• 한국환경농학회지
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• 제25권4호
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• pp.371-381
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• 2006

New proteomic techniques have been pioneered extensively in recent years, enabling the high-throughput and systematic analyses of cellular proteins in combination with bioinformatic tools. Furthermore, the development of such novel proteomic techniques facilitates the elucidation of the functions of proteins under stress or disease conditions, resulting in the discovery of biomarkers for responses to environmental stimuli. The ultimate objective of proteomics is targeted toward the entire proteome of life, subcellular localization biochemical activities, and the regulation thereof. Comprehensive analysis strategies of proteomics can be classified into three categories: (i) protein separation via 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification via either Edman sequencing or mass spectrometry (MS), and (iii) proteome quantitation. Currently, MS-based proteomics techniques have shifted from qualitative proteome analysis via 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, toward quantitative proteome analysis. In vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes. protein-labeling tagging with isotope-coded affinity tags, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope-labeled amino acids can be in vivo labeled into live culture cells via metabolic incorporation. MS-based proteomics techniques extend to the detection of the phosphopeptide mapping of biologically crucial proteins, which ale associated with post-translational modification. These complementary proteomic techniques contribute to our current understanding of the manner in which life responds to differing environment.

• 동의생리병리학회지
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• 제23권4호
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• pp.888-894
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• 2009

It has been reported that silibinin, a natural polyphenolic flavonoid, induces cell death in various cancer cell types. However, the underlying mechanisms by which silibinin induces apoptosis in human glioma cells are poorly understood. The present study was therefore undertaken to examine the effect of silibinin on glioma cell apoptosis and to determine its underlying mechanism in human glioma cells. Apoptosis was estimated by FACS analysis. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (${\Psi}m$) were measured using fluorescence dyes DCFH-DA and $DiOC_6$(3), respectively. Cytochrome c release from mitochondria and caspase-3 activation were estimated by Western blot analysis using specific antibodies. Exposure of cells to 30 mM silibinin induced apoptosis starting at 6 h, with increasing effects after 12-48h in a time-dependent manner. Silibinin caused ROS generation and disruption of ym, which were associated with the silibinin-induced apoptosis. The silibinin-induced ROS generation and disruption in ym were prevented by inhibitors of mitochondrial electron transport chain. The hydrogen peroxide scavenger catalase blocked ROS generation and apoptosis induced by silibinin. Silibinin induced cytochrome c release into cytosolic fraction and its effect was prevented by catalase and cyclosporine A. Silibinin treatment caused caspase-3 activation, which was inhibited by DVED-CHO and cyclosporine A. Pretreatment of caspase inhibitors also protected against the silibinin-induced apoptosis. These findings indicate that ROS generation plays a critical role in the initiation of the silibinin-induced apoptotic cascade by mediation of the mitochondrial apoptotic pathway including the disruption of ${\Psi}m$, cytochrome c release, and caspase-3 activation.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7409-7414
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• 2015

Loop-mediated isothermal amplification (LAMP) developed by Notomi et al. (2000) has made it possible to amplify DNA with high specificity, efficiency and rapidity under isothermal conditions. The ultimate products of LAMP are stem-loop structures with several inverted repeats of the target sequence and cauliflower-like patterns with multiple loops shaped by annealing between every other inverted repeats of the amplified target in the similar strand. Because the amplification process in LAMP is achieved by using four to six distinct primers, it is expected to amplify the target region with high selectivity. However, evaluation of reaction accuracy or quantitative inspection make it necessary to append other procedures to scrutinize the amplified products. Hitherto, various techniques such as turbidity assessment in the reaction vessel, post-reaction agarose gel electrophoresis, use of intercalating fluorescent dyes, real-time turbidimetry, addition of cationic polymers to the reaction mixture, polyacrylamide gel-based microchambers, lateral flow dipsticks, fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assays and nanoparticle-based colorimetric tests have been utilized for this purpose. In this paper, we reviewed the best-known techniques for evaluation of LAMP amplicons and their applications in molecular biology beside their advantages and deficiencies. Regarding the properties of each technique, the development of innovative prompt, cost-effective and precise molecular detection methods for application in the broad field of cancer research may be feasible.

