RESUMEN
DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.
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Cilios , ARN Helicasas DEAD-box , Modelos Animales de Enfermedad , Neoplasias Hepáticas , Ribonucleasa III , Animales , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/deficiencia , Cilios/patología , Cilios/metabolismo , Ratones , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Mutación , Hígado/patología , Hígado/metabolismo , Conductos Biliares/patologíaRESUMEN
Triple negative breast cancer (TNBC) is responsible for significant number of breast cancer-associated deaths because of lacking of successful molecular-targeted therapy. To explore a therapeutic target for TNBC, we performed a siRNA-mediated knockdown screening and identified Polo-like kinase 1 (PLK1) as a potential therapeutic target for TNBC. Knockdown of PLK1 as well as a small compound inhibitor for PLK1, BI-2536, induced G2/M arrest and created polyploid cell population, shown by increased DNA content and nuclear size. Inhibition of PLK1 eventually triggered apoptosis in multiple TNBC cell lines. In addition, we confirmed that PLK1 was significantly overexpressed in the tissues from TNBC patients compared with the tissues of normal mammary glands and benign breast tumors. Our data indicated that PLK1 plays a pivotal role in the regulation of mitosis of TNBC cells. Although future in vivo studies are warranted, targeting PLK1 by a selective inhibitor such as BI-2536 can be an attractive molecular-targeted therapy for TNBC.
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Proteínas de Ciclo Celular , Terapia Molecular Dirigida , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , Neoplasias de la Mama Triple Negativas/metabolismo , Mama/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen/métodos , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , Quinasa Tipo Polo 1RESUMEN
Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.
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Neoplasias de la Mama/terapia , Receptores ErbB/genética , Exosomas/metabolismo , MicroARNs/administración & dosificación , Animales , Secuencia de Bases , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Femenino , Humanos , Ratones , Microscopía Inmunoelectrónica , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone ß subunit gene (Fshß) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LßT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LßT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshß, it is possible that LHX2 controls the synthesis of FSH at the transcription level.
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Hormona Folículo Estimulante de Subunidad beta/genética , Regulación de la Expresión Génica , Proteínas con Homeodominio LIM/metabolismo , Porcinos/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Células CHO , Línea Celular Tumoral , Clonación Molecular , Cricetinae , Hormona Folículo Estimulante de Subunidad beta/biosíntesis , Proteínas con Homeodominio LIM/genética , Datos de Secuencia Molecular , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Regarding the process of uveitis development, many past studies have used the experimental autoimmune uveoretinitis (EAU) and other animal models to observe histologically the infiltration of inflammatory cells and the process of lesion progression. However, no detailed study of the process of clearance of infiltrated inflammatory cells from the eye has been reported. The purpose of this study was to investigate the process of clearance of polymorphonuclear leukocytes (PMNs) using an experimental hypopyon model. PMNs obtained from ascites of SD rat were injected into the anterior chamber of SD rats. The process of PMNs clearance was evaluated by serial photography and 3D optical coherence tomography (3D-OCT), and histological changes were observed simultaneously. The hypopyon heights regressed from 1.04±0.06 mm at 1h (day 0) to 0.45±0.07 mm at day 1, and 0 mm at day 3 after PMNs injection. When the hypopyon heights at the three time points were compared, significant differences were found between groups (P<0.05). The hypopyon volumes also decreased from 1.46±0.07 mm(3) at 1h to 1.16±0.09 mm(3) at 2h, and 0.83±0.04 mm(3) at 3h after PMN injection. When the hypopyon volumes at the three time points were compared, significant differences were found between groups (P<0.05). Light micrographs of inferior segment of the eyeball revealed dense PMNs in the chamber angle at 1h after PMNs injection and many PMNs in the iris stroma and vessels, as well as at the episcleral and subconjunctival tissues around limbus at 3h and day 1 after PMNs injection. Light micrographs of superior segment of the eyeball at 3h after injection revealed PMNs in the episcleral and subconjunctival vessels. Electron micrographs of inferior segment of the eyeball at 3h after PMNs injection revealed dense PMNs with slightly condensed nuclei in the anterior chamber, as well as in the iris stroma and vessels. In conclusion, in the experimental hypopyon model, PMNs injected into the anterior chamber were cleared from the eye mainly through the iris stroma and vessels, as well as the episcleral and subconjunctival tissues around limbus.
