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Effects of Changes in Glycosylation Sites on Secretion of Recombinant Human Erythropoietin in Cultured CHO Cells

  • Lee, H. G;Lee, P. Y.;Lee, Y. K.;Kim, S. J.;H. K. Chung;M. K. Seo;Park, J. K.;K. S. Min;W. K. Chang
    • Korean Journal of Animal Reproduction
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    • v.27 no.4
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    • pp.299-307
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    • 2003
  • The effects of additions/deletions in glycosylated residues of recombinant human EPO (rhEPO) produced in CHO-K1 on their secretion were examined. hEPO cDNA was amplified from human liver mRNA and cloned into the pCR2.1 TOPO. Using overlapping-extension site-directed mutagenesis method, glycosylation sites at 24th, 38th, 83rd, and 126th were respectively or accumulatively removed by substituting its asparagine (or serine) with glutamine. To add novel glycosylation sites, 69 and 105th leucine was mutated to asparagine. Mutant and wild type rhEPO constructs were cloned into the pcDNA3 expression vector with CMV promoter and transfected into CHO cell line, CHO-K1, to produce mutant rhEPO mutant rhEPO proteins. Enzyme-linked immunosorbant assay (ELISA) and Western analysis with monoclonal anti-EPO antibody were performed using supernatants of the cultures showing transient and stable expressions respectively. Addition of novel glycosylation reduced rhEPO secretion dramatically while deletion mutants had little effect except some double deletion mutants ($\Delta$24/83 and $\Delta$38/83) and triple mutant ($\Delta$24/38/83). This fact suggests that not single but combination of changes in glycosyl groups affect secretion of rhEPO in cell culture, possibly via changes in their conformations.

HIPI Controller of IPMSM Drive using ALM-FNN (ALM-FNN을 이용한 IPMSM 드라이브의 HIPI 제어기)

  • Ko, Jae-Sub;Choi, Jung-Sik;Chung, Dong-Hwa
    • Journal of the Korean Institute of Illuminating and Electrical Installation Engineers
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    • v.23 no.8
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    • pp.57-66
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    • 2009
  • The conventional fixed gain PI controller is very sensitive to step change of command speed, parameter variation and load disturbances. The precise speed control of interior permanent magnet synchronous motor(IPMSM) drive becomes a complex issue due to nonlinear coupling among its winding currents and the rotor speed as well as the nonlinear electromagnetic developed torque. Therefore, there exists a need to tune the PI controller parameters on-line to ensure optimum drive performance over a wide range of operating conditions. This paper proposes hybrid intelligent-PI(HIPI) controller of IPMSM drive using adaptive learning mechanism(ALM) and fuzzy neural network(FNN). The proposed controller is developed to ensure accurate speed control of IPMSM drive under system disturbances and estimation of speed using artificial neural network(ANN) controller. The PI controller parameters are optimized by ALM-FNN at all possible operating condition in a closed loop vector control scheme, The validity of the proposed controller is verified by results at different dynamic operating conditions.

Evaluation of Cytotoxicity for Immunity Rejection of US11, hDAF and FasL Transgene-Transfected Cells

  • Kang, Jung Won;Shin, Hyeon Yeong;Oqani, Reza K.;Lin, Tao;Lee, Jae Eun;Kim, So Yeon;Lee, Joo Bin;Jin, Dong Il
    • Reproductive and Developmental Biology
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    • v.41 no.3
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    • pp.57-63
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    • 2017
  • Xenotransplantation is proposed as a solution to the problem of organ shortage. However, transplantation of xenogeneic organs induces an antigen-antibody reaction in ${\alpha}$-1,3-gal structure that are not present in humans and primates, and thus complement is also activated and organs die within minutes or hours. In this study, we used FasL gene, which is involved in the immune response of NK cell, and US11, which suppresses MHC Class I cell membrane surface expression, to inhibit cell mediated rejection in the interspecific immunity rejection, and also hDAF(CD55) was introduced to confirm the response to C3 complement. These genes were tranfeced into Korean native pig fetal fibroblasts using pCAGGS vector. And cytotoxicity of NK cell and human complement was confirmed in each cell line. The US11 inhibited the cytotoxicity of NK cell and, in addition, the simultaneous expression of US11 and Fas ligand showed excellent suppress to T-lymphocyte cytotoxicity, hDAF showed weak resistance to cytotoxicity of natural killer cell but not in CD8+ CTLs. Cytotoxicity study with human complement showed that hDAF was effective for reducing complement reaction. In this studies have demonstrated that each gene is effective in reducing immune rejection.

