• BMB Reports
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• 제31권1호
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• pp.106-110
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• 1998

We have employed a DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) assay system to characterize the enzyme activity of the endonuclease secreted by human B lymphoblastic IM9 cells. Experimental results clearly demonstrated that the endonuclease activity of IM9 cell culture medium is distinct from that of DNase I in the DNA-native-PAGE assay system. Immunoprecipitation analysis using anti-DNase I antibody showed that the secreted endonuclease is not recognized by the antibody. The secreted endonuclease was estimated using supercoiled plasmid DNA as a substrate. The pH optimum required for the catalytic activity was determined to be in the range of pH 6.6-7.4. No significant difference in the endonuclease secretion was observed by stimulation of the IM9 cells with interferon-${\gamma}$ or interleukin-$1{\beta}$.

• 대한한의학회지
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• 제25권1호
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• pp.72-84
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• 2004

Objectives : he purpose of this study was to examine the genetic variations and changes of gene expression in the human constitutions. Methods : To analysis variations of individual gene expression, we had selected three groups of volunteers. In each group have a typical constitutional characteristics. By this rime we are analyzed their gene expression patterns by using DNA chip. Results : we can acquire a new information of standard for human constitutions. 1. The 21 genes under express and 3 genes over express in So-Yang constitution 2. The 18 genes under express and 18 genes over express in So-Eum constitution 3. The 16 genes under express and 2 genes over express in Tae-Eum constitution Conclusions : Constitution, QSCCII, Character, Genome, DNA chip.

• Archives of Pharmacal Research
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• 제17권3호
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• pp.199-203
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• 1994

Several lines evidence indicate that microtubule depolymerization initiates DNA synthesis or enhances the effects of serum or purified growth factors in many types of fibroblasts. Yet little is known about the intracellular events responsible for the mitogenic effect of microtubule disrupting agents. The effects of antitubulin agents on DNA synthesis in sparse and dense cultures in the presence or absence of serum and possible involvement of G-proteins in their mitotic action were examined. In these studies, colchicine by itself appeared to be mitogenic only for confluent quiesecent human lung fibroblasts. In sparse culture, however, colchicine inhibited serum-stimulated DNA synthesis. Colcemid, another antitubulin agent, showed similar effects of growth inhibition and stimulation in sparse and confluent cultures while lumicolhicine, inactive colchicine, did not. The mitogenic effect of two antitubulin agents, colchicine and colcemid, was partially inhibited by pertussis toxin. These data suggest that microtubular integrity is associated with the expression of either negative or positive control on DNA synthesis and mitogenic effect of antitubulin agents may be partially mediated by pertussis toxin-sensitive G protein.

• 대한약리학회지
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• 제24권1호
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• pp.1-10
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• 1988

카테콜아민 생합성에 관여하는 마지막 효소인 phenylethanolamine N-methyltransferase는 Norepinephrine을 epinephrine으로 전환시키는 중요한 효소이다. PNMT효소의 발현은 epinephrine 신경세포의 발현에 필수적이다. 따라서 PNMT유전자를 크로닝하여 그 구조를 결정하고, 유전자 발현연구를 하는 것은 상당히 중요한 일이다. 그러나 최근에 저자가 bovine cDNA를 처음으로 분리하여 그 구조를 보고한 것 외에는 아직까지 인간 PNMT cDNA나, 전체 genomic DNA의 분리 보고는 없다. 이에 저자들은 인간 PNMT유전자의 전체구조와 여러 종(species) 사이의 진화적인 관계를 규명하기 위해서 human genomic library(Charon 4A)를 만들고, 이 library 이용하여 bovine cDNA를 probe로 13.1 Kb길이의 genomic clone을 분리 크로닝하는데 성공하였다. 이 유전자는 두개의 EcoRI site가 포함되어 있어서, EcoRI제한효소에 의해서 7.5 Kb, 5.0 Kb,0.6 Kb로 분리되었으며, Southern과 dot blot 실험 에서 보면 5.0 Kb와 0.6 Kb에 exon이 흩어져 존재하고 있으며, 7.5 Kb는 flanking sequence로 판명되었다.

