• The Korean Journal of Physiology and Pharmacology
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• v.9 no.6
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• pp.333-339
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• 2005

B/K protein is a novel protein containing double C2-like domains. We examined the specific signaling pathway that regulates the transcription of B/K in PC12 cells. When the cells were treated with forskolin ($50{\mu}M$), B/K mRNA and protein levels were time-dependently decreased, reaching the lowest level at 3 or 4 hr, and thereafter returning to the control level. Chemicals such as dibutyryl-cAMP, cellpermeable cyclic AMP (cAMP) analogue and CGS21680, adenosine receptor $A_{2A}$ agonist, also repressed the B/K transcription. However, 1,9-dideoxyforskolin did not show inhibitory effect on B/K transcription, suggesting direct involvement of cAMP in the forskolin-induced inhibition of B/K transcription. Effect of forskolin, dibutyryl cAMP and CGS21680 was significantly reduced in PKA-deficient PC12 cell line (PC12-123.7). One cAMP-response element (CRE)-like sequence (B/K CLS) was found in the promoter region of B/K DNA, and electrophoretic mobility shift assay indicated its binding to CREM and CREB. Forskolin significantly suppressed the promoter activity in CHO-K1 cells transfected with the constructs containing B/K CLS, but not with the construct in which B/K CLS was mutated (AC：TG). Taken together, we suggest that the transcription of B/K gene in PC12 cells may be regulated by PKA-dependent mechanism.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1563-1570
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• 2010

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1${\alpha}$ gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by ${\beta}$-galactosidase assay for lacZ and Western blot analysis for hER${\alpha}$. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest ${\beta}$-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hER${\alpha}$ gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hER${\alpha}$ and CXAU1-2XERE was the most effective system for the $E_2$-dependent induction of the ${\beta}$-galactosidase activity. This system showed the highest ${\beta}$-galactosidase activity at $10^{-6}\;M\;E_2$, and the activity could be detected at even the concentration of $10^{-10}\;M\;E_2$. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.

• Microbiology and Biotechnology Letters
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• v.26 no.1
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• pp.15-22
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• 1998

The dnrF gene, responsible for conversion of aklavinone to $\varepsilon$-rhodomycinone via C-11 hydroxylation, was mapped in the daunorubicin gene cluster of Streptomyces peucetius subsp. caesius ATCC 27952, close to drrAB, one of the anthracycline resistance genes. To characterize the enzymatic properties of the aklavinone 11-hydroxylase, the dnrF gene was overexpressed in Escherchia coli. The pET-22(+) plasmid which has the T7 promoter under the control of lacUV5 gene was used for the overexpression of the dnrF gene, and the recombinant plasmid pET213 that contains the dnrF gene linked to the T7 promoter of pET-22b(+) was introduced into the E. coli BL2l. When the expression of the dnrF gene was induced by IPTG at the final concentration of 1 mM, the induced protein could be detected in SDS-PAGE only in insoluble precipitate. The insoluble protein was electroeluted from the gel and used for the preparation of antiserum in mice. Various culture conditions were tested to maximize the expression of the aklavinone 11-hydroxylase in soluble form. The enzymatic activity was checked by the bioconversion experiment, and the protein was confirmed by the SDS-PAGE and the Western blot analysis. From the analysis of the data, it was concluded that the culture induced with IPTG at the final concentration of 0.02 mM at 37$^{\circ}C$ yielded the best productivity of active form of enzyme.

• Biomolecules & Therapeutics
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• v.17 no.1
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• pp.70-78
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• 2009

