To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.METHODSFor this purpose, we used rat Sprague Dawley and various colon carcinoma ce...To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.METHODSFor this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method.RESULTSCRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels.CONCLUSIONOur findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.展开更多
In this paper, the author focuses on spaces used for personal hygiene--the bathroom. The paper begins with a description of the hygienic movement in the late 19th century. At that time, urinating took place in semi-pu...In this paper, the author focuses on spaces used for personal hygiene--the bathroom. The paper begins with a description of the hygienic movement in the late 19th century. At that time, urinating took place in semi-public spaces outside the dwelling. Today, the WC has moved well into the dwelling, and in many dwellings, the bathroom has developed as the most private space. Thus, the bathroom can be regarded as the last domain of privacy in today＇s housing, and in a number of new dwellings this quality is exploited in new ways. The development of ＂space for hygiene＂ in the 20th century will be studied by analyzing the spatial organization of dwellings.展开更多
In the search for new anti-tumor agents, exploiting features such as the flexibility of coordination modes of metals have become an alternative strategy for synthesizing pharmaceuticals. It has been shown that the Cu...In the search for new anti-tumor agents, exploiting features such as the flexibility of coordination modes of metals have become an alternative strategy for synthesizing pharmaceuticals. It has been shown that the CuDP （copper（II）, doxycycline, and 1,10-phenanthroline） complex cleaves DNA strands by an oxidative mechanism and by intercalating the major groove, resulting in a cytotoxic action. The objective of this study was to assess the mutageinc/recombinogenic effects of the CuDP complex in vivo using the SMART （Somatic Mutation and Recombination Test） in Drosophila melanogaster. Treatments were carried out with third instar larvae at the standard cross and high bioactivation cross using three concentrations of CuDP （6.92, 13.84 or 27.67 mM）. The mutagenic doxorubicin （0.4 mM） was used as a positive control and reverse osmosis water as a negative control. For each compound, marked trans-heterozygous and balanced heterozygous individuals were analyzed to determine the mutational and recombinogenic events occurring in the cells, We found that CuDP significantly increased the frequencies of mutant cells in both standard and high bioactivation crosses, mostly by induction of recombination. These data show that CuDP is a direct recombinogenic agent that is independent of bioactivation,展开更多
基金Supported by grants from Association pour la Recherche sur le Cancer,Ligue Nationale contre le Cancer,No.GEFLUC and No.ESPOIR
文摘To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation.METHODSFor this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method.RESULTSCRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels.CONCLUSIONOur findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.
文摘In this paper, the author focuses on spaces used for personal hygiene--the bathroom. The paper begins with a description of the hygienic movement in the late 19th century. At that time, urinating took place in semi-public spaces outside the dwelling. Today, the WC has moved well into the dwelling, and in many dwellings, the bathroom has developed as the most private space. Thus, the bathroom can be regarded as the last domain of privacy in today＇s housing, and in a number of new dwellings this quality is exploited in new ways. The development of ＂space for hygiene＂ in the 20th century will be studied by analyzing the spatial organization of dwellings.
文摘In the search for new anti-tumor agents, exploiting features such as the flexibility of coordination modes of metals have become an alternative strategy for synthesizing pharmaceuticals. It has been shown that the CuDP （copper（II）, doxycycline, and 1,10-phenanthroline） complex cleaves DNA strands by an oxidative mechanism and by intercalating the major groove, resulting in a cytotoxic action. The objective of this study was to assess the mutageinc/recombinogenic effects of the CuDP complex in vivo using the SMART （Somatic Mutation and Recombination Test） in Drosophila melanogaster. Treatments were carried out with third instar larvae at the standard cross and high bioactivation cross using three concentrations of CuDP （6.92, 13.84 or 27.67 mM）. The mutagenic doxorubicin （0.4 mM） was used as a positive control and reverse osmosis water as a negative control. For each compound, marked trans-heterozygous and balanced heterozygous individuals were analyzed to determine the mutational and recombinogenic events occurring in the cells, We found that CuDP significantly increased the frequencies of mutant cells in both standard and high bioactivation crosses, mostly by induction of recombination. These data show that CuDP is a direct recombinogenic agent that is independent of bioactivation,
