Screening of Novel Antibiotics Inhibiting Bacterial Peptide Deformylase(PDF) : 세균의 Peptide Deformylase(PDF)를 저해하는 새로운 항균물질의 스크리닝

김현주 Handong Global Uniersity 2005 국내석사

Peptide deformylase (PDF)는 세균의 단백질 생합정 과정에 있어 필수적인 효소인 반면 포유동물의 세포에서는 요구되어지지 않기에 새로운 항생 물질의 개발을 위한 좋은 target으로 알려져 있다. 세균에서의 단백질 생합성은 formylated methionine 로 시작되며 peptide가 합성되는 동안 formyl group은 peptide deformylase (PDF)에 의해 제거되어지고 mature단백질이 생성된다. 본 연구에서는 pET-14b 백터에 클로닝한 Staphylococcus aureus 6538p의 pof 유전자를 Escherichia coli BL2l(DE3) 에 형절전환시켰다. PDF 단백질은 Isopropy1-β-D-thiogalactopyranoside (IPTG)의 유도에 의해 대량 발현되어졌고 N-말단에 histine 이 연속적으로 6개가 붙어있는 fusion 단백질인 PDf 는 nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography를 이용하여 순수 분리되어졌다. 순수 분리 정제된 PDf 효소의 활성을 확인하기 위해, PDF/FDH coupled assay 방법을 응용하였다. PDf의 기질로 사용되는 formy-Methionine-Alanine-Serine (fMAS)로부터 peptide deformylase (PDF)에 의해 생성된 formate 가 다시 formate dehydrogenase (FDH)에 의해 산화 되어지는데, 이때 부산물로 생성되는 NADH의 양을 340 nm에서 측정함으로써 PDF의 활성정도를 간접적으로 측정되었다. 75,000 개의 화합물 은행과 토양 미생물 배양액이 본 연구에서 확립된 PDF 저해제 검색계를 이용하여 평가되어졌다. 그 결과 약 150개의 화합물 (0.2 %)이 25 μM에서 50 % 이상의 저해 효과를 보였으며,그 중 12개는 Staphylococcus aureus에 대해 항균 효과도 보였다. 이들 증 4개는 컴퓨터 모델링을 통해 PDF와 결합한 구조를 추측해 보았다. Peptide deformylase (PDF) is essential and unique to bacteria for cytoplasm protein synthesis, but not required in mammalian cells, thus making it an attractive target for the discovery of novel antibacterial drugs. Protein synthesis in eubacteria begins with a formulated methionine, and the formal group of the nascent polapeptide is removed by peptide deformalase (PDF) to produce a mature protein. In this study, a def gene from Staphylococcus aureus 6538p was cloned in pET-14b vector and transformed into Escherichia coli BL21(DE3). PDF protein was overexposed by addition of Isopropal-β-D-thiogalactoparanoside (IPTG). NH_(2)-terminal His-tagged PDF protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography. Enzymatic activity of purified 6xHis-tagged PDF was tested on the substrate, formal-Methionine-Alanine-Serine (fMAS), by formats dshadrogsnass-couplsd spectrometric assay. For the discovery of new PDF inhibitors from chemical libraries and culture booths of soil bacteria, a target-oriented screening system ' a 96-well plate was developed. About 75,000 commercial chemical libraries were tested in this screening system. About 150 chemicals (0.2 %) inhibited more than 50 % PDF enzyme activity at 25 μM, and twelve chemicals among them have antibacterial activity also against Staphylococcus aureus. The structure of PDF-inhibitor complex was proposed by computer modeling.

Screening of Novel Antibiotics Inhibiting Bacterial Peptide Deformylase(PDF)

Chan, U Zar Li Handong Global Uniersity 2004 국내석사

Design of liquid storage tanks needs special considerations on the complex interaction between the structure and the liquid during earthquakes. Earthquake may cause severe sloshing in a liquid storage tank resulting from the interaction. This sloshing gives dynamic loading effects on the tank structure. The loading effects are classified as general effects and local effects. General effects include the increase in hydraulic dynamic pressure on the wall, which have been well considered in the current design. However, local slashing effects, as impact loads acting on localized parts of structures, have not yet considered in the current design of liquid storage tanks. This research deals with the localized effects of sloshing impact loads acting on tank structures in liquid storage tanks during earthquakes. In order to achieve the goal of this research, at first, the phenomenon of sloshing has been investigated. Then as a preliminary study, theoretical concepts on nonlinear dynamic analysis have also been investigated. As the next step, nonlinear dynamic structural analyses have been carried out to figure out the effects of the localized impact loads due to sloshing and to examine the safety level of the tank structure. Based on the analytical results, the current design has been evaluated. The results show that the localized sloshing load intensity corresponding to the earthquake level specified in Korea Design Code, is almost negligible and the effect of the localized impact pressure is in the safe side.

