoma Mucinoso/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Procedimientos Quirúrgicos de Citorreducción , Hipertermia Inducida , Neoplasias Peritoneales/terapia , Seudomixoma Peritoneal/terapia , Uraco , Adenocarcinoma Mucinoso/química , Adenocarcinoma Mucinoso/patología , Adulto , Anciano , Factor de Transcripción CDX2 , Cisplatino/administración & dosificación , Supervivencia sin Enfermedad , Femenino , Proteínas de Homeodominio/análisis , Humanos , Infusiones Parenterales , Queratina-20/análisis , Queratina-7/análisis , Masculino , Persona de Mediana Edad , Mitomicina/administración & dosificación , Mucina 5AC/análisis , Mucina 2/análisis , Neoplasias Peritoneales/química , Neoplasias Peritoneales/patología , Seudomixoma Peritoneal/metabolismo , Seudomixoma Peritoneal/patología , Transactivadores/análisisRESUMEN
Endogenous ceramide plays an important role in the palmitate (Palm) impairment of proinsulin gene expression in pancreatic islet ß-cells. Changes in the liposoluble ceramide levels not only depend on metabolic enzymes but also on its transport to subcellular sites in response to Palm stimuli. In this study, we show that suppression of ceramide transport protein (CERT) mRNA with small interfering RNA contributed to intracellular ceramide accumulation in response to chronic Palm exposure and impairment of proinsulin gene expression, similar to the effect of inhibiting ceramide scavenging enzyme sphingomyelin synthase (SMS). High dose Palm treatment increased protein kinase D (PKD)-induced phosphorylation of CERT and its dysfunction. Intracellular accumulation of ceramide was associated with reduction of PDX-1 nuclear localization and MafA protein levels and stimulation of CCAAT/enhancer binding protein ß (C/EBP ß) expression. These conditions also corresponded with a reduction of PDX-1 and MafA and an increase of C/EBP ß binding to the insulin promoter. Furthermore, down-regulation of C/EBP ß could block ceramide impairment of proinsulin gene expression. The results reveal that Palm-mediated dysfunction of ceramide transport may contribute to intracellular ceramide accumulation and result in dysfunction of pancreatic beta cells by affecting binding of transcription factors to the insulin promoter.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Palmitatos/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Ceramidasas/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/metabolismo , Insulina/genética , Lectinas Tipo C/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Oxidorreductasas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transactivadores/análisis , Transactivadores/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
INTRODUCTION: Stem cell lines are usually grown in medium containing animal products. Fetal bovine serum (FBS) is an important additive for cell growth; however, the allergenic potential and the possibility of contamination when we use a medium containing serum would be a barrier to transplantation and consequently to the introduction of cell therapy methods into clinical applications. METHODS: Dental mesenchymal cells were isolated and expanded in vitro and maintained in 4 different serum-free media (SFMs): SFM#1 (ITS-X, embryotrophic factor [ETF]); SFM#2 (ITS-X); SFM#3 (ETF); and SFM#4 (ETF, sodium pyruvate, ascorbic acid, fibroblast growth factor [FGF-a], acidic). Viability, proliferative, and immunocytochemical tests for the cells were performed by using 4 stem cell markers (CD44H, CK19, nestin, and P63) for ectoderm, mesoderm, and endoderm. RESULTS: Viability tests showed a significant difference between the control and SFMs in both deciduous tooth pulp cells (DTPCs) and wisdom tooth pulp cells (WTPCs). However, all SFMs demonstrated 84%-90% viability, whereas the control showed 90%-93%. In both DTPCs and WTPCs, SFM#1 had the highest proliferation rate among the 4 SFMs. Immunocytochemistry stained positive stem cell markers most intensely in cells cultured with SFM#1. Furthermore, all stem cell markers for ectoderm, mesoderm, and endoderm were expressed in the cells cultured with SFM#1. CONCLUSIONS: SFM#1 showed an acceptable survival rate, the highest proliferation rate, and the strongest expression of all the stem cell markers. SFM#1 proved to be a suitable medium for the culture of human dental pulp stem cells and to preserve pluripotency in differentiation.
