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1.
Eur Radiol ; 26(1): 95-102, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25911616

RESUMEN

OBJECTIVE: The efficiency of endovascular liver gene transfer in pigs is evaluated by comparing two models of retrograde catheterization: single lobe catheterization with portal inflow (open procedure) versus whole liver isolation with portal and inferior vena cava blockage (close procedure). METHODS: Percutaneous endovascular catheterization was performed in pigs. Open procedure (n = 3): 8Fr balloon catheter placement in a suprahepatic branch through the jugular vein. Closed procedure (n = 3): simultaneous catheterization of the intrahepatic portal vein (transhepatic catheterization, 10Fr balloon catheter), the supra- and infrahepatic cava veins (8Fr balloon catheters through the jugular and femoral veins). In both models, 200 ml of hAAT DNA solution (20 µg/ml) were retrogradely injected at 20 ml/s. Tissue samples (8 per liver) were obtained 14 days later and the exogenous DNA, RNA and protein per cell were quantified. Blood samples were collected periodically for transaminase determination in all the animals. RESULTS: The open procedure achieved lower (approx. 1000-fold) DNA delivery, resulting in a significantly lower (p < 0.001) gene transcription (> 100-fold). The closed model also achieved a higher translation index, although differences were smaller (p < 0.001). CONCLUSIONS: Portal inflow blockage increased the delivery, transcription and translation indexes, significantly improving the final procedure efficacy when compared with an open procedure. KEY POINTS: Endovascular hydrodynamic pig liver gene transfer: open procedure versus closed procedure. Open procedure resulted in much lower DNA delivery than closed model. Open procedure reached significantly lower gene transcription index. Translation index with closed model was higher than with the open.


Asunto(s)
Proteínas Portadoras/genética , ADN/genética , Procedimientos Endovasculares/métodos , Técnicas de Transferencia de Gen , Hepatopatías/terapia , Perfusión/métodos , Animales , Femenino , Terapia Genética/métodos , Humanos , Hígado , Porcinos
2.
J Gene Med ; 16(7-8): 179-92, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25092576

RESUMEN

BACKGROUND: Hydrodynamic gene delivery has proved an efficient strategy for nonviral gene therapy in the murine liver but it has been less efficient in pigs. The reason for such inefficiency remains unclear. The present study used a surgical strategy to seal the whole pig liver in vivo. METHODS: A solution of enhanced green fluorescent protein (eGFP) DNA was injected under two different venous injection conditions (anterograde and retrograde), employing flow rates of 10 and 20 ml/s in each case, with the aim of identifying the best gene transfer conditions. The gene delivery and information decoding steps were evaluated by measuring the eGFP DNA, mRNA and protein copy number 24 h after transfection. In addition, gold nanoparticles (diameters of 4 and 15 nm) were retrogradely injected (10 ml/s) to observe, by electron microscopy, the ability of the particle to access the hepatocyte. RESULTS: The gene delivery level was higher with anterograde injection, whereas the efficacy of gene expression was better with retrograde injection, suggesting differences in the decoding processes. Thus, retrograde injection mediates gene transcription (mRNA copy/cell) equivalent to that of intermediate expression proteins but the mRNA translation was lower than that of rare proteins. Electron microscopy showed that nanoparticles within the hepatocyte were almost exclusively 4 nm in diameter. CONCLUSIONS: The results suggest that the low activity of mRNA translation limits the final efficacy of the gene transfer procedure. On the other hand, the gold nanoparticles study suggests that elongated DNA conformation could offer advantages in that the access of 15-nm particles is very limited.


Asunto(s)
Hígado/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Transfección/métodos , Animales , Permeabilidad de la Membrana Celular , Femenino , Oro/química , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Hígado/citología , Nanopartículas del Metal/química , Tamaño de la Partícula , Plásmidos/genética , ARN Mensajero/metabolismo , Sus scrofa , Transcripción Genética
3.
J Biol Chem ; 286(6): 4150-64, 2011 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-21118818

