RESUMEN
Eosinophilic gastroenteritis (EGE) is a rare disease which belongs to primary eosinophilic gastrointestinal disorders (primary EGIDs), characterized by an accumulation of eosinophils in the gastrointestinal (GI) tract and is strongly associated with atopy and allergy. The clinical presentations vary depending on the site and depth of eosinophilic intestinal infiltration. Radiology pictures may show irregular thickening of the folds, but these findings can also be present in other conditions like inflammatory bowel disease and lymphoma. The endoscopic appearance is also nonspecific. The definite diagnosis requires biopsy for histological evidence of GI eosinophilic infiltration and clinicians make the diagnosis in correlation with and by exclusion of other possible causes of eosinophilic infiltration. Because EGE is a rare disease, the treatment is based on limited case reports and clinicians' experience. Corticosteroids are the mainstay of therapy. The prognosis of EGE is relatively good when patients receive timely and proper treatment.
Asunto(s)
Corticoesteroides/uso terapéutico , Endoscopía del Sistema Digestivo , Enteritis/diagnóstico , Eosinofilia/diagnóstico , Gastritis/diagnóstico , Intestino Delgado/patología , Estómago/patología , Acetatos/uso terapéutico , Biopsia , Cromolin Sódico/uso terapéutico , Ciclopropanos , Enteritis/tratamiento farmacológico , Eosinofilia/tratamiento farmacológico , Gastritis/tratamiento farmacológico , Antagonistas de los Receptores Histamínicos H1/uso terapéutico , Humanos , Factores Inmunológicos/uso terapéutico , Inmunosupresores/uso terapéutico , Infliximab/uso terapéutico , Intestino Delgado/diagnóstico por imagen , Cetotifen/uso terapéutico , Antagonistas de Leucotrieno/uso terapéutico , Mercaptopurina/uso terapéutico , Quinolinas/uso terapéutico , Estómago/diagnóstico por imagen , Sulfuros , Tomografía Computarizada por Rayos XRESUMEN
Acute promyelocytic leukemia (APL) is predominantly characterized by chromosomal translocations between the retinoic acid receptor, alpha (RARA) gene and the promyelocytic leukemia (PML) or promyelocytic leukemia zinc finger (PLZF) gene. In APL cells with PML/RARA fusions, arsenic trioxide and all-trans retinoic acid treatments specifically target the fusion protein for proteasome-dependent degradation, thereby promoting cellular differentiation and clinical remission of disease. In contrast, APL cells expressing PLZF/RARA fusion proteins are largely resistant to similar treatments and prognosis for patients with this translocation is poor. Understanding the molecular mechanisms regulating PLZF/RARA protein stability would provide novel therapeutic targets for PLZF/RARA-associated APL. Toward this end, we have performed an RNAi-based screen to identify factors affecting PLZF/RARA stability. Among the factors identified was the ubiquitin-specific peptidase 37 (USP37). We showed that USP37 interacted with PLZF/RARA through the PLZF moiety and sustained PLZF/RARA steady state levels. Domain mapping study revealed that N-terminal domain of USP37 is required for the PLZF/RARA interaction and protein regulation. Furthermore, overexpression or depletion of USP37 caused an increase or decrease of PLZF/RARA protein half-life, correlating with down- or upregulation of PLZF/RARA poly-ubiquitination, respectively. By PLZF/RARA-transduced primary mouse hematopoietic progenitor cells, we demonstrated that Usp37 knockdown alleviated PLZF/RARA-mediated target gene suppression and cell transformation potential. Altogether, our findings of USP37-modulating PLZF/RARA stability and cell transformation suggest that USP37 is a potential therapeutic target for PLZF/RARA-associated APL.