Coptis japonica (C. japonica) is a perennial medicinal plant that has anti-inflammatory activity. C. japonica contains numerous biologically active alkaloids including berberine, palmatine, epi-berberine, and coptisine. The most well-known anti-inflammatory principal in C. japonica is berberine. For example, berberine has been implicated in the inhibition of iNOS induction by cytokines in microglial cells. However, the efficacies of other alkaloids components on microglial activation were not investigated yet. In this study, we investigated the effects of three alkaloids (palmatine, epi-berberine and coptisine) from C. japonica on lipopolysaccharide (LPS)-induced microglial activation. BV2 microglial cells were immunostimulated with LPS and then the production of several inflammatory mediators such as nitric oxide (NO), reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) were examined as well as the phosphorylation status of Erk1/2 mitogen activated protein kinase (MAPK). Palmatine and to a lesser extent epi-berberine and coptisine, significantly reduced the release of NO, which was mediated by the inhibition of LPS-stimulated mRNA and protein induction of inducible nitric oxide synthase (iNOS) from BV2 microglia. In addition to NO, palmatine inhibited MMP-9 enzymatic activity and mRNA induction by LPS. Palmatine also inhibited the increase in the LPS-induced MMP-9 promoter activity determined by MMP-9 promoter luciferase reporter assay. LPS stimulation increased Erk1/2 phosphorylation in BV2 cells and these alkaloids inhibited the LPS-induced phosphorylation of Erk1/2. The anti-inflammatory effect of palmatine in LPS-stimulated microglia may suggest the potential use of the alkaloids in the modulation of neuroinflammatory responses, which might be important in the pathophysiological events of several neurological diseases including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD) and stroke.

• Journal of Life Science
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• v.14 no.1
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• pp.154-162
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• 2004

Thrombospondin-1 (TSP-1), a negative regulator in tumor growth and angiogenesis, is cell-type specifically regulated and at transcriptional level by external stimuli. Previously, we found that phorbol 12-myristate 13-acetate (PMA) suppressed TSP-1 expression in porcine aortic endothelial (PAE) cell, but enhanced in hepatoma cell line, Hep 3B cell. A region between -767 and -723 on the tsp-1 promoter was defined as a responsive site to the suppression in PAE cell. eased on the previous results, the molecular mechanism of TSP-1 expression was determined by characterizing interactions between cis-elements and trans-factors using three overlapped oligonucleotide probes, oligo a-1 (from -767 to -738), a-2 (-759 to -730) and a-3 (-752 to -723). The results from electromobility shift assay showed that PMA-induced suppression of TSP-1 transcription in PAE cell might be caused via a negative regulator binding to the region from -752 to -730 and additionally generated by lacking two positive regulators binding to the sites from -767 to -760 and from -752 to -730. Especially, PMA enhanced the binding ability of the negative regulator to the site from -752 to -730 in PAE cell, but anti-c-Jun did not affected its binding ability.

• KSBB Journal
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• v.28 no.1
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• pp.1-6
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• 2013

Curcumin has diverse anticancer activities that lead to tumor growth inhibition of cancer cells and induction of apoptosis. Curcumin is involved in the regulation of multiple genes via transcription factors including NF-${\kappa}B$, STATs, AP1, and SP. Notch signaling plays critical roles in maintaining the balance between cell proliferation, differentiation and apoptosis, and thereby may contribute to the development of various cancers involving breast cancer. This study was to investigate the effects of curcumin on Notch1 gene expression and to explore the underlying mechanism. Here, we found that curcumin decreased the levels of Notch1 mRNA and protein in MDA-MB-231 human breast cancer cells, along with the downregulation of Sp family genes (Sp1, Sp2, Sp3, and Sp4). The repressive effect of curcumin on Notch1 gene transcription was confirmed by performing Notch1 promoter-driven reporter assay and three Sp-binding sites were identified on Notch1 promoter that may act as curcumin-respose elements. Moreover, treatment with mitramycin A, a specific Sp inhibitor, decreased the levels of Notch1 mRNA and protein in human breast cancer cells. Taken together, our results indicate that Notch1 gene expression is downregulated by curcumin, at least in part, through the suppression of Sp family, which may lead to apoptosis in human breast cancer cells.

• Applied Biological Chemistry
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• v.39 no.3
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• pp.195-199
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• 1996

We have cloned several E. coli sfs genes which stimulate mal gene expression with $crp^{{\ast}1}$). One the genes (pPVC2) was sequenced and potential CRP binding site is located in the upstream of the putative promoter in the regulatory region. In order to investigate the regulation of the sfs1 gene by the cAMP-CRP complex, we have constructed the sfs-lacZ fusion gene in this research. The overall transcriptional stimulations of sfs1 gene in the presence cAMP were confirmed by ${\beta}-galactosidase$ activity and Western blot analysis of sfs1-lacZ fusion gene. Transcriptional regulation by cAMP-CRP was also confirmed by Northern blot analysis. End-labelled DNA of the DNA fragment in sfs1 regulation region were used for gel retardation assay to examine the CRP-DNA complex in the presence of cAMP. Results here indicate that CRP binding site in the regulatory region of sfs1 gene is positive regulator for the expression of sfs1 gene.

