• Asian Pacific Journal of Cancer Prevention
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• 제16권9호
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• pp.3667-3671
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• 2015

Invasion and metastasis is the major cause of tumor recurrence, difficulty for cure and low survival rate. Excavating key transcription factors, which can regulate tumor invasion and metastasis, are crucial to the development of therapeutic strategies for cancers. PU.1 is a master hematopoietic transcription factor and a vital regulator in life. Here, we report that, compared to adjacent non-cancerous tissues, expression of PU.1 mRNA in metastatic hepatocellular carcinoma (HCC), but not primary HCC, was significantly down-regulated. In addition, levels of PU.1 mRNA in metastatic hepatoma cell lines MHCC97L and MHCC97H were much lower than in non-metastatic Hep3B cells. Transwell invasion assays after PU.1 siRNA transfection showed that the invasion of hepatoma cell lines was increased markedly by PU.1 knockdown. Oppositely, overexpression of PU.1 suppressed the invasion of these cells. However, knockdown and overexpression of PU.1 did not influence proliferation. Finally, we tried to explore the potential mechanism of PU.1 suppressing hepatoma cell invasion. ChIP-qPCR analysis showed that PU.1 exhibited a high binding capacity with miR-615-5p promoter sequence. Overexpression of PU.1 caused a dramatic increase of pri-, pre- and mature miR-615-5p, as well as a marked decrease of miR-615-5p target gene IGF2. These data indicate that PU.1 inhibits invasion of human HCC through promoting miR-615-5p and suppressing IGF2. These findings improve our understanding of PU.1 regulatory roles and provided a potential target for metastatic HCC diagnosis and therapy.

• Asian Pacific Journal of Cancer Prevention
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• 제13권8호
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

• 한국환경보건학회지
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• 제28권4호
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• pp.6-11
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• 2002

3.3'-Dichlorobenzidine(DCB) has be shown carcinogenic in several animals, and the development of non-invasive biomonitoring method in workers exposed with it is a very important subject. DNA adduct is a good biomarker for biomonitoring about carcinogens exposure, and lymphocytes is a good non-invasive samples. So we studied to analyze metabolites in blood lymphocytes of female Sprague-Dawley rats exposed orally with DCB(20, 30, and 40 mg/kg wt.) for 3 weeks. For analysis of them, we isolated DNA adducts from blood lymphocytes by using the enzymes method in /sup 32/P-postlabeling, and measured them by using gas chromatography/mass spectrometry-selected ion monitoring(GC/MS-SIM). 4-aminobiphenyl and phenanthrene-d/sub 10/ were added as internal standard for blank sample. Standard metabolites of DCB were synthesized with using pyridine and acetic acid which were promoter and controller in acetylation of DCB. And they were used for calibration curve. Our results showed two kinds of metabolites in DNA adducts of blood lymphocytes. They were N-acetyl 3,3'-dichlorobenzidine(acDCB) and N,N'-diacetyl 3,3'-dichiorobenzidine(di-acDCB ). They were combined with DNA at the same time as an acetyl of it was removed. So we measured DCB and acDCB for two kinds of metabolites in DNA adducts of blood lymphocytes. Our results showed the levels of DCB were 1.46∼2.26 times more than that of acDCB. And also the levels of metabolites in 20, 30 and 40 mg/kg wt. were gradually increased with going days from 1st to 3rd week. They are 1.66, 1.38 and 0.90 times in total metabolites, 1.76, 1.49 and 1.02 times in DCB, and 1.51, 1.22 and 1.28 times in acDCB. In conclusion, the results of this study showed DCB exposed to rats formed DNA adduct in blood lymphocytes after acetylated to N-acetyl 3.3'-dichloro benzidine(acDCB) and N,N'-diacetyl 3,3'-dichlorobenzidine(di-acDCB), and they could be analyzed by us ing gas chromatography/mass spectrometry-selected ion monitoring(GC/MS-SIM).

• Asian Pacific Journal of Cancer Prevention
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• 제15권8호
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• pp.3495-3501
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• 2014

Melanoma-associated antigen (MAGE) family genes have been considered as potentially promising targets for anticancer immunotherapy. MAGED4 was originally identified as a glioma-specific antigen. Current knowledge about MAGED4 expression in glioma is only based on mRNA analysis and MAGED4 protein expression has not been elucidated. In the present study, we investigated this point and found that MAGED4 mRNA and protein were absent or very lowly expressed in various normal tissues and glioma cell line SHG44, but overexpressed in glioma cell lines A172,U251,U87-MG as well as glioma tissues, with significant heterogeneity. Furthermore, MAGED4 protein expression was positively correlated with the glioma type and grade. We also found that the expression of MAGED4 inversely correlated with the overall methylation status of the MAGED4 promoter CpG island. Furthermore, when SHG44 and A172 with higher methylation were treated with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR) reactivation of MAGED4 mRNA was mediated by significant demethylation in SHG44 instead of A172. However, 5-AZA-CdR treatment had no effect on MAGED4 protein in both SHG44 and A172 cells. In conclusion, MAGED4 is frequently and highly expressed in glioma and is partly regulated by DNA methylation. The results suggest that MAGED4 might be a promising target for glioma immunotherapy combined with 5-AZA-CdR to enhance its expression and eliminate intratumor heterogeneity.

