RESUMO
The development of zinc finger nuclease (ZFN) technology has enabled the genetic engineering of the rat genome. The ability to manipulate the rat genome has great promise to augment the utility of rats for biological and pharmacological studies. A Wistar Hannover rat model lacking the multidrug resistance protein Mdr1a P-glycoprotein (P-gp) was generated using a rat Mdr1a-specific ZFN. Mdr1a was completely absent in tissues, including brain and small intestine, of the knockout rat. Pharmacokinetic studies with the Mdr1a P-gp substrates loperamide, indinavir, and talinolol indicated that Mdr1a was functionally inactive in the blood-brain barrier and intestine in Mdr1a(-/-) rats. To identify possible compensatory mechanisms in Mdr1a(-/-) rats, the expression levels of drug-metabolizing enzyme and transporter-related genes were compared in brain, liver, kidney, and intestine of male and female Mdr1a(-/-) and control rats. In general, alterations in gene expression of these genes in Mdr1a(-/-) rats seemed to be modest, with more changes in female than in male rats. Taken together, our studies demonstrate that the ZFN-generated Mdr1a(-/-) rat will be a valuable tool for central nervous system drug target validation and determining the role of P-gp in drug absorption and disposition.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Engenharia Genética/métodos , Dedos de Zinco/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Animais , Endonucleases , Feminino , Expressão Gênica , Genoma , Masculino , Ratos , Ratos Transgênicos , Ratos Wistar , Distribuição TecidualRESUMO
Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11ß-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11ß-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.
Assuntos
Tecido Adiposo/metabolismo , Síndrome Metabólica/genética , Transativadores/metabolismo , Ativação Transcricional , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Células 3T3-L1 , Tecido Adiposo/patologia , Animais , Pressão Sanguínea , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Fragmentação do DNA , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica , Resistência à Insulina , Metabolismo dos Lipídeos , Masculino , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Camundongos , Camundongos Transgênicos , Músculos/metabolismo , Músculos/patologia , Fenótipo , Tetraciclina/metabolismo , Transativadores/genética , Transfecção , TransgenesRESUMO
Wilms tumor (WT) is the most common solid childhood tumors all over the world. MicroRNAs (miRs) contribute to tumorigenesis of various cancers through targeting gene. The present study investigated the vital role of miR-194-5p and its underlying mechanism in the progression of WT. Immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated downregulation of miR194-5p and upregulation of Crk, in WT tissues compared to adjacent normal tissues. Transfection with miR-194-5p mimics into nephroblastoma cells showed a significant decline in cell migration and invasion, which was detected by Transwell assay. Luciferase assay confirmed that Crk was a direct target gene of miR-194-5p. More important, the mesenchymal to epithelial transition (EMT) biomarkers containing E-cadherin, N-cadherin and Zeb1 were examined by Western blot, and revealed that miR-194-5p mimics decreased the levels of N-cadherin and Zeb1 but increased E-cadherin, which suggested that miR-194-5p inhibited EMT. Crk knockdown could reverse the increased nephroblastoma cell invasion, migration and EMT caused by miR-194-5p inhibitor. Interestingly, qRT-PCR and Western blot analysis showed that overexpression of miR-194-5p deactivated HGF/c-Met/Scr signaling pathway via targeting Crk. In conclusion, miR-194-5p inhibited nephroblastoma cell metastasis and EMT in the progression of WT by targeting Crk. Thus, miR-194-5p might be a potential target in WT particularly for the prevention of metastasis and EMT.
Assuntos
Transição Epitelial-Mesenquimal , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Tumor de Wilms/genética , Tumor de Wilms/patologia , Sequência de Bases , Linhagem Celular Tumoral , Pré-Escolar , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Lactente , Masculino , MicroRNAs/genética , Metástase Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-met/metabolismo , Quinases da Família src/metabolismoRESUMO
A series of [XN5]- (X = O, S, Se, Te) compounds has been examined with ab initio and Density Functional Theory (DFT) methods. The five-membered nitrogen ring series of structures are global minima and may exist or be characterized due to their significant dissociation barriers (29.7-32.7 kcal mol(-1)). Nucleus-independent chemical shifts (NICS) criteria and the presence of (4n+2) pi-electrons confirmed that the five-membered nitrogen ring in their structures exhibits characteristics of aromaticity. Thus, the strong stability of the five-membered nitrogen ring structures may be attributed partially to their aromaticity.