• Tuberculosis and Respiratory Diseases
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• v.58 no.6
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• pp.590-599
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• 2005

Object : Cigarette smoking is a major cause of mucus hypersecretion, which is a pathophysiological feature of many inflammatory airway diseases. Mucins, which are an important part of the airway mucus, are synthesized from the Muc gene in airway epithelial cells. However, the signaling pathways for cigarette smoke-induced mucin synthesis are unknown. The aim of this study was to determine the signal pathway for smoking induced Muc5ac gene expression. Methods : A549 cells were cultured and transiently transfected with the Muc5ac promoter fragment. These cells were stimulated with 5% cigarette smoke extract (CSE) alone or with CSE after a pretreatment with various signal transduction pathway inhibitors (AG1478, PD98059 and SB203580). The Muc5ac promoter activity was examined using the luciferase reporter system, and the level of phosphorylated EGFR, ERK1/2, p38 MAPK and JNK were all examined using Western blot analysis. Muc5ac mRNA expression was also examined using reverse transcriptase polymerase chain reactions (RT-PCR). Results : 1. The peak level of luciferase activity of the Muc5ac promoter was observed at 5% concentration and after 3 hours of incubation with the CSE. The level of EGFR phosphorylation and the luciferase activity of the transfected cells caused by the CSE were significantly suppressed by AG1478 or PD98059 (P<0.01). 2. CSE phosphorylated ERK1/2 or p38 MAPK but not JNK. The Muc5ac mRNA expression level was increased by the CSE but that was suppressed by PD98059 or AG1478. 3. The CSE-induced phosphorylation of ERK1/2 was blocked by PD98059 and that of p38 MAPK was blocked by either PD98059 or SB203580. Either PD98059 or SB203580 suppressed the luciferase activity of the transfected cells (P<0.0001). Conclusion : The Muc5ac mRNA expression level was increased by the CSE. The increased CSE-induced transcriptional activity was mediated via EGF receptor activation, which led to ERK1/2 and p38 MAPK phosphorylation.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.39 no.1
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• pp.75-81
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• 2013

Chrysin (5,7-dihydroxyflavone) is a natural flavonoid found in various plants and foods such as propolis and honey. It has been reported that chrysin has various biological effects including antioxidant, anti-aging, anti-inflammatory and anti-cancer. In this study, we investigated the effect of chrysin on the transcriptional activity of VDR in human epidermal keratinocytes by performing dual-luciferase assay. Chrysin significantly induced the transcriptional activity of VDR in a concentration-dependent manner. The VDR mRNA expression was investigated by quantitative real time PCR and chrysin increased the VDR mRNA expression in normal human epidermal keratinocytes. We also found that chrysin increased the expression of keratinocyte differentiation markers such as keratin 10, involucrin and filaggrin. Therefore, the results suggest that chrysin can stimulate the differentiation of human keratinocytes by increasing transcriptional activity of VDR.

• Proceedings of the Korean Society of Toxicology Conference
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• 2001.10a
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• pp.202-202
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• 2001

Stable MCF-7-ERE cells, in which pERE-Luc reporter gene has been stably integrated into the genome of the MCF-7 cells, were used to detect the estrogenic activity of various dietary flavonoids.in either pure chemical or mixtures. Estradiol (E2) induced luciferase activity in dose dependent manner and this activity was inhibited by tamoxifen (Tam) concomitant treatment.(omitted)

• Korean Journal of Plant Resources
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• v.29 no.5
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• pp.620-624
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• 2016

The Wnt/β-catenin signaling pathway is mandatory in adipogenesis. In this study, we investigated the applicability of functional materials for the treatment of obesity by examining Wnt/β-catenin pathway reporter activity related to adipocyte differentiation inhibiting with korean native plant extracts. The luciferase activity of HEK 293-TOP cells increased the reporter activity approximately 152% and 130% by treatment with Sanguisorba officinalis and Thuja orientalis, respectively. Ricinus communis were represented about 90% higher activity, two samples(Rosa rugosa and Sophorae Flos) showed 80% higher activity than the control. Three samples of plant extracts (Zanthoxylum piperitum, Pueraria thunbergiana, Solanum nigrum) were about 70% higher activity compared with the non-treated control. Cytotoxicity of plant extracts was not detected in the rat neural stem cells. These results suggest that the selected eight plant extracts are safe compounds. Our findings indicate that Wnt/β-catenin pathway reporter activity could be used for high throughput screening system. In addition, the plant extracts selected as candidates for adipocyte differentiation inhibiting may be potential therapeutic agents for obesity, it will be exploring the possibility of developing an anti-obesity materials through further experiments with selected plant extracts.

