RESUMO
Development of the eyelid requires coordination of the cellular processes involved in proliferation, cell size alteration, migration, and cell death. C57BL/6J-corneal opacity (B6-Co) mice are mutant mice generated by the administration of N-ethyl-N-nitrosourea (100 mg/kg). They exhibit the eyelids open at birth phenotype, abnormal round cell shape from tightened F-actin bundles in leading edge keratinocytes at E16.5, and gradual corneal opacity with neovessels. The tip of the leading edge in B6-Co mice did not move forward, and demonstrated a sharp peak shape without obvious directionality. Analysis of the biological characteristics of B6-Co mice demonstrated that abnormal migration of keratinocytes could affect eyelid development, but proliferation and apoptosis in B6-Co mice had no effect. Mutant gene mapping and sequence analysis demonstrated that in B6-Co mice, adenosine was inserted into the untranslated regions, between 3030 and 3031, in the mRNA 3'-terminal of Fgf10. In addition, guanine 7112 was substituted by adenine in the Mtap1B mRNA, and an A2333T mutation was identified in Mtap1B. Quantitative real-time polymerase chain reaction analysis showed that expression of the Hbegf gene was significantly down-regulated in the eyelids of B6- Co mice at E16.5, compared to B6 mice. However, the expression of Rock1, Map3k1, and Jnk1 genes did not show any significant changes. Abnormal keratinocyte migration and down-regulated expression of the Hbegf gene might be associated with impaired eyelid development in B6-Co mice.
Assuntos
Córnea/metabolismo , Neovascularização da Córnea/genética , Opacidade da Córnea/genética , Pálpebras/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Queratinócitos/metabolismo , Regiões 3' não Traduzidas , Actinas/genética , Actinas/metabolismo , Animais , Movimento Celular , Polaridade Celular , Proliferação de Células , Forma Celular , Córnea/anormalidades , Córnea/crescimento & desenvolvimento , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Embrião de Mamíferos , Etilnitrosoureia , Pálpebras/anormalidades , Pálpebras/crescimento & desenvolvimento , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutagênicos , Fenótipo , Cultura Primária de CélulasRESUMO
Transgene silencing, which is common in transgenic plants and animals, limits the generation and application of genetically modified organisms, and is associated with the exogenous gene copy number, the methylation status of its promoters, and histone modification abnormalities. Here, we analyzed the expression of the exogenous gene DsRed and the methylation status of its cytomegalovirus (CMV) promoter in six healthy transgenic cashmere goats and transgenic nuclear donor cells. The CMV promoter exhibited high methylation levels (74.4-88.2%) in four of the goats, a moderate methylation level (58.7%) in one, and a low methylation level (21.2%) in one, while the methylation level of the transgenic nuclear donor cells was comparatively low (14.3%). DsRed expression was negatively correlated with promoter methylation status. Transgenic cashmere goats carried one to three copies of the CMV promoter fragment and one to six copies of the DsRed fragment, but copy number showed no obvious correlation with DsRed expression. After treatment with the methylation inhibitor 5-azacytidine, DsRed expression in transgenic goat cells significantly increased and CMV promoter methylation significantly decreased; this indicated an inverse correlation between promoter methylation status and DsRed expression. After treatment with the histone deacetylase inhibitor trichostatin A, DsRed expression increased, indicating that an abnormal histone modification in transgenic goats is also involved in exogenous gene silencing. These findings indicate the potential of trichostatin A and 5-azacytidine to rescue the biological activity of silenced exogenous transgenes in adult-derived transgenic cells under culture conditions.
Assuntos
Azacitidina/farmacologia , Cabras/genética , Histonas/genética , Ácidos Hidroxâmicos/farmacologia , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Citomegalovirus/genética , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Feminino , Dosagem de Genes , Expressão Gênica , Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteínas Luminescentes/agonistas , Proteínas Luminescentes/antagonistas & inibidores , Proteínas Luminescentes/metabolismo , Masculino , TransgenesRESUMO
A simple, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of auraptene, a constituent isolated from Fructus aurantii with potential to combat Alzheimer's disease, in rat plasma. Rat plasma samples were pretreated by protein precipitation with methanol. The analytes were separated by a Waters Sun Fire C18 column (50 mm x 2 mm, 5 µm) and eluted with 1:1000 methanol and formic acid/water (v/v) mobile phase with a flow rate of 0.5 mL/min. Multiple reaction monitoring was used to monitor the transition of the deprotonated auraptene molecule with an m/z of 299.3 [M+H](+), to the product ion with an m/z of 162.9 [M+H](+). Progesterone, with an m/z of 315.2â 96.9 was used as an internal standard. The limits of detection and of quantification of auraptene in the rat plasma were 1 and 5 ng/mL, respectively. The method was linear in the concentration range of 20- 2000 ng/mL with coefficient correlation of 0.9956. After auraptene (100 mg/kg, p.o.) administration, the maximum plasma concentration and the time taken to reach maximum concentration were 1719.5 ± 384.3 g/mL and 108.0 ± 25.3 min, respectively. The elimination half-life was 108.0 ± 25.3 for auraptene (100 mg/kg, p.o.) and 3.0 ± 0 min for auraptene (2 mg/kg, i.v.). The oral bioavailability was about 8.5%.
Assuntos
Análise Química do Sangue/métodos , Cumarínicos/sangue , Espectrometria de Massas/métodos , Animais , Disponibilidade Biológica , Análise Química do Sangue/normas , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Cumarínicos/farmacocinética , Masculino , Espectrometria de Massas/normas , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
microRNA-218 (miR-218) is a vertebrate-specific miRNA that plays a crucial role in tumorigenesis and tumor progression. This study analyzed the miR-218 expression level and clinical significance in pancreatic cancer. One hundred and seven pairs of pancreatic cancer and adjacent normal tissues were analyzed by quantitative reverse-transcriptase polymerase chain reaction. The correlation between miR-218 expression and clinicopathological characters was determined by the two-sample Student t-test. The survival correlations were analyzed by the Kaplan-Meier method and Cox proportional hazards model. The relative expression of miR-218 in pancreatic cancer tissues (2.63 ± 1.59) was significantly lower than that in matched noncancerous pancreatic tissues (6.52 ± 2.50, P < 0.001). The low expression of miR-218 in the pancreatic cancer tissues were strongly correlated with the TNM classification (P = 0.02), distant metastasis (P = 0.001), and tumor differentiation (P = 0.003). The low level of miR-218 expression was significantly correlated with the shorter overall survival time of pancreatic cancer patients (5-year overall survival rate: 7.5 vs 34.9%; log-rank test: P < 0.001). Multivariate analyses confirmed that a low level of miR-218 expression was an independent predictor of poor prognosis in pancreatic cancer patients (Hazard ratio: 7.24; 95% confidence interval: 2.01-18.28; P = 0.007). Our findings suggested a significant downregulation in the expression of miR-218; this might have considerable potential value in the prognosis for pancreatic cancer.