• 동의생리병리학회지
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• 제23권3호
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• pp.581-589
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• 2009

The water extract of Dohongsamul-tang(DHSMT) has been traditionally used in treatment of ischemic heart and brain diseases in Oriental Medicine. However, little is known about the mechanism by which DHSMT protects C6 glial cells from glucose deprevation induced damages. Therefore, this study was designed to evaluate the protective effects of DHSMT on 2-deoxy-D-glucose induced autophagy of C6 glial cells. Autophagic phenotype is evaluated by fluorescence microscopy and flow cytometry with specific biological staining dyes, including monodansylcadaverine and acridine orange, as well as Western blot analysis with microtubule-associated protein 1 light chain 3(LC3) and Beclin-1. Treatment with 2-deoxy-D-glucose significantly resulted in a decrease of the viability of C6 glial cells and increase of the extracellular LDH release in a dose and time-dependent manner. However, pretreatment with DHSMT protected C6 glial cells from glucose deprivation with 2-deoxy-D-glucose. The author also observed the fact that autophagy phenotype occurred by 2-deoxy-D-glucose in C6 glial cells. Pretreatment with 3-MA, a pharmacological inhibitior of autophagy, abolished the formation of acidic vesicle organelle in C6 glial cells treated with 2-deoxy-D-glucose. However, pretreatment with DHSMT inhibited the formation of autophagic phenotypes, including formation of acidic vesicle organelle, and increase of the expression of LC-3 II Beclin-1 proteins in C6 glial cells treated with 2-deoxy-D-glucose. Taken together, these data suggest that DHSMT is able to protect C6 glial cells from glucose deprivation with marked inhibition of autophagy formation.

• Journal of Plant Biotechnology
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• 제37권2호
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• pp.196-204
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• 2010

With the completion of genome sequencing of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. The recent techniques of proteomics have been advanced quickly so that the high-throughput and systematic analyses of cellular proteins are enabled in combination with bioinformatics tools. Furthermore, the development of proteomic techniques helps to elucidate the functions of proteins under stress or diseased condition, resulting in the discovery of biomarkers responsible for the biological stimuli. Ultimate goal of proteomics orients toward the entire proteome of life, subcellular localization, biochemical activities, and their regulation. Comprehensive analysis strategies of proteomics can be classified as three categories: (i) protein separation by 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification by either Edman sequencing or mass spectrometry (MS), and (iii) quanitation of proteome. Currently MS-based proteomics turns shiftly from qualitative proteome analysis by 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, to quantitative proteome analysis. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. The in vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes, protein-labeling tagging with isotope-coded affinity tag, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope labeled amino acid can be in vivo labeled into live culture cells through metabolic incorporation. MS-based proteomics extends to detect the phosphopeptide mapping of biologically crucial protein known as one of post-translational modification. These complementary proteomic techniques contribute to not only the understanding of basic biological function but also the application to the applied sciences for industry.

• 동의생리병리학회지
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• 제19권6호
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• pp.1585-1593
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• 2005

The genetic effects of restraint stress challenge on HPA axis and the therapeutic effect of Boshimgeonbi-tang on the stress were studied with cDNA microarray analyses, RT-PCR on hypothalamus using an immobilization-stress mice as an animal model. Male CD-1 mice were restrained in a tightly fitted and ventilated vinyl holder for 2hrs once a day, and this challenge was repeated for seven· consecutive days. In the change of body weight it showed that the Boshimgeonbi-tang is effected recovery on weight loss caused by the immobilization-stress. Seven days later, total RNA was extracted from the organs of the mouse, body-labeled with $CyDye^{TM}$ fluorescence dyes and then hybridized to CDNA microarray chip. Scanning and analyzing the array slides were carried out using GenePix4000 series scanner and GenePix $Pro^{TM}$ analyzing program, respectively. The expression profiles of 109 genes out of 6000 genes on the chip were significantly modulated in hypothalamus by the immobilization stress. Energy metabolism-, lipid metabolism-, apoptosis-, stress protein, transcriptional factor, and signal transduction-related genes were transcriptionally activated whereas DNA repair-, protein biosysthesis-, and structure integrity-related genes were down-regulated in hypothalamus. The 58 genes were up-regulated by the mRNA expression folds of 1.5 to 7.9. and the 51 genes were down-regulated by 1.5 - 5.5 fold. The 11 genes among them were selected to confirm the expression profiles by RT-PCR. The mRNA expression levels of Tnfrsf1a (apoptosis), Calm2 (cell cycle), Bag3 (apoptosis), Ogg1 (DNA repair), Aatk (apoptosis), Dffa (apoptosis), Fkbp5 (protein folding) were restored to the normal one by the treatment of Boshimgeonbi-tang.

