• Environmental Analysis Health and Toxicology
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• 제22권4호
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• pp.357-366
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• 2007

To investigate the possible enhancement of genotoxicity in stress environment, we examined the of effect of genotoxic material in oxidative stress-induced condition using human tell line. Human lymphoblast cell line, TK6 was treated with hydrogen peroxide ($H_2O_2$) for induction of oxidative stress, and treated with N'-methyl-N'-nitroguanidine (MNNG), af a genetoxic material. We carried out MTS assay to set treatment doses. TK6 was treated with $H_2O_2$ at 6.75 (low dote) or $13.5\;{\mu}M$ (high dose) for 2 h, and treated with MNNG af 0.117 (low dose), 0.234 (middle dose), $0.468\;{\mu}M$ (high dose) for 2 h. As results, a treatment of MNNG induced DNA dam age as dose dependently. And TK6 treated with $H_2O_2$ at low as well as high dose followed by MNNG treatment showed higher DNA damage compared to MNNG alone treated groups. Malondialdehyde, as a marker of lipid peroxidation was increased in $H_2O_2$ and MNNG treated groups. Real-time RT-PCR analyses for expression of several antioxidative enzymes showed that catalase mRNA and glutathione peroxidase 1 mRNA expression were decreased in $H_2O_2$ and MNNG treated groups. Taken together, we conclude that genotoxicity induced by MNNG is enhanced in a condition of oxidative stress induced by $H_2O_2$ and it suggests that it should be associated with induction of lipid peroxidation and decrease of antioxidant enzymes.

• IMMUNE NETWORK
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• 제1권1호
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• pp.45-52
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• 2001

Background: Many types of cancer become resistant to current chemotherapeutic and radiotherapeutic intervention. To overcome this situation application of gene therapy by the introduction of suicide genes followed by their prodrugs may be promising. A viral enzyme, Herpes simplex thymidine kinase (HSV-tk), which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining prodrug-activating gene therapy and irradiation might result in enhanced antitumor effects. Methods: The HSV-tk gene was cloned into the retroviral vector, pLXSN and established the clones producing retroviruses carrying the HSV-tk gene. The carcinoma cell line, HCT116 and Huh-7 were transduced with high-titer recombinant retroviruses. These cell lines were treated with ganciclovir before or after irradiation for the defining combinational effect of suicide gene therapy and radiotherapy. Results: The titers of cloned PA3 17 amphotropic retroviruses ranged from 4 to 6 X $10^6CFU/ml4$. After selectional periods, the expression of HSV-tk was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). The growth of cells expressing HSV-tk was inhibited as increase of GCV dose after 48 hr and the growth inhibitory effect of GCV was much higher after 72 hr. When the cells transduced with HSV-tk gene were exposed to radiation, the growth inhibitory effect of GCV was significantly increased, as compared with non-transduced parental cells. Conclusions: The results suggest that the addition of HSV-tk gene therapy to standard radiation therapy may improve the effectiveness of treatment for solid tumors.

• 대한의생명과학회지
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• 제11권2호
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• pp.165-173
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• 2005

A potent demethylating agent, 5-Azacytidine (5-AzaC) has been widely used as in many studies on DNA methylation, regulation of gene expression, and cancer biology. The mechanisms of the demethylating activity were known to be formation of complex between DNA and DNA methyltransferase (MTase), which depletes cellular MTase activity. However, 5-AzaC can also induce hypermethylation of a transgene in a transgenic cell line, G12 cells and it was explained as a result of defense mechanisms to inactivate foreign gene(s) somehow. This finding evoked the question that whether the phenomenon of hypermethylation induced by 5-AzaC is limited to the transgene or it can be occurred in endogenous gene(s). In order to answer the question, mutagenicity test of 5-AzaC and molecular characterization of mutants obtained from the test were performed using an endogenous gene, thymidine kinase (tk) in Chinese hamster V79 cells. When V79 and V79-J3 subclone cells were treated with 1, 2.5 ,5, $10{\mu}M$ of 5-AzaC for 48 hours, their maximum mutant frequencies were revealed as $6\times10^{-3}\;at\;5{\mu}M$(350-fold induction over background) and $8\times10^{-3}\;at\;2.5{\mu}M$ (l,800-fold induction over background) respectively. Since the induction rates were too high to be induced by true mutations, many trifluorothymidine (TFT)-resistant $(TFT^R)$ cells were subjected to Northern blot analysis to check the presence of tk transcripts. Surprisingly, all clones tested possessed the transcripts in a similar level, that implicates the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the gene in spite of unusually high mutation frequency. In addition, it has shown that the TK activity in the pool of 5-AzaC-induced $TFT^R$ cells has about a half of that in spontaneously-induced $TFT^R$ cells or in non-selected parental V79-J3 cells. This result suggests that the mechanism(s) underlying the TFT-resistance between spontaneously occurred and 5-AzaC-induced cells may be different. These findings have shown that the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the tk gene, and 5-AzaC may be induced by one or combined pathways among many drug resistance mechanisms. The exact mechanisms for the 5-AzaC-induced $TFT^R$ phenotype remain to elucidate.

