• Journal of Soil and Groundwater Environment
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• v.17 no.3
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• pp.49-58
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• 2012

Batch experiments using indigenous and commercialized adventive microorganisms were performed to investigate the feasibility of the biodegradation process for the diesel contaminated soil, which was taken in US Military Camp 'Hialeah', Korea. TPH concentration of the soil was determined as 3,819 mg/kg. Four indigenous microorganisms having high TPH degradation activity were isolated from the soil and by 16S rRNA gene sequence analysis, they were identified as Arthrobacter sp., Burkholderia sp., Cupriavidus sp. and Bacillus sp.. Two kinds of commercialized solutions cultured with adventive microorganisms were also used for the experiments. Various biodegradation conditions such as the amount of microorganism, water content and the temperature were applied to decide the optimal bioavailability condition in the experiments. In the case of soils without additional microorganisms (on the natural attenuation condition), 35% of initial TPH was removed from the soil by inhabitant microorganisms in soil for 30 days. When the commercialized microorganism cultured solutions were added into the soil, their average TPH removal efficiencies were 64%, and 54%, respectively, which were higher than that without additional microorganisms. When indigenous microorganisms isolated from the contaminated soil were added into the soil, TPH removal efficiency increased up to 95% (for Bacillus sp.). According to the calculation of the average biodegradation rates for Bacillus sp., the remediation goal (87% of the removal efficiency: 500 mg/kg) for the soil would reach within 24 days. Results suggested that TPH removal efficiency of biodegradation by injecting indigenous microorganisms is better than those by injecting commercialized adventive microorganisms and only by using the natural attenuation.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2000.11a
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• pp.13-16
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• 2000

Microorganisms have been widely employed for the production of useful bioproducts including primary metabolites such as ethanol, succinic acid, acetone and butanol, secondary metabolites represented by antibiotics, proteins, polysaccharides, lipids and many others. Since these products can be obtained in small quantities under natural condition, mutation and selection processes have been employed for the improvement of strains. Recently, metabolic engineering strategies have been employed for more efficient production of these bioproducts. Metabolic engineering can be defined as purposeful modification of cellular metabolic pathways by introducing new pathways, deleting or modifying the existing pathways for the enhanced production of a desired product or modified/new product, degradation of xenobiotics, and utilization of inexpensive raw materials. Metabolic flux analysis and metabolic control analysis along with recombinant DNA techniques are three important components in designing optimized metabolic pathways, This powerful technology is being further improved by the genomics, proteomics, metabolomics and bioinformatics. Complete genome sequences are providing us with the possibility of addressing complex biological questions including metabolic control, regulation and flux. In silico analysis of microbial metabolic pathways is possible from the completed genome sequences. Transcriptome analysis by employing ONA chip allows us to examine the global pattern of gene expression at mRNA level. Two dimensional gel electrophoresis of cellular proteins can be used to examine the global proteome content, which provides us with the information on gene expression at protein level. Bioinformatics can help us to understand the results obtained with these new techniques, and further provides us with a wide range of information contained in the genome sequences. The strategies taken in our lab for the production of pharmaceutical proteins, polyhydroxyalkanoate (a family of completely biodegradable polymer), succinic acid and me chemicals by employing metabolic engineering powered by genomics, proteomics, metabolomics and bioinformatics will be presented.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.963-967
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• 2014

Previous studies have suggested anti-tumor effects of asiatic acid in some human cancer cell lines. This agent is reported to increase the levels of $p21^{WAF1/CIP1}$ in human breast cancer cell lines. However, the molecular mechanisms have not been established. Here we report that asiatic acid up-regulates $p21^{WAF1/CIP1}$ protein expression but not the level of $p21^{WAF1/CIP1}$ mRNA in HepG2 human hepatoma cells. Furthermore, we found that the asiatic acid induced increase of $p21^{WAF1/CIP1}$ protein was associated with decreased phosphorylation (ser-146) of $p21^{WAF1/CIP1}$. Knockdown of NDR1/2 kinase, which directly phosphorylates $p21^{WAF1/CIP1}$ protein at ser-146 and enhances its proteasomal degradation, increased the levels of $p21^{WAF1/CIP1}$ protein and eliminated the regulation of $p21^{WAF1/CIP1}$ stability by asiatic acid. At the same time, the expression of NDR1/2 kinase decreased during treatment with asiatic acid in HepG2 cells. Moreover, asiatic acid inhibited the proliferation of HepG2 cells, this being attenuated by knockdown of $p21^{WAF1/CIP1}$. In conclusion, we propose that asiatic acid inhibits the expression NDR1/2 kinase and promotes the stability of $p21^{WAF1/CIP1}$ protein through attenuating NDR1/2 dependent phosphorylation of $p21^{WAF1/CIP1}$ in HepG2 cells.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.804-810
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• 1999

