• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1174-1178
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• 2011

Exopolysaccharides (EPSs) are microbial polysaccharides that are released outside of the bacterial cell wall. There have been few studies on EPS-producing lactic acid bacteria that can enhance macrophage activity and the underlying signaling mechanism for cytokine expression. In the current study, EPS-overproducing Lactobacillus (L.) paracasei KB28 was isolated from kimchi and cultivated in conditioned media containing glucose, sucrose, and lactose. The whole bacterial cells were obtained with their EPS being attached, and the cytokine-inducing activities of these cells were investigated. Gas chromatography analysis showed the presence of glucose, galactose, mannose, xylose, arabinose, and rhamnose in EPS composition. EPS-producing L. paracasei KB28 induced the expression of tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-6, and IL-12 in mouse macrophages. This strain also caused the degradation of $I{\kappa}B{\alpha}$ and phosphorylation of the major MAPKs: Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK)1/2. The use of pharmacological inhibitors showed that different signaling pathways were involved in the induction of TNF-${\alpha}$, IL-6 and IL-12 by L. paracasei KB28. Our results provide information for a better understanding of the molecular mechanisms of the immunomodulatory effect of food-derived EPS-producing lactic acid bacteria.

• Journal of Society of Preventive Korean Medicine
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• v.5 no.1
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• pp.144-150
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• 2001

The cytotoxic activity of Cratalariae sessiliflorae on cultured NIH 3T3 fibroblasts and human oral epithelioid carcinoma cells (KB) were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT) colorimetric method These fractions of methanol extract of Cratalariae sessiliflorae showed inhibitory effect in vitro in the milligram range against KB cell lines. In general, the antitumor activities of these fractions were does-dependent over the milligram range. The comparison of IC50 values of these fractions in tumor cell lines showed that their susceptibility to these fractions decrease in the following order: Fr. 4> Fr. 6> Fr. 10> Fr. 2> Fr. 11> Fr. 3> Fr. 8> Fr. 7> Fr. 9> Fr. 1> Fr. 5 by the MTT assay. These fractions were tested for their cytotoxic effects on NIH 3T3 fibroblasts using MTT assay. They exhibited potent cytotoxic activities in vitro in the milligram range against NIH 3T3 fibroblasts. In general, the cytotoxic activities of these fractions were does-dependent over the milligram range. The comparison of CD50 values of these fractions in NIH 313 fibroblasts shows that their susceptibility to these fractions in decrease the following order: Fr. 10> Fr. 9> Fr. 2 = Fr. 4> Fr. 8> Fr. 11> Fr. 1 = Fr. 7> Fr. 3> Fr. 5 = Fr. 6 by the MTT assay. These results suggests that fraction 5 has the most growth - inhibitory activity against KB cell lines.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

• Journal of Life Science
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• v.14 no.6 s.67
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• pp.1023-1027
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• 2004

The RAD3 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. An yeast RAD3 gene has been previously isolated by functional complementation. In order to identify the RAD3 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD3 DNA, and then isolated RAD3 homologous DNA from C. cinereus chromosome. The RAD3 homolog DNA was contained in 3.2 kb DNA fragment. Here, we report the results of characterization of a fungus C. cinereus homolog to the yeast RAD3 gene. Southern blot analysis confirmed that the C. cinereus chromosome contains the RAD3 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from the C. cinereus cells were hybridized with the 3.4 kb PvuII DNA fragment of the S. cerevisiae RAD3 gene, transcripts size of 2.8 kb were detected. In order to investigate whether the increase of the amount of transcripts by DNA damaging agent, transcript levels were examined after treating agents to the cells. The level of transcripts were not increased by untraviolet light (UV). This result indicated that the RAD3 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the HRD3 gene is essential for viability of the cells and DNA repair function. These observations suggest an evolutionary conservation of other protein components with which HRD3 interacts in mediating its DNA repair and viability functions.

