• Proceedings of the Korean Society of Applied Pharmacology
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• 2006.04a
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• pp.123-135
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• 2006

Alzheimer's disease (AD) is the most common form of dementia in the aging population and is clinically characterized by a progressive loss of cognitive abilities. Pathologically, it is defined by the appearance of senile plaques - extracellular insoluble, congophilic protein aggregates composed of amyloid $\beta$ (A$\beta$) and neurofibrillary tangles (NFTs) - inyracellular lesions consisting of paired helical filaments from hyperphosphorylated cytoskeletal tau protein as described by Alois Alzheimer a century ago. These hallmarks still serve as the major criteria for a definite diagnosis of the disease. Consequently, one of the key strategy for drug development in this disease area focuses on reducing the concentration of cerebral A$\beta$ plaque by using substances that inhibit A$\beta$ fibril formation. We focused on developing inhibitors by synthesizing several kinds of aromatic molecules. The synthetic compounds were initially screened to evaluate the effective compound by tioflavin T fluorescence assay. The selected effective compounds were tested cytotoxicity and protective effect from A$\beta$-induced neuronal toxicity by cell based MTT assay with HT22 hippocampal neurons. The BBB permeability on effectors was also tested in in vitro co-culture model(HUVEC/C6 cell line). The behavior test wea carried out in mutant APP/PS1 transgenic mouse model of Alzheimer's disease. And inhibition of A$\beta$ fibril formation by the effective compound was monitored with transmitted electron microscopic images.

• Bulletin of the Korean Chemical Society
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• v.18 no.7
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• pp.761-766
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• 1997

Phospholipase C from Clostridium perfringens is known to catalyze the hydrolysis of phospholipids in biological membranes. In this study, a simple and sensitive method for assaying phospholipase C was developed by using liposomes entrapping calcein as a fluorescent marker. Phospholipase C-induced lysis of liposomes was determined by measuring the fluorescence intensity of calcein released out from liposomes, Various liposomes with different compositions were prepared by reverse-phase evaporation method to investigate the effect of liposomal composition on the lytic activity of phospholipase C. The calcein-entrapping efficiency of liposomes was affected by the chain length of fatty acid in phosphatidylcholine constituting liposomes. The lytic activity of phospholipase C was the highest against liposomes prepared with eggPC. The lytic activity decreased with increasing chain length of fatty acid in phosphatidylcholine. Incorporation of cholesterol more than 20% into the liposomal bilayer inhibited the phospholipase C-induced lysis. The lysis of liposomes was more greatly increased by the addition of 10 mM of calcium. The lytic activity of phospholipase C was also affected by the surface charge of liposomes. Taken together, it was concluded that reverse-phase evaporation vesicles composed of dipalmitoylphosphatidylcholine and cholesterol in the molar ratio of 9 : 1 allowed to detect the lowest concentration of phospholipase C (0.10 μg/assay volume). This study suggested that the use of liposomes can provide a simple, sensitive and inexpensive method for assaying phospholipase C.

• Research in Plant Disease
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• v.28 no.2
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• pp.92-97
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• 2022

Apple stem grooving virus (ASGV) is a high-risk viral pathogen that infects many types of fruit trees, especially pear and apple, and causes serious economic losses across the globe. Thus, rapid and reliable detection assay is needed to identify ASGV infection and prevent its spread. A reliable reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed, optimize, and evaluated for the coding region of coat protein of ASGV in pear leaf. The developed RT-LAMP facilitated the simple screening of ASGV using visible fluorescence and electrophoresis. The optimized reaction conditions for the RT-LAMP were 63℃ for 50 min, and the results showed high specificity and 100-fold greater sensitivity than the reverse transcription polymerase chain reaction. In addition, the reliability of the RT-LAMP was validated using field-collected pear leaves. Furthermore, the potential application of paper-based RNA isolation, combined with RT-LAMP, was also evaluated for detecting ASGV from field-collected samples. These assays could be widely applied to ASGV detection in field conditions and to virus-free certification programs.

