BACKGROUND Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease(IBD),causing an excessive pro-inflammatory cytokines expression.Tristetraprolin(TTP),an mRNA binding protei...BACKGROUND Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease(IBD),causing an excessive pro-inflammatory cytokines expression.Tristetraprolin(TTP),an mRNA binding protein,plays a role in regulating the inflammatory factors,recognizing specific sequences on the 3’untranslated region of cytokine mRNAs.TTP activity depends on its phosphorylation state:the unphosphorylated TTP degrades pro-inflammatory cytokine mRNAs;on the contrary,the phosphorylated TTP fails to destabilize mRNAs furthering their expression.The phospho-TTP forms a complex with the chaperone protein 14-3-3.This binding could be one of the factors that promote intestinal inflammation as a cause of disease progression.AIM To assess if TTP phosphorylation has a role in paediatric IBD.METHODS The study was carried out on a cohort of paediatric IBD patients.For each patient enrolled,a specimen of inflamed and non-inflamed colonic mucosa was collected.Furthermore,the experiments were conducted on macrophages differentiated from blood samples of the same patients.Macrophages from healthy donors’blood were used as controls.Co-immunoprecipitation assay and immunoblotting analyses were performed to observe the formation of the phospho-TTP/14-3-3 complex.In the same samples TNF-αexpression was also evaluated as major factor of the pro-inflammatory activity.RESULTS In this work we studied indirectly the phosphorylation of TTP through the binding with the chaperone protein 14-3-3.In inflamed and non-inflamed colon mucosa of IBD paediatric patients immunoblot assay demonstrated a higher expression of the TTP in inflamed samples respect to the non-inflamed;the coimmunoprecipitated 14-3-3 protein showed the same trend of expression.In the TNF-αgene expression analysis higher levels of the cytokine in inflamed tissues compared to controls were evident.The same experiments were conducted on macrophages from IBD paediatric patients and healthy controls.The immunoblot results demonstrated a high expression of both TTP and co-immunoprecipitated 14-4-3 protein in IBD-derived macrophages in comparison to healthy donors.TNF-αprotein levels from macrophages lysates showed the same trend of expression in favour of IBD paediatric patients compared to healthy controls.CONCLUSION In this work,for the first time,we describe a relation between phospho-TTP/14-3-3 complex and IBD.Indeed,a higher expression of TTP/14-3-3 was recorded in IBD samples in comparison to controls.展开更多
AIM: To examine the effect of intravitreal adenoviral vector-mediated tristetraprolin(Ad-TTP) on VEGF m RNA expression in a rat model of laser-induced choroidal neovascularization.METHODS: Ad-TTP was prepared using a ...AIM: To examine the effect of intravitreal adenoviral vector-mediated tristetraprolin(Ad-TTP) on VEGF m RNA expression in a rat model of laser-induced choroidal neovascularization.METHODS: Ad-TTP was prepared using a commercial kit. Retinal laser-induced photocoagulation(10 spots per eye) was performed on rats in this experimental choroidal neovascularization(CNV) model. Rats were divided into four groups: control(single intravitreal injection of balanced salt solution, n =10), laser-induced CNV(photocoagulation only, n =20), laser-induced CNV plus Ad-TTP injection(photocoagulation plus a single intravitreal Ad-TTP injection, n =20) and Ad-TTP injection only(n =10). Changes in choroidal morphology were evaluated in ten rats in the laser only and the laser plus Ad-TTP groups. Two weeks after laser injury, the size of CNV was calculated by perfusion with high-molecular-weight fluorescein isothiocyanate(FITC)-dextran. VEGF m RNA expression in retina-choroid tissue from ten rats in each group was measured by reverse transcription polymerase chain reaction(RT-PCR). RESULTS: Two weeks after treatment, the area of laser-induced CNV was reduced by approximately 60% in the rats given the Ad-TTP injection compared with that in the laser-only group. There was a tendency toward decreased VEGF m RNA expression in the Ad-TTP injection groups.CONCLUSION: A single intravitreal injection of Ad-TTP significantly suppressed CNV size in this experimental laser-induced CNV model. Ad-TTP injection also decreased VEGF m RNA expression compared with that inthe laser-induced CNV group. The present study is meaningful as the first study to investigate the effect of tristetraprolin delivered via intravitreal injection.展开更多
