• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1022-1027
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• 2005

Based on plasmid display technology by the complexes of fusion protein and the encoding plasmid DNA, an in vitro selection method for high affinity DNA-binding protein was developed and experimentally demonstrated. The GAL4 DNA-binding domain (GAL4 DBD) was selected as a model DNA-binding protein, and enhanced green fluorescent protein (EGFP) was used as an expression reporter for the selection of target proteins. Error prone PCR was conducted to construct a mutant library of the model. Based on the affinity decrease with increased salt concentration, mutants of GAL4 DBD having high affinity were selected from the mutant protein library of protein-encoding plasmid complex by this method. Two mutants of (Lys33Glu, Arg123Lys, Ile127Lys) and (Ser47Pro, Ser85Pro) having high affinity were obtained from the first generation mutants. This method can be used for rapid in vitro selection of high affinity DNA-binding proteins, and has high potential for the screening of high affinity DNA-binding proteins in a sequence-specific manner.

• Journal of Pharmaceutical Investigation
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• v.38 no.3
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• pp.183-189
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• 2008

The present study compared the feasibility of Caco-2 and MDCK cells as an efficient in-vitro model for the drug classification based on Biopharmaceutics Classification System (BCS) as well as an in-vitro model for drug interactions mediated by P-gp inhibition or P-gp induction. Thirteen model drugs were selected to cover BCS Class I{\sim}IV$ and their membrane permeability values were evaluated in both Caco-2 and MDCK cells. P-gp inhibition studies were conducted by using vinblastine and verapamil in MDCK cells. P-gp induction studies were also performed in MDCK cells using rifampin and the P-gp expression level was determined by western blot analysis. Compared to Caco-2 cells, MDCK cells required shorter period of time to culture cells before running the transport study. Both Caco-2 and MDCK cells exhibited the same rank order relationship between in-vitro permeability values and human permeability values of all tested model compounds, implying that those in-vitro models may be useful in the prediction of human permeability (rank order) of new chemical entities at the early drug discovery stage. However, in the case of BCS drug classification, Caco-2 cells appeared to be more suitable than MDCK cells. P-gp induction by rifampin was negligible in MDCK-cells while MDCK cells appeared to be feasible for P-gp inhibition studies. Taken all together, the present study suggests that Caco-2 cells might be more applicable to the BCS drug classification than MDCK-cells, although MDCK cells may provide some advantage in terms of capacity and speed in early ADME screening process.

• BMB Reports
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• v.56 no.11
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• pp.594-599
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• 2023

A number of therapeutic drugs have been developed from functional chemicals found in plants. Knowledge of plants used for medicinal purposes has historically been transmitted by word of mouth or through literature. The aim of the present study is to provide a systemic platform for the development of lead compounds against breast cancer based on a traditional medical text. To verify our systematic approach, integrating processes consisted of text mining of traditional medical texts, 3-D virtual docking screening, and in vitro and in vivo experimental validations were demonstrated. Our text analysis system identified rutin as a specific phytochemical traditionally used for cancer treatment. 3-D virtual screening predicted that rutin could block EGFR signaling. Thus, we validated significant anti-cancer effects of rutin against breast cancer cells through blockade of EGFR signaling pathway in vitro. We also demonstrated in vivo anti-cancer effects of rutin using the breast cancer recurrence in vivo models. In summary, our innovative approach might be proper for discovering new phytochemical lead compounds designing for blockade of malignant neoplasm including breast cancer.

• Horticultural Science & Technology
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• v.33 no.6
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• pp.883-890
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• 2015

The soilborne fungus Fusarium oxysporum f. sp. lilii (Fol) is a serious threat to all lily cultivars, especially infecting bulbs and flowers. It has become increasingly important to develop varieties resistant against the bulb rot disease. Genetic diversity of cultivars and reliable screening methods are required for this purpose. Here, an efficient in vitro screening system for evaluating resistance to Fol in 38 in vitro-grown lily plants was investigated. Various factors including culture conditions of Fol, inoculum density, appropriate plant materials, inoculation method and duration, and incubation period of plant materials after inoculation were combined to optimize the screening method. As a result, we suggest optimal conditions for an in vitro screening system for the selection of Fol-resistant lily cultivars as follows. Fol was grown on potato dextrose agar (PDA) medium for 6 days at $25^{\circ}C$ in darkness and used as working inoculation. Spore suspensions were prepared (inoculum density: $1.0{\times}10^4$ $spores{\cdot}mL^{-1}$), and then leaf segments $1.5{\times}2.0cm^2$ were inoculated by dipping for 22 hours at $25^{\circ}C$ in dark. Later, leaves were cultured on 0.6% agar plates at $25^{\circ}C$ and 50% humidity with a photoperiod of 16 hours light/8 hours dark (fluence rate of $40{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$) to examine the progress of bulb rot. After 7 days, disease levels were classified into indices 1 (no symptom) to 6 (serious bulb rot). Soil inoculation of Fol carried out with resistant or susceptible lily cultivars that had been selected through in vitro screening confirmed the reproducibility of results. Therefore, the in vitro screening method established in this study is efficient and reliable for selection of lily cultivars resistant against bulb rot disease.