There is a possibility that carvone, a monoterpene from spearmint (Mentha spicata), could induce the bph degradative pathway and genes in Alcaligenes eutrophus H850, which is a known Gram-negative PCB degrader with a broad substrate specificity that was thoroughly investigated with Arthrobacter sp. BIB, a Gram-positive PCB degrader. The strains BIB and H850 were unable to utilize and grow on the plant terpene [(R)-(-)-carvone] (50ppm) to be recognized as a sole carbon source. Nevertheless, the carvone did induce 2,3-dihydroxybiphenyl 1,2-dioxygenase (encoded by bphC) in the strain B lB, as observed by a resting cell assay that monitors accumulation of a yellow meta ring fission product from 4,4'-dichlorobiphenyl (DCBp). The monoterpene, however, did not appear to induce the meta cleavage pathway in the strain H850. Instead, an assumption was made that the strain might be using an alternative pathway, probably the ortho-cleavage pathway. A reverse transcription (RT)-PCR system, utilizing primers designed from a conserved region of the bphC gene of Arthrobacter sp. M5, was employed to verify the occurrence of the alternative pathway. A successful amplification (182bp) of mRNA transcribed from the N-terminal region of the bphC gene was accomplished in H850 cells induced by carvone (50ppm) as well as in biphenyl-growth cells. It is, therefore, likely that H850 possesses a specific PCB degradation pathway and hence a different substrate specificity compared with B1B. This study will contribute to an elucidation of the dynamic aspects of PCB bioremediation in terms of roles played by PCB degraders and plant terpenes as natural inducer substrates that are ubiquitous and environmentally compatible.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5747-5751
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• 2014

Background: Coptisine, an isoquinoline alkaloid extracted from Coptidis rhizoma, has many biological activities such as antidiabetic, antimicrobial and antiviral actions. However, whether coptisine exerts anti-cancer metastasis effects remains unknown. Materials and Methods: Effects of coptisine on highly metastatic human breast cancer cell MDA-MB-231 proliferation were evaluated by trypan blue assay and on cell adhesion, migration and invasion by gelatin adhesion, wound-healing and matrigel invasion chamber assays, respectively. Expression of two matrix metalloproteinases (MMPs), MMP-9, MMP-2 and their specific inhibitors tissue inhibitor of metalloproteinase 1 (TIMP-1) and tissue inhibitor of metalloproteinase 2 (TIMP-2) were analyzed by RT-PCR. Results: Coptisine obviously inhibited adhesion to an ECM-coated substrate, wound healing migration, and invasion through the matrigel in MDA-MB-231 breast cancer cells. RT-PCR revealed that coptisine reduced the expression of the ECM degradation-associated gene MMP-9 at the mRNA level, and the expression of TIMP-1 was upregulated in MDA-MB-231 cells, while the expression of MMP-2 and its specific inhibitor TIMP-2 was not affected. Conclusions: Taken together, our data showed that coptisine suppressed adhesion, migration and invasion of MDA-MB-231 breast cancer cells in vitro, the down-regulation of MMP-9 in combination with the increase of TIMP-1 possibly contributing to the anti-metastatic function. Coptisine might be a potential drug candidate for breast cancer therapy.

• Biomolecules & Therapeutics
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• v.19 no.1
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• pp.27-32
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• 2011

The activation of microglia induces the overproduction of inflammatory mediators that are responsible for the neurodegenerative disorders including Alzheimer's disease and Parkinson's disease. The large amounts of prostaglandin $E_2$ ($PGE_2$) produced by inducible cyclooxygenase (COX-2) is one of the main inflammatory mediators that can contribute to neurodegeneration. The inhibition of COX-2 thus may provide therapeutic strategy for the treatment of neurodegenerative diseases. From the activity-guided purification of EtOAc soluble fraction of Siegesbeckia glabrescens, four compounds were isolated as inhibitors of $PGE_2$ production in LPS-activated microglia. Their structures were determined as 3, 4'-dimethylquercetin (1), 3, 7-dimethylquercetin (2), 3-methylquercetin (3) and 3, 7, 4'-trimethylquercetin (4) by the mass and NMR spectral data analysis. The compounds 1-4 showed dose-dependent inhibition of $PGE_2$ production in LPS-activated microglia with their $IC_{50}$ values of 7.1, 4.9, 4.4, $12.4\;{\mu}M$ respectively. They reduced the expression of protein and mRNA of COX-2 through the inhibition of I-${\kappa}B{\alpha}$ degradation and NF-$\kappa}B$ activity that were correlated with the inactivation of p38 and ERK. Therefore the active compounds from Siegesbeckia glabrescens may have therapeutic effects on neuro-inflammatory diseases through the inhibition of overproduction of $PGE_2$ and suppression of COX-2 overexpression.

