• The Korean Journal of Nuclear Medicine Technology
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• v.23 no.1
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• pp.35-39
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• 2019

Purpose Hepatitis B virus (hepatitis B virus, HBV) infection is a worldwide major public health problem and it is known as a major cause of chronic hepatitis, liver cirrhosis and liver cancer. And serologic tests of hepatitis B virus is essential for diagnosing and treating these diseases. In addition, with the development of molecular diagnostics, the detection of HBV DNA in serum diagnoses HBV infection and is recognized as an important indicator for the antiviral agent treatment response assessment. We performed HBeAg assay using Immunoradiometric assay (IRMA) and Chemiluminescent Microparticle Immunoassay (CMIA) in hepatitis B patients treated with antiviral agents. The detection rate of HBV DNA in serum was measured and compared by RT-PCR (Real Time - Polymerase Chain Reaction) method Materials and Methods HBeAg serum examination and HBV DNA quantification test were conducted on 270 hepatitis B patients undergoing anti-virus treatment after diagnosis of hepatitis B virus infection. Two serologic tests (IRMA, CMIA) with different detection principles were applied for the HBeAg serum test. Serum HBV DNA was quantitatively measured by real-time polymerase chain reaction (RT-PCR) using the Abbott m2000 System. Results The detection rate of HBeAg was 24.1% (65/270) for IRMA and 82.2% (222/270) for CMIA. Detection rate of serum HBV DNA by real-time RT-PCR is 29.3% (79/270). The measured amount of serum HBV DNA concentration is $4.8{\times}10^7{\pm}1.9{\times}10^8IU/mL$($mean{\pm}SD$). The minimum value is 16IU/mL, the maximum value is $1.0{\times}10^9IU/mL$, and the reference value for quantitative detection limit is 15IU/mL. The detection rates and concentrations of HBV DNA by group according to the results of HBeAg serological (IRMA, CMIA)tests were as follows. 1) Group I (IRMA negative, CMIA positive, N = 169), HBV DNA detection rate of 17.7% (30/169), $6.8{\times}10^5{\pm}1.9{\times}10^6IU/mL$ 2) Group II (IRMA positive, CMIA positive, N = 53), HBV DNA detection rate 62.3% (33/53), $1.1{\times}10^8{\pm}2.8{\times}10^8IU/mL$ 3) Group III (IRMA negative, CMIA negative, N = 36), HBV DNA detection rate 36.1% (13/36), $3.0{\times}10^5{\pm}1.1{\times}10^6IU/mL$ 4) Group IV(IRMA positive, CMIA negative, N = 12), HBV DNA detection rate 25% (3/12), $1.3{\times}10^3{\pm}1.1{\times}10^3IU/mL$ Conclusion HBeAg detection rate according to the serological test showed a large difference. This difference is considered for a number of reasons such as characteristics of the Ab used for assay kit and epitope, HBV of genotype. Detection rate and the concentration of the group-specific HBV DNA classified serologic results confirmed the high detection rate and the concentration in Group II (IRMA-positive, CMIA positive, N = 53).

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.6
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• pp.805-812
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• 2010

DNA damage including base modifications, loss of base and breaks in DNA strands can occur by exposure to irradiation, smoking and several components of food. Unrepaired DNA damage is known to lead to cellular dysfunction, cell death, cancer, and other diseases such as arteriosclerosis and diabetes. The protective effect of garlic on oxidative stress induced DNA damage has been reported recently. In this study, we investigated the protective effect of garlic extracts prepared by different processing methods (raw garlic extracts, RGE; grilled garlic extracts, GGE; pickled garlic extracts, PGE) on leukocytic DNA damage using comet assay. Human leukocytes were incubated with ethanol and methanol extract of garlic at various concentrations (1, 5, 10, 50 ${\mu}g$/mL), followed by oxidative stimuli (200 ${\mu}M$ $H_2O_2$ or 200 ${\mu}M$ 4-hydroxynonenal (HNE)). The methanol and ethanol extracts of RGE, GGE, and PGE showed inhibitory activities of DNA damage induced by $H_2O_2$ or HNE. Especially methanol extract of RGE ($ED_{50}$; 13.3 ${\mu}g$/mL) had a higher antigenotoxic effect on $H_2O_2$ induced DNA damage than those of GGE (23.5 ${\mu}g$/mL) or PGE (24.5 ${\mu}g$/mL). HNE induced DNA damage tended to be effectively inhibited by the lower concentration of all garlic extracts. Therefore, garlic might have protective effects against oxidative DNA damage regardless of processing methods (raw, grilled, pickled) which are the general consumed forms of garlic in Korea.

