• Title/Summary/Keyword: Quencher-free

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Label/Quencher-Free Detection of Exon Deletion Mutation in Epidermal Growth Factor Receptor Gene Using G-Quadruplex-Inducing DNA Probe

  • Kim, Hyo Ryoung;Lee, Il Joon;Kim, Dong-Eun
    • Journal of Microbiology and Biotechnology
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    • v.27 no.1
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    • pp.72-76
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    • 2017
  • Detection of exon 19 deletion mutation in the epidermal growth factor receptor (EGFR) gene, which results in increased and sustained phosphorylation of EGFR, is important for diagnosis and treatment guidelines in non-small-cell lung cancer. Here, we have developed a simple and convenient detection system using the interaction between G-quadruplex and fluorophore thioflavin T (ThT) for discriminating EGFR exon 19 deletion mutant DNA from wild type without a label and quencher. In the presence of exon 19 deletion mutant DNA, the probe DNAs annealed to the target sequences were transformed into G-quadruplex structure. Subsequent intercalation of ThT into the G-quadruplex resulted in a light-up fluorescence signal, which reflects the amount of mutant DNA. Due to stark differences in fluorescence intensity between mutant and wild-type DNA, we suggest that the induced G-quadruplex structure in the probe DNA can report the presence of cancer-causing deletion mutant DNAs with high sensitivity.

Effects of Antioxidants on the Photosynsthesis and Carbohydrates/Saponin Contents in Panax ginseng Leaves (인삼잎의 광합성과 탄수화물.사포닌 함량에 미치는 항산화제의 효과)

  • 양덕조;김용해
    • Journal of Ginseng Research
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    • v.18 no.3
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    • pp.175-181
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    • 1994
  • We studied the folilar wiping effects of antioxidants (ascorbate, glutathione and sodium azide), which effectively inhibited the chlorophyll bleaching or completely recorved the early stage of photosynthesis of Panax ginseng C.A. Meyer, on photosynthesis, stomatal resistance, free sugar, starch, and total saponin contents of ginseng under the excess light intensity (45 kLux) during 6 days. Ascorbate and glutathione, endogenous antioxidant, recovered photosynehtsis and stomatal resistance, and reduced the photoinhibition by the excess light intensity (45 kLux) on free sugar, starch and total saponin contents. But sodium azide, exogenous $^{1}O_2$ quencher, showed negative effect. Therefore, we assumed that carbohydrates and saponin metabolisms of ginseng by antioxidants (ascorbate, glutathione) were normal. For the reduction of inhibition by excess light in ginseng a program for the higher activation of antioxidants and antioxidative enzymes in ginseng leaf will be desirable. Key words Antioxidants, ascorbate, glutathione, Photoinhibition, ginseng.

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Effect of Number and Location of Amine Groups on the Thermodynamic Parameters on the Acridine Derivatives to DNA

  • Kwon, Ji Hye;Park, Hee-Jin;Chitrapriya, Nataraj;Han, Sung Wook;Lee, Gil Jun;Lee, Dong Jin;Cho, Tae-Sub
    • Bulletin of the Korean Chemical Society
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    • v.34 no.3
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    • pp.810-814
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    • 2013
  • The thermodynamic parameters for the intercalative interaction of structurally related well known intercalators, 9-aminoacridine (9AA) and proflavine (PF) were determined by means of fluorescence quenching study. The fluorescence intensity of 9AA decreased upon intercalation to DNA, poly[$d(A-T)_2$] and poly[$d(G-C)_2$]. A van't Hoff plot was constructed from the temperature-dependence of slope of the ratio of the fluorophore in the absence and presence of a quencher molecule with respect to the quencher concentration, which is known as a Stern-Volmer plot. Consequently, the thermodynamic parameters, enthalpy and entropy change, for complex formation was calculated from the slope and y-intercept of the van't Hoff plot. The detailed thermodynamic profile has been elucidated the exothermic nature of complex formation. The complex formation of 9AA with DNA, poly[$d(A-T)_2$] and poly[$d(G-C)_2$] was energetically favorable with a similar negative Gibb's free energy. On the other hand, the entropy change appeared to be unfavorable for 9AA-poly[$d(G-C)_2$] complex formation, which was in contrast to that observed with native DNA and poly[$d(A-T)_2$] cases. The equilibrium constant for the intercalation of PF to poly[$d(G-C)_2$] was larger than that to DNA, and was the largest among sets tested despite the most unfavorable entropy change, which was compensated for by the largest favorable enthalpy. The favorable hydrogen bond contribution to the formation of the complexes was revealed from the analyzed thermodynamic data.