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Técnicas de Cultivo de Célula , Medio de Cultivo Libre de Suero , Pulpa Dental/citología , Células Madre Mesenquimatosas/fisiología , Animales , Ácido Ascórbico/farmacología , Biomarcadores/análisis , Bovinos , Recuento de Células , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Pulpa Dental/efectos de los fármacos , Ectodermo/citología , Endodermo/citología , Factor 1 de Crecimiento de Fibroblastos/farmacología , Humanos , Receptores de Hialuranos/análisis , Insulina/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteínas de Filamentos Intermediarios/análisis , Queratina-19/análisis , Células Madre Mesenquimatosas/efectos de los fármacos , Mesodermo/citología , Tercer Molar/citología , Proteínas del Tejido Nervioso/análisis , Nestina , Ácido Pirúvico/farmacología , Selenio/farmacología , Diente Primario/citología , Transactivadores/análisis , Factores de Transcripción , Transferrina/farmacología , Proteínas Supresoras de Tumor/análisisRESUMEN
Recent studies demonstrated that PNZIP and its homologs encode a special cyclase and play an important role in chlorophyll biosynthesis in higher plants. To investigate the molecular mechanism governing the PNZIP gene, the PNZIP promoter was isolated and analyzed. Deletion analysis indicated that G-box is an important element in the regulation of the reporter gene expression. Further mutation assay demonstrated that G-box and GATACT elements are necessary and sufficient for the high and tissue-specific expression of the GUS gene. Using yeast one-hybrid screening, we have isolated a novel tobacco bZIP protein, NtbZIP, which can specifically recognize the G-box of the PNZIP promoter. The NtbZIP protein shares a limited amino acid homology to Arabidopsis ABI5 and AtAREB1 and very low homology to other bZIP proteins. Northern blot analysis showed that the NtbZIP gene is not induced by exogenous ABA and is expressed in different tobacco organs. Cotransformation assays showed that the NtbZIP protein could activate the transcription of the GUS gene driven by the PNZIP promoter. Transgenic tobaccos analysis demonstrated that constitutively expressing antisense NtbZIP gene resulted in a lower NTZIP synthesis and reduced chlorophyll levels. We suggest that NTZIP is a target gene of NtbZIP, which is involved in the regulation of chlorophyll biosynthesis.
Asunto(s)
Nicotiana/genética , Oxigenasas/genética , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Oxigenasas/biosíntesis , Fotosíntesis/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/metabolismo , Elementos de Respuesta , Análisis de Secuencia , Distribución Tisular , Nicotiana/enzimología , Nicotiana/metabolismo , Transactivadores/análisisRESUMEN
Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5.6 and 11.1-mmol/L concentrations; (v) slowed cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity.
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Glucemia/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Taurina/farmacología , Animales , Glucemia/metabolismo , Calcio/metabolismo , Suplementos Dietéticos , Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/análisis , Homeostasis/efectos de los fármacos , Insulina/sangre , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Masculino , Ratones , Fosforilación/efectos de los fármacos , Receptor de Insulina/metabolismo , Taurina/sangre , Transactivadores/análisis , Tirosina/metabolismoRESUMEN
During the infection of plants, Agrobacterium tumefaciens introduces several Virulence proteins including VirE2, VirF, VirD5 and VirE3 into plant cells in addition to the T-DNA. Here, we report that double mutation of virF and virE3 leads to strongly diminished tumor formation on tobacco, tomato and sunflower. The VirE3 protein is translated from a polycistronic mRNA containing the virE1, virE2 and virE3 genes, in Agrobacterium. The VirE3 protein has nuclear localization sequences, which suggests that it is transported into the plant cell nucleus upon translocation. Indeed we show here that VirE3 interacts in vitro with importin-alpha and that a VirE3-GFP fusion protein is localized in the nucleus. VirE3 also interacts with two other proteins, viz. pCsn5, a component of the COP9 signalosome and pBrp, a plant specific general transcription factor belonging to the TFIIB family. We found that VirE3 is able to induce transcription in yeast when bound to DNA through the GAL4-BD. Our data indicate that the translocated effector protein VirE3 is transported into the nucleus and there it may interact with the transcription factor pBrp to induce the expression of genes needed for tumor development.
