• Title/Summary/Keyword: Indicator assay

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A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay

  • Kim, Yong Hyun;Sathiyanarayanan, Ganesan;Kim, Hyun Joong;Bhatia, Shashi Kant;Seo, Hyung-Min;Kim, Jung-Ho;Song, Hun-Seok;Kim, Yun-Gon;Park, Kyungmoon;Yang, Yung-Hun
    • Journal of Microbiology and Biotechnology
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    • v.25 no.12
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    • pp.2110-2115
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    • 2015
  • A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

Highly catalysis Zinc MOF-loaded nanogold coupled with aptamer to assay trace carbendazim by SERS

  • Jinling Shi;Jingjing Li;Aihui Liang;Zhiliang Jiang
    • Advances in nano research
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    • v.14 no.4
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    • pp.313-327
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    • 2023
  • Zinc metal organic framework (MOFZn)-loaded goad nanoparticles (AuNPs) sol (Au@MOFZn), which was characterized by TEM, Mapping, FTIR, XRD, and molecular spectrum, was prepared conveniently by solvothermal method. The results indicated that Au@MOFZn had a very strong catalytic effect with the nanoreaction of AuNPs formation between sodium oxalate (SO) and HAuCl4. AuNPs in the new indicator reaction had a strong resonance Rayleigh scattering (RRS) signal at 370 nm. The indicator AuNPs generated by this reaction, which had the most intense surface enhanced Raman scattering (SERS) peak at 1621 cm -1. The new SERS/RRS indicator reaction in combination with specific aptamer (Apt) to fabricate a sensitive and selective Au@MOFZn catalytic amplification-aptamer SERS/RRS assay platform for carbendazim (CBZ), with SERS/RRS linear range of 0.025-0.5 ng/mL. The detection limit was 0.02 ng/mL. Similarly, this assay platform has been also utilized to detect oxytetracycline (OTC) and profenofos (PF).

Study on the Clinical Validity of Sperm Penetration Assay (Sperm Penetration Assay의 임상적 타당성에 관한 연구)

  • Pang, Myung-Geol;Oh, Sun-Kyung;Shin, Chang-Jae;Kim, Jung-Gu;Moon, Shin-Yong;Chang, Yoon-Seok;Lee, Jin-Yong
    • Clinical and Experimental Reproductive Medicine
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    • v.20 no.1
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    • pp.1-7
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    • 1993
  • The present study was designed to test the validity of the semen analysis(S/A) and the sperm penetration assay(SPA) as a prognostic indicator of male fertility in 123 patients undergoing in vitro fertilization(IVF). We attempted to correlate the traditional semen parameters or the extent of sperm penetration in SPA with the results of human IVF rate or cleavage rate. Poor correlation was found between the results of S/A and human IVF rate(sensitivity, 80.6% ;specificity, 46.7%; positive predictive value, 91.6%;negative predictive value, 25%). Conversely, good correlation was found between the results of SPA and human IVF rate(sensitivity, 100% ; specificity, 80% ;positive predictive value, 97.3% ;negative predictive value, 100%). Our results corroborate the conclusion that SPA can be a valuable tool as a prognostic indicator of male fertilizing ability.

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Estimating dehalogenation reactivity of nanoscale zero-valent iron by simple colorimetric assay by way of 4-chlorophenol reduction

  • Mines, Paul D.;Kaarsholm, Kamilla M.S.;Droumpali, Ariadni;Andersen, Henrik R.;Hwang, Yuhoon
    • Environmental Engineering Research
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    • v.25 no.2
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    • pp.197-204
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    • 2020
  • A number of different nanoscale zero-valent iron (nZVI) materials have been prepared and compared depending on the desired properties for the particular application, but different physicochemical properties of this prepared nZVI make it difficult to universally compare and standardize them to the same scale. In this study, we aimed to demonstrate a simple microplate-based colorimetric assay using 4-chlorophenol as an indicator with respect to the remediation of real treatment targets, such as trichloroethylene (TCE), 1,1,1-trichloroethane (TCA), and atrazine. Effect of nickel contents on 4-chlorophenol reduction was successfully investigated by the miniaturized colorimetric assay. In the same manner, the effect of nickel contents on dehalogenation of TCE, TCA, and atrazine was investigated and the pseudo-first-order kinetic constants were compared with the results for 4-chlorophenol. The similar pattern could be observed between 4-chlorophenol reduction obtained by colorimetric assay and TCE, TCA, atrazine reduction obtained by a traditional chromatographic method. The reaction kinetics does not match perfectly, but the degree of reaction can be estimated. Therefore, the colorimetric assay can be a useful and simple screening tool to determine nZVI reactivity toward halogenated organics before it is applied to a particular remediation site.

