• 대한한방내과학회지
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• 제27권1호
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• pp.178-187
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• 2006

Objectives : Peroxynitrite $(ONOO^-),\;{\cdot}O_2^-$ and nitric oxide (NO) are cytotoxic species that can oxidize several cellular components such as proteins, lipids and DNA. It has been implicated in the aging process and age-related disease such as Alzheimer's disease, rheumatoid arthritis, cancer and atherosclerosis. The aim of this study was to investigate $ONOO^-$ scavengactivities, and that of its precursors, NO and ${\cdot}O_2^-$ of Samjunghwan. Methods : To investigate $ONOO^-,\;NO,\;{\cdot}O_2^-$ scavenging activities, fluorescent probes, namely 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), 4,5-diaminofluorescein (DAF-2) and dihydrorhodamine 123 (DHR 123) were used. Results : Samjunghwan markedly scavenged authentic $ONOO^-,\;{\cdot}O_2^-$ and NO. It also inhibited $ONOO^-$ induced by .02' and NO which are derived from SIN-I. Furthermore, Samjunghwan inhibited $ONOO^-$, ${\cdot}O_2^-$ and NO generation in LPS-treated ICR mouse kidney postmitochondria. Conclusions : These results suggest that Samjunghwan is an effective $ONOO^-,\;{\cdot}O_2^-$ and NO scavenger, and that this substance has a potential role as an inhibitor of aging process, and in therapy against age-related diseases.

• International Journal of Industrial Entomology and Biomaterials
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• 제17권1호
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• pp.121-124
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• 2008

The caspase family proteins play an important role in programmed cell death (apoptosis). To date, the expression profiles of the caspase family genes in Bombyx mori (Bm) are poorly known. In this study, we examined the expression profiles of two novel Bm caspase family genes (ice-2 and ice-5), the potential change of the mitochondrial membrane and the morphology in Bm cells after stimulation of ultraviolet (UV) irradiation. The results showed the potential change of the mitochondrial membrane occurred at 5 hours after UV irradiation treatment. Analysis of fluorescent quantitative RT-PCR demonstrated that both the ice-2 and ice-5 might be involved in UV induced apoptosis in Bm cells. Notably, after UV irradiating, expression pattern of ice-2 and ice-5 were remarkably different. The ice-2 gene was highly expressed at two time points, 0.5 and 5 hours after UV stimulating, while the expression level of ice-5 only peaked at 5 hours after UV stimulating. It indicated that apoptosis induced by UV irradiation was involved in the mitochondrial pathway and the two isoforms of Bm ice may act but play different role during the apoptosis of Bm cells.

• BMB Reports
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• 제51권10호
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• pp.526-531
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• 2018

Protein-Protein Interactions (PPIs) play essential roles in diverse biological processes and their misregulations are associated with a wide range of diseases. Especially, the growing attention to PPIs as a new class of therapeutic target is increasing the need for an efficient method of cell-based PPI analysis. Thus, we newly developed a robust PPI assay (SeePPI) based on the co-translocation of interacting proteins to the discrete subcellular compartment 'processing body' (p-body) inside living cells, enabling a facile analysis of PPI by the enriched fluorescent signal. The feasibility and strength of SeePPI (${\underline{S}}ignal$ ${\underline{e}}nhancement$ ${\underline{e}}xclusively$ on ${\underline{P}}-body$ for ${\underline{P}}rotein-protein$ ${\underline{I}}nteraction$) assay was firmly demonstrated with FKBP12/FRB interaction induced by rapamycin within seconds in real-time analysis of living cells, indicating its recapitulation of physiological PPI dynamics. In addition, we applied p53/MDM2 interaction and its dissociation by Nutlin-3 to SeePPI assay and further confirmed that SeePPI was quantitative and well reflected the endogenous PPI. Our SeePPI assay will provide another useful tool to achieve an efficient analysis of PPIs and their modulators in cells.

• Natural Product Sciences
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• 제18권1호
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• pp.26-31
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• 2012

Actinidia arguta (Actinidiaceae) has been reported to have several pharmacological effects such as anti-inflammatory, anti-allergic, and anti-oxidant activities. The present study investigated the protective activity of an ethanol extract from the leaf and stem of A. arguta against glutamate-induced neurotoxicity using cultured rat cortical neurons. Exposure of cultured cortical neurons to $500{\mu}M$ glutamate for 12 h triggered neuronal cell death. A. arguta inhibited glutamate-induced neuronal death and apoptosis, which were measured by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining, respectively. The increase of pro-apoptotic proteins, Bax and c-caspase-3, in glutamate-treated neurons was significantly inhibited by treatment with A. arguta. A. arguta also inhibited $500{\mu}M$ glutamate-induced elevation of intracellular calcium concentration ($[Ca^{2+}]_i$) and reactive oxygen species (ROS) generation, which were measured by fluorescent dyes, Fluo-4 AM and $H_2DCF$-DA, respectively. These results suggest that A. arguta may prevent glutamate-induced apoptotic neuronal death by inhibiting $[Ca^{2+}]_i$ elevation and ROS generation and, therefore, may have a therapeutic role for the prevention of neurodegeneration in cerebral ischemic diseases.