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Cámara Anterior/metabolismo , Modelos Animales de Enfermedad , Neutrófilos/metabolismo , Uveítis Supurativa/metabolismo , Animales , Cámara Anterior/patología , Imagenología Tridimensional , Cinética , Neutrófilos/patología , Ratas , Ratas Sprague-Dawley , Tomografía de Coherencia Óptica , Uveítis Supurativa/patologíaRESUMEN
INTRODUCTION: Mesenchymal stem cells (MSCs) are promising therapeutic tools in regenerative medicine. In particularly adipose tissue derived MSC (AMSC) has powerful potential for the therapeutics of rheumatoid arthritis (RA) because these cells can control immune balance. RA systemically occurs autoimmune disease. Interestingly, IL-1 receptor antagonist deficient (IL-1ra-/-) mice induce inflammation in joints like RA. In RA therapy, although AMSC improves the inflammation activity, it is little known to play roles of extracellular microvesicles (EV) for improvement of RA. To clarify the MSC-derived EVs are involved amelioration mechanisms for RA by themselves, we examined the functional effects of development for RA by AMSC-EVs. METHODS: We isolated AMSCs derived mice adipose tissue and purified EVs from the culture supernatant of AMSCs. To examine whether EVs can improve RA, we administrated EVs or AMSCs to IL-1ra knockout mice as RA model mice. We analyzed EVs-included factor by western blot methods and RA improvement effect by ELISA. RESULTS: In this study, we showed that the swellings of joints on mice in wild type AMSC and that in AMSC-EVs decreased than that in IL-1ra-/- mice-AMSC-EVs and in none-treated. We detected IL-1ra expression in AMSC-EVs in wild type mice but not that in IL-1ra-/- mice. Proinflammatory cytokine expression changes in mice showed in AMSCs and AMSC-EVs, but no apparent differences cytokine expressions were detected in IL-1ra-/- mice. CONCLUSIONS: In this study, we concluded that MSCs might improve RA by the transferring of factors such as IL-1ra, which are included their MSC derived- EVs.
RESUMEN
Mixtures of Pt clusters dispersed on gamma-Al(2)O(3) and additional gamma-Al(2)O(3) led to much higher DME combustion turnover rates than on the individual components or on Pt clusters supported on non-acidic oxides.
RESUMEN
The fusion oncoproteins, TLS-CHOP and EWS-CHOP, are characteristic markers for myxoid and round cell liposarcomas (MLS/RCLS). Especially, the peptide sequence of 26 amino acids corresponding to the normally untranslated CHOP exon 2 and parts of exon 3 (5'-UTR) is a unique structure for these chimeric proteins. In this report, we have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. These antibodies reacted with TLS-CHOP fusion protein, but not reacted with normal TLS and CHOP proteins by Western blot analysis. In addition, one of the antibodies also recognized the chimeric oncoprotein in archival paraffin-embedded tissue samples of MLS/RCLS. The oncoprotein was detectable by the antibody even in the paraffin-embedded tissue samples whose mRNAs were too degraded to be detected by a nested reverse transcription-polymerase chain reaction-based assay. Thus, the molecular assay using the novel antibody is expected to be one of the most sensitive diagnostic assays for MLS/RCLS.
