• 약학회지
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• 제48권2호
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• pp.147-152
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• 2004

A wide variety of cancer chemotherapeutic agents have been shown to induce programmed cell death (PCD, APOPTOSIS) in various tumor cell lines in vitro. cis-Malonato [(4R,5R)-4,5-bis(aminomethyl)-2-isoprpopyl-1,3-dioxolane] platinum(II) (heptaplatin), which is a new drug approved by KFDA in 1999, in a novel platinum-based antitumor agent with clinical potential against stomach cancer and the 3rd generation of the cisplatin. This study was performed to know how heptaplatin and cisplatin and sunpla (mixture of heptaplatin and mannitol) affect on SK-MEL-28 cell line, and how they induce the apoptosis. At EM analysis, the morphology of the cell was changed by treatment of the cisplatin, heptaplatin and sunpla. Apoptotic body formed around plasma membrane, and chromatin condensation represented in nucleus. This phenomenon is one of the characteristic of the apoptosis. The DNA of SK-MEL-28 cell line truncated by cisplatin and sunpla treatment was identified on 2% agarose gel electrophoresis. TUNEL assay was performed to know whether SK-MEL-28 cell die as apoptosis or necrosis by cisplatin, heptaplatin and sunpla. At this result, fluorescence intensity increased according to increase of time and concentration. Therefore, it was identified that cislatin, heptaplatin and sunpla induced apoptosis. Fas expressed on SK-MEL-28 cell membrane by cisplatin, heptaplatin and sunpla was identified by using flow cytometer and the expression of bcl-2(anti-apoptotic gene) decreased according to increase of concentration of the cisplatin, heptaplatin and sunpla. Cisplatin, heptaplatin and sunpla induced apoptosis against SK-MEL-28 cell line, and the apoptotic mechanism was identified as Fas-mediated apoptosis and decreased bcl-2 expression.

• 동의생리병리학회지
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• 제17권5호
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• pp.1243-1250
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• 2003

This study was designed to elucidate the synergistic cytotoxic mechanisms of the co-treatment of adriamycin and Paljinhangahm-dan in human hepatoma malignant cancer cell line, HepG2. The combination of adriamycin and the ethanol extract of Paljinhangahm-dan synergistically augmented the cytotoxicity of Adriamycin and Paljinhangahm-dan in HepG2 cells. The cytotoxicity of two drugs was revealed as apoptosis characterized by DNA fragmentaton in agarose gel electrophoresis. The apoptotic cytotoxicity of adriamycin and Paljinhangahm-dan was accompanied by the cleavage of procaspase -3 protease and PARP. Of note, anti apoptotic Bcl2 protein was obviously decreased, but Fas was dramatically increased in HepG2 cells co-treated with Adriamycin and Paljinhangahm -dan. In addition, through 2-D gel electorphoresis, we observed that the expression levels of a lot of proteins were obviously changed by the status of drug treatments. This results suggest that the synergistic cytotoxicity of the co-treatment of adriamycin and Paljinhangahm-dan might be caused by the changes of the expression levels of a lot of proteins which play pivotal roles in cell survival or death.

• 한국잠사곤충학회지
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• 제38권1호
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• pp.36-41
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• 1996

AcNPV 와 BmNPV의 배양세포주에서의 동시감염에 의해 선발된 재조합 바이러스 RecS-A6는 그 다각체 외부 형태가 모바이러스와 다를뿐만 아니라 배양 세포주에 따라서도 그 형태에 차이가 있었다. 이러한 다각체의 특징적인 형태가 나타나는 요인을 다각체 단백질 유전자를 중심으로 조사한 결과 RecS-A6는 AcNPV 와 BmNPV의 다각체 단백질 유전자를 모두 갖고 있는 것이 확인되었으며, 또한 RecS-A6의 다각체를 단백질 전기영동하여 분석한 결과 RecS-A6의 다각체를 단백질 전기영동하여 분석한 결과 AcNPV와 BmNPV의 다각체 단백질이 모두 다각체 형성에 이용되었음을 확인할 수 있었다.

• 한국축산식품학회지
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• 제31권6호
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• pp.851-857
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• 2011

This study was conducted to develop a real time reverse transcriptase-PCR (RT-PCR) method for the detection of viable Enterobacteriaceae in milk using primers based on the genes of ribosomal proteins S11 and S13 and to determine effects of heating and subsequent treatments on the threshold cycle (Ct) of the real time RT-PCR. Total RNA was isolated from 17 strains of bacteria including 11 strains of Enterobacteriaceae suspended in milk using a modified Tri reagent method. SYBR Green Master Mix was added to the RNA and the mixture was subjected to the real time RT-PCR. The Cts of eleven type strains of the Enterobacteriaceae in milk ($10^7$ cells) in the real time RT-PCR ranged from 21.5 to 24.6. However, the Cts of Pseudomonas fluorescens, Acinetobacter calcoaceticus, and three gram-positive bacteria were more than 40. The real time RT-PCR detected as low as $10^3$ cells in agarose gel electrophoresis. The Cts increased from 22.0 to 34.2 when milk samples contaminated with Escherichia coli ($10^7$ cells/mL) were heated at $65^{\circ}C$ for 30 min. In addition, subsequent incubation at $37^{\circ}C$ for 6 and 24 h increased the Cts further up to 36.2 and 37.2, respectively. Addition of RNase A to the bacterial suspension obtained from the heated milk and subsequent incubation at $37^{\circ}C$ for 1 h increased the Cts to more than 40. The results of this study suggests that pretreatment of bacterial cells heated in milk with RNase A before RNA extraction might enhance the ability to differentiate between viable and dead bacteria using real time RT-PCR.

