• Biomolecules & Therapeutics
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• 제18권2호
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• pp.152-158
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• 2010

Melanin concentrating hormone is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G-protein coupled receptor family, especially plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. Although fluorescence-based calcium mobilization assay platform has been one of the most widely accepted tools for receptor research and drug discovery, fluorescence interference and shallow assay window limit their application in high throughput screening and have led to a growing interest in alternative, luminescence-based technologies. Herein, a luminescence-based functional assay system for the MCH1 receptor was developed and validated with the mitochondrial targeted aequorin. Aequorin based functional assay system for MCH1 presented excellent Z' factor (0.8983) and high signal-to-noise ratio (141.9). The nonpeptide MCH1 receptor antagonist, SNAP 7941 and GSK 803430, exhibited $IC_{50}$ values of 0.62 ${\pm}$ 0.11 and 12.29 ${\pm}$ 2.31 nM with excellent correlation coefficient. These results suggest that the aequorin based assay system for MCH1 is a strong alternative to the traditional GPCR related tools such as radioligand binding experiments and fluorescence functional determinations for the compound screening and receptor research.

• Animal cells and systems
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• 제13권2호
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• pp.97-103
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• 2009

Recently, great advance is achieved in the field of genome-wide functional screening using cell-based assay. Here, we briefly introduce well-established and typical cell-based assays of GPCR and some parameters which should be considered for genome-wide functional screening. Because of characters and importance of GPCR as drug targets, several ways of assay systems were devised. Among them, high-content screening (HCS) that is based on the analysis of image by confocal microscope is becoming favorite choice. The advances in this technology have been driven exclusively by industry for their convenience. Now, it is turn for academy to define more detail signaling networks via HCS using cDNA or siRNA libraries at genome-wide level. By isolating novel signaling mediators using cDNA or siRNA library, and postulating them as new candidates for therapeutic target, more understanding about life science and more increased chances to develop therapeutics against human disease will be achieved.

• 한국식품과학회지
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• 제32권4호
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• pp.747-754
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• 2000

Comet assay를 이용하여 방사선 조사육의 조사여부 및 조사량을 판별해내는 방법을 개발하기 위해 1-10 kGy의 감마선 조사량으로 조사한 육조직에서 일어나는 DNA 손상을 측정하였다. Comet assay에 있어 최적의 comet image를 얻기 위해 세포 분리, 세포 lysis 및 전기영동에 대한 여러 조건들을 적용하여 최적의 조건을 확립하였다. DNA 손상도는 관찰되는 comet의 평균 tail length와 tail length에 의해 구분된 4 손상 등급에의 분포, 그리고 그 분포비율에 의해 본 연구에서 제시한 식에 의해 계산한 relative damage index(RDI) 값 등으로 비교 판정하였다. 평균 tail length와 RDI 값은 조사량이 증가함에 따라 증가하여 DNA 손상도가 증가됨을 나타내었으며, 평균 tail length으로는 조사량 간의 차이를 명확히 분별하기가 어려웠던 반면 RDI 값에 의하면 조사여부 및 조사량을 판별하기에 적합함을 알 수 있었다. 국내산 한우육과 수입 냉장육에 대하여 blind test를 실시한 결과 수입육이 높은 DNA 손상도를 나타내었는데 그 RDI 값은 방사선 조사에 의한 값보다는 비교적 낮은 것이어서 수입육의 DNA 손상은 방사선 조사가 아닌 저온처리의 결과인 것으로 판단되었다. 본 연구의 결과로부터 Comet assay가 우육의 방사선 조사여부 및 조사량 판별에 유용한 기술로 응용될 수 있을 것으로 판단되었다.

• The Plant Pathology Journal
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• 제35권4호
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• pp.372-380
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• 2019

Pseudomonas syringae pv. actinidiae (Psa) is by far the most important pathogen of kiwifruit. Sustainable expansion of the kiwifruit industry requires the use of Psa-tolerant or resistant genotypes for the breeding of tolerant cultivars. However, the resistance of most existing kiwifruit cultivars and wild genotypes is poorly understood, and suitable evaluation methods of Psa resistance in Actinidia have not been established. A unique in vitro method to evaluate Psa resistance has been developed with 18 selected Actinidia genotypes. The assay involved debarking and measuring the lesions of cane pieces inoculated with the bacterium in combination with the observation of symptoms such as callus formation, sprouting of buds, and the extent to which Psa invaded xylem. Relative Psa resistance or tolerance was divided into four categories. The division results were consistent with field observations. This is the first report of an in vitro assay capable of large-scale screening of Psa-resistance in Actinidia germplasm with high accuracy and reproducibility. The assay would considerably facilitate the breeding of Psa-resistant cultivars and provide a valuable reference and inspiration for the resistance evaluation of other plants to different pathogens.

