• 한국양식학회지
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• 제11권4호
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• pp.457-463
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• 1998

미꾸라지(Misgrunus mizolepis)의 DNA로부터 클론된 핵기질 부착부위(MAR)를 포함하는 발현벡터인 pUC19N6-luc 벡터를 구성하였다. 이를 물고기 CHSE-214 세포주에 electroporation으로 transfection 시킨 후 유전자의 발현율, 벡터의 copy 수 및 염색체내 삽입 양상을 luciferase 활성도 분석, PCR 및 Southern blotting를 통해 분석하였다. 대조군 발현벡터에 luciferase 유전자는 전형적인 transient 발현양상을 나타내는데 비해, 미꾸라지 MAR가 포함된 pUC19N6-luc 벡터의 luciferase 유전자의 발현은 transfection 후 5일째부터 급격히 증가하는 양상을 보였다. Transfection된 CHSE-214 세포내에서 pUC19N6-luc 벡터는 대조군 벡터에 비해 높은 copy 수를 유지하였으나, 염색체내 삽입은 거의 비슷한 시간에 일어났다. 결론적으로 transfection 후 시간경과에 따른 pUC19N6-luc 벡터내의 luciferase 유전자의 발현 증가에 미치는 MAR의 효과는 벡터 copy수 증가 때문이 아니라, 염색체내 삽입후 형성되는 전사활성구조의 형성에 기인하는 것으로 판단된다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권2호
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• pp.1155-1157
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• 2013

Cancer is a highly complex medical problem with ramifications for public health throughout the world. Most studies have mainly focused on change in the nuclei as being aetiologically responsible. Few have examined the relationship between the cytoplasm and cancer, despite the fact that research has indicated that the cytoplasmic environment is an important factor for cellular differentiation and that the genetic information provided by the nucleus is entirely dependent on this environment for its expression. Gene mutations may be the result, rather than the cause of carcinogenesis. We submit a new concept - "short base sequences" (50-500 bps, including DNA or RNA sequences) in the cytoplasm which could play an important role in carcinogenesis. This is a new theory to explain the origin of the cancer.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2006년도 수산관련학회 공동학술대회 발표요지집
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• pp.476-478
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• 2006

To analyze subcellular localization of betanodavirus protein B2, a plasmid expressing Betanodavirus protein B2 fused to enhanced green fluorescent protein (EGFP-Nl) was constructed. The transient expression of full-length B2 fused to EGFP in GF cells confirmed the equal distribution of protein B2 between cytoplasm and nucleus. However, transfection of N-terminal half of the B2 revealed that this truncated form predominantly localized to the cytoplasm. By using several deletion mutants and point mutants, we determined the regions and/or motif responsible for the subcellular localization of betanodavirus.

• BMB Reports
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• 제42권9호
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• pp.568-573
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• 2009

Fatty acid oxidation (FAO) defects cause abnormal lipid accumulation in various tissues, which provides an opportunity to uncover novel genes that are involved in lipid metabolism. During a gene expression study in the riboflavin deficient induced FAO disorder in the chicken, we discovered the dramatic increase in mRNA levels of an uncharacterized gene, ANKRD9. No functions have been ascribed to ANKRD9 and its orthologs, although their sequences are well conserved among vertebrates. To provide insight into the function of ANKRD9, the expression of ANKRD9 mRNA in lipidperturbed paradigms was examined. The hepatic mRNA level of ANKRD9 was repressed by thyroid hormone ($T_3$) and fasting, elevated by re-feeding upon fasting. However, ANKRD9 mRNA level is reduced in response to apoptosis. Transient transfection assay with green fluorescent protein tagged- ANKRD9 showed that this protein is localized within the cytoplasm. These findings point to the possibility that ANKRD9 is involved in intracellular lipid accumulation.

• Biomolecules & Therapeutics
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• 제24권4호
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• pp.380-386
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• 2016

Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-${\kappa}B$-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of ${\beta}$-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular ${\beta}$-catenin protein but not mRNA. The inhibition of proteasome by MG132 and $GSK3{\beta}$ inhibition by SB216763 blocked silymarin-mediated downregulation of ${\beta}$-catenin. In addition, silymarin increased phosphorylation of ${\beta}$-catenin and a point mutation of S33Y attenuated silymarin-mediated ${\beta}$-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of ${\beta}$-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells.

• IMMUNE NETWORK
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• 제16권3호
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• pp.176-182
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• 2016

CCR3 is a chemokine receptor that mediates the accumulation of allergic inflammatory cells, including eosinophils and Th2 cells, at inflamed sites. The regulatory sequence of the CCR3 gene, contains two Runt-related transcription factor (RUNX) 1 sites and two PU.1 sites, in addition to a functional GATA site for transactivation of the CCR3 gene. In the present study, we examined the effects of the cis-acting elements of RUNX1 and PU.1 on transcription of the gene in EoL-1 eosinophilic cells and Jurkat T cells, both of which expressed functional surface CCR3 and these two transcription factors. Introduction of RUNX1 siRNA or PU.1 siRNA resulted in a modest decrease in CCR3 reporter activity in both cell types, compared with transfection of GATA-1 siRNA. Cotransfection of the two siRNAs led to inhibition in an additive manner. EMSA analysis showed that RUNX1, in particular, bound to its binding motifs. Mutagenesis analysis revealed that all point mutants lacking RUNX1- and PU.1-binding sites exhibited reduced reporter activities. These results suggest that RUNX1 and PU.1 participate in transcriptional regulation of the CCR3 gene.