RESUMEN

MicroRNAs (miRNAs) are an emerging class of non-coding endogenous RNAs involved in multiple cellular processes, including cell differentiation. Treatment with retinoic acid (RA) results in neural differentiation of neuroblastoma cells. We wanted to elucidate whether miRNAs contribute to the gene expression changes induced by RA in neuroblastoma cells and whether miRNA regulation is involved in the transduction of the RA signal. We show here that RA treatment of SH-SY5Y neuroblastoma cells results in profound changes in the expression pattern of miRNAs. Up to 42 different miRNA species significantly changed their expression (26 up-regulated and 16 down-regulated). Among them, the closely related miR-10a and -10b showed the most prominent expression changes. Induction of miR-10a and -10b by RA also could be detected in LA-N-1 neuroblastoma cells. Loss of function experiments demonstrated that miR-10a and -10b are essential mediators of RA-induced neuroblastoma differentiation and of the associated changes in migration, invasion, and in vivo metastasis. In addition, we found that the SR-family splicing factor SFRS1 (SF2/ASF) is a target for miR-10a -and -10b in HeLa and SH-SY5Y neuroblastoma cells. We show here that changes in miR-10a and -10b expression levels may regulate SFRS1-dependent alternative splicing and translational functions. Taken together, our results give support to the idea that miRNA regulation plays a key role in RA-induced neuroblastoma cell differentiation. The discovery of SFRS1 as direct target of miR-10a and -10b supports the emerging functional interaction between two post-transcriptional mechanisms, microRNAs and splicing, in the neuronal differentiation context.


Asunto(s)
Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Receptores Inmunológicos/metabolismo , Tretinoina/farmacología , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Diferenciación Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Embrión de Pollo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/genética , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Proteínas de Unión al ARN , Receptores Inmunológicos/genética , Factores de Empalme Serina-Arginina
4.
Am J Physiol Heart Circ Physiol ; 303(3): H368-76, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22685168

RESUMEN

Downregulation of ß(1)- adrenergic receptors (ß(1)-ARs) and increased expression/function of G-protein-coupled receptor kinase 2 (GRK2) have been observed in human heart failure, but changes in expression of other ARs and GRKs have not been established. Another unresolved question is the incidence of these compensatory mechanisms depending on heart failure etiology and treatment. To analyze these questions, we quantified the mRNA/protein expressions of six ARs (α(1A), α(1B), α(1D), ß(1), ß(2), and ß(3)) and three GRKs (GRK2, GRK3, and GRK5) in left (LV) and right ventricle (RV) from four donors, 10 patients with ischemic cardiomyopathy (IC), 14 patients with dilated cardiomyopathy (DC), and 10 patients with nonischemic, nondilated cardiopathies (NINDC). We correlated the changes in the expressions of ARs and GRKs with clinical variables such as left ventricular ejection fraction (LVEF) and left ventricular end-systolic and left ventricular end-diastolic diameter (LVESD and LVEDD, respectively). The main findings were 1) the expression of the α(1A)-AR in the LV positively correlates with LVEF; 2) the expression of GRK3 and GRK5 inversely correlates with LVESD and LVEDD, supporting previous observations about a protective role for both kinases in failing hearts; and 3) ß(1)-AR expression is downregulated in the LV and RV of IC, in the LV of DC, and in the RV of NINDC. This difference, better than an increased expression of GRK2 (not observed in IC), determines the lower LVEF in IC and DC vs. NINDC.


Asunto(s)
Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/genética , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Isquemia Miocárdica/complicaciones , Miocardio/química , Receptores Adrenérgicos/análisis , Adulto , Análisis de Varianza , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Cardiomiopatía Dilatada/tratamiento farmacológico , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/fisiopatología , Femenino , Genotipo , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/fisiopatología , Fenotipo , ARN Mensajero/análisis , Receptores Adrenérgicos/genética , España , Volumen Sistólico , Función Ventricular Izquierda
5.
J Pharmacol Exp Ther ; 328(3): 982-90, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19060223