• Nutrition Research and Practice
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• 제11권2호
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• pp.105-113
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• 2017

BACKGROUND/OBJECTIVES: A high-fat diet (HFD) induces obesity, which is a major risk factor for cardiovascular disease and cancer, while a calorie-restricted diet can extend life span by reducing the risk of these diseases. It is known that health effects of diet are partially conveyed through epigenetic mechanism including DNA methylation. In this study, we investigated the genome-wide hepatic DNA methylation to identify the epigenetic effects of HFD-induced obesity. MATERIALS AND METHODS: Seven-week-old male C57BL/6 mice were fed control diet (CD), calorie-restricted control diet (CRCD), or HFD for 16 weeks (after one week of acclimation to the control diet). Food intake, body weight, and liver weight were measured. Hepatic triacylglycerol and cholesterol levels were determined using enzymatic colorimetric methods. Changes in genome-wide DNA methylation were determined by a DNA methylation microarray method combined with methylated DNA immunoprecipitation. The level of transcription of individual genes was measured by real-time PCR. RESULTS: The DNA methylation statuses of genes in biological networks related to lipid metabolism and hepatic steatosis were influenced by HFD-induced obesity. In HFD group, a proinflammatory Casp1 (Caspase 1) gene had hypomethylated CpG sites at the 1.5-kb upstream region of its transcription start site (TSS), and its mRNA level was higher compared with that in CD group. Additionally, an energy metabolism-associated gene Ndufb9 (NADH dehydrogenase 1 beta subcomplex 9) in HFD group had hypermethylated CpG sites at the 2.6-kb downstream region of its TSS, and its mRNA level was lower compared with that in CRCD group. CONCLUSIONS: HFD alters DNA methylation profiles in genes associated with liver lipid metabolism and hepatic steatosis. The methylation statuses of Casp1 and Ndufb9 were particularly influenced by the HFD. The expression of these genes in HFD differed significantly compared with CD and CRCD, respectively, suggesting that the expressions of Casp1 and Ndufb9 in liver were regulated by their methylation statuses.

• 생명과학회지
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• 제8권4호
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• pp.400-409
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• 1998

Formation of adduct was studied in benzo(a)pyrene(BP)- and doxorubicin(Dx)-treated human NC-37 cells and isolated nuclei. Major adducts formed were determined by fluorescence absorption spectrophotometery and DNA-lin-ked protein assay. When isolated nuclei were exposed to carcinogens BP and DMBA, and anticancer drugs m-AMSA, ellipticine and Dx, varying degrees of adduct formation occured between DNA-protein complex and these drugs. When the mixture was centrifuged 1.7 M sucrose solution, binding BP and DMBA appeared to be similar between the sediment and the supernatant. When the sediment was centrifuged again with 0.35% polymin-P, the amount of BP bound was 2-fold greater in the protein(1077$\pm$55cpm) than in DNA fraction (470$\pm$20cpm), whereas that of DMBA was 1.6-fold greater in the DNA than in protein fraction. In the case of m-AMSA, ellipticine and Dx, the amount of binding was slightly greater in supernatant than in sediment in centrifugation with 1.7 M sucrose, and more than 3 times greater in the DNA- than in protein- fraction in centrifugation with 0.35% polymin P. DNA fractions which associated with a subset of nonhistone chromosomal protein were isolated from NC-37 cells exposed to $^{3}$H-BP and $^{14}$C-Dx. They were separated into two distince components DNA-S and DNA-P by centrifugation with 2M Nacl chromatin extraction. The results indicated that the amount of $^{3}$H-BP bound was 6.0-fold greater in DNA-P as compared with DNA-S, while that of $^{14}$C-Dx binding appreaed to be 6.2-fold greater in DNA-S than in DNA-P fraction. When $^{3}$H-BP binding wasdetermined in the presence of cold Dx, the amount of binding was reduced only in the DNA-P fraction, indicating that the interaction between DNA and protein is decreased. Gene expression by these drugs, BP treated cells were increased to compare with nomal cells but reduced by treatment with BP-Dx. These results suggest that the protein moiety which tightly bound to DNA-P fraction may play an important role in the regulation of gene expression.