Asunto(s)
Agrobacterium tumefaciens/patogenicidad , Proteínas Bacterianas/fisiología , Tumores de Planta/microbiología , Transactivadores/fisiología , Agrobacterium tumefaciens/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Canales Iónicos/biosíntesis , Canales Iónicos/genética , Carioferinas/metabolismo , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/genética , Proteínas Nucleares/análisis , Cebollas/química , Tumores de Planta/genética , Transactivadores/análisis , Transactivadores/genética , Transcripción Genética , Levaduras/genéticaRESUMEN
AIM: To investigate the effect of atorvastatin (Lipitor), a commonly used drug for dyslipidaemia in experimental autoimmune uveitis (EAU). METHODS: 48 B10-RIII mice were immunised with human interphotoreceptor retinoid binding protein (IRBP) peptide p161-180. They were divided into three groups of 16 each and treated orally once daily for 14 days; group one received phosphate buffered saline (control group), group two received 1 mg/kg of atorvastatin (low dose group), and group three received 10 mg/kg (high dose). On day 14 lymph nodes, spleens, and right eyes were harvested. RNA was extracted from lymph nodes for RNase protection assay (RPA) to determine proinflammatory (IL-1 alpha and IL-1 beta), Th1 (TNF-alpha, IL-2, IL-12), and Th2 (IL-4, IL-5, and IL-10) cytokine levels. Protein was extracted from spleens for western blot to detect the expression of phosphorylated signal transducer and activator of transcription (STAT) 4 and STAT6. The severity of inflammation in enucleated eyes was graded by a masked observer. Paired t test was performed for the mean difference in histological scoring between treated groups and the immunised control group. RESULTS: Surprisingly, atorvastatin did not modulate the immune response. The proinflammatory cytokines, IL-1 alpha and IL-1 beta, and Th1 cytokines, TNF-alpha and IL-2, were upregulated equally in control and atorvastatin treated groups. IL-12 and Th2 cytokines were not upregulated in all three groups. Western blot analysis showed high levels of phosphorylated STAT4, but not STAT6 protein in the control and atorvastatin treated groups. Mean differences in histological scoring between treated groups and the immunised control group were not statistically significant. CONCLUSIONS: Atorvastatin treatment had no effect on Th1 and Th2 cytokine transcription. Although histological grading suggested mildly decreased inflammation in the high dose treated group, the equivalence of cytokine expression in all groups suggests that the statins may not modulate IRBP induced uveoretinitis.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Pirroles/farmacología , Uveítis/tratamiento farmacológico , Animales , Atorvastatina , Enfermedades Autoinmunes/inmunología , Western Blotting/métodos , Proteínas de Unión al ADN/análisis , Interleucina-1/análisis , Interleucina-2/análisis , Ratones , Ratones Endogámicos , Modelos Animales , Factor de Transcripción STAT4 , Factor de Transcripción STAT6 , Células TH1/inmunología , Células Th2/inmunología , Transactivadores/análisis , Factor de Necrosis Tumoral alfa/análisis , Uveítis/inmunologíaRESUMEN
Mice with mutations in the Wnt coreceptor low-density lipoprotein receptor-related protein-6 (LRP6) have a smaller and severely disorganized dorsal thalamus and lack thalamocortical projections. Using molecular markers, we showed that most dorsal thalamic and epithalamic neurons were missing, and most of the major dorsal thalamic nuclei were not identifiable. However, the ventral thalamus was essentially unaffected, although the dorsal thalamic defect leads to rostral displacement of portions of the ventral thalamus. Analysis of younger embryos showed that epithalamic and dorsal thalamic neurons were not produced at early stages of development, whereas ventral thalamic neurons were still produced. These defects were accompanied by improper formation of the boundary between dorsal and ventral thalamus, the zona limitans interthalamica (ZLI). Furthermore, the expression of an early marker of posterior forebrain development that marks the compartment from the midbrain-hindbrain junction to the ZLI (including the future dorsal thalamus, pretectum, and midbrain) was disrupted, supporting the idea that diencephalic development is abnormal from very early in embryogenesis. This study provides compelling in vivo evidence that thalamic development requires normal activity of the LRP6-mediated canonical Wnt signaling pathway.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Receptores de LDL/fisiología , Tálamo/embriología , Animales , Proteínas del Citoesqueleto/fisiología , Diencéfalo/anomalías , Diencéfalo/embriología , Edad Gestacional , Proteínas Hedgehog , Proteínas Relacionadas con Receptor de LDL , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Noqueados , Morfogénesis , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/fisiología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal/fisiología , Núcleos Talámicos/anomalías , Núcleos Talámicos/embriología , Tálamo/anomalías , Transactivadores/análisis , Transactivadores/deficiencia , Transactivadores/fisiología , Proteínas Wnt , Proteína Wnt-5a , beta CateninaRESUMEN