• Journal of Life Science
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• v.26 no.2
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• pp.174-180
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• 2016

Dicumarol is a coumarin derivative isolated from sweet clover (Melilotus alba), and has anti-coagulant activity with the inhibitory activity of NAD(P)H quinone oxidoreductase1 (NQO1). NQO1 catalyzes the two-electron reduction of quinones to hydroquinones. Dicumarol competes with NAD(P)H for binding to NQO1, resulting in the inhibition of NQO1 enzymatic activity. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). However, the effects of dicumarol on metalloproteinase (MMP)-9 expression and activity are not investigated here. This study investigated whether dicumarol inhibits MMP-9 expression and activity in PMA-treated human renal carcinoma Caki cells. Dicumarol markedly inhibited the PMA-induced MMP-9 mRNA expression and MMP-9 activity. NF-κB and AP1 promoter activity, which is important in MMP-9 expression, also decreased in dicumarol-treated cells. Furthermore, dicumarol markedly suppressed the ability of PMA-mediated migration in Caki cells. When the relevance of NQO1 in the dicumarol-mediated inhibitory effect on PMA-induced MMP9 activity was elucidated, knock-down of NQO1 with siRNA was found to have no effect on PMA-induced MMP9 activity, suggesting that the stimulating effect of dicumarol on PMA-induced MMP9 activity is independent of NQO1 activity. Taken together, the present studies suggested that dicumarol may inhibit PMA-induced migration via down-regulation of MMP-9 expression and activity.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.895-903
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• 2005

Thrombospondin-1 (TSP-1), a negative regulator in tumor growth and angiogenesis, is cell-type specifically regulated under pathological conditions or by extracellular stimuli, and the regulation of TSP-1 gene expression is important for developing new approaches in tumor therapy. Mistletoe is a parasitir plant that have been used for immunomodulation and antitumor therapy. Helixor A is an aqueous part of mistletoes extract. Here we showed that TSP-1 expression was significantly induced at both mRNA and protein levels in the Hepatocarcinorna cell line (Hep3B) and primary bovine endothelial cell line (BAE) exposed to Helixor A. Our promoter analysis confirmed that the expression of TSP-1 gene was regulated by Helixor A at the transcriptional level. In cell invasion assay, the conditioned media obtained from treatment of these cells significantly reduced the number of invasive cells and also inhibited capillary-like tube formation of BAE cells on Matrigel. Moreover, the inhibitory efforts of the conditioned media on cell invasion and tube formation were reversed by blocking with anti-TSP-1 neutralizing antibodies, suggesting that TSP-1 is involved in Helixor A-indured antiangiogenic effect. Taken together, our results suggest that Helixor A have an antiangiogenic effects through upregulation of TSP-1.

• Korean Journal of Food Science and Technology
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• v.48 no.1
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• pp.66-71
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• 2016

Delphinidin is a blue-red pigment and one of the major anthocyanins in plants. It plays an important role in anti-oxidant, anti-inflammatory, anti-mutagenic and anti-cancer properties. In this study, we investigated the inhibitory effects of delphinidin on vascular endothelial growth factor (VEGF) gene expression, an important factor involved in angiogenesis and tumor progression in human prostate cancer. Delphinidin decreased levels of epidermal growth factor (EGF)-induced VEGF mRNA expression in PC-3M cells. The expression of the EGF-induced hypoxia inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) and signaling transducer and activator of transcription 3 (STAT3) proteins, which are the major transcription factors for VEGF, were inhibited by delphinidin. In addition, delphinidin decreases HRE-promoter reporter gene activity, suggesting that delphinidin can suppress the transcription of HIF-$1{\alpha}$ under EGF induction, leading to a decrease in the expression of VEGF. Delphinidin specifically suppressed the phosphorylation of Akt, p70S6K, and 4EBP1, but not the phosphorylation of EGFR. Therefore, our results suggest that delphinidin may inhibit human prostate cancer progression and angiogenesis by inhibiting HIF-$1{\alpha}$, STAT3 and VEGF gene expression.