• Environmental Mutagens and Carcinogens
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• v.24 no.3
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• pp.121-127
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• 2004

Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounds such as policyclic aromatic hydrocarbon(PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in Hepa-I and MCF-7 cells, 5' flanking DNA of human CYP1B1 was cloned into pGL3 basic vector containing luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydrozyestradiol that is considered as carcinogenic metabolite. Luciferase activity was induced about 20 folds over that control by 1 nM TCDD (2,3,7,8-tetrachloto-p-dioxin). Recent industrialized society, human has been widely been exposed to widespread environmental contaminants such as PAHs(polycyclic aromatic hydrocarbon) that are originated from the imcomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR(aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarker for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. We have used the United State of America EPA selected 13 different PAHs, PAHs mixtures and extracts from environmental samples to evaluate the bioassay system. We examined effects of PAHs on the CYP1B1-luciferase reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene and dibenzo(a, h)anthracene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. Acenaphthene, anthracene, benzo(b)fluoranthene, fluorene, fluoranthene, anphthanlene, pyrene, phenanthrene and carbazole were weak responders in MCF-7 cells. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA.

• Environmental Mutagens and Carcinogens
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• v.24 no.4
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• pp.198-205
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• 2004

Cytochrome P4501B1(CYP1B1) is known to be inducible by xenobiotic compounds such as policyclic aromatic hydrocarbon(PAH) and dioxins such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin(TCDD). And these induction of CYP1B1 is also regulated by many categories of chemicals. In order to investigate the effects of several chemicals on CYP1B1 gene expression in Hepa-I and MCF-7 cells, 5' flanking DNA of human CYP1B1 was cloned into pGL3 basic vector containing luciferase gene, and then transfected into these cells. After treatment of chemicals, the luciferase activity was measured. CYP1B1 enzyme metabolize PAHs and estradiol. CYP1B1 metabolize estradiol to 4-hydrozyestradiol that is considered as carcinogenic metabolite. Recent industrialized industrialized society, human has been widely been exposed to widespread environmental contaminants such as PAHs(polycyclic aromatic hydrocarbon) that are originated from the imcomplete combustion of hydrocarbons. PAHs are known to be ligands of the AhR(aryl hydrocarbon receptor). Induction of cytochrome P4501B1(CYP1B1) in cell culture is widely used as a biomarker for PAHs. Therefore we have studied the effect of PAHs in the human breast cancer cells MCF-7 to evaluate bioactivity of PAHs. We have used the United State of America EPA selected 13 different PAHs, PAHs mixtures and extracts from environmental samples to evaluate the bioassay system. We examined effects of PAHs on the CYP1B1-luciferase reporter gene and CYP1B1 mRNA level. Benzo(k)fluoranthene and dibenzo(a, h)anthracene showed strong response to CYP1B1 promoter activity stimulation, and also CYP1B1 mRNAs increase in MCF-7 cells in a concentration-dependent manner. RT-PCR analysis indicated that PAHs significantly up-regulate the level of CYP1B1 mRNA. Some flavonoids such as genistein, daidzein, chrysin, naringenin and morin were also investigeted. These flavonoids decreased B(k)F infuced luciferase activity at low concentration. But, these flavonoids exhibited stimulatory effect at high concentration.

• Molecular & Cellular Toxicology
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• v.4 no.1
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• pp.11-15
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• 2008

The expression of neuronal nitric oxide synthase (nNOS) is regulated by various spliced first exons (exon 1a-1i), sharing differentially common exon 2 in diverse human tissues. The highly complex structure and regulation of human nNOS gene gave limitations of information for the precise mechanism of nNOS regulation. In the present study, we report that the repeats of polymorphic dinucleotides $(GT)^nA(TG)^n$ repeats located in just upstream to the exon 1f in human nNOS gene play suppressive role in transcription, as shown in the characteristics of Z-DNA motif in other genes. In neuronal and trophoblast cells transfected transiently with luciferase construct without dinucleotide repeats at the 5'-flanking region of exon 1f in nNOS gene, the luciferase activity was increased markedly. However, the presence of the dinucleotide repeats dramatically suppressed the luciferase activity to the basal level, and which was dependent on the length of $(GT)^n$ and $(TG)^n$ repeats. More importantly, we found the polymorphisms in the length of dinucleotide repeats in human. Furthermore, we show for the first time here that there is a significant association of the lengths of polymorphic dinucleotide $(GT)^n$ and $(TG)^n$ repeats with the risk of schizophrenia.

