• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2000년도 International Symposium on Bioinformatics
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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.

• BMB Reports
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• 제38권4호
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• pp.414-419
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• 2005

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

• BMB Reports
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• 제34권4호
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• pp.316-321
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• 2001

Dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1) catalyzes the reduction of DHA to reduced ascorbate (AsA) using glutathione (GSH) as the electron donor in order to maintain an appropriate level of ascorbate in plant cells. To analyze the physiological role of DHAR in environmental stress adaptation, we developed transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants that express a human DHAR gene isolated from the human fetal liver cDNA library in the chloroplasts. We also investigated the DHAR activity, levels of ascorbate, and GSH. Two transgenic plants were successfully developed by Agrobacterium-mediated transformation and were confirmed by PCR and Southern blot analysis. DHAR activity and AsA content in mature leaves of transgenic plants were approximately 1.41 and 1.95 times higher than in the non-transgenic (NT) plants, respectively In addition, the content of oxidized glutathione (GSSG) in transgenic plants was approximately 2.95 times higher than in the NT plants. The ratios of AsA to DHA and GSSG to GSH were changed by overexpression of DHAR, as expected, even though the total content of ascorbate and glutathione was not significantly changed. When tobacco leaf discs were subjected to methyl viologen at $5\;{\mu}M$, $T_0$ transgenic plants showed about a 50% reduction in membrane damage compared to the NT plants.

• BMB Reports
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• 제34권6호
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• Asian-Australasian Journal of Animal Sciences
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• 제21권9호
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• pp.1233-1237
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• 2008

The genetic variability within and among three deer species in Malaysia, namely Cervus nippon (sika), Cervus timorensis (rusa) and Cervus unicolor (sambar), were evaluated using the RAPD technique. The DNA extracted from the buffy coat of 34 sika, 38 rusa and 9 sambar were analysed using ten primers that gave bands which showed good resolution. The primers generated 164 RAPD markers in total, and these ranged in size from 150 to 900 bp. The percent of polymorphism of the bands generated per primer ranged from 66.66-93.33% for rusa, 36.84-61.14% for sambar and 52.38-100% for sika. The overall percent polymorphism observed for the 164 RAPD markers was 99.39%. The results revealed five exclusive, monomorphic markers for sambar and one exclusive, monomorphic marker for sika; none was observed for rusa. However, these cannot be declared as markers for the identification of the species without analysis of more samples, populations and species. The means of within population genetic distances, based on Dice's and Jaccard's similarity indices, were similar for the rusa (0.383 and 0.542, respectively) and sika (0.397 and 0.558, respectively) populations with the sambar population being the least variable (0.194 and 0.323, respectively). The Dice based genetic distances within the species ranged from 0.194 to 0.397 and the genetic distances among the species were 0.791-0.911. The genetic distances based on Dice's and Jaccard's similarity indices between the rusa and sambar were 0.556 and 0.713, between the rusa and sika populations were 0.552 and 0.710, and between sambar and sika were 0.622 and 0.766, respectively.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.564-570
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• 2016

The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using $300{\mu}g/mL$ G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5131-5136
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• 2012

Cancer is one of the major health problems worldwide and its current treatments have a number of undesired adverse side effects. Natural compounds may reduce these. Currently, a few plant products are being used to treat cancer. In this study, goniothalamin, a natural occurring styryl-lactone extracted from Goniothalamus macrophyllus, was investigated for cytotoxic properties against cervical cancer (HeLa), breast carcinoma (MCF-7) and colon cancer (HT29) cells as well as normal mouse fibroblast (3T3) using MTT assay. Fluorescence microscopy showed that GTN is able to induce apoptosis in HeLa cells in a time dependent manner. Flow cytometry further revealed HeLa cells treated with GTN to be arrested in the S phase. Phosphatidyl serine properties present during apoptosis enable early detection of the apoptosis in the cells. Using annexin V/PI double staining it could be shown that GTN induces early apoptosis on HeLa cells after 24, 48 and 72 h. It could be concluded that goniothalamin showing a promising cytotoxicity effect against several cancer cell lines including cervical cancer cells (HeLa) with apoptosis as the mode of cell death induced on HeLa cells by Goniothalamin was.

• Asian Pacific Journal of Cancer Prevention
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• 제13권3호
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• pp.1019-1024
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• 2012

Background: Infections with human papillomavirus (HPV) are highly prevalent among sexually active young women in India. However, not much is known about the incidence of type-specific human papillomavirus (HPV) infections and their patterns of persistence, especially in the Indian context. Objective: The objective of this study was to evaluate the rate of acquisition and persistence of HPV types in young women. Methods: Women residing in an urban slum in Delhi (n=1300) were followed for 24 months at 6 monthly intervals. Exfoliated cervical cells collected at each visit were tested for the presence of HPV DNA. Genotyping was performed using the reverse line blot assay. Results: The incidence rate for any HPV type was calculated to be 5 per 1000 women-months. Among high risk HPV types, HPV16 had the highest incidence rate followed by HPV59, HPV52 and HPV18, i.e., 3.0, 0.58, 0.41 and 0.35 women per 1000 women-months respectively. The persistence rate was higher for high-risk than low-risk HPV types. Among low-risk types, HPV42, HPV62, HPV84 and HPV89 were found to persist. Whereas almost all high risk types showed persistence, the highest rate was found in women with HPV types 16, 45, 67, 31, 51 and 59. The persistence rate for HPV16 infection was 45 per 1000 women-months. Conclusion: Incident HPV infections and high risk HPV type-specific persistence were found to be high in our study population of young married women. Understanding the patterns of HPV infection may help plan appropriate strategies for prevention programs including vaccination and screening.

• Asian Pacific Journal of Cancer Prevention
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• 제14권5호
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• pp.3001-3003
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• 2013

Objective: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shown to have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector for Syk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. Methods: A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vector pLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant Syk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. After selection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive ability were examined. Results: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stably in the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulated significantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05). Conclusions: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibit their invasive ability in vitro.

• 한국수산과학회지
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• 제50권6호
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• pp.714-720
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• 2017

Exposure to ultraviolet (UV) radiation is associated with the development of adverse effects in skin. Among the three types of UV rays, UV-B causes the most damaging effects, inducing sunburn and penetrating the outer skin, resulting in DNA mutations and skin cancer. The objective of this study was to formulate a UV-protective film by incorporating Ecklonia cava extracts. Cells covered with the film were exposed to UV-B (50, 80, and $100mJ/cm^2$). To determine the protective effects of the film, we evaluated cell viability, intracellular ROS generation, and apoptosis. We found that all E. cava extracts absorbed UV light and exhibited protective effects against UV-B-induced photodamage. Among the protective films examined in this study, that incorporating an E. cava 70% ethanol extract (70EX) formed the most effective protection against UV-B in HaCaT cells. These findings suggest that the application of film containing E. cava extract could prevent UV-B-induced photodamage, and offer protection against the detrimental effects of UV radiation, thus maintaining physiological condition.