Asunto(s)
Anticuerpos Monoclonales , Liposarcoma/diagnóstico , Proteínas de Fusión Oncogénica/inmunología , Proteína EWS de Unión a ARN/inmunología , Proteína FUS de Unión a ARN/inmunología , Factor de Transcripción CHOP/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Western Blotting , Niño , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Adhesión en Parafina , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Methane is shown to react with ethene over In-loaded ZSM-5 to higher hydrocarbons such as propene and toluene at around 673 K. Such methane conversion is not catalyzed by proton-exchanged ZSM-5 (H-ZSM-5) under the same conditions, only C2H4 being converted to higher hydrocarbons. By using 13C-labeled methane (13CH4) as a reactant, the reaction paths for the formation of propene, benzene and toluene were examined. 13C-labeled propene (13CC2H6) is formed by the reaction of 13CH4 with C2H4. The lack of 13C-labeled benzene revealed that propene is not transformed to benzene, which instead originates entirely from C2H4. The 13C atom is inserted both into the methyl group and benzene ring in the toluene formed. This indicates that toluene is formed by two reaction paths; the reaction of 13CC2H6 with butenes formed by the dimerization of C2H4 and the reaction of benzene with 13CH4. The existence of the latter path was proved by the direct reaction of 13CH4 with benzene. The reaction of methane with benzene was also carried out in a continuous flow system over In-loaded ZSM-5. The reaction afforded 7.6% and 0.9% yields of toluene and xylenes, respectively, at 623 K.
RESUMEN
RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal of evidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents. However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression of angiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing through RNAi without activating TLRs or RIG-I-like receptor signaling. In addition, we confirmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration (AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies against blinding choroidal neovascularization due to AMD.
RESUMEN
A variety of cells release membrane vesicles, such as exosomes and microvesicles (MVs), that are thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents throughout the body. There have been considerable efforts to use MVs as diagnostic or prognostic markers because their composition is reflective of minor physiological changes. Furthermore, recent studies demonstrate that MVs derived from infected cells and tumors contribute to disease pathogenesis. This review presents an overview of the potential roles of MVs with respect to clinical diagnosis and disease pathogenesis.
Asunto(s)
Comunicación Celular/fisiología , Membrana Celular/metabolismo , Exosomas/metabolismo , Presentación de Antígeno , Biomarcadores/metabolismo , Enfermedades Transmisibles/diagnóstico , Enfermedades Transmisibles/fisiopatología , Humanos , Neoplasias/diagnóstico , Neoplasias/patología , PronósticoRESUMEN
The development and differentiation of the pituitary gland progress through spatial and temporal expressions of many transcription factors. Transcription factor HESX1, which begins to be expressed in the Rathke's pouch at the early stage of pituitary development, acts as a transcription repressor. Another transcription factor, PROP1, which is a pituitary-specific factor and important for the determination of the differentiation of pituitary hormone-producing cells, appears later than HESX1 and is assumed to block the action of HESX1. Both factors are members of the homeodomain family, and the amino acid residue at the 50th position of the homeodomain is glutamine (Gln-50). We recently observed that both factors share the same target sequence through different binding profiles. Hence, using random oligonucleotides and an electrophoretic mobility-shift assay, we have examined the DNA-binding preference of HESX1 by a determination of its binding sequence. HESX1 binds as a monomer to a TAATT motif but not to a TAAT motif. In the presence of PROP1, HESX1 develops to bind to an inverted TAAT motif by forming a heterodimer. Thus, the formation of a heterodimer between HESX1 and PROP1 provides a condition in which, in the early pituitary primordium, HESX1 alters its repressive role to an active one by forming a heterodimer with newly appearing PROP1 so that PROP1 finally replaces HESX1 to advance to the middle stage of pituitary development.