• Natural Product Sciences
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• v.21 no.4
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• pp.240-247
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• 2015

Viridicatol (1) has previously been isolated from the extract of the marine-derived fungus Penicillium sp. SF-5295. In the course of further biological evaluation of this quinolone alkaloid, anti-inflammatory effect of 1 in RAW264.7 and BV2 cells stimulated with lipopolysaccharide (LPS) was observed. In this study, our data indicated that 1 suppressed the expression of well-known pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and consequently inhibited the production of iNOS-derived nitric oxide (NO) and COX-2-derived prostaglandin E2 ($PGE_2$) in LPS stimulated RAW264.7 and BV2 cells. Compound 1 also reduced mRNA expression of pro-inflammatory cytokines such as $interleukin-1{\beta}$ ($IL-1{\beta}$), interleukin-6 (IL-6), and tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$). In the further evaluation of the mechanisms of these anti-inflammatory effects, 1 was shown to inhibit nuclear factor-kappa B ($NF-{\kappa}B$) pathway in LPS-stimulated RAW264.7 and BV2 cells. Compound 1 blocked the phosphorylation and degradation of inhibitor kappa B $(I{\kappa}B)-{\alpha}$ in the cytoplasm, and suppressed the translocation of $NF-{\kappa}B$ p65 and p50 heterodimer in nucleus. In addition, viridicatol (1) attenuated the DNA-binding activity of $NF-{\kappa}B$ in LPS-stimulated RAW264.7 and BV2 cells.

• Molecules and Cells
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• v.21 no.3
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• pp.381-388
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• 2006

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heatshocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

• Biomolecules & Therapeutics
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• v.20 no.1
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• pp.50-56
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• 2012

WIN-34B showed analgesic and anti-inflammatory effects in various animal models of pain and osteoarthritis. However, the molecular mechanism by which WIN-34B inhibits pain and inflammation in vivo remains to be elucidated. We investigated the molecular mechanisms of the actions of WIN-34B using various in vitro models using fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA FLSs), RAW264.7 cells and peritoneal macrophages. WIN-34B inhibited the level of IL-6, $PGE_2$, and MMP-13 in IL-$1{\beta}$-stimulated RA FLSs in a dose-dependent manner. The mRNA levels were also inhibited by WIN-34B. The level of $PGE_2$, NO, IL-$1{\beta}$, and TNF-${\alpha}$ were inhibited by WIN-34B at different concentrations in LPS-stimulated RAW264.7 cells. The production of NO and $PGE_2$ was inhibited by WIN-34B in a dose-dependent manner in LPS-stimulated peritoneal macrophages. All of these effects were comparable to the positive control, celecoxib or indomethacin. I${\kappa}B$B signaling pathways were inhibited by WIN-34B, and the migration of NF-${\kappa}B$ into the nucleus was inhibited, which is consistent with the degradation of $I{\kappa}B-{\alpha}$. Taken together, the results suggest that WIN-34B has potential as a therapeutic drug to reduce pain and inflammation by inhibiting the production of pro-inflammatory mediators.

• Journal of Microbiology
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• v.34 no.2
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• pp.192-197
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• 1996

L-Xylosone was detected as its quinoxaline derivative in the degradation solution of dehydro-L-ascorbic acid. The production rate of L-xylosone was much faster in aerated phosphate-cirate buffer (pH 5. 6) than in pure water. L-Xylosone and dehydro-L-ascorbic acid were identified in the crude extracts of Saccharomyces cerevisiae. The concentration of L-xylosone in the crude cell extracts was calculated to be about 0.2 nmol $(mg protein)^{-1}$. When L-xylosone was added to asynchronous culture of S. cerevisiae, it inhibited primarily the synthesis of protein and RNA. Examination of the effect of L- xylosone on synchronous culture of the yeast indicated that L-xylosone inhibited the initiation of DNA replication and that the cells were arrested at $G_1$, stage of cell division cycle. These results suggested a possibility that L-xylosone can act as an inhibitor of cell growth.