• 생약학회지
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• 제48권3호
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• pp.173-178
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• 2017

We investigated the efficacy of single compound of plant extract in coxsackievirus B3 (CVB3) infection. CVB3 is a main cause of Hand-foot-mouth diseases (HFMD) and viral myocarditis in children and adult. Several single compounds of plant extract were purified by HPLC and tested as antiviral drug candidate. Among them, kaempferol was selected to effective anti-enterovirus compound by HeLa cells survival assay. CVB3 infected HeLa cells were treated with kaempferol ($100{\mu}g/ml-100ng/ml$) and their antiviral effect was confirmed. After 16 hours of treatment, HeLa cells were lysed and proteins were extracted for western blot analysis. CVB3 viral capsid protein VP1 production and transcription factor eIF4G-1 cleavage was significantly decreased in $100{\mu}g/ml$ kaempferol treatment. Virus replication was observed by virus RNA amplification. Kaempferol strongly reduced virus positive and negative strand RNA amplification. Moreover, MAPK signal induced by CVB3 infection, pERK and pmTOR, kaempferol treatment significantly inhibited the activity. Plant extract single compound, kaempferol, is a strong candidate to be developed non-toxic anti-enterovirus treatment agent.

• 생명과학회지
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• 제7권4호
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• pp.392-401
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• 1997

폴리오바이러스는 바이러스들 중에서도 특히 커기가 작은 바이러스로서 피막(coat)을 둘러싸는 막(envelop) 이 없다. 폴리오바이러스는 (+) 가닥의 단일 RNA 게놈을 갖는데 이는 한 개의 해독판 (open reading frame)을 이용하여 다단백전구체를 만든 후 바이러스 자체의 단백질분해효소에 의해 스스로 잘라져서 궁극적으로느 특이한 기능을 갖는 여러개의 단백질이 된다. P1 다단백질전구체로부터 만들어지는 단백질들은 바이러스의 피막을 구성하는 성분이다. 단백질분해효소인 2A에 의한 최초의 절단은 구조단백질 P1 전구체와 구조단백질이 아닌 P2-P3간을 분리시켜준다. 단백질분해효소 2A는 진핵세포 판독개시인자(translation initiation factor) 4F의 한 subunit인 숙주단백질 p220의 절단에 간접으로 참여한다. 이 단백질의 절단은 캡(cap)에 의존하는 숙주세포의 대부분의 판독을 차단하게 되며 이는 판독에 사용되는 숙주세포의 모든 기구들을 캡에 의존하지 않는 폴리오바이러스 NA 특유의 판독을 위해 전적으로 사용할 수 있게 해준다. 2B, 2C, 2BC 단백질의 기능에 대해서는 많이 알려져 있지 않다. 2B, 2C, 2BC와 3CD 단백질들은 바이러스로 인해 만들어지는 소낭(vesicle)의 복제복합체에 함유되어 있으므로 바이러스의 RNA 복제시 중요한 역할을 함을 암시해준다. 새로이 만들어진 모든 바이러스 RNA는 VPg와 공유결합으로 연결되어 있다. VPg는 3AB로부터 만들어진 아미노산 22개 짜리의 폴리펩타이드이다. 3C와 3CD는 단백질분해소로 다단백질 전구체의 대부분의 절단부위를 잘라준다. 3C단백질은 숙주의 전사인자를 불활성화 시킴으로써 RNA polymer II와 III에 의한 전사를 저해한다. 3D는 RNA의존선RNA 중합효소이다. 폴리오바이러스는 (+)가닥 RNA 바이러스의 일반적인 복제양식을 따른다. 즉 (+) 가닥 RNA는 이와 상보적인 (-)가닥 RNA로 전사되고 이는 다시 (+)가닥 RNA의 합성을 위한 주형으로 사용된다. 폴리오바이러스의 RNA 합성은 세포내막에서 일어나는 데 RNA 복제에 요구되는 주형 RNA와 이때 필요한 단백질들이 어떤 방법으로 세포내막에서 모일 수 있는지는 아직 밝혀진 것이 적다. 바이러스입자의 형성은 세포막의 RNA 복제가 들어가는 데 피막단백질이 (+)가닥 RNA을 인식하는 표지 즉 packaging singal에 대해서는 거의 알려져 있지 않다. 폴리오바이러스 감염 후 첫 바이러스입자가 만들어지기 까는 약 6시간이 소요된다.