• KSBB Journal
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• 제11권1호
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• pp.37-45
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• 1996

한국의 남해안에서 한천 분해능이 뛰어난 해양 미 생물을 분리하여 통정한 결과 Pseudomonas 속으로 판명되었으며, 본 연구에서는 이 균을 Halophilic P Pseudomonas sp. W7이라 명영하였다. 이 균주는 호염성 세균으로, 한천의 존재 하에서 높은 효소활성 을 가지는 extracellular agarase를 생산해 내었다. 이 extracellular agarase를 DEAE-Cellulose ani- on exchange chromatography와 gel filtration을 통해 정제하였으며, 정제된 agarase는 SDS-PAGE 를 통해 약 89KDa의 분자량을 지니는 single protein band염을 확인하였다. 한편 agarase의 대량생 산을 위하여 host cell Eo coli JM83과 vector pUC19를 이용하여 gene cloning을 행하였다. Pseudomonas sp. W7의 chromosomal DNA가 삽 입 된 균주 중에 서 agarase activity를 나타내는 E. coli JM83/pSWl과 Eo coli JM83/pSW3를 선멸하 였으며, 전기영통 실험결과 이들은 각각 307Kb, 3.0 K Kb의 chromosomal 단편을 지니고 있음을 확인하였 다. 또한 agarase 유전자가 압입된 변이 균주(B)는 inclusion body 형태의 interacellular agarase를 세포 내에 축적하고 있음이 확인되었다.

• 한국양식학회지
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• 제19권2호
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• pp.67-76
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• 2006

Genomic DNA isolated from two Porphyra species, P. tenera and P. dentate from Wando located on the southern coast of Korean peninsula was amplified by PCR reaction. The amplified products were separated by agarose gel electrophoresis (AGE) with decamer primer and stained with ethidium bromide. The eight arbitrarily selected primers OPA-04, OPA-06, OPB-01, OPB-08, OPB-10, OPB-11, OPB-14 and OPC-10 generated the shared loci, polymorphic, and specific loci. The size of DNA bands varies from 100 bp to 2,200 bp. The complexity of the banding patterns varies dramatically between the primers and two Porphyra species. A total of 528 loci observed were identified in P. tenera and 443 in P. dentata: 22 polymorphic loci (4.2%) in P. tenera and 30 (6.8%) in P. dentata. 154 shared loci observed, the average 19.3 per primer, were identified in P. tenera and 143 loci, the aver-age 17.9 per primer, in P. dentata species. The number of specific loci in P. tenera and P. dentata was 73 and 77, respectively. The average bandsharing value was $0.623{\pm}0.008$ with P. tenera and $0.560{\pm}0.009$ within P. dentata. The average bandsharing value between two Porphyra species was $0.408{\pm}0.004$, ranged from 0.305 to 0.564. The dendrogram obtained by the eight primers indicates four genetic clusters. The genetic distance between two Porphyra species ranged from 0.076 to 0.627. The individual no. 02 of P. tenera was genetically closely related to no. 01 of P. tenera(genetic distance=0.082). Especially, two entities between the individual DENTATA no.21 and DENTATA no. 19 of P. dentata showed the longest genetic distance (0.627) in comparison with other individuals used. In this study, RAPD-PCR analysis has revealed the significant genetic distance between two Porphyra species pairs (P<0.001).

• BMB Reports
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• 제38권4호
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• pp.391-398
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• 2005

3-hydrogenwadaphnin (3-HK) is a new daphnane-type diterpene ester isolated from Dendrostellera lessertii with strong anti-tumoral activity in animal models and in cultures. Here, prolonged effects of this new agent on proliferation and viability of several different cancerous cell lines were evaluated. Using [$^3H$]thymidine incorporation, it was found that the drug inhibited cell proliferation and induced G1/S cell cycle arrest in leukemic cells 24 h after a single dose treatment. The cell viability of Jurkat cells was also decreased by almost 10%, 31% and 40% after a single dose treatment (7.5 nM) at 24, 48 and 72 h, respectively. The drug-treated cells were stained with acridine orange/ethidium bromide to document the chromatin condensation and DNA fragmentation. These observations were further confirmed by detection of DNA laddering pattern in the agarose gel electrophoresis of the extracted DNA from the treated cells. Treatment of K562 cells with the drug at 7.5, 15 and 30 nM caused apoptosis in 25%, 45% and 65% of the cells, respectively. Exogenous addition of $25-50\;{\mu}M$ guanosine and/or deoxyguanosine to the cell culture of the drug-treated cells restored DNA synthesis, released cell arrest at G1/S checkpoint and decreased the apoptotic cell death caused by the drug. These observations were not made using adenosine. However, the drug effects on K562 cells were potentiated by hypoxanthine. Based on these observations, perturbation of GTP metabolism is considered as one of the main reasons for apoptotic cell death by 3-HK.

• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1615-1619
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• 2015

Background: Promoter hypermethylation mediated gene silencing of tumor suppressor genes is considered as most frequent mechanism than genetic aberrations such as mutations in the development of cancers. BRD7 is a single bromodomain containing protein that functions as a subunit of SWI/SNF chromatin-remodeling complex to regulate transcription. It also interacts with the well know tumor suppressor protein p53 to trans-activate genes involved in cell cycle arrest. Loss of expression of BRD7 has been observed in breast cancers and nasopharyngeal carcinomas due to promoter hypermethylation. However, the genetic status of BRD7 in oral squamous cell carcinomas (OSCCs) is not known, although OSCC is one of the most common among all reported cancers in the Indian population. Hence, in the present study we investigated OSCC samples to determine the occurrence of hypermethylation in the promoter region of BRD7 and understand its prevalence. Materials and Methods: Genomic DNA extracted from biopsy tissues of twenty three oral squamous cell carcinomas were digested with methylation sensitive HpaII type2 restriction enzyme that recognizes and cuts unmethylated CCGG motifs. The digested DNA samples were amplified with primers flanking the CCGG motifs in promoter region of BRD7 gene. The PCR amplified products were analyzed by agarose gel electrophoresis along with undigested amplification control. Results: Methylation sensitive enzyme technique identified methylation of BRD7 promoter region seventeen out of twenty three (74%) well differentiated oral squamous cell carcinoma samples. Conclusions: The identification of BRD7 promoter hypermethylation in 74% of well differentiated oral squamous cell carcinomas indicates that the methylation dependent silencing of BRD7 gene is a frequent event in carcinogenesis. To the best of our knowledge, the present study is the first to report the occurrence of BRD7and its high prevalence in oral squamous cell carcinomas.

• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1599-1603
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• 2015

Background: Epigenetic silencing of tumor suppressor genes due to promoter hypermethylation is one of the frequent mechanisms observed in cancers. Hypermethylation of several tumor suppressor genes involved in cell cycle regulation has been reported in many types of tumors including oral squamous cell carcinomas. LATS1 (Large Tumor Suppressor, isoform 1) is a novel tumor suppressor gene that regulates cell cycle progression by forming complexes with the cyclin dependent kinase, CDK1. Promoter hypermethylation of the LATS1 gene has been observed in several carcinomas and also has been linked with prognosis. However, the methylation status of LATS1 in oral squamous cell carcinomas is not known. As oral cancer is one of the most prevalent forms of cancer in India, the present study was designed to investigate the methylation status of LATS1 promoter and associate it with histopathological findings in order to determine any associations of the genetic status with stage of differentiation. Materials and Methods: Tumor chromosomal DNA isolated from biopsy tissues of thirteen oral squamous cell carcinoma biopsy tissues were subjected to digestion with methylation sensitive HpaII enzyme followed by amplification with primers flanking CCGG motifs in promoter region of LATS1 gene. The PCR amplicons were subsequently subjected to agarose gel electrophoresis along with undigested amplification control. Results: HpaII enzyme based methylation sensitive PCR identified LATS1 promoter hypermethylation in seven out of thirteen oral squamous cell carcinoma samples. Conclusions: The identification of LATS1 promoter hypermethylation in seven oral squamous cell carcinoma samples (54%), which included one sample with epithelial dysplasia, two early invasive and one moderately differentiated lesions indicates that the hypermethylation of this gene may be one of the early event during carcinogenesis. To the best of our knowledge, this is the first study to have explored and identified positive association between LATS1 promoter hypermethylation with histopathological features in oral squamous cell carcinomas.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.1869-1872
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• 2012

Background: To assess the diagnostic potential of tumor-associated high molecular weight DNA in stool samples of 32 colorectal cancer (CRC) patients compared to 32 healthy Malaysian volunteers by means of polymerase chain reaction (PCR). Methods: Stool DNA was isolated and tumor-associated high molecular weight DNA (1.476 kb fragment including exons 6-9 of the p53 gene) was amplified using PCR and visualized on ethidium bromide-stained agarose gels. Results: Out of 32 CRC patients, 18 were positive for the presence of high molecular weight DNA as compared to none of the healthy individuals, resulting in an overall sensitivity of 56.3% with 100% specificity. Out of 32 patients, 23 had tumor on the left side and 9 on the right side, 16 and 2 being respectively positive. This showed that high molecular weight DNA was significantly (p = 0.022) more detectable in patients with left side tumor (69.6% vs 22.2%). Out of 32 patients, 22 had tumors larger than 1.0 cm, 18 of these (81.8%) being positive for long DNA as compared to not a single patient with tumor size smaller than 1.0 cm (p <0.001). Conclusion: We detected CRC-related high molecular weight p53 DNA in stool samples of CRC patients with an overall sensitivity of 56.3% with 100% specificity, with a strong tumor size dependence.