RESUMEN

In human and animal hypertension models, increased activity of G-protein-coupled receptor kinase (GRK) 2 determines a generalized decrease of beta-adrenergic vasodilatation. We analyzed the possibility of differential changes in the expression and functionality of alpha(1A), alpha(1B), alpha(1D), beta(1), beta(2), and beta(3)-ARs also being involved in the process. We combined the quantification of mRNA levels with immunoblotting and functional studies in aortas of young and adult spontaneously hypertensive rats (SHRs) and their controls (Wistar Kyoto). We found the expression and function of beta(1)-adrenoceptors in young prehypertensive SHRs to be higher, whereas a generalized increase in the expression of the six adrenoceptors and GRK2 was observed in aortas of adult hypertensive SHRs. alpha(1D)- and beta(3)-adrenoceptors, the subtypes that are more resistant to GRK2-mediated internalization and mostly expressed in rat aorta, exhibited an increased functional role in hypertensive animals, showing two hemodynamic consequences: 1) an increased sensitivity to the vasoconstrictor stimulus accompanied by a decreased sensitivity to the vasodilator stimulus (alpha(1D)-ARs are the most sensitive to agonists, and beta(3)-ARs are the least sensitive to agonists); and 2) a slower recovery of the basal tone after adrenergic stimulus removal because of the kinetic characteristic of the alpha(1D) subtype. These functional changes might be involved in the greater sympathetic vasoconstrictor tone observed in hypertension.


Asunto(s)
Aorta/fisiología , Hipertensión/fisiopatología , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos beta/fisiología , Animales , Aorta/fisiopatología , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Frecuencia Cardíaca/fisiología , Humanos , Modelos Biológicos , ARN Mensajero/genética , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores Adrenérgicos alfa 1/fisiología , Sístole/fisiología , Regulación hacia Arriba , Vasodilatación
6.
Biochem J ; 409(2): 417-28, 2008 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-17937659

RESUMEN

The structural ground underlying the pH-dependency of the dimer-tetramer transition of Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of wild-type and site-directed mutants of recombinant lectins. Synthetic genes coding for the full-length alpha-chains of the seed lectins of Dioclea guianensis (termed r-alphaDguia) and Dioclea grandiflora (termed r-alphaDGL) were designed and expressed in Escherichia coli. This pioneering approach, which will be described in detail in the present paper, yielded recombinant lectins displaying carbohydrate-binding activity, dimer-tetramer equilibria and crystal structures indistinguishable from their natural homologues. Conversion of the pH-stable tetrameric r-alphaDGL into a structure exhibiting pH-dependent dimer-tetramer transition was accomplished through mutations that abolished the interdimeric interactions at the central cavity of the tetrameric lectins. Both the central and the peripheral interacting regions bear structural information for formation of the canonical legume lectin tetramer. We hypothesize that the strength of the ionic contacts at these sites may be modulated by the pH, leading to dissociation of those lectin structures that are not locked into a pH-stable tetramer through interdimeric contacts networking the central cavity loops.


Asunto(s)
Dioclea/metabolismo , Lectinas de Plantas/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Intrones , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad
7.
Mol Endocrinol ; 21(10): 2391-402, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17595318

RESUMEN

Retinoic acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidylinositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAPK signaling pathways through a rapid, nongenomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor, retinoic acid receptor (RAR), on the basis of the pharmacological profile of the activation, loss, and gain of function experiments with mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells, and the physical association between liganded RAR and PI3K activity. The association of RAR with the two subunits of PI3K was differentially regulated by the ligand. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARalpha and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR proved to be relevant for PI3K activation. A chimerical RAR fusing c-Src myristylation domain to the N terminus of RARalpha (Myr-RARalpha) was targeted to plasma membrane. Transfection of Myr-RARalpha to mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells and COS-7 cells results in strong activation of the PI3K signaling pathway, although both in the absence as well in the presence of RA. Our results support a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signaling complex involving RAR and the subunits of PI3K.


Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Línea Celular Tumoral , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/análisis , Receptores de Ácido Retinoico/agonistas , Receptores de Ácido Retinoico/análisis , Receptor alfa de Ácido Retinoico , Transducción de Señal , Tretinoina/farmacología
8.
Oncogene ; 24(43): 6533-44, 2005 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-16007183