Asunto(s)
Transformación Celular Neoplásica/genética , Endopeptidasas/genética , Leucemia Promielocítica Aguda/genética , Proteínas de Fusión Oncogénica/genética , Animales , Trióxido de Arsénico , Arsenicales , Diferenciación Celular/genética , Endopeptidasas/metabolismo , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Promielocítica Aguda/patología , Ratones , Proteínas de Fusión Oncogénica/metabolismo , Óxidos , Estabilidad Proteica , Translocación Genética/genéticaRESUMEN
Promyelocytic leukemia (PML) protein is a tumor suppressor with complicated action mechanisms not yet fully understood. In this study, we found that the nuclear factor of activated T cell (NFAT) is an unexpected partner of PML: PML specifically enhanced the transcription activation of NFAT. In PML-null mouse embryonic fibroblasts, no transcription activity of NFAT could be detected. There was a selective requirement of PML isoform in NFAT activation: PML-I and PML-VI, but not PML-IV, increased NFAT transactivation. PML specifically promoted the expression of many, but not all, NFAT-targeted genes. We found a specific binding of PML to NFATc. The interaction of PML with NFATc in vivo was further confirmed by chromatin immunoprecipitation and DNA affinity precipitation assay analysis. The unexpected coupling of PML with NFAT reveals a novel mechanism underlying the diverse physiological functions of PML.
Asunto(s)
Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Cloranfenicol O-Acetiltransferasa/metabolismo , Cromatina/genética , Cromatina/metabolismo , Humanos , Células Jurkat , Cinética , Leucemia Promielocítica Aguda/enzimología , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Factores de Transcripción/genética , Transcripción Genética , Activación Transcripcional , Proteínas Supresoras de Tumor/genéticaRESUMEN
SUMO (small ubiquitin-related modifier) modification is emerging as an important post-translational control in transcription. In general, SUMO modification is associated with transcriptional repression. Although many SUMO-modified transcription factors and co-activators have been identified, little is known about the mechanism underlying SUMOylation-elicited transcriptional repression. Here, we summarize that SUMO modification of transcription factors such as androgen receptor, glucocorticoid receptor, Smad4 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] co-activator results in the recruitment of a transcriptional co-repressor Daxx, thereby causing transcriptional repression. Such a SUMO-dependent recruitment of Daxx is mediated by the interaction between the SUMO moiety of SUMOylated factors and Daxx SUMO-interacting motif. Interestingly, the transrepression effect of Daxx on these SUMOylated transcription factors can be relieved by SUMOylated PML (promyelocytic leukaemia) via altering Daxx partition from the targeted gene promoter to PML nuclear bodies. Because Daxx SUMO-interacting motif is a common binding site for SUMOylated factors, a model of competition for Daxx recruitment between SUMOylated PML and SUMOylated transcription factors was proposed. Together, our findings strongly suggest that Daxx functions as a SUMO reader in the SUMO-dependent regulation of transcription and subnuclear compartmentalization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Transcripción Genética , Compartimento Celular , Proteínas Co-Represoras , Chaperonas Moleculares , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Transcripción Genética/fisiologíaRESUMEN
In this study, we report the cloning and characterization of a novel human Ste20-related kinase that we designated MST4. The 416 amino acid full-length MST4 contains an amino-terminal kinase domain, which is highly homologous to MST3 and SOK, and a unique carboxy-terminal domain. Northern blot analysis indicated that MST4 is highly expressed in placenta, thymus, and peripheral blood leukocytes. Wild-type but not kinase-dead MST4 can phosphorylate myelin basic protein in an in vitro kinase assay. MST4 specifically activates ERK but not JNK or p38 MAPK in transient transfected cells or in stable cell lines. Overexpression of dominant negative MEK1 or treatment with PD98059 abolishes MST4-induced ERK activity, whereas dominant-negative Ras or c-Raf-1 mutants failed to do so, indicating MST4 activates MEK1/ERK via a Ras/Raf-1 independent pathway. HeLa and Phoenix cell lines overexpressing wild-type, but not kinase-dead, MST4 exhibit increased growth rate and form aggressive soft-agar colonies. These phenotypes can be inhibited by PD98059. These results provide the first evidence that MST4 is biologically active in the activation of MEK/ERK pathway and in mediating cell growth and transformation.