• Microbiology and Biotechnology Letters
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• v.37 no.1
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• pp.17-23
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• 2009

The gene encoding Thermus thermophilus HJ6 DNA polymerase (Tod) was cloned and sequenced. The open reading frame (ORF) of the Tod gene was composed of 2,505 nucleotides and encoded a protein (843 amino acids) with a predicted molecular weight of 93,795 Da. The deduced amino acid sequence of Tod showed 98% and 86% identities to the Thermus thermophilus HB8 DNA pol and Thermus aquaticus DNA pol, respectively, The Tod gene was expressed under the control of the bacteriophage $\lambda$ promoters PR and PL on the expression vector pJLA503 in Escherichia coli strain BL21 (DE3) codon plus. The expressed enzyme was purified by heat treatment, $HiTrap^{TM}$ Q column, and $HiPrep^{TM}$ Sephacryl S-200 HR 26/60 column chromatographies. The optimal temperature and pH for DNA polymerase activity were found to be $75{\sim}80^{\circ}C$ and 9.0, respectively. The optimal concentrations of $Mg^{2+}$ and $Mn^{2+}$ were 2.5 mM and 1 mM, respectively. The enzyme activity was activated by divalent cations, and was inhibited by monovalent cations. The result of the PCR experiment with Tod DNA polymerase indicates that this enzyme might be useful in DNA amplification and PCR-based applications.

• Proceedings of the Microbiological Society of Korea Conference
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• 2004.05a
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• pp.47-50
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• 2004

The defenses against free radical damage include specialized repair enzymes that correct oxidative damages in DNA, and detoxification systems such as superoxide dismutases. These defenses may be coordinated genetically as global responses. We hypothesized that the expression of the SOD and the DNA repair genes would inhibit DNA damage under oxidative stress. Therefore, the protection of E. coli mutants deficient in SOD and DNA repair genes $(sod^-\;xth^-\;and\;nfo^-)$ was demonstrated by transforming the mutant strain with a plasmid pYK9 which encoded Photobacterium leiognathi CuZnSOD and human AP endonuclease. The results show that survival rates were increased in $sod^+\;xth^-\;nfo^+$ cells compared to $sod^-\;xth^-\;ap^+,\;sod^-\;xth^-\;ap^-,\;and\;sod^+\;xth^-\;ap^-$ cells under oxidative stress generated from 0.1 mM Paraquat or 3 mM $H_2O_2$. The data suggested that, at least, SOD and DNA repair enzymes may have collaborate protection and repair of the damaged DNA. Additionally, both enzymes are required for protection against free radicals.

• The Korean Journal of Zoology
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• v.38 no.1
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• pp.87-95
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• 1995

한국산 담수어류의 잉어목, 황어아과(Leuciscinae) 어류 2속 5종의 계통적 유연관계를 구명하기 위하여 mtDNA분석을 실시하였다 6 base를 인지하는 10개의 제한효소를 처리하여 얻어진 강tDNA의 크기는 16.5-17.5 Kb였으며 Bcl I, Bgl I, Bgl II, Hin dIII, Pvu II, Xba I 등은 종간 차이가 뚜렷하였다. 각종의 집단간 mtDNA분화정도는 매우 낮았으나(p=1% 미만) M. oxyephalus의 무주집단과 제주집단은 예리적으로 큰 차이를 보였다(p=5.3%) Moroco속의 종간 분화정도를 비교한 결과 M. oxycephafus와 M. lagowsk서 사이가 평균 f=7.2%로 근면관계가 제일 가까웠고 M. keumkang과 M. semotilus는 타종들과 근연관계가 제일 멀었다. Moroco속과 Phoxinus속간의 평균 유전적 분화정도는 f=13.7%로 현저한 차이를 보였다. Brown 등(1979)의 공식을 이용하여 이들 황어아과 2속 5종의 분화시기를 추정한 결과 이들은 후기 선신세(Pliocene)와 흥적세(Pleistocene) 사이에 분화된 것으로 추정되었으며 이 결과는 동위효소 연구에서 얻어진 결파(Yang and Min, 1989)와 잘 일치한다.