Screening of Chemopreventive Activities of Traditional Medicinal Plants Using Quinone Reductase Assay and the Comet assay : Quinone Reductase 활성분석 및 Comet assay 를 통한 약용식물의 암예방 활성 탐색

김해홍 Handong Global Uniersity 2003 국내석사

81가지 중국 전통 약재의 암예방효과를 quinone reductase assay 와 comet assay 통해서 탐색했다. Phase II enzymes의 유도는 암예방의 중요한 기작이다. 중국 전통 약재의 80% methanol 추출물의 quinone reductase 활성에 대한 유도 정도를 murine hepatoma (Hepa 1c1c7)로 측정했다. 81 가지 약재중에 독활, 후박, 상기생의 활성 유도 정도가 250% 이상으로 발견되었다. 독활, 후박, 상기생을 투여해서 얻은 최고 유도 정도는 401.9%, 270.5% 와 301.8% 였다. 이러한 QR 유도 활성은 QR inducers, t-butylhydroquinone ( 유도활성 170.1%) 와 β-naphthoflavone ( 유도활성320.0%) 보다 강력했다. 또 독활의 항유전독성 효과는 comet assay를 이용하여 mouse embryo fibroblase 3T3 cells로 관찰되었다. 그런데 독활은 H2O2인한 세포 DNA 손상에 아무 효과를 나타냈지 않았다. 그래서 독활의 암예방 효과는 antioxidative 기작 때문이 아니라 detoxification의 작용 때문인 것이라고 생각 된다. Chemopreventive activities of 81 traditional medicinal plants were screened using quinone reductase assay and Comet assay. Induction of phase II enzymes is a major mechanism of chemoprevention. The induction levels of quinone reductase (QR) activity in cultured murine hepatoma (Hepa 1c1c7) cells by 80%-methanol extracts of traditional medicinal plants were measured. Among the tested 81 plants, the extracts of Aralia continentalis, Magnolia obovata, and Viscum album were found to induce QR activities more than 250%. The maximum induction levels obtained were 401.9%, 270.5%, and 301.8% by treatments of the extracts of A. continentalis (318㎍/ml), M. obovata (53.8㎍/ml) and V. album (80.6㎍/ml), respectively. These QR induction activities were more potent than those of the known QR inducers, t-butylhydroquinone (170.1%) and β-naphthoflavone (320.0%). In addition, the antigenotoxic effect of A. continentalis in mouse embryo fibroblase 3T3 cells was investigated by the single cell gel electrophoresis assay (Comet assay). A. continentalis did not show any protection effect from DNA damage in cells caused by H2O2. It was concluded from the results that the chemopreventive activity of A. continentalis was due to an activation of detoxification mechanism, not to any antimutagenic or antioxidative mechanisms.

Cytotoxicity of recombinant immunotoxin composed of anti-carcinoembryonic antigen(CEA) single chain Fv(scFv) and recombinant lectin A chain of Korean mistletoe(Viscum album coloractum) in colorectal tumor : 항-배아암종항원 단일쇄Fv와 한국산 겨우살이의 렉틴 A쇄로 이루어진