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Secuencias de Aminoácidos , Proteínas de Homeodominio , Hipófisis , Estructura Cuaternaria de Proteína , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Hipófisis/anatomía & histología , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Represoras , Porcinos , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We cloned a porcine ortholog of homeodomain transcription factor Msx1 from the porcine pituitary cDNA library. The amino acid sequence of Msx1 shows high conservation among mammalian species. RT-PCR for porcine fetal and postnatal pituitaries showed that Msx1 is already expressed at early fetal day 40, decreases to a low level before birth and then remarkably decreases after birth. On the other hand, Msx1 expression was observed in all pituitary-derived cell line tested, with most in a gonadotrope lineage LbetaT4. Transfection assay demonstrated that Msx1 markedly repressed the basal Cga and Fshb gene expression, while Lhb expression was affected slightly. Taken together, Msx1 may play a role in repressing gene expression in the fetal and postnatal periods.
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Clonación Molecular , Factor de Transcripción MSX1/genética , Porcinos/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/aislamiento & purificación , Factor de Transcripción MSX1/aislamiento & purificación , Factor de Transcripción MSX1/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Hipófisis/embriología , Hipófisis/metabolismo , Homología de Secuencia de Aminoácido , Porcinos/embriologíaRESUMEN
This study aimed to identify protein(s) that bind(s) to the highly AT-rich sequence of porcine Fshb promoter region -852/-746 (named Fd2) by the Yeast One-Hybrid Cloning System and finally a paired related homeodomain transcription factor, Prx2, known as a key factor for skeletogenesis was cloned. RT-PCR analysis of fetal and postnatal porcine pituitaries demonstrated that Prx2 starts to be expressed at around fetal days 40-50 just before the beginning of Lhb-expression and that the level of Prx2 increases after birth. Immunohistochemical analysis of the prepubertal porcine pituitary revealed that some Prx2-positive cells overlap some Lh beta-positive cells. Transient transfection assay using non-pituitary CHO cells and pituitary tumor-derived LbetaT2 cells revealed that Prx2 plays a cell-type dependent role in modulation of the Fshb promoter, showing stimulation in CHO cells and repression in LbetaT2 cells via the regions of Fd2 and -596/-239. The binding ability of Prx2 to the regions of Fd2 and -596/-239 was confirmed by electrophoretic mobility shift assay. DNase I footprinting revealed that broad regions of Fd2 were bound by Prx2 and that -596/-239 contained seven Prx2-binding sites. The SELEX method using a random N15-mer oligonucleotide pool demonstrated that Prx2 monomer binds to a TAATT motif, which is present in Fd2 and -596/-239. However, the binding of Prx2 to TAATT with a single molecule and its inverted repeat with two molecules could not induce transcriptional activation, indicating that the Prx2-dependent transcriptional modulation demonstrated in cultured cells is not introduced by Prx2 alone. Thus, this study demonstrated for the first time that Prx2 is expressed in the pituitary gland and at least in a part of gonadotropes in which Prx2 may play a role in repression of the Fshb gene.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hipófisis/embriología , Hipófisis/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Clonación Molecular , Cricetinae , Cricetulus , Ensayo de Cambio de Movilidad Electroforética , Femenino , Hormona Folículo Estimulante/genética , Hormona Folículo Estimulante/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Embarazo , Regiones Promotoras Genéticas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The aim of this study was to characterize the promoter activity of the porcine pituitary glycoprotein hormone common alpha gene (Cga) promoter (-1059/+12) and the role of LIM homeodomain transcription factor Lhx3. A transfection assay using Chinese hamster ovary (CHO) cells showed that the -1059/-101 region of the Cga promoter definitely responds to Lhx3 and that the -1059/-240 region exhibits a high basal transcriptional level in a pituitary-derived cell line, LbetaT2. A DNA binding and DNase I footprinting assay demonstrated that Lhx3 has seven binding sites in the -1059/+12 region of Cga, including a pituitary glycoprotein hormone basal element (PGBE) known as a LIM homeodomain factor-binding site. A transfection assay of the sequence of Lhx3-binding sites fused with minimal promoter vector confirmed their Lhx3-dependent stimulations in LbetaT2 cells. RT-PCR analysis of porcine pituitary ontogeny demonstrated that porcine Lhx3 showed striking changes of expression in both sexes during the fetal period but a stable high level of expression after birth. Thus, the porcine Cga promoter is regulated by Lhx3 through seven sites in the distal and proximal regions.