• Archives of Pharmacal Research
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• v.22 no.5
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• pp.474-478
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• 1999

Modulation of unscheduled DNA synthesis by dehydroepiandrosterone (DHEA) after exposure to various chemical carcinogens was investigated in the primary rat hepatocytes. Unscheduled DNA synthesis was induced by treatment of such direct acting carcinogens as methly methanesulfonate (MMS) and ethyl methanesulfonate (EMS) or procarcinogens including benzo(a)pyrene (BaP) and 7, 12-dimethylbenz(a)anthracene (DMBA). Unscheduled DNA synthesis was determined by measuring [methyl-3H]thymidine radioactivity incorporated into nuclear DNA of hepatocytes treated with carcinogens in the presence or absence of DHEA. Hydroxyurea $(5{\times}10^{-3} M)$was added to growth medium to selectively suppress normal replication. DHEA at concentrations ranging from $(1{\times}10^{-6} M)$ to$(5{\times}10^{-4} M)$ did not significantly inhibit unscheduled DNA synthesis induced by either MMS $(1{\times}10^{-4} M)$ or EMS $(1{\times}10^{-2} M)$. In contrast, DHEA-significantly inhibited unscheduled DNA synthesis induced by BaP $(6.5{\times}10^{-5} M)$ and DMBA.$(2{\times}10^{-5} M)$. DHEA-induced hepatotoxicity in rats was examined using lactate dehydrogenase (LDH) release as an indicator of cytotoxicity. DHEA exhibit no significant increase in LDH release compared with the control at 18 h. These data suggest that nontoxic concentration of DHEA does not affect the DNA excision repair process, but it probably influence the enzymatic system responsible for the metabolic activation of procarcinogens and thereby decreases the amount of the effective DNA adducts formed by the ultimate reactive carcinogenic species.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.159-172
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• 1998

사람 성장호르몬 수용체(hGHR) cDNA는 PCR방법에 의하여 fagment로서 보고되어진 바 있으나, liver cDNA로 부터 전장을 cloning한 보고는 없는 실정으로 본 연구에서는 기능을 가진 약 4.6kbp의 cDNA hGHR을 cloning 하는데 성공하였다. 먼저 cloning하기 위하여 human liver mRNA와 human breast cancer tissue로부터 회수한 mRNA를 RT-PCR방법에 의하여 human cDNA library와 cloning에 필요한 probe를 제작하였다. human library mRNA는 GT-PCR방법에 의하여 증폭하여 증폭되어진 산물은 λZAP Vector를 이용하여 cDNA library를 구축하였고,screeing을 위하여 임 보고 되어진 hGHR fragment native sequence를 기초로 N-terminal부분의 primer를 설계하여 950bp의 probe를 얻는데 성공하였다. 이 probe를 이용하여 준비된 human liver cDNA library로부터 2.5$\times$10 6개의 plaque로부터 6개의 positive clone을 획득하였고, 이들중 poly Asignal인 "AATAAA"를 포함하고 있는 가장 긴 약 3.8kbp의 clone을 sequencing한 결과 open reading frame을 포함하고 있었으나, 5'부분의 결손되어 있었다. 그리하여 이 부분은 human breast cancer tissue로 부터 회수한 mRNA를 RT-PCR에 의하여 증폭하였고, sequencing결과 이미 보고되어진 native hGHR와 비교한 결과 하나의 nucleotide가 silent mutation으로 판명되었다.한편 human liver cDNA library로부터 cloning한 3.8cp의 positive clone의 5'end의 결손된 부분에 silent mutation된 PCR 산물을 연결함으로써 native hGHR와 유사한 cDNA hGHR subcloning에 성공하였다. 이러한 cDNA hGHR의 clone이 function을 가지고 있는지를 검토하기 위하여 eukaryotic 발현 vector인 pCXN2에 의거 ligation한 후 chinese hamster ovary cell[CHO-KI]에 transfect를 실시하였다. Dexamethasone은 첨가하지 않고 hGH만의 존재하에서 이들 cell을 배양시키고 cell menbrane에서 발현 여부를 판정키 위하여 hGHR monocloual antibody를 사용하여 flow cytometery해석을 실시하는 한편 125I-hGH binding assay에 의하여 hGH binding activity를 측정하였다. 최종적으로 GH signal transduction의 target genedf으로 알려져 있는 serine protease inhibitor 2.1(Spi 2.1) gene의 promotor activity를 검토한 결과 hGHR을 transfect한 CHO Cell에 있어서 hGH의 농도에 의존적으로 증가되었다. 따라서 본 실험에서 cloning한 cDNA hGHR는 native hGHR와 같은 기능을 가지는 것으로 판명되었다.것으로 판명되었다.

