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소변으로 EPO를 분비하는 형질전환 돼지생산

  • 박진기;이연근;민관식;임기순;성환후;양병철;이창현;이향흔;김진회
    • Proceedings of the KSAR Conference
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    • 2001.03a
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    • pp.55-55
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    • 2001
  • Erythropoietin(EPO)는 적혈구 세포 증식, 분화 및 생존에 있어서 가장 중요한 요인이다. 또한, 빈혈성저산소증에 있어서도 EPO가 중요한 역할을 한다고 알려져 있다. 태아에서 EPO 생산부위는 간이라고 알려져 있으나, 임신 120-140일에 신장으로 이동하기 시작하여 출생 후 약 40일경 이후에는 완전히 신장에서만 분비한다 EPO단백질의 분비는 오전 8시에 가장 낮고 오후 8시에 가장 높은 2중 리듬의 형태로 발현되어진다. EPO는 27개의 leader sequence와 165개의 아미노산으로 총 193개의 아미노산으로부터 분비된다. EPO단백질의 분자량은 18 kDa이나, 약 40%의 당쇄가 첨가되어있는 당단백질으로서 분자량은 30 kDa이다 N-linked 당쇄 3개(Asn-24, 38 및 83)와 O-linked 당쇄 1개(Ser 126)의 첨가부위가 존재하며, 2개의 disulfide bridges(7-161번, 29-33번)를 형성하고 있다. 이러한 당쇄의 수식은 EPO의 대사에 있어서 매우 중요하다. EPO를 가축의 소변으로부터 생산하기 위하여 생쥐의 3.6 kb UII promoter 하류에 genome hEPO와 SV 40 poly A를 연결하여 형질전환용 발현 벡터를 구축하였으며, 과배란 유기로 채란되어진 돼지의 1-세포기 수정란의 웅성전핵에 유전자를 미세주입기로 주입 후 즉시 대리모에 이식하였다. 66두에 미세주입된 1572개의 수정란을 외과적 방법으로 이식, 평균 23개의 수정란을 이식하였다. 생산된 자돈 112두중 2두(3-5, 3-15번)에서 PCR양성반응(304, 567bp)을 나타내어, 2두의 돼지로부터 소변을 회수하였다. 회수된 소변을 이용 Elisa방법으로 EPO를 분석한 결과 3-5번 돼지에서만 분만 후 지속적으로 EPO농도가 증가되었다. EPO의 최고농도는 1.1 IU/$m\ell$였으며, 이러한 결과는 CHO 세포에서의 500-1000 IU/$m\ell$의 생산량보다도 약 500-1000배정도 낮은 수준이었다. 이상을 종합하여 보면, 1) 가축에서도 생리활성물질을 소변에서 생산할 수 있는 UII promoter의 활용가능성을 제시하였으며, 2) 현재로서는 EPO의 발현량이 너무 낮아, 사용된 생쥐의 promoter를 보완할 필요성이 있다고 사료된다. 그러나, UII promoter를 이용하여 생리활성 물질을 생산할 수 있는 형질전환 돼지 생산의 성공은, 앞으로 형질전환 가축을 이용하는 활용 면에서도 더욱 더 활발할 것으로 기대된다.

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Induction of Apoptosis in Glioma Cells and Upregulation of Fas Expression Using the Human Interferon-β Gene

  • Guo, Yan;Wang, Gan;Gao, Wen-Wei;Cheng, Shi-Wen;Wang, Ren;Ju, Shi-Ming;Cao, He-Li;Tian, Heng-Li
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.6
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    • pp.2837-2840
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    • 2012
  • We investigated whether IFN-${\beta}$ inhibits the growth of human malignant glioma and induces glioma cell apoptosis using the human IFN-${\beta}$ gene transfected into glioma cells. A eukaryonic expression vector ($pSV2IFN{\beta}$) for IFN-${\beta}$ was transfected into the glioma cell line SHG44 using liposome transfection. Stable transfection and IFN-${\beta}$ expression were confirmed using an enzyme-linked immunosorbent assay (ELISA). Cell apoptosis was also assessed by Hoechst staining and electron microscopy. In vivo experiments were used to establish a SHG44 glioma model in nude mice. Liposomes containing the human IFN-${\beta}$ gene were injected into the SHG44 glioma of nude mice to observe glioma growth and calculate tumor size. Fas expression was evaluated using immunohistochemistry. The IFN-${\beta}$ gene was successfully transfected and expressed in the SHG44 glioma cells in vitro. A significant difference in the number of apoptotic cells was observed between transfected and non-transfected cells. Glioma growth in nude mice was inhibited in vivo, with significant induction of apoptosis. Fas expression was also elevated. The IFN-${\beta}$ gene induces apoptosis in glioma cells, possibly through upregulation of Fas. The IFN-${\beta}$ gene modulation in the Fas pathway and apoptosis in glioma cells may be important for the treatment of gliomas.