RESUMEN

1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3) has antitumor activity in addition to its classical action on calcium metabolism and bone tissue biology. It is thought to regulate the expression of multiple target genes and thus modulate processes critical for tumor growth and metastases. Here we show that 1alpha,25(OH)2D3 differentially regulates the expression of Id1 and Id2 genes, members of a family of transcriptional regulators of cell proliferation and differentiation. 1alpha,25(OH)2D3 induced epithelial differentiation in SW480-ADH human colon carcinoma cell line by promoting expression of the proteins implicated in adherent junction formation, including E-cadherin, and by inhibiting beta-catenin transcriptional activity. 1alpha,25(OH)2D3 activated the human Id1 gene promoter and rapidly induced Id1 RNA and protein. Ectopic overexpression of Id1 was not sufficient to induce E-cadherin, which was critical for the morphological changes induced by 1alpha,25(OH)2D3 in SW480-ADH cells. Conversely, Id2 transcription rate, RNA and protein levels were decreased by 1alpha,25(OH)2D3. Id2 downregulation by 1alpha,25(OH)2D3 mediated the antiproliferative effect of 1alpha,25(OH)2D3 on SW480-ADH cells. In addition, we showed that 1alpha,25(OH)2D3 changed the levels of the inducer of angiogenesis, vascular endothelial growth factor and the potent antiangiogenic factor thrombospondin-1, leading to a balanced change in the angiogenic potential of SW480-ADH human colon carcinoma cells.


Asunto(s)
Carcinoma/genética , Neoplasias del Colon/genética , Proteínas de Unión al ADN/genética , Neovascularización Patológica/tratamiento farmacológico , Proteínas Represoras/genética , Factores de Transcripción/genética , Vitamina D/análogos & derivados , Animales , Cadherinas/efectos de los fármacos , Cadherinas/metabolismo , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Proteínas del Citoesqueleto/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteína 1 Inhibidora de la Diferenciación , Proteína 2 Inhibidora de la Diferenciación , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Proteínas Represoras/efectos de los fármacos , Transactivadores/efectos de los fármacos , Transactivadores/metabolismo , Factores de Transcripción/efectos de los fármacos , Células Tumorales Cultivadas , Vitamina D/farmacología , beta Catenina
9.
FEBS J ; 273(17): 3962-74, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16934035

RESUMEN

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407+/-15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (betaalpha)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.


Asunto(s)
Acetilglucosamina/metabolismo , Quitinasas/química , Fabaceae/enzimología , Hemaglutininas/química , Lectinas de Plantas/química , Semillas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Quitinasas/genética , Quitinasas/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , ADN Complementario/aislamiento & purificación , Fabaceae/genética , Hemaglutininas/genética , Hemaglutininas/metabolismo , Datos de Secuencia Molecular , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Unión Proteica , Semillas/genética
10.
Front Biosci ; 10: 1932-45, 2005 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-15769675

RESUMEN

Excessive dietary fat and cholesterol exacerbate atherosclerosis. To obtain unbiased insight into the early pathological changes induced by fat feeding in the artery wall, we used high-density microarrays to generate transcriptional profiles of aortic tissue from two groups of atherosclerosis-prone apolipoprotein E-null mice: controls maintained on standard chow and experimental animals exposed short-term to a Western-type diet, a regimen which produced severe hypercholesterolemia without significant development of atheromas. By applying rigorous selection criteria, we identified 311 genes differentially regulated by these dietary conditions. The set of diet-regulated genes exhibited striking functional relationships and represented both novel and known regulatory networks implicated in injury of the artery wall, including cell adhesion genes, histocompatibility antigen and major histocompatibility complex genes, flavin-containing monooxygenases, interferon-regulated genes, small inducible cytokines, collagen and procollagen genes, and complement system components. Further examination of genes identified by this study will provide insights into the molecular mechanisms by which high-fat cholesterol-rich dietary regime initiates pathological alterations in healthy arteries.


Asunto(s)
Apolipoproteínas E/fisiología , Aterosclerosis/metabolismo , Dieta Aterogénica , Perfilación de la Expresión Génica , Edad de Inicio , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/etiología , Aterosclerosis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción Genética
11.
J Mol Biol ; 322(2): 325-37, 2002 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-12217694

RESUMEN

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn(2+) and Hg(2+), two known PSA inhibitors.