• 한국자원식물학회지
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• 제22권4호
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• pp.323-329
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• 2009

기능성 옥수수 수염을 가지는 옥수수 계통을 선발하기 위해 다양한 자식계통과 F1을 대상으로 옥수수 수염의 항산화 성분과 항산화능을 측정하였다. 측정결과, 총 페놀 함량, 총 플라보노이드 함량의 경우 각각 980${\sim}$2,420 mg 100 $g^{-1}$, 1,532${\sim}$3,274 mg 100 $g^{-1}$의 범위를 나타내었다. 안토시아닌의 고유영역인 517 nm에서의 흡광도는 0.05에서 0.76까지의 범위를 보였다. 옥수수 수염의 항산화 성분과 DPPH, ABTS, FRAP assay에서 얻어진 항산화능간의 상관관계를 분석한 결과, ABTS와 FRAP assay에서 옥수수 수염의 항산화 활성과 페놀성물질 및 안토시아닌간의 상관계수가 유의하게 높았다. 옥수수 수염의 안토시아닌을 추출하는 용매가 산성이라는 점과 안토시아닌의 DPPH 용액과의 가시영역 파장에서의 간섭을 고려할 때, DPPH 보다는 ABTS assay나 FRAP assay가 더 적합한 항산화능 측정 방법이 될 것이다. 이들 중 특히 ABTS assay는 옥수수 수염의 주요한 기능성 성분인 안토시아닌과 플라보노이드 및 페놀성 물질과 모두 높은 상관성을 보여 옥수수 수염의 종합적인 항산화능을 평가하고 우수 계통을 선발하는데 적합한 측정 방법이 될 것으로 사료된다. 본 실험에서 안토시아닌 함량과 페놀 함량이 높고 ABTS assay와 FRAP assay에서 모두 높은 항산화능을 보여준 S15은 고 기능성 품종 육종 및 옥수수 수염을 이용한 기능성 소재 개발에 이용될 수 있을 것이다. 또한 옥수수 수염의 항산화능 측정에 대한 항산화 실험의 적합성 연구는 우수한 옥수수 수염을 선발하는데 도움이 될 것이다.

• Journal of Korean Medical Science
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• 제33권51호
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• pp.340.1-340.14
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• 2018

Background: Various pneumococcal vaccines have been evaluated for immunogenicity by opsonophagocytic assay (OPA). A multiplexed OPA (MOPA) for 13 pneumococcal serotypes was developed by Nahm and Burton, and expanded to 26 serotypes in 2012. The development of new conjugate vaccines with increased valence has necessitated expanded MOPAs to include these additional serotypes. In this study, we validated this expanded MOPA platform and applied to measure antibodies against 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F) in human sera. Methods: All materials, including serum, complement, bacterial master stocks, and HL-60 cells, were evaluated for assay optimization. Following optimization, the assay was validated for accuracy, specificity, and intra- and inter-assay precision with sera from adult donors following standard protocols. The assay was applied to evaluate functional antibodies of 42 sera immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23). Results: The expanded MOPA platform was specific for all serotypes, with the exception of serotype 20. The assay results were highly correlated with those obtained from single-serotype OPA, indicating acceptable accuracy. The coefficients of variation were 7%-24% and 13%-39% in tests of intra- and inter-assay precision, respectively, using three quality-control samples. A MOPA that included 11 additional serotypes in the PPV23 was established and validated with respect to accuracy, specificity, and precision. The opsonic indices of immune sera were obtained using this validated assay. Conclusion: The expanded MOPA will be useful for evaluation of the immunogenicity of PPV23 and future conjugate vaccine formulations.

• Preventive Nutrition and Food Science
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• 제8권1호
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• pp.54-60
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• 2003

To develop a functional chungkookjang; the anticancer effects of chungkookjangs prepared with different varieties of soybeans, starters, fermentation periods and seasoning additive ratios; were studied against AGS human gastric adenocarcinoma cells using the MTT assay, at different stages chungkookjang processing. The chungkookjang samples exerted different antiproliferative effects according to the variety of soybeans used. The chungkookjangs manufactured with soybean var. manrikong exhibited the highest cytotoxicity against AGS human cancer cells. The chungkookjangs fermented with rice straw and B. licheniformis strongly inhibited the growth of the AGS human cancer cells. All fermented chungkookjangs had a strong inhibitory effect on the growth of the cancer cells; however, the non-fermented soybean (chungkookjang) showed a low inhibition rate. The fermented chungkookjangs mixed with red pepper powder (RPP) and garlic exhibited strong antiproliferative effect against the cancer cells, and chungkookjang prepaved with 1.1 % RPP and 1.1 % garlic showed the highest cytotoxicity against the cancer cells. The functional chungkookjang fermented with soybean variety of manrikong and B. licheniformis for 3 days at 4$0^{\circ}C$ and then mixed with 7.9% salt, 1.l% RPP and 1.1% garlic, exhibited a higher antiproliferative effect than the chungkookjangs prepared by traditional or modified methods, according to the MTT assay. The functional chungkookjang exhibited a similar anticancer effect to the traditional doenjang. These results indicate that the fermentation period and the ratio of seasoning additives, as well as the variety of soybeans and starter cultures may affect the degree of the anticancer effect of chungkookjang.