Apoptosis in Human Corneal Epithelial cells induced by Exhausted Medium (Exhausted Medium에 의한 각막상피 세포의 세포고사 유도)

  • Kim, Jae-Min
    • Journal of Korean Ophthalmic Optics Society
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    • v.5 no.1
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    • pp.83-87
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    • 2000
  • To investigate exhausted-medium-induced apoptosis in human corneal epithelial(HCE) cells, this study was performed DNA gel electrophoresis, M30 CytoDEATH staining and FAS-FAS ligand ELISA. SV-40 transfected cells were grown to confluency in culture for 7days. The supernatant was harvested and filtered with $0.22{\mu}m$ filter paper. Fresh HCE cells were exposed to the filtered exhausted medium for 1~2 days. Apoptotic cells were prepared for DNA extraction and run the agarose gel for DNA ladder pattern. M30 CytoDEATH was used a tool for easy and reliable determination of very early apoptosis in HCE cells. The control and exhausted medium were assayed for soluble FAS/FAS ligand protein by ELISA. HCE cells exposed to exhausted medium showed a typical DNA ladder pattern. Sporadic M30 CytoDEATH positive cells were detected among HCE cells exposed to exhausted medium. Soluble FAS/FAS ligand levels were not elevated in the exhausted medium compared to the fresh medium control. This study suggests that possible mechanism of exhausted medium induced apoptosis does not include the FAS-FAS ligand system.

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The Induction of Human Corneal Epithelial Apoptosis by Serum-free Medium (무 혈청배지에 의한 각막상피 세포의 세포고사)

  • Ra, Myung Suk;Kim, Jai-Min
    • Journal of Korean Ophthalmic Optics Society
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    • v.4 no.1
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    • pp.1-6
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    • 1999
  • The corneal epithelium is constantly shed and apoptosis may play an important role in this turn-over. We sought to define that serum-free medium was able to induce apoptosis of corneal epithelial cells. SV-40 transfected human corneal epithelial(HCE) cells were grown to 70% confluency in culture. Serum-free medium was added to cells and the cells incubated for 1, 2, 3, or 6 days. Apoptosis of cells at different times was assessed by staining cells with Giemsa or Hoechst 33342 and measuring DNA fragmentation using the TUNEL assay. HCE cells exposed to serum-free medium demonstrated a high incidence of apoptosis, which increased over time to $50{\pm}4%$ after 3 days. They also stained positively with TUNEL assay. Serum-free medium caused time dependent apoptosis of HCE cells. Thus, serum-like nutrient might be important in corneal epithelial cell homeostasis.

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The Characteristics and Spatio-temporal Distribution of Fish Schools during Summer in the Marine Ranching Area (MRA) of Yeosu using Acoustic Data (음향 자료를 이용한 하계 여수 바다목장 해역에서 어군의 시·공간 분포와 특징)