Asunto(s)
Caballos , Calicreínas/química , Calicreínas/aislamiento & purificación , Antígeno Prostático Específico/química , Semen/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Calicreínas/antagonistas & inhibidores , Calicreínas/metabolismo , Masculino , Mercurio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Antígeno Prostático Específico/antagonistas & inhibidores , Estructura Secundaria de Proteína , Alineación de Secuencia , Especificidad por Sustrato , Zinc/metabolismo
12.
Mol Endocrinol ; 18(2): 303-11, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14615607

RESUMEN

The skin-targeted overexpression of the glucocorticoid receptor (GR) in transgenic mice dramatically impairs the inflammatory responses to tumor promoter agents and suppresses skin tumor development. The antiinflammatory, rapid effects of corticosteroids are partially exerted through interference of GR with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in several tissues, a highly relevant pathway in the mouse skin tumor progression process. In this work, we aimed to elucidate whether a cross-talk mechanism between GR and PI3K/Akt occurred in intact skin as well as the biological relevance of this interaction during skin tumorigenesis. We report that, in transgenic mice overexpressing the receptor, GR physically associated with p85 alpha/PI3K in skin, resulting in decreased Akt and I kappa B kinase activity. GR activation by dexamethasone in normal mouse skin also decreased Akt activity within minutes, whereas cotreatment with the GR antagonist RU486 abolished dexamethasone action. Indeed, GR exerted a nongenomic action because keratinocyte transfection with a transcriptionally defective receptor mutant still decreased PI3K and Akt activity. Moreover, GR coexpression greatly reduced the accelerated growth of malignant tumors and increased Akt activity induced by Akt-transfected keratinocytes, as shown by in vivo tumorigenic assays. Overall, our data strongly indicate that GR/PI3K-Akt cross-talk constitutes a major mechanism underlying the antitumor effect of glucocorticoids in skin.


Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Glucocorticoides/metabolismo , Piel/metabolismo , Piel/patología , Administración Tópica , Animales , Pruebas de Carcinogenicidad , Células Cultivadas , Dexametasona/administración & dosificación , Dexametasona/farmacología , Femenino , Antagonistas de Hormonas/farmacología , Quinasa I-kappa B , Técnicas In Vitro , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Ratones Desnudos , Ratones Transgénicos , Mifepristona/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Receptores de Glucocorticoides/genética , Transducción de Señal , Piel/efectos de los fármacos
13.
Thromb Res ; 135(1): 183-91, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25466848

RESUMEN

INTRODUCTION: An inverse relationship has been reported between decreased postoperative Antithrombin (AT) plasmatic levels and the incidence of complications. We hypothesized that Nuclear Hormone Receptors could modulate the expression of SERPINC1, encoding AT, through a Hormone Regulatory Element present in its promoter, and thus hormone analogs could be a pharmacological complement in surgical procedures to activate endogenous AT synthesis. MATERIALS AND METHODS: The expression of SERPINC1 was analyzed in HepG2 cells by quantitative RT-PCR and Western Blot. Two studies were conducted with (a) patients submitted to cardiac surgery with cardiopulmonary bypass receiving (n =17) or not (n=321) glucocorticoids (GCs) as part of their pharmacological treatment, and (b) patients who received (n =20) or not (n=16) GCs as part of their surgery (exodontia or knee arthroscopic, respectively). AT activity in plasma was determined by Innovance Antithrombin Test on a BCS XP System hemostasis analyzer. RESULTS: 13 nuclear hormone receptor ligands were assayed, being GW4064 (FXR ligand) the most potent activator. Retinoids, activating RXR, and GCs (Dexamethasone, cortisone and methylprednisolone) also resulted in increased AT expression. Chronic GC treatment mitigates the decreased AT activity observed after cardiac surgery. In patients who received two acute GC doses, pre-operative and post-operative AT activity was similar, whereas a significant decrease was observed after surgery in untreated patients. CONCLUSIONS: Whereas retinoids and FXR ligands are investigational compounds, regulation of AT by GCS could have a higher potential for translation to clinical practice, pre-conditioning the patient against complications related to reduced AT levels. Larger prospective studies are needed to define the exact role of GCs and their potential clinical utility in cardiac surgery.


