• Archives of Plastic Surgery
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• 제34권6호
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• pp.671-678
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• 2007

Purpose: Venous malformation(VM) which often causes pain and discomfort is the most common type of vascular malformations. Although it is presented with disfigured appearance and associated soft tissue or skeletal hypertrophy, the molecular bases of VMs are poorly understood. Differentially expressed genes(DEGs) of VMs were investigated to illuminate the molecular mechanism of the disease entity. Methods: Gene expressions of VM patients' subcutaneous tissue were studied in comparison with normal persons' by $GeneFishing^{TM}$ technique using the annealing control primers (ACPs) to identify DEGs. Candidate genes were sequenced and screened by basic local alignment search tool (BLAST) afterwards. Results: Among seventy DEGs identified, forty DEGs which had shown significantly different expression pattern were sequenced. Twenty eight out of 40 were up-regulated while 12 were down-regulated. BLAST searches revealed that 37 were known genes and 3 were unknown genes. Many genes were involved in the differentiation and remodeling of smooth muscle cells, opposed to the previous hypothesis that a lot of angiogenetic genes would be involved. Furthermore, several transcription factors and related genes, as well as cell signaling and metabolism regulators, were up regulated. Conclusion: It suggests that analysis of DEGs in VMs provide basic knowledge about its pathophysiology. and new therapeutic approaches.

• Journal of Microbiology and Biotechnology
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• 제22권8호
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• pp.1133-1140
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• 2012

Ribonucleic acid interference (RNAi) inhibits the expression of target genes in a sequence-specific manner, and shows potential for gene knockdown in filamentous fungi, in which the locus-specific gene knockout occurs in low frequency. In this study, the function of the repressor of cellulase expression I (ACEI) was verified in Trichoderma koningii (T. koningii) YC01 through RNAi, and ace1-silenced strains with improved cellulase productivity were obtained. An expression cassette that transcribed the interfering double-stranded RNA (dsRNA) of ace1 was constructed and transformed into T. koningii, and the transformants, in which the expression of ace1 was successfully silenced, were selected. As a result of the ace1 gene silencing, the expression levels of the main cellulase and xylanase genes were elevated, and the enhanced production of total proteins, cellulase, and xylanase was observed in the cultivation. In addition, the down-regulation of ace1 resulted in an increasing expression of xyr1, but no clear variation in the expression of cre1, which suggested that ACEI acted as a repressor of the xyr1 transcription, but was not involved in the regulation of the cre1 expression. The results of this work indicate that ace1 is a valid target gene for enhancing enzyme production in T. koningii, and RNAi is an appropriate tool for improving the properties of industrial fungi.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1606-1613
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• 2015

Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivo-induced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae.

• 한국환경독성학회:학술대회논문집
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• 한국환경독성학회 2002년도 추계국제학술대회
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• pp.171-171
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• 2002

Recent industrial society has human widely exposed to PAHs that are comming from the incomplete combustion of organic material as widerspread environmetal contaminants. Biological activities of PAHs are not known although PAHs are considered as carcinogens. PAHs in the mammalian cells affect CYP1A1 gene expression as well as other phase II drug metabolizing enzymes as UDPGT, NMOR etc. The mechanism of action of PAHs has been studied extensively, however it is not clear how PAHs turn on CYP1A1 in human breast cancer. Our labolatory have been studied the effect of PAHs in the human breast cancer cell lind MCF7. In this study, we examined the ZR-75-1 human breast cancer cells as a new system to evaluate bioactivity of PAHs. ZR-75-1 human breast cancer cell line has been estabilished from the breast cnacer patient, has estrogen receptors and progesteron receptors. We have been able to estbilish long term culture system of this cells then used for the study to observe the effect of PAHs. We demonstrate that PAHs induced the transcription of an aryl hydrocarbon-responsive reporter vector containing the CYP1A1 promoter and 7-ethoxyresolufin O-deethylase(EROD) activity of CYP1A1 enzyme in a concentration-dependant manner. RT-PCR analysises indicated that PAHs significantly up-regulate the constitutive level of CYP1A1 mRNA. Apparently, ZR-75-1 cells have Aryl hydrocarbon recetors, therefore it would be good experimental tool to study the cross-talk between PAHs and steroid actions.