• Asian-Australasian Journal of Animal Sciences
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• 제29권1호
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• pp.126-133
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• 2016

A gene from Actinomyces sp. Korean native goat (KNG) 40 that encodes an endo-${\beta}$-1,4-glucanase, EG1, was cloned and expressed in Escherichia coli (E. coli) $DH5{\alpha}$. Recombinant plasmid DNA from a positive clone with a 3.2 kb insert hydrolyzing carboxyl methyl-cellulose (CMC) was designated as pDS3. The entire nucleotide sequence was determined, and an open-reading frame (ORF) was deduced. The ORF encodes a polypeptide of 684 amino acids. The recombinant EG1 produced in E. coli $DH5{\alpha}$ harboring pDS3 was purified in one step using affinity chromatography on crystalline cellulose and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymogram analysis of the purified enzyme revealed two protein bands of 57.1 and 54.1 kDa. The amino terminal sequences of these two bands matched those of the deduced ones, starting from residue 166 and 208, respectively. Putative signal sequences, a Shine.Dalgarno-type ribosomal binding site, and promoter sequences related to the consensus sequences were deduced. EG1 has a typical tripartite structure of cellulase, a catalytic domain, a serine-rich linker region, and a cellulose-binding domain. The optimal temperature for the activity of the purified enzyme was $55^{\circ}C$, but it retained over 90% of maximum activity in a broad temperature range ($40^{\circ}C$ to $60^{\circ}C$). The optimal pH for the enzyme activity was 6.0. Kinetic parameters, $K_m$ and $V_{max}$ of rEG1 were 0.39% CMC and 143 U/mg, respectively.

• 한국균학회지
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• 제36권2호
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• pp.189-195
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• 2008

Aspergillus nidulans는 빛이 없는 조건에서는 유성분화가 주로 일어나고 빛이 있는 조건에서는 유성분화가 억제되고 대신 무성분화가 유도된다. 빛에 의해서 유성분화가 억제되는 것은 빛에 반응하여 유성 또는 무성분화를 조절하는 유전자가 있다는 것을 시사한다. 따라서 빛에 의해서 조절되는 유전자를 연구하기 위하여 광 조건하에서 유성분화를 하는 silA98 돌연변이를 분리하였으며, 이를 보완하는 유전자를 분리 및 분석하고자 A. nidulans의 AMA-NotI genomic library로부터 silA98 돌연변이를 상보하는 유전자 silA를 분리하였다. silA 유전자의 예상 ORF는 2,388 bp의 염기로 구성되어지고 795개의 아미노산을 암호화하고 있었다. 이 유전자는 Saccharomyces cerevisiae의 ARO80 유전자와 상동성을 보이며 SilA 단백질의 N 말단에는 약 51.9%의 상동성을 가지는 ${Zn_2}{Cys_6}$ motif를 지니고 있었다. silA 유전자 결손돌연변이주는 광 존재 하에서뿐만 아니라 고농도의 sorbitol에서도 유성분화가 유도되었다. 이는 silA 유전자가 빛과 고삼투 조건에서 유성분화를 억제하는 조절과정에 관여하고 있음을 의미한다. silA 유전자를 niiA promoter로 과다 발현시켰을 때의 형질은 야생형과 큰 차이를 보이지 않았다.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.

• Journal of Animal Science and Technology
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• 제44권6호
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• pp.677-684
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• 2002

