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Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice 总被引:7,自引:0,他引:7
Many phase II protective genes contain a cis -acting enhancer region known as the antioxidant response element (ARE). Increased expression of these genes contributes to the protection of cells from oxidative stress. Transgenic reporter mice were created that carry in their genome the core ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene. Primary cortical cultures derived from these mice were treated with tBHQ resulting in a dose-dependent increase in hPAP activity. Histochemical staining for hPAP activity was observed in both glia and neurons from tBHQ-treated cultures. The tBHQ-mediated increase in hPAP was not affected by the antioxidant glutathione monoethyl ester (GSHEE), whereas the increase in hPAP following DEM treatment was completely blocked by GSHEE. Pre-treatment of cultures with the PI3-kinase inhibitor LY 294002 demonstrated a dose-dependent decrease in tBHQ-induced hPAP activity. In addition, the tBHQ-mediated expression of ARE-driven genes in primary cortical cultures was blocked by LY 294002. Interestingly, basal expression of Nrf2 was also inhibited by LY 294002. We theorize that increased levels of genes controlled by the ARE are important for cellular protection against oxidative stress. These ARE-hPAP transgenic mice will be an important in vivo model for testing our hypothesis. 相似文献
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Shan Y Lambrecht RW Hong Lu T Bonkovsky HL 《Archives of biochemistry and biophysics》1999,372(2):224-229
Heme oxygenase catalyzes the first and rate-controlling step of heme catabolism. Induction of heme oxygenase-1 can be caused by numerous factors, including heme, other metalloporphyrins, transition metal ions, heat shock, ultraviolet light, phorbol esters, sodium arsenite, and phenylarsine oxide (PAO). Induction of this enzyme may protect cells from oxidative damage. Using heme oxygenase-1 promoter/reporter gene constructs, we have previously reported that the sodium arsenite-mediated induction of heme oxygenase-1 in chick embryo liver cells and chicken hepatoma (LMH) cells involves an AP-1 element. We have now investigated whether the PAO-mediated induction of heme oxygenase-1 also involves an AP-1 element. Primary cultures of chick embryo liver cells were transiently transfected with heme oxygenase-1 promoter/reporter gene constructs, treated with PAO, and reporter gene activities were measured. We found that the PAO-mediated increase in reporter gene activity was dose- and time-dependent. This activity was decreased by prior treatment with N-acetylcysteine. Studies with mutated constructs showed that both an AP-1 element and a metal responsive element are involved in the PAO-mediated induction of the heme oxygenase-1 reporter construct. Electrophoretic mobility shift assays showed that nuclear proteins from PAO-treated cells had increased binding to an AP-1 probe, and that this increase was abrogated by N-acetylcysteine. These findings support the hypothesis that the PAO-mediated induction of heme oxygenase-1 is caused by activation of AP-1 and MRE/cMyc elements and may involve nuclear proteins whose states of phosphorylation determine binding to regulatory elements, and thus the level of expression of heme oxygenase-1. 相似文献
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The ability to unambiguously mark a cell's genotype is essential for studies in which genetically distinct cell populations must be distinguished from one another in vivo. One approach to this challenge has been the creation of transgenic mice expressing a transgene marker that is easily detectable, with no background staining. Multiple transgenic mouse strains bearing constructs with different combinations of promoter elements and coding sequences have been described, each with its own advantages and limitations. In this report we describe the use of an 800-bp promoter fragment isolated from the beta(geo) integration site in ROSA26 mice to target expression of two marker genes. We demonstrate that the ROSA26 promoter directs ubiquitous expression of human placental alkaline phosphatase and enhanced green fluorescent protein during embryonic and postnatal development in mouse and rat. We further demonstrate the general utility of these transgenes for marking donor cells in transplantation studies. 相似文献
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Lefevre P Tripon E Plumelet C Douaire M Diot C 《Biochemical and biophysical research communications》2001,280(1):25-31
