• Biomedical Science Letters
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• v.15 no.1
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• pp.1-8
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• 2009

Human genomic DNA is continuously attacked by oxygen radicals originated from cellular metabolic processes and numerous environmental carcinogens. 2-deoxyribonolactone (dL) is a major type of oxidized abasic (AP) lesion implicated in DNA strand scission, mutagenesis, and formation of covalent DNA-protein crosslink (DPC) with DNA polymerase (Pol) ${\beta}$. We show here that human DNA polymerase (Pol)${\iota}$ and mitochondrial $Pol{\gamma}$ give rise to stable DNA-protein crosslink (DPC) formation that is specifically mediated by dL lesion. $Pol{\gamma}$ mediates DPC formation at the incised dL residue by its 5'-deoxyribose-5-phosphate (dRP) lyase activity, while $Pol{\gamma}$ cross links with dL thorough its intrinsic dRP lyase and AP lyase activities. Reactivity in forming dL-mediated DPC was significantly higher with $Pol{\gamma}$ than with $Pol{\iota}$. DPC formation by $Pol{\gamma}$, however, can be reduced by an accessory factor of $Pol{\gamma}$ holoenzyme that may attenuate deleterious effects of crosslink adducts on mitochondrial DNA. Comparative kinetic analysis of DPC formation showed that the rate of DPC formation with either $Pol{\iota}$ or $Pol{\gamma}$ was lower than that with $Pol{\beta}$. These results revealed that the activity of catalytic lyase in DNA polymerases determine the efficiency of DPC formation with dL damages. Irreversible crosslink formation of such DNA polymerases by dL lesions may result in a prolonged strand scission and a suicide of DNA repair proteins, both of which could pose a threat to the genetic and structural integrity of DNA.

• Archives of Pharmacal Research
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• v.21 no.5
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• pp.537-542
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• 1998

to more effectively drive immune responses toward antigen-specific T helper type 2 (Th2) cell-mediated responses, we constructed a mammalian expression vetor (oPVA/IL4) carrying a fused gene in which the ovalbumin (OVA) cDNA was covalently linked to murine interleukin-4 (IL-4) cDNA. A biologically active OVA/IL4 DNA, as demonstrated by Wes tern blotting and cytokine bioassay. In tramuscular injection of BALB/c mice with the pOVA/IL4 DNA increased both the production of OVA-specific IL-4 by CD$4^{+}$ T cells and the ratio of anti-OVA lgG1 to anti-OVA lgG2a isotypes, while the injection with the pOVA DNA alone, or with the mixture of the pOVA and pIL4 DNA did no or little increase. furthermore, the OVA-specific, Th2 cell-mediated immune responses were significantly enhanced by multiple injections with the pOVA/IL4 DNA. These studies indicate that the direct linkage of an OVA gene to an IL-4 gene in the expression plasmid confines the effects of IL-4 to the OVA-specific cells, efficiently driving the immune response toward OVA-specific, Th2 cell-mediated responses.

• Bulletin of the Korean Chemical Society
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• v.28 no.1
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• pp.95-98
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• 2007

Polyethylenimine (PEI) has been widely investigated for delivery of DNA into cells. It was previously reported that there were at least two types of cytotoxicity in PEI-mediated gene delivery, immediate and delayed toxicities. PEI-mediated gene delivery protocols use net cationic complexes with an excess of PEI to maintain equilibrium between the complexed and dissociated forms in solution. In this study, toxicity of free PEI or PEI/ DNA complex was investigated. Human embryonic kidney 293 cells were incubated with free PEI or PEI/DNA complex for 4 hrs. Then, the cells were analyzed at 6, 24, 48, and 96 hrs after the incubation. In MTT assay, the viability of the cells incubated with PEI/DNA complex was continuously decreased with time, while that of the cells incubated with free PEI was not. On the contrary, the expression level of the luciferase gene increased gradually along with time. Release of DNAs from the complexes for transcription produces free PEIs in the cells. This process may proceed slowly due to high charge density of PEI and may be related to delayed toxicity. In addition, apoptotic cells were observed only in the cells incubated with the PEI/DNA complex from 24 hrs after the incubation. The results suggest that PEI/DNA complex contributes to the delayed toxicity by inducing apoptosis and that the delayed toxicity may be related to decomplexation of the complexes in the cells.

• Journal of Life Science
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• v.24 no.4
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• pp.428-436
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• 2014

Loop-mediated isothermal amplification (LAMP) technique relies on autocycling strand displacement DNA sysnthesis without template denaturation steps under isothermal conditions. LAMP has more advantages than modern PCR, as it requires only basic laboratory equipment like an isothermal water bath, oven, and heating cabinet. Hence, in this study, five random primers were designed with Streptococcus parauberis, shikimate kinase Arok gene sequences (Genbank accession number: CP0024711). Two primers were selected based on the ladder pattern. Other optimum reaction conditions like temperature, time, and sensitivity were established and confirmed with conventional SYBR-green PCR. Results confirmed that the designed random primers were species specific, without any non-target DNA amplification under isothermal conditions. Hence, this study suggests that LAMP techniques could be used in the diagnosis of fish pathogen, specifically S. parauberis.

• The Korean Journal of Pharmacology
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• v.23 no.1
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• pp.45-49
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• 1987

Employing the approach to isolate the genes expressed preferentially in cytolytic T cell (CTL) but not in other types of cell, 3 CTL-specific cDNAs were recently cloned. To characterize these cDNA clones in relation to CTL activation, their expression pattern after T cell antigen receptor (TCR) or interleukin 2 (IL-2) stimulation were investigated by RNA blot analysis of cloned CTL L3 cells. Transcripts level of two cDNA clones were markedly elevated by TCR stimulation but not by IL-2. In addition, transcripts expression of both clones were abrogated by cyclosporin A treatment. These results indicated that gene activation mediated by TCR is distinct from that mediated by IL-2 and imply that those two unidentified cDNA clones are related to TCR-mediated, IL-2-independent but cyclosporin A-sensitive pathway for CTL activation.