Asunto(s)
Transformación Celular Neoplásica , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias/etiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Secuencia de Aminoácidos , División Celular , Clonación Molecular , Humanos , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias/patología , Filogenia , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Previously, we have demonstrated that oxidative stress or Ras/ERK activation leads to the transcriptional repression of alpha-subunit of epithelial Na(+) channel (ENaC) in lung and salivary epithelial cells. Here, we further investigated the coordinated molecular mechanisms by which alpha-ENaC expression is regulated. Using both stable and transient transfection assays, we demonstrate that the overexpression of high mobility group protein I-C (HMGI-C), a Ras/ERK-inducible HMG-I family member, represses glucocorticoid receptor (GR)/dexamethasone (Dex)-stimulated alpha-ENaC/reporter activity in salivary epithelial cells. Northern analyses further confirm that the expression of endogenous alpha-ENaC gene in salivary Pa-4 cells is suppressed by an ectopic HMGI-C overexpression. Through yeast two-hybrid screening and co-immunoprecipitation assays from eukaryotic cells, we also discovered the interaction between HMGI-C and PIAS3 (protein inhibitor of activated STAT3 (signal transducer and activator of transcription 3)). A low level of ectopically expressed PIAS3 cooperatively inhibits GR/Dex-dependent alpha-ENaC transcription in the presence of HMGI-C. Reciprocally, HMGI-C expression also coordinately enhances PIAS3-mediated repression of STAT3-dependent transactivation. Moreover, overexpression of antisense HMGI-C construct is capable of reversing the repression mediated by Ras V12 on GR- and STAT3-dependent transcriptional activation. Together, our results demonstrate that Ras/ERK-mediated induction of HMGI-C is required to effectively repress GR/Dex-stimulated transcription of alpha-ENaC gene and STAT3-mediated transactivation. These findings delineate a network of inhibitory signaling pathways that converge on HMGI-C.PIAS3 complex, causally associating Ras/ERK activation with the repression of both GR and STAT3 signaling pathways in salivary epithelial cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Células Epiteliales/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Glándulas Salivales/metabolismo , Canales de Sodio/metabolismo , Transactivadores/antagonistas & inhibidores , Transcripción Genética , Proteínas ras/metabolismo , Animales , Sitios de Unión , Northern Blotting , Western Blotting , Línea Celular , ADN Complementario/metabolismo , Dexametasona/farmacología , Regulación hacia Abajo , Canales Epiteliales de Sodio , Proteína HMGA2 , Modelos Biológicos , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Ratas , Receptores de Glucocorticoides/biosíntesis , Factor de Transcripción STAT3 , Transducción de Señal , Activación Transcripcional , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The Epstein-Barr virus-encoded early protein, BHRF1, is a structural and functional homologue of the anti-apoptotic protein, Bcl-2. There is accumulating evidence that BHRF1 protects a variety of cell types from apoptosis induced by various external stimuli. To identify specific proteins from normal epithelial cells that interact with BHRF1 and that might promote or inhibit its anti-apoptotic activity, we screened a yeast two-hybrid cDNA library derived from human normal foreskin keratinocytes and identified a cellular gene encoding human prenylated rab acceptor 1 (hPRA1). The interaction of hPRA1 with BHRF1 was confirmed using glutathione S-transferase pull-down assays, confocal laser scanning microscopy, and co-immunoprecipitation. Two regions of PRA1, amino acids 30-53 and the carboxyl-terminal 21 residues, are important for BHRF1 interactions and two regions of BHRF1, amino acids 1-18 and 89-142, including the Bcl-2 homology domains BH4 and BH1, respectively, are crucial for PRA1 interactions. PRA1 expression interferes with the anti-apoptotic activity of BHRF1, although not of Bcl-2. These results indicate that the PRA1 interacts selectively with BHRF1 to reduce its anti-apoptotic activity and might play a role in the impeding completion of virus maturation.