• BMB Reports
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• 제54권2호
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• pp.98-105
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• 2021

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8177-8186
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• 2016

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta ($pol{\beta}{\Delta}_{208-304}$) specific for ovarian cancer. $Pol{\beta}{\Delta}_{208-304}$ has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. $Pol{\beta}{\Delta}_{208-304}$ cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards $H_2O_2$ and UV when compared with HeLa cells alone. It has been shown that cell death in $Pol{\beta}{\Delta}_{208-304}$ transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1321-1324
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• 2012

Background: DNA polymerase beta ($pol{\beta}$) is a key enzyme in the base excision repair pathway. It is 39kDa protein, with two subunits, one large subunit of 31 kDa having catalytic activity between exon V to exon XIV, and an 8 kDa smaller subunit having single strand DNA binding activity. Exons V to VII have double strand DNA binding activity, whereas exons VIII to XI account for the nucleotidyl transferase activity and exons XII to XIV the dNTP selection activity. Aim: To examine the association between $pol{\beta}$ polymorphisms and the risk of ovarian cancer, the present case control study was performed using 152 cancer samples and non-metastatic normal samples from the same patients. In this study, mutational analysis of $pol{\beta}$ genomic DNA was undertaken using primers from exons IX to XIV - the portion having catalytic activity. Results: We detected alteration in DNA polymerase beta by SSCP. Two specific heterozygous point mutations of $pol{\beta}$ were identified in Exon 9:486, A->C (polymorphism 1; 11.18%) and in Exon 11:676, A->C (polymorphism 2; 9.86%). The correlation study involving polymorphism 1 and 4 types of tissue showed a significant correlation between mucinous type with a Pearson correlation value of 4.03 (p=0.04). The association among polymorphism 2 with serous type and stage IV together have shown Pearson ${\chi}^2$ value of 3.28 with likelihood ratio of 4.4 (p=0.07) with OR =2.08 (0.3-14.55). This indicates that there is a tendency of correlation among polymorphism 2, serous type and stage IV, indicating a risk factor for ovarian cancer. Conclusion: Hence, the results indicate that there is a tendency for $pol{\beta}$ polymorphisms being a risk factor for ovarian carcinogenesis in India.

• 한국발생생물학회지:발생과생식
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• 제20권2호
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• pp.141-147
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• 2016

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (${\gamma}H2AX$), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2003년도 추계국제학술대회
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• pp.159-159
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• 2003

Comet assay is a potential monitoring tool because DNA strand breakage may be produced by a wide range of agents. The comet assay, also called the single-cell gell electrophoresis (SCGE) assay, is rapid and sensitive method for the detection of DNA damage in cells. This study was performed for the identification of DNA damage in the cells from flounders and clams exposed to PAHs. As a control experiments, flounder and clam cells were exposed to $H_2O$$_2$. The cells exposed to $H_2O$$_2$ were displayed a typical nuclei movement DNA damage of cells were significantly increased when the isolated cells from the blood of flounders and the tissue of clams were in vitro exposed to the different concentrations (5, 10, 50, 100 ppb) of five kinds of PAHs (benzo[a]pyrene, pyrene, fluoranthene, anthrancene, and phenanthrene). For the in vivo test, flounders and clams were exposed to the different concentrations of BaP for 4 days. The results showed that DNA strand breakage was effected by the concentration of BaP and the duration of exposure. In high concentration of BaP, the mean tail lengths of nuclei was longer than it In low concentration, while the mean size of head DNA decreased. In this research, both in vitro and in vivo genotoxicity of PAHs could be biomonitored by the comet assay. Especially, clams and flounders seem to be useful as materials for monitoring genotoxic damage by comet assay.