• Journal of Physiology & Pathology in Korean Medicine
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• v.24 no.4
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• pp.586-591
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• 2010

Chemokine and Growth Factor are major mediumtors of immuno-inflammatory pathway. The purpose of this study is to investigate whether productions of Chemokine and Growth Factor in lipopolysaccharide (LPS)-induced mouse macrophage RAW 264.7 cells are modulated by Gallic acid (GA), which is easily founded in tannin-containing natural materials such as red wine, green tea, grape juice, and Corni Fructus. Productions of Chemokine and Growth Factor were analyzed by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on $xMAP^{(R)}$ (multi-analyte profiling beads) technology. At first, cell culture supernatant was obtained after treatment with LPS and GA for 24 hour. Then, the antibody-conjugated beads were added and incubated for 30 minutes. After incubation, detection antibody was added and incubated for 30 minutes. And Strepavidin-conjugated Phycoerythrin (SAPE) was added. After incubation for 30 minutes, the level of SAPE fluorescence was analyzed on Bio-plex Suspension Array System. Based on fluorescence intensity, concentrations of Chemokine and Growth Factor were determined. The results of the experiment are as follows. GA significantly inhibited the production of interferon-inducible protein (IP)-10, keratinocyte-derived chemokine(KC), and vascular endothelial growth factor (VEGF) in LPS-induced RAW 264.7 cells at the concentration of 25, 50, 100, 200 uM (p<0.05). GA significantly inhibited the production of monocyte chemoattractant protein-1(MCP-1) and macrophage-colony stimulating factor(M-CSF) in LPS-induced RAW 264.7 cells at the concentration of 50, 100, 200 uM (p<0.05). GA diminished the production of granulocyte macrophage-colony stimulating factor (GM-CSF) in LPS-induced RAW 264.7 cells. But GA did not show the inhibitory effect on the production of leukemia inhibitory factor (LIP) and macrophage inflammatory protein (MIP)-2 in LPS-induced RAW 264.7 cells. These results suggest that GA has the immuno-modulating activity related with its inhibitory effects on the production of IP-10, KC, MCP-1, VEGF, and M-CSF in LPS-induced macrophages.

• Natural Product Sciences
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• v.10 no.2
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• pp.74-79
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• 2004

Dietary flavonoids are currently receiving considerable attention in developing novel cancer-preventive approaches because of their potential capacities to actively induce apoptosis of cancer cells. In our previous report, a flavonoid fraction, which consisted mainly of protocatechuic acid, fustin, fisetin, sulfuretin, and butein and named RCMF (RVS chloroform-methanol fraction), was prepared from a crude acetone extract of Rhus verniciflua Stokes (RVS) that is traditionally used as food additive and herbal medicine. In this study, we evaluated the effects of the RCMF on cell proliferation and apoptosis using SV40-transformed tumorigenic hepatocytes, BNL SV A.8. Tritium uptake assay showing the proliferative capacity of the cells was strongly suppressed in the presence of RCMF. This anti-proliferative effect was further confirmed through trypan blue exclusion. RCMF-mediated suppression of cell growth was verified to be apoptotic, based on the increase in DNA fragmentation, low fluorescence intensity in nuclei after propidium iodide staining, and the appearance of DNA laddering. Collectively, this study demonstrated that RCMF can be approached as a potential agent that is capable of significantly inhibiting cell growth of hepatic cancer cells.

• Proceedings of the Korean Society of Toxicology Conference
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• 2002.11b
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• pp.176-176
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• 2002

In order to investigate whether the induction of micronucleus and aneuploidy in human lymphocytes by Hydroquinone (HQ) is associated with genetic polymorphisms of CYP1A1, GSTM1, GSTT1, NQO1 gene, the cytokinesis-block micronucleus (CBMN) assay in combination with fluorescence in situ hybridization (FISH) technique using specific centromeric probes for chromosome 7 and 8 and PCR-RFLP based genotyping for 30 healthy people were performed.(omitted)

• Annals of Clinical Microbiology
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• v.18 no.2
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• pp.37-43
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• 2015

Background: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens. Methods: The cross-reactivity of MTB- and NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method. Results: The MTB- and NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade. Conclusion: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears.