BACKGROUND: The ubiquitin-proteasome pathway is important in regulating protein signaling pathways that are involved in tumorigenesis. beta-transducin repeat-containing proteins (beta-TrCP) are components of the ubiquitin ligase complex targeting beta-catenin and IkappaBalpha for proteasomal degradation and are thus a negative regulator of Wnt/beta-catenin signaling and a positive regulator of NF-kappaB signaling. We analyzed expression of beta-TrCP in colorectal cancers and its association with types of beta-catenin subcellular localization, an indirect measure of activation. METHODS: Levels of beta-TrCP1 mRNA and protein were measured by quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively, in samples of tumor and normal tissues from 45 patients with colorectal cancer. Types of beta-catenin activation (diffuse or invasion edge) and NF-kappaB activation were examined by immunohistochemistry. Apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) assay. All statistical tests were two-sided. RESULTS: Compared with the beta-TrCP1 levels in normal tissues, 25 (56%) of 45 tumors had increased beta-TrCP1 mRNA and protein levels. Of the 22 (49%) tumors with beta-catenin activation, 12 had the diffuse type (i.e., nuclear accumulation throughout the tumor) and 10 had the invasion edge type (i.e., nuclear accumulation predominantly in the tumor cells that formed the invasion edge). Increased beta-TrCP1 levels were statistically significantly associated with beta-catenin activation (P =.023) and decreased apoptosis (P =.035). beta-TrCP accumulated in the nuclei of tumor cells that contained increased levels of beta-TrCP1 mRNA and the active form of NF-kappaB. Higher levels of beta-TrCP1 mRNA were detected in primary tumors of patients who had metastases (0.960 arbitrary units, 95% confidence interval = 0.878 to 1.042) than in the tumors of patients who did not (0.722 arbitrary units, 95% confidence interval = 0.600 to 0.844; P =.016). CONCLUSION: In colorectal cancer, increased expression of beta-TrCP1 is associated with activation of both beta-catenin and NF-kappaB, suggesting that the integration of these signaling pathways by increased beta-TrCP expression may contribute to an inhibition of apoptosis and tumor metastasis.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas del Citoesqueleto/metabolismo , FN-kappa B/metabolismo , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Adenocarcinoma/química , Adenocarcinoma/patología , Anciano , Anciano de 80 o más Años , Apoptosis , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , Proteínas del Citoesqueleto/análisis , ADN Complementario/análisis , ADN de Neoplasias/análisis , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Persona de Mediana Edad , FN-kappa B/análisis , Pruebas de Precipitina , ARN Mensajero/análisis , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/análisis , Ubiquitina-Proteína Ligasas/análisis , beta Catenina , Proteínas con Repetición de beta-Transducina/análisis , Proteínas con Repetición de beta-Transducina/genéticaRESUMEN
PROBLEM: The inflammatory-anti-inflammatory cytokine network is thought to play a critical role in regulated progression and termination of pregnancy. The aim of this study was to evaluate the effects of interferon (IFN)-gamma on the expression of Cyclooxygenase (COX)-2 and production of prostaglandin E(2) (PGE(2)) in the human placenta from term and preterm labor deliveries. METHOD OF STUDY: Placental explant culture system was used. COX-2 expression was determined by complementary techniques of immunohistochemistry and Western blotting. Released IFN-gamma and PGE(2) by placental explants were measured by enzyme-linked immunosorbent assay. Signal transducer and activator of transcription 1 (STAT1) phosphorylation was evaluated by Western blotting using a specific antibody. RESULTS: IFN-gamma was poorly detected in the placenta but was significantly expressed in decidual tissues from both term and preterm pregnancies as detected by immunohistochemistry. IFN-gamma significantly inhibited COX-2 expression and PGE(2) release in cultured placental explants from term and preterm labor deliveries. This effect most likely occurred in a STAT1-dependent manner as this regulatory protein was phosphorylated in response to IFN-gamma. IFN-gamma receptor (IFN-gammaR) was expressed in normal early pregnancy placental samples. However, its expression was significantly reduced in placental samples from term and preterm deliveries. Of interest, IFN-gammaR was expressed in placentas from term and preterm labor deliveries after 24 hr in culture. CONCLUSIONS: Our data suggest that the human placenta is an important site for IFN-gamma-mediated repression of COX-2 expression and PGE2 production, implying that functional withdrawal of IFN-gamma may be involved in the onset of term or preterm labor.