• Toxicological Research
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• v.33 no.2
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• pp.107-118
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• 2017

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.

• Bulletin of the Korean Chemical Society
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• v.31 no.6
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• pp.1609-1614
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• 2010

Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Standard in vitro test was chosen using a rat brain homogenate as the source of AChE. Screening of reactivation potency was performed with two concentration of reactivator (1000 ${\mu}M$ and 10 ${\mu}M$). Results were compared to established reactivators pralidoxime, methoxime, HI-6, trimedoxime and obidoxime. More than 30 novel reactivators performed equal or better reactivation ability of POX-inhibited AChE compared to currently used reactivators. The structure-activity relationship for reactivators of paraoxon-inhibited AChE was developed.

• Bulletin of the Korean Chemical Society
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• v.34 no.1
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• pp.231-236
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• 2013

Some cytotoxicity studies for the interpretation of the interaction between nanoparticles and cells are non-mechanistic and time-consuming. Therefore, non-biological screening methods, which are faster and simpler than in-vivo and in-vitro methods, are required as alternatives to current cytotoxicity tests. Here, we proposed a simple screening method for the analysis of the interaction between several AgNPs (bare-, citrate-, and polyvinylpyrrolidone-coating) and dye-containing vesicles acting as a biomimetic cell-membrane. The interaction between AgNPs and vesicles could be evaluated readily by UV-vis spectra. Absorbance deviation in UV-vis spectra revealed a large attraction between neighboring particles and vesicles. This was confirmed by (Derjagin, Landau, Verwey, and Overbeek) theory and DMF (dark-field microscopy) analysis. This proposed method might be useful for analyzing the cytotoxicity of nanoparticles with cell-membranes instead of in vitro or in vivo cytotoxicity tests.

• Mycobiology
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• v.28 no.4
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• pp.190-192
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• 2000

Antifungal bacteria for biological control of plant diseases or production of novel antibiotics to plant pathogens were isolated in 1997 from various soils of Ansung, Chunan, Koyang, and Paju in Korea. Sixty-four bacterial strains pre-screened from approximately 1,400 strains were tested on V-8 juice agar against eight plant pathogenic fungi using in vitro bioassay technique for inhibition of mycelial growth. Test pathogens were Alternaria mali, Colletotrichum gloeosporioides, C. orbiculare, Fusarium oxysporum f. sp. cucumerinum, F. oxysporum f. sp. lycopersici, Magnaporthe grisea, Phytophthora capsici, and Rhizoctonia solani. A wide range of antifungal activity of bacterial strains was found against the pathogenic fungi, and strain RC-B77 showed the best antifungal activity. Correlation analysis between inhibition of each fungus and mean inhibition of all eight fungi by 64 bacterial strains revealed that C. gloeosporioides would be best appropriate for detecting bacterial strains producing antibiotics with potential as biocontrol agents for plant pathogens.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1097-1104
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• 2012

Background/Aim: Pristimerin isolated from Celastrus and Maytenus spp can inhibit proteasome activity. However, whether pristimerin can modulate cancer metastasis is unknown. Methods: The impacts of pristimerin on the purified and intracellular chymotrypsin proteasomal activity, the levels of regulator of G protein signaling 4 (RGS 4) expression and breast cancer cell lamellipodia formation, and the migration and invasion were determined by enzymatic, Western blot, immunofluorescent, and transwell assays, respectively. Results: We found that pristimerin inhibited human chymotrypsin proteasomal activity in MDA-MB-231 cells in a dose-dependent manner. Pristimerin also inhibited breast cancer cell lamellipodia formation, migration, and invasion in vitro by up-regulating RGS4 expression. Thus, knockdown of RGS4 attenuated pristimerin-mediated inhibition of breast cancer cell migration and invasion. Furthermore, pristimerin inhibited growth and invasion of implanted breast tumors in mice. Conclusion: Pristmerin inhibits proteasomal activity and increases the levels of RGS4, inhibiting the migration and invasion of breast cancer cells.

• YAKHAK HOEJI
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• v.50 no.1
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• pp.52-57
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• 2006

The activation of cyclin dependent kinase 4 (CDK4) is found in more than half of all human cancers. Therefore CDK4 is an attractive target for the development of a novel anticancer agent. For mass screening of CDK4 inhibitor, we set up in vitro kinase assay for CDK4 activity using a cyclin D1-CDK4 fusion protein, which is constitutively active and exhibits enhanced stability. From the screening of representative compound library of Korea Chemical Bank, we found that 7-chloro-4-nitro-benzo[1,2,5]oxadiazole 1-oxide (FBP-1248) selectively inhibited CDK4 activity in vitro by ATP competitive manner. This compound prevented the phosphorylation of retinoblatsoma tumor suppressor protein, Rb, and inhibited cell growth through cell cycle arrest. In summary, we developed an efficient assay system for CDK4 activity in vitro and identified the CDK4 inhibitory compound, FBP-1248.