• Clinical and Experimental Pediatrics
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• 제46권4호
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• pp.335-339
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• 2003

목 적 : 폐 표면 활성제 단백질-A는 폐 표면 활성제의 생리작용과 구조에 관여하며, 폐의 숙주방어와 염증반응에 주된 역할을 하고 있다. 스테로이드 치료는 미숙아의 분만이 예측되는 산모에 대해서 미숙아의 급성 호흡곤란 증후군 등을 예방할 목적으로, 또는 미숙아의 만성 폐질환을 예방하기 위하여 널리 사용되고 있다. 현재까지 인간에서 두 가지의 SP-A 유전자 및 여러 종류의 대립형질이 밝혀져 있고, 덱사메타손 치료에 서로 다른 반응을 보이고 있으며, SP-A 3'UTR이 관여 할 가능성이 있다는 연구가 발표되었다. 저자들은 가장 많은 빈도를 보이는 8가지 SP-A 대립형질에서 덱사메타손 처치에 대하여 서로 다른 반응이 3'UTR에 의하여 이루어지는지를 규명하고자 연구를 하였다. 방 법 : 리포터 유전자로서 luciferase를 사용하여 8종류의 SP-A 대립형질의 3'UTR을 포함하는 구성체를 만들었다. 이 구성체들은 SV40 promotor에 의하여 조절되었으며, SP-A를 생산하는 H441 세포내로 형질도입 되었다. 덱사메타손 처치 후 리포터 유전자의 활성도를 측정하였으며, 대조군에 따라 표준화되었다. 결 과 : 덱사메타손 처치 시 $6A^3$, 6A, 1A 대립 유전자에서 리포터 유전자의 활성 반응이 유의하게 감소하였다. 결 론 : SP-A 유전자 변이 중 덱사메타손 처치에 대한 유전자 표현 반응이 서로 다르다는 사실은 스테로이드 치료 시 미숙아의 개개인의 SP-A 유전자 변이에 따라 고려하여 사용해야함이 타당할 것으로 생각된다.

• 치과방사선
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• 제28권1호
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• pp.245-259
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• 1998

Cellular transforming genes have been identified in a number of different tumor cell lines and tumor types. A significant number of these oncogenes belong to the ras gene family. The ras gene family consists of three closely related genes:H-ras, K-ras and N-ras which code for a related 21 kDa protein. Mutations in codon 12, 13 and 61 of one of the three ras genes convert these genes into acute oncogenes. The presence of H-ras gene mutations has important prognostic implications in various tumors. Each genomic DNA was isolated from tumors induced by implantation with DMBA, or by treatment with DMBA -implantation/irradiation. When genome DNA was transfected into NIH 3T3 cells and investigated by two-step PCR-RFLP, the fOllowing results were concluded: 1. Transformation foci developed in two groups when the genome DNA of two experimental groups were transfected into NIH 3T3 cells. 2. Transformation efficiency was 0.01-0.02 foci/㎍DNA in the experimental group with the DMBA-implantation, 0.01-0.03 foci/㎍lgDNA in the experimental group with the DMBA-implantation/irradiation according to results of transfection assay. 3. When the point mutation of H-ras gene was investigated by a two-step PCR-RFLP, there was 13.9％ (5/36) in the experimental group with the DMBA implantation, 15.4 ％ (6/39) in the experimental group with the DMBA -implantation/irradiation. 4. The point mutation in codon 12 and 61 of H-ras was 5.6％(2/36) and 8.3％(3/36) in the experimental group with the DMBA implantation. 5. The point mutation in codon 12 and 61 of H-ras gene was 7.7％(3/39) in the experimental group with the DMBA -implantation/irradiation.

• Molecules and Cells
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• 제37권4호
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• pp.322-329
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• 2014

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the ${\gamma}$-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.

• The Korean Journal of Physiology and Pharmacology
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• 제10권6호
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• pp.349-354
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• 2006

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. The application of recombinant reporter plasmid such as the firefly luciferase gene has proven to be a very effective method to detect these chemicals. The bioassay system, CALUX, is sensitive in directly detecting AhR-agonists from a variety of environmental and biologic materials. However, responses of the AhR-dependent bioassays are dependent on the cell types used. Thus, we developed a sensitive bioassay using the recombinant mouse hepatoma cell (Hepa1c1c7) for the determination of dioxins. The recombinant cell line was stably transfected with firefly luciferase reporter gene (pGudLuc1.1). The transfected cells showed the highest induction of luciferase activity at 4.5 hr and a decrease beyond this time point. The system showed the highest sensitivity of detection ever reported. Upon TCDD exposure cells showed 2 fold increase at 10 pM and 7 fold increase at 100 pM, respectively. The passage number after the transfection played an important role in the sensitivity. The increase of passage number tended to increase the sensitivity of the cells up to 15. The media without phenol red showed a higher induction rate than with phenol red, suggesting the preferable use of phenol red-free media for the bioassay. Since each of the assays has unique characteristics that make them suitable for some screening applications and not others, development of sensitive bioanalytical methods based on a variety of cellular systems in a key to the successful determination of dioxins. The bioassay system developed in this study will contribute to further development of successful screening the AhR agonists among the environmental mixture. In addition, the rapid and sensitive nature of this cellular system can be applied as a valuable tool to screen the dioxin-like moieties among the prodrugs at the initial stage, thereby expediting the new drug discovery.