• Journal of Life Science
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• v.24 no.3
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• pp.323-328
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• 2014

MicroRNAs comprise a family of small noncoding RNAs that modulate physiological processes, including adipogenesis. MicroRNA-17 (miR-17) promotes adipocyte differentiation and enhances lipid accumulation. The transcriptional regulation of miR-17 during adipogenesis remains unknown. In this study, we investigated whether miR-17 is a target of peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$), which is a key regulator of adipogenesis. The levels of miR-17 and the expression of $PPAR{\gamma}$ increased after the induction of adipocyte differentiation. Three putative peroxisome proliferator response elements (PPREs) were identified in the miR-17 promoter region. Using chromatin immunoprecipitation and luciferase reporter assays, we observed the interaction of $PPAR{\gamma}$ with the miR-17 promoter. Mutagenesis experiments showed that the -677/-655 region of the miR-17 promoter could function as a PPRE site. These results suggest that $PPAR{\gamma}$ is essential for transcriptional activation of the miR-17 gene, thereby contributing to understanding the molecular mechanism of adipogenesis in adipocytes.

• Journal of Life Science
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• v.20 no.5
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• pp.655-661
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• 2010

To elucidate the mechanism underlying the suppressive regulation of hST8Sia I expression in retinoic acid (RA)-induced SK-MEL-2 cells, we characterized the promoter region of the hST8Sia I gene. Functional analysis of the 5‘-flanking region of the hST8Sia I gene by the transient expression method showed that the -1146 to -646 region, which contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kB, functions as the RA-repressive promoter in SK-MEL-2 cells. Site-directed mutagenesis and ChIP analyses indicated that the NF-kB binding site at -731 to -722 is crucial for the RA-induced repression of hST8Sia I in SK-MEL-2 cells. In addition, the transcriptional activity of hST8Sia I suppressed by RA in SK-MEL-2 cells was strongly inhibited by extracellular signal-regulated protein kinase (ERK) inhibitor U0126 and protein kinase C (PKC) inhibitor GO6976, as determined by RT-PCR and luciferase assay of hST8Sia I promoter containing the -1146 to -646 regions. These results suggest that RA markedly modulates transcriptional regulation of hST8Sia I gene expression through the PKC/ERK signal pathway in SK-MEL-2 cells.

• Journal of Life Science
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• v.28 no.12
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• pp.1432-1437
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• 2018

Clara cell secretory protein (CCSP) plays an important role in protecting the lungs from inflammation. This research focuses on identifying the cis-element for binding the repressor of CCSP gene expression. A DNase I footprinting experiment revealed three protected regions between -812 and -768 bp (45 bp) of the mCCSP promoter. One motif (D3: GCCTGGGAA) was 100% conserved across rat, hamster, and human. The addition of excess amounts of the D3 motif exhibited high competition within that 45 bp range in an electrophoretic mobility shift assay. However, when mutated D3 ($G{\underline{AA}}TG{\underline{TT}}AA$) was used, the competition was significantly reduced. This demonstrates that the D3 motif within that 45 bp region of the mCCSP promoter is an important site for the protein-DNA interaction. Transient transfection assays with -756 Luc resulted in highly decreased expression of CCSP than those with -812 Luc, suggesting that the 45 bp could function as a binding site for the repressor. Co-transfection of KLF4 exhibited significant repression of the -812 Luc but not the -768 Luc which clearly shows that KLF4 might function as a repressor for the CCSP gene and also suggests that the D3 motif is strongly involved in the binding of KLF4. In addition, when anti-KLF4 antibody was added, super-shifted bands were observed. This result demonstrates that KLF4 could function as a repressor by binding to this 45 bp region of the CCSP promoter and that the D3 motif might be involved in the specific binding of KLF4.

• Food Science and Preservation
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• v.13 no.6
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• pp.796-802
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• 2006

In order to investigate the anti-inflammatory and cellular protective effects of Atractomorpha lata Motschulsky, Oxya japonica japonica Thunberg and Stethophyma magister Rehn, we first examined hydrogen peroxide-induced cytotoxicity in SH-SY5Y cells as well as antioxidant assays including DPPH and FRAP assays. We found that water, ethanol and methanol extracts of Oxya japonica japonica Thunberg and Stethophyma magister Rehn had potentials to anti-oxidant activity and especially water extract of Oxya japonica japonica Thunberg exhibited the most potent protective effects against $H-2O_2$-induced cytotoxicity in SH-SY5Y cells by MTT assay. Taken Together, these findings indicate that water extract of Oxya japonica japonica Thunberg could be useful insect resources for agrobiotechnological or oriental medicinal purposes.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.15-19
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• 2004

The expression of a chimeric transgene (nos-npt II) has been examined in 9 years old transgenic poplars (Populus koreana x P. nigra) growing in a nursery. The expression of the gene in twenty six independentely transformed plants were examined by 1) enzyme (NPT II) assay, 2) RT-PCR, and 3) resistance to kanamycin. High NPT II activities in young leaves of all the transformed plants were found even without a selection pressure for antibiotics for 9 years. However, the activity varied with the positions of leaves in the stem in that young leaves showed higher activity than did mature tissues. When leaf segments were cultured in the presence of 150 mg/l kanamycin, only those from young leaves produced vigorously growing callus. However, as in the case of NPTII assay, the leaf segments from mature leaves did not form callus well on the media. RT-PCR with nptII specific primers also showed that amplification products were observed only when RNAs from young tissues were used. The total RNA gel showed that while RNA in young leaves are relatively stable and in a large quantity, those in old leaves were mostly degraded. All the above results suggest that the gene is transcriptionally active only in young tissue even though it is attached to a constituitive promoter. Therefore, the expression of foreign gene in poplar plants seemed to be affected by the metabolic state of the cells and thus vary greatly with the developmental stages and the age of tissue.

• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1279-1284
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• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.3
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• pp.314-319
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• 2017

Chlorogenic acid, a well-known polyphenol, and its derivatives, ester of caffeic acid on quinic acid moiety, are abundant in coffee, tea, fruits, and various vegetables. This study examined the effects of cryptochlorogenic acid (CCA) on osteoblast differentiation. CCA-induced mRNA expression levels of osteogenic genes in MC3T3E1 and C3H10T1/2 cells were determined by RT-PCR and qPCR. CCA regulated expression of key osteogenic genes in the early stage of differentiation, including distal-less homeobox 5 (Dlx5), DNA-binding protein inhibitor (Id1), and runt-related transcription factor 2 (Runx2). These results suggest that CCA may enhance osteoblast differentiation through expression of osteogenic genes such as Id1, Dlx5, and Runx2, especially in the early stage.

• Environmental Mutagens and Carcinogens
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• v.22 no.3
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• pp.175-182
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• 2002

Endocrine disruptors (EDs) are the chemicals that affect endocrine systems through activation or inhibition of steroid hormone response. It is necessary to have a good system to evaluate rapidly and accurately endocrine-disrupting activities of suspected chemicals and their degradation products. The key targets of EDs are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We constructed a co-expression system of Gal4p DNA binding domain (DBD)- ligand binding domain of human estrogen receptor $\alpha$ or $\beta$, and Gal4p transactivation domain (TAD)-co-activator AIB-1, SRC-1 or TIF-2 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of CYC1 promoter and Gal4p binding site (GAL4 upstream activating sequence, GAL4$_{UAS}$). Expression of this reporter gene was dependent on the presence of estrogen or EDs in the culture medium. We found that the two-hybrid system with combination of the hER$\beta$ LBD and co-activator SRC-1 was most effective in the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by those chemicals correlated with their estrogenic activities measured by other assay systems, indicating that this assay system is efficient and reliable for measuring estrogenic activity. The data in this research demonstrated that the yeast detection system using steroid hormone receptor and co-activator is a useful tool for identifying chemicals that interact with steroid receptors.s.

• Journal of Microbiology
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• v.40 no.4
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• pp.313-318
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• 2002

To perform the prophylactic study of a vaccine derived from human papillomavirus (HPV) using Balb/c mice, we produced virus like particles consisting of HPV capsid protein L1 which has been reported to induce significant humoral and cellular immunity using various animal model systems. In order to produce HPV16 VLPs, the cDNA of L1 capsid protein in HPV type 16, obtained by polymerase chain reaction, was inserted into yeast expression vector, YEG$\alpha$-HIR525 under the control of GAL10 promoter. The transformation of YEG$\alpha$-HPV16 L1 was performed into the yeast Saccharomyces cerevisiae Y2805 by the lithium acetate method and the yeast clone expressing the highest level of L1 capsid protein of human papillomavirus type 16 was selected by Western blot analysis using anti-HPV16 L1 antibody. The purification of HPV16 VLP has been performed by the ultracentrifugation and gel-filtration methods. To validate the vaccine efficacy of the purified HPV16 VLPs and investigate the properties of HPV16 VLPs to induce humoral immunity, ELISA assay was performed. A significantly increased production of anti-HPV16 VLP antibodies was observed in sera from immunized mice. The neutralization activity of antibodies in the sera from the vaccinated mice was demonstrated by a rapid and simple assay to detect hemagglutihation inhibition activity.

• Korean Journal of Microbiology
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• v.47 no.2
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• pp.158-162
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• 2011

Amino acid sequence alignment shows that $\underline{P}$roteus $\underline{m}$irabilis $\underline{t}$ranscription $\underline{r}$egulator (PMTR) has cystein sequence homology at metal binding domain to CueR (copper resistance) protein, which conserves two cysteins (Cys 112 and Cys 120 in PMTR). Gel shift assay revealed that PMTR protein bound to promoter region of Escherichia coli copA (copper-translocating P-type ATPase) and Proteus mirabilis atpase (putative copper-translocating P-type ATPase) genes except that of E. coli zntA (zinc-translocating P-type ATPase) gene. DNase I protection experiment indicated that PMTR protein protected the region over -35 box and close to -10 box. DNase I hypersensitive bases were shown at C and A bases of labeled template strand and at G and C bases of labeled non-template strand of DNA. These hypersensitive bases were appeared in other metalloregulatory proteins of MerR family, which suggests protein-induced DNA bending.