• Molecules and Cells
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• 제21권3호
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• pp.367-375
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• 2006

We previously identified a novel cancer/testis antigen gene CAGE by screening cDNA expression libraries of human testis and gastric cancer cell lines with sera of gastric cancer patients. CAGE is expressed in many cancers and cancer cell lines, but not in normal tissues apart from the testis. In the present study, we investigated its role in the motility of cells of two human cancer cell lines: HeLa and the human hepatic cancer cell line, SNU387. Induction of CAGE by tetracycline or transient transfection enhanced the migration and invasiveness of HeLa cells, but not the adhesiveness of either cell line. Overexpression of CAGE led to activation of ERK and p38 MAPK but not Akt, and inhibition of ERK by PD98059 or p38 MAPK by SB203580 counteracted the CAGE-promoted increase in motility in both cell lines. Overexpression of CAGE also resulted in a reduction of ROS and an increase of ROS scavenging, associated with induction of catalase activity. Inhibition of ERK and p38 MAPK increased ROS levels in cells transfected with CAGE, suggesting that ROS reduce the motility of both cell lines. Inhibition of ERK and p38 MAPK reduced the induction of catalase activity resulting from overexpression of CAGE, and inhibition of catalase reduced CAGE-promoted motility. We conclude that CAGE enhances the motility of cancer cells by activating ERK and p38 MAPK, inducing catalase activity, and reducing ROS levels.

• International Journal of Industrial Entomology and Biomaterials
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• 제28권1호
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• pp.10-18
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• 2014

Baculoviruses base vectors come to be regarded as methods for in vivo gene delivery and transient expression to the silkworm. In the case of silkworm, B. mori, two types of baculoviruses, AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV (Bombyx mori nuclear polyhedrosis virus), are potentially applicable as vectors. Recently, AcNPV showed promising results with some silkworm strains despite different host-specificities. We searched for a highly-permissive silkworm strain in the B. mori stocks of Kyungpook National University that could produce high levels of recombinant protein. Seventy strains were screened using the recombinant AcNPV/BmA3-Luc virus. Based on the measured luciferase activity, the strains could be divided into three groups, high-, middle-, and low-permissive strains, according to their relative recombinant protein expression levels. At 48 hours post-injection, the luciferase activity in the high-permissive strains was 500-fold greater than that of the low-permissive strains. At 72 hours post-injection, a significant elevation in luciferase activity was observed in the hemocytes of all strains. Then, based on the above results, the High Permissive Strain (HPS) S10 and the Low Permissive Strain (LPS) S39 were pick up and was carried out Dot blotting, RT-PCR and Real time PCR.

• 대한본초학회지
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• 제26권1호
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• pp.65-74
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• 2011

Objectives : This study was undertaken to verify the modulation mechanism of Gyeongshingangjeehwan18 (GGEx18) in ob/ob male mice. Methods : Eight-week old mice (wild-type C57BL/6J and ob/ob) were used for all experiments. Wild-type C57BL/6J mice were used as lean control and obese ob/ob mice were randomly divided into 5 groups : obese control, GGEx15 (Ephedra sinica Stapf + Rheum palmatum L.), GGEx16 (Ephedra sinica Stapf + Laminaria japonica Aresch), GGEx17 (Rheum palmatum L. + Laminaria japonica Aresch), and GGEx18 (Ephedra sinica Stapf + Laminaria japonica Aresch + Rheum palmatum L.). After mice were treated with several kinds of GGEx for 11 weeks, the mRNA expression of peroxisome proliferator-activated receptor (PPAR) target genes and uncoupling protein (UCP) were measured. In addition, $PPAR{\alpha}$ and $PPAR{\beta}$ transactivation was examined in NMu2Li hepatocytes, C2C12 myocytes, and 3T3-L1 preadipocytes using transient transfection assays. Results : 1. Hepatic $PPAR{\alpha}$ target genes, such as ACOX and VLCAD mRNA levels were significantly increased by GGEx18 compared with obese controls. In skeletal muscle, LCAD mRNA expression was stimulated by GGEx16, GGEx17, and GGEx18, whereas MCAD mRNA expression by GGEx17 and GGEx18. $PPAR{\beta}$ target LPL mRNA levels were also increased by GGEx16, GGEx17, and GGEx18 in skeletal muscle, but adipose LPL mRNA levels were decreased. In addition, GGEx18 upregulated UCP mRNA expression in skeletal muslce. 2. $PPAR{\alpha}$ reporter gene expression was increased by GGEx18 in NMu2Li cells compared with vehicle. $PPAR{\alpha}$ and $PPAR{\beta}$ reporter activities were also increased by all GGEx treatments in C2C12 and 3T3-L1 cells. Conclusions : These results suggest that GGEx can act as $PPAR{\alpha}$ and $PPAR{\beta}$ activators, and that GGEx may regulate obesity by stimulating $PPAR{\alpha}$, $PPAR{\beta}$, and UCP activity. Of the 4 compositions, GGEx18 seems to be most effective in improving obesity and lipid disorders.