  • Yoon, Eun-A;Hwang, Doo-Jin;Kim, Ho-Sang;Lee, Kyung-Seon
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.47 no.3
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    • pp.283-291
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    • 2014
  • This study assessed dominant fish species, and the characteristics and spatio-temporal distribution of fish schools using acoustic and catch data in the marine ranching area (MRA) of Yeosu in July and August 2013. Acoustic data were collected using a 200-kHz dual beam transducer, and catch data were analyzed through auction data generated by a set net installed in the MRA. More fish schools were detected by acoustic methods in July than in August. The temporal distribution of fish schools differed between July and August, but, many schools demonstrated a high mean volume scattering strength (SV) around artificial reefs. Additionally, the characteristics of fish schools detected by echograms and the species caught by set nets differed between July and August. The dominant fish species were Engraulis japonicus, Pampus argenteus, Scomberomorus niphonius, and Pampus echinogaster in July, and approximately 85% of the catch in August consisted of Scomberomorus niphonius. Therefore, hydro-acoustic tools are useful for estimating fish school characteristics in large areas over a short period. To determine species, it is important to conduct net sampling surveys during the acoustic surveys. However, if a database of fish school characteristics organized by species is constructed through continuous study, it could be possible to identify fish species through acoustic methods alone.

Evaluation of the effective dose and image quality of low-dose multi-detector CT for orthodontic treatment planning (3차원 안모분석을 위한 저선량 Multi-detector CT 영상의 유효선량 및 화질 평가)

  • Chung, Gi-Chung;Han, Won-Jeong;Kim, Eun-Kyung
    • Imaging Science in Dentistry
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    • v.40 no.1
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    • pp.15-23
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    • 2010
  • Purpose : This study was designed to compare the effective doses from low-dose and standard-dose multi-detector CT (MDCT) scanning protocols and evaluate the image quality and the spatial resolution of the low-dose MDCT protocols for clinical use. Materials and Methods : 6-channel MDCT scanner (Siemens Medical System, Forschheim, Germany), was used for this study. Protocol of the standard-dose MDCT for the orthodontic analysis was 130 kV, 35 mAs, 1.25 mm slice width, 0.8 pitch. Those of the low-dose MDCT for orthodontic analysis and orthodontic surgery were 110 kV, 30 mAs, 1.25 mm slice width, 0.85 pitch and 110 kV, 45 mAs, 2.5 mm slice width, 0.85 pitch. Thermoluminescent dosimeters (TLDs) were placed at 31 sites throughout the levels of adult female ART head and neck phantom. Effective doses were calculated according to ICRP 1990 and 2007 recommendations. A formalin-fixed cadaver and AAPM CT performance phantom were scanned for the evaluation of subjective image quality and spatial resolution. Results : Effective doses in ${\mu}Sv$ ($E_{2007}$) were 699.1, 429.4 and 603.1 for standard-dose CT of orthodontic treatment, low-dose CT of orthodontic analysis, and low-dose CT of orthodontic surgery, respectively. The image quality from the low-dose protocol were not worse than those from the standard-dose protocol. The spatial resolutions of both standard-dose and low-dose CT images were acceptable. Conclusion : From the above results, it can be concluded that the low-dose MDCT protocol is preferable in obtaining CT images for orthodontic analysis and orthodontic surgery.

A Study on the 2-D distribution of Dynamic Poisson's Ratio using 3-C Geophones (3성분 지오폰을 이용한 동포아송비의 2차원 분포 연구)

  • Hong, Myung-Ho;Hwang, Yoon-Gu;Cho, Cheol-Hee;Lee, Yoon-Jung;Kim, Ki-Young
    • 한국지구물리탐사학회:학술대회논문집
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    • 2005.05a
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    • pp.223-226
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    • 2005
  • In order to acquire 3 components data which has the good signal to noise ratio with only one shot, 3-C geophones were used, As a result, the vertical component showed the distinct first arrival of P-wave, and the horizontal component was improved the signal to noise ratio of S-wave, while was attenuated P-wave. The 2-D Poisson's ratio section was computed from P- and S-wave cell velocities included velocity tomograms of the P- and S-waves. The Poisson's ratio values were computed in the range of $0.2{\~}0.3$. With one shot, we can obtain 2-D distribution of dynamic Poisson's ratio as well as velocity tomograms of P- and S-waves.

