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Expression of the lux Genes in Escherichia coli for the Basis of Development of Biosensor (바이오센서 개발을 위한 발광 유전자의 대장균에서의 발현 시스템 분석)

  • Cho, Mi-Mi;Kim, Young-Doo;Kang, Kyung-Sook;Kim, Sook-Kyung;Yang, In-Chul;Park, Sang-Ryoul;Lee, Chan-Yong
    • Korean Journal of Microbiology
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    • v.45 no.4
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    • pp.419-424
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    • 2009
  • To provide the basis of biosensor based on the lux genes from bioluminescent bacteria of Photobacterium leiognathi and Vibrio harveyi, we test the expression of lux genes in several strains of Escherichia coli. The expression of the recombinant plasmid of PlXba.pT7-3, containing all lux genes requiring for light emission without adding substrate, in E. coli 43R was so strong to see the blue-green light in single colony as well as in the alginate immobilized cell. In addition, the light intensity was decreased by adding heavy metal ion such as cadmium and zinc ions. These result raise the possibility that a biosensor can be developed using the lux genes system.

Auranofin Enhances Sulforaphane-Mediated Apoptosis in Hepatocellular Carcinoma Hep3B Cells through Inactivation of the PI3K/Akt Signaling Pathway

  • Hwangbo, Hyun;Kim, So Young;Lee, Hyesook;Park, Shin-Hyung;Hong, Su Hyun;Park, Cheol;Kim, Gi-Young;Leem, Sun-Hee;Hyun, Jin Won;Cheong, Jaehun;Choi, Yung Hyun
    • Biomolecules & Therapeutics
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    • v.28 no.5
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    • pp.443-455
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    • 2020
  • The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.

Purification and Characterization of Polyphenol Oxidase from Flammulina velutipes (팽나무버섯 polyphenol oxidase의 정제 및 특성)

  • Pyo, Han-Jong;Son, Dae-Yeul;Lee, Chan
    • Korean Journal of Food Science and Technology
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    • v.34 no.4
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    • pp.552-558
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    • 2002
  • Polyphenol oxidase from Flammulina velutipes was purified and characterized. Purification of polyphenol oxidase was achieved by ammonium sulfate precipitation, Superdex G-200 gel filtration chromatography, Phenyl superose affinity chromatography, Mono-Q anion exchange chromatography and Superdex S-200 gel filtration chromatography on FPLC. After these purification steps specific activity of purified polyphenol oxidase increased to 199.1 units/mg. Polyphenol oxidase from F. velutipes was composed of a single polypeptide with molecular weight of about 40 kDa. Optimum pH and temperature for the enzyme reaction were found to be 6.0 and $25^{\circ}C$, respectively. The activity of the enzyme gradually decreased at acidic pH between 3 and 5, and the enzyme lost its activity at alkaline pH between 8 and 10. This enzyme exhibited high substrate specificity to o-diphenols. Km-values for L-DOPA and caffeic acid were found to be 3.97 mM and 1.78 mM, respectively. 2-mercaptoethanol, L-ascorbic acid, sodium bisulfite, EDTA and $Mg^{2+}$ inhibited the activity of pholyphenol oxidase and $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$ and $Ni^{2+}$ increased enzyme activity. The activity of enzyme was well maintained at $-70^{\circ}C$ for over 4 months, and at $-20^{\circ}C$ for 1 months.

Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo

  • Zhang, Jian;Kang, Min-Jung;Kim, Myung-Jin;Kim, Mi-Eun;Song, Ji-Hyun;Lee, Young-Min;Kim, Jung-In
    • Nutrition Research and Practice
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    • v.2 no.4
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    • pp.200-203
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    • 2008
  • Obesity has become a worldwide health problem. Orlistat, an inhibitor of pancreatic lipase, is currently approved as an anti-obesity drug. However, gastrointestinal side effects caused by Orlistat may limit its use. In this study the inhibitory activities of dandelion (Taraxacum officinale) against pancreatic lipase in vitro and in vivo were measured to determine its possible use as a natural anti-obesity agent. The inhibitory activities of the 95% ethanol extract of T. officinale and Orlistat were measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate at concentrations of 250, 125, 100, 25, 12.5 and $4\;{\mu}g/ml$. To determine pancreatic lipase inhibitory activity in vivo, mice (n=16) were orally administered with com oil emulsion (5 ml/kg) alone or with the 95% ethanol extract of T. officinale (400 mg/kg) following an overnight fast. Plasma triglyceride levels were measured at 0, 90, 180, and 240 min after treatment and incremental areas under the response curves (AUC) were calculated. The 95% ethanol extract of T. officinale and Orlistat, inhibited, porcine pancreatic lipase activity by 86.3% and 95.7% at a concentration of $250\;{\mu}g/ml$, respectively. T. officinale extract showed dose-dependent inhibition with the $IC_{50}$ of $78.2\;{\mu}g/ml$. A single oral dose of the extract significantly inhibited increases in plasma triglyceride levels at 90 and 180 min and reduced AVC of plasma triglyceride response curve (p<0.05). The results indicate that T. officinale exhibits inhibitory activities against pancreatic lipase in vitro and in vivo. Further studies to elucidate anti-obesity effects of chronic consumption of T. officinale and to identify the active components responsible for inhibitory activity against pancreatic lipase are necessary.