Secondary Carotenoid Accumulation in Haematococcus (Chlorophyceae): Biosynthesis, Regulation, and Biotechnology

  • Jin Eon-Seon;Lee Choul-Gyun;Polle Jurgen E.W.
    • Journal of Microbiology and Biotechnology
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    • v.16 no.6
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    • pp.821-831
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    • 2006
  • Unicellular green algae of the genus Haematococcus have been studied extensively as model organisms for secondary carotenoid accumulation. Upon environmental stress, such as strong irradiance or nitrogen deficiency, unicellular green algae of the genus Haematococcus accumulate secondary carotenoids in vesicles in the cytosol. Because secondary carotenoid accumulation occurs only upon specific environmental stimuli, there is speculation about the regulation of the biosynthetic pathway specific for secondary carotenogenesis. Because the carotenoid biosynthesis pathway is located both in the chloroplast and the cytosol, communication between both cellular compartments must be considered. Recently, the induction and regulation of astaxanthin biosynthesis in microalgae received considerable attention because of the increasing use of this secondary carotenoid as a source of pigmentation for fish aquaculture, as a component in cancer prevention, and as a free-radical quencher. This review summarizes the biosynthesis and regulation of the pathway, as well as the biotechnology of astaxanthin production in Haematococcus.

Protective Effect of Astaxanthin Produced by Xanthophyllomyces dendrorhous Mutant on Indomethacin-Induced Gastric Mucosal Injury in Rats

  • Kim, Jeong-Hwan;Choi, Seok-Keun;Lim, Wang-Jin;Chang, Hyo-Ihl
    • Journal of Microbiology and Biotechnology
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    • v.14 no.5
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    • pp.996-1003
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    • 2004
  • Nonsteroidal anti-inflammatory drugs such as indomethacin induce severe gastric mucosal damage in humans and rodents. In the present study, the in vivo protective effect of astaxanthin on indomethacin-induced gastric lesions in rats was investigated. The test groups were injected with indomethacin (25 mg/kg) after the oral administration of astaxanthin (25 mg/kg) for 1, 2, and 3 days, while the control group was treated only with indomethacin. Thiobarbituric acid reactive substances in the gastric mucosa, as an index of lipid peroxidation, increased significantly after indomethacin administration and this increase was inhibited by oral administration of astaxanthin. In addition, pretreatment with astaxanthin resulted in a significant increase of the activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-px). Histologic examination clearly revealed acute gastric mucosal lesions induced by indomethacin in the stomach of the control group, but were not observed in that of the test group. These results indicate that astaxanthin activates SOD, catalase, and GSH-px, and removes the lipid peroxides and free radicals induced by indomethacin. It is evident that astaxanthin acts as a free radical quencher and antioxidant, and is an effective molecule in the remedy of gastric mucosal lesions.

Research Method of Fatty Acids Transfer between Phospholipid Model Membranes (인지질 모델막에서의 지방산 이동에 관한 연구 방법)

