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1.
Microvasc Res ; 80(3): 365-71, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20478315

RESUMEN

BACKGROUND: Ischemic preconditioning (IP) and intermittent clamping (IC) increase the ischemic tolerance of the liver. The underlying mechanisms are not completely understood. Heat shock proteins protect cellular integrity in stress and have been discussed as mediators in preconditioning. IP and IC in rat livers were compared with respect to HSP induction and postischemic microcirculation. METHODS: All animals were exposed to 70min of partial warm liver ischemia. Different clamping protocols were used: in control animals (C) 70min continuous ischemia was applied. IP was performed by 5min ischemia and 10min reperfusion before the 70min ischemia time. In IC-groups, ischemia time of 70min was divided into four intervals. Each group included 21 animals with 3 different reperfusion intervals; either 30min, 12 or 36h. Intravital microscopy was performed after 30min of reperfusion. AST-levels and HSP induction were analysed 90min, 12 and 36h after reperfusion. RESULTS: IP and IC significantly improved sinusoidal perfusion (IP: 83.4±2.8%; IC: 84.4±4.6% vs. C: 60.4±3.9%; p<0.001) and leucocyte adherence in sinusoids (IP: 51.9±12.0, IC: 40.9±4.7 vs. C: 90.1±17.7/mm(2) liver surface; p<0.001) and postsinusoidal venules. AST-levels were minimized in IP and IC compared to controls (12h after reperfusion: IP: 969±934U/l, IC: 675±562U/l vs. C: 2373±792U/l; p=0.004). In the course of reperfusion HSP70 protein expression doubled between 90min and 12h in IC (0.529±0.227 vs. 0.992±0.246; p<0.05) and control-groups (0.572±0.314 vs. 1.106±0.309; p<0.05) whereas it remained unchanged in the IP-group (0.437±0.383 vs. 0.412±0.439; n.s.). CONCLUSION: Microcirculation is similarly preserved by IP and IC. The early protection derived by IP prevents further induction of HSP70 in opposite to IC. Therefore, IP may offer a more comprehensive protection against I/R on a cellular and transcriptional level.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Precondicionamiento Isquémico , Hígado/irrigación sanguínea , Microvasos/metabolismo , Daño por Reperfusión/prevención & control , Animales , Aspartato Aminotransferasas/sangre , Adhesión Celular , Constricción , Modelos Animales de Enfermedad , Proteínas HSP70 de Choque Térmico/genética , Leucocitos/inmunología , Circulación Hepática , Masculino , Microcirculación , Microscopía Fluorescente , Microvasos/inmunología , Microvasos/fisiopatología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Factores de Tiempo , Regulación hacia Arriba , Isquemia Tibia/efectos adversos
2.
Genomics ; 93(3): 213-20, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19059335

RESUMEN

The Alternative Splicing and Transcript Diversity database (ASTD) gives access to a vast collection of alternative transcripts that integrate transcription initiation, polyadenylation and splicing variant data. Alternative transcripts are derived from the mapping of transcribed sequences to the complete human, mouse and rat genomes using an extension of the computational pipeline developed for the ASD (Alternative Splicing Database) and ATD (Alternative Transcript Diversity) databases, which are now superseded by ASTD. For the human genome, ASTD identifies splicing variants, transcription initiation variants and polyadenylation variants in 68%, 68% and 62% of the gene set, respectively, consistent with current estimates for transcription variation. Users can access ASTD through a variety of browsing and query tools, including expression state-based queries for the identification of tissue-specific isoforms. Participating laboratories have experimentally validated a subset of ASTD-predicted alternative splice forms and alternative polyadenylation forms that were not previously reported. The ASTD database can be accessed at http://www.ebi.ac.uk/astd.


Asunto(s)
Empalme Alternativo/genética , Bases de Datos Genéticas , Animales , Sistemas de Administración de Bases de Datos , Humanos , Almacenamiento y Recuperación de la Información/métodos , Ratones , Ratas , Reproducibilidad de los Resultados , Programas Informáticos , Interfaz Usuario-Computador
4.
Carcinogenesis ; 26(9): 1503-12, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15888491

RESUMEN

The serine/threonine kinase AKT plays a critical role in controlling the balance between cell survival and apoptosis. Several reports implicated AKT in the molecular pathogenesis of different human malignancies and overexpression of AKT was recently demonstrated to be an early event in colorectal carcinogenesis. We report here the identification of nine putative Tcf/Lef-binding elements (TBEs) upstream to the ATG initiation site of the AKT1 gene. Four of these TBEs are located upstream of the transcriptional start, whereas five TBEs are situated in Exon 1 of the AKT1 gene. Accordingly, we hypothesized that AKT1 expression might be regulated by Wnt/beta-catenin signaling. To elucidate the regulation of AKT expression in colon cancer cells, we generated reporter constructs containing the luciferase gene under the control of different regions derived from the AKT1 promoter/enhancer. Transient expression of the constructs in colorectal cancer (CRC) cell lines resulted in significant activation of the reporter gene. Luciferase was stimulated 20- to 50-fold in SW480, SW948 and HCT116 CRC cells. In contrast, the AKT1 promoter/enhancer constructs showed only a weak response in 293 embryonic kidney cells. Coexpression of a constitutively active beta-catenin mutant in colon cancer cells further enhanced reporter gene activation from the AKT1 promoter/enhancer, whereas it was downregulated by introduction of either wild-type APC or dnTcf-4. In addition, immunohistochemical staining of tumor sections derived from CRC patients showed elevated expression levels of AKT1, correlating with enhanced cytoplasmic/nuclear expression of beta-catenin. In summary our data suggest that beta-catenin/Tcf contributes to the transcriptional regulation of the AKT1 gene.


