• Toxicological Research
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• v.9 no.2
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• pp.217-224
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• 1993

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a well-known dopamine neuron-specific toxin. But the involvement of oxidative damage in the pathogenesis of MPTP-induced parkinsonism is still uncertain. In this study, by using 2',7',-dichlorofluorescin diacetate(DCFH-DA) that detects intracellular oxidative processes, the effect of MPTP on dichlorofluorescein fluorescence in dissociated cells from fetal rat mesencephalon in culture was investigated. At 7th day in culture, cells were loaded with DCFH-DA, and exposed to 1 mM MPTP or MPP+. MPTP induced dichlorofluorescein-fluorescence which was peaked at 3 min and mostly faded away 30 min after MPTP treatment.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.6
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• pp.461-467
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• 2005

Background. 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, succinimidyl ester mixed (CFSE) is the fluorescent labelling agent of living cells and used to trace the cells in vivo after transplatnation of various cells. The CFSE labelled cells can maintain fluorescence for up to 7 days after labelling. The MC3T3-E1 cell line (MC3T3) has been used for many studies about osteoblast, which is well known as a mouse preosteoblast. So the CFSE would be used to trace the transplanted MC3T3. However there are few reports about CFSE labelling of MC3T3. This study is aimed to know about adequate concenturation and incubation time of CFSE to MC3T3. Materials and methods. The MC3T3 was incubated in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ using ${\alpha}$-minimal essential medium (${alpha}$-MEM) containing10% FBS and gentamycin. Ten mM CFSE solution in dimethylsulphoxide (DMSO: 1%) was diluted with phosphate buffered saline (PBS) and final concentration of culture medium was, respectively, 5, 10, 15, 20, 25 and 30 ${{\mu}M$. Then the MC3T3 was incubated with CFSE in a humidified atmosphere of 95% air with 5% $CO_2$ at $37^{\circ}C$ for 5, 10, 15, 20, 25, 30, 35, 40 and 45 minutes in each concentration. The fluorescence of CFSE labelled cells was analysed with a inverted fluorescence microscope. The duration of cell labelling was also studied. Trypan blue dye exclusion test was done for cell viability. Results. For concentration between 5 and 10 ${\mu}M$, CFSE did not significantly label the MC3T3 in vitro. The destruction of MC3T3 was observed at the concentration of 20 ${\mu}M$. In the concentration of 15 ${\mu}M$, the best labelling was obtained at an incubation period between 15 and 30 minutes. The MC3T3 labelled with an incubation period of 15 minutes at 15 ${\mu}M$ was still fluorescent 7 days after CFSE labelling. The mean cell viability was 95.93%. Conclusion. These results suggests an incubation period of 15 minutes at 15 ${\mu}M$ of CFSE provides best labelling of MC3T3 in vitro.

• Journal of the Korean Wood Science and Technology
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• v.46 no.3
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• pp.253-259
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• 2018

To determine the depth of preservative penetration in ACQ treated wood, the degree of penetration of Cu was measured. In this study, we developed a DDAC coloring method to investigate the penetration depth of DDAC, which is one of the active ingredient of ACQ, into wood. The following conclusions were obtained. The DDAC component reacts with a 2', 7'-dichlorofluorescein indicator and results in a deep orange color. This orange coloring reaction appears not only in DDAC solutions but also in ACQ treated wood tissues in which DDAC is present. It is possible to visually verify that DDAC has better wood penetration than Cu in the spruce, which is an refractory wood species, by the DDAC coloring method developed this study. In addition to the results, it is necessary to investigate the difference in penetration of Cu and DDAC for other wood species with poor preservative penetration.

