• Molecules and Cells
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• v.19 no.1
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• pp.114-123
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• 2005

Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were $A{\rightarrow}G$ transition (15/33) and $G{\rightarrow}A$ transition (8/33). The predominant base substitution, $A{\rightarrow}G$ transition (11/27) and $G{\rightarrow}A$ transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.

• Molecules and Cells
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• v.41 no.9
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• pp.830-841
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• 2018

Recent studies have indicated that microRNAs (miRNAs) play an important role in hepatocellular carcinoma (HCC) progression. In this study, we showed that miR-766-3p was decreased in approximately 72% of HCC tissues and cell lines, and its low expression level was significantly correlated with tumour size, TNM stage, metastasis, and poor prognosis in HCC. Ectopic miR-766-3p expression inhibited HCC cell proliferation, colony formation, migration and invasion. In addition, we showed that miR-766-3p repressed Wnt3a expression. A luciferase reporter assay revealed that Wnt3a was a direct target of miR-766-3p, and an inverse correlation between miR-766-3p and Wnt3a expression was observed. Moreover, Wnt3a up-regulation reversed the effects of miR766-3p on HCC progression. In addition, our study showed that miR-766-3p up-regulation decreased the nuclear ${\beta}-catenin$ level and expression of Wnt targets (TCF1 and Survivin) and reduced the level of MAP protein regulator of cytokinesis 1 (PRC1). However, these effects of miR-766-3p were reversed by Wnt3a up-regulation. In addition, PRC1 upregulation increased the nuclear ${\beta}-catenin$ level and protein expression of TCF1 and Survivin. iCRT3, which disrupts the ${\beta}-catenin-TCF4$ interaction, repressed the TCF1, Survivin and PRC1 protein levels. Taken together, our results suggest that miR-766-3p down-regulation promotes HCC cell progression, probably by targeting the Wnt3a/PRC1 pathway, and miR-766-3p may serve as a potential therapeutic target in HCC.

• Microbiology and Biotechnology Letters
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• v.42 no.4
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• pp.347-353
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• 2014

${\beta}$-Glucosidase from Aspergillus usamii KCTC 6954 was used to convert ginsenoside Rb1 to compound K, which has a high bio-functional activity. The enzymatic activities during culturing for 15 days were determined using ${\rho}$-nitrophenyl-${\beta}$-glucopyranoside. The growth rate of the strain and the enzymatic activity were maximized after 6 days (IU; $175.93{\mu}M\;ml^{-1}\;min^{-1}$). The activities were maximized at $60^{\circ}C$ in pH 6.0. During culturing, Rb1 was converted to Rd after 9 d and then finally converted to compound K at 15 d. In the enzymatic reaction, Rb1 was converted to the ginsenoside Rd within 1 h of reaction time and compound K could be detected after 8 h. As a result, this study demonstrates that $Rb1{\rightarrow}Rd{\rightarrow}F2{\rightarrow}$compound K is the main metabolic pathway catalyzed by ${\beta}$-glucosidase and that ${\beta}$-glucosidase is a feasible option for the development of specific bioconversion processes to obtain minor ginsenosides such as Rd and compound K.

• Journal of Korean Society of Environmental Engineers
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• v.27 no.5
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• pp.491-498
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• 2005

J sewage treatment plant (WWTP) in Busan has used methanol as an external carbon source for the biological denitrification process. Methanol is widely used. but rather expensive and very dangerous in handling. Therefore, it has been required that the economic alternative carbon source must be developed. By-product from a fine chemical industry can be Purified by removing high molecular weight substances using the ultrafilter membrane separation process and RBDCOD fraction becomes $98{\sim}99%$ of COD substances in the purified by-product. The purified by-product containing three types of alcohols, methanol, prophylenglycol and methoxypropanol; showed similar chemical characteristics to the methanol, a main external carbon source, in biodegradation pathway. Shown above, the compatibility between main and alternative carbon sources has been achieved. Also very short or no adaptation period is necessary in the case of exchanging these carbon sources. The compatibility between external carbon sources is an essential element for stabilizing WWTP operations. During the full-scale application test of the by-product, the alternative carbon source line got on par with the treatment efficiency of the methanol line. With the test result, J-WWTP changed methanol to a fine chemical by-product, in two out of three J-WWTP lines. Moreover, it is expected that 55.4% of the external carbon source cost reduction can be achieved in the alternative carbon source applied lines.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.4
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• pp.483-489
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• 2005

