• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.19 no.4
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• pp.223-231
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• 2014

In order to prevent the spread of non-indigenous aquatic species through the ballast water in commercial ships, International Maritime Organization (IMO) adopted in 2004 the International Convention for Control and Management of Ship's Ballast Water and Sediments. The Convention mandates treatment of ballast water for most transoceanic voyages and its confirmation of treatment is made with plankton live/dead assay. Fluorescein diacetate assay (FDA), which produces bright green light for live phytoplankton, has been a de facto standard method to determine the survival of marine plankton, but its staining efficacy has been in dispute. In the present study, we examined the limitation of FDA, and compared its efficacy with Neutral red (NR) staining, another promising assay and widely used especially for zooplankton mortality. For all phytoplankton species studied in the present study, except Ditylum brightwellii, the staining efficiency was <50% with FDA. The green FDA fluorescence interfered with phytoplankton autofluorescence in most samples. In contrast, NR assay stained over 90% of both phytoplankton and zooplankton species tested in this study. FDA assay also showed that green FDA fluorescence rapidly faded when phytoplankton cells were exposed to microscope light. Both FDA and NR assay were negative on formalin-killed individuals of both phytoplankton and zooplankton species. Our results suggest that NR assay is more effective for determining the survival of marine plankton and can be applied to test the efficacy of ballast water treatment.

• Korean Journal of Environmental Agriculture
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• v.28 no.4
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• pp.435-441
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• 2009

In the present study, we analyzed the defensin protein deduced from Korean radish (Raphanus sativus L.) seeds.To express the genes in E. coli, we constructed a recombinant expression vector with a defensin gene, named rKRs-AFP gene isolated from Korean radish seeds. Over expressed rKRs-AFP proteins was separated by SDS-PAGE to determine the purity, and protein concentration was determined by the Bradford method. Antifungal activity was assessed by disk assay method against the tested fungi. As a result, when 500 mL of cell culture were disrupted by sonicator, 32.5 mg total proteins were obtained. The purified protein showed a single band on SDS-PAGE with estimated molecular weight about 6 KDa, consistent with the molecular mass calculated from the deduced amino acid sequence. The purified rKRs-AFP protein showed remarkable antifungal activities against several fungi including Aspergillus niger, Botrytis cinerea causing the gray mold disease, and Candida albicans. In field tests using the purified rKRs-AFP protein, the protein showed the reducing activity of disease spot and the mitigating effect of spreading of disease like agrichemicals. The immuno-assay of rKRs-AFP protein showed that the purified protein entirely accumulated at B. cinerea cytoplasm through the hyphal septa shown by fluorescence imaging. There was no fluorescence inside the cell, when the hypha was incubated without the protein. These all results indicate that the recombinant rKRs-AFP proteins can be utilized as a potential antifungal drug to control harmful plant fungal pathogens.

• Nuclear Engineering and Technology
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• v.25 no.2
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• pp.255-258
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• 1993

A method for analysis of trace uranium in urine sample was studied using a time-resolved $N_2$-laser-induced fluorimetry. The Fluran solution was found to be efficient to mask the chloride ions which are known to quench uranium fluorescence in the fluorimetric assay of uranium in urine. This improved method made the sample preparation much simpler than other conventional ones. The fluorescence intensities at 1% urine mixture with 10% Fluran aqueous solution showed good linearities in the concentration range of 10-500 ppb(before dilution).

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.5
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• pp.1196-1201
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• 2008

A examination of the kinetic properties of UDP-glucose : (1,3)-$\beta$-glucan (callose) synthase from mung bean seedings (Sorbus cortex) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Deoxynojirimycin increased the inhibitory effect of (1,3)-$\beta$-glucan synthase at the concentration-dependent manner by fluorescence assay. The inhibitory effect of Fr. 2-16 (97.15%) showed higher than that of deoxynojirimycin (80.63%). Fr. 2-3 inhibited the growth of the Candida albicans at 1 mm inhibition zone by disk diffusion method. These results suggest that Sorbus cortex extract can be used as a stable antifungal material.

• Biomolecules & Therapeutics
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• v.19 no.1
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• pp.1-8
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• 2011

G-protein coupled receptors (GPCRs) constitute an important class of drug targets and are involved in every aspect of human physiology including sleep regulation, blood pressure, mood, food intake, perception of pain, control of cancer growth, and immune response. Radiometric assays have been the classic method used during the search for potential therapeutics acting at various GPCRs for most GPCR-based drug discovery research programs. An increasing number of diverse small molecules, together with novel GPCR targets identified from genomics efforts, necessitates the use of high-throughput assays with a good sensitivity and specificity. Currently, a wide array of high-throughput tools for research on GPCRs is available and can be used to study receptor-ligand interaction, receptor driven functional response, receptor-receptor interaction,and receptor internalization. Many of the assay technologies are based on luminescence or fluorescence and can be easily applied in cell based models to reduce gaps between in vitro and in vivo studies for drug discovery processes. Especially, cell based models for GPCR can be efficiently employed to deconvolute the integrated information concerning the ligand-receptor-function axis obtained from label-free detection technology. This review covers various platform technologies used for the research of GPCRs, concentrating on the principal, non-radiometric homogeneous assay technologies. As current technology is rapidly advancing, the combination of probe chemistry, optical instruments, and GPCR biology will provide us with many new technologies to apply in the future.