조준석 Handong Global Uniersity 2005 국내석사

The mistletoe lectin has so strong anti-cancer effect and immune control ability that it has been used as an injection widely in trial of cancer drug supplement since 1920s (19, 20). The Korean mistletoe (viscum album coloratum), which is different kind of the European mistletoe, has stronger effect of anti-tumor and increase immunomodulating reaction than the European mistletoe (21-23). Though the several good abilities of lectin from Korean mistletoe, there hasn’t been reported any research for the development of immunotoxin. We made successfully recombinant immunotoxin, which has recombinant lectin A chain as toxin part. Many recombinant immunotoxins have been produced in E.coli as inclusion bodies. Usually the purification of inclusion bodies are performed by being solubilized in Gu-HCl, reduced with dithioerythritol and refolded by dilution in a refolding buffer containing arginine to prevent aggregation, and oxidized and reduced with glutathione to facilitate redox shuffling. And then the active monomeric protein is purified from the refolding solution by ion exchange and size exclusion chromatography (24, 25). However, the refolding process does not always produce completely native protein (26) and several attempts to refold immunotoxins under various conditions yielded very low amounts with poor specific activity (27). Sometimes the refolded proteins are aggregated (28, 29). It is advantageous to produce proteins soluble in E.coli for subsequent manipulation (30). We used the AKTATM prime with HiTrapTM FPLC refolding program system, which is already set for inclusion bodies purification especially for enzymes and succeed in development of soluble immunotoxin without complex and long ways of refolding steps. Although we reduced complex methods and taking time to generate recombinant immunotixns, there were still some problems to be improved for the best and efficient yield. First, the culture conditions of expression in E.coli should have adjusted for immunotoxin to be expressed as soluble protein. The bacterial expressed proteins went soluble from or inclusion body depending on kinds of host, degree, bacterial concentration of primary induction and concentration of IPTG and so on. If we get the best condition of expression for soluble form of immunotoxin, it should be easier for purification and refolding and higher for the yield rate. Second, we should have figured out exact concentration value of purified immunotoxins. We certainly checked the binding ability before and after purification and the cytotoxicity for specific antigen. The concentration of immunotoxin caused the decrease and increase of cytotoxicity depended on dilution rate. The purified immunotoxin was concentrated by Centrifugal Concentration System (Vision Scientific) but it was out of range in BCA assay. It might be related to the rate of yield and rate of loss while performing each step of purification and refolding. Third, the low yield rate should have improved. The expression of new immunotoxins was surely confirmed by SDS-PAGE in comparing with negative control. Usually bacterial over-expression is seen at a glance because expression band was so thicker than the others. In our research, new band was appeared at exact molecular size of immunutoxin we expected but it wasn’t pretty thick like usual over-expression band of other proteins. It doesn’t mean very high yield rate of immunotoxins. With the improvement of problems we mentioned above, the further studies were expected such as in vitro assay for development of more efficient and powerful immunotoxin without side effect to be used in clinical trial. To prove the various possibility of using recombinant lectin A chain from Korean mistletoe, the strategy to connect it with another antibody about another tumors is under plan.

