• Archives of Pharmacal Research
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• v.27 no.9
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• pp.906-911
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• 2004

In order to evaluate estrogenic compounds in natural products, an in vitro detection system was established. For this system, the human breast cancer cell line MCF7 was stably trans-fected using an estrogen responsive chloramphenicol acetyltransferase (CAT) reporter plas-mid yielding MCF7/pDsCAT-ERE119-Ad2MLP cells. To test the estrogenic responsiveness of this in vitro assay system, MCF7/pDsCAT-ERE119-Ad2MLP cells were treated with various concentrations of 17f3-estradiol. Treatments of 10$^{-8}$ to 10$^{-12}$ M 17$\beta$-estradiol revealed significant concentration dependent estrogenic activities compared with ethanol. We used in vitro assay system to detect estrogenic effects in Puerariae radix and Ginseng radix Rubra extracts. Treat-ment of 500 and 50 $\mu\textrm{g}$/ml of Puerariae radix extracts increased the transcriptional activity approximately 4- and 1.5-fold, respectively, compared with the ethanol treatment. Treatment of 500, 50, and 5 $\mu\textrm{g}$/ml of Ginseng radix Rubra extracts increased the transcriptional activity approximately 3.2-,2.7, and 1.4-fold, respectively, compared with the ethanol treatment. These observations suggest that Puerariae radix and Ginseng radix Rubra extracts have effective estrogenic actions and that they could be developed as estrogenic supplements.

• Korean Journal of Plant Resources
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• v.23 no.3
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• pp.233-241
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• 2010

In vitro elimination of Sweet potato leaf curl virus (SPLCV) from infected sweet potato is difficult due to low number of virus-free plants obtained from meristem tip culture and long growth period required for the virus detection. In this study, efficient production of the SPLCV-free sweet potato by in vitro therapy coupled with a PCR assay for virus detection was investigated. Infected shoots cultured on Murashige and Skoog medium were treated at three different temperatures for 7 weeks followed by meristem tip culture on the medium with or without ribavirin at 50 mg/L. The regenerated plantlets were tested for virus infection by a PCR assay. The results showed that the both heat- and cold-treatments, and addition of the ribavirin did not have significant effect on efficiency of the virus elimination. The meristem size, however, greatly affected the survival rate. Meristems sized over 0.4 mm survived better than smaller ones (0.2-0.3 mm). The PCR assay was approved to be a rapid, sensitive and reliable for the SPLCV detection in regenerated plantlets. Therefore, combination of cultivating meristem tips sized 0.4-0.5 mm on the medium at $22^{\circ}C$ without ribavirin and detection of SPLCV in the regenerated plantlets by the PCR assay was an efficient system for the SPLCV elimination from infected sweet potato.

• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1323-1329
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• 2021

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

• Archives of Pharmacal Research
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• v.25 no.6
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• pp.903-909
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• 2002

Research in the cytokine field has grown exponentially in recent years, and the validity of such studies relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Transforming growth factor (TGF)-$\beta$, a hormonally active polypeptide found in normal and transformed tissue, is a potent regulator of cell growth and differentiation. The most widely used bioassay for TGF-$\beta$ is the inhibition of the proliferation of mink lung epithelial cells. Though detection of [$^3$H]thymidine incorporation is more sensitive than the MTT assay, it presents some disadvantages due to the safety and disposal problems associated with radioisotopes. In this study, we attempted to ascertain the experimental conditions which could be used for measuring the in vitro biological activity of TGF-$\beta$ in a safer and more sensitive way compared with the currently available methods. We compared the commonly used method, the MTT assay, to the XTT assay using different parameters including cell number, incubation time and the wave length used for detecting the product. We examined the anti-proliferative activities of TGF-$\beta$ in three different cell lines: Mv-1-Lu mink lung epithelial cells, MCF10A human breast epithelial cells and H-ras-transformed MCF10A cells. Herein, we present an experimental protocol which provides the most sensitive method of quantifying the biological activity of TGF-$\beta$, with a detection limit of as low as 10 pg/ml: Mv-1-Lu or H-ras MCF10A cells ($1{\times}10^5/well$) were incubated with TGF-$\beta$ at $37^{\circ}C$ in a humidified $CO_2$ incubator for 24 hr followed by XTT treatment and determination of absorbance at 450 or 490 nm. Our results may contribute to the establishment of an in vitro bioassay system, which could be used for the satisfactory quantitation of TGF-$\beta$.

• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.1-6
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• 1990

We established an in vitro system of central serotonergic neurons by culturing dissociated rat embryonic (El4) brainstem cells to 14 days in vitro and monitored the serotonergic neuronal growth by measuring 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents in the cells with hish performance liquid chromatography with electrochemical detection (HPLC-EC). We studied also tile effects of various drugs on the contents of 5-HT and 5-HIAA, confirming in vivo reports. The 5-HT content (13 ng/mg protein) and 5-HT turnover rate (17 pmol/mg protein/h) at 14 days in vitro were in good agreement with those reported in the adult rat brain. The 5-HT content was more easily depleted with p-chlorophenylalanine, a tryptophan hydroxylase inhibitor than with NSD 1015 (3-hydroxybenzylhydrazine), an aromatic L-amino acid decarboxylase (AADC) inhibitor. Incubation of the cultures with tryptophan or 5-hydroxytryptophan increased the rate of serotonin formation implying that neither tryptophan hydroxylase nor AADC is saturated with its amino acid substrate in this in vitro system . The 5-HT content was depleted by reserpine. The 5-HT and 5-HIAA contents were increased and decreased, respectively, by monoamine oxidase inhibitors. All the above results indicate that the biochemical properties of the central serotonergic neurons in this culture system reflect reliably those of central serotonergic neurons in vivo. We suggest that measuring 5-HT and 5-HIAA contents in the primary cultured dissociated brainstem-cells with HPLC-EC is useful in the study of pharmacology as well as toxicolgy of the central serotonergic neurons.