• The Korean Journal of Food And Nutrition
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• v.30 no.3
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• pp.531-538
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• 2017

This study was performed to investigate the effects of garlic on uncoupling protein 2 (UCP2) transcriptional regulation of UCP2-luciferase transgenic mice fed on a high fat diet to induce obesity. To examine the transcriptional regulation of UCP2, we generated transgenic mice with a UCP2 promoter (-1,830/+30 bp) containing luciferase as a reporter gene. UCP2-luciferase transgenic mice were fed a 45% high-fat diet for 8 weeks to induce obesity. Subsequently, mice were maintained on either a high-fat control diet (TG-CON), or high-fat diets supplemented with 2% (TG-GL2) or 5% (TG-GL5) garlic for a further 8 weeks. Dietary garlic reduced body weight and energy efficiency ratio in the TG-GL5 group, compared to the TG-CON group. Furthermore, garlic supplementation significantly decreased white adipose tissue fat mass and plasma levels of triglycerides, total cholesterol, and leptin in the TG-GL2 and TG-GL5 groups, compared to the TG-CON group. Specifically, UCP2 promoter activity in metabolic tissues such as liver, white adipose tissue, brown adipose tissue, and skeletal muscle was increased by garlic supplementation. These results suggest that dietary garlic was partially associated with an increase of UCP2 transcriptional activity in metabolic tissues for decreasing obesity.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.2
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• pp.157-164
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• 1999

To determine molecular mechanisms of Aquaporin-CD (AQP2) gene regulation, the promoter region of the AQP2 gene was examined by transiently transfecting a promoter-luciferase reporter fusion gene into mouse renal collecting duct cell lines such as mIMCD-3, mIMCD-K2, and M-1 cells, and NIH3T3 mouse embryo fibroblast cells. PCR-Southern analysis reveals that mIMCD-3 and mIMCD-K2 cells express AQP2, but M-1 and NIH3T3 cells do not, and that the treatment with cpt-cAMP $(400\;{\mu}M)$) or forskolin/isobutylmethylxanthine (IBMX) increased the AQP2 expression in IMCD cells. In both IMCD and NIH3T3 cells, the constructs containing the promoter of AQP2 gene showed promoter activities, indicating lack of tissue-specific element in the 1.4 kb 5'-flanking region of the mouse AQP2 gene. Luciferase activity in the IMCD cells transfected with the construct containing 5-flanking region showed responsiveness to cpt-cAMP, indicating that the 1.4 kb 5'-flanking region contains the element necessary for the regulatory mechanism by cAMP. The promoter-luciferase constructs which do not have a cAMP-responsible element (CRE) still showed the cAMP responsiveness in IMCD cells, but not in NIH3T3 cells. Increase in medium osmolarity did not affect AQP2 promoter activity in mIMCD-K2 cells. These results demonstrate that AQP2 gene transcription is increased with cAMP treatment through multiple motifs including CRE in the 5'-flanking region of the gene in vitro, and the regulatory mechanism may be important for in vivo regulation of AQP2 expression.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.6
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• pp.636-642
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• 2007

Background: CpG DNA plays an important role in immune cell function. This study examined whether the temporal control of toll-like receptor (TLR)9 by CpG DNA can regulate the expression of matrix metalloproteinase-9(MMP-9). Methods and materials: Macrophages were cultured in the presence of 10% FBS. For the various MMP genes analysis, RT-PCR and real-time PCR were performed. In addition, zymography assay performed for the MMP activity. The phosphorylation assay did for the ERK1/2 and NF${\kappa}B$ activation, and luciferase promoter assay was for the NF${\kappa}B$ activity. Results: CpG DNA induced the mRNA expression of MMP-2, MMP-9, and MMP-13, but not of MMP-7, MMP-8, and MMP-12, in a time-dependent manner. Especially, the mRNA expression of MMP-9 was strongly induced by CpG DNA using real-time RT-PCR. The TLR9 inhibitor, chloroquine, suppressed CpG DNA-induced MMP-9 expression and its activity. Moreover, CpG DNA induced the phosphorylation of ERK and the inhibition of ERK by U0126 suppressed CpG DNA-induced MMP-9 expression and its activity. CpG DNA stimulated $I{\kappa}B-{\alpha}$ degradation and luciferase activity. In addition, pretreatment of SN-50, the inhibitor of NF${\kappa}B$, strongly blocked the CpG DNA-induced MMP-9 expression and activity. Conclusion: These observations suggest that CpG DNA may play important roles in the activation of macrophages by regulating the production of MMP-9 via the sequential TLR9-ERK-NF${\kappa}B$ signaling pathway.