Asunto(s)
Proteínas del Citoesqueleto/fisiología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/fisiología , Apoptosis , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Neoplasias del Colon , Neoplasias Colorrectales , Proteínas del Citoesqueleto/genética , Elementos de Facilitación Genéticos , Genes Reporteros , Humanos , Riñón , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-akt , Transactivadores/genética , Activación Transcripcional , beta Catenina
5.
World J Gastroenterol ; 11(9): 1303-16, 2005 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-15761968

RESUMEN

AIM: To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs. METHODS: We used two strategies (SSH suppression subtractive hybridization and hybridization of cDNA arrays) to determine early changes in gene expression profiles in a rat model of partial WI/R, comparing postischemic and adjacent nonischemic liver lobes. Differential gene expression was verified (WI/R; 1 h/2 h) and analyzed in more detail after warm ischemia (1 h) in a reperfusion time kinetics (0, 1, 2 and 6 h) and compared to untreated livers by Northern blot hybridizations. Protein expression was examined on Western blots and by immunohistochemistry for four differentially expressed target genes (Hsp70, Hsp27, Gadd45a and IL-1rI). RESULTS: Thirty-two individual WI/R target genes showing altered RNA levels after confirmation by Northern blot analyzes were identified. Among them, six functionally uncharacteristic expressed sequences and 26 known genes (12 induced in postischemic liver lobes, 14 with higher transcriptional expression in adjacent nonischemic liver lobes). Functional categories of the verified marker genes indicate on the one hand cellular stress and tissue damage but otherwise activation of protective cellular reactions (AP-1 transcription factors, apoptosis related genes, heat shock genes). In order to assign the transcriptional status to the biological relevant protein level we demonstrated that Hsp70, Hsp27, Gadd45a and IL-1rI were clearly up-regulated comparing postischemic and untreated rat livers, suggesting their involvement in the WI/R context. CONCLUSION: This study unveils a WI/R response gene set that will help to explore molecular pathways involved in the tissue damage after WI/R. In addition, these genes especially Hsp70 and Gadd45a might represent promising new candidates indicating WI/R liver damage.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Hígado/fisiología , Hibridación de Ácido Nucleico/métodos , Daño por Reperfusión/genética , Daño por Reperfusión/fisiopatología , Animales , Proteínas de Ciclo Celular/genética , Expresión Génica , Proteínas de Choque Térmico HSP27 , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Calor , Cinética , Hígado/patología , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Receptores de Interleucina-1/genética , Receptores Tipo I de Interleucina-1 , Daño por Reperfusión/patología , Factor de Transcripción AP-1/fisiología , Transcripción Genética/fisiología
6.
Int J Cancer ; 114(1): 46-57, 2005 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-15523676

RESUMEN

The growth-regulatory interplay between ganglioside GM1 on human SK-N-MC neuroblastoma cells and an endogenous lectin provides a telling example for glycan (polysaccharide) functionality. Galectin-1 is the essential link between the sugar signal and the intracellular response. The emerging intrafamily complexity of galectins raises the question on defining extent of their structural and functional overlap/divergence. We address this problem for proto-type galectins in this system: ganglioside GM1 as ligand, neuroblastoma cells as target. Using the way human galectin-1 interacts with this complex natural ligand as template, we first defined equivalent positioning for distinct substitutions in the other tested proto-type galectins, e.g., Lys63 vs. Leu60/Gln72 in galectins-2 and -5. As predicted from our in silico work, the tested proto-type galectins have affinity for the pentasaccharide of ganglioside GM1. In contrast to solid-phase assays, cell surface presentation of the ganglioside did not support binding of galectin-5, revealing the first level of regulation. Next, a monomeric proto-type galectin (CG-14) can impair galectin-1-dependent negative growth control by competitively blocking access to the shared ligand without acting as effector. Thus, the quaternary structure of proto-type galectins is an efficient means to give rise to functional divergence. The identification of this second level of regulation is relevant for diagnostic monitoring. It might be exploited therapeutically by producing galectin variants tailored to interfere with galectin activities associated with the malignant phenotype. Moreover, the given strategy for comparative computational analysis of extended binding sites has implications for the rational design of galectin-type-specific ligands.


Asunto(s)
Gangliósido G(M1)/metabolismo , Galectinas/metabolismo , Neuroblastoma/metabolismo , Secuencia de Aminoácidos , Animales , Pollos , Galectina 1/metabolismo , Galectina 2/metabolismo , Galectina 3/metabolismo , Galectina 4/metabolismo , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Células Tumorales Cultivadas
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