Asunto(s)
Interferón gamma/fisiología , Isoenzimas/metabolismo , Placenta/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Adolescente , Adulto , Western Blotting , Ciclooxigenasa 2 , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Decidua/química , Dinoprostona/análisis , Dinoprostona/metabolismo , Femenino , Humanos , Inmunohistoquímica , Interferón gamma/análisis , Interferón gamma/farmacología , Isoenzimas/análisis , Trabajo de Parto , Proteínas de la Membrana , Trabajo de Parto Prematuro , Fosforilación , Placenta/química , Placenta/efectos de los fármacos , Embarazo , Primer Trimestre del Embarazo/metabolismo , Segundo Trimestre del Embarazo/metabolismo , Prostaglandina-Endoperóxido Sintasas/análisis , Receptores de Interferón/análisis , Receptores de Interferón/metabolismo , Factor de Transcripción STAT1 , Transactivadores/análisis , Transactivadores/metabolismo , Receptor de Interferón gammaRESUMEN
The epidermis of birds differs from that of mammals by the presence of intracellular lipid droplets and the absence of sebaceous glands. We describe here the cultivation of chicken epidermal keratinocytes; these cells cannot be grown in medium supplemented with the usual fetal bovine serum even in the presence of supporting 3T3 cells, but they can grow from single cells in the presence of supporting 3T3 cells and 10% chicken serum. As revealed by their cell structure, their protein composition, and their gene expression, chicken keratinocytes possess the general properties of mammalian keratinocytes. They ultimately undergo in culture a process of terminal differentiation in which their nucleus is destroyed and a cornified envelope is formed. Chicken keratinocytes also show important properties that mammalian keratinocytes do not possess: they accumulate neutral lipids, usually in the form of a single perinuclear droplet; they accumulate carotenoids; they synthesize beta-keratins; and their multiplication requires a non-lipid factor, present in chicken serum but not in fetal calf serum. The lipid-synthesizing function of sebocytes in mammals is carried out by the keratinocytes themselves in birds. The availability of cultured chicken keratinocytes should allow studies that were hitherto impossible such as the tracing of the keratinocyte lineage during development of the chicken embryo and the investigation of the complete life cycle of viruses that require specific chicken keratinocyte products (such as Marek's disease virus).
Asunto(s)
Técnicas de Cultivo de Célula , Pollos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinas/biosíntesis , Lípidos/biosíntesis , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Células Cultivadas , Pollos/metabolismo , Proteínas de Unión al ADN , Evolución Molecular , Genes Supresores de Tumor , Queratinocitos/ultraestructura , Queratinas/análisis , Queratinas/genética , Lípidos/análisis , Datos de Secuencia Molecular , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Alineación de Secuencia , Transactivadores/análisis , Transactivadores/metabolismo , Factores de Transcripción , Proteínas Supresoras de TumorRESUMEN
The regulation of TRH gene expression in the paraventricular nucleus of the hypothalamus (PVH) by leptin is critical for normal function of the thyroid axis in rodents and humans. The TRH neuron in the PVH expresses both leptin and melanocortin-4 receptors, suggesting that both signaling systems may regulate TRH gene expression in vivo. Indeed, the TRH promoter responds to both of these signaling pathways in cell culture through identified cis-acting elements, which include signal transducer and activator of transcription (STAT) 3 and cAMP-response element binding protein binding sites that mediate leptin and melanocortin responses, respectively. To determine whether leptin signaling can directly target the TRH promoter in vivo, we developed a chromatin immunoprecipitation assay to use on leptin-treated animals. After a single injection of leptin in fasting animals, we could detect a significant increase in TRH gene expression in the PVH that correlated well with the induction of phosphorylated-STAT3 in the hypothalamus. Furthermore, using a STAT3 antibody, we could immunoprecipitate the STAT-binding site containing regions of both the TRH promoter and the promoter of the suppressor of cytokine signaling-3 gene, another well-defined target of leptin action. In contrast, upstream regions of these promoters that lack STAT sites were not precipitated. Taken together these experiments demonstrate that STAT3 mediates transcriptional effects of leptin in vivo and that the TRH promoter is a likely direct site of leptin action. In addition, these experiments demonstrate that chromatin immunoprecipitation can be used to characterize leptin-signaling in vivo.