• 대한한방내과학회지
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• 제28권1호
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• pp.80-91
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• 2007

Objectives : Peroxynitrite (ONOO-), superoxide anion radical (?O2-) and nitric oxide (NO) are cytotoxic because they can oxidize several cellular components such as proteins, lipids and DNA. They have been implicated in the aging process, and age-related diseases such as Alzheimer's disease, rheumatoid arthritis, cancer and atherosclerosis. The aim of this study was to investigate Sabohwan's activity for scavenging ONOO- and its precursors. NO and ?02-. Methods : For this study, the fluorescent probes, namely 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), 4.5-diaminofluorescein (DAF-2) and dihydrorhodamine 123 (DHR 123) were used. Results : Sabohwanblocked tert-butylhydroperoxide (t-BHP)-induced cell death in a dose-dependent fashion. It scavenged t-BHP-induced ONOO-, NO and ?O2- in YPEN cells. Sabohwan inhibited the generation of ONOO-, NO and ?O2- in the lipopolysaccharide (LPS)-treated mouse kidney postmitochondria both in vitro and in vivo. The lipid peroxide level increased and glutathione level decreased in the LPS-treated mice, whereas the ones in the Sabohwanadministered group among the LPS-treated mice reversed toward their natural levels. Conclusions : These results suggest that Sabohwanis an effective ONOO-, ?O2- and NO scavenger, and thereby it might have a potential role as a therapy against the aging process and age-related diseases.

• Bulletin of the Korean Chemical Society
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• 제35권2호
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• pp.425-430
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• 2014

Amyloidogenic proteins often exhibit fibrillar polymorphism through alternative assembly processes, which has been considered to have possible pathological implications. Here, firefly luciferase (LUC) is shown to induce amyloid polymorphism of ${\alpha}$-synuclein, the major constituent of Lewy bodies found in Parkinson's disease, by acting as a novel template. The drastically accelerated fibrillation kinetics of ${\alpha}$-synuclein with LUC required the nucleation center produced by the active enzyme of LUC. Fluorescent dye binding, transmission electron microscopy, and Fourier transformed infrared spectroscopy revealed the morphologically distinctive amyloid fibrils of ${\alpha}$-synuclein prepared in the absence or presence of LUC. As the altered morphological characteristics became inherent to the mature fibrils, those properties were inherited to next-generations via nucleation-dependent fibrillation process. The seed control, therefore, would be an effective means to modify amyloid fibrils with different biochemical characteristics. In addition, the LUC-directed amyloid fibrillar polymorphism also suggests that other cellular biomolecules including enzymes in general are able to diversify amyloid fibrils, which could be self-propagated with diversified biological activities, if any, inside cells.

• KSBB Journal
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• 제31권1호
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• pp.33-39
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• 2016

Developing the method for the selection of animal cell line producing therapeutic monoclonal antibody (mAb) is invaluable as its market is rapidly growing. Although the quality of produced mAb is as important as quantity, however there is no method developed for the selective screening of cell lines on the basis of both quantity and quality. From recent reports, the ratio of light and heavy chain mRNAs of mAb in the cell is a key parameter for the indication of product quality. Therefore, it is obvious that developing the novel method that can detect both light and heavy chain mRNAs in single live cell will provide unprecedented opportunities in bio-industry. Here, we have constructed oligonucleotide probes, molecular beacons for the detection of light or heavy chain mRNAs, respectively, in the live cells producing mAbs. Both beacons showed increased fluorescent intensity after transient transfection of plasmid expressing mAbs analyzed by fluorometer. Flow cytometric analysis clearly demonstrated that both molecular beacons can simultaneously detect the expression of light and heavy chain mRNAs of mAb in the same cell. The technique described in the thesis provides the new direction and concept for developing the method for the smart selection of cell lines producing recombinant proteins including therapeutic mAbs.