The basic experiments for the fabrication of the SPUDT type Inter using the SFIT type filter (SFIT형태를 이용한 SPUDT형 필터제작에 관한 기초실험)

  • You, Il-Hyun
    • Journal of the Korea Institute of Information and Communication Engineering
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    • v.11 no.10
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    • pp.1916-1923
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    • 2007
  • We have studied to obtain the SAW filter for the passband was formed on the Langasite substrate and was evaporated by Aluminum-Copper alloy and thin we performed computer-simulated by simulator. We cm fabricate that the block weighted type IDT as an input transducer of the filter and the withdrawal weighted type IDT as an output transducer of the filter from the results of our computer-simulation. Also, we have performed to obtain the properly design conditions about phase shift of the SAW filter for WCDMA. We have employed that the number of pairs of the input and output IDT are 50 pairs and the thickness and the width of reflector are $5000\;{\AA}$ and $3.6{\mu}m$ respectively. And we have employed that the distances from the hot electrode to the reflector are $2.0{\mu}m$, $2.4{\mu}m$ and the distance from the hot electrode to the ground is $1.5{\mu}m$ respectively. Frequency response of the fabricated SAW filter has the property that the center frequency is about 190MHz and bandwidth at the 3dB is probably 7,8MHz. And we could obtain that return loss is less then -18dB, ripple characteristics is probably 3dB and triple transit echo is less then -25dB after when we have matched impedance.

Reduced Graphene Oxide / Polyaniline Composite Material for Supercapacitor Electrode (환원된 그래핀 옥사이드/폴리아닐린 복합재료 기반의 슈퍼커패시터용 전극 제조)

  • Jeong, Hyeon Taek;Kim, Se Hyun;Ahn, Won Jun;Choi, Jae Yong;Park, Hyeon Young;Kim, Chang Hyun;Kim, Yong Ryeol
    • Journal of the Korean Applied Science and Technology
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    • v.35 no.4
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    • pp.1088-1095
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    • 2018
  • In this study, reduced graphene oxide/polyaniline composite was fabricated tomaximize their advantages with electrochemical performances and use as a electrodematerial for supercapcaitor. Polyaniline as an electrode material was synthesized bychemical polymerization of aniline monomer and reduced graphene oxide wasintroduced to prepare composite with polyaniline without any pre-treatment. Thereduced graphene oxide, polyaniline and their composite electrodes were fabricatedon gold coated PET(polyethylene terephthalate) substrate through spray coatingmethod which can also apply to industrial scale. we have also prepared reducedgraphene oxide and polyaniline single material electrode to compare theirelectrochemical properties with reduced graphene oxide/polyaniline composite electrode. We have analyzed and compared electrochemical properties of eachelectrodes by using cyclic voltammetry(CV), galvanostaticcharge-discharge(GCD) and electrochemical impedancespectroscopy(EIS) at same condition. As a result, reduced graphene oxide /polyaniline composite electrode showed higher capacitance value more thanpolyaniline and reduced graphene oxide electrode, respectively. Internal resistanceof reduce graphene oxide/polyaniline composite electrode was 24% and 58% lessthan polaniline and reduced graphene oxide electrode respectively. These resultsconsidered that reduced graphene oxide/polyaniline composite electrode has potential ability and enable to apply flexible energy storage and wearable devices.

Exocyclic GpC DNA methyltransferase from Celeribacter marinus IMCC12053 (Celeribacter marinus IMCC12053의 외향고리 GpC DNA 메틸트랜스퍼라아제)

  • Kim, Junghee;Oh, Hyun-Myung
    • Korean Journal of Microbiology
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    • v.55 no.2
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    • pp.103-111
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    • 2019
  • DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, carries out $N^6$-adenine or $N^4$-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate. Celeribacter marinus IMCC12053 from the Alphaproteobacterial group was isolated from a marine environment. Single molecule real-time sequencing method (SMRT) was used to detect the methylation patterns of C. marinus IMCC12053. Gibbs motif sampler program was used to observe the conversion of adenosine of 5'-GANTC-3' to $N^6$-methyladenosine and conversion of $N^4$-cytosine of 5'-GpC-3' to $N^4$-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and $N^4$-cytosine methyltransferase was cloned. IPTG inducer was used to confirm the methylation activity of DNA methylase, and cloned into a pQE30 vector using dam-/dcm- E. coli as the expression host. The genomic DNA and the plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes that were sensitive to methylation, to confirm the methylation activity. These methylases protected the restriction enzyme site once IPTG-induced methylases methylated the chromosome and plasmid, harboring the DNA methylase. In this study, cloned exocyclic DNA methylases were investigated for potential use as a novel type of GpC methylase for molecular biology and epigenetics.