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras , Proteínas Fúngicas/fisiología , Herpesvirus Humano 4/fisiología , Receptores de Superficie Celular , Proteínas Virales/fisiología , Línea Celular , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP , Humanos , Proteínas de la Membrana , Pruebas de Precipitina , Unión Proteica , Proteínas de Transporte Vesicular , Proteínas Virales/metabolismoRESUMEN
The optimal activation of cAMP-responsive element binding protein (CREB), similar to the full activation of T lymphocytes, requires the stimulation of both CD3 and CD28. Using a reporter system to detect interaction of CREB and CREB-binding protein (CBP), in this study we found that CREB binds to CBP only by engagement of both CD3 and CD28. CD3/CD28-promoted CREB-CBP interaction was dependent on p38 mitogen-activated protein kinase (MAPK) and calcium/calmodulin-dependent protein kinase (CaMK) IV in addition to the previously identified extracellular signal-regulated kinase pathway. Extracellular signal-regulated kinase, CaMKIV, and p38 MAPK were also the kinases involved in CREB Ser(133) phosphorylation induced by CD3/CD28. A reconstitution experiment illustrated that optimum CREB-CBP interaction and CREB trans-activation were attained when these three kinase pathways were simultaneously activated in T cells. Our results demonstrate that coordinated activation of different kinases leads to full activation of CREB. Notably, CD28 ligation activated p38 MAPK and CaMKIV, the kinases stimulated by CD3 engagement, suggesting that CD28 acts by increasing the activation extent of p38 MAPK and CaMKIV. These results support the model of a minimum activation threshold for CREB-CBP interaction that can be reached only when both CD3 and CD28 are stimulated.
Asunto(s)
Antígenos CD28/fisiología , Complejo CD3/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/inmunología , Transactivadores/metabolismo , Animales , Proteína de Unión a CREB , Calcio/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Activación Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Células Jurkat , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosforilación , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Linfocitos T/enzimología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Activación Transcripcional/inmunología , Transfección , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Etk, also named Bmx, is a member of the Tec tyrosine kinase family, which is characterized by a multimodular structure including a pleckstrin homology (PH) domain, an SH3 domain, an SH2 domain, and a catalytic domain. The signaling mechanisms regulating Etk kinase activity remain largely unknown. To identify factor(s) regulating Etk activity, we used the PH domain and a linker region of Etk as a bait for a yeast two-hybrid screen. Three independent clones encoding protein-tyrosine phosphatase D1 (PTPD1) fragments were isolated. The binding of PTPD1 to Etk is specific since PTPD1 cannot associate with either the Akt PH domain or lamin. In vitro and in vivo binding studies demonstrated that PTPD1 can interact with Etk and that residues 726-848 of PTPD1 are essential for this interaction. Deletion analysis of Etk indicated that the PH domain is essential for PTPD1 interaction. Furthermore, the Etk-PTPD1 interaction stimulated the kinase activity of Etk, resulting in an increased phosphotyrosine content in both factors. The Etk-PTPD1 interaction also increased Stat3 activation. The effect of PTPD1 on Etk activation is specific since PTPD1 cannot potentiate Jak2 activity upon Stat3 activation. In addition, Tec (but not Btk) kinase can also be activated by PTPD1. Taken together, these findings indicate that PTPD1 can selectively associate with and stimulate Tec family kinases and modulate Stat3 activation.
