The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Arthur F J Ram - One of the best experts on this subject