• BMB Reports
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• 제37권5호
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• pp.538-545
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• 2004

A new CRT binding factor (CBF) gene designated Cbcbf25 was cloned from Capsella bursa-pastoris, a wild grass, by the rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbcbf25 was 898 bp with a 669 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 223 amino acids. The predicted CbCBF25 protein contained a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and a possible acidic activation domain in the C-terminal region. Bioinformatic analysis revealed that Cbcbf25 has a high level of similarity with other CBF genes like cbf1, cbf2, and cbf3 from Arabidopsis thaliana, and Bncbf5, Bncbf7, Bncbf16, and Bncbf17 from Brassica napus. A cold acclimation assay showed that Cbcbf25 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. Our study implies that Cbcbf25 is an analogue of other CBF genes and may participate in cold-response, by for example, controlling the expression of cold-regulated genes or increasing the freezing tolerance of plants.

• Clinical and Experimental Reproductive Medicine
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• 제30권1호
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• pp.47-55
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• 2003

Objectives: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. Methods: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at $21{\sim}23$ days of age. Ovaries were collected $1{\sim}3$ days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for $1{\sim}2$ days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 IU PMSG. Some mice received a single intraperitoneal injection of 10 IU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. Results: Treatment of immature mice with diethylstilbestrol (DES) for $24{\sim}48$ h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for $24{\sim}48$ h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. Interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at $6{\sim}9$ h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. Conclusion: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.

• 생명과학회지
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• 제25권9호
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• pp.1043-1050
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• 2015

신경과흥분은 신경세포의 수지돌기 말단부에 있는 흥분성 수용체에 대한 과도한 자극에 의해서 신경세포가 손상을 받는 현상으로 transcription factor의 발현을 유도하여 통증을 유발하는 자극, 학습, 발작, 흥분, 신경변성, 저산소성 국소빈혈, 뇌신경손상, 신경절제, 약제내성 등의 원인이 된다. 신경과흥분은 정상농도 이상의 NMDA에 의해서도 유발되는데 본 논문에서는 mouse의 복강으로 과량의 NMDA를 투여하여 소뇌에서 RT-PCR 방법으로 Inducible transcription factors (Egr-1, c-jun, JunB, FosB) mRNAs의 상대적 발현량을 비교하였다. NMDA를 투여한 군에서 inducible transcription factors (Egr-1, C-Jun, JunB, FosB)가 투여량과 시간의 경과에 따라 다양한 발현의 변화를 보였으며, NMDA투여 후 일정한 시간에서 투여한 양에 대한 변화는 체중 g 당 5 μg의 NMDA투여한 경우에 현저한 변화가 나타났다. 조사한 transcription factor 중에서 JunB의 발현 변화가 다른 transcription factor보다 두드러지게 나타났다. NMDA 투여량이 일정할 때 투여 후 경과 시간에 따른 발현양상은 투여 후 24시간이 경과한 후에 발현의 변화가 두드러지게 증가하는 경향을 나타내었고 대부분 이 48시간 경과 후 발현이 최고치에 도달하였다. 이러한 결과는 과흥분이 유도된 소뇌에서의 유전자 발현의 변화를 2D-gel 또는 microarray와 같은 방법을 이용하여 세포 내의 전체 단백질 혹은 유전자의 변화를 관찰함으로써 NMDA 수용체의 과흥분에 의한 뇌세포의 사멸에 관련된 기전을 밝힐 수 있는 좋은 자료가 될 수 있을 것으로 기대된다.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.149-156
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• 1999

