AIM: To investigate the(-)-epigallocatechin-3-gallate(EGCG) binding to transforming growth factor-β(TGF-β) type Ⅱ receptor(TGFRⅡ).METHODS: The expression of α-smooth muscle actin(α-SMA) was used as a marker for ...AIM: To investigate the(-)-epigallocatechin-3-gallate(EGCG) binding to transforming growth factor-β(TGF-β) type Ⅱ receptor(TGFRⅡ).METHODS: The expression of α-smooth muscle actin(α-SMA) was used as a marker for fibrotic change inhuman lung fibroblast MRC-5 cells. The α-SMA expression level was determined by western blotting and immunohistological analysis. We examined whether the anti-fibrotic effects of EGCG on MRC-5 cells was dependent on antioxidant mechanism by using edaravone and N-acetylcysteine(NAC). The suppression effects of EGCG on Smad2/3 activation were studied by confocal fluorescence microscopy. The binding of EGCG to recombinant TGFRⅡ protein was analyzed by immunoprecipitation and affinity chromatography.RESULTS: When MRC-5 cells were treated with TGF-β, EGCG decreased the expression of α-SMA in a dose dependent manner, whereas catechin did not influence the α-SMA expression in the cells. Except for EGCG, antioxidant compounds(e.g., edaravone and NAC) had no effects on the TGF-β-induced α-SMA expression. Nuclear localization of phosphorylated Smad2/3 was observed after TGF-β treatment; however, EGCG treatment attenuated the nuclear transportation of Smad2/3 in the presence or absence of TGF-β. After a TGFRⅡ expression vector was introduced into COS-7 cells, cell lysates were untreated or treated with EGCG or catechin. The immunoprecipitation experiments using the lysates showed that EGCG dose-dependently bound to TGFRⅡ and that catechin did not at all. Affinity chromatography study indicated that EGCG would bind to TGFRⅡ.CONCLUSION: Our results demonstrate that EGCG interacts with TGFRⅡ and inhibits the expression of α-SMA via the TGF-β-Smad2/3 pathway in human lung fibroblast MRC-5 cells.展开更多
The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modul...The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modulation of bacterial colonization on the tooth surface. Previously, we found a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding the proline-rich protein P-B. To examine whether E. coli genomic DNA is present in bovine genomic DNA, we constructed a plasmid library from the bovine DNA. Although results so far have failed to indicate any such presence in the bovine nucleotides examined, experiments using the polymerase chain reaction (PCR) revealed unusual amplification of nucleotides. As an initial step of the study on possible occurrence of E. coli-derived nucleotide sequence in bovine genomic DNA of P-B, we examined the structure of the PCR products generated by unexpected amplification. The determined structure of the PCR products suggested that when the two single strand chains that grow by reading the sequence of the respective template reached a hybridizable short nucleotide structure, they became hybridized and subsequent elongation was continued by reading the sequence of the counter chain that had been elongated by reading the template. It is possible that elongation of the chain was interrupted once before the completion of amplification due to the template’s palindrome region which had formed a double strand structure during the PCR process. Such an unusual amplification made possible under certain conditions in a DNA sequence may be one of the mechanisms for the genetic recombination found in our previous study.展开更多
文摘AIM: To investigate the(-)-epigallocatechin-3-gallate(EGCG) binding to transforming growth factor-β(TGF-β) type Ⅱ receptor(TGFRⅡ).METHODS: The expression of α-smooth muscle actin(α-SMA) was used as a marker for fibrotic change inhuman lung fibroblast MRC-5 cells. The α-SMA expression level was determined by western blotting and immunohistological analysis. We examined whether the anti-fibrotic effects of EGCG on MRC-5 cells was dependent on antioxidant mechanism by using edaravone and N-acetylcysteine(NAC). The suppression effects of EGCG on Smad2/3 activation were studied by confocal fluorescence microscopy. The binding of EGCG to recombinant TGFRⅡ protein was analyzed by immunoprecipitation and affinity chromatography.RESULTS: When MRC-5 cells were treated with TGF-β, EGCG decreased the expression of α-SMA in a dose dependent manner, whereas catechin did not influence the α-SMA expression in the cells. Except for EGCG, antioxidant compounds(e.g., edaravone and NAC) had no effects on the TGF-β-induced α-SMA expression. Nuclear localization of phosphorylated Smad2/3 was observed after TGF-β treatment; however, EGCG treatment attenuated the nuclear transportation of Smad2/3 in the presence or absence of TGF-β. After a TGFRⅡ expression vector was introduced into COS-7 cells, cell lysates were untreated or treated with EGCG or catechin. The immunoprecipitation experiments using the lysates showed that EGCG dose-dependently bound to TGFRⅡ and that catechin did not at all. Affinity chromatography study indicated that EGCG would bind to TGFRⅡ.CONCLUSION: Our results demonstrate that EGCG interacts with TGFRⅡ and inhibits the expression of α-SMA via the TGF-β-Smad2/3 pathway in human lung fibroblast MRC-5 cells.
文摘The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modulation of bacterial colonization on the tooth surface. Previously, we found a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding the proline-rich protein P-B. To examine whether E. coli genomic DNA is present in bovine genomic DNA, we constructed a plasmid library from the bovine DNA. Although results so far have failed to indicate any such presence in the bovine nucleotides examined, experiments using the polymerase chain reaction (PCR) revealed unusual amplification of nucleotides. As an initial step of the study on possible occurrence of E. coli-derived nucleotide sequence in bovine genomic DNA of P-B, we examined the structure of the PCR products generated by unexpected amplification. The determined structure of the PCR products suggested that when the two single strand chains that grow by reading the sequence of the respective template reached a hybridizable short nucleotide structure, they became hybridized and subsequent elongation was continued by reading the sequence of the counter chain that had been elongated by reading the template. It is possible that elongation of the chain was interrupted once before the completion of amplification due to the template’s palindrome region which had formed a double strand structure during the PCR process. Such an unusual amplification made possible under certain conditions in a DNA sequence may be one of the mechanisms for the genetic recombination found in our previous study.
