• Proceedings of the PSK Conference
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• 2003.10b
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• pp.169.1-169.1
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• 2003

Recently development of cell-based assay systems which are useful in molecular cell biology and drug discovery attracts significant attention. Here, we introduce a new technologies for monitoring enzyme activity and its inhibition inside living cells. Among various enzymes, proteases are important targets for studying various biological and disease-related processes such as viral infections, apoptosis and Alzheimer's disease. In this study, a sensitive cell-based protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells is introduced. (omitted)

• BMB Reports
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• v.43 no.7
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• pp.468-473
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• 2010

Previously, various inhibitors of cell wall synthesis induced the drp35 gene of Staphylococcus aureus efficiently. To determine whether drp35 could be exploited in antistaphylococcal drug discovery, we cloned the promoter of drp35 ($P_d$) and developed different biological assay systems using an engineered S. aureus strain that harbors a chromosomally-integrated $P_d$ - lacZ transcriptional fusion. An agarose-based assay showed that $P_d$ is induced not only by the cell wall-affecting antibiotics but also by rifampicin and ciprofloxacin. In contrast, a liquid medium-based assay revealed the induction of $P_d$ specifically by the cell wall-affecting antibiotics. Induction of $P_d$ by sublethal concentrations of cell wall-affecting antibiotics was even assessable in a microtiter plate assay format, indicating that this assay system could be potentially used for high-throughput screening of new cell wall-inhibiting compounds.

• Biomolecules & Therapeutics
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• v.15 no.4
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• pp.261-265
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• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1384-1391
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• 2006

The signal transduction system is one of the most important devices involved in maintaining life, and many protein kinases are included in the cellular signal transduction system. Finding a protein kinase inhibitor is very valuable, as it can be used to study cell biology and applied to pharmaceuticals. For the efficient and rapid screening of protein kinase inhibitors, two assay systems were combined; the nonradioactive protein kinase assay system that uses an FITC-labeled IRS-2 peptide and the cell-based paper disc assay system that uses Streptomyces griseus as the indicator strain. Among 330 kinds of herb extracts tested, the extract of Dalbergia odorifera exhibited the strongest inhibitory activity in the two assay systems and was selected for further isolation. Based on solvent extraction and many steps of chromatography, seven compounds were finally separated to homogeneity and their structures determined by $^{1}H$ and $^{13}C$ NMR spectroscopies. Four were to be flavonoids and identified as butin ($C_{15}H_{12}O_5$, Mw=272.07), 3'-hydroxymelanetin ($C_{16}H_{12}O_6$, Mw=300.06), liquiritigenin ($C_{15}H_{12}O_4$, Mw=256.07), and 2'-hydroxyformononetin ($C_{16}H_{12}O_{5}$, Mw=284.07). 3'-Hydroxymelanetin inhibited the phosphorylation of the GSK3 protein by Akt to 37% at a concentration of $10{\mu}g/ml$ and showed the strongest cytotoxicity ($ED_{50}<50{\mu}g/ml$) against the human cancer cell line HCT116. Under the same conditions, liquiritigenin also inhibited the phosphorylation of GSK3 by Akt to 26%, and its cytotoxicity against the HCT116 cell line was lower than $100{\mu}g/ml$.

• Korean Journal of Microbiology
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• v.42 no.3
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• pp.195-198
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• 2006

Lethal toxin is a critical virulence factor of anthrax. It is composed two protein: protective antigen (PA) and lethal factor (LF). PA binds to specific cell surface receptors and, forms a membrane channel that mediates entry of LF into the cell. LF is a zinc-dependent metalloprotease, which cleaves MKKs [MAPK (mitogen-activated protein kinase) kinases] at peptide bonds very close to their N-termini. In this study, we suggest application of cell-based assays in the early phase of drug discovery, with a particular focus on the use of yeast cells. We constructed MEK1 expression system in yeast to determine LF activity and approached cell-based assay system to screen inhibitors, in which the results covering the construction of LF-substrate in yeast expression vector, expression, and LF-mediated proteolysis of substrate were described. These results could provided the basic steps in design of cell-based assay system with the high efficiency, rapidly and easy way to screening of inhibitors.