포유류의 자궁조직은 발정주기를 통하여 역동적으로 변화하고 있으며 이러한 자궁조직의 분화는 시상하부-뇌하수체-생식소를 잇는 축에 의해 조절되는 스테로이드 호르몬에 의해 이루어진다. 그러나 에스트로겐 (E)이 어떤 유전자를 발현하여 자궁 내의 변화를 일으키는지는 아직 자세히 알려지지 않고 있다. 본 연구는 난소를 절제한 성숙한 흰쥐에 E을 처리한 후 자궁조직 내에서 c-fos, c-jun 및 hsp25 mRNA의 발현 변화를 Northern blot analysis방법을 사용하여 연구한 것이다. c-fos및 c-jun 암원유전자의 mRNA발현은 E처리 후 1시간 이전부터 증가하기 시작하며, 3시간 이내에 최고치에 도달한 후 급격히 감소하여 기저수준으로 되돌아갔다. 반면에 hsp25 mRNA수준은 E처리 후 3시간 대에서 최고치를 나타내나 증가된 발현량이 서서히 감소하며 12시간이 지난 후 까지도 정상대조군에 비해 높은 수준으로 유지되었다. 이러한 E의 영향이 선별적인지를 검증하기 위하여 E의 길항제인 tamoxifen을 사전처리하고 E을 추가로 처리하여 c-fos, c-jun및 hsp25 mRNA의 발현이 최고치에 이르렀던 3시간대에 자궁조직을 얻어 각각의 유전자 발현량을 조사한 결과 E에 의해 증가되었던 c-fos, c-jun 및 hsp25 mRNA의 수준이 억제됨을 확인하였다. 이러한 결과는 E이 자궁조직에 영향을 미치는데 초기의 일시적인 변화를 보이는 암원유전자인 c-fos 및 c-jun이 중요한 역할을 하리라는 것을 시사하며 hsp25의 경우는 좀더 늦은 반응에 관여하거나 c-fos및 c-jun에 의하여 간접적으로 조절을 받을 수도 있음을 보여 주는 것으로 사료된다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제32권1호
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• pp.36-41
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• 2006

Objective. The objective of this study was to examine the hypothesis that inflammatory synovial fluid from TMJ internal derangement initiates a transient increase in intracellular calcium concentration ([$Ca^{2+}$]i) in chondrocytes and the induced Ca2+ signaling affects iNOS/COX-2 gene expression patterns following exposure to inflamed synovial fluid. Materials and Methods. Two female adult patients with symptoms of TMD who agreed to participate in the study were selected for this study. Immortalized human juvenile costal chondrocyte C-28/I2 was grown to 80% confluency and synovial fluids from two patients were added respectively to culture media for 24 hours at the concentration of 100ng/10ml. Confocal laser scanning microscope (CLSM) was used to examine changes of intracellular calcium concentration ([$Ca^{2+}$]i). RT-PCR was performed to identify the expression profile of IL-1${\alpha}$, iNOS, COX-2. Results. Increased [$Ca^{2+}$]i was observed in chondrocytes subjected to inflamed synovial fluid compared to control cultures and in respective cultures exposed to inflamed synovial fluids from each patient, IL-1${\beta}$, COX-2 mRNA were detected. However, in neither case iNOS mRNA was expressed. IL-1${\alpha}$, COX-2, and iNOS mRNA were expressed in control culture. Conclusion. Our results show that immortalized chondrocytes cultured with inflamed synovial fluids from patients diagnosed as disc displacement without reduction and limitation in mouth opening showed increased calcium concentration and expression of COX-2 while inhibiting the production of iNOS, which in turn could adversely affect the chondrocytes in at least short term by hindering physiologic role of NO against inflammatory cascades. These findings suggest that inflamed synovial fluid may differentially regulate the transcriptomes of relevant inflammatory mediators, especially iNOS/COX-2 axis in chondrocytes through adjusting calcium transients.