Ultra-Wideband Ranging in Non-LoS channel : Non-LoS 채널환경에 기반을 둔 초광대역 신호 거리추정 연구

조영훈 Handong Global Uniersity 2006 국내석사

UWB 위치추적시스템은 UWB신호만의 높은 시간 분해능으로 인해, 고정밀 위치추적이 가능하다. 그러나 실내 장애물로 의한 도착 신호의 지연은 신호의 이동시간으로 위채인식에 적지 않은 오차를 줄 수가 있다. 본 논문은 Non-LoS 채널조건에서의 신호 지연현상이 위치인식의 핵심인 거리추정에 주는 영향과 확률적 모델에 근거한 거리추정오차를 예측성능을 주제로 다룬다. 첫째로 Non-LoS조건에서의 신호의 지연현상이 위치인식에 주는 영향을 보기 위해서, 두 가지 위치인식 기법인 TDoA와 ToA를 사용하여 실내건물에서의 위치인식 성능을 비교하여 보았다. 그 결과 거리정보를 사용한 ToA가 상대적인 지연시간정보를 사용하는 TDoA보다는 성능이 나은 방식임을 알게 되었으며, 이로서 신호지연이 위치인식 성능에 저하요인이 됨을 알 수 있었다. 다음은 거리오차를 추정하는 기법과 추적하는 기법을 소개한다. [6]에서 소개된 방식은 수신신호의 세기와 지연시간 2 개의 정보를 토대로, Non-LoS 조건의 거리오차를 추정하는 기법을 제안하고 있다. 본 논문에서는 지연시간 정보 1개로만 오차를 추정하는 방식을 시도하여 보았다. 이를 위해서, 신호 지연시간의 확률적 성향 특성에 관한 데이터가 필요하였고, 이런 데이터를 확보하기 위해서, 실내 도면에서 가장 빨리 도착한 신호의 이동경로를 찾아내는 Ray-tracing 시뮬레이터를 제작하였다. 시뮬레이터를 사용하여, 5개의 실내도면의 지연시간에 대한 최단 경로와 지연시간정보를 저장하였고, 히스토그램화 하였다. 오차추정에 사용하기 위해서 수학식으로 변환할 필요가 있었으며, Log-normal pdf를 사용하여, 히스토그램과 근접한 수식을 만들었다. 이 log-normal 수식을 사용하여, 2가지 오차를 보상하는 방식을 소개하며, 첫째는 MAP, ML, MMSE을 통해 오차를 추정하는 방식과 두 번째는 Kalman flter를 사용하여 오차를 추적해가는 방식을 제안한다. 구한 Log-normal 수식을 사응하여, 구할 수 있늘 최소 오차추정 평균은 0.23ns^(2)이었으며, MAP, ML과 MMSE은 각각 63, 45, 44 ns^(2)이었다. Kalman filter는 Monte-carlo simulation을 사용하였으며, estimation error는 2.3 ns^(2)이 나왔다. Kalman filter의 측정 오차를 Log-normal pdf의 Conditional mean 을 사용하였으며, measurement variance는 conditional variance를 사용하였다. Kalman filter,를 사용한 에러오차 추적 방식은 측정한 거리를 conditional mean 으로 사용하였는데, 측정한 거리가 커질 수록 커지는 그래프를 보여주었다. 그러나 LoS의 경우에는 이 conditional mean와 coditional variance를 0으로 정하도록 예의를 주는 알고리즘이 필요하다. 본 논문에서는 LoS와 Non-LoS 채널 신호를 구분 할 수 있는 UWB 거리추정 송수신기를 제안한다. Threshold-controller와 shift-register를 두어서, 첫 수신 펄스신호의 shift-register 샘플 bit 위치와 최대 크기로 수신된 펄스신호의 위치를 비교한다. Threshold값을 올리면서, 첫 수신 펄스신호의 값을 LoS조건에서의 첫 수신 펄스신호의 크기를 Look-up table과 비교한다. 첫 신호의 크기가 -10dB 이상 차이나면, Non-LoS 선호로 간주한다. Shift-register는 또한 Non-LoS의 경우, 거리오차를 예측하기 위해서 첫 신호와 최대 크기로 수신된 펄스의 위치의 차를 사용해서 위치오차를 예측하는 방식으로도 사용될 수 있다. 그 밖에도, 본 논문에서 소개하는 송수신기는 doppler 효과나 고속으로 신호동기를 유지하는데 강한 잇점이 있다. 또한 저전력이면서 설계가 용이한, 디지털 로직 펄스 생성기와 고속 통신에서 실시간 동기를 위한 push-pull 메카니즘을 소개한다. UWB location awareness system provides an inch-accuracy positioning performance due to UWB's high time resolution. However, an excess delay of a signal due to nonplus condition causes a considerable ranging error. In this paper, we firstly discuss about an effect of nonplus condition to a ranging error. Viewing the effect of ranging error due to excess delay requires a performance of positioning method. ToA and TDoA method was under test to view the positioning error for nonplus condition. The result was that ToA using range information gave a better location awareness performance than TDoA applying relative arrival time of different reference signal. Based on this test, it has been proven that a ranging error due to excess delay considerable positioning error. Secondly, the paper deals with estimating ranging error and estimation performance based on the statistical model. We introduce ranging error estimation and tracking method. Reference [6] suggests range error estimation using two parameter; received power and excess delay. We suggest technique of estimating of ranging error using only one parameter; excess delay. This estimation requires a statistical model of the excess delay. To collect massive excess delay data within limited time, we used simulation method ray-tracing simulator with indoor map excess delay simulation. Ray-tracing simulator is a tool which finds the shortest path in nonplus condition. Electromagnetic parameters for three lousy media are collected using experiment data; wall, glass and wood. Five selected indoor maps were collected and reformed with colormap versus lousy media parameter. Each map was under simulation, collecting measured range and its ranging error, The data is then expressed in statistical form; histogram. to use the statistical model in ranging error estimation requires the model transformed into mathematical expression. Approximation in form of Log-iiorural pdf is done. Using this approximated equation, two kinds of ranging error estimation methods are performed; ranging error estimation based on MAP, ML, MMSE and ranging error tracking based on Kalman filter algorithm. CRLB, the lowest estimation error variance that can be achieved by approximated equation, is 0.23ns^(2). Three error estimation variances are 63ns^(2) for MAP, 48n^(2) for ML and 44ns^(2) for MMSE. Estimation performance was not satisfactory. Next we attempt ranging error tracking method was to improve the estimation performance. We used basic kalman filter. It requires two data; prediction and measurement. In our Kalman filter estimation, we employed E[Excess-delay/R] of approximated pdf as ranging error measurement and E[{Excess-delay/R}^(2)] as measurement variance. Both conditional mean and variance have increasing in value with measured range. Error estimation performance of the kalman tracking method was done by Monte-carlo simulation. Average error variance of the Kalman filter ranging error tracking is 2.3 ns^(2). This data is based on with considering LoS and Non-LoS detection. Kalman tracking can be improved if UWB ranging system has exception handling on LoS signal from Non-LoS signal. If the system detects a signal to be LoS signal, then Kalman filter zeros conditional mean and variance of a measured range without range information. Ground rule of the first arrived signal power for LoS condition is fixed with Fries formula. LoS the first arrived signal power with measured range data can be listed as Look-up table or His formula with measured range. The nonplus first arrived signal power is always less than the LoS first arrived signal. Such transceiver is introduced. It is embedded with a threshold-controller to detect signal power and shift-register to store a signal location. Shift-register can also be used to estimate range based on the difference between the 1st arrived signal and maximum power signal location. Moreover, the transceiver employs a special mechanism which is robust to a doppler effect and fast synchronization. Lastly, a transceiver is embedded with a simple digital pulse generator and UWB band modulator low power consumption and push-pull mechanism for fine tuning.