• Preventive Nutrition and Food Science
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• 제14권1호
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• pp.21-28
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• 2009

In this study, we focused on natural water-soluble antioxidants from Tetraselmis suecica (T. suecica) and Chlorella ellipsoidea (C. ellipsoidea). They were prepared by enzymatic digestion using five carbohydrases (Viscozyme, Celluclast, AMG, Termamyl and Ultraflo) and five proteases (Protamex, Alcalase, Flavourzyme, Neutrase, and Kojizyme), and the potential antioxidant activity of each was assessed. Most enzymatic digests from T. suecica had a higher radical scavenging activity than those from C. ellipsoidea. Among the enzymatic digests, Kojizyme digest from T. suecica exhibited the highest effect on DPPH radical scavenging. Viscozyme (30.2%) and Neutrase (34.6%) digests from T. suecica exhibited higher hydroxyl radical scavenging activity. Kojizyme digest from T. suecica (81.5%) had strong alkyl radical scavenging activity. Neutrase (61.9%) and Kojizyme (61.5%) digest from T. suecica possessed the highest effects on hydrogen peroxide scavenging. Among the tested samples, Neutrase (TN) and Kojizyme (TK) digests from T. suecica showed the highest antioxidant activity (DPPH, alkyl radical, hydrogen peroxide). Therefore, TN and TK digests were selected for use in the further experiments. Those digests showed enhanced cell viability against $H_2O_2$-induced oxidative damage, and relatively good hydrogen peroxide scavenging activity in an African green monkey kidney (Vero) cell line. These results suggested that an enzymatic digestion will be an effective way for the production of a potential water-soluble antioxidant from a microalgae, T. suecica.

• Radiation Oncology Journal
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• 제21권3호
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• pp.227-237
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• 2003

목적: 만성 골수성 백혈병 세포인 K562 세포주는 방사선 및 다양한 항암제에 대한 apoptosis에 저항성을 가진다. 지난 연구에서 K562 세포는 방사선에 대하여 내성반응을 보이며, 세포내 PTK의 작용을 억제하고자 방사선 조사와 함께 투여한 herbimycin A (HMA)에 의하여 방사선에 대한 apoptosis와 같은 감수성반응이 유도되는 반면, genistein에 의하여 방사선에 대한 apoptosis 반응이 저해됨을 확인하였다. 본 연구에서는 타이로신 인산화효소 억제에 의한 K562 세포의 방사선 반응변화를 조절하는 신호전달경로를 조사하였다. 대상 및 방법: K562 세포를 지수증식기의 세포들만 선택하여 실험에 이용하였다. 방사선조사는 6 MeV 선형가속기(Clinac 1800C, Varian)를 이 용하여 $200\~300$ cGy/min 선량률로 $0.5\~12 $ Gy를 균일하게 조사하였다. HMA와 genistein은 각각 $0.25/muM,\;25\muM$을 방사선 조사 후 즉시 투여하였다. 실험에서 신호전달 경로로 abl kinase, MAPK family, NF-kB, c-fos, c-myc, thymidine kinase1 (TK1) 등에서의 단백질 또는 유전자 발현 및 활성을 조사하였다. 또한 약제 투여에 따른 유전자 발현차이(differential gene expression)를 조사하였다. 결과: Abl kinase의 발현 및 활성 변화를 조사하였으나 PTK 저해제에 의한 방사선 유도 세포사의 변화와의 연관성을 찾을 수 없었다. 세포 생존 및 사멸의 신호전달체계에서 주요 조절과정인 MAPK family의 관여 여부 확인에서 방사선으로 인한 SAPK/JNK의 활성화의 유도가 관찰되었으나, PTK 저해제에 따른 변화는 없었으며, 또한 MAPK/ERK와 p38 MAPK 활성은 모든 조건에서 변함 없이 일정하였다. 전사인자 활성화에 대한 조사에서 방사선 조사와 함께 genistein을 투여한 경우에 NF-kB활성이 증가하였다. 유전자 발현 차이의 조사에서 genistein 투여에 의한 TK 1 유전자 발현 및 단백질 활성이 증가하였다. 결론: PTK 억제에 의한 K562 세포의 방사선에 대한 반응 변화는 bcrabl kinase 활성과는 무관하게 진행되며, MAPK family 경로 외의 다른 경로를 통한 전사인자 활성화 과정이 연관되어 있음을 확인하였다.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 춘계학술대회
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• pp.185-185
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• 2003