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Preliminary Evaluation of the Activity Concentration Limits for Consumer Goods Containing NORM

  • Jang, Mee;Chung, Kun Ho;Ji, Young Yong;Lim, Jong Myung;Kang, Mun Ja;Choi, Guen Sik
    • Journal of Radiation Protection and Research
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    • v.41 no.2
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    • pp.101-104
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    • 2016
  • Background: To protect the public from natural radioactive materials, the 'Act on safety control of radioactive rays around living environment" was established in Korea. There is an annual effective dose limit of 1 mSv for products, but the activity concentration limit for products is not established yet. Materials and Methods: To suggest the activity concentration limits for consumer goods containing NORM, in this research, we assumed the "small room model" surrounding the ICRP reference phantom to simulate the consumer goods in contact with the human bodies. Using the Monte Carlo code MCNPX, we evaluate the effective dose rate for the ICRP reference phantom in a small room with dimension of phantom size and derived the activity concentration limit for consumer goods. Results and Discussion: The consumer goods have about 1600, 1200 and $19000Bq{\cdot}kg^{-1}$ for $^{226}Ra$, $^{232}Th$ and $^{40}K$, and the activity concentration limits are about six times comparing with the values of building materials. We applied the index to real samples, though we did not consider radioactivity of $^{40}K$, indexes of the some samples are more than 6. However, this index concept using small room model is very conservative, for the consumer goods over than index 6, it is necessary to reevaluate the absorbed dose considering real usage scenario and material characteristics. Conclusion: In this research, we derived activity concentration limits for consumer goods in contact with bodies and the results can be used as preliminary screening tool for consumer goods as index concept.

p53-mediated Inhibitory Mechanism on HIV-1 Tat is Likely to be Associated with Tat-Phosphorylation (p53에 의한 HIV-1 Tat 활성억제와 인산화관련 가능성 연구)

  • Byune, Hee-Sun;Lee, Sang-Gu;Bae, Yong-Soo
    • The Journal of Korean Society of Virology
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    • v.28 no.1
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    • pp.39-52
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    • 1998
  • HIV-1 tat, a strong transactivator, is essential for the HIV-1 replication and AIDS progression. The Tat function is markedly inhibited by human anti-oncogene p53. This work was initiated to identify the p53-associated inhibitory mechanism on tat-mediated transactivation. Inhibitory function of p53 was confirmed by co-transfection of tat-expressing Jurkat cells with LTR-CAT plasmid, or H3T1 cells (LTR-CAT integrated HeLa cells) with different ratio of pSV-tat/pCDNA-p53 plasmids. Results from the direct protein-protein interaction between soluble p53 and tat, and yeast two-hybrid experiments showed that the co-suppression mechanism is unlikely to be due to the direct interaction. CAT activity was not affected by tat in Jurkat cells which were transfected with p53-promoter-CAT or p53-enhancer-CAT, suggesting that the tat-mediated p53 suppression is not directly associated with p53-promoter. Finally, we have tested protein kinase activity in p53-tranfected Jurkat cells, which might phosphorylate HIV-1 tat, resulting in inhibition of tat function. Some of our data lead us to assume that the p53-mediated tat inhibition is likely to be associated with p53-associated, signaling-mediated phosphorylation of tat, resulting in the dysfunction of tat. This study is now under investigation.

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Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction

  • Guo, Yijie;Fukuda, Tomokazu;Nakamura, Shuichi;Bai, Lanlan;Xu, Jun;Kuroda, Kengo;Tomioka, Rintaro;Yoneyama, Hiroshi;Isogai, Emiko
    • Asian-Australasian Journal of Animal Sciences
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    • v.28 no.2
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    • pp.273-279
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    • 2015
  • Leptospiral lipopolysaccharide (L-LPS) has shown potency in activating toll-like receptor 2 (TLR2) in pig fibroblasts (PEFs_NCC1), and causes the expression of proinflammatory cytokines. However, the stimulation by L-LPS was weak eliciting the function of TLR2 sufficiently in pig innate immunity responses during Leptospira infection. In this study, the immune response of pig embryonic fibroblast cell line (PEFs_SV40) was investigated and was found to be the high immune response, thus TLR2 is the predominate receptor of L-LPS in pig cells. Further, we found a strategy using the antibody against L-LPS, to prevent L-LPS interaction with TLR2 in pig cells which could impact on immune activation.