Use of the Comet Assay to Assess DNA Damage in Hemocytes and Gill of Oyster(Crassostrea gigas) Exposed to Pyrene and Benzo(a)pyrene (Pyrene과 Benzo(a)pyrene에 노출된 굴의 혈구세포과 아가미 세포에서의 DNA손상 측정을 위한 Comet assay의 이용)

  • 김기범;배세진
    • Journal of Aquaculture
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    • v.16 no.3
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    • pp.196-201
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    • 2003
  • Sessile organisms such as the oyster Crassostrea gigas have been given much attention as a potential biomonitoring indicator to assess the impact of toxicants on aquatic organism. In this study, we exposed cells isolated from gill of oyster (Crassostrea gigas) to hydrogen peroxide in vitro. In addition oysters were in vivo exposed to pyrene and benzo(a)pyrene at various concentrations for 2 weeks. Comet assay was used to detect DNA single strand breaks and to investigate the application of this technique as a tool for aquatic biomonitoring. Hydrogen peroxide increased DNA single strand break with increasing concentration after 30 minutes exposure in vitro. Pyrene and benzo(a)pyrene caused DNA damage only at very high concentration (100 $\mu\textrm{g}$/L or 1000 $\mu\textrm{g}$/L) at two week exposure in vivo. DNA damage was relatively higher at hemocyte than at gill. It suggested that metabolized PAHs are transferred to hemolymph from digestive gland which have a relatively high enzyme activity, and attacked the DNA of hemocyte, while gill accumulated PAHs without degrading them to their metabolites due to low enzyme activity at gill. Both in vitro and in vivo exposure experiments showed that the comet assay is an effective tool on screening whether the organism are exposed to genotoxic contaminants.

Studies on the microbiological assay method for tabtoxin produced in pseudomonas syringae pv. tabaci (Pseudomonas syringae pv. tabaci가 생산하는 tabtoxin의 미생물학적 검색방법에 관한 연구)

  • 백형석;구재관;전홍기
    • Korean Journal of Microbiology
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    • v.27 no.3
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    • pp.310-315
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    • 1989
  • Tabtoxin produced in Pseudomonas syringae pv. tabace irreversibly inhibits its known physiological target, glutamine synthetase so that causes wildfire disease on leaves of host plant. In this study, we examined a rapid and sensitive microbiological method for tabtoxin assay in several media. In minimal A agar medium nd minimal glucose agar medium, growth inhibition zone of Agrobacterium tumefaciens was larger than that of other indicator strain. However, mostly, growth inhibition zone of indicator strains on the minimal glucose agar medium was smaller than that of on the miniaml A agar medium. In complex agar medium, growth inhibithiton zone was not observed in all the tested indicator strains. Pseudomonas syringae pv. tabaci produced more tabtoxin according to the incubation time. When glutamine was added to the minimal glucose agar medium, growth inhkbition zone of Agrobacterium tumefaciens was reduced.

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Protective effect of Korean diet food groups on lymphocyte DNA damage and contribution of each food group to total dietary antioxidant capacity (TDAC) (한식 식품군의 in vitro 총 항산화능 (TDAC)과 ex vivo DNA 손상 보호효과와의 관련성)