• Bulletin of the Korean Chemical Society
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• v.30 no.11
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• pp.2655-2660
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• 2009

A breed of cattle, i.e., Korean cattle (Hanwoo), was identified based on the DNA mobilities of their microsatellites (MSs) by capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector. The MS markers were used for the accurate identification of species-specific genes. The DNA mobilities of the MS markers of Hanwoo and Holstein were measured using a CGE system with a fused-silica capillary (inner diameter of 75 ${\mu}m$, outer diameter of 365 ${\mu}m$, and total length of 50 cm). The capillary was dynamically coated with 1.0% (w/v) polyvinylpyrrolidone ($M_r$ = 1,000,000) and then filled with a mixture of 1.3% (w/v) poly(ethylene oxide) ($M_r$ = 600,000) and 1.9% (w/v) poly(ethylene oxide) (Mr = 8,000,000) as a sieving gel matrix. The species-specific genes of Hanwoo and Holstein were clearly distinguished within 33 min. This CGE assay technique is expected to be a useful analytical method for the fast and accurate identification of breeds of cattle.

• Journal of Pharmaceutical Investigation
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• v.35 no.1
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• pp.51-55
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• 2005

A high-performance liquid chromatographic method was employed for the determination of bumetanide in human plasma. After addition of internal standard (IS, naproxen) and acidification of the plasma with 1 M hydrochloric acid, the drug and IS were extracted into dichloromethane. The organic phase was back-extracted into 1 M sodium bicarbonate solution and 50 ${\mu}l$ of the aqueous phase was injected onto a reversed-phase C18 column with a mobile phase consisting of methanol: water: glacial acetic acid = 65 : 35 : 1. The samples were detected utilizing a fluorescence detector (excitation wavelength 235 nm, emission wavelength 405 nm). The method was specific and validated with a lower limit of 5 ng/mL. Intra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification. The applicability of the method was demonstrated by analysis of plasma after oral administration of a single 2 mg dose to 24 healthy subjects. From the plasma bumetanide concentration vs. time curves, the mean AUC was $246.5{\pm}73.8\;ng{\cdot}hr/mL$ and $C_{max}$ of $132.1{\pm}40.9$ ng/mL reached 1.2 hr after administration. The mean biological half-life of burnet ani de was $1.1{\pm}0.2$hr. Based on the results, this simple and validated assay method could readily be used in any pharmacokinetic or bioequivalence studies using humans.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.17 no.11
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• pp.640-647
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• 2016

By minimizing fluorescence interference phenomena, aequorin-based luminescence technology can provide a relatively sensitive detection platform with integration of $G{\alpha}16$ protein in order to track internal calcium mobilization by G protein-coupled receptors (GPCR). In this type of cell-based functional assay format, it is essential to optimize the transfection process of a receptor and $G{\alpha}16$ protein. For this study, corticotropin releasing factor receptor subtype 2(CRF2) was set as a model system to generate three stable cells with CRF2 and $G{\alpha}16$ in addition to transiently transfected cells under three different conditions. Agonist (sauvagine) and antagonist (K41498) responses in those cells were analyzed to develop the optimum transfection process. As a result, the effective signal ratio in the dose response experiments of sauvagine and K41498 were at least 10-fold higher (z'=0.77) in CRF2-$G{\alpha}16$ stable cells. For the transient transfection cells, stable expression of $G{\alpha}16$ prior to the CRF2 represented a two-fold higher signal (z'=0.84) than the other cases of transient transfection. In conclusion, for the utilization of transient transfection processes to develop a cell-based GPCR functional assay system, it is suggested to introduce various target receptors after stable expression of $G{\alpha}16$ protein.