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Overexpression and purification of recombinant lysozyme from Agrius convolvuli expressed as inclusion body in Escherichia coli

  • Park, Soon-Ik;Yoe, Sung Moon
    • Animal cells and systems
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    • v.16 no.6
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    • pp.455-461
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    • 2012
  • Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

Measurement of Nonlinear Time-variant Source Characteristics of Intake and Exhaust Systems in Fluid Machines

  • Jang Seung-Ho;Ih Jeong-Guon
    • The Journal of the Acoustical Society of Korea
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    • v.24 no.3E
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    • pp.87-89
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    • 2005
  • The acoustical sources of intake and exhaust systems in fluid machines are often characterized by the source impedance and strength using linear frequency-domain modeling. In the case of the sources which are nonlinear and time-variant, however, the source parameters were sometimes incorrectly obtained. In this paper, the source model and direct measurement technique are modified in order to evaluate the effect due to nonlinear and periodically time-varying source character as well as the linear property of the reflectivity of in-duct fluid machine source. With a priori known kinematical information of the source, the types of nonlinear time-variant terms can be presumed by a simple physical model, in which there is practically no restriction on the form of the model. The concept of source impedance can be extendable by introducing the linear frequency response function for each nonlinear or time-variant input. Extending the conventional method and adapting the reverse MISO technique, it is possible to develop a direct method that can deal with the nonlinear time-variant source parameters. The proposed direct method has a novel feature that there is no restriction on the probability or spectral natures of the excited sound pressure data. The present method is verified by the simulated measurements for simplified fluid machines. It is thought that the proposed method would be useful in predicting the insertion loss or the radiated sound level from intake or exhaust systems.

The effect of thermodynamic stability of casting solution on the membrane inversion process morphology and permeation properties in phase inversion process

  • Kim, Jeong-Hoon;Lee, Kew-Ho
    • Proceedings of the Membrane Society of Korea Conference
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    • 1995.04a
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    • pp.26-27
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    • 1995
  • Most of synthetic polymeric membranes used in ultrafiltration, reverse osmosis and microfiltration processes are prepared by phase inversion(or phase separation) technique. In this technique, a homogeneous polymer solution is cast into thin film or hollow fiber shape and then immersed into a nonsolvent coagulant bath. The exchange of solvent and nonsolvent across the interface between casting solution and coagu!ant can make the casting solution phase-separate and form a membrane with a symmetric or asymmetric structure. Because of importance of this technique in membrane field, many investigations have been dedicated to elucidate the mechanism of membrane formation by phase inversion technique.[1-10] These investigation have suggested that the structure formation and permeation properties of phase inversion membrane depend on the variables such as the nature and content of casting solution and coagulant, temperature of casting solution and coagulant, and the diffusional exchange rate of solvent and nonsolvent etc. which can be related to the thermodynamic and kinetic properties of the casting system. The variables such as the nature and content of casting solution can also be the important factor affecting the structure formation and permeation property of the phase inversion membrane.

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Expression and Purification of a Cathelicidin-Derived Antimicrobial Peptide, CRAMP

  • Park Eu-Jin;Chae Young-Kee;Lee Jee-Young;Lee Byoung-Jae;Kim Yang-Mee
    • Journal of Microbiology and Biotechnology
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    • v.16 no.9
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    • pp.1429-1433
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    • 2006
  • Application of recombinant protein production and particularly their isotopic enrichment has stimulated development of a range of novel multidimensional heteronuclear NMR techniques. Peptides in most cases are amenable to assignment and structure determination without the need for isotopic labeling. However, there are many cases where the availability of $^{15}N$ and/or $^{13}C$ labeled peptides is useful to study the structure of peptides with more than 30 residues and the interaction between peptides and membrane. CRAMP (Cathelicidin-Related AntiMicrobial Peptide) was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. CRAMP was successfully expressed as a GST-fused form in E. coli and purified using affinity chromatography and reverse-phase chromatography. The yield of the CRAMP was 1.5 mg/l 1. According to CD spectra, CRAMP adopted ${\alpha}$-helical conformation in membrane-mimetic environments. Isotope labeling of CRAMP is expected to make it possible to study the structure and dynamic properties of CRAMP in various membrane systems.