Effect of Short Circuit Current Enhancement in Solar Cell by Quantum Well Structure and Quantitative Analysis of Elements Using Secondary Ion Mass Spectrometry (양자우물구조에 의한 태양전지 단락전류 증가 효과와 이차이온 질량분석법에 의한 원소 정량 분석)

  • Kim, Junghwan
    • Applied Chemistry for Engineering
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    • v.30 no.4
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    • pp.499-503
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    • 2019
  • Characteristics of solar cells employing a lattice matched GaInP/GaAs quantum well (QW) structure in a single N-AlGaInP/p-InGaP heterojunction (HJ) were investigated and compared to those of solar cells without QW structure. The epitaxial layers were grown on a p-GaAs substrate with $6^{\circ}$ off the (100) plane toward the <111>A. The heterojunction of solar cell consisted of a 400 nm N-AlGaInP, a 590 nm p-GaInP and 14 periods of a 10 nm GaInP/5 nm GaAs for QW structure and a 800 nm p-GaInP for the HJ structure (control cell). The solar cells were characterized after the anti-reflection coating. The short-circuit current density for $1{\times}1mm^2$ area was $9.61mA/cm^2$ for the solar cell with QW structure while $7.06mA/cm^2$ for HJ control cells. Secondary ion mass spectrometry and external quantum efficiency results suggested that the significant enhancement of $J_{sc}$ and EQE was caused by the suppression of recombination by QW structure.

Development of Voltammetric Nanobio-incorporated Analytical Method for Protein Biomarker Specific to Early Diagnosis of Lung Cancer (폐암 조기 진단을 위한 단백질 바이오마커 측정용 전압-전류법 기반의 나노바이오 분석법 개발)

  • Li, Jingjing;Si, Yunpei;Nde, Dieudonne Tanue;Lee, Hye Jin
    • Applied Chemistry for Engineering
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    • v.32 no.4
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    • pp.461-466
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    • 2021
  • In this article, a portable and cost-effective voltammetric biosensor with nanoparticles was developed for the measurements of heterogeneous nuclear ribonucleoprotein A1 protein (hnRNP A1) biomarker which can potentially be used for lung cancer diagnosis. Gold nanoparticles were first electrodeposited onto screen printed carbon electrode (SPCE) followed by immobilizing a single stranded DNA aptamer specific to hnRNP A1 onto the electrode surface. Ethanolamine was also used when immobilizing DNA aptamer on the surface to prevent signals from non-specific adsorption events. Sequential injection of hnRNP A1 biomarker and anti-hnRNP A1 conjugated with alkaline phosphatase (ALP) onto the aptamer chip surface allows to form the sandwich complex of DNA aptamer/hnRNP A1/ALP-anti-hnRNP A1 on the electrode surface which further reacted with 4-aminophenyl phosphate (APP). The electrocatalytic reaction of the enzyme, ALP, and the substrate, APP, resulting in the oxidative current response changes at -0.05 and -0.17 V (vs. Ag/AgCl) against the hnRNP A1 concentration was measured using cyclic and differential pulse voltammetry, respectively. The Au nanoparticles-integrated voltammetric biosensor was applied to analyze human normal serum solutions possibly suggesting potential applicability for lung cancer diagnosis.

A Study on Inhibition of Bacterial Membrane Formation in Biofilm formed by Acne Bacteria in Valine through Property Analysis (물성 분석을 통한 Valine 의 여드름균 바이오필름 내부 세균막 형성 억제 연구)

  • Song, Sang-Hun;Hwang, Byung Woo;Son, Seongkil;Kang, Nae-Gyu
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.47 no.2
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    • pp.163-170
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    • 2021
  • This study was conducted to create a technology to remove acne bacteria with human-friendly materials. First, the Cutibacterium acnes (C. acnes) were adsorbed to the mica disc to grow, and then the biofilm was checked through an atomic microscope to see if the biofilm had grown. Based on the topographic image, the shape changed round, the size was 17% longer on average, and the phase value of the resonance frequency separating materials was observed as a single value, the biofilm grown by covering the extracellular polymeric substrate (EPS). As a result of processing 50 mM of amino acids in the matured biofilm, the concentration of C. acnes decreased when valine, serine, arginine and leucine were treated. Scanning with nanoindentation and AFM contact modes confirmed that the hardness of biofilms treated with Valine (Val) increased. This indicates that an AFM tip measured cell which may have more solidity than that of EPS. The experiment of fluorescent tagged to EPS displays an existence of EPS at the condition of 10 mM Val, but an inhibition of growth of EPS at the 50 mM Val. Number of C. acnes was also reduced above 10 mM of Val. Weak adhesion of biofilm generated from an inhibition of EPS formation seems to induce decrease of C. acnes. Accordingly, we elucidated that Val has an efficiency which eliminates C. acnes by approach of an inhibition of EPS.