Asunto(s)
Regulación de la Expresión Génica , Hormonas Glicoproteicas de Subunidad alfa/biosíntesis , Hormonas Glicoproteicas de Subunidad alfa/genética , Proteínas de Homeodominio/biosíntesis , Animales , Sitios de Unión , Células CHO , Línea Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Proteínas con Homeodominio LIM , Ratones , Estructura Terciaria de Proteína , Porcinos , Factores de TranscripciónRESUMEN
LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line L beta T2 was carried out using the pituitary alpha GSU (glycoprotein hormone alpha-subunit) promoter (-1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, alpha GSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT-PCR (reverse transcription-PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in L beta T4 and GH3 cells with only small amounts in L beta T2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT-PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on alpha GSU gene expression in the pituitary glands.
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Hormonas Glicoproteicas de Subunidad alfa/genética , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Hipófisis/metabolismo , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas con Dominio LIM , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Hipófisis/crecimiento & desarrollo , Hormonas Hipofisarias/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Sus scrofa , Factores de Transcripción/metabolismoRESUMEN
CArG binding factor A (CBF-A) is a transcription factor first isolated from mouse C2 myogenic cells. Several lines of evidence indicate that CBF-A is also present in the anterior pituitary lobe and participates in the process of development and cell transformation. This study was performed to clone porcine CBF-A and to investigate its roles in the porcine anterior pituitary lobe. The predicted amino acid sequence of porcine CBF-A showed a unique insertion of five TG-repeats in the N-terminal region in comparison with those of other mammals, whereas the other regions appeared to be mostly conserved including two RNA recognition motifs in the middle region. Investigation of the expression of CBF-A gene during porcine pituitary development by RT-PCR showed an exclusive and temporary decrease in expression level shortly after birth in both sexes that was gradually but insufficiently restored. The expression of fluorescence protein-fused CBF-A in CHO cells demonstrated that CBF-A is located in the nuclei. We examined whether CBF-A regulates the expression of pituitary hormone genes in CHO cells and found that CBF-A significantly stimulated the promoter activity of growth hormone and prolactin by about 2-fold but did not stimulate the LHbeta gene. The specific DNA binding ability of porcine CBF-A was examined using serial oligonucleotides, CArG box and CC(W)0-6GG (W=A or T). As a result, porcine CBF-A was shown to have a high binding affinity for double- and single-stranded CC(W)6GG but no affinity for the known sequences of the CBF-A-target genes. Accordingly, this study demonstrated that porcine CBF-A may play a role in regulating at least two pituitary hormone genes, GH and PRL.
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Clonación Molecular , Proteínas de Unión al ADN/genética , Expresión Génica , Adenohipófisis/metabolismo , Porcinos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/química , ADN/metabolismo , ADN Complementario/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Femenino , Regulación de la Expresión Génica , Hormona del Crecimiento/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Adenohipófisis/embriología , Adenohipófisis/crecimiento & desarrollo , Prolactina/genética , Homología de SecuenciaRESUMEN