• Environmental Analysis Health and Toxicology
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• v.11 no.1_2
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• pp.49-57
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• 1996

The production of reactive oxygen species on addition of hexavalent chromium (potassium dichromate, $K_2Cr_2O_7$ ) to lung cells in culture was studied using flow cytometer analysis. A Coulter Epics Profile flow cytometer was used to detect the formation of reactive oxygen species after $K_2Cr_2O_7$ was added to A549 cells grown to confluence. The cells were loaded with the dye, 2',7'-dichlorofluorescein diacetate, after which cellular esterases removed the acetate groups and the dye was trapped intracellularly. Reactive oxygen species oxidized the dye, with resultant fluorescence. Increased doses of Cr(VI) caused increasing fluorescence (10-fold higher than background at 200 gM). Addition of Cr(III) compounds, as the picolinate or chloride, caused no increased fluorescence. Electron paramagnetic resonance (EPR) spectroscopic studies indicated that three (as yet unidentified) spectral "signals" of the free radical type were formed on addition of 20, 50, 100 and 200 gM Cr(VI) to the A549 cells in suspension. Two other EPR 'signals" with the characteristics of Cr(V) entities were seen at field values lower than the standard free radical value. radical value.

• Fisheries and Aquatic Sciences
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• v.14 no.3
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• pp.179-185
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• 2011

The antioxidant activities of a methanolic extract of Eisenia bicyclis and its organic solvent fractions, including dichloromethane ($CH_2Cl_2$), ethyl acetate (EtOAc), n-butanol (n-BuOH), and water ($H_2O$) fractions, were investigated. Scavenging activities against DPPH, hydroxyl, superoxide anion, and peroxynitrite radicals were evaluated using electron spin resonance spectrometry; intracellular reactive oxygen species (ROS) were evaluated by a 2',7'-dichlorofluorescein diacetate assay using RAW264.7 mouse macrophages. The antioxidant activities of the individual fractions were: EtOAc>n-BuOH>$CH_2Cl_2$ >$H_2O$. The EtOAc fraction exhibited strong radical scavenging activity and a significantly reduced ROS level in RAW264.7 cells. Moreover, the phenolic contents of the extract and fractions followed the same order as their radical scavenging activities. Our results indicate that E. bicyclis is a valuable natural source of antioxidants that may be applicable to the functional food industry.

• Preventive Nutrition and Food Science
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• v.19 no.4
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• pp.261-267
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• 2014

As a part of ongoing research to elucidate and characterize antioxidant and anti-inflammatory nutraceuticals, solvent-partitioned fractions from Spergularia marina were tested for their ability to scavenge radicals and suppress inflammation. The results of the 2',7'-dichlorofluorescein diacetate assay indicate that solvent-partitioned fractions from S. marina scavenged intracellular radicals in $H_2O_2$-stimulated mouse macrophages. The tested fractions decreased the generation of nitric oxide (NO) and the expression of inflammation mediators, namely, inducible nitric oxide synthase (iNOS) and interleukin (IL)-6, by lipopolysaccharide (LPS)-induced mouse macrophages, indicating that S. marina decreases inflammation. Among all tested fractions [i.e., $H_2O$, n-buthanol (n-BuOH), 85% aqueous methanol (aq. MeOH), and n-hexane], the 85% aq. MeOH fraction showed the strongest antioxidant and anti-inflammatory response. The 85% aq. MeOH fraction scavenged 80% of the free radicals produced by $H_2O_2$-induced control cells. In addition, NO production was 98% lower in 85% aq. MeOH fraction-treated cells compared to LPS-induced control cells. The mRNA expression of iNOS and IL-6 was also suppressed in 85% aq. MeOH fraction-treated cells. The results of the current study suggest that the phenolic compound components of S. marina are responsible for its antioxidant and anti-inflammatory effects.

• Applied Biological Chemistry
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• v.38 no.5
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• pp.478-483
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• 1995

To investigate the constituents of antimutagenic factor from brown rice, methanol extracts were fractionated into ether, ethylacetate, buthanol and aqueous fractions. The ether fractions showed distinct antimutagenic effect and active spot were selected by silica gel chromatography. The specific activity of active spot decreased with isolation of the active components from the methanol extract. Qualitative analyses of the active spot by using various spraying reagents revealed that ninhydrin and orcinol did not develop colored reactions. But, ferric chloride, 2,7-dichlorofluorescein, antimony pentachloride, phosphomolybdic acid, bromothymol blue and rhodamine 6G led to colored reactions. These results suggested that the consitituents of active material were neither polar nor nitrogen-containing compounds and that they may contain phenolic compounds and fatty acid derivatives. Main compounds of the active spot were analyzed to be o-hydroxy benzyl alcohol(saligenin), octanoic acid(caprylic acid), 9,12-cis-octadecadienoic acid(linoleic acid), 11-cis-octadecenoic acid(oleic acid), hexadecanoic acid(palmitic acid), 1H-indole-2-carboxylic acid and 1,2-benzenedicarboxylic acid(phthalate) in GC/Mass spectrum, and antimutagenicity of these active compounds using standard regeant was reconfirmed in S. typhimurium reversion assay.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.61-69
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• 1993