Asunto(s)
Leptina/farmacología , Regiones Promotoras Genéticas/genética , Hormona Liberadora de Tirotropina/genética , Animales , Cromatina , ADN/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipotálamo/química , Técnicas de Inmunoadsorción , Ratones , Ratones Obesos , Obesidad/genética , Obesidad/metabolismo , Núcleo Hipotalámico Paraventricular/química , Fosforilación , Proteínas Represoras/genética , Factor de Transcripción STAT3 , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/análisis , Transactivadores/metabolismo , Factores de Transcripción/genéticaRESUMEN
Chemopreventive activity by retinoic acid (RA) has been demonstrated previously in rat colon. The spontaneous tumourigenesis in the Min/+ mouse, which harbours a germline mutation in the tumour suppressor gene adenomatous polyposis coli (Apc), is characterized by inactivation of Apc, nuclear accumulation of beta-catenin and the enhanced expression of specific genes activated by T cell factor (TCF)/beta-catenin signalling. Recently it was reported that beta-catenin interacts with retinoic acid receptor in a retinoid-dependent manner, reducing beta-catenin/TCF regulated transcription. Our hypothesis was therefore that dietary supplementation with all-trans RA may inhibit the Apc-driven tumourigenesis in Min/+ mice. Surprisingly, in two different experiments the results showed that dietary RA significantly stimulated both the formation and growth of small intestinal tumours. In the first experiment Min/+ mice were exposed to 50 mg 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine/kg bodyweight at day 3-6 after birth and then treated with 50 mg/kg dietary RA in 1-3 weeks from the age of 2 weeks. In the second experiment the mice were not treated with carcinogen, and the diet was supplemented with 5 or 10 mg/kg RA from the age of 4 weeks until termination of the experiment at 11 weeks. Immunohistochemical studies revealed no differences in beta-catenin, cyclin D1 or proliferating cell nuclear antigen staining following RA treatment. There was no intestinal toxicity in mice fed 10 mg/kg RA, indicating that the increased tumourigenesis in Min/+ mice is a specific effect of all-trans RA.
Asunto(s)
Genes APC , Mutación de Línea Germinal , Neoplasias Intestinales/inducido químicamente , Tretinoina/toxicidad , Animales , Peso Corporal , Ciclina D1/análisis , Proteínas del Citoesqueleto/análisis , Suplementos Dietéticos , Femenino , Imidazoles/toxicidad , Neoplasias Intestinales/química , Neoplasias Intestinales/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Antígeno Nuclear de Célula en Proliferación/análisis , Transactivadores/análisis , beta CateninaRESUMEN
Similar to leptin, ciliary neurotrophic factor (CNTF) suppresses appetite and selectively reduces body fat in leptin-deficient ob/ob mice. To assess the relative importance of specific regions of the hypothalamus in mediating these effects, we administered a CNTF analogue (CNTFAx15) or leptin to mice made obese by administration of gold thioglucose (GTG), which destroys a well-defined portion of the medial basal hypothalamus. CNTFAx15 treatment reduced appetite and body weight in obese GTG-lesioned C57BL/6 mice, whereas leptin failed to effect similar changes regardless of whether treatment was initiated before or after the lesioned mice had become obese. Because leptin does not reduce food intake or body weight in most forms of obesity (a condition termed 'leptin resistance'), we also investigated the actions of leptin in GTG-lesioned leptin-deficient (ob/ob) mice. By contrast to C57BL/6 mice, leptin treatment reduced food intake and body weight in GTG-lesioned ob/ob mice, although the effect was attenuated. To further compare the neural substrates mediating the anorectic actions of leptin and CNTF, we determined the patterns of neurone activation induced by these proteins in the hypothalamus of intact and GTG-lesioned mice by staining for phosphorylated signal transducer and activator of transcription 3 (pSTAT3). CNTFAx15 stimulated robust pSTAT3 signalling in neurones of the medial arcuate nucleus in both intact and lesioned C57BL/6 and ob/ob mice. Leptin administration stimulated pSTAT3 signalling in only a few neurones of the medial arcuate nucleus in intact or lesioned C57BL/6 mice, but elicited a robust response in intact or lesioned ob/ob mice. By contrast to CNTFAx15, leptin treatment also resulted in prominent activation of STAT3 in several areas of the hypothalamus outside the medial arcuate nucleus. This leptin-induced pSTAT3 signal was at least as prominent in intact and GTG-lesioned C57BL/6 mice as it was in ob/ob mice, and thus was not correlated with appetite suppression or weight loss. These results indicate that the medial arcuate nucleus is a key mediator of appetite suppression and weight loss produced by CNTF and leptin, whereas GTG-vulnerable regions play a role only in leptin-induced weight loss. Other regions of hypothalamus in which pSTAT3 signal is induced by leptin may regulate energy metabolism through mechanisms other than appetite reduction.
