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Establishment of a new cell-based assay to measure the activity of sweeteners in fluorescent food extracts.

ABSTRACT: Taste receptors have been defined at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca(2+) concentration using Ca(2+)-sensitive fluorescent dyes. However, this method cannot be applied to food-derived samples that contain fluorescent substances. To establish an assay system that would be applicable to fluorescent samples, we tested the use of Ca(2+)-sensitive photoproteins, such as aequorin and mitochondrial clytin-II, as Ca(2+) indicators in a human sweet taste receptor assay. Using these systems, we successfully detected receptor activation in response to sweetener, even when fluorescent compounds coexisted. This luminescence-based assay will be a powerful tool to objectively evaluate the sweetness of food-derived samples even at an industry level.

SUBMITTER: Toda Y

PROVIDER: S-EPMC3217308 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Establishment of a new cell-based assay to measure the activity of sweeteners in fluorescent food extracts.

Toda Yasuka Y Okada Shinji S Misaka Takumi T

Journal of agricultural and food chemistry 20111020 22

Taste receptors have been defined at the molecular level in the past decade, and cell-based assays have been developed using cultured cells heterologously expressing these receptors. The most popular approach to detecting the cellular response to a tastant is to measure changes in intracellular Ca(2+) concentration using Ca(2+)-sensitive fluorescent dyes. However, this method cannot be applied to food-derived samples that contain fluorescent substances. To establish an assay system that would be ...[more]

PMID: 21981007

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Project description:Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye-based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose-dependent, which may reflect changes in the reduction properties of the media. Our food color-based colorimetric assay has several advantages, including lower cost, irreversibility, and not requiring constant monitoring , over the commonly used 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for determining laccase activity. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression.IMPORTANCECryptococcus neoformans is present in the environment, and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently, cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor for C. neoformans. Developing a simple and less costly assay to determine the laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.

Dataset Information

Establishment of a new cell-based assay to measure the activity of sweeteners in fluorescent food extracts.

Publications

Establishment of a new cell-based assay to measure the activity of sweeteners in fluorescent food extracts.

Similar Datasets

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