Asunto(s)
Antitrombinas/metabolismo , Glucocorticoides/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Retinoides/farmacología , Anciano , Secuencia de Bases , Puente Cardiopulmonar , Estudios de Cohortes , Femenino , Células Hep G2 , Humanos , Isoxazoles/farmacología , Ligandos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Unión al ARN/metabolismo , Receptores X Retinoide/metabolismo , Resultado del Tratamiento
14.
J Pharm Sci ; 93(6): 1641-8, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15124220

RESUMEN

The intrinsic absorption of salbutamol in different intestinal segments of the rat was measured and related with the corresponding intestinal P-glycoprotein (P-gp) expression levels. The apparent absorption rate constants (k(a), h(-1)) observed in each fraction by means of the "in situ" rat gut absorption method after perfusion of a 0.29-mM isotonic solution of salbutamol were used as absorption indexes. In a separate series of studies, a semiquantitative analysis of the mRNA expression of P-gp by means of polymerase chain reaction and Western blot with an antibody raised against the P-gp were also performed. The "in situ" k(a) values determined in the different segments (h(-1)) showed that the absorption is not homogeneous along the intestinal tract, that is, 0.499 +/- 0.054 for colon, 0.474 +/- 0.052 for the proximal segment, 0.345 +/- 0.014 for the mean, and 0.330 +/- 0.023 for the distal fraction. Addition of verapamil to the perfusion fluid did provide a better absorption of salbutamol in the distal segment. The analysis of the mRNA expression and levels of P-gp showed that the enzyme content in each section of the intestine was inversely related to salbutamol absorption.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Albuterol/análisis , Albuterol/metabolismo , Absorción Intestinal/fisiología , Animales , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar
15.
Blood Coagul Fibrinolysis ; 24(4): 454-7, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23343694

RESUMEN

We recently reported prospective results from a cohort of patients scheduled for elective cardiac surgery with cardiopulmonary bypass (CPB) in which most baseline clinical parameters of patients and surgery outcomes failed to demonstrate relationships with post-CPB antithrombin (AT) activity. In this extension study, a larger sample size (250 patients) was analyzed following general linear models. Patients' sociodemographic and pre-CPB clinical data as well as pre/post-CPB AT activity and outcomes were collected. There was a significant decrease of post-CPB AT activity (95.6 ± 13.7-64.6 ± 12.1%; P < 0.001). Univariate and multivariate analyses revealed that a decrease of approximately 1% post-CPB AT activity may be expected per 3 years increase in patient's age. Univariate analysis showed that post-CBP AT activity was inversely related to the need for transfusions, acute renal failure and occurrence of any complication (re-intervention, low cardiac output, arrhythmia, lung dysfunction, stroke, acute renal failure, mesenteric ischemia and re-hospitalization; P < 0.05). Multivariate analysis adjusted for age and pre-CPB AT did not show statistical significance. Odds ratio (OR) less than 1 was observed in most outcomes (0.8 on average), which suggested a reduction of the probability for an increase of 10% in post-CBP AT. Our results confirm the role of low postsurgery AT activity influencing outcomes in patients undergoing CPB.


Asunto(s)
Lesión Renal Aguda/sangre , Antitrombina III/metabolismo , Arritmias Cardíacas/sangre , Gasto Cardíaco Bajo/sangre , Puente Cardiopulmonar , Complicaciones Posoperatorias/sangre , Factores de Edad , Anciano , Análisis de Varianza , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento
16.
Life Sci ; 93(2-3): 108-15, 2013 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-23770231

RESUMEN

AIMS: The present study investigates the expression and clinical relevance of the constitutive NO synthases in heart and peripheral blood mononuclear cells (PBMCs) obtained from heart failure patients. MAIN METHODS: mRNA and protein levels (qRT-PCR and immunoblot) of eNOS and nNOS were determined in: i) Left ventricle (LV, n=4) and PBMCs (n=10) from healthy donors; ii) LV, right ventricle (RV) and PBMCs of heart failure (HF) patients (n=32); and iii) biopsies and PBMCs of the HF patients after cardiac transplant (n=15). KEY FINDINGS: Expression of constitutive NOS isoforms in heart exhibits wide variability in HF patients, but this variability was not related to aetiology, disease severity, concomitant pathologies or drug regimes. A significantly increased eNOS expression was found in LV from HF patients without vs. with pulmonary hypertension. Overall, higher eNOS expression in this chamber was associated with lower pulmonary arterial pressure. Furthermore, a higher eNOS expression in HF is associated with smaller LV diameter, whereas, a higher post-transplant eNOS expression is related to greater cardiac distensibility. In the RV, nNOS increased after transplant. The positive correlation found between the nNOS expression in the LV of HF patients and the cardiac index suggests a role for this isoform in facilitating cardiac work. A decreased expression of eNOS was observed in PBMCs from HF patients vs. healthy donors, which recovers after transplant. SIGNIFICANCE: A selective up-regulation of the cardiac expression of each NOS isoform in the failing heart, which is not mirrored by PBMCs, is related to an improved health status.