Since we had previously demonstrated that siRNAs to tristetraprolin (TTP) markedly inhibited insulin stimulation of hepatic HMG-CoA reductase (HMGR) transcription, we investigated the effects of transfecting rat liver...Since we had previously demonstrated that siRNAs to tristetraprolin (TTP) markedly inhibited insulin stimulation of hepatic HMG-CoA reductase (HMGR) transcription, we investigated the effects of transfecting rat liver with TTP constructs. We found that transfecting diabetic rats with TTP did not increase HMGR transcription but rather led to modest inhibition. We then investigated whether co-transfection with protein kinase B, hepatic form (AKT2), might lead to phosphorylation and result in activation of HMGR transcription. We found that this treatment resulted in near complete inhibition of transcription. Transfection with peroxisome proliferator-activated receptor g coactivator (PGC-1a) also inhibited HMGR transcription. These results show that although TTP is needed for activation of HMGR transcription, it cannot by itself activate this process. AKT2 and PGC-1a, which mediate the activation of gluconeogenic genes by insulin, exert the opposite effect on HMGR.展开更多
目的研究elavl1/HuR对斑马鱼肠神经系统早期发育的影响。方法应用免疫组化方法检测elavl1、HuC/HuD在野生型斑马鱼肠道的表达。挑选25个斑马鱼胚胎,应用Western Blot法检测elavl1在受精后24~96 h野生型斑马鱼中的蛋白表达水平。在野生...目的研究elavl1/HuR对斑马鱼肠神经系统早期发育的影响。方法应用免疫组化方法检测elavl1、HuC/HuD在野生型斑马鱼肠道的表达。挑选25个斑马鱼胚胎,应用Western Blot法检测elavl1在受精后24~96 h野生型斑马鱼中的蛋白表达水平。在野生型斑马鱼胚胎处于1细胞期时分别显微注射4 ng、8 ng elavl1a morpholino和8 ng control morpholino,挑选25个受精后24 h的胚胎采用Western Blot法检测elavl1的蛋白表达,免疫组化检测受精后72 h、96 h的胚胎斑马鱼肠道神经元的分布情况。结果elavl1在斑马鱼全身都有表达,而HuC/HuD在第四天斑马鱼肠道部位出现表达;在受精后24~96 h时期,elavl1在斑马鱼中的蛋白表达量随斑马鱼生长而增加,且受精后96 h时elavl1蛋白量明显高于受精后24 h、48 h、72 h,差异有统计学意义(P<0.05);分别注射4 ng、8 ng elavl1a Mo和8 ng Con Mo后,发现注射8 ng Con Mo组elavl1蛋白含量高于其他两组,且差异有统计学意义(P<0.05);敲降elavl1a后免疫组化显示在受精后72 h时三组都不能在肠道末端明显观察到神经元,受精后96 h时可以观察到elavl1a Mo 8 ng组和elavl1a Mo 4 ng组较Con Mo 8 ng组斑马鱼肠神经元数量有所减少,每组随机抽取5条斑马鱼统计肠道末端到前面四个体节长度的肠道神经元数量,发现elavl1a Mo 8 ng组和elavl1a Mo 4 ng组较Con Mo 8 ng组斑马鱼肠神经元数量差异有统计学意义(P<0.05)。结论elavl1a是斑马鱼肠神经发育过程中重要基因,敲降elavl1a会减少肠神经元细胞数量。展开更多
基金Supported by the Italian Ministry of Health projects Ricerca Corrente1/17 and 21/17(Institute for Maternal and Child Health IRCCS Burlo Garofolo)
文摘BACKGROUND Intestinal inflammation and epithelial injury are the leading actors of inflammatory bowel disease(IBD),causing an excessive pro-inflammatory cytokines expression.Tristetraprolin(TTP),an mRNA binding protein,plays a role in regulating the inflammatory factors,recognizing specific sequences on the 3’untranslated region of cytokine mRNAs.TTP activity depends on its phosphorylation state:the unphosphorylated TTP degrades pro-inflammatory cytokine mRNAs;on the contrary,the phosphorylated TTP fails to destabilize mRNAs furthering their expression.The phospho-TTP forms a complex with the chaperone protein 14-3-3.This binding could be one of the factors that promote intestinal inflammation as a cause of disease progression.AIM To assess if TTP phosphorylation has a role in paediatric IBD.METHODS The study was carried out on a cohort of paediatric IBD patients.For each patient enrolled,a specimen of inflamed and non-inflamed colonic mucosa was collected.Furthermore,the experiments were conducted on macrophages differentiated from blood samples of the same patients.Macrophages from healthy donors’blood were used as controls.Co-immunoprecipitation assay and immunoblotting analyses were performed to observe the formation of the phospho-TTP/14-3-3 complex.In the same samples TNF-αexpression was also evaluated as major factor of the pro-inflammatory activity.RESULTS In this work we studied indirectly the phosphorylation of TTP through the binding with the chaperone protein 14-3-3.In inflamed and non-inflamed colon mucosa of IBD paediatric patients immunoblot assay demonstrated a higher expression of the TTP in inflamed samples respect to the non-inflamed;the coimmunoprecipitated 14-3-3 protein showed the same trend of expression.In the TNF-αgene expression analysis higher levels of the cytokine in inflamed tissues compared to controls were evident.The same experiments were conducted on macrophages from IBD paediatric patients and healthy controls.The immunoblot results demonstrated a high expression of both TTP and co-immunoprecipitated 14-4-3 protein in IBD-derived macrophages in comparison to healthy donors.TNF-αprotein levels from macrophages lysates showed the same trend of expression in favour of IBD paediatric patients compared to healthy controls.CONCLUSION In this work,for the first time,we describe a relation between phospho-TTP/14-3-3 complex and IBD.Indeed,a higher expression of TTP/14-3-3 was recorded in IBD samples in comparison to controls.