To understand the molecular mechanisms that regulate intramuscular fat deposition and its release, cDNA clones expressed in adipose tissues of Korean cattle were identified by differential screening from adipose tissue cDNA library. By partial nucleotide sequencing of 486 clones and a search for sequence similarity in NCBI nucleotide databases, 245 clones revealed unique clones. By a functional grouping of the clones, 14% of the clones were categorized to metabolism and enzyme-related group (stearoyl CoA desaturase, lactate dehydrogenase, fatty acid synthase, ATP citrate lyase, lipoprotein lipase, acetyl CoA synthetase, etc), and 6% to signal transduction/cell cycle-related group (C/EBP, cAMP-regulated phosphoprotein, calmodulin, cyclin G1, cyclin H, etc), and 4% to cytoskeleton and extracellular matrix components (vimentin, ankyrin 2, gelosin, syntenin, talin, prefoldin 5). The obtained 245 clones will be useful to study lipid metabolism and signal transduction pathway in adipose tissues and to study obesity in human. Some clones were subjected to full-sequencing containing open reading frame. The cDNA clone of bovine homolog of human prefoldin 5 gene had a total length of 959 nucleotides coding for 139 amino acids. Comparison of the deduced amino acid sequences of bovine prefoldin 5 with those of human and mouse showed over 95% identity. The cDNA clone of bovine homolog of human ubiquitin-like/S30 ribosomal fusion protein gene had a total length of 484 nucleotides coding for 133 amino acids. Comparison of the deduced amino acid sequences of bovine ubiquitin-like/S30 ribosomal fusion protein gene with those of human, rat and mouse showed over 97% identity. The cDNA clone of bovine homolog of human proteolipid protein 2 mRNA had a total length of 928 nucleotides coding for 152 amino acids. Comparison of the deduced amino acid sequences of bovine proteolipid protein 2 with those of human and mouse showed 87.5% similarity. The cDNA clone of bovine homolog of rat thymosin beta 4 had a total length of 602 nucleotides coding for 44 amino acids. Comparison of the deduced amino acid sequences of bovine thymosin beta 4 gene with those of human, mouse and rat showed 93.1% similarity. The cDNA clone of bovine homolog of human myotrophin mRNA had a total length of 790 nucleotides coding for 118 amino acids. Comparison of the deduced amino acid sequences of bovine myotrophin gene with those of human, mouse and rat showed 83.9% similarity. The functional role of these clones in adipose tissues needs to be established.

• Journal of Ginseng Research
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• v.41 no.3
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• pp.392-402
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• 2017

Background: Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. Methods: Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. $H_2O_2$ productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusion: Of the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-$\text\tiny L$-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.

• Journal of Ginseng Research
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• v.44 no.1
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• pp.58-66
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• 2020

