• BMB Reports
• /
• v.41 no.3
• /
• pp.230-235
• /
• 2008

LRP15 is a novel gene cloned from lymphocytic cells, and its function is still unknown. Bioinformatic data showed that LRP15 might be regulated by DNA methylation and had an important role in DNA repair. In this study, we investigate whether the expression of LRP15 is regulated by DNA methylation, and whether overexpression of LRP15 increases efficiency of DNA repair of UV-induced DNA damage in HeLa cells. The results showed (1) the promoter of LRP15 was hypermethylated in HeLa cells, resulting a silence of its expression. Gene expression was restored by a demethylating agent, 5-aza-2'-deoxycytidine, but not by a histone deacetylase inhibitor, trichostatin A; (2) overexpression of LRP15 inhibited HeLa cell proliferation, and the numbers of cells in the G2/M phase of the cell cycle in cells transfected with LRP15 increased about 10% compared with controls; (3) cyclin B1 level was much lower in cells overexpressing LRP15 than in control cells; and (4) after exposure to UV radiation, the LRP15-positive cells showed shorter comet tails compared with the LRP15-negative cells. From these results we conclude that the expression of LRP15 is controlled by methylation in its promoter in HeLa cells, and LRP15 confers resistance to UV damage and accelerates the DNA repair rate.

• Korean Journal of Microbiology
• /
• v.39 no.3
• /
• pp.141-147
• /
• 2003

The simultaneous expression levels of antifungal activity related genes was analyzed by DNA microarray. We constructed DNA chips contained 2,000 randomly digested genome spots of the antifungal bacterium of Bacillus lentimorbus WJ5 and compared its quantitative aspect with 7 antifungal activity deficient mutants induced by gamma radiation ($^{60}Co$). From the analysis of microarray hybridization by the Gene Cluster (Michael Eisen, Stanford Univ.), totally 408 genes were expressed and 20 genes among them were significantly suppressed in mutants. pbuX (xanthine permease, K222), ywbA (phosphotransferase system enzyme II, K393), ptsG (PTS glucose specific enzyme II ABC component, K877), yufO (ABC transporter (ATP-binding protein), K130l), and ftsY (signal recognition particle (docking protein), K868) were simultaneously down-regulated in all mutants. It suggested that they were supposed to be related to the antifungal activity of B. lentimorbus WJ5.

• Journal of Life Science
• /
• v.15 no.5 s.72
• /
• pp.685-691
• /
• 2005

Growth and DNA synthesis inhibitory effects of doenjang methanol extract and its solvent fractions on AGS human gastric adenocarcinoma cells, Hep 3B human hepatocellular carcinoma cells, HT-29 human colon cancer cells and MG-63 human osteosarcoma cells were studied. The treatment of doenjang methanol extract ($ 200{\mu}g/ml $) with the AGS, Hep 3B, HT-29 and MG-63 cancer cells after 6 days of incubation inhibited the growth of cancer cells by $32\%$, $51\%$, $84\%$ and $33\%$, respectively. To separate active compounds of doenjang, doenjang methanol extract was fractionated with dichloromethane, ethylacetate, and buthanol. Among the solvent fractions, the dichloromethane and ethylacetate fractions showed the highest growth inhibitory effects on various cancer cells. For example, the dichloromethane and ethylacetate fractions ($200a{\mu}g/ml$) sig-nificantly inhibited the growth of various cancer cells by $89\∼96\%$ and$62\∼86\%$, respectively. DNA synthesis of AGS and Hep 3B cancer cells was significantly inhibited by adding dichloromethane fraction ($200{\mu}g/ml$) up to $94\%$ and $80\%$, respectively. Similarly, the ethylacetate fraction ($ 200\mug/ml $) showed a $ 95\% $ inhibition rate of DNA synthesis in AGS cells. These results suggest that the dichloromethane and ethylacetate fractions have specific active compounds, which will explain this anticancer effect of doenjang.