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Apetito/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/fisiología , Aurotioglucosa , Factor Neurotrófico Ciliar/análogos & derivados , Leptina/administración & dosificación , Animales , Peso Corporal/efectos de los fármacos , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/fisiología , Resistencia a Medicamentos , Ingestión de Alimentos , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Neuronas/química , Neuronas/fisiología , Obesidad/etiología , Obesidad/fisiopatología , Fosforilación , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/análisis , Transactivadores/fisiología , Aumento de Peso/efectos de los fármacosRESUMEN
BACKGROUND: Germinated barley foodstuff (GBF), which contains glutamine-rich protein and hemicellulose-rich fiber, exhibits therapeutic effects in ulcerative colitis; however, its mechanism is still under investigation. The aim of this study was to evaluate the anti-inflammatory effects of GBF on colitis in terms of the epithelial inflammatory response. METHODS: Mice with dextran sulfate sodium-induced colitis were used. The effects of GBF on the colitis were evaluated by measuring the body weight; disease activity; mucosal damage (histology, mucosal inflammatory parameters, nuclear factor kappa B [NFkB] activation, and signal transducer and activator of transcription 3 [STAT3]); serum interleukin 6 (IL-6) level; cecal short-chain fatty acids (SCFAs); and bile acid contents. RESULTS: GBF significantly prevented disease activity and body weight loss after induction of colitis. Serum IL-6 level and mucosal STAT3 expression were also significantly attenuated, with a conspicuous reduction of mucosal damage; NFkB activity showed the same tendency. Cecal butyrate content was significantly higher and, interestingly, GBF mice had lower bile acid concentrations than the control group. CONCLUSIONS: GBF has the potential to reduce the epithelial inflammatory response by depressing STAT-3 expression and inhibiting NFkB binding activity. These effects may be brought about by an increase of butyrate production and adsorption of bile acids.
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Colitis/dietoterapia , Hordeum , Fitoterapia/métodos , Preparaciones de Plantas/uso terapéutico , Animales , Butiratos/metabolismo , Colitis/patología , Proteínas de Unión al ADN/análisis , Femenino , Germinación , Interleucina-6/sangre , Mucosa Intestinal/química , Intestino Delgado/microbiología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/análisis , Factor de Transcripción STAT3 , Transactivadores/análisisRESUMEN
In songbirds, the initiation of song behaviour and the neural substrate of this system are highly influenced by gonadal steroids. Receptors for gonadal steroid hormones, such as androgens and oestrogens, have been localized within select nuclei of the song system. An important step in steroid receptor action is the recruitment of nuclear receptor coactivators. The coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), has been implicated in both androgen and oestrogen receptor transactivation. Although the role of CBP in transcriptional mechanisms has been widely studied, little is known about CBP expression in the brain. The association between the distribution of CBP and oestrogen receptors in the hippocampus has been related to long-term memory. However, the distribution of brain CBP has not been related to the expression of gonadal steroid receptors in a system as relevant to reproductive behaviour as the avian song system. Western immunoblotting of European starling (Sturnus vulgaris) brain tissue reveals a band at 265 kDa. Immunohistochemical localization of CBP in starling brain indicates wide, but heterogeneous expression. CBP-immunoreactive (CBP-ir) cells define the boundaries of song control nuclei. In HVc (sometimes called the High Vocal Center) and the robust nucleus of the archistriatum (RA), there is a higher density of CBP-ir cells within the boundaries of these nuclei than in adjacent neostriatum or archistriatum, for HVc and RA, respectively. We also report that the distribution of CBP-ir cells varies among different nuclei within the song control system. CBP-ir cells within area X (also a part of the song system) and HVc are densely packed into clusters, whereas cells can be easily discriminated in RA. CBP is also highly expressed in hypothalamic areas, indicating that areas rich in steroid receptors also contain CBP. These data suggest that CBP is important for modulating transcriptional activities in the song system and other sites in the songbird brain that express gonadal steroid receptors.