Background: The biological and pharmacological effects of BST204, a fermented ginseng extract, have been reported in various disease conditions. However, its molecular action in metabolic disease remains poorly understood. In this study, we identified the antiadipogenic activity of BST204 resulting from its inhibition of the S6 kinase 1 (S6K1) signaling pathway. Methods: The inhibitory effects of BST204 on S6K1 signaling were investigated by immunoblot, nuclear fractionation, immunoprecipitation analyses. The antiadipogenic effect of BST204 was evaluated by measuring mRNA levels of adipogenic genes and by chromatin immunoprecipitation and quantitative real-time polymerase chain reaction analysis. Results: Treatment with BST204 inhibited activation and nuclear translocation of S6K1, further decreasing the interaction between S6K1 and histone H2B in 10T1/2 mesenchymal stem cells. Subsequently, phosphorylation of H2B at serine 36 (H2BS36p) by S6K1 was reduced by BST204, inducing an increase in the mRNA expression of Wnt6, Wnt10a, and Wnt10b, which disturbed adipogenic differentiation and promoted myogenic and early osteogenic gene expression. Consistently, BST204 treatment during adipogenic commitment suppressed the expression of adipogenic marker genes and lipid drop formation. Conclusion: Our results indicate that BST204 blocks adipogenesis of mesenchymal stem cells through the inhibition of S6K1-mediated histone phosphorylation. This study suggests the potential therapeutic strategy using BST204 to combat obesity and musculoskeletal diseases.

• Molecules and Cells
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• v.21 no.2
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• pp.206-212
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• 2006

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and $p16^{INK4a}$ functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and $p16^{INK4a}$ pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.

• Molecules and Cells
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• v.43 no.3
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• pp.236-250
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• 2020

Currently, many available anti-cancer therapies are targeting apoptosis. However, many cancer cells have acquired resistance to apoptosis. To overcome this problem, simultaneous induction of other types of programmed cell death in addition to apoptosis of cancer cells might be an attractive strategy. For this purpose, we initially investigated the inhibitory role of TRIP-Br1/XIAP in necroptosis, a regulated form of necrosis, under nutrient/serum starvation. Our data showed that necroptosis was significantly induced in all tested 9 different types of cancer cell lines in response to prolonged serum starvation. Among them, necroptosis was induced at a relatively lower level in MCF-7 breast cancer line that was highly resistant to apoptosis than that in other cancer cell lines. Interestingly, TRIP-Br1 oncogenic protein level was found to be very high in this cell line. Up-regulated TRIP-Br1 suppressed necroptosis by repressing reactive oxygen species generation. Such suppression of necroptosis was greatly enhanced by XIAP, a potent inhibitor of apoptosis. Our data also showed that TRIP-Br1 increased XIAP phosphorylation at serine87, an active form of XIAP. Our mitochondrial fractionation data revealed that TRIP-Br1 protein level was greatly increased in the mitochondria upon serum starvation. It suppressed the export of CypD, a vital regulator in mitochondria-mediated necroptosis, from mitochondria to cytosol. TRIP-Br1 also suppressed shikonin-mediated necroptosis, but not TNF-α-mediated necroptosis, implying possible presence of another signaling pathway in necroptosis. Taken together, our results suggest that TRIP-Br1/XIAP can function as onco-proteins by suppressing necroptosis of cancer cells under nutrient/serum starvation.

• Environmental Mutagens and Carcinogens
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• v.26 no.4
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• pp.125-132
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• 2006

Previous studies showed that epigallocatechin gallate(EGCG) have substantial effects of suppressing the N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-initiated cell transformation process on the bases of foci formation frequency and loss of anchorage dependency. In this study we tried to clarify the molecular mechanism of suppressing the cell transformation process. Mouse cell line balb/c 3T3 A31-1-1 was exposed 2 days to MNNG followed by 15 days 12-O-tetradecanoylphorbol-13-acetate(TPA) treatment for our transformation process. EGCG was added after the time point of 24 hours exposure to TPA and incubated for 19 days. 2029 genes were selected in our transformation process that showed fold change value of 1.5 or more in the microarray gene expression analysis covering the mouse full genome. These genes were found to be involved mainly in the cell cycle pathway, focal adhesion, adherens junction, TGE-$\beta$ signaling, apoptosis, lysine degradation, insulin signaling, ECM-receptor interaction. Among the genes, we focused on the 631 genes(FC>0.5) reciprocally affected by EGCG treatment. Our study suggest that EGCG down-regulate the gene expressions of up stream signaling factors such as nemo like kinase with MAPK activity and PI3-Kinase, Ras GTPase and down stream factors such as cyclin D1, D2, H, T2, cdk6.