• The Plant Pathology Journal
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• 제27권2호
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• pp.148-155
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• 2011

Our previous sequence and phylogenetic analyses of the Korean Rice stripe virus (RSV) suggested possible genetic reassortment of RNA segments, but whether this RNA variation contributed to the recent RSV outbreaks in Korea is yet unclear. To further clarify these RSV-RNA segment variations, we developed a reverse transcription-polymerase reaction/restriction enzyme (RT-PCR/RE) analysis-based method. We identified five REs, including DraI, EcoR1, NdeI/AseI, and SpeI, that could differentiate RSV RNA 1-4 subtypes, respectively. Our RT-PCR/RE results provided a clear pattern of RNA reassortment, i.e., different groups of isolates having their RNA segments derived from two to three different RSV ancestors, such as from Eastern and Southwestern Chinese or Japanese M and T isolates. We also found that the migratory small brown planthopper from Eastern China caught by aerial net traps that possesses RSV-RNA3 genotypes corresponds mainly to Eastern China, with a few for Southwestern China based on RT-PCR/RE, sequence and phylogenetic analyses, indicating that RSV populations in Eastern China may also have strong RNA variation. The development of an RE analysisbased method proved a useful epidemiological tool for rapid genotyping and identification of mixed infections by RSV strain and by different subtype.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.25-30
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• 2002

내분비계 장애물질은 다양한 동물의 생식과 배아의 발생에 위해한 영향을 미친다고 보고되었다. 이러한 초기 배아의 비정상 발생 중 대표적인 것이 할구파편이다. 본 연구에서는 착상 전 생쥐 초기 배아를 PCB, BPA, DDT에 체내, 체외에서 각각 노출시켰다. 내분비계 장애 물질에 노출시켰던 배아는 할구파편, 불규칙한 할구와 깨짐, 일부 파괴된 투명대 배아 등의 다양한 형태학적 비정상 양상을 보였다. 착상 전 생쥐 초기배아의 세포괴사에 관여하는 유전자를 조사하기 위하여 RT-PCR을 사용하여 mRNA수준에서 평가하였다. Bcl-2, bad 그리고 bax유전자 발현 정도를 대조군과 내분비계 장애물질 처리군으로 나누어 비교하였다. Bcl-2유전자는 내분비계 장애물질 처리군에서 낮아지는 경향을 보였고, bad와bix는 내분비계 장애물질 처리군에서 보다 높은 경향을 보였다. 이러한 연구 결과는 착상 전 생쥐 초기배아를 체내와 체외에서 내분비계 장애물질에 노출시킴으로써 발생적 독성물질을 신속히 판단할수 있는 유용한 수단이 될 수 있을 것으로 생각한다.

• 한국동물위생학회지
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• 제34권2호
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• pp.111-116
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• 2011

Artificial insemination (AI) of swine is a very useful reproductive tool and that offers convenience in the Korean swine industry. Since many viruses have been reported to be excreted through boar semen, we investigated the presence of antibodies and antigens against viruses causing reproductive failure in semen of boar in 349 semen samples collected from six Korean AI centers. Viral antigens were detected by polymerase chain reaction (PCR) or reverse transcription-PCR predominantly. The results was as follows. The major reproductive failure causing factor was porcine circovirus type 2 (PCV2), followed by porcine reproductive and respiratory syndrome virus (PRRSV) ($X^2$=166.64, P<0.001). PCV2 and PRRSV, Japanese encephalitis virus (JEV), encephalomyocarditis virus (EMCV) was detected in 73 samples (20.9%), 44 samples (12.6%), 4 samples (1.1%), 3 samples (0.9%), respectively and porcine parvovirus in one sample (0.3%) Classical swine fever virus (CSFV), bovine viral diarrhea virus and Aujeszky's disease virus (ADV) were not detected. Enzyme-linked immunosorbent assay was carried out in 111 serum samples from three AI centers. In most pigs, antibodies response was showed prominently in CSFV (105 sera, 94.6%) ($X^2$=82.580, P<0.001), followed by, in PRRSV (100 sera, 90.1%), PCV2 (92 sera, 90.1%), and PPV (8 sera, 82.9%). ADV antibody was not detected. Thus, the experimental results will be used for the base data, with respect to the state of viral stillbirth in general pig farms, as well as AI centers and breeding farms in Korea.

• Clinical and Experimental Reproductive Medicine
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• 제41권1호
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• pp.1-8
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• 2014

Objective: Estrogen related receptor ${\beta}$ (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotencyrelated genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. Methods: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without $2{\mu}g/mL$ CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. Results: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. Conclusion: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

• 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.

• Molecules and Cells
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• 제39권7호
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• pp.530-535
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• 2016

Large conductance calcium-activated potassium (BK) channels participate in many important physiological functions in excitable tissues such as neurons, cardiac and smooth muscles, whereas the knowledge of BK channels in bone tissues and osteoblasts remains elusive. To investigate the role of BK channels in osteoblasts, we used transcription activator-like effector nuclease (TALEN) to establish a BK knockout cell line on rat ROS17/2.8 osteoblast, and detected the proliferation and mineralization of the BK-knockout cells. Our study found that the BKknockout cells significantly decreased the ability of proliferation and mineralization as osteoblasts, compared to the wild type cells. The overall expression of osteoblast differentiation marker genes in the BK-knockout cells was significantly lower than that in wild type osteoblast cells. The BK-knockout osteoblast cell line in our study displays a phenotype decrease in osteoblast function which can mimic the pathological state of osteoblast and thus provide a working cell line as a tool for study of osteoblast function and bone related diseases.