• Environmental Analysis Health and Toxicology
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• v.14 no.1_2
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• pp.1-12
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• 1999

Recently, several new methods for the detection of genetic damages in vitro and in vivo based on molecular biological techniques were introduced according to the rapid progress in toxicology combined with cellular and molecular biology. Among these methods, mouse lymphoma thymidine kanase (tk) gene forward mutation assay, single cell gel electrophoresis (comet assay) and transgenic animal and cell line model as a target gene of lac I (Big Blue) and lac Z (Muta Mouse) gene mutation are newly introduced based on molecular toxicological approaches. The mouse lymphoma tk$\^$+/-/ gene assay (MOLY) using L5178Y tk$\^$+/-/ mouse lymphoma cell line is one of the mammalian forward mutation assays, and has many advantages and more sensitive than hprt assay. The target gene of MOLY is a heterozygous tk$\^$+/-/ gene located in 11 chromosome, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. The comet assay is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakages in mammalian cells, Also, transgenic animal and cell line models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease process, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Also in vivo acridine orange supravital staining micronucleus assay by using mouse peripheral reticulocytes was introduced as an alternative of bone marrow micronucleus assay. In this respect, there was an International workshop on genotoxicity procedure (IWGTP) supported by OECD and EMS (Environmental Mutagen Society) at Washington D. C. in March 25-26, 1999. The objective of IWGTP is to harmonize the testing procedures internationally, and to extend to finalization of OECD guideline, and to the agreement of new guidelines under the International Conference of Harmonization (ICH) for these methods mentioned above. Therefore, we introduce and review the principle, detailed procedure, and application of MOLY, comet assay, transgenic mutagenesis assay and supravital staining micronucleus assay.

• Microbiology and Biotechnology Letters
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• v.47 no.1
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• pp.69-77
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• 2019

Screening microorganisms that can produce glucan hydrolases for industrial, environmental, and biomedical applications is important. Herein, we describe a novel approach to perform glucan hydrolase screening-based on analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) spectra-which involves degradation of the oligo- and polysaccharides. As a proof-of-concept study, glucan hydrolases that could break down glucans made of several glucose units were used to demonstrate the MALDI-MS-based enzyme assay. First, the enzyme activities of ${\alpha}$-amylase and cellulase on a mixture of glucan oligosaccharides were successfully discriminated, where changes of the MALDI-MS profiles directly reflected the glucan hydrolase activities. Next, we validated that this MALDI-MS-based enzyme assay could be applied to glucan polysaccharides (i.e., pullulan, lichenan, and schizophyllan). Finally, the bacterial glucan hydrolase activities were screened on 96-well plate-based platforms, using cell lysates or samples of secreted enzyme. Our results demonstrated that the MALDI-MS-based enzyme assay system would be useful for investigating bacterial glucoside hydrolases in a high-throughput manner.

• Biomolecules & Therapeutics
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• v.19 no.1
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• pp.134-139
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• 2011

Sphingosine kinase (SPHK) has a central role to control cell death and cell proliferation, which is suggested as a sphingolipid rheostat by regulating the levels between ceramide and sphingosine 1-phosphate (S1P). Therefore, physiological regulators of SPHK will be a good candidate to develop a new targeted drug. For this purpose, a series of synthetic pseudoceramides were tested by SPHK assay either cell-based or cell-free system. K10PC-5 strongly inhibited SPHK, while K6PC-5 activated SPHK in cell-free system. Specifically, K6PC-5 activated SPHK under the co-treatment with $50\;{\mu}M$ dimethylsphingosine (DMS), a SPHK inhibitor. Collectively, we developed a simple SPHK assay system to find SPHK regulatory pseudoceramide compounds, K10PC-5 and K6PC-5 which may be useful to cancer treatment or immune regulation like FTY720, a synthetic sphingolipid mimetic compound.

• Environmental Mutagens and Carcinogens
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• v.24 no.2
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• pp.79-84
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• 2004

Three synthetic chemicals, benzoyl chloride, 2-propyn-l-ol, and 2-phenoxy ethanol were selected for genotoxicity testing, based on production quantity and available genotoxic data. In our previous report, benzoyl chloride induced chromosomal aberrations in Chinese hamster lung (CHL) fibroblast in vitro with and without metabolic activation, while 2-propyn-l-ol and 2-phenoxy ethanol induced only with metabolic activation. To compare the genotoxicity of chromosome aberration assay, the single cell gel electrophoresis (comet) assay subjected using CHL cells. As a result, statistically significant differences of tail moment values of benzoyl chloride, 2-propyn-1-ol, and 2-phenoxy ethanol were observed compared with control values on almost all concentrations with S9 or without S9 metabolic activation system. This results suggest that genotoxic results of the comet assay and the chromosome aberration assay show correlationship of genotoxicity in the CHL fibroblast. In summary, the positive result of chromosome aberration of benzoyl chloride, 2-propyn-l-ol, and 2-phenoxy ethanol was also induced DNA damages in comet assay with same cell line. Consequently, comet assay will be useful and more accurate tool to detect and to confirm the genotoxicity especially DNA damages in CHL fibroblast.