• Korean journal of applied entomology
• /
• v.54 no.2
• /
• pp.91-97
• /
• 2015

The sweetpotato whitefly, Bemisia tabaci, is the major insect pest that transmitted over 100 plant viruses including tomato yellow leaf curl virus (TYLCV) of tomato plant as virus vector in the world. In this study, cDNA library of whitefly was constructed using Gateway system for selecting target gene in order to control of B. tabaci using virus-induced gene silencing (VIGS) vector with RNAi. First of all, when using oligo d(T) rimer, the calculated titer of cDNA library was confirmed with $1.4{\times}10^4$ clones and average insert sizes was confirmed with 1 kb. However, insert size was very big for construction of cDNA. Otherwise, when using attB-N25 random primer and sonication for 6 sec, the calculated titer of cDNA library was confirmed with $1.04{\times}10^5$ clones. But mostly insert band wasn't identified on the electrophoresis, because it seemed that insert size is too small (${\leq}100bp$), also the size of identified insert was somewhat big. Finally, when using oligo d(T) primer and sonication for 1 sec, cDNA insert of whitefly was appropriated for VIGS with 300-600 bp. However, cDNA sequence included a poly A and titer was very low to $5.2{\times}10^2$ clones. It was supposed that heat shock transformation was used instead of electro-transformation. It is considered that when constructing cDNA library for using VIGS vector, (1) random primer should be used for First strand cDNA synthesis in order to remove poly A and (2) sonication for 1 sec should be performed in order to get appropriated insert size and (3) electro-transformation should be performed in order to improve transformation efficiency.

• Food Science of Animal Resources
• /
• v.28 no.3
• /
• pp.276-282
• /
• 2008

It is estimated that over 80% of deer antlers produced in the world are consumed in Korea. However, mislabeling or fraudulent replacement of costly antlers with cheaper ones is one of the most common problems in the domestic antler market. Therefore, there is a great need for the development of technology to identify species of antlers. This study was carried out to develop an accurate and reliable method for the identification and authentication of species or subspecies of antlers using DNA sequence analysis and comparison of mitochondrial cytochrome band D-loop region genes among antlers of five deer species, Cervus elaphus sibericus, Cervus elaphus canadensis, Cervus nippon, Cervus elaphus bactrianus and Rangifer tarandus. A variable region of cytochrome band D-loop genes was amplified using PCR with specifically designed primers and sequenced directly. The cytochrome band D-loop region genes showed different DNA sequences between the species of antlers and thus it is possible to differentiate between species on the basis of sequence variation. To distinguish between reindeer (Rangifer tarandus) antlers and other deer antlers, PCR amplicons of the cytochrome b gene were digested with the restriction enzymes NlaIV and TaqI, respectively, which generates a species-specific DNA profile of the reindeer. In addition, samples of 32 sliced antlers labeled Cervus elaphus sibericus from commercial markets were collected randomly and the mt DNA D-loop region of these antler samples was sequenced. Among the antler samples investigated, only 62.5% were from Cervus elaphus sibericus, and others were from Cervus elaphus bactrianus (25.0%), elk (Cervus elaphus canadensis) and reindeer (Rangifer tarandus). Our results suggest that DNA sequencing of mt DNA and PCR-RFLP methods using NlaIV and TaqI enzymes are useful for the identification and discrimination of deer antler species by routine analysis.

• Tuberculosis and Respiratory Diseases
• /
• v.65 no.6
• /
• pp.476-486
• /
• 2008

Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates $NF-{\kappa}B$ in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate $NF-{\kappa}B$ in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of $NF-{\kappa}B$ activation using proteasome inhibitor MG132 which blocks $I{\kappa}B{\alpha}$ degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study $NF-{\kappa}B$-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgG ${\kappa}-NF-{\kappa}B$ luciferase construct. To investigate DNA binding of $NF-{\kappa}B$ activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 ($3{\mu}M$) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced $NF-{\kappa}B$ transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of $NF-{\kappa}B$ activated by TRAIL and supershift with p65 antibody. $I{\kappa}B{\alpha}$ degradation was proven by western blot. MG132 completely blocked both TRAIL-induced $NF-{\kappa}B$ dependent luciferase activity and DNA binding of $NF-{\kappa}B$. Conclusion: This results suggest that inhibition of $NF-{\kappa}B$ can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.