Succession and Heterogeneity of Plant Community in Mt. Yongam, Kwangnung Experimental Forest (광릉내 용암산 식물군집의 천이와 이질성)

  • You, Young-Han;Kwang-Je Gi;Dong-Uk Han;Young-se Kwak;Joon-He Kim
    • The Korean Journal of Ecology
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    • v.18 no.1
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    • pp.89-97
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    • 1995
  • In order to study the successional trend and the heterogeneity of forest community, we investigated DBH frequency distribution of dominant tree species and the changes of several community indicies including ${\beta}-diversity\;({\beta}_t)$ along a belt transect in Mt. Yongam, Kwangnung Experimental Forest, which has been preserved for about 530 years. Quercus serrata, Carpinus laxiflora, and C. cordata were the three dominant species and their DBH frequency distribution showed a reverse J-shaped form, so these species seem to maintain by themselves. Dominancediversity curve had a lognormal distribution. d and H'for pooled quadrats were 0.13 and 1.09, respectively, but these indices within each quadiat varied with the range of 0.13 to 0.57 and 0.5 to 1.09, respectively. The value of ${\beta}_t$ along the belt transect ranged from 0.14 to 0.42. These results suggest that this forest community is in the stable climax stage but the components experience a heterogeneous microsuccession.

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Performance Analysis of a Baseband Noncoherent Code Tracking Loop for DS-CDMA Systems (CDMA 시스템용 기저 대역 비동기식 동기 추적 회로의 성능 분석)

  • 이경준;박형래;채수환
    • The Journal of Korean Institute of Electromagnetic Engineering and Science
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    • v.8 no.6
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    • pp.645-655
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    • 1997
  • In this paper, the performance of the noncoherent code tracking loop designed at baseband for CDMA applications is analyzed in detail and is confirmed by computer simulations. Analytical closed-form formula for jitter variance is derived for AWGN channel environments as a function of pulse shaping filter, timing offset, signal-to-interference ratio, and loop filter coefficients. The design issue of the loop filter is also addressed with emphasis on the second-order tracking loop. Finally, the performance of the designed tracking loop is examined by computer simulations for both AWGN and Rayleigh fading channels, when applied to the reverse link of the coherent CDMA system for IMT-2000 designed by ETRI.

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Long-Term Stability for Co-Electrolysis of CO2/Steam Assisted by Catalyst-Infiltrated Solid Oxide Cells

  • Jeong, Hyeon-Ye;Yoon, Kyung Joong;Lee, Jong-Ho;Chung, Yong-Chae;Hong, Jongsup
    • Journal of the Korean Ceramic Society
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    • v.55 no.1
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    • pp.50-54
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    • 2018
  • This study investigated the long-term durability of catalyst(Pd or Fe)-infiltrated solid oxide cells for $CO_2$/steam co-electrolysis. Fuel-electrode supported solid oxide cells with dimensions of $5{\times}5cm^2$ were fabricated, and palladium or iron was subsequently introduced via wet infiltration (as a form of PdO or FeO solution). The metallic catalysts were employed in the fuel-electrode to promote $CO_2$ reduction via reverse water gas shift reactions. The metal-precursor particles were well-dispersed on the fuel-electrode substrate, which formed a bimetallic alloy with Ni embedded on the substrate during high-temperature reduction processes. These planar cells were tested using a mixture of $H_2O$ and $CO_2$ to measure the electrochemical and gas-production stabilities during 350 h of co-electrolysis operations. The results confirmed that compared to the Fe-infiltrated cell, the Pd-infiltrated cell had higher stabilities for both electrochemical reactions and gas-production given its resistance to carbon deposition.