한우 성장단계 특이발현 유전자 탐색에 관한 연구(장 등, 2002)에서 선정된 후보유전자 중, EPV 20, TCTP 및 aldolase A를 비롯하여 24개월령 cDNA probe에 대하여 강한 signal을 나타낸 ADRP 유전자의 발현양상을 분석하기 위하여 성장단계별 한우 등심조직 시료를 사용하여 semiquantitative RT-PCR 및 northern blot 분석을 실시하였다. EPV 20 유전자는 한우 등심조직에서 성장단계에 따른 발현량 차이가 거의 없는 결과를 근거로, 근육조직에서 항상 발현되는 유전자로 판단하였다. 또한 발달단계에 따라 발현량 차이가 있는 것으로 보고된 aldolase A 유전자 역시 뚜렷한 발현량 차이는 없었으며, aldolase A의 muscle specific promoter와 근육분화 관련 transcription factor에 관한 연구를 통하여 근육분화 및 근육수축에 있어 aldolase A의 역할 및 조절작용을 밝힐 수 있을 것으로 사료된다. 성장관련 단백질로 알려져 있는 TCTP의 발현양상을 분석한 결과, 한우 등심조직에서 성장함에 따라 발현량이 약간 증가하는 양상을 나타내었다. 이와 같은 발현양상 분석결과로 TCTP 유전자는 성장과 관련된 기능이 있지만 동물의 성장에 있어 직접적으로 관여하지는 않을 것으로 판단된다. 지방세포 분화의 초기단계에서 발현량이 급증하고 lipid droplet 형성에 관여하며 지방축적과도 관련이 있는 것으로 보고된 ADRP 유전자의 transcript의 크기는 약 1.82 kb 이었고, 24개월령 한우 등심조직에서 발현량이 급격히 증가하였으며, 30개월령 조직에서는 감소하는 양상을 나타내었다. 이와 같은 결과로부터 ADRP 유전자를 한우 성장단계 특이발현 유전자로 선정하였으며, ADRP 유전자의 발현양상은 근육내 지방축적과 관련이 있을 것으로 추정하였다. 이후에는 발현조절 기작의 해명 및 근육내 지방축적과의 관련성 분석을 위하여 ADRP 유전자의 전체적인 구조 및 전사조절 인자 분석을 비롯하여 다른 지방대사 및 지방축적 관련 유전자와의 상호작용 및 다형성 탐색분석에 관한 연구가 필요 할 것으로 판단하였다.

• 한국임상수의학회지
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• 제26권2호
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• pp.123-129
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• 2009

동맥관 개존증(PDA)은 동맥관의 불완전한 폐쇄로 인해 하행 대동맥과 폐동맥 사이에 비정상적인 단락이 형성된 것이다. 최근 인간의 유전자 연구에서 전사인자 AP-2 beta(TFAP2B)의 유전자 변이가 PDA 증후적 사례를 초래함을 발견하였다. TFAP2B 유전자의 변이는 사람의 PDA와 같은 특정 선천성 심장 기형과 관련되어 있다. 본 연구에서는 PDA에 이환된 개의 유전자 스크리닝을 하기 위해 개 TFAP2B유전자의 전체 exon 부위를 분리하였다. 개 TFAP2B유전자는 사람의 TFAP2B와 아미노산 서열이 매우 유사하였다. PDA에 이환된 말티즈견의 TFAP2B유전자 스크리닝에서 단일 c.936+203G>A 염기 변화가 발견되었다. 그러나 대조군의 유전자 스크리닝에서도 동일한 염기 변화가 발견되었다. 이 염기의 변화는 인트론 지역에 위치해 있었으며 이환되지 않은 대조군 개에서 발견된 것으로 보아 TFAP2B는 말티즈 견의 유전성 PDA와 다른 종의 PDA 환자를 초래하지 않을 것으로 보인다. 향후 더 많은 샘플을 모으고 PDA에 이환된 다양한 종과 다른 선천성 심장 기형을 가진 환축에서 TFAP2B를 스크리닝하는 연구가 필요하다.

• Journal of Plant Biotechnology
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• 제34권4호
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• pp.299-305
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• 2007

산화스트레스 유도성 SWPA2 프로모터 조절하에 항산화 효소 SOD와 APX 유전자를 동시에 엽록체에 발현시킨 형질전환 감자 (품종 수미)를 대상으로 methyl viologen (MV) 처리에 의해 유도되는 산화스트레스 내성을 잎절편체, 소식물체 및 식물체 수준에서 조사하였다. 잎 절편에 $3{\mu}M$ MV를 처리하였을 때 SSA 식물체의 잎절편체는 비형질전환 (NT) 식물체에 비해 40% 정도 상해를 적게 받았다. 소식물체 수준에서 MV에 의한 산화스트레스 내성을 조사하기 위하여 SSA감자 shoot을 $0.3{\mu}M$ MV 첨가 배지에 배양하였을 때 뿌리의 생장에서 내성이 나타났다. 또한 온실에서 4주 생장한 식물체에 $350{\mu}M$ MV를 처리하였을 경우에도 SSA 식물체는 NT 식물체에 비해 약 75% 손상을 적게 입은 것으로 나타내었다. 추후 SSA 식물체를 이용하여 건조, 고온 등의 복합재해에 내성을 분석이 진행되어야 할 것이며 그 결과 복합스트레스 내성 감자 품종 (수미)을 개발할 수 있을 것으로 기대한다.