Asunto(s)
Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Animales , Células COS , Proteínas de Unión al ADN/fisiología , Humanos , Janus Quinasa 2 , Fosforilación , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas no Receptoras , Proteínas Tirosina Quinasas/fisiología , Factor de Transcripción STAT3 , Transactivadores/fisiología , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
Etk (also called Bmx) is a member of the Btk tyrosine kinase family and is expressed in a variety of hematopoietic, epithelial, and endothelial cells. We have explored biological functions, regulators, and effectors of Etk. Coexpression of v-Src and Etk led to a transphosphorylation on tyrosine 566 of Etk and subsequent autophosphorylation. These events correlated with a substantial increase in the kinase activity of Etk. STAT3, which was previously shown to be activated by Etk, associated with Etk in vivo. To investigate whether Etk could mediate v-Src-induced activation of STAT3 and cell transformation, we overexpressed a dominant-negative mutant of Etk in an immortalized, untransformed rat liver epithelial cell line, WB, which contains endogenous Etk. Dominant-negative inactivation of Etk not only blocked v-Src-induced tyrosine phosphorylation and activation of STAT3 but also caused a great reduction in the transforming activity of v-Src. In NIH3T3 cells, although Etk did not itself induce transformation, it effectively enhanced the transforming ability of a partially active c-Src mutant (c-Src378G). Furthermore, Etk activated STAT3-mediated gene expression in synergy with this Src mutant. Our findings thus indicate that Etk is a critical mediator of Src-induced cell transformation and STAT3 activation. The role of STAT3 in Etk-mediated transformation was also examined. Expression of Etk in a human hepatoma cell line Hep3B resulted in a significant increase in its transforming ability, and this effect was abrogated by dominant-negative inhibition of STAT3. These data strongly suggest that Etk links Src to STAT3 activation. Furthermore, Src-Etk-STAT3 is an important pathway in cellular transformation.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Unión al ADN/fisiología , Células Epiteliales/fisiología , Genes src/fisiología , Proteínas Tirosina Quinasas/fisiología , Transactivadores/fisiología , Animales , Células Epiteliales/patología , Humanos , Proteínas de la Membrana/fisiología , Ratas , Factor de Transcripción STAT3 , Transducción de Señal/fisiologíaRESUMEN
Etk/BMX is a non-receptor protein tyrosine kinase that requires a functional phosphatidylinositol 3-kinase via the pleckstrin homology domain to be activated by cytokine. In the present study, a conditionally active form of Etk was constructed by fusing the hormone-binding domain of estrogen receptor (ER) to an amino terminus truncated form of Etk, PHDelta1-68Etk, to generate DeltaEtk:ER. In stably transfected Pa-4DeltaEtk:ER cells, the activity of DeltaEtk:ER was stimulated within minutes by the treatment of DeltaEtk:ER stimulant, estradiol, and sustained for greater than 24 h. A robust induction in the phosphorylation of signal transducers and activators of transcription (STAT) proteins, including STAT1, STAT3, and STAT5, was accompanied with DeltaEtk:ER activation. Moreover, the conditionally activated Etk stimulated STAT1- and STAT5-dependent reporter activities by approximately 160- and approximately 15-fold, respectively, however, elicited only a modest STAT3-mediated reporter activation. Qualitatively comparable results were obtained in lung A549 cells, indicating that DeltaEtk:ER inducible system could function in an analogous fashion in different epithelial cells. Furthermore, we demonstrated that Etk activation alone augmented cyclin D1 promoter/enhancer activity via its STAT5 response element in both Pa-4DeltaEtk:ER and A549 cells. Altogether, these findings support the notion that the activation of Etk kinase is sufficient to transactivate STAT-mediated gene expression. Hence, our inducible DeltaEtk:ER system represents a novel approach to investigate the biochemical events following Etk activation and to evaluate the contribution by kinase activation of Etk alone or in conjunction with other signaling pathway(s) to the ultimate biological responses.