• Korean Journal of Poultry Science
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• v.50 no.4
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• pp.261-266
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• 2023

The skeletal muscles of livestock play a crucial role as protein sources for humans, and the consumption of poultry meat is steadily increasing worldwide. Numerous genes, including myogenic regulatory factors, are involved in myogenesis, and precise regulation of them is essential. In this study, genes specifically expressed in muscles were selected, and their promoters were cloned and analyzed. The analysis of gene expression in various tissues of animals revealed that many genes exhibited specific expression patterns in skeletal muscles, with TNNT3, TNNC2, and MYF6 genes showing similar patterns in poultry. The promoter regions of three genes were amplified by polymerase chain reaction to sizes of 1.2 kb, 1.03 kb, and 1.43 kb, respectively. These fragments were then inserted at the front of the enhanced green fluorescent protein gene in vectors. It was confirmed that the sequences of three promoters closely matched the chicken genome sequences. Upon introducing vectors with each promoter into QM7 quail muscle cells, all three promoters successfully induced the expression of the green fluorescent protein. The brightness of the green fluorescence in each promoter was approximately seven times dimmer compared to the control, CMV-IE promoter. It is predicted that more than 230 transcription factors can bind to each promoter, especially various transcription factors expressed in muscles, including myogenic regulatory factors such as MYF5, MYOD, and MYOG. These promoters can be valuable for studying gene expression in poultry muscle cells, and further research is needed to precisely investigate the regulatory region of gene expression in promoters.

• Molecules and Cells
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• v.38 no.11
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• pp.1007-1012
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• 2015

EphA7 is a key molecule in regulating the development of the dien- and mesencephalon. To get insight into the mechanism of how EphA7 gene expression is regulated during the dorsal specification of the dien- and mesencephalon, we investigated the cis-acting regulatory sequence driving EphA7 to the dorsal midline of the dien- and mesencephalon. Transgenic LacZ reporter analysis, using overlapping EphA7 BACs, was used to narrow down the dorsal midline-specific enhancer, revealing the 25.3 kb genomic region as the enhancer candidate. Strikingly, this genomic DNA was located far downstream of the EphA7 transcription start site, +302.6 kb to +327.9 kb. Further enhancer mapping, using comparative genomic analysis and transgenic methods, showed that the 187 bp genomic DNA alone, approximately 305 kb downstream of the EphA7 transcription start site, was sufficient to act as the dorsal midline-specific enhancer of EphA7. Importantly, our results indicate that the 187 bp dorsal midline-specific enhancer is critically regulated by homeobox transcription factors during the development of the dien- and mesencephalon.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.28 no.1
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• pp.127-143
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• 1998

The author evaluated the effects of taxol, a microtubular inhibitor, as a possible radiation sensitizer and the production of prostaglandins on three human cancer cell lines(KB, RPMI-2650 and SW-13) and one murine cell line(L929). Each cell line was divided into four groups (control, taxol only, radiation only and combination of taxol and radiation). The treatment consisted of a single irradiation of 10Gy and graded doses (5, 50, 100, 200, 300, 500 nM) of taxol for a 24-h period. The cytotoxicity of taxol alone was measured at 1 day after(1-day group) and 4 days after(4-day group) the treatment. The survival ratio of cell was analyzed by MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-dimethyl tetrazolium bromide) test. Prostaglandins(PGE2 and PGI2) were measured in the culture medium by a radioimmunoassay. The results obtained were as follows. 1. There was a significantly increased cytotoxicity of KB cells in 4-day group than those in I-day group. There was a high correlation between doses of taxol and cell viability in both groups(l-day group R=0.82741, 4-day group R=0.84655). 2. There was a significantly increased cytotoxicity of RPMI -2650 cells treated with high concentration of taxol in 4-day group than those in I-day group. Also there was a high correlation between doses of taxol and cell viability in 4-day group(R=0.93917). 3. There was a significantly increased cytotoxicity of SW-13 cells treated with high concentration of taxol in 4-day group than those in 1-day group. However no high correlation was observed between doses of taxol and cell viability in both groups(1-day group R=0.46362, 4-day group R=0.65425). 4. There was a significantly increased cytotoxicity of L929 cells treated with low concentration of taxol in 4-day group than those in 1-day group. At the same time, there was a low correlation between doses of taxol and cell viability in both groups(1-day group R=0.34237, 4-day group R=0.23381). 5. In I-day group of L929 cells, higher cytotoxicities were observed in the groups treated with 500 nM taxol than given 10 Gy radiation alone. L929 cells in I-day group alone showed a radiosensitizing effect by taxol.. 6. In addition to L929 cells, all cancer cells treated with a combination of taxol and radiation in 4-day group appeared to have some fragmented nuclei and to float on the medium. In addition, L929 cells appeared to be more confluent. 7. The level of PGE2 production was the highest in the contol KB cells. This appeared to increase in every experimental group of all three cancer cells except L929 cells. There was a significantly increased production of PGE2 in SW -13 cells treated with a combination taxol and radiation compared to the other experimental groups. 8. The level of PGE2 production in the control group of RPMI-Z650 cells was the highest. This appeared to increase in every experimental group of all cells except in SW-13 cells. This also increased significantly in RPMI-2650 cells treated with a combination of taxol and radiation compared to the other experimental groups.

