Phosphorylation of β-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition （EMT）. Introducing mimic peptide of tyrosine residue 654 domain of β-catenin into cells may i...Phosphorylation of β-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition （EMT）. Introducing mimic peptide of tyrosine residue 654 domain of β-catenin into cells may influence phosphorylation of β-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell permeable peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic peptide of β-catenin （PM） with fused penetratin was constructed, purified and then detected for the penetration of the mimic peptide into human renal tubular epithelial cells （HK-2）. The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of β-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.展开更多
Corticotropin-releasing factor(CRF), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to inve...Corticotropin-releasing factor(CRF), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN(0.1, 1 and 10 mu M) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1(CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2(CRF-R2) antagonist antisauvagine-30(anti-Svg-30). The results also showed that UCN caused a rapid peak increase inCa2+(i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase inCa2+(i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN onCa2+(i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value ofCa2+(i)(P < 0.01). Taken together, our present study suggested that UCN induced the increase of Ca2+(i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation. Copyright (c) 2008 S. Karger AG, Basel.展开更多
AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-...AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide.RAW264.7 cells were incubatedwith these antibodies either before or after ricin exposure.The changes in cytotoxicity were estimated by MTT assay.Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies.Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION:Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for postexposure treatment of ricin intoxication.展开更多
基金the National Natural Sciences Foundation of China (No. 30370657).
文摘Phosphorylation of β-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition （EMT）. Introducing mimic peptide of tyrosine residue 654 domain of β-catenin into cells may influence phosphorylation of β-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell permeable peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic peptide of β-catenin （PM） with fused penetratin was constructed, purified and then detected for the penetration of the mimic peptide into human renal tubular epithelial cells （HK-2）. The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of β-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.
文摘Corticotropin-releasing factor(CRF), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. The purpose of this study was to investigate the effect of urocortin (UCN), a 40-amino-acid CRF family peptide, on degranulation and intracellular calcium of rat lung mast cells. The activation and degranulation of mast cells were observed by Toluidine blue staining and transmission electron microscope. The intracellular calcium was investigated using confocal laser scanning microscopy and flow cytometry. The results indicated that all the three different concentrations of UCN(0.1, 1 and 10 mu M) significantly induced the activation and degranulation of rat lung mast cells in vitro. This effect was markedly blocked by selective CRF receptor 1(CRF-R1) antagonist antalarmin, but not by specific CRF receptor 2(CRF-R2) antagonist antisauvagine-30(anti-Svg-30). The results also showed that UCN caused a rapid peak increase inCa2+(i) at point of 300s after UCN treatment, followed by a decrease to a sustained plateau phase. The peak increase inCa2+(i) induced by UCN was significantly inhibited by antalarmin, but not by anti-Svg-30. This effect of UCN onCa2+(i) in rat lung mast cells was also found by flow cytometry. Regression analysis revealed a positive correlation between mast cells degranulation extent and the maximum value ofCa2+(i)(P < 0.01). Taken together, our present study suggested that UCN induced the increase of Ca2+(i) and degranulation of rat lung mast cells through CRF-R1. These findings may have implications for the pathophysiology of allergic and inflammatory lung disorders such as asthma, which is closely associated with mast cell activation and degranulation. Copyright (c) 2008 S. Karger AG, Basel.
基金Supported by NIEHS Center Grant ES00260(Tchou-Wong KM)NIAID R21 Grant AI059476(Tchou-Wong KM)from the National Institutes of Health
文摘AIM:To evaluate the ability of anti-ricin A-chain antibodies,delivered intracellularly,to protect against ricininduced cytotoxicity in RAW264.7 cells. METHODS:Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide.RAW264.7 cells were incubatedwith these antibodies either before or after ricin exposure.The changes in cytotoxicity were estimated by MTT assay.Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS:Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies.Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION:Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for postexposure treatment of ricin intoxication.