• Bulletin of the Korean Chemical Society
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• v.26 no.3
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• pp.379-383
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• 2005

This research aims to develop DNA chip array without an indicator. We fabricated microelectrode array by photolithography technology. Several DNA probes were immobilized on an electrode. Then, indicator-free target DNA was hybridized by an electrical force and measured electrochemically. Cyclic-voltammograms (CVs) showed a difference between DNA probe and mismatched DNA in an anodic peak. Immobilization of probe DNA and hybridization of target DNA could be confirmed by fluorescent. This indicator-free DNA chip microarray resulted in the sequence-specific detection of the target DNA quantitatively ranging from $10^{-18}\;M\;to\;10^{-5}$ M in the buffer solution. This indicator-free DNA chip resulted in a sequence-specific detection of the target DNA.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.7-12
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• 1996

Reproducible the random amplified polymorphic DNAs(RAPDs) patterns were obtained in the two silkworm strains(J111, Galwon) by adjusting concentration optimized of Taq DNA polymerase(one unit), dNTP(200$\mu$M), MgCl2(1.5mM) and template DNA(30ng). In addition, anealing temperature ranging 35$^{\circ}C$ to 42$^{\circ}C$ by the adjusted condition was investigated and fixed at 35$^{\circ}C$ in this study. Variation among individuals and between male and female of Jam 113 strain was not authorized. DNA polymorhpisms among silkworms were authorized by five RAPD markers using OPM04 random primer. Using the primer showing polymorhpims between parents(J111, Galwon) in thirty three individuals, RAPD-PCR for F2 analysis was performed and segregated 3 : 1 in the F2 population. Consequently, RAPDs detected in the parents were obtained as genetic markers, which can be used for construction of genetic map for this industrially particular insect, silkworm Bombyx mori

• Korean Journal of Microbiology
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• v.25 no.2
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• pp.94-102
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• 1987

Rat liver LDH A-cDNA has been isolated from a .lambda.gt11-rat lover cDNA library and partially characterized. The size of the isolated rat liver LDH A-cDNA if shown to be 1.6Kb and restriction enzyme sites for the rat liver LDH A-cDNA are also mapped. 682-nucleotide sequence coding for 3'-end of rat liver LDH A-cDNA has been analyzed and compared to the nucleotide sequence of the same region of rat $C_6$-glioma cell LDH A-cDNA which has been cloned from the hormonally stimulated cell cultures. The result shows that 177 nucleotide sequences coding for the C-terminal 59-amino acids are identical but 505 nucleotide sequences of 3'-nontranslated region of the two LSH A-cDNA exhibit characteristic differences in thier nucleotide sequences. Computer analysis for the folding structures for 3'-end 400 nucleotide sequences of the two LDH A-cDNA shows a possibility implying that the two LDH A-mRNAs isolated from different tissues of rats may have different half life and therefore their translational efficiency may be different. It has been previously demonstrated that isoproterenol stimulated rat $C_6$ -glioma cell cultures produce LDH A-mRNA showing 2 to 3-fold longer half life in comparison to that of noninduced LHD A-mRNA. The result therefore support for the idea that hormonally stimulated rat $C_6$-glioma cells may produce LDH A-mRNA containing different nucleotide sequences at the 3'-end nontranslated region by which the hormonally induced LDH A-mRNA could have more stable secondary mRAN structure in comparison to that of noninduced LDH A-mRNA.