Purpose: The transforming growth factor-beta (TGF-β) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-β signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro.
Methods: The inhibitory effects of cholest-4-en-3-one on TGF-β-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-β receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation.
Results: Cholest-4-en-3-one attenuated TGF-β signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-β-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling.
Conclusions: Our results suggest that cholest-4-en-3-one inhibits TGF-β signaling may be due, in part to the translocation of TGF-β receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-β deficiency. 相似文献
Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment. 相似文献
Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of
derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase
inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES
cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect
of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented
in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell
derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established
ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and
germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells.
During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection
and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with
Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which
is essential for reducing variability in the results obtained from different cell lines. 相似文献
The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species. 相似文献
The response of suspension-cultured pear (Pyrus communis cv Bartlett) cells to heat stress was studied using three viability tests: regrowth (culture growth during 10 days after stress); triphenyltetrazolium chloride reduction; and electrolyte leakage. Critical (50% injury) temperatures for a 20-minute exposure were 42°, 52°, and 56°C, respectively, for these viability tests. Electrolyte leakage had the lowest temperature coefficient. Heat stress inhibition of triphenyltetrazolium chloride reducing capacity was much greater if the viability test was conducted 3 days, rather than immediately, after the stress treatment. Consistent with a major role for indirect metabolic strain in heat injury, treatment with 3.6 micromolar cycloheximide and heat stress (20 minutes at 43°C) affected culture regrowth similarly. We conclude that the measurements of direct response are not adequate substitutes for regrowth tests in assessing heat injury to cultured plant cells. 相似文献