• Fisheries and Aquatic Sciences
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• 제24권1호
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• pp.1-9
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• 2021

Human prostate cancer is the second most frequently diagnosed cancer worldwide, and its incidence rate continues to increase. Advanced prostate cancer is more difficult to treat than early forms due to its chemotherapy resistance. There is need for more effective agents that can inhibit the progression of advanced prostate cancer. Demethoxyfumitremorgin C (DMFTC) was isolated from the fermentation extract of the marine fungus Aspergillus fumigatus. Antiproliferative activity of DMFTC against human prostate cancer PC3 cells was examined through cell cycle analysis by flow cytometry, the fluorescent nuclear imaging analysis with propidium iodide (PI), and proteins expression related to cell cycle arrest and apoptosis were investigated via Western blotting. DMFTC inhibited PC3 cells growth through G1 phase cell cycle arrest and apoptosis induction. It activated the tumor suppressor p53 and the Cdk inhibitor p21, which regulate the cell progression into the G1 phase. Additionally, PI-positive late apoptotic non-viable cells were increased and the expression levels of the G1-positive downstream regulators cyclin D, cyclin E, Cdk2, and Cdk4 were decreased by DMFTC treatment. These results suggest that DMFTC induces G1 arrest and apoptosis induction through regulation of p53/p21-dependent cyclin-Cdk complexes, and it may be a useful therapeutic agent for the treatment of human advanced prostate cancer.

• Journal of Microbiology and Biotechnology
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• 제31권2호
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• pp.233-239
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• 2021

Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF domain that they contain can bind bilin(s) autocatalytically via heterologous recombination and then fluoresce, with potential applications as biomarkers and biosensors. Here, we report that a novel red/green CBCR GAF domain, SPI1085g2 from Spirulina subsalsa, covalently binds both phycocyanobilin (PCB) and phycoerythrobilin (PEB). The PCB-binding GAF domain exhibited canonical red/green photoconversion with weak fluorescence emission. However, the PEB-binding GAF domain, SPI1085g2-PEB, exhibited an intense orange fluorescence (λabs.max = 520 nm, λfluor.max = 555 nm), with a fluorescence quantum yield close to 1.0. The fluorescence of SPI1085g2-PEB was selectively and instantaneously quenched by copper ions in a concentration-dependent manner and exhibited reversibility upon treatment with the metal chelator EDTA. This study identified a novel PEB-binding cyanobacteriochrome-based fluorescent protein with the highest quantum yield reported to date and suggests its potential as a biosensor for the rapid detection of copper ions.

• 생명과학회지
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• 제28권12호
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• pp.1416-1423
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• 2018

Biotin에 강한 결합 친화력($K_D=10^{-14}M$)과 함께 streptavidin의 tetramer 특징은 VHH 항체를 streptavidin에 융합시키게 하여 biotinylated horseradish peroxidase를 사용하는ELISA 와Western blot analysis 등의 면역분석법에서 VHH 항체의 항원결합력을 증가시키는데 응용 가능하다. 이를 응용하기 위해 우리는 Streptomyces avidinii 염색체 DNA로부터 PCR을 통해 streptavidin유전자를 증폭하고 이를 green fluorescent protein항원에 특이적으로 결합하는 8B9 VHH 항체유전자에 융합시켰다. 대장균에서 수용성 융합단백질로 발현시키기 위해 pUC119 플라스미드에 기초한 발현시스템을 사용하였다. 즉 lacZ promoter를 사용하여 IPTG에 의해 단백질발현을 유도하게 하였으며, 아미노말단에 pelB leader를 두어 발현된 단백질의 periplasmic space로 이동하게 하여 수용성 단백질형태의 분비를 촉진하였으며 카르복시말단에 6개의 polyhistidine tags를 두어 $Ni^+$-NTA-agarose column을 사용하여 발현된 단백질을 정제하였다. Streptavidin이 biotin에 강하게 결합함으로 대장균에 독성을 나타냄에도 불구하고 본 수용성 융합단백질은 성공적으로 발현되었다. SDS-PAGE에서 가열하는 경우 변성되어 30.6 kDa를, 가열하지 않는 경우에는 자연 상태의 122.4 kDa을 나타내었다. 이는 8B9 VHH항체에 융합된 streptavidin moiety에 의해 monomer subunit가 비공유결합으로 tetramerization됨을 제시해준다. 또한 본 융합단백질은 ELISA와 Westernblot analysis에서 보여진 것처럼 parental streptavidin과 유사한 biotin결합력과 green fluorescent protein항원 결합력을 모두 나타내었다. 결론적으로 streptavidin에 융합된 8B9 VHH 항체형태의 융합단백질은 대장균에서 수용성 tetramer로 성공적으로 발현 및 정제되었으며 biotin과 green fluorescent protein 항원에 동시에 결합함으로써 tetrameric and bifunctional VHH 항체제조의 가능성을 제시해주었다.