Prediction of Strength and Propagation Characteristics of Supersonic Flight Sonic Boom (초음속 비행체의 소닉붐 강도와 전파 특성 예측)

  • Jung, Suk Young;Ha, Jae-hyoun;Lee, Younghwan;Jin, Hyeon
    • Journal of the Korean Society for Aeronautical & Space Sciences
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    • v.48 no.7
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    • pp.497-504
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    • 2020
  • A technique was developed for analysis on sonic boom created by supersonic flight and for prediction of its sound level and atmospheric propagation characteristics. It is of great importance to anticipate sound level of sonic boom because it causes environmental issue. For that purpose, the simplified sonic-boom prediction method was applied to calculate sound pressure according to physical properties and flight information of the object and distance to measurement site, in this study. Propagation characteristics of shock wave emanated from a flying object was analyzed by using line-of-sight vector and ray tracing method which dealt with refraction of wave due to atmospheric density distribution along altitude. Predicted results agreed well with measured data from real flight.

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.

A Study for the Border line Extraction technique of City Spatial Building by LiDAR Data (LiDAR 데이터와 항공사진의 통합을 위한 사각 빌딩의 경계점 설정)

  • Yeon, Sang-Ho;Lee, Young-Wook
    • Proceedings of the Korea Contents Association Conference
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    • 2007.11a
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    • pp.27-29
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    • 2007
  • The visual implementation of 3-dimensional national environment is focused by the requirement and importance in the fields such as, national development plan, telecommunication facility deployment plan, railway construction, construction engineering, spatial city development, safety and disaster prevention engineering. The currently used DEM system using contour lines, which embodies national geographic information based on the 2-D digital maps and facility information has limitation in implementation in reproducing the 3-D spatial city. Moreover, this method often neglects the altitude of the rail way infrastructure which has narrow width and long length. There it is needed to apply laser measurement technique in the spatial target object to obtain accuracy. Currently, the LiDAR data which combines the laser measurement skill and GPS has been introduced to obtain high resolution accuracy in the altitude measurement. In this paper, we first investigate the LiDAR based researches in advanced foreign countries, then we propose data a generation scheme and an algorithm for the optimal manage and synthesis of railway facility system in our 3-D spatial terrain information. For this object, LiDAR based height data transformed to DEM, and the realtime unification of the vector via digital image mapping and raster via exactness evaluation is transformed to make it possible to trace the model of generated 3-dimensional railway model with long distance for 3D tract model generation.

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Feature information fusion using multiple neural networks and target identification application of FLIR image (다중 신경회로망을 이용한 특징정보 융합과 적외선영상에서의 표적식별에의 응용)

  • 선선구;박현욱
    • Journal of the Institute of Electronics Engineers of Korea SP
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    • v.40 no.4
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    • pp.266-274
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    • 2003
  • Distance Fourier descriptors of local target boundary and feature information fusion using multiple MLPs (Multilayer perceptrons) are proposed. They are used to identify nonoccluded and partially occluded targets in natural FLIR (forward-looking infrared) images. After segmenting a target, radial Fourier descriptors as global shape features are defined from the target boundary. A target boundary is partitioned into four local boundaries to extract local shape features. In a local boundary, a distance function is defined from boundary points and a line between two extreme points. Distance Fourier descriptors as local shape features are defined by using distance function. One global feature vector and four local feature vectors are used as input data for multiple MLPs to determine final identification result of the target. In the experiments, we show that the proposed method is superior to the traditional feature sets with respect to the identification performance.

