• Title/Summary/Keyword: targeting

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Efficient Gene Targeting using Nuclear Localization Signal (NLS) and Negative Selection Marker Gene in Porcine Somatic Cells

  • Kim, Hye Min;Lee, Sang Mi;Park, Hyo Young;Kang, Man-Jong
    • Reproductive and Developmental Biology
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    • v.38 no.2
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    • pp.71-77
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    • 2014
  • The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

Identification of N-terminal amino acids of ApPDE4 involved in targeting to plasma membrane and cellular morphological change by expression of N-terminal peptide (원형질막 타기팅에 필요한 ApPDE4의 N-말단의 아미노산 서열 분석 및 발현에 의한 형태적 변화)

  • Kim, Kun-Hyung;Jun, Yong-Woo;Lee, Jin-A;Jang, Deok-Jin
    • Analytical Science and Technology
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    • v.26 no.1
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    • pp.106-112
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    • 2013
  • PDE plays an important role in cAMP-mediated cellular signaling within the cells. The proper targeting of each PDE is mediated by unique N-terminal of each PDE isoform. It has been recently reported that supershort-, short- and long-forms of PDE4 in Aplysia were cloned in Aplysia. Long-form of ApPDE4 was localized at plasma membrane and presynaptic terminal in Aplysia sensory neurons. However, it remains elusive which part of ApPDE4 is minimal region for the proper targeting and what are the effects on the cell functions. Here, we identified that N-terminal 13 amino acids of ApPDE4 long-form is minimal regions for the plasma membrane targeting. In addition, overexpression of ApPDE4(N20)-mRFP could induce morphological changes in HEK293T cells. Interestingly, mRFP-$PLC{\delta}1$(PH), which selectively binds to PI4,$5P_2$, could induce morphological changes in similar with that by ApPDE4(N20)-mRFP. These results suggested that binding of ApPDE4(N20) to lipids including PI4,$5P_2$ might be responsible for targeting of ApPDE4 to plasma membrane and morphological changes in HEK293T cells.

A Study on HILS for Performance Analysis of Airborne EOTS for Aircraft (항공기용 EOTS 성능분석을 위한 HILS시스템 구축에 관한 연구)

  • Chun, Seungwoo;Baek, Woonhyuk;La, Jongpil
    • Journal of the Korea Society of Computer and Information
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    • v.18 no.12
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    • pp.55-64
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    • 2013
  • In this paper, the HILS (Hardware In-the-Loop Simulation) system to analyze and to verify the performance of the targeting pod is addressed. The main functions of the targeting pod is acquiring and tracking targets to guide a LGB (Laser Guided Bomb) to the targets. For the analysis of targeting pod, the real time simulate images generation of IR and daylight cameras, sever control technology, and the analysis of laser transfer characteristics are necessary. For the real time image generation and the laser transfer characteristics analysis, off-the-shelf SDK(Software Development Kit) OKTAL-SE is used. For the servo controller, well-proven mechanism in the previous program is applied to increase servo control accuracy. To analyze the performance of a targeting pod in a realistic environment, 1553B, ARINK818 interface and etc. which are actually implemented in real combat aircrafts are applied in the system. By using the developed HILS system, the performance of currently operating targeting pods in real combat aircrafts can be analyzed and predicted. Additionally, the relationship between overall system performance and each module performance can be analyzed, the currently developed HILS system is expected to be a very useful tool to generate system development requirements of targeting pods and to reduce any possible future development risks.

Effects of G-Rh2 on mast cell-mediated anaphylaxis via AKT-Nrf2/NF-κB and MAPK-Nrf2/NF-κB pathways

  • Xu, Chang;Li, Liangchang;Wang, Chongyang;Jiang, Jingzhi;Li, Li;Zhu, Lianhua;Jin, Shan;Jin, Zhehu;Lee, Jung Joon;Li, Guanhao;Yan, Guanghai
    • Journal of Ginseng Research
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    • v.46 no.4
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    • pp.550-560
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    • 2022
  • Background: The effect of ginsenoside Rh2 (G-Rh2) on mast cell-mediated anaphylaxis remains unclear. Herein, we investigated the effects of G-Rh2 on OVA-induced asthmatic mice and on mast cell-mediated anaphylaxis. Methods: Asthma model was established for evaluating airway changes and ear allergy. RPMCs and RBL-2H3 were used for in vitro experiments. Calcium uptake, histamine release and degranulation were detected. ELISA and Western blot measured cytokine and protein levels, respectively. Results: G-Rh2 inhibited OVA-induced airway remodeling, the production of TNF-α, IL-4, IL-8, IL-1β and the degranulation of mast cells of asthmatic mice. G-Rh2 inhibited the activation of Syk and Lyn in lung tissue of OVA-induced asthmatic mice. G-Rh2 inhibited serum IgE production in OVA induced asthmatic mice. Furthermore, G-Rh2 reduced the ear allergy in IgE-sensitized mice. G-Rh2 decreased the ear thickness. In vitro experiments G-Rh2 significantly reduced calcium uptake and inhibited histamine release and degranulation in RPMCs. In addition, G-Rh2 reduced the production of IL-1β, TNF-α, IL-8, and IL-4 in IgE-sensitized RBL-2H3 cells. Interestingly, G-Rh2 was involved in the FcεRI pathway activation of mast cells and the transduction of the Lyn/Syk signaling pathway. G-Rh2 inhibited PI3K activity in a dose-dependent manner. By blocking the antigen-induced phosphorylation of Lyn, Syk, LAT, PLCγ2, PI3K ERK1/2 and Raf-1 expression, G-Rh2 inhibited the NF-κB, AKT-Nrf2, and p38MAPK-Nrf2 pathways. However, G-Rh2 up-regulated Keap-1 expression. Meanwhile, G-Rh2 reduced the levels of p-AKT, p38MAPK and Nrf2 in RBL-2H3 sensitized IgE cells and inhibited NF-κB signaling pathway activation by activating the AKT-Nrf2 and p38MAPK-Nrf2 pathways. Conclusion: G-Rh2 inhibits mast cell-induced allergic inflammation, which might be mediated by the AKT-Nrf2/NF-kB and p38MAPK-Nrf2/NF-κB signaling pathways.