Asunto(s)
Pulmón/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Glándulas Salivales/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Activación Enzimática , Células Epiteliales/metabolismo , Pulmón/citología , Fosforilación , Proteínas Tirosina Quinasas/genética , Ratas , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/metabolismo , Glándulas Salivales/citología , Factores de Transcripción/metabolismo , Tirosina/metabolismoRESUMEN
The signals originating from transforming growth factor beta/activin/bone morphogenetic proteins (BMPs) are transduced by a set of evolutionarily conserved family of Smad proteins which, upon activation, directly translocate to the nucleus where they may activate transcription. Smad proteins of different species contain conserved amino- (N) and carboxy- (C) terminal domains separated by a proline-rich linker. Human, Drosophila, and Xenopus Smad1 all have been shown to mediate the biological effects of BMP-4 in Xenopus embryos. We have investigated the functional domains of human Smad1 (hSmad1) using the Xenopus embryo system. Dorsal injection of hSmad1 RNA into the 4-cell-stage embryos results in embryonic ventralization. Since the C-terminus of Smads has been shown to mediate the transcriptional activity, whereas this activity is masked by the presence of the N-terminus, we tested the effect of a hSmad1 construct lacking the C-terminal domain [hSmad1(N)] in the Xenopus embryo system. Surprisingly, we found that hSmad1(N) not only synergizes with hSmad1 in embryonic ventralization, but induces ventralization by itself. Ectopic expression of a dominant negative BMP receptor (DN-BR) as well as neural inducers noggin and chordin induce neurogenesis in the animal cap, which is inhibited by co-expression of either hSmad1 or hSmad1(N). Ventral expression of DN-BR induces formation of a second body axis at tailbud stage, which is also prevented by hSmad1 and hSmad1(N). It has recently been reported that calmodulin interacts with the N-terminal domain of Smad proteins. We demonstrate that the ventralizing activity of hSmad1 and hSmad1(N) is markedly inhibited by calmodulin. Thus, calmodulin acts as a Smad1 inhibitor. A model is proposed to accomodate these findings.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Animales , Tipificación del Cuerpo , Calmodulina/metabolismo , Proteínas de Unión al ADN/química , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Sistema Nervioso/embriología , Proteínas Smad , Proteína Smad1 , Transactivadores/química , Proteínas de Xenopus , Xenopus laevis/embriologíaRESUMEN
OBJECTIVE: To compare infrared thermometry with rectal thermometry as a method of assessing core body temperature in dogs and to assess the effect of otitis externa on external ear canal temperature (EECT). DESIGN: Prospective study. ANIMALS: 650 dogs without history or clinical signs of otitis externa and 85 dogs with recurrent or chronic otitis externa. PROCEDURE: Rectal temperature was measured, using a mercury thermometer. External ear canal temperature was measured, using an infrared tympanic thermometer. RESULTS: Measurements of body temperature at the 2 sites did not agree. Sensitivity and specificity of infrared thermometry in detecting fever, as determined by rectal thermometry, were 69.7 and 84.6%, respectively. Use of methods to predict rectal temperature from EECT did not improve the accuracy of infrared thermometry. Otitis externa significantly influenced EECT. CLINICAL IMPLICATIONS: Use of infrared thermometry as a replacement for rectal thermometry in assessing core body temperature in dogs was unsatisfactory. The 2 methods for measuring body temperature were not interchangeable in dogs.
Asunto(s)
Temperatura Corporal , Enfermedades de los Perros/metabolismo , Conducto Auditivo Externo/metabolismo , Otitis Externa/veterinaria , Termómetros/veterinaria , Animales , Perros , Femenino , Rayos Infrarrojos , Modelos Lineales , Masculino , Otitis Externa/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Termómetros/normasRESUMEN
Diets high in simple carbohydrates and low in fats lead in the mammalian liver to induction of a set of enzymes involved in lipogenesis. This induction occurs, in part, through transcriptional mechanisms that lead to elevated levels of the mRNA for these enzymes. For most of the lipogenic enzymes, an increase in glucose metabolism is required to trigger the transcriptional response. The intracellular mediator of this signaling pathway is unknown, although evidence suggests either glucose-6-phosphate or xylulose-5-phosphate. Studies to map the regulatory sequences of lipogenic enzyme genes involved in the transcriptional response have been performed for the L-type pyruvate kinase, S14, and acetyl-coenzyme A carboxylase genes. These studies have identified the DNA sequences necessary to link the signal generated by carbohydrate metabolism to specific nuclear transcription factors.