Asunto(s)
Insuficiencia Cardíaca/enzimología , Trasplante de Corazón , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Femenino , Ventrículos Cardíacos/enzimología , Ventrículos Cardíacos/fisiopatología , Humanos , Leucocitos Mononucleares/enzimología , Masculino , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo III/genética , Valor Predictivo de las Pruebas
17.
Eur J Pharmacol ; 641(1): 41-8, 2010 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-20511116

RESUMEN

Among the three alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) a peculiar intracellular localization and poor coupling to membrane signals of cloned alpha(1D)-adrenoceptor have been reported. In addition, the alpha(1L)-adrenoceptor (low affinity for prazosin), a functional phenotype of alpha(1A), has been described. The purpose of this work was to analyze the expression, cellular localization and coupling to membrane signalling (inositol phosphate accumulation) of alpha(1)-adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (alpha(1D)>alpha(1B)>>alpha(1A)). alpha(1)-Adrenoceptors were also detected by immunoblotting, revealing alpha(1A)- and alpha(1B)-adrenoceptors to be predominantly expressed in the membrane fraction and the alpha(1D)-adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of alpha(1D)-adrenoceptor in tissue homogenates, whereas only alpha(1A)- and alpha(1B)-subtypes were detected in membranes. The proportion of alpha(1A)-adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (alpha(1A/L))-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The alpha(1A/L)-adrenoceptor was heavily implicated in the inositol phosphate response, while the alpha(1D)-subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of alpha(1D)-adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the alpha(1L)-adrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the alpha(1A)-adrenoceptor coupled to G-protein.


Asunto(s)
Corteza Cerebral/metabolismo , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Animales , Unión Competitiva , Membrana Celular/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Citosol/metabolismo , Femenino , Regulación de la Expresión Génica , Guanosina Trifosfato/química , Guanosina Trifosfato/farmacología , Fosfatos de Inositol/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
18.
J Hypertens ; 28(6): 1281-9, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20216086

RESUMEN

OBJECTIVE: The objective of our work was to analyze if changes in the expression of beta-adrenoceptors (beta-ARs) and G-protein-coupled receptor kinases (GRKs) in human lymphocytes - a practical surrogate for myocardial or vascular cells - are related to the hypertensive state and its clinical consequences. METHODS: Real-time quantitative RT-PCR was employed to evaluate the expression of the three beta-ARs (beta1, beta2, beta3) and three GRKs (GRK2, GRK3, GRK5) in human lymphocytes obtained from both normotensive and hypertensive patients, some of whom had been treated with blockers of the renin-angiotensin system. Office blood pressure, 24-h ambulatory blood pressure, urinary albumin excretion and serum biochemical profile were also recorded. RESULTS AND CONCLUSIONS: beta1-AR expression levels were higher in circulating lymphocytes from hypertensive patients (2-DeltaDeltaCt = 2.135 +/- 0.4252*, vs. control group), but this difference was not observed when these patients were treated with blockers of the renin-angiotensin system. beta1-AR levels directly correlated (r2 = 0.5711, P = 0.0185) with urinary albumin excretion in microalbuminuric patients, which relates alterations of this receptor to cardiovascular risk. An inverse correlation was observed between the expression levels of beta2-AR and diastolic blood pressure (r2 = 0.2078, P = 0.0031), suggesting that beta2-AR levels in lymphocytes mirror their expression in vascular cells, in which beta2-AR-mediated relaxation regulates vascular resistance. mRNA levels for GRK3 were inversely correlated with systolic and diastolic blood pressure (day, night and 24 h), which suggests a protective role for GRK3 in the regulation of human blood pressure, as supported by previous findings in transgenic mice.