To develope the novel anti-allergic drug, many sophoricoside derivatives were synthesized. Among these derivatives, JSH-II-3, JSH-Ⅵ-3, JSH-Ⅶ-3, and JSH-Ⅷ-3 were selected and subjected to high throughput toxicity screening (HTTS) because they revealed strong IL-5 inhibitory activity and limitation of quantity. Mouse lymphoma thymidine kinase (tk$\^$+/-/) gene assay (MOLY) and single cell gel electrophoresis (Comet) assay in mammalian cells were used as HTTS tool in our laboratory. In MOLY assay, JSH-Ⅶ-3 at 50 ∼ 6 $\mu\textrm{g}$/ml concentrations was not shown significant mutagenic effect in the absence and presence of S-9 metabolic activation system. However, the concentration of ISH-II-3, 38 $\mu\textrm{g}$/ml, induced increased mutation frequency (MF) in the presence of S-9 metabolic activation system. Also in comet assay, DNA damage was not observed in JSH-Ⅵ-3 and JSH-Ⅶ-3, wherase concentration of 32.8 $\mu\textrm{g}$/ml in JSH-II-3 and 13.9 $\mu\textrm{g}$/ml in JSH-Ⅶ-3 were induced DNA damage in the absence of S-9 metabolic activation system. Therefore, we suggest that JSH-Ⅵ-3 and JSH-Ⅶ-3 have no genotoxic effects but JSH-II-3 and JSH-Ⅷ-3 induce some mutagenicity and DNA strand breaks in mouse lymphoma cell line used this study.

• 한국지반환경공학회 논문집
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• 제6권4호
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• pp.47-58
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• 2005

지하공간의 효율적인 이용을 위하여 여러 분야에서 활발한 연구와 개발이 이루어지고 있으며 최근 들어 해안지역의 대심도 굴착을 통한 지하공간 활용방안에 관한 연구와 시공이 지속적으로 이루어지고 있다. 해안지역 및 매립지반의 침투류 거동은 조위영향에 의한 부정류 흐름이 발생되므로 부정류 흐름해석의 지배인자인 불포화토의 함수특성에 대한 연구가 요구된다. 본 연구에서는 불포화 함수특성에 따른 대심도 굴착지역의 침투류 거동을 분석하기 위하여 해안 준설매립지역 토사에 대한 함수특성시험을 수행하였으며, 불포화 함수특성에 따라 침투류 해석을 수행하여 실제 계측된 침투유량과 비교 분석하였다. 해안 매립지 토사에 대한 함수특성시험 결과 및 함수특성 계수에 대한 분석결과, Fredlund and Xing의 경험방정식이 해안 매립지반 토사에 대하여 높은 정확도로 실험결과 값을 나타내었다. 함수특성을 고려한 침투해석 결과, 부정류해석 결과가 정류해석 결과에 비하여 양수시험 결과 및 실제 계측된 용수량과 근접한 침투유량이 도출되어 해안 준설매립지반과 같이 불포화토 특성이 지배적인 지반에 대해서는 함수특성을 고려한 부정류 침투해석을 적용하여야 할 것으로 판단된다.