Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was construc...Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was constructed and used to transfect humanhepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor(AhR)-meditated luciferase gene expression. The reliability of luciferase induction in thiscell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection timewas examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. Result The results suggested that theluciferase activity in recombinant cells was peaked at about 4 h and then decreased to astable activity by 14 h after TCDD treatment. The detection limit of this cell line was0.11pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, thedetection time is 68 h shorter and the detection procedure is also simpler.展开更多
Brassica napus(cv.Madrigal)seedlings pre-treated with ascospores of Leptosphaeria biglobosa or foliar sprays of either acibenzolar-S-methyl(ASM)or menadione sodium bisulphite(MSB)were chal- lenge inoculated with L.mac...Brassica napus(cv.Madrigal)seedlings pre-treated with ascospores of Leptosphaeria biglobosa or foliar sprays of either acibenzolar-S-methyl(ASM)or menadione sodium bisulphite(MSB)were chal- lenge inoculated with L.maculans ascospores and assessed for phoma leaf spot development and tissue morphology and gene expression responses to infection.Rates of increase in phoma leaf spot area 8―21 d after challenge inoculation were significantly greater on water pre-treated plants than on plants pre-treated with L.biglobosa,ASM or MSB on both pre-treated leaves(local effect)and younger leaves without pre-treatment(systemic effect).Ninety-six h after challenge inoculation,the invasive hyphae of L.maculans were encircled by rings of necrotic mesophyll cells on leaves pre-treated with L. biglobosa,ASM or MSB but not those pre-treated with water.Quantification of transcript levels of genes commonly used as markers of the major defence signalling pathways(PDF1.2,PR-1,NPR1,APX, CHB4)0–96 h after L.maculans challenge inoculation showed expression patterns indicating prefer- ential activation of the jasmonate/ethylene pathway and involved induction of NPR1 locally and sys- temically in leaves of plants pre-treated with L.biglobosa ascospores.展开更多
Expression of cellular genes is regulated by binding of transcription factors to their promoter, either activating or inhibiting transcription of a gene. Particularly interesting is the case when the expressed protein...Expression of cellular genes is regulated by binding of transcription factors to their promoter, either activating or inhibiting transcription of a gene. Particularly interesting is the case when the expressed protein regulates its own transcription. In this paper, the features of this self-regulating process are investigated. In the presented model here, the gene can be in two states. Either a protein is bound to its promoter or not. The steady state distributions of protein during and just before switching from one state to the next state are analyzed. Moreover, a powerful numerical method based on the corresponding master equation to compute the protein distribution in the steady state is presented and compared to an already-existing method. Additionally the special case of self-regulation, in which protein can only be produced, if one of these proteins is bound to the promoter region, is analyzed. Furthermore, a self-regulating gene is compared to a similar gene, which also has two states and produces the same amount of proteins but is not regulated by its protein-product.展开更多
文摘Objective To improve the chemically-activated luciferase expression (CALUX)bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms ofDLCs. Method A recombinant vector was constructed and used to transfect humanhepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor(AhR)-meditated luciferase gene expression. The reliability of luciferase induction in thiscell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection timewas examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. Result The results suggested that theluciferase activity in recombinant cells was peaked at about 4 h and then decreased to astable activity by 14 h after TCDD treatment. The detection limit of this cell line was0.11pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%.Conclusion The luciferase induction is 30-fold more sensitive than EROD induction, thedetection time is 68 h shorter and the detection procedure is also simpler.
文摘应用化学诱导荧光素酶表达基因法(chemical activated luciferase gene expression,CALUX)调研了小清河(济南段)主要支流的多种内分泌干扰物综合激素效应,明确了4类内分泌干扰物的含量水平及分布特征。结果表明,调研河流的二噁英类、雌激素类和肾上腺皮质激素类的年平均毒性当量浓度范围为0.020~0.040 ng 2,3,7,8-TCDD TEQ·L^(-1)、0.007~0.050 ng E2-eq·L^(-1)和1.8~9.6 ng Dex-eq·L^(-1)。二噁英类和雌激素类的含量水平相对较低,雄激素类内分泌干扰物的污染风险较低,肾上腺皮质激素类是其内分泌干扰物污染的最大贡献者。二噁英类检出水平无明显的季节性和区域性差别,雌激素类季节性变化明显,春、冬两季检出率高,肾上腺皮质激素类分布的季节性分布不明显,但区域性分布规律明显,农业区支流的检出频次和含量水平明显低于居民区和商业区支流。
基金Supported by a Rothamsted International Fellowship to LIU ShengYi,a Royal Society China Fellowship to LIU RenHu,the UK Biotechnology and Biological Sciences Research Council(BBSRC),Department for Environment,Food and Rural Affairs(Defra),the Chadacre Trust,UK and the Ministry of Agriculture,P.R.China
文摘Brassica napus(cv.Madrigal)seedlings pre-treated with ascospores of Leptosphaeria biglobosa or foliar sprays of either acibenzolar-S-methyl(ASM)or menadione sodium bisulphite(MSB)were chal- lenge inoculated with L.maculans ascospores and assessed for phoma leaf spot development and tissue morphology and gene expression responses to infection.Rates of increase in phoma leaf spot area 8―21 d after challenge inoculation were significantly greater on water pre-treated plants than on plants pre-treated with L.biglobosa,ASM or MSB on both pre-treated leaves(local effect)and younger leaves without pre-treatment(systemic effect).Ninety-six h after challenge inoculation,the invasive hyphae of L.maculans were encircled by rings of necrotic mesophyll cells on leaves pre-treated with L. biglobosa,ASM or MSB but not those pre-treated with water.Quantification of transcript levels of genes commonly used as markers of the major defence signalling pathways(PDF1.2,PR-1,NPR1,APX, CHB4)0–96 h after L.maculans challenge inoculation showed expression patterns indicating prefer- ential activation of the jasmonate/ethylene pathway and involved induction of NPR1 locally and sys- temically in leaves of plants pre-treated with L.biglobosa ascospores.
文摘Expression of cellular genes is regulated by binding of transcription factors to their promoter, either activating or inhibiting transcription of a gene. Particularly interesting is the case when the expressed protein regulates its own transcription. In this paper, the features of this self-regulating process are investigated. In the presented model here, the gene can be in two states. Either a protein is bound to its promoter or not. The steady state distributions of protein during and just before switching from one state to the next state are analyzed. Moreover, a powerful numerical method based on the corresponding master equation to compute the protein distribution in the steady state is presented and compared to an already-existing method. Additionally the special case of self-regulation, in which protein can only be produced, if one of these proteins is bound to the promoter region, is analyzed. Furthermore, a self-regulating gene is compared to a similar gene, which also has two states and produces the same amount of proteins but is not regulated by its protein-product.