• Bulletin of the Korean Chemical Society
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• v.29 no.7
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• pp.1311-1314
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• 2008

Bacillus halodurans O-methyltransferase (BhOMT) is an S-adenosylmethionine dependent methyltransferase. In our previous study, three dimensional structure of the BhOMT has been determined by comparative homology modeling and automated docking study showed that two hydroxyl groups at 3'- and 4'-position in Bring and structural rigidity of C-ring resulting from the double bond characters between C2 and C3 of flavonoid, were key factors for interaction with BhOMT. In the present study, BhOMT was cloned and expressed. Binding assay was performed on purified BhOMT using fluorescence experiments and binding affinity of luteolin, quercetin, fisetin, and myricetin were measured in the range of $10^7$. Fluorescence quenching experiments indicated that divalent cation plays a critical role on the metal-mediated electrostatic interactions between flavonoid and substrate binding site of BhOMT. Fluorescence study confirmed successfully the data obtained from the docking study and these results imply that hydroxyl group at 7-position of luteolin, quercetin, fisetin, and myricetin forms a stable hydrogen bonding with K211 and carboxyl oxygen of C-ring forms a stable hydrogen bonding with R170. Hydroxyl group at 3'-and 4'-position in the B-ring also has strong $Ca^{2+}$ mediated electrostatic interactions with BhOMT.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.118-123
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• 2008

A new method for antimicrobial susceptibility testing of vitro-cultured bacteria on an ordinary fluorescence spectrometer was developed. The viable bacteria reduced 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to produce insoluble particles that displayed intense resonance scattering light. The assay showed a linear relationship between the number of viable bacteria and the intensity of resonance scattering light. Dead bacteria were unable to reduce MTT. Methicillin-resistant Staphylococcus aureus exposed to flavonoids from Marchantia convoluta showed a flavonoids concentration-dependent inhibition of the ability to reduce MTT. In the assay, less than 12 h was required to attain susceptibility results and fewer bacteria were utilized than in traditional methods. The RLS technique could, in combination with the MTT assay, be a rapid and sensitive measuring method to determine the in vitro activity of new antimicrobials.

• Archives of Pharmacal Research
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• v.23 no.5
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• pp.441-445
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• 2000

Achiral-chiral column switching HPLC assay was developed to allow the separation and quantification of the enantiomers of terbutaline in human plasma by means of fluorescence detection. Plasma samples were prepared by solid-phase extraction with sep-pak silica, followed by HPLC assay. The enantiomers of terbutaline and the internal standard were separated from the biological matrix on a silica column, and the two enantiomers were resolved and quantified on a Sumichiral OA-4900 column. The two columns were connected by a switching valve equipped with silica trap column, The trap column was used to concentrate the terbutaline in the eluent from the achiral column before back flushing onto the chiral phase. For each enantiomers, the assay was linear between 2.5-125 ng/$m\ell$ (r=0.9999) and detection limit was 1.0 ng/$m\ell$ .

• Journal of the East Asian Society of Dietary Life
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• v.17 no.3
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• pp.393-401
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• 2007

This study was performed to investigate the antioxidant activity of Korean ginseng using an ORAC(Oxygen Radical Absorbance Capacity) assay. Four fractions each (80% ethanol, ethyl acetate, water saturated 1-butanol, and water) were obtained from different ginseng samples (White Ginseng: ; 6 yrs-., 5 yrs-., ; Cork Ginseng: ; 5 yrs-., 4 yrs-.). The saponin content of each fraction was quantified by LC/MS, and the antioxidant capacity of the ginseng was measured by the ORAC assay. The ORAC method, which was recently validated using automatic liquid handling systems, has been adapted for manual handling with the use of a conventional fluorescence microplate reader. Furthermore, the ORAC assay provides a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxy radical, which is the exiting and emission of 2,2'-Azobis (2-methylpropionamidine)-dihychloride (AAPH). As a result of our experiments, ginsenosides Rg1 and Rb1 were the two major saponins found in the ginseng samples, and Rc, Rb2, Re, Rd, Rg3, and Rh1 were detected in a small quantities. For the antioxidant capacities of the fractions (80% ethanol, ethyl acetate, butanol, and water), we found that the organic solvent fraction had similar antioxidant capacities, and were higher than the capacity of the water fraction. When determining the similarities in each fraction, only the ethyl acetate fraction showed similarity compared to other fractions (p>0.05). The antioxidant capacity of ginseng may come from phenolic compounds and some nonpolar saponins. However, based on the results of this study, we hypothesize that some acidic polysaccharides and other biological components may contribute to its antioxidant capacity. Additional research is required to determine other possible biological response modifiers that contribute to the antioxidant capacity of ginseng.

• KSBB Journal
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• v.22 no.2
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• pp.67-72
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• 2007

This study investigates the optimal method of administrating 5-aminolevulinic acid (5-ALA) in the context of fluorescence detection by analyzing protoporphyrin IX (PpIX) fluorescence in the cultured normal and cancer cells. 5-ALA was injected as a photosensitizer to the lung cancer cells (A549, NCI-H460) and normal lung cells (HeI299). Hel299, A549, and NCI-H460 cells were incubated with various concentrations of 5-ALA ($0\sim800{\mu}g/mL$). The accumulation of PpIX induced by 5-ALA was observed in A549, NCI-H460 and Hel299 cells. The cell viability was estimated by means of the MTT assay. Formation of PpIX was measured by fluorescence spectroscopy. Especially, formation of PpIX in cancer cells was higher than normal cells. This study suggests that the difference of PpIX induced in normal and cancer cells treated with 5-ALA may use by means of fluorescence diagnosis for cancer.