• Journal of Radiation Protection and Research
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• 제20권2호
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• pp.71-78
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• 1995

본 연구에서는 유전자 손상회복에 관여하는 단백질을 이용하여 돌연변이 생성을 억제시키는 물질로서 알려진 cobaltous chloride가 유전자 손상회복에 미치는 영향을 연구하므로서 방사선으로 인한 손상방지 및 방사선 방어효과에 대한 적용가능성을 평가하였다. Cobaltous chloride가 RecA 단백질의 기능에 미치는 영향을 조사한 결과 RecA 단백질에 의한 DNA strand exchange 반웅에 있어 cobaltous chloride 처리로 RecA 단백질이 $_{ss}DNA$로 부터 SSB 단백질과 더 효과적으로 경쟁함으로써 안정된 $RecA-_{ss}DNA$ complex의 형성을 유도하고, 증가된 ATPase활성에 의한 ATP 가수분해로 손상된 DNA의 회복이 촉진될 수 있다는 사실을 입증 해주고 있다. 또한 RecA단백질은 UV에 의해 손상된 supercoiled DNA에 더 효과적으로 결합됨이 관찰되었으며 UV 선량과도 상관관계가 있음을 확인하였다. 따라서 이와 같은 연구결과들은 방사선으로 인한 유전적인 손상방지 및 방사선 방어효과에 관한 연구에 적용될 수 있을 것으로 기대된다.

• 대한화장품학회지
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• 제24권1호
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• pp.87-99
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• 1998

알킬화제인 MMS에 의한 유전독성의 감소에 미치는 홍삼추출물의 영향을 배양 NIH3T3 세포계에서 분석하였다. 1mM의 MMS를 30분간 처리한 후 정상 배지에서 배양한 시간간격에 따라 세포의 생존률은 증가하였는데 홍삼추출물이 함유된 배지에서 배양한 경우는 약 17%정도 증가한 생존률을 보였다. MMS 처리 후 감소된 DNA 복제가 정상배지 배양시간에 따라 증가하는 정도도 홍삼추출물을 후처리할 경우 현저한 증가를 보였다. MMS 상해를 회복하기 위한 절제회복능은 홍삼추출물을 처리할 경우 유의미한 증가를 보였다. 이러한 절제회복과정 중 효소에 의한 절제단계가 홍삼추출물 처리에 의해 활성화됨을 단사절단 분석을 통하여 규명하였다. 자외선 상해의 경우와 비해서 MMS 상해의 절제단계를 활성화하는 홍삼의 효과는 약간 떨어지는 것으로 보이나, MMS 상해회복의 전과정에 대한 홍삼의 효과는 자외선의 경우와 유사하였으며, MMS 상해에 의한 DNA 복제억제의 완화나 세포생존률은 자외선의 그것들보다 홍삼에 의해 더 증가된 측면을 보였다. 이상의 결과는 홍삼추출물이 MMS 상해의 절제회복에 유의미한 증가를 보이며 따라서 유전독성을 감소시키는 항노화제로써 사용할 수 있음을 시사한다.

• Bulletin of the Korean Chemical Society
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• 제23권8호
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• pp.1106-1109
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• 2002

Studies on subtle spacer ligand effects of AgCF3SO3 with 3,3'-Py2X (X = O vs S) have been carried out. The reaction of AgCF3SO3 with 3,3'-Py2O and 3,3'-Py2S produces [Ag(CF3SO3)(3,3'-Py2O)] and [Ag(3,3'-Py2S)] (CF3SO3), respectively. Crystallographic characterization of [Ag(CF3SO3)(3,3'-Py2O)] (monoclinic P1, a =8.405(2) $\AA$, b = 10.714(2) $\AA$, c = 18.031(2) $\AA$, $\alpha=$ 77.36(2), $\beta=107.83(2)^{\circ}$, $\gamma=$ 66.92(2), V = 1438.0(5) $\AA3$ , Z =2,R = 0.0486) reveals that the skeletal structure is an anion-bridged double-strand. The double-strands are packed like a plywood. The framework of [Ag(3,3'-Py2S)](CF3SO3) (orthorhombic Pcab, a = 17.330(2) $\AA$, b = 8.640(1) $\AA$, c = 19.933(6) $\AA$, V = 2985(1) $\AA3$ , Z =8, R = 0.0437) is a sinusoidal single-strand. The formation of each coordination polymer appears to be primarily associated with the donating ability and the confor ma-tional energy barrier of the spacer ligands. Thermal analyses indicate that [Ag(CF3SO3)(3,3'-Py2O)] and [Ag(3,3'-Py2S)](CF3SO3) are stable up to 250 $^{\circ}C$ and 210 $^{\circ}C$, respectively. For the anion exchangeability, the nature of the spacer ligand is more significant factor than the distance of silver(Ⅰ)···triflate.