The identity and reversibility of the elementary steps required for catalytic combustion of dimethyl ether (DME) on Pt clusters were determined by combining isotopic and kinetic analyses with density functional theory estimates of reaction energies and activation barriers to probe the lowest energy paths. Reaction rates are limited by C-H bond activation in DME molecules adsorbed on surfaces of Pt clusters containing chemisorbed oxygen atoms at near-saturation coverages. Reaction energies and activation barriers for C-H bond activation in DME to form methoxymethyl and hydroxyl surface intermediates show that this step is more favorable than the activation of C-O bonds to form two methoxides, consistent with measured rates and kinetic isotope effects. This kinetic preference is driven by the greater stability of the CH3OCH2* and OH* intermediates relative to chemisorbed methoxides. Experimental activation barriers on Pt clusters agree with density functional theory (DFT)-derived barriers on oxygen-covered Pt(111). Measured DME turnover rates increased with increasing DME pressure, but decreased as the O2 pressure increased, because vacancies (*) on Pt surfaces nearly saturated with chemisorbed oxygen are required for DME chemisorption. DFT calculations show that although these surface vacancies are required, higher oxygen coverages lead to lower C-H activation barriers, because the basicity of oxygen adatoms increases with coverage and they become more effective in hydrogen abstraction from DME. Water inhibits reaction rates via quasi-equilibrated adsorption on vacancy sites, consistent with DFT results indicating that water binds more strongly than DME on vacancies. These conclusions are consistent with the measured kinetic response of combustion rates to DME, O2, and H2O, with H/D kinetic isotope effects, and with the absence of isotopic scrambling in reactants containing isotopic mixtures of 18O2-16O2 or 12CH3O12CH3-13CH3O13CH3. Turnover rates increased with Pt cluster size, because small clusters, with more coordinatively unsaturated surface atoms, bind oxygen atoms more strongly than larger clusters and exhibit lower steady-state vacancy concentrations and a consequently smaller number of adsorbed DME intermediates involved in kinetically relevant steps. These effects of cluster size and metal-oxygen bond energies on reactivity are ubiquitous in oxidation reactions requiring vacancies on surfaces nearly saturated with intermediates derived from O2.
RESUMEN
We report a tumor in an 80-year-old man that was difficult to distinguish from other tumors, i.e., small cell carcinoma of the lung, PNET/Ewing tumor, malignant lymphoma, or malignant melanoma (amelanotic), and which was finally identified as cutaneous neuroendocrine carcinoma using immunohistochemical and ultrastructural methods. Autopsy did not show any tumors in the lungs, excluding the possibility of small cell carcinoma of the lung. Immunohistochemistry tests gave negative results for LCA, UCHL-1, CD3, and CD20, thereby excluding malignant lymphoma, and the negative results for S-100 protein and HMB-45 ruled out malignant melanoma. The possibility of PNET/Ewing sarcoma was also excluded because of negativity for CD99. In addition, the ultramicrostructure showed intercellular junctional complexes and neuroendocrine granules, indicating that the tumor had characteristics of both epithelial and neuroendocrine tissues. We therefore diagnosed the primary carcinoma of the skin as cutaneous neuroendocrine carcinoma.
Asunto(s)
Carcinoma Neuroendocrino/ultraestructura , Neoplasias Cutáneas/ultraestructura , Anciano de 80 o más Años , Carcinoma Neuroendocrino/diagnóstico , Diagnóstico Diferencial , Resultado Fatal , Humanos , Inmunohistoquímica , Masculino , Neoplasias Cutáneas/diagnósticoRESUMEN
A Ti-based oxysulfide, Sm(2)Ti(2)S(2)O(5), was studied as a visible light-driven photocatalyst. Under visible light (440 nm < or = lambda < or = 650 nm) irradiation, Sm(2)Ti(2)S(2)O(5) with a band gap of approximately 2 eV evolved H(2) or O(2) from aqueous solutions containing a sacrificial electron donor (Na(2)S-Na(2)SO(3) or methanol) or acceptor (Ag(+)) without any noticeable degradation. This oxysulfide is, therefore, a stable photocatalyst with strong reduction and oxidation abilities under visible-light irradiation. The electronic band structure of Sm(2)Ti(2)S(2)O(5) was calculated using the plane-wave-based density functional theory (DFT) program. It was elucidated that the S3p orbitals constitute the upper part of the valence band and these orbitals make an essential contribution to the small band gap energy. The conduction and valence bands' positions of Sm(2)Ti(2)S(2)O(5) were also determined by electrochemical measurements. It indicated that conduction and valence bands were found to have satisfactory potentials for the reduction of H(+) to H(2) and the oxidation of H(2)O to O(2) at pH = 8. This is consistent with the results of the photocatalytic reactions.