A Microtiter-Based Fluorescence Assay for (1,3)-β-Glucan Synthases

Shedletzky, Esther; Unger, Christoph; Delmer, Deborah P.

doi:10.1006/abio.1997.2162

Cited by 98 publications

(72 citation statements)

References 11 publications

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“…Activity of β(1,3)-glucan synthase present in MMF was measured with uridine-5′-diphospho-d-glucose as a substrate, using the microtitre plate fluorescence method described by Shedletzky et al 16 , based on the quantification of the aniline blue-glucan complex. The fluorescence was measured with the microplate reader at the excitation wavelength of 400 nm and the emission wavelength of 460 nm.…”

Section: Preparation Of the Mixed Membrane Fraction And Glucan Synthamentioning

confidence: 99%

Chemical reactivity and antimicrobial activity of N-substituted maleimides

Salewska

Boros-Majewska

Łącka

et al. 2011

Journal of Enzyme Inhibition and Medicinal Chemistry

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Section: Preparation Of the Mixed Membrane Fraction And Glucan Synthamentioning

confidence: 99%

Chemical reactivity and antimicrobial activity of N-substituted maleimides

Salewska

Boros-Majewska

Łącka

et al. 2011

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…In order to gain insight into the mode of action of PLME, we preliminarily examined the effect of the drug on the glucan production in membrane fractions of A. fumigatus. Glucan production was estimated using a fluorometric assay using aniline blue dye, which specifically binds to glucan to yield fluorescence [13]. The membrane fractions of A. fumigatus were prepared by the method of Shedletzaky et al [13].…”

Section: Fig 2 Effects Of Plme On Hyphal Morphology In Aspergillus Fmentioning

confidence: 99%

“…Glucan production was estimated using a fluorometric assay using aniline blue dye, which specifically binds to glucan to yield fluorescence [13]. The membrane fractions of A. fumigatus were prepared by the method of Shedletzaky et al [13]. The reaction mixture contained 50 mM Tris/HCl (pH 7.5), 20 m M GTP, 4 mM EDTA, 0.5% Brij 35, 6.6% glycerol, 0.2 mM UDP-glucose, and the membrane fraction.…”

Section: Fig 2 Effects Of Plme On Hyphal Morphology In Aspergillus Fmentioning

confidence: 99%

“…The reaction was terminated by the addition of 0.2 volume of 6.0 M NaOH. Glucan produced was solubilized at 80°C for 30 minutes followed by the addition of aniline blue mixture [13]. Fluorescence from glucan -aniline blue complex was quantified at an excitation wavelength of 400 nm and an emission wavelength of 460 nm.…”

Section: Fig 2 Effects Of Plme On Hyphal Morphology In Aspergillus Fmentioning

confidence: 99%

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Identification of Phoslactomycin E as a Metabolite Inducing Hyphal Morphological Abnormalities in Aspergillus fumigatus IFO 5840

et al. 2007

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In our survey for antifungal compounds, a fermentation broth of Streptomyces sp. HA81-2 was found to inhibit the in vitro growth of Aspergillus fumigatus IFO 5840 accompanied by hyphal morphological abnormalities. One of the isolated antibiotics was identified as phoslactomycin E based on LC-MS and NMR spectral data. In a preliminary assay using the membrane fractions of A. fumigatus, phoslactomycin E was found to inhibit the activity of 1,3-b glucan synthase.Keywords antifungal activity, phoslactomycin E, Aspergillus fumigatus, hyphal morphological abnormalities, 1,3-b glucan synthase Opportunistic fungal infections are a major cause of serious morbidity and mortality in immunocompromised patients. However, there are few drugs for antifungal chemotherapy of deep-seated mycosis. As both human and fungal cells are eukaryotes, it is difficult to develop antifungal drugs without side effects in human hosts. Current targets are limited to ergosterol, cell wall components, biosynthetic pathways of cell wall and cytosine deaminase [1,2]. Therefore, there is a great need for novel antifungal drugs with new mechanisms of action. Morphological deformation of fungal hyphae is often induced by antifungal drugs [3]. As this phenomenon is specific to fungi, it could be used to screen for selective antifungal antibiotics [4]. In addition, this hyphal deformation is expected to reflect the disruption of cell wall biosynthesis, which is a specific target of antifungal agents. Moreover, investigating the mechanism of antifungal-induced hyphal morphological abnormalities is potentially useful for understanding the morphogenesis of fungal hyphae.In the course of our search for antifungal antibiotics, we found that a fermentation broth of Streptomyces sp. HA81-2 exhibited antifungal activity against Aspergillus fumigatus IFO 5840 accompanied by hyphal abnormalities. The isolated active fraction was identified as phoslactomycin E (PLME) [5] based on LC-MS and NMR spectral data. We herein report the isolation procedures for PLME, its molecular characteristics, and its induction of morphological changes in fungal cells.Active principles were isolated as follows; fermentation broth was filtered, the filtrate was applied on Diaion HP-20 column, and then eluted using an increasing gradient of aq MeOH (10ϳ100%). The 80ϳ90% MeOH fractions were concentrated in vacuo to afford crude extract, which was partitioned between water and CHCl 3 . Purification of

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“…Callose accumulation has been analyzed by fluorescence microscopic observation of callose deposits stained by aniline blue on the leaves/tissues (Ellinger et al 2013;Oliveira-Garcia and Deising 2013). Although fluorescence spectroscopic determination of solubilized callose using aniline blue was also reported for the analysis of callose synthase activity in membrane preparations (Shedletzky et al 1997), we found it is difficult to apply the spectroscopic method for plant materials because of the high background of the alkaline extract of plant leaves.…”

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confidence: 99%

Quantification of stimulus-induced callose spots on plant materials

Kohari

Yashima

Desaki

et al. 2016

Plant Biotechnology

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Plants are known to synthesize and accumulate callose, a β-1,3-linked glucan, in response to pathogen infection, elicitor treatment and wounding. Although callose deposition has often been used as an index of defense responses in plant, its quantification is laborious and difficult. We have developed a simple computer program to quantify callose spots on microscopic images based on multiple Rotational Morphological Processing (RMP), which is suitable for the detection of biological spots with intricate morphology. The program developed in this study is useful for the detection of callose spots on various biological backgrounds, making it possible to compare the responses of plant materials with different genetic background or different stimuli. Validity of this program was supported by comparing the results obtained by the new method and conventional manual counting. This program can be applicable to wide range of images taken by various microscopic systems and also in variable formats.

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A Microtiter-Based Fluorescence Assay for (1,3)-β-Glucan Synthases

Cited by 98 publications

References 11 publications

Chemical reactivity and antimicrobial activity of N-substituted maleimides

Chemical reactivity and antimicrobial activity of N-substituted maleimides

Identification of Phoslactomycin E as a Metabolite Inducing Hyphal Morphological Abnormalities in Aspergillus fumigatus IFO 5840

Quantification of stimulus-induced callose spots on plant materials

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