  • 임병순;김혜경;김을상
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.26 no.4
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    • pp.743-750
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    • 1997
  • Direct measurement of the kinetics of free fatty acid transfer between phospholipid model membrane is technically limited by the rapid nature of the transfer process. Separation of membrane-bound fatty acid by centrifugation has shown that although the equilibrium distribution of free fatty acid is determined by this method, fatty acid transfer occurs too rapidly for accurate kinetic measurements. Recently fluorescence resonance energy transfer(FRET) assay has been developed to examine transfer of fatty acids between membranes. Donor membranes which has fluorescent fatty acid, anthroyloxy fatty acid(AOFA), is mixed with acceptor membranes which has non-interchangeable fluorescent quencher, nitrobenzo-xadiazol(NBD), using stopped flow apparatus. As the fluorescent fatty acids transfer from donor membrane to acceptor membrane, fluorescence intensity would be decreased and the rate and degree of fatty acid transfer can be analyzed. Fatty acid transfer between micelles is more complicated because of bile salt. Therefore in experiments with micelles, fluorescence self quenching assay is used. At high concentrations, a fluorophore tends to quench its own fluorescence causing a reduction in fluorescence intensity. Donor micelles contained self quenching concentrations of fluorophore and acceptor micelles had no fluorophore. Upon mixing of donor and acceptor micelles, the rate of transfer of the fluorophore from the donor to the acceptor was measured by monitoring the release in self quenching when its concentration in donor decreased over time.

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Inhibitory Effect of Capsaicin against Carcinogen-induced Oxidative Damage in Rats

  • Yu, Ri-Na;Park, Min-Ah;Kawada, Teruo;Kim, Byung-Sam;Han, In-Seob;Yoo, Hoon
    • Preventive Nutrition and Food Science
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    • v.7 no.1
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    • pp.67-71
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    • 2002
  • Capsaicin (trans-8-methyl-N-vanillyl-6-nonenarnide), a major pungent component of hot pepper, is known to exert antioxidative properties. In this study, we investigated the protective effects of capsaicin against chemical carcinogen-induced oxidative damage in rats. Male Sprague Dawley rats weighting 230~250 g were treated with chemical carcinogens such as 2-nitropropane (2NP) or n-methyl-N'-nitro-N-nitrosoguanidine (MNNG) after (or before) the administration of capsaicin at doses of 0.5, 1,5 mg/kg. The level of lipid peroxidation in rat liver was estimated by measuring the amounts of thiobarbituric acid reactive substances. The degree of oxidative DNA damage was evacuated by measuring a DNA adduct, 8-hydroxydeoxyguanosine (8-OHdG), in urine. Antioxidative activities of capsaicin and its metabolites in vitro were determined by the measurement of DPPH (1,1-diphenyl-2-picrylhydrazyl), a radical quencher. Significant inhibition of 2-NP induced lipid peroxidation was observed in the liver of the rat when treated with capsaicin. MNNG-induced urinary excretion of 8-OHdG was decreased by capsaicin treatment. Capsaicin and its metabolites inhibited net only the formation of free radicals, but also lipid peroxidation in vitro. Our results show that capsaicin may function as a free radical scavenger against chemical carcinogen-induced oxidative cellular damage in vivo. The observed antioxidative activities of capsaicin may play an important role in the process of chemoprevention.

Spectroscopic Studies on the Mechanism of Interaction of Vitamin $B_{12}$ with Bovine Serum Albumin

  • Kamat, B.P.;Seetharamappa, J.
    • Journal of Photoscience
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    • v.11 no.1
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    • pp.29-33
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    • 2004
  • The mechanism of interaction of cyanocobalamin (CB) with bovine serum albumin (BSA) has been investigated by spectrofluorometric and circular dichroism methods. Association constant for the CB-BSA system showed that the interaction is non-covalent in nature. Binding studies in the presence of an hydrophobic probe, 8-anilino-l-naphthalene sulphonic acid, sodium salt (ANS) showed that there is hydrophobic interaction between CB and ANS and they do not share common sites in BSA. Stern-Volmer analysis of fluorescence quenching data showed that the fraction of fluorophore (protein) accessible to the quencher (CB) was close to unity indicating thereby that both tryptophan residues of BSA are involved in drug-protein interaction. The rate constant for quenching, greater than $10^{10}$ $M^{-1}$ $s^{-1}$, indicated that the drug binding site is in close proximity to tryptophan residue of BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of CB to BSA involves hydrophobic bonds predominantly. Significant increase in concentration of free drug was observed for CB in presence of paracetamol. Circular dichroism studies revealed the change in helicity of BSA due to binding of CB to BSA.

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