• Journal of Preventive Medicine and Public Health
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• 제45권4호
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• pp.235-243
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• 2012

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10433-10438
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• 2015

Background: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children and represents approximately 25% of cancer diagnoses among those younger than 15 years of age. Aim and Objectives: This study investigated substitutions in the ATP synthase subunit 6 gene of mitochondrial DNA (mtDNA) as a potential diagnostic biomarker for early detection and diagnosis of acute lymphoblastic leukemia. Based on mtDNA from 23 subjects diagnosed with acute lymphoblastic leukemia, approximately 465 bp of the ATP synthase subunit 6 gene were amplified and sequenced. Results: The sequencing revealed thirty-one mutations at 14 locations in ATP synthase subunit 6 of mtDNA in the ALL subjects. All were identified as single nucleotide polymorphisms (SNPs) with a homoplasmic pattern. The mutations were distributed between males and females. Novel haplotypes were identified in this investigation: haplotype (G) was recorded in 34% in diagnosed subjects; the second haplotype was (C) with frequency of 13% in ALL subjects. Neither of these were observed in control samples. Conclusions: These haplotypes were identified for the first time in acute lymphoblastic leukemia patients. Five mutations able to change amino acid synthesis for the ATP synthase subunit 6 were associated with acute lymphoblastic leukemia. This investigation could be used to provide an overview of incidence frequency of acute lyphoblastic leukemia (ALL) in Saudi patients based on molecular events.

• Molecular & Cellular Toxicology
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• 제2권1호
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• pp.35-47
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• 2006

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) are well known as the most toxic environmental compound in these days. Many researches are reported that dioxin produces multiple toxic effects, such as endocrine toxicity, reproductive toxicity, immunotoxicity and cancer. In this study, we carried to discover novel evidence for previously unknown gene expression patterns in human exposed to dioxin by using radioactive cDNA microarray. 548 workers who were divided into experimental and control groups according to their urinary Naphthol levels were enrolled in our study. Blood mRNA in human was isolated, and the gene expression profiles were analyzed by cDNA microarray. Gene expression analysis identified 52 genes which exhibited a significant change. In our study, most notably, genes involved in cell cycle, cell proliferation, signal transduction and apoptosis in human exposed to dioxin, such as CCND3, TSHR, and EFRN5, were up-regulated. In the current study, we observed gene expression of people that are exposed to dioxin using radioactive cDNA microarray. Through these results, we suggest when objects are exposed to toxic compounds, such as dioxin, the radioactive cDNA microarray may be using in sensitively detecting of cancerous change.

• Journal of Photoscience
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• 제9권2호
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• pp.479-481
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• 2002

UV-induced DNA damage causes cell killing and mutations leading to carcinogenesis. In normal human cells, UV damage such as cyclobutane pyrimidine dimers (CPDs) and primidine-prymidone (6-4) photoproducts are mainly repaired by nucleotide excision repair mechanism. The molecular processes have been well characterized recently. To know the influence of mitochondrial genome on the nucleotide excision repair mechanism against CPDs, we comparatively examined the production of CPDs by UVC irradiation and their repair kinetics in human cells completely lacking mitochondrial DNA (mtDNA) and the parental HeLa S cells. Whole DNA extracted from the cells exposed to UVC was treated with T4-endonuclease V to break the phosphodiester bond adjacent to CPDs. The DNA was electrophoresed in a denaturing agarose gel, which was visualized by ethidium bromide staining. The relative amount of CPDs was determined by image analysis using NIH Image software. MtDNA- less (rho-O) cells were apparently more sensitive to UVC than HeLa S cells, while the level of induction of CPDs in rho-O and HeLa cells was comparable. The repair of CPDs was less efficient in rho-O cells compared with HeLa cells. The residual amount of CPDs after 24-h repair was larger in rho-O cells than in HeLa cells where more than 90 % of CPDs were repaired by then. The non-repaired CPDs would lead to apoptosis in rho-O cells. These results suggest that mitochondrial genome may contribute to some ATP-dependent steps in nucletide excision repair by supplying sufficient ATP which is generated through a respiratory chain in mitochondria.