On-line Signature Verification Using Fusion Model Based on Segment Matching and HMM (구간 분할 및 HMM 기반 융합 모델에 의한 온라인 서명 검증)

  • Yang Dong Hwa;Lee Dae-Jong;Chun Myung-Geun
    • Journal of the Korean Institute of Intelligent Systems
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    • v.15 no.1
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    • pp.12-17
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    • 2005
  • The segment matching method shows better performance than the global and points-based methods to compare reference signature with an input signature. However, the segment-to-segment matching method has the problem of decreasing recognition rate according to the variation of partitioning points. This paper proposes a fusion model based on the segment matching and HMM to construct a more reliable authentic system. First, a segment matching classifier is designed by conventional technique to calculate matching values lot dynamic information of signatures. And also, a novel HMM classifier is constructed by using the principal component analysis to calculate matching values for static information of signatures. Finally, SVM classifier is adopted to effectively combine two independent classifiers. From the various experiments, we find that the proposed method shows better performance than the conventional segment matching method.

Up-regulation of Thy-1 Promotes Invasion and Metastasis of Hepatocarcinomas

  • Cheng, Bian-Qiao;Jiang, Yi;Li, Dong-Liang;Fan, Jing-Jing;Ma, Ming
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.4
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    • pp.1349-1353
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    • 2012
  • Increasing evidence has revealed that thy-1 was a potential stem cell marker of liver cancer, but no data have been shown on how thy-1 regulates the pathophysiology of liver cancer, such as proliferation, apoptosis, invasion and migration. We previously demonstrated that thy-1 was expressed in about 1% of hepg2 cells, thy-1+hepg2 cells, but not thy-1-, demonstrating high tumorigenesis on inoculation $0.5{\times}10^5$ cells per BACA/LA mouse after 2 months. In the present study, our results showed that higher expression of thy-1 occurs in 72% (36/50 cases) of neoplastic hepatic tissues as compared to 40% (20/50 cases) of control tissues, and the expression of thy-1 is higher in poorly differentiated liver tumors than in the well-differentiated ones. In addition, thy-1 expression was detected in 85% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis or hepatitis. There was a significant negative correlation between thy-1expression and E-cadherin expression (a marker of invasion and migraton), but not between thy-1 expression and AFP expression in all the liver cancer and blood samples. We further investigated the relationship between thy-1 and E-cadherin in liver cancer hepg2 cell line which was transfected with pReceiver-M29/thy-1 eukaryotic expression vector followed by aspirin treatment. Lower expression of E-cadherin but higher expressions of thy-1 were detected in hepg2 cells transfected with pReceiver-M29/thy-1. Taken together, our study suggested that thy-1 probably regulates liver cancer invasion and migration.

RNAi-induced K-Ras Gene Silencing Suppresses Growth of EC9706 Cells and Enhances Chemotherapy Sensitivity of Esophageal Cancer

  • Wang, Xin-Jie;Zheng, Yu-Ling;Fan, Qing-Xia;Zhang, Xu-Dong
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.12
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    • pp.6517-6521
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    • 2012
  • To analyze the growth, proliferation, apoptosis, invasiveness and chemotherapy sensitivity of EC9706 cells after K-Ras gene silencing, an expression carrier pSilencer-siK-Ras was constructed, and the EC9706 cell line was transfected using a liposome technique. Six groups were established: Control, siRNA NC (transfected with empty vector pSilencer2.1); Ras siRNA (transfected with pSilencer-siK-Ras2); Paclitaxel; Paclitaxel + siRNA NC; and Ras siRNA + Paclitaxel. After the treatment, RT-PCR, Western blotting, MTT assay, flow cytometry and the Transwell technique were used to assess expression of K-Ras mRNA and protein in EC9706 cells, as well as cell growth, proliferation, apoptosis and invasiveness. The effect of Paclitaxel chemotherapy was also tested. pSilencer-siK-Ras2 effectively down-regulated expression of K-Ras mRNA and protein in EC9706 cells, growth being significantly inhibited. Flow cytometry indicated obvious apoptosis of cells in the experimental group, with arrest in the G1 phase; cell migration ability was also reduced. After pSilencer-siK-Ras2 transfection or the addition of Paclitaxel, EC9706 cells were suppressed to different extents; the suppressive effect was strengthened by combined treatment. The results suggested that RNAi-induced K-Ras gene silencing could enhance chemotherapy sensitivity of esophageal cancer.