Asunto(s)
Hipotálamo/química , Hipotálamo/fisiología , Proteínas Nucleares/análisis , Pájaros Cantores/fisiología , Transactivadores/análisis , Vocalización Animal/fisiología , Factores de Edad , Animales , Western Blotting , Proteína de Unión a CREB , Inmunohistoquímica , Masculino , Núcleo Hipotalámico Paraventricular/química , Núcleo Hipotalámico Paraventricular/fisiología , Área Preóptica/química , Área Preóptica/fisiología , Núcleo Hipotalámico Ventromedial/química , Núcleo Hipotalámico Ventromedial/fisiologíaRESUMEN
The basal cell-specific cytokeratin antibody (34betaE12) is widely used to aid in the diagnosis of cancer in challenging prostate needle biopsies (NBX) and transurethral resections of the prostate (TURP). Because prostate carcinoma (PCa) lacks basal cells, the absence of basal cell as determined by 34betaE12 can aid in the confirmation of a histologically suspicious lesion. However, false-negative staining occurs because of patchy cytoplasmic staining, making a definitive diagnosis difficult. A recently identified basal cell marker p63, a p53 homologue, stains basal cell nuclei but not secretory cells. The aim of this study is to determine if the p63 antibody offers any clinically useful advantage over 34betaE12 in the diagnosis of challenging atypical prostate lesions. Ninety-four cases, comprised of 25 consecutive prostate NBX and 2 TURP with an atypical suspicious focus, 55 NBX cases of histologically unequivocal PCa and 12 TURP specimen removed for benign prostate hyperplasia, were stained with the monoclonal antibodies 34betaE12 and 4A4 anti-p63. Basal cell staining intensity, percentage basal cell-positive glands in benign, malignant, and atypical foci, and number of benign glands not staining were evaluated for 34betaE12 and p63 stains. A total of 67 prostate NBX cases, including one TURP, were diagnosed with PCa, 1 atypical small acinar proliferation, 10 benign, and 4 cases excluded because of lost tissue on step sections. None of the 67 PCa NBX cases demonstrated 34betaE12 or p63 immunoreactivity (100% specific). Whereas 57 of 108 (53%) prostate NBX cores from 78 cases demonstrated a similar percentage of basal cell staining for both antibodies, 45 of 108 (41%) NBX cores demonstrated a higher percentage of p63 basal cell staining in benign glands. Only 6 of 108 NBX (6%) cores had a higher percentage of basal cell staining with 34betaE12 (Wilcoxon signed rank test, p <0.0001). Lack of basal cell staining in more than two benign glands occurred in 25 of 108 (23%) and 10 of 108 (9%) prostate NBX cores stained with 34betaE12 and p63, respectively. In the vast majority of atypical cases, both 34betaE12 and p63 staining differences were not clinically significant, except in 2 of 27 (7%) cases p63 offered diagnostic utility beyond the 34betaE12 immunostain. p63 in these cases demonstrated discontinuous but strong staining in atypical glands and adjacent benign glands, whereas 34betaE12 failed to stain optimally in this critical area. For 12 TURP cases the mean percentage basal cell positivity in benign glands was 75% and 95% for 34betaE12 and p63, respectively (p = 0.006). Lack of basal cell staining in more than two glands occurred in 12 of 12 (100%) and 2 of 12 (17%) TURP specimens stained with 34betaE12 and p63, respectively (p <0.0001). In summary, 34betaE12 and p63 are highly specific for basal cells and therefore are negative in areas of PCa. p63 is more sensitive than 34betaE12 in staining benign basal cells, particularly for TURP specimens, offering slight advantage over 34betaE12 in diagnostically challenging cases. p63 may be used as an alternative to 34betaE12 stain for difficult prostate lesions.
Asunto(s)
Adenocarcinoma/patología , Biomarcadores de Tumor , Queratinas , Proteínas de la Membrana , Fosfoproteínas , Neoplasias de la Próstata/patología , Transactivadores , Adenocarcinoma/química , Adenocarcinoma/cirugía , Biomarcadores de Tumor/análisis , Biopsia con Aguja , Proteínas de Unión al ADN , Técnica del Anticuerpo Fluorescente Indirecta , Genes Supresores de Tumor , Humanos , Técnicas para Inmunoenzimas , Queratinas/análisis , Masculino , Peso Molecular , Fosfoproteínas/análisis , Próstata/química , Próstata/patología , Próstata/cirugía , Neoplasias de la Próstata/química , Neoplasias de la Próstata/cirugía , Reproducibilidad de los Resultados , Coloración y Etiquetado , Transactivadores/análisis , Factores de Transcripción , Resección Transuretral de la Próstata , Proteínas Supresoras de TumorRESUMEN
We have previously reported that a specialized subpopulation of astrocytes in the arcuate nucleus of the hypothalamus show an unusually intense immunoreactivity for brain fatty acid binding protein (bFABP). Since bFABP has been shown to regulate the activity of an enzyme, fatty acid synthase, that has a potent influence upon the regulation of feeding by the hypothalamus, it was of interest to determine if bFABP + astrocytes are positioned to potentially influence the activity of feeding-regulating neurones. In this study, we examined the anatomical relationship between specialized arcuate astrocytes immunoreactive for bFABP and feeding-regulating neurones that are responsive to leptin and which are immunoreactive for the transcription factor STAT3. The results show that both cell types are abundant in the arcuate nucleus of the hypothalamus and are frequently closely adjacent to each other. This study provides an anatomical basis for the possibility that specialized arcuate astrocytes regulate the function of leptin-sensitive, feeding-regulating neurones of the arcuate nucleus.