Asunto(s)
Albuminuria/metabolismo , Presión Sanguínea , Quinasa 3 del Receptor Acoplado a Proteína-G/metabolismo , Linfocitos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Femenino , Humanos , Linfocitos/enzimología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
19.
Mol Endocrinol ; 23(11): 1799-814, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19812389

RESUMEN

Retinoic acid (RA) signaling is mediated by the retinoic acid receptor (RAR), belonging to the nuclear hormone receptor superfamily. In addition to its classical transcriptional actions, RAR also mediates rapid transcription-independent (nongenomic) actions, consisting in the activation of signal transduction pathways, as the phosphatidyl-inositol-3-kinase or the ERK MAPK-signaling pathways. RA-induced rapid transcription-independent actions play a role in different physiological contexts. As an effort toward understanding the functions of those rapid actions on signaling elicited by RA, we have identified nuclear proteins the phosphorylation state of which is rapidly modified by RA treatment in neuroblastoma cells, using a proteomic approach. Our results show that RA treatment led to changes in the phosphorylation patterns in two families of proteins: 1) those related to chromatin dynamics in relation to transcriptional activation, and 2) those related to mRNA processing and, in particular, mRNA splicing. We show that treatment of neuroblastoma cells with RA leads to alteration of the regulation of pre-mRNA splicing and mRNA translation. Thus, our results underscore novel functions for the rapid signaling elicited by RAR in the regulation of mRNA processing. We conclude that RA activation of signaling pathways can indeed regulate mRNA processing as part of a cellular response orchestrated by the nuclear receptor RAR.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas Nucleares/química , Biosíntesis de Proteínas , Proteómica/métodos , Empalme del ARN , ARN Mensajero/metabolismo , Tretinoina/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Cromatina/química , Humanos , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Plásmidos/metabolismo , Transducción de Señal
20.
Am J Physiol Heart Circ Physiol ; 289(5): H1923-32, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15951348

RESUMEN

The mRNA levels for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D, were quantified by real-time RT-PCR in arteries from Wistar rats. The alpha1D-adrenoceptor was prominent in both aorta (79.0%) and mesenteric artery (68.7%), alpha1A predominated in tail (61.7%) and small mesenteric artery (73.3%), and both alpha1A- and alpha1D-subtypes were expressed at similar levels in iliac artery. The mRNA levels of the alpha1B-subtype were a minority in all vessels (1.7-11.1%). Concentration-response curves of contraction in response to phenylephrine or relaxation in response to alpha1-adrenoceptor antagonists on maximal sustained contraction induced by phenylephrine were constructed from control vessels and vessels pretreated with 100 micromol/l chloroethylclonidine (CEC) for 30 min. The significant decrease in the phenylephrine potency observed after CEC treatment together with the inhibitory potency displayed by 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspiro (4,5) decane-7-dionedihydrochloride} (BMY-7378, an alpha1D-adrenoceptor antagonist) confirm the relevant role of alpha1D-adrenoceptors in aorta and iliac and proximal mesenteric arteries. The potency of 5-methylurapidil (an alpha1A-adrenoceptor antagonist) and the changes in the potency of both BMY-7378 and 5-methylurapidil after CEC treatment provided evidence of a mixed population of alpha1A- and alpha1D-adrenoceptors in iliac and distal mesenteric arteries. The low potency of prazosin (pIC50 < 9) as well as the high 5-methylurapidil potency in tail and small mesenteric arteries suggest the main role of alpha1A/alpha1L-adrenoceptors with minor participation of the alpha1D-subtype. The mRNA levels and CEC treatment corroborated this pattern and confirmed that the alpha1L-adrenoceptor could be a functional isoform of the alpha1A-subtype.


Asunto(s)
Arterias/metabolismo , ARN Mensajero/biosíntesis , Receptores Adrenérgicos alfa 1/biosíntesis , Receptores Adrenérgicos alfa 1/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Cartilla de ADN , Relación Dosis-Respuesta a Droga , Arteria Ilíaca/efectos de los fármacos , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Fenilefrina/farmacología , Isoformas de Proteínas , Ratas , Ratas Wistar , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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