Hepatic Targeting of Acyclovir Using Asialofetuin as a Drug Carrier (아시알로페투인을 약물수송체로 이용한 아시클로버의 간표적화)

  • Yong, Chul-Soon;Son, Sung-Ho;Jun, Chul-Soo;Oh, Doo-Man
    • Journal of Pharmaceutical Investigation
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    • v.24 no.4
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    • pp.217-225
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    • 1994
  • With the purpose of improving the therapeutic index of $[^3H]$ acyclovir (ACV) in the treatment of chronic hepatitis B infection, asialofetuin (AF) which after selective interaction with Ashwell's receptor specifically enters into hepatocytes, was chosen as a carrier system for hepatic targeting. This drug was first converted to its monophosphate (ACVMP), which was subsequently activated by water soluble carbodiimide to conjugate with ${\varepsilon}-NH_2$ groups of Iysine residues of AF. The molar ratio of ACVMP to AF in the conjugate was 3.9. In rats, elimination of ACVMP-AF conjugate after i.v. injection showed two phase elimination kinetics. Initial apparent elimination rate constant in rats was $0.191\;min^{-1}$ which was greater than that of ACV. The elimination rate constant from terminal phase was $0.021\;min^{-1}$. Area under the total radioactivities versus time curve was found to be several times larger in liver than in other organs (spleen, intestine, lung and kidney) after i.v. administration of the conjugate labelled in the drug moiety. The above results suggested that ACVMP-AF conjugate was rapidly taken up by hepatocytes and could be a useful hepatic targeting system.

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Development of Worm-like Polymeric Drug Carriers with Multiple Ligands for Targeting Heterogeneous Breast Cancer Cells

  • Lee, A-Hyeong;Oh, Kyung-Taek;Baik, Hye-Jung;Lee, Bo-Reum;Oh, Young-Taik;Lee, Don-Haeng;Lee, Eun-Seong
    • Bulletin of the Korean Chemical Society
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    • v.31 no.8
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    • pp.2265-2271
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    • 2010
  • In this study, wormorm-like polymeric micelles were construted from poly(L-lactic acid)-b-poly(ethyelen glycol) (PLLA-b-PEG) block copolymers via worm-like (or cylindrical) self- assembly that consisted of a relatively long PLLA block ($M_n$ 7K Daltons) at the core and a relatively short PEG block ($M_n$ 2K Daltons) as the shell. Several cancer-targeting moieties (such as folate, cobalamin, and cyclic arginine-glycine-aspartic (RGD) peptide) were chemically coupled with the succinylated or maleimided PEG block of PLLA-b-PEG to act as a cancer cell-specific targeting ligand for breast cancer. The worm-like micelles with muplite cancer cell-specific ligands proved to be successful in recognizing different breast cancer cells at once. This has the potential to aid in cancer-specific drug delivery and to be used as an effective treatment for breast cancer.

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

  • Oh, Doo-Byoung
    • BMB Reports
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    • v.48 no.8
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    • pp.438-444
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    • 2015
  • Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444]

A Content Analysis of Web sites from a Perspective of Children's Online Privacy Protection (아동 대상 인터넷 사이트의 개인정보보호 실태와 개선 방향)

  • Kim, So-Ra;Rhee, Kee-Choon
    • Journal of the Korean Home Economics Association
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    • v.45 no.8
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    • pp.127-142
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    • 2007
  • The objective of this study was to discuss child privacy issues at Web sites targeting children, Specifically, the study examined l)types of information required for join the membership, 2)whether privacy policies at Web sites for children abide by privacy guideline, and 3)specific examples of recommendable privacy policy and problematic privacy policy from Web sites, Total of 305 Web sites targeting children were used for content analysis, Selected Web sites included recommended sites by Korea Council of Children's Organizations and food business Web sites, The results showed that more than 70% of Web sites required private information when children join the membership. Most of these Web sites provided mailing service for children. Generally, Web sites showed problems in parents' approval procedures. Also, privacy policies at Web sites frequently omitted purpose specification principle and the security safeguard principle. Regulating online service provides and marketers targeting children would be necessary for protecting child privacy. Further, education program targeting parents and children could help them make right choices to protect children's online privacy.

Domestic Clinical Studies Related to the Exercise Program Targeting Farmers : a Systematic Review (농업인을 대상으로 한 운동프로그램 관련 국내 임상연구에 대한 체계적 문헌고찰)

  • Kook, Kil-Ho;Lee, Eun-Jung;Oh, Min-Seok
    • Journal of Korean Medicine Rehabilitation
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    • v.21 no.4
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    • pp.77-86
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    • 2011
  • Objectives: This study aims to evaluate domestic clinical studies related to the exercise program targeting farmers. Methods: Five Korean databases were seamed for prospective clinical trials of exercise program targeting farmers from 2001 to August, 2011. Only studies conducted in Korean language were searched. Results: Thirteen clinical studies were included among 3374 studies searched. Six were observational studies, four were non-randomized controlled trials, and three were randomized controlled trials. All of included studies reported favorable effects of exercise group compared to baseline of controlled group. Conclusions: We found various mistake in the applications of statistical methodologies in the studies of exercise program targeting farmers. It is necessary for more randomized controlled trials to evaluate effect of exercise program target farmers.