• Bulletin of the Korean Chemical Society
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• 제28권5호
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• pp.783-790
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• 2007

The immobilization of proteins and their molecular interactions on various polymer-modified glass substrates [i.e. 3-aminopropyltriethoxysilane (APTS), 3-glycidoxypropyltrimethoxysilane (GPTS), poly (ethylene glycol) diacrylate (PEG-DA), chitosan (CHI), glutaraldehyde (GA), 3-(trichlorosilyl)propyl methacrylate (TPM), 3'-mercaptopropyltrimethoxysilane (MPTMS), glycidyl methacrylate (GMA) and poly-l-lysine (PL).] for potential applications in a nanoarray protein chip at the single-molecule level was evaluated using prismtype dual-color total internal reflection fluorescence microscopy (dual-color TIRFM). A dual-color TIRF microscope, which contained two individual laser beams and a single high-sensitivity camera, was used for the rapid and simultaneous dual-color detection of the interactions and colocalization of different proteins labeled with different fluorescent dyes such as Alexa Fluor® 488, Qdot® 525 and Alexa Fluor® 633. Most of the polymer-modified glass substrates showed good stability and a relative high signal-to-noise (S/N) ratio over a 40-day period after making the substrates. The GPTS/CHI/GA-modified glass substrate showed a 13.5-56.3% higher relative S/N ratio than the other substrates. 1% Top-Block in 10 mM phosphate buffered saline (pH 7.4) showed a 99.2% increase in the blocking effect of non-specific adsorption. These results show that dual-color TIRFM is a powerful methodology for detecting proteins at the single-molecule level with potential applications in nanoarray chips or nano-biosensors.

• 동의생리병리학회지
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• 제17권6호
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• pp.1393-1403
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• 2003

The genetic effects of restraint stress challenge on HPA axis and the therapeutic effect of Boshimgeonbi-Tang on the stress were studied with cDNA microarray analyses on hypothalamus using an immobilization-stress mouse as stress model. Male CD-1 mice were restrained in a tightly fitted and ventilated vinyl holder for 2hours once a day, and this challenge was repeated for seven consecutive days. The body weights of the immobilization-stress mice were diminished about 25 percent degree as compared to normal ones. Seven days later, total RNA was extracted from the organs of the mouse, body-labeled with CyDye/sup TM/ fluorescence dyes (Amersham Bioscience Co., NJ), and then hybridized to cDNA microarray chip. Scanning and analyzing the array slides were carried out using GenePix 4000 series scanner and GenePix Pro/sup TM/ analyzing program, respectively. The expression profiles of 109 genes out of 6000 genes on the chip were significantly modulated in hypothalamus by the immobilization stress. Energy metabolism-, lipid metabolism-, apoptosis- and signal transduction-related genes were transcriptionally activated whereas DNA repair-, protein biosynthesis-, and structure integrity-related genes were down-regulated in hypothalamus. The 58 genes were up-regulated by the mRNA expression folds of 1.5 to 7.9. and the 51 genes were down-regulated by 1.5 - 3.5 fold. The 20 genes among them were selected to confirm the expression profiles by RT-PCR. The mRNA expression levels of Tnfrsf1a (apoptosis), Calm2 (cell cycle), Bag3 (apoptosis), Hspe1 (protein folding), Aatk (apoptosis), Dffa (apoptosis), Itgb1 (cell adhesion), Vcam1 (cell adhesion), Fkbp5 (protein folding), BDNF (neuron survival) were restored to the normal one by the treatment of Boshimgeonbi-Tang.