In chicken, adiposity is influenced by hepatic stearoyl-CoA desaturase (SCD) 1. This gene is up-regulated by low-fat high-carbohydrate diet and down-regulated by addition of polyunsaturated fatty acids (PUFA). In this study, we present evidence for an inhibition of chicken SCD1 expression by PUFA using reporter gene constructs in transient transfection assays. This inhibition does not involve the peroxisome proliferator-activated receptor pathway, in contrast with what has been observed in rodents. We were able to localise a PUFA as well as an insulin response element within the -372/+125 bp region of the promoter. Sequence analyses of this region allowed identification of several cis-regulatory elements: A sterol regulatory element (SRE) and a juxtaposed NF-Y element which have been shown to be involved in the regulation of mouse SCD genes by PUFA. In addition, we identified an overlapping Sp1/USF motif, which was described to play a role in insulin/glucose and PUFA regulation of fatty synthase, ATP-citrate-lyase, and leptin genes. These data provide the first characterisation of the chicken SCD1 promoter and putative cis-sequences involved in the regulation of this gene by PUFA and insulin. 相似文献
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Inhibition of HIV-LTR gene expression by oligonucleotides targeted to the TAR element. 总被引:15,自引:4,他引:11
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T Vickers B F Baker P D Cook M Zounes R W Buckheit Jr J Germany D J Ecker 《Nucleic acids research》1991,19(12):3359-3368
All human immunodeficiency virus mRNAs contain a sequence known as TAR (trans-activating responsive sequence). The TAR element forms a stable RNA stem-loop structure which binds the HIV tat (trans-activator) protein and mediates increased viral gene expression. In principle, molecules which bind to the TAR RNA structure would inhibit trans-activation by perturbing the native RNA secondary structure. We have constructed a series of phosphodiester and phosphorothioate antisense oligonucleotides which specifically bind to the HIV TAR element. Specific binding to the TAR element was demonstrated in vitro with enzymatically synthesized TAR RNA. The TAR-directed phosphorothioates inhibited trans-activation in a sequence-dependent fashion in a cell culture model using an HIV LTR/human placental alkaline phosphatase gene fusion and tat protein supplied in trans. The molecules also inhibited HIV replication in both acute and chronically infected viral assays, but without sequence specificity. We have constructed a series of vectors consisting of the MMTV promoter and 5'-untranslated region of four different mRNAs, including the TAR region, to study the effect of TAR on gene expression in heterologous systems. The results suggest that, in the absence of the HIV LTR, the TAR element has a repressive effect on gene expression, which is relieved by tat. 相似文献
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Both Pit-1 and the estrogen receptor are required for estrogen responsiveness of the rat prolactin gene 总被引:10,自引:0,他引:10
R N Day S Koike M Sakai M Muramatsu R A Maurer 《Molecular endocrinology (Baltimore, Md.)》1990,4(12):1964-1971
To examine the functional relationship between distinct cis-active elements within the distal enhancer region of the rat PRL gene, we have used deletional and mutational analysis of that region in transient transfection studies in GH3 pituitary tumor cells. Results from these studies demonstrate that the region of the PRL distal enhancer containing the Pit-1-binding sites is critical not only for enhancer activity and the response to cAMP, but also for the response to estradiol. An interaction of the estrogen receptor with factors conferring basal enhancer activity is suggested by studies with a mutant distal enhancer region in which the PRL estrogen response element was converted to a palindromic estrogen response element. To directly examine potential interactions, cotransfection studies using PRL distal enhancer reporter gene constructs and expression vectors for Pit-1 and rat estrogen receptor were performed in two heterologous cell lines. The activity of the reporter gene under the control of the PRL distal enhancer linked to either the thymidine kinase promoter or the PRL proximal promoter was not significantly altered by cotransfection with the Pit-1 expression vector in COS-1 or RAT-1 cells. Coexpression of these reporter constructs and an expression vector for estrogen receptor resulted in only a slight response to estradiol. However, when both Pit-1 and estrogen receptor were cotransfected with the distal enhancer reporter gene, a marked induction was observed in response to estradiol, and this activity was dependent upon the concentration of the Pit-1 expression vector.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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