  • Lee, Min Young;Han, Jeong-Hwa;Kang, Myung-Hee
    • Journal of Nutrition and Health
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    • v.49 no.5
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    • pp.277-287
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    • 2016
  • Purpose: This study was performed to compare total phenolic contents, in vitro antioxidant capacity, and reduction effect of Korean food groups on ex vivo DNA damage in human cells and analyze correlations between each indicator. Methods: Vegetable foods in the Korean diet based the results of the KNHANES V-2 (2011) were classified into 10 food groups: cereals, fruits, vegetables, nuts, kimchi, seaweeds, potatoes, mushrooms, legumes, and oils. Eighty-four foods constituted more than 1% of the total intake in each food group and finally designated as vegetable foods in the Korean diet. Total phenolic content of each food group was measured. Further, in vitro antioxidant capacity was measured based on DPPH radical scavenging assay, TEAC assay, and $ORAC_{ROO{\cdot}}$ assay. Ex vivo DNA damage in human lymphocytes was assessed using comet assay. Results: Total phenolic contents of food groups of the Korean diet increased in the order of mushrooms, fruits, vegetables, seaweeds, and kimchi. Meanwhile, antioxidant rankings of food groups as mean values from the three in vitro test methods increased in the order of mushrooms, seaweeds, vegetables, kimchi, and fruits. Protection against ex vivo DNA damage in human lymphocytes was highest in mushrooms, followed by vegetables, fruits, seaweeds, and kimchi. The rankings of the food groups for total phenolic content, in vitro DAC, and ex vivo DNA protection activity were similar, and correlations between each indicator were significantly high. Conclusion: Mushrooms, fruits, vegetables, and seaweeds among the tested food groups in the Korean diet showed high total phenolic contents, in vitro antioxidant capacities, and protection against DNA damage. Correlations between each indicator in terms of total phenolic content, in vitro antioxidant capacity, and ex vivo DNA protection between each food group were found to be particularly high.

Comparison between the antioxidant properties of environment-friendly agro-produce and processed food

  • Kim, Soo-Mi;Kim, Joo-Hee;Son, Myoung-Jin;Kim, Se-Young;Rico, Catherine;Kang, Mi-Young
    • Proceedings of the Korean Society of Organic Agriculture Conference
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    • 2009.12a
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    • pp.316-316
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    • 2009
  • This study was conducted to investigate the effects of environment-friendly materials on garlic cultivation, and the methods for processing garlic at proper ripening stage to increase its antioxidant properties and market value. The functional properties of garlic and processed black garlic were analyzed in vitro and ex vivo. The ethanol extracts of garlic and black garlic were used in the antioxidant, linoleic acid autooxidation, lipid oxidation and RAW264.7 indicator cell using NO induced activity analyses. Also, toxicity tests by RBL-2H3 and RAW264.7 indicator cell using MTT assay were compared. Obesity inhibition test of garlic and black garlic were also studied.

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Identification of 3'-Hydroxymelanetin and Liquiritigenin as Akt Protein Kinase Inhibitors

  • Yang Hye-Young;Lee Hong-Sub;Ko Jong-Hee;Yeon Seung-Woo;Kim Tae-Yong;Hwang Bang-Yeon;Kang Sang-Sun;Chun Jae-Sun;Hong Soon-Kwang
    • Journal of Microbiology and Biotechnology
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    • v.16 no.9
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    • pp.1384-1391
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    • 2006
  • The signal transduction system is one of the most important devices involved in maintaining life, and many protein kinases are included in the cellular signal transduction system. Finding a protein kinase inhibitor is very valuable, as it can be used to study cell biology and applied to pharmaceuticals. For the efficient and rapid screening of protein kinase inhibitors, two assay systems were combined; the nonradioactive protein kinase assay system that uses an FITC-labeled IRS-2 peptide and the cell-based paper disc assay system that uses Streptomyces griseus as the indicator strain. Among 330 kinds of herb extracts tested, the extract of Dalbergia odorifera exhibited the strongest inhibitory activity in the two assay systems and was selected for further isolation. Based on solvent extraction and many steps of chromatography, seven compounds were finally separated to homogeneity and their structures determined by $^{1}H$ and $^{13}C$ NMR spectroscopies. Four were to be flavonoids and identified as butin ($C_{15}H_{12}O_5$, Mw=272.07), 3'-hydroxymelanetin ($C_{16}H_{12}O_6$, Mw=300.06), liquiritigenin ($C_{15}H_{12}O_4$, Mw=256.07), and 2'-hydroxyformononetin ($C_{16}H_{12}O_{5}$, Mw=284.07). 3'-Hydroxymelanetin inhibited the phosphorylation of the GSK3 protein by Akt to 37% at a concentration of $10{\mu}g/ml$ and showed the strongest cytotoxicity ($ED_{50}<50{\mu}g/ml$) against the human cancer cell line HCT116. Under the same conditions, liquiritigenin also inhibited the phosphorylation of GSK3 by Akt to 26%, and its cytotoxicity against the HCT116 cell line was lower than $100{\mu}g/ml$.