In order to search for new antibiotics from anaerobic bacteria, a large number of samples from domestic soil were collected and processed by apropriate methods. A potential strain, Streptococcus sp. An-21-1, was found to produce antimicrobial compounds. The Results were as follows; 1. During fermentation, the bacteria grew rapidly up to 20hr, thereafter entered the death phase. The optimal temperature and pH for the bacterial growth were $37^{\circ}C$ and pH 7.0, respectively. 2. Antibiotics were purified from culture broth by solvent extraction, silica gel column chromatography and Sepadex L.H 20 column. 3. Physicochemical properties of Ap-1 and Ap-2 were similar ; Their melting points were between $234-237^{\circ}C$. Color reactions of ninhydrin, 2,7-dichlorofluorescein, 4-dimethylaminobenzaldehyde, Dragendroffs reagent and 20% $H_2SO_4$, were positive. Therefore, we assumed that these antibiotics have amine group, immine group, alkaloid, and lipid components. These were stable to heat. UV spectrophotometry showed two peaks at 210 nm and 260 nm. From above results, we assumed these antibiotics are belong to the peptide antibiotic family.

• Archives of Pharmacal Research
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• v.27 no.2
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• pp.194-198
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• 2004

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human degenerative diseases such as cancer, aging, arteriosclerosis, and rheumatism. Much attention has been focused on the development of safe and effective antioxidants. To discover sources of antioxidative activity in marine algae, extracts from 17 kinds of seaweed were screened for their inhibitory effect on total ROS generation in kidney homogenate using 2',7'-dichlorofluorescein diacetate (DCFH-DA). ROS inhibition was seen in three species: UIva pertusa, Symphyocladia latiuscula, and Ecklonia stolonifera. At a final concentration of 25 $\mu\textrm{g}$/mL, U. pertusa inhibited 85.65$\pm$20.28％ of total ROS generation, S. latiscula caused 50.63$\pm$0.09％ inhibitory, and the Ecklonia species was 44.30$\pm$7.33％ inhibition. E. stolonifera OKAMURA (Lam-inariaceae), which belongs to the brown algae, has been further investigated because it is commonly used as a foodstuff in Korea. Five compounds, phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5), isolated from the ethyl acetate soluble fraction of the methanolic extrclct of E. stolonifera inhibited total ROS generation.

• Korean Journal of Veterinary Research
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• v.62 no.1
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• pp.3.1-3.7
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• 2022

Disulfiram (DSF) is a marketed drug to treat patients with alcohol dependence by inhibiting aldehyde dehydrogenase. Over the last few decades, DSF has been shown to have anticancer effects through different mechanisms. Moreover, this effect can be elevated when used with copper (Cu). Subsequent studies have been conducted on various cancers, but few on lymphoma. This study investigated the anticancer effects of DSF on lymphoma and how this effect changed when treated with Cu. DSF synergistically decreased the metabolic activity of EL4 lymphoma cells when combined with Cu. At 1 µM of DSF alone, the metabolic activity of EL4 cells decreased by 49% compared to the control, whereas it decreased by 87% with a DSF + CuCl₂ treatment. Rhodamine 123 and 2',7'-dichlorofluorescein diacetate staining showed that DSF induced the reduction of the mitochondrial membrane potential and promoted the production of reactive oxygen species. In particular, the combined treatment of DSF + Cu induced cell death based on multiple assays, including annexin V-fluorescein isothiocyanate/propidium iodide staining. Overall, DSF has anticancer effects on lymphoma cells and exhibits synergistic effects when combined with Cu. This study provides some valuable information to broaden the use of DSF in clinics and basic research.