• International Journal of Oral Biology
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• v.33 no.1
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• pp.25-31
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• 2008

Inhibition of proteasome activity may reduce many types of cancer, so it's pathway is effective in cancer as well as in clinical fields. Here the author has carried out experiment targeting on the elevation of apoptosis in oral cancer cells by combination of proteasome inhibitor, lactacystin, and DNA replication inhibitor, etoposide. The growth of KB cells was measured by MTT methods and apoptosis was analyzed by DNA fragmentation and Hochest nucleus staining. The proteasome activity was analyzed by fluorescent tagged peptide and cellular protein expression was detected by Western hybridization. Though lactacystin and etoposide inhibited KB cell growth alone, but low combined doses inhibited cell growth more strongly and induced apoptosis. The proteasome activity was also seriously inhibited by the combination of both chemicals. Tumor suppressor proteins and apoptosis inducing proteins were highly increased under the combination of both chemicals. From above studies we can conclude that proteasome inhibitors may be used for the treatment of oral cancer and proteasome inhibitors with DNA replication inhibitors may be effective in clinical trials of oral cancer.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.27 no.1
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• pp.107-122
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• 1997

Radiation sensitivity data was generated for two human cancer cell lines(KB, RPMI 2650) and human primary gingival fibroblast was tested three times using a viable cell number counting with a hemocytometer, MTT(3-[4,5-Dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide) assay, and LDH(Lactate dehydrogenase) assay. Single irradiation of 2, 4, 6, 10, 15, 20Gy were applied to the tumor cell lines and the primary cultured gingival fibroblast The two fractions of 4Gy and 10Gy were seperated with a 4 hour time interval. The irradiation was done with 241.5cGy/min dose rate using /sup 137/Cs MK cell irradiator at room temperature. The obtained results were as followed : 1. There was significantly different viable cell numbers as the amount of radiation dose on the tested cells were cell number counted with a hemocytometer. In fractions, there were more viable cells remaining. 2. Phase-contrast microscopically, radiation-induced morphologic changes were pronounced on the tumor cells, however, almost no differences on the gingival fibroblast. 3. There was significantly different absorbance at 2Gy on RPMI 2600, 4Gy on KB and GF in MTT assay. In fractions, the absorbance was significantly higher on KB. 4. The level of extracellular LDH activity in the experimental group was significantly higher in the 2-4Gy than the control group. 5. The total level of extracellular and intracellular LDH activity was decreased as increased amounts of radiation dose was applied.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.631-636
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• 1999

The thermostable endo-chitosanase gene from the isolated strain Bacillus sp. KFB-C108 was identified on the basis of a phylogenetic analysis of the 16S rRNA gene sequence, and was cloned into plasmid pUCl8 using E. coli $DH5\alpha$ as the host strain. Positive clones carrying recombinant plasmids (pKCHO I and pKCHO II) containing chitosanase activity were selected using the direct activity staining method. Detailed physical maps showed the two plasmid inserts were identical except that the KCHO II insert (2.6 kb) was 1.8 kb smaller than that of the KCHO I. The recombinant plasmids were analyzed to determine the essential region for chitosanase activity, and a 1.3-kb fragment (KCHO-6) was subcloned into pTrc99A using the EcoRI and BamHI sites to construct pTrc99A/KCHO-6(pTrEB13). The resulting plasmid exerted high chitosanase activity upon transformation of E. coli $DH5{\alpha}cells$, overproducing about 20 times more in the cloned cells than in the wild-type cells. The cloned chitosanase protein exhibited the same molecular weight and catalytic activity similar to those of Bacillus sp. KFB-C108. The cloned enzyme was an endo-type that produced a chitosan tetramer as the major reaction product; however, it produced no monomers or dimers.