• 대한전기학회논문지:전기물성ㆍ응용부문C
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• 제48권5호
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• pp.285-295
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• 1999

Electroluminescent(EL) devices based on organic materials have been of great interest due to their possible applications for large-area flat-panel displays. They are attractive because of their capability of multi-color emission, and low operation voltage. An approach to realize such device characteristics is to use active layers of lanthanide complexes with their inherent extremely sharp emission bands in stead of commonly known organic dyes. In general, organic molecular compounds show emission due to their $\pi$-$\pi*$ transitions resulting in luminescence bandwidths of about 80 to 100nm. Spin statistic estimations lead to an internal quantum efficiency of dye-based EL devices limited to 25%. On the contrary, the fluorescence of lanthanide complexes is based on an intramolecular energy transfer from the triplet of the organic ligand to the 4f energy states of the ion. Therefore, theoretical internal quantum efficiency is principally not limited. In this study, Powders of TPD, $Eu(TTA)_3(phen) and AlQ_3$ in a boat were subsequently heated to their sublimation temperatures to obtain the growth rates of 0.2~0.3nm/s. Organic electrolumnescent devices(OELD) with a structure of $glass substrate/ITO/Eu(TTA)_3(phen)/AI, glass substrate/ITO/TPD/Eu(TTA)_3(phen)/AI and glass substrate/ITO/TPD/Eu(TTA)_3(phen)/AIQ_3AI$ structures were fabricated by vacuum evaporation method, where aromatic diamine(TPD) was used as a hole transporting material, $Eu(TTA)_3(phen)$ as an emitting material, and Tris(8-hydroxyquinoline)Aluminum$(AlQ_3)$ as an electron transporting layer. Electroluminescent(EL) and current density-voltage(J-V) characteristics of these OELDs with various thickness of $Eu(TTA)_3(phen)$ layer were investigated. The triple-layer structure devices show the red EL spectrum at the wavelength of 613nm, which is almost the same as the photoluminescent(PL) spectrum of $Eu(TTA)_3(phen)$.It was found from the J-V characteristics of these devices that the current density is not dependent on the applied field, but on the electric field.

• Clinical and Experimental Pediatrics
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• 제46권1호
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• pp.56-66
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• 2003

목 적 : 제대정맥은 모체의 혈액을 태아로 운반하여 산소와 영양물질을 공급하는 유일한 통로이다. 이러한 제대 혈류의 장애가 있을 시 자궁내 성장제한, 임신성 고혈압 등을 초래할 수 있다. 제대-태반 혈관의 내피세포 손상을 유발하는 원인 중 amiloride 유도체들을 중심으로 내피세포에 미치는 amiloride 유도체들의 작용을 밝히고, 세포 내 이온농도 변화와 apoptosis 간의 관계를 규명하고자 하였다. 방 법 : 인간 제대정맥 내피세포는 Clonetics로부터 구입하였으며, 내피세포 성장에 필요한 여러 성장인자가 포함된 배지에서 배양하였다. MTT 방법과 flow cytometry 방법을 이용하여 세포독성 효과 및 apoptosis를 확인하였다. 세포 내 이온농도 변화를 측정하기 위해서는 각각의 목적에 적합한 형광염료들을 세포 내에 부하시켜 놓은 뒤, 형광 현미경과 연결된 영상분석장치를 이용하여 관찰하였다. 결 과 : 1) Amiloride 유도체들은 농도 의존적으로 HUVEC의 사멸을 나타내었으며, 각각의 정도는 HMA($IC_{50}$; $11.2{\mu}M$), MIA($13.6{\mu}M$)>EIPA($30.8{\mu}M$)>>amiloride($106{\mu}M$) 순이었다. 2) 세포주기 분석결과 apoptosis의 특징적인 sub $G_0/G_1$ ploidy peak를 나타냈으며, 이러한 작용은 caspase 억제제에 의해 감소되었다. 3) Annexin-V와 propidium iodide 이중 염색 결과 apoptosis로 진행된 세포의 비율(73.0%)을 대조군(11.3%)에 비해 현저히 증가시켰다. 4) HMA에 의한 apoptosis 효과는 세포외액의 pH를 높이거나, $NH_4Cl$을 투여하여 세포내액의 pH를 높일 경우 크게 증가하였다. 5) HMA는 농도 의존적으로 세포내 주요 이온인 $K^+$ 및 $Cl^-$의 세포내 농도를 감소시켰으며, 이러한 효과 역시 세포외액의 pH를 높일수록 현저하게 증가하였다. 결 론 : 이상의 결과들로 미루어 볼 때, HUVEC에서 amiloride 유도체들에 의한 apoptosis 과정에 세포내 주요 이온 농도 감소가 일부 관여하고 있을 것이라 생각된다.