基金Supported by Biomedical Research Institute Fund (GNUHBRIF-2013-0002) from the Gyeongsang National University Hospital
文摘AIM: To examine the effect of intravitreal adenoviral vector-mediated tristetraprolin(Ad-TTP) on VEGF m RNA expression in a rat model of laser-induced choroidal neovascularization.METHODS: Ad-TTP was prepared using a commercial kit. Retinal laser-induced photocoagulation(10 spots per eye) was performed on rats in this experimental choroidal neovascularization(CNV) model. Rats were divided into four groups: control(single intravitreal injection of balanced salt solution, n =10), laser-induced CNV(photocoagulation only, n =20), laser-induced CNV plus Ad-TTP injection(photocoagulation plus a single intravitreal Ad-TTP injection, n =20) and Ad-TTP injection only(n =10). Changes in choroidal morphology were evaluated in ten rats in the laser only and the laser plus Ad-TTP groups. Two weeks after laser injury, the size of CNV was calculated by perfusion with high-molecular-weight fluorescein isothiocyanate(FITC)-dextran. VEGF m RNA expression in retina-choroid tissue from ten rats in each group was measured by reverse transcription polymerase chain reaction(RT-PCR). RESULTS: Two weeks after treatment, the area of laser-induced CNV was reduced by approximately 60% in the rats given the Ad-TTP injection compared with that in the laser-only group. There was a tendency toward decreased VEGF m RNA expression in the Ad-TTP injection groups.CONCLUSION: A single intravitreal injection of Ad-TTP significantly suppressed CNV size in this experimental laser-induced CNV model. Ad-TTP injection also decreased VEGF m RNA expression compared with that inthe laser-induced CNV group. The present study is meaningful as the first study to investigate the effect of tristetraprolin delivered via intravitreal injection.
文摘Since we had previously demonstrated that siRNAs to tristetraprolin (TTP) markedly inhibited insulin stimulation of hepatic HMG-CoA reductase (HMGR) transcription, we investigated the effects of transfecting rat liver with TTP constructs. We found that transfecting diabetic rats with TTP did not increase HMGR transcription but rather led to modest inhibition. We then investigated whether co-transfection with protein kinase B, hepatic form (AKT2), might lead to phosphorylation and result in activation of HMGR transcription. We found that this treatment resulted in near complete inhibition of transcription. Transfection with peroxisome proliferator-activated receptor g coactivator (PGC-1a) also inhibited HMGR transcription. These results show that although TTP is needed for activation of HMGR transcription, it cannot by itself activate this process. AKT2 and PGC-1a, which mediate the activation of gluconeogenic genes by insulin, exert the opposite effect on HMGR.
基金supported by grants from Guangzhou Major Scienceand Technology Projects(2011Y1-00017-3)Nanyang Qi Wei Microecological Gene Science and Technology Development Co.,Ltd~~
文摘目的研究elavl1/HuR对斑马鱼肠神经系统早期发育的影响。方法应用免疫组化方法检测elavl1、HuC/HuD在野生型斑马鱼肠道的表达。挑选25个斑马鱼胚胎,应用Western Blot法检测elavl1在受精后24~96 h野生型斑马鱼中的蛋白表达水平。在野生型斑马鱼胚胎处于1细胞期时分别显微注射4 ng、8 ng elavl1a morpholino和8 ng control morpholino,挑选25个受精后24 h的胚胎采用Western Blot法检测elavl1的蛋白表达,免疫组化检测受精后72 h、96 h的胚胎斑马鱼肠道神经元的分布情况。结果elavl1在斑马鱼全身都有表达,而HuC/HuD在第四天斑马鱼肠道部位出现表达;在受精后24~96 h时期,elavl1在斑马鱼中的蛋白表达量随斑马鱼生长而增加,且受精后96 h时elavl1蛋白量明显高于受精后24 h、48 h、72 h,差异有统计学意义(P<0.05);分别注射4 ng、8 ng elavl1a Mo和8 ng Con Mo后,发现注射8 ng Con Mo组elavl1蛋白含量高于其他两组,且差异有统计学意义(P<0.05);敲降elavl1a后免疫组化显示在受精后72 h时三组都不能在肠道末端明显观察到神经元,受精后96 h时可以观察到elavl1a Mo 8 ng组和elavl1a Mo 4 ng组较Con Mo 8 ng组斑马鱼肠神经元数量有所减少,每组随机抽取5条斑马鱼统计肠道末端到前面四个体节长度的肠道神经元数量,发现elavl1a Mo 8 ng组和elavl1a Mo 4 ng组较Con Mo 8 ng组斑马鱼肠神经元数量差异有统计学意义(P<0.05)。结论elavl1a是斑马鱼肠神经发育过程中重要基因,敲降elavl1a会减少肠神经元细胞数量。
