• BMB Reports
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• v.33 no.4
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• pp.294-299
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• 2000

Proteolytic processing of the dengue virus serotype 2 polyprotein precursor is catalyzed by a host signal peptidase and a virus encoded two-component protease consisting of the nonstructural proteins, NS2B and NS3. We expressed in Escherichia coli the NS2B, NS3(pro) and NS2B-NS3 proteins from the dengue virus type 2 strain 16681 as N-terminal fusions with a hexahistidine affinity tag under the control of the inducible trc promoter. All fusion proteins were purified to >90% purity by detergent extraction of inclusion bodies and a single step metal chelate chromatography. Proteins were refolded on-column and recovered with yields of 0.5, 6.0 and 1.0 mg/l of E. coli culture that was grown to $OD_{600}=1.0$ for NS2B, NS3(pro) and NS2B-NS3, respectively. Purified proteins gave strong signals in Western blots using $Ni^{2+}-nitrilotriacetic$ acid as a probe for the presence of the polyHis tag. During the purification process, $(His)_{6}NS2B-NS3$ was apparently not autoproteolytically cleaved at the NS2B/NS3 site.

• BMB Reports
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• v.35 no.2
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• pp.206-212
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• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1651-1658
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• 2020

Since Zika virus (ZIKV) was first detected in Uganda in 1947, serious outbreaks have occurred globally in Yap Island, French Polynesia and Brazil. Even though the number of infections and spread of ZIKV have risen sharply, the pathogenesis and replication mechanisms of ZIKV have not been well studied. ZIKV, a recently highlighted Flavivirus, is a mosquito-borne emerging virus causing microcephaly and the Guillain-Barre syndrome in fetuses and adults, respectively. ZIKV polyprotein consists of three structural proteins named C, prM and E and seven nonstructural proteins named NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 in an 11-kb single-stranded positive sense RNA genome. The function of individual ZIKV genes on the host innate immune response has barely been studied. In this study, we investigated the modulations of the NF-κB promoter activity induced by the MDA5/RIG-I signaling pathway. According to our results, two nonstructural proteins, NS2A and NS4A, dramatically suppressed the NF-κB promoter activity by inhibiting signaling factors involved in the MDA5/RIG-I signaling pathway. Interestingly, NS2A suppressed all components of MDA5/RIG-I signaling pathway, but NS4A inhibited most signaling molecules, except IKKε and IRF3-5D. In addition, both NS2A and NS4A downregulated MDA5-induced NF-κB promoter activity in a dosedependent manner. Taken together, our results suggest that NS2A and NS4A signifcantly antagonize MDA5/RIG-I-mediated NF-κB production, and these proteins seem to be controlled by different mechanisms. This study could help understand the mechanisms of how ZIKV controls innate immune responses and may also assist in the development of ZIKV-specific therapeutics.

• Applied Chemistry for Engineering
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• v.25 no.4
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• pp.396-400
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• 2014

The pore surface of mesoporous materials, SBA-15 and MCM-41 were functionalized with organosilanes, 3-aminopropyltrimethoxysilane (1NS) and N-[(3-trimethoxysilyl)propyl]ethylenediamine (2NS) via grafting method. $(n-BuCp)_2ZrCl_2$ and methylaluminoxane (MAO) were impregnated on the surface-functionalized mesoporous materials for the application to ethylene polymerization. In the case of SBA-15/2NS/$(n-BuCp)_2ZrCl_2$ supported Zr and Al contents decreased as grafted 2NS content increased. However, in the case of MCM-41/2NS/$(n-BuCp)_2ZrCl_2$ supported Al content decreased, but Zr content increased as grafted 2NS content increased. The polymerization activity of SBA-15/2NS/$(n-BuCp)_2ZrCl_2$ increased as the amount of grafted 2NS increased. Increase in the amount of grafted 2NS should caused decrease in pore volume and diameter. Consequently, it decreased the amount of supported metallocene and MAO in general. However, the smaller pore-sized MCM-41 could have lower supported MAO content due to its large molecular size in case that MCM-41 was surface-functionalized with 2NS. Therefore, the supported metallocene content could increase and its polymerization activity was higher than that of SBA-15.