• Environmental Analysis Health and Toxicology
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• v.21 no.2 s.53
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• pp.127-138
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• 2006

In urban areas, diesel exhaust particles (DEP) are probably a major component of particulate matters, especially in Korea where drive many diesel vehicles. The aim of this study was to investigate genotoxic effects of DEP using single ceil gel electrophoresis. In order to evaluate the mechanisms of DEP genotoxicity, the rat microsome mediated and DNA repair enzyme treated comet assays together with conventional comet assay were performed. Total diesel particles (DEPT) was collected without site fractionation from diesel engine bus and dichloromethane extract was obtained. The organic extract of DEPT revealed DNA damage itself in NIH/3T3 cells. The level of DNA breaks plus oxidative DNA lesions and microsome mediated DNA damage was assessed by modified single cell gel eletrophoresis. DEPT was able to induce oxidative DNA damage as well as microsome mediated DNA damage. Vitamin C as an model antioxidant reduced DNA damage in endonuclase III treated comet assay. One of flavonoid, galangin as a CYP1A1 inhibitor. reduced DNA damage in the presence of S-9 mixture. $DEP_T$ is the sources of oxidative stress, but antioxidants can significantly reduce oxidative DNA dmage. And $DEP_T$ may contain indirect mutagens which can be inhibited by CYP1A1 inhibitors. The ethanol extracts of the mixed vegetables (BV) or the mixed fruits (BF) were evaluated for their in vitro antigenotoxic effects. BV and BF showed potent Inhibitory effects against DEPT induced DNA damage with oxidative DNA lesions and in the prescence of S-9 mixture. These results indicate that BV and BF could prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P4501A1 in cell culture.

• Journal of Microbiology and Biotechnology
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• v.24 no.9
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• pp.1232-1237
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• 2014

Lycopene, which is a well-known red carotenoid pigment, has been drawing scientific interest because of its potential biological functions. The current study reports that lycopene acts as a bactericidal agent by inducing reactive oxygen species (ROS)-mediated DNA damage in Escherichia coli. Lycopene treatment elevated the level of ROS-in particular, hydroxyl radicals ($^*OH$)-which can damage DNA in E. coli. Lycopene-induced DNA damage in bacteria was confirmed and we also observed cell filamentation caused by cell division arrest, an indirect marker of the DNA damage repair system, in lycopene-treated E. coli. Increased RecA expression was observed, indicating activation of the DNA repair system (SOS response). To summarize, lycopene exerts its antibacterial effects by inducing $^*OH$-mediated DNA damage that cannot be ameliorated by the SOS response. Lycopene may be a clinically useful adjuvant for current antimicrobial therapies.

• BMB Reports
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• v.46 no.4
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• pp.225-229
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• 2013

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. In this study, we investigated whether ferritin enhances DNA cleavage by the reaction of MG with lysine. When plasmid DNA was incubated with MG and lysine in the presence of ferritin, DNA strand breakage was increased in a dose-dependent manner. The ferritin/MG/lysine system-mediated DNA cleavage was significantly inhibited by reactive oxygen species (ROS) scavengers. These results indicated that ROS might participate in the ferritin/MG/lysine system-mediated DNA cleavage. Incubation of ferritin with MG and lysine resulted in a time-dependent release of iron ions from the protein molecules. Our data suggest that DNA cleavage caused by the ferritin/MG/lysine system via the generation of ROS by the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin.

• YAKHAK HOEJI
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• v.51 no.4
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• pp.239-245
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• 2007

In order to evaluate the genotoxicity of airborne particulate matter extracted with dichloromethane (APE), the rat microsome mediated (S-9) or DNA repair enzyme treated Comet assays were performed using the single cell gel electrophoresis in A549 human lung carcinoma cells. It was found that the cells interacting with APE showed more DNA single-strand breaks relative to untreated cells. The genotoxicity of APE was increased with the treatment of S-9 mixture. Microsome mediated DNA damage was inhibited by CYP1Al inhibitor, quercetin. The APE also showed oxidative DNA damage evaluated by endonuclease III treatment. Oxidative DNA damage of APE was inhibited by antioxidants such as vita- min C and Trolox. We also found that the vegetables or fruits extract may reduce APE-induced genotoxicity by their anti- oxidant activity and CYP1A1 inhibition.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.204-204
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• 2022

Transformant construction using protoplasts requires less sample preparation time than particle bombardment and Agrobacterium-mediated transfection. There are two protoplast transfection methods: the PEG-mediated transfection method and the Lipofectamine transfection method. When Lipofectamine is mixed with DNA, Lipofectamine surrounds DNA like a cell membrane because of the positive charge of Lipofectamine. The Lipofectamine-DNA complex makes DNA insertion into cells easier. Fectin has similar functions to lipofectamine and is less expensive than lipofectamine. The 3D-fectin technology has been highlighted in animal cell transfection. Therefore, we performed PEG-mediated transfection, Lipofectamine transfection, and 3D-pectin transfection with a GFP construct. Protoplasts were isolated using the first leaf of "Bobwhite" after 4 hours of incubation in an isolation Buffer (cellulase + macerozyme). Protoplasts transformed by each method were cultured for 48 hours, and then GFP fluorescence expression was confirmed under confocal microscopy. GFP signals were detected in PEG-mediated transfection and Lipofectamine transfection. And the GFP signals were also detected in protoplasts to which 3D-fectin technology was applied, suggesting that 3D-fectin technology can be used for plant protoplast transfection.