• Journal of Ginseng Research
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• 제41권2호
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• pp.169-179
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• 2017

Background: Ginsenosides are the main pharmacological components of Panax ginseng root, which are thought to be primarily responsible for the suppressing effect on oxidative stress. Methods: 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and oxygen radical absorption capacity were applied to evaluate the antioxidant activities of the ginsenosides. Human embryonic kidney 293 (HEK-293) cells were incubated with ginsenosides extracted by pulsed electric field (PEF) and solvent cold soak extraction (SCSE) for 24 h and then the injury was induced by $40{\mu}M$ $H_2O_2$. The cell viability and surface morphology of HEK-293 cells were studied using MTS assay and scanning electron microscopy, respectively. Dichloro-dihydro-fluorescein diacetate fluorescent probe assay was used to measure the level of intracellular reactive oxygen species. The intracellular antioxidant activities of ginsenosides were evaluated by cellular antioxidant activity assay in HepG2 cells. Results: The PEF extracts displayed the higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and stronger oxygen radical absorption capacity (with an oxygen radical absorption capacity value of $14.48{\pm}4.04{\mu}M\;TE\;per\;{\mu}g/mL$). The HEK-293 cell model also suggested that the protective effect of PEF extracts was dose-dependently greater than SCSE extracts. Dichloro-dihydro-fluorescein diacetate assay further proved that PEF extracts are more active (8% higher than SCSE extracts) in reducing intracellular reactive oxygen species accumulation. In addition, scanning electron microscopy images showed that the HEK-293 cells, which were treated with PEF extracts, maintained more intact surface morphology. Cellular antioxidant activity values indicated that ginsenosides extracted by PEF had stronger cellular antioxidant activity than SCSE ginsenosides extracts. Conclusion: The present study demonstrated the antioxidative effect of ginsenosides extracted by PEF in vitro. Furthermore, rather than SCSE, PEF may be more useful as an alternative extraction technique for the extraction of ginsenosides with enhanced antioxidant activity.

• 한국식품과학회지
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• 제31권4호
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• pp.1071-1076
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• 1999

본 연구에서는 김치발효에 관여하는 우세 발효균들의 집락을 분리하고, 발암원인 MNNG에 대하여 이들 분리주들이 갖는 DNA 손상 억제효과를 조사하였다. 이를 위해서 세포의 DNA 손상 및 복구를 측정하는데 이용되는 매우 신속하고 감도가 높은 Single Cell Gel Electrophoresis(SCGE 법, 또는 comet assay)를 사용하였다. 배추김치에서 분리한 3개의 분리주중에서 B-3를 제외한 B-1과 B-2는 양성 대조구와 비교하였을 때 우수한 항유전 독성효과를 나타내었으며(p<0.01), 율무김치의 3 개의 분리주(Y-1, Y-2, Y-3)와 1개의 분리주(Y-4)도 각각 항유전 독성효과를 나타내었다(p<0.05, p<0.01). 총각김치의 5 개 분리주중에서 3개의 분리주와 깍두기의 9개의 분리주중에서 2 개의 분리주들도 각각 항유전 독성효과를 나타내었다(p<0.05, p<0.01).

• 한국자원식물학회지
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• 제29권5호
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• pp.588-597
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• 2016

Little attention has been paid to the functional aspect of the flower petal of Paeonia lactiflora, compared to that of its root. To determine the components of flower petal of Paeonia lactiflora, we conducted the Fourier transform ion cyclotron resonance (FT-ICR) MASS spectrophotometric analysis. We detected the 24 different types of ingredients from the 70% ethanol extracts of flower petal of peonia lactiflora cv. ‘Red Charm’. The main compounds were quercetin glucopyranosides, methyl gallate, paonioflolol and kaemperol glucopyranosides. We further tested its functional activity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the extracts was 87.9-90.4% at 0.1mg/ml. This result showed that these flower extracts have approximately 5-fold stronger antioxidant potential than a previous report with root extracts (Bang et al. 1999). The result of tyrosinase inhibition assay of Paeonia lactflora extract was almost similar to that of arbutin except significantly higher effect in the coral sunset extract at 0.1% concentration. Hyaluronidase inhibition assay showed 76.5% inhibition at 5% concentration of this flower extract, indicating that Peaonia lactiflora flower extracts have the major anti-inflammatory, anti-oxidant and brightening effects. Taken together, these results suggest these three Paeonia lactiflora species extracts might provide the basis to develop a new natural brightening agent.