• Environmental Mutagens and Carcinogens
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• v.21 no.1
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• pp.14-22
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• 2001

The detection of many synthetic chemicals used in industry that may pose a genetic hazard in our environment is of great concern at present. In this respect, administrative authorities has great concern to regulate and to evaluate the chemical hazard to environment and human health. The clastogenicity of 28 synthetic chemicals was evaluated in Chinese hamster lung fibroblast cells in vitro. Glycidylacrylate which is one of the most cytotoxic chemical among 28 chemicals tested revealed clastogenicity in the range of 0.31-1.25 $\mu\textrm{g}$/$m\ell$ both in the presence and absence of metabolic activation system. Neopentyl glycol (340-1360 $\mu\textrm{g}$/$m\ell$) also revealed weak positive result both in the presence and absence of metabolic activation system. Cyanoguanidine (/$420.5-841 $\mu\textrm{g}$m\ell$) and N-butylchloride ($231.5-926 $\mu\textrm{g}$/m\ell$) revealed weak positive result only in the absence of S-9 metabolic activation system. Nevertheless total aberration percentages of N-butylchloride in the presence of metabolic activation system, and 3,4＇-dichlorobenztrifluoride in the absence of S-9 metabolic activation revealed above 5% aberration, there is no statistical significance. From the results of chromosomal aberration assay with 28 synthetic chemicals in Chinese hamster lung cells, glycidylacrylate (CAS No. 106-90-0), neopentyl glycol (CAS No. 126-30-7), N-butyl chloride (CAS No. 109-69-3) and cyanoguanidine (CAS No. 461-58-5) revealed positive clastogenic results in this study.

• Environmental Mutagens and Carcinogens
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• v.22 no.4
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• pp.215-222
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• 2002

The detection of many synthetic chemicals used in industry that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health. In this respect, to regulate and to evaluate the chemical hazard will be important to environment and human health. The clastogenicity of 17 synthetic chemicals was evaluated in Chinese hamster lung fibroblast cells in vitro. Two most cytotoxic chemicals, dodecyl methacrylate (CAS No. 142-90-5) and 2-ethylhexyl methacrylate (CAS No. 688-84-6), among 17 chemicals tested revealed no clastogenicity in the range of 0.0165-0.066 $\mu\textrm{g}$/$m\ell$ and 0.006-0.024 $\mu\textrm{g}$/$m\ell$ both in the presence and absence of metabolic activation system, respectively. All 17 chemicals revealed no significant induction of chromosomal aberration both in the presence and absence of metabolic activation system in this assay. From the results of chromosomal aberration assay with 17 synthetic chemicals in Chinese hamster lung cells in vitro, we did not observed positive clastogenic results in this study.

• 한국가시화정보학회:학술대회논문집
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• 2007.11a
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• pp.105-108
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• 2007

To diagnose the vascular diseases from the viewpoint of hemodynamics, we need detailed quantitative hemodynamic information of related blood flows with a high spatial resolution of tens micrometer and a high temporal resolution in the order of millisecond. For investigating in-vivo hemodynamic phenomena of vascular circulatory diseases, a new diagnosing technique combining a medical radiography and PIV method was newly developed. This technique called 'Angiographic PIV system' consists of a medical X-ray tube, an X-ray CCD camera, a shutter module for generating double pulse-type X-ray, and a synchronizer. Through several preliminary tests, the feasibility of the Angiographic PIV technique was verified. For in-vivo applications to real blood flows, we developed tracer microcapsules, which were optimized to this system, made of a contrast material of iodine and a matrix material of PVA (polyvinylpyrrolidone). In near future, the Angiographic PIV technique will be used for understanding hemodynamic phenomena of vascular diseases and for their early detection.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.635-636
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• 2005

• Molecules and Cells
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• v.45 no.7
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• pp.502-511
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• 2022

Bacterial volatile compounds (BVCs) exert beneficial effects on plant protection both directly and indirectly. Although BVCs have been detected in vitro, their detection in situ remains challenging. The purpose of this study was to investigate the possibility of BVCs detection under in situ condition and estimate the potentials of in situ BVC to plants at below detection limit. We developed a method for detecting BVCs released by the soil bacteria Bacillus velezensis strain GB03 and Streptomyces griseus strain S4-7 in situ using solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME-GC-MS). Additionally, we evaluated the BVC detection limit in the rhizosphere and induction of systemic immune response in tomato plants grown in the greenhouse. Two signature BVCs, 2-nonanone and caryolan-1-ol, of GB03 and S4-7 respectively were successfully detected using the soil-vial system. However, these BVCs could not be detected in the rhizosphere pretreated with strains GB03 and S4-7. The detection limit of 2-nonanone in the tomato rhizosphere was 1 µM. Unexpectedly, drench application of 2-nonanone at 10 nM concentration, which is below its detection limit, protected tomato seedlings against Pseudomonas syringae pv. tomato. Our finding highlights that BVCs, including 2-nonanone, released by a soil bacterium are functional even when present at a concentration below the detection limit of SPME-GC-MS.