• Journal of Sensor Science and Technology
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• v.12 no.1
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• pp.1-9
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• 2003

CsI single crystals doped with lithium, potassium or rubidium were grown by using Czochralski method at Ar gas atmosphere. The energy resolutions of CsI(Li:0.2 mole%), CsI(K:0.5 mole%) and CsI(Rb:1.5 mole%) scintillators were 14.5%, 15.9% and 17.0% for $^{137}Cs$(0.662 MeV), respectively. The energy calibration curves of CsI(Li), CsI(K) and CsI(Rb) scintillators were linear for $\gamma$-ray energy. The time resolutions of CsI(Li:0.2 mole%), CsI(K:0.5 mole%) and CsI(Rb:1.5 mole%) scintillators measured by CFT(constant-fraction timing method) were 9.0 ns, 14.7 ns and 9.7 ns, respectively. The fluorescence decay times of CsI(Li:0.2 mole%) scintillator had a fast component and slow one of ${\tau}_1=41.2\;ns$ and ${\tau}_2=483\;ns$, respectively. The fluorescence decay times of CsI(K:0.5 mole%) scintillator were ${\tau}_1=47.2\;ns$ and ${\tau}_2=417\;ns$. And the fluorescence decay times of CsI(Rb:1.5 mole%) scintillator were ${\tau}_1=41.3\;ns$ and ${\tau}_2=553\;ns$. The phosphorescence decay times of CsI(Li:0.2 mole%), CsI(K:0.5 mole%) and CsI(Rb:1.5 mole%) scintillators were 0.51 s, 0.57 s and 0.56 s, respectively.

• Polymer(Korea)
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• v.39 no.1
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• pp.169-173
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• 2015

Metallocene was supported on the silica, which was functionalized with aminosilanes such as aminopropyltrimethoxysilane (1NS) or N-[3-(trimethoxysilyl)propyl]ethylenediamine (2NS), and ionic liquids such as 1-butyl-4-methylpyridinium chloride (Cl), tributylmethylammonium chloride (Amm), benzyldimethyltetradecylammonium chloride (Ben), 1-butyl-1-methylpyrrolidinium chloride (Pyr), and then ethylene polymerizations were performed. The Zr contents of $SiO_2/1NS/IL/(n-BuCp)_2ZrCl_2$ and $SiO_2/2NS/IL/(n-BuCp)_2ZrCl_2$ were lower than those of only aminosilane-treated silicas. However, the polymerization activity of $SiO_2/1NS/IL/(n-BuCp)_2ZrCl_2$ was higher than that of $SiO_2/1NS/(n-BuCp)_2ZrCl_2$. The polymerization activity of $SiO_2/2NS/IL/(n-BuCp)_2ZrCl_2$ was lower than that of $SiO_2/2NS/(n-BuCp)_2ZrCl_2$ due to much lower Zr content.

• Molecules and Cells
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• v.22 no.1
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• pp.13-20
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• 2006

Hepatitis C virus (HCV) non-structural protein 5A protein (NS5A), which consists of three functional domains, is involved in regulating viral replication, interferon resistance, and apoptosis. Recently, the three-dimensional structure of the domain 1 was determined. However, currently the molecular basis for the domains 2 and 3 of HCV NS5A is yet to be defined. Toward this end, we expressed, purified the domain 2 of the NS5A (NS5A-D2), and then performed biochemical and structural studies. The purified domain 2 was active and was able to bind NS5B and PKR, biological partners of NS5A. The results from gel filtration, CD analysis, 1D $^1H$ NMR and 2D $^1H-^{15}N$ heteronuclear single quantum correlation (HSQC) spectroscopy indicate that the domain 2 of NS5A appears to be flexible and disordered.