Asunto(s)
Carbohidratos de la Dieta/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Lípidos/biosíntesis , ATP Citrato (pro-S)-Liasa/genética , Acetil-CoA Carboxilasa/genética , Animales , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/metabolismo , Ácido Graso Sintasas/genética , Glucosa/metabolismo , Islotes Pancreáticos/fisiología , Transducción de SeñalRESUMEN
The Escherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selection. This assay, designated the "split-hybrid system," contains a two-component reporter. The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream of HIS3. Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression. TetR subsequently binds to the tet operators, blocking the expression of HIS3 and preventing yeast growth in media lacking histidine. The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133. Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains. The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP. These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction. The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein-protein interactions.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Nucleares/metabolismo , Mutación Puntual , Selección Genética , Transactivadores , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína de Unión a CREB , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca de Genes , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Fosforilación , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genéticaRESUMEN
A growth hormone (GH)-inducible nuclear factor (GHINF) from rat liver has been purified to near homogeneity. On SDS-polyacrylamide gel electrophoresis and UV-cross-linking, a major band of mass approximately 93 kDa and a minor band of approximately 70 kDa are detected in the purified fraction. DNase I footprinting using purified GHINF yields a protected region of -149/-115 on the rat serine protease inhibitor 2.1 (Spi 2.1) promoter encompassed within the growth hormone response element (GHRE). Mutational analysis demonstrated that GHINF binds synergistically to two gamma-interferon-activated sites (GAS) within the GHRE, with the 3' element being the pivotal binding domain. Functional assays show that both GAS elements are necessary for full GH response. GHINF has no immunoreactivity with either a C-terminal Stat1 antibody or an N-terminal Stat3 antibody, while cross-reacting with a C-terminal Stat5 monoclonal antibody. GHINF will bind to two GAS elements from the Stat5 binding region of the beta-casein gene. These studies indicate that GHINF is a Stat5-related factor binding synergistically to two GAS elements to activate Spi 2.1 transcription.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Hormona del Crecimiento/farmacología , Interferón gamma/farmacología , Proteínas de la Leche , Proteínas Nucleares/fisiología , Inhibidores de Serina Proteinasa/genética , Transactivadores/fisiología , Animales , Secuencia de Bases , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Mutación , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT1 , Factor de Transcripción STAT5 , Transcripción GenéticaRESUMEN
Regulatory sequences involved in the transcriptional induction of the rat S14 gene in response to increased glucose metabolism in the hepatocyte were investigated and compared with those of the liver-type pyruvate kinase (L-PK) gene. The carbohydrate response element (ChoRE) of the S14 gene was found to consist of two motifs related to the consensus binding site for the c-myc family of transcription factors, CACGTG. These two motifs are separated by five base pairs, a similar arrangement to that found in the L-PK ChoRE. In its natural context, the S14 ChoRE requires a novel accessory factor to support the full response glucose. This factor, as well as the factor hepatic nuclear factor-4, are both capable of binding to the L-PK gene to enhance its carbohydrate regulation. The need for an accessory factor for supporting the glucose response can be overcome in two ways. First, multimers of the ChoREs of either the L-PK or S14 genes can function independently to support the glucose response. Second, mutations in the S14 ChoRE that create a perfect match to the consensus CACGTG motif at each locus no longer require an accessory factor site. The spacing of the two CACGTG motifs, but not the nature of the bases within the spacer, are critical for control. These observations suggest that a carbohydrate responsive factor binds to both motifs in a highly specific spatial orientation to confer the response to increased carbohydrate metabolism.