Automatic Local Update of Triangular Mesh Models Based on Measurement Point Clouds (측정된 점데이터 기반 삼각형망 곡면 메쉬 모델의 국부적 자동 수정)

  • Woo, Hyuck-Je;Lee, Jong-Dae;Lee, Kwan-H.
    • Korean Journal of Computational Design and Engineering
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    • v.11 no.5
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    • pp.335-343
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    • 2006
  • Design changes for an original surface model are frequently required in a manufacturing area: for example, when the physical parts are modified or when the parts are partially manufactured from analogous shapes. In this case, an efficient 3D model updating method by locally adding scan data for the modified area is highly desirable. For this purpose, this paper presents a new procedure to update an initial model that is composed of combinatorial triangular facets based on a set of locally added point data. The initial surface model is first created from the initial point set by Tight Cocone, which is a water-tight surface reconstructor; and then the point cloud data for the updates is locally added onto the initial model maintaining the same coordinate system. In order to update the initial model, the special region on the initial surface that needs to be updated is recognized through the detection of the overlapping area between the initial model and the boundary of the newly added point cloud. After that, the initial surface model is eventually updated to the final output by replacing the recognized region with the newly added point cloud. The proposed method has been implemented and tested with several examples. This algorithm will be practically useful to modify the surface model with physical part changes and free-form surface design.

Distinctive pH Dependence and Substrate Specificity of Peptide Hydrolysis by Human Stromelysin-1 (Stromelysin-1에 의한 펩타이드 가수분해에서 pH와 기질특이성 연구)

  • ;Marianne V. Sorensen
    • Journal of Life Science
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    • v.10 no.2
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    • pp.210-217
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    • 2000
  • A kinetic profile of the catalytic domain of stromelysin-1 (SCD) using the fluorescent peptide substrate has been determined by the stopped-flow technique. The pH profile has a pH optimum of about 5.5 with an extended shoulder above pH 7. Three pKa values, 5.0, 5.7, and 9.8 are found for the free enzyme state and two pH independent Kcat/Km values of 4.1$\times$104 M-1 s-1 and 1.4$\times$104 M-1 s-1 at low and high pH, respectively. The profile is quite different in shape with other MMP family which has been reported, having broad pH optimum with two pKa values. The substrate specificity of SCD towards fluorescent heptapeptide substrates has been also examined by thin layer chromatography. The cleavage sites of the substrates have been identified using reverse-phase HPLC method.SCD cleaves Dns-PLA↓L↓WAR and Dns-PLA↓L↓FAR at two positions. However, the Dns-PLA↓LRAR, Dns-PLE↓LFAR, adn Dns-PLSar↓LFAR are cleaved exclusively at one bond. The double cleavages of Dns-PLALWAR and Dns-PLALFAR by SCD are in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis agrees with the structural data that the S1' pocket of SCD is deeper than that of matriysin. The differences observed between SCD and matrilysin may form the basis of understanding the structural relationships and substrate specificities of the MMP family in vivo.

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Evidence for Volatile Memory in Plants: Boosting Defence Priming through the Recurrent Application of Plant Volatiles

  • Song, Geun Cheol;Ryu, Choong-Min
    • Molecules and Cells
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    • v.41 no.8
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    • pp.724-732
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    • 2018
  • Plant defence responses to various biotic stresses via systemic acquired resistance (SAR) are induced by avirulent pathogens and chemical compounds, including certain plant hormones in volatile form, such as methyl salicylate and methyl jasmonate. SAR refers to the observation that, when a local part of a plant is exposed to elicitors, the entire plant exhibits a resistance response. In the natural environment, plants are continuously exposed to avirulent pathogens that induce SAR and volatile emissions affecting neighbouring plants as well as the plant itself. However, the underlying mechanism has not been intensively studied. In this study, we evaluated whether plants "memorise" the previous activation of plant immunity when exposed repeatedly to plant defensive volatiles such as methyl salicylate and methyl jasmonate. We hypothesised that stronger SAR responses would occur in plants treated with repeated applications of the volatile plant defence compound MeSA than in those exposed to a single or no treatment. Nicotiana benthamiana seedlings subjected to repeated applications of MeSA exhibited greater protection against Pseudomonas syringae pv. tabaci and Pectobacterium carotovorum subsp. carotovorum than the control. The increase in SAR capacity in response to repeated MeSA treatment was confirmed by analysing the defence priming of the expression of N. benthamiana Pathogenesis-Related 1a (NbPR1a) and NbPR2 by quantitative reverse-transcription PCR compared with the control. We propose the concept of plant memory of plant defence volatiles and suggest that SAR is strengthened by the repeated perception of volatile compounds in plants.