• Proceedings of the Korea Information Processing Society Conference
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• 2015.10a
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• pp.322-324
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• 2015

네트워크 시뮬레이터인 NS-2(Network Simulator 2)는 다양한 인터넷 프로토콜을 분석 할 수 있는 이산 사건 시뮬레이터로 데이터 처리량과 패킷 지연 및 전력 소모 등을 구할 수 있다. 그러나 NS-2를 사용하여 실험 시 네트워크 토폴로지의 설정이 변경 될 때마다 Tcl 스크립트를 통해 단일적으로 밖에 대응하지 못한다는 불편함이 존재한다. 이 문제를 해결하기 위해 본 논문에서는 멀티프로세싱 기법의 NS-2 Batch Job 시스템을 제안한다. 쉘(Bash) 스크립트로 NS-2 내부 구조의 간섭 없이 자동화 일괄 작업(Batch Job) 시스템을 모듈 구조로 구현 및 적용시켰다. 또한 실험의 시간 효율을 극대화시키기 위해 멀티프로세싱 기법을 이용하여, 하드웨어 성능의 부하가 걸리지 않는 선에서 NS-2 시뮬레이션을 다중 처리할 수 있도록 만들었다. 성능 비교분석 결과, 제안하는 Batch Job 시스템을 적용하면 기존에 NS-2를 이용한 실험에 걸리는 시간에 비해 소요시간이 평균 48% 감소한 결과를 볼 수 있다. 이는 하드웨어 성능이 향상된다면, 부하가 걸리지 않는 상한까지 더 많은 개수의 NS-2 프로세스를 실행시킬 수 있기 때문에 더욱 큰 시간 효율을 보여줄 수 있다.

• Bulletin of the Korean Chemical Society
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• v.20 no.3
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• pp.301-306
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• 1999

Hepatitis C virus (HCV) has been known to be an enveloped virus with a positive strand RNA genome and the major agent of the vast majority of transfusion associated cases of hepatitis. For viral replication, HCV structural proteins are first processed by host cell signal peptidases and NS2/NS3 site of the nonstructural protein is cleaved by a zinc-dependent protease NS2 with N-terminal NS3. The four remaining junctions are cleaved by a separate NS3 protease. The solution conformations of NS4B/5A, NS5A/5B substrates and NS5A/5B inhibitor have been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data suggested that the both NS5A/5B substrate and inhibitor appeared to have a folded tum-like conformation not only between P1 and P6 position but also C-terminal region, whereas the NS4B/5A substrate exhibited mostly extended conformation. In addition, we have found that the conformation of the NS5A/5B inhibitor slightly differs from that of NS5A/5B substrate peptide, suggesting different binding mode for NS3 protease. These findings will be of importance for designing efficient inhibitor to suppress HCV processing.

• Journal of Microbiology and Biotechnology
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• v.33 no.5
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• pp.600-606
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• 2023

Dengue virus (DENV) is a widespread arbovirus. To efficiently establish infection, DENV evolves multiple strategies to hijack the host innate immune response. Herein, we examined the inhibitory effects of DENV serotype 2 (DENV2) nonstructural proteins on RIG-I-directed antiviral immune response. We found that DENV2 NS2A, NS2B, NS4A, and NS4B significantly inhibited RIG-I-mediated IFN-β promoter activation. The roles of NS2B in RIG-I-directed antiviral immune response are unknown. Our study further showed that NS2B could dose-dependently suppress RIG-I/MAVS-induced activation of IFN-β promoter. Consistently, NS2B significantly decreased RIG-I- and MAVS-induced transcription of IFNB1, ISG15, and ISG56. Mechanistically, NS2B was found to interact with MAVS and IKKε to impair RIG-I-directed antiviral response. Our findings demonstrated a previously uncharacterized function of NS2B in RIG-I-mediated antiviral response, making it a promising drug target for anti-DENV treatments.