• 대한소아치과학회지
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• 제28권2호
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• pp.238-246
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• 2001

본 실험은 탁월한 골유도능으로 관심의 대상이 되고 있는 BMP의 세포내 신호 전달자로 알려진 Smad 1과 Smad 5가 조골세포 초기 분화표지인자인 ALP 유전자의 발현에 미치는 영향 및 그 조절기전을 알아보고자 하였다. BMP 처리 없이도 Smad에 의해 ALP가 발현되는가를 알아보기 위해 Smad 1과 Smad 5가 각각 stably transfection된 C2C12 세포를 3일간 배양후 histochemical assay를 하였고, Smad 1과 Smad 5의 expression vector와 ALP promoter vector를 transient co-transfection한 후 ALP promoter activity를 측정하였다. Smad에 의한 BMP의 효과를 알아보기 위해서 100ng/ml의 BMP-2를 처리한 군과 처리하지 않은 군으로 나누어 세포를 배양한후 ALP 유전자의 발현을 northern blot analysis로 확인 하였다. Smad가 ALP 유전자의 발현을 직접적으로 조절하는가를 알아보기 위해서는 단백질 합성억제제인 cycloheximide를 전처리하여 ALP 유전자의 발현을 northern blot analysis하였다. 이상의 실험결과 다음과 같은 결론을 얻었다. $\cdot$ Smad 1과 Smad 5가 과발현된 세포에서는 BMP 처리없이도 ALP가 발현된다. $\cdot$ Smad 1과 Smad 5가 과발현된 세포에서 BMP 처리후 ALP 발현 증가율이 대조군 보다 현저히 높게 나타나 Smad가 BMP 효과를 증가시킨다는 것을 알 수 있다. $\cdot$ Smad는 새로운 단백질의 합성을 통해 ALP 유전자를 발현시킨다.

• 생명과학회지
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• 제16권2호
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• pp.289-296
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• 2006

경제적으로 중요한 식용작물인 콩(Glycine max)의 초기형 질전환 효율증대를 위하여 연구를 수행하였다. 높은 초기형 질전환 조건 확립을 위하여 기초실험으로 Agrobacterium 감 염에 순응적인 국내 콩 품종을 스크린 하였으며, 적정한 agar 농도와 선발항생제 hygromycin 농도 규명, 상처방법, explant의 치상방향, 호르몬 전처리, 액체배지 선발 등 형질 전환 초기효율에 미치는 영향을 transient GUS 분석을 통하여 알아보았다. Agrobacterium 감염에 순응적인 품종을 선발하기 위하여 32개의 국내 콩 장려품종을 스크린한 결과, 일품검정콩, 은하콩, 만리콩, 대원콩 등 14개의 순응형 품종을 선발하였으며, 효율적인 제균제로는 cefotaxime이, 효율적인 선발항생제로서는 hygromycin이 선택되었다. Hygromycin 농도는 10-15 ppm이 효율적이었고, agar 농도 0.6-0.8%, explant의 치상방향은 향배축(adaxial side)을 down하여 치상하였을 때가 높은 GUS ($\beta$-glucuronidase)발현빈도를 나타냈고 상처방법은 scalpel보다. 동양침을 사용하였을 때 높게 나타났다. 형질전환 체계확립을 위하여 호르몬 전처리를 한 결과, BA 5 ppm 과 10 ppm을 처리하였을 때 후기 선발에서 높은 생존율을 보였고, 전처리 후에 액체배지에서 선발을 하였을 때 비형질 전환체(escape)의 빈도를 크게 낮출 수 있었다. 또한 여러 조건의 결과를 비교하여 볼 때, 국내 콩 품종 중에서 형질전환을 위한 재료로써는 은하콩이 가장 적합한 것으로 생각된다.