• Journal of Periodontal and Implant Science
• /
• v.38 no.sup2
• /
• pp.343-354
• /
• 2008

Purpose: Aggregatibacter actinomycetemcomitans is associated with localized aggressive periodontitis. It produces cytolethal distending toxin (CDT), which induces cell cycle arrest in the G2/M phase. The CDT holotoxin is composed of CdtA, CdtB, and CdtC. CdtB has structural homology to human DNase I and is an active component of the CDT complex acting as a DNase. In particular, the pattern homology seen in the CdtB subunit has been associated with specific DNase I residues involved in enzyme catalysis, DNA binding, and metal ion binding. So, to study the functions and regulation of recombinant CdtB, we made up a quantity of functional recombinant CdtB and tested it in relation to the metal ion effect. Materials and Methods: We constructed the pET28a-cdtB plasmid from A. actinomycetemcomitans Y4 by genomic DNA PCR and expressed it in the BL21 (DE3) Escherichia coli system. We obtained the functional recombinant CdtB by the refolding system using the dialysis method and then analyzed the DNase activity and investigated the metal ion effect from plasmid digestion. Results: The recombinant CdtB subunit was expressed as the inclusion bodies. We were able to obtain functional recombinant CdtB subunit using refolding system. We confirmed that our refolded recombinant CdtB had DNase activity and was influenced by the metal ions $Mg^{2+}$ and $Ca^{2+}$. Conclusion: We suggest that the factors influencing recombinant CdtB may contribute to CDT associated diseases, such as periodontitis, endocarditic, meningitis, and osteomyelitis.

• The Plant Pathology Journal
• /
• v.34 no.6
• /
• pp.490-498
• /
• 2018

Panicle blight and seed rot disease caused mainly by Burkholderia glumae and Burkholderia gladioli is threatening rice cultivation worldwide. The bacteria have been reported as seed-borne pathogens from rice. Accurate detection of both pathogens on the seeds is very important for limiting the disease dissemination. Novel primer pairs targeting specific molecular markers were developed for the robust detection of B. glumae and B. gladioli. The designed primers were specific in detecting the target species with no apparent cross-reactions with other related Burkholderia species at the expected product size. Both primer pairs displayed a high degree of sensitivity for detection of B. glumae and B. gladioli separately in monoplex PCR or simultaneously in duplex PCR from both extracted gDNA and directly preheated bacterial cell suspensions. Limit of detection was as low as 0.1 ng of gDNA of both species and $3.86{\times}10^2cells$ for B. glumae and $5.85{\times}10^2cells$ for B. gladioli. On inoculated rice seeds, the designed primers could separately or simultaneously detect B. glumae and B. gladioli with a detection limit as low as $1.86{\times}10^3cells$ per rice seed for B. glumae and $1.04{\times}10^4cells$ per rice seed of B. gladioli. The novel primers maybe valuable as a more sensitive, specific, and robust tool for the efficient simultaneous detection of B. glumae and B. gladioli on rice seeds, which is important in combating rice panicle blight and seed rot by early detection and confirmation of the dissemination of pathogen-free rice seeds.

• Korean Journal of Food Science and Technology
• /
• v.33 no.6
• /
• pp.669-675
• /
• 2001

The objective of this study was to examine the effects of antioxidant vitamins on the formation of $AFB_{1}-DNA$ adduct and $AFB_{1}-inducing$ cellular oxidative damage. Intraperitoneal(i.p.)injections of 10 mg/kg vitamin C(VC) and 63.8 mg/kg vitamin E(VE) were repeatedly administrated 4 times with 2 days interval to 6 week old male ICR mice. After one hour of vitamin treatments, 0.4 mg/kg $AFB_1$ was injected in $AFB_1$ plus vitamin treated groups by same way. On the other hands, $AFB_1$ treated group was only injected with $AFB_1$ by the same method described above without vitamins. According to quantitative analysis of the $AFB_1$ in mice serum by indirect competitive ELISA, 12.28 and 18.78 ng/mL were detected in $AFB_1-treated$ groups, but 7.60 and 4.85 ng/mL in $AFB_1$ plus VC and VE treated groups, respectively. 23.78, 25.48 ng/mL of $AFB_1-DNA$ adduct were detected in mice liver of $AFB_1$treated groups, while 5.26, 7.81 ng/mL in $AFB_1$ plus VC and VE treated groups, respectively. Consequently, the differences in the concentrations of $AFB_1$ related materials between vitamin treated and non-treated groups were significant. Immunohistochemistry revealed brownish infiltration of $AFB_1$ around central vein and sinusoid in $AFB_1-treated$ group. This manifestation was distinctly reduced in $AFB_1$ plus VC and VE treated groups.

• Proceedings of the Korean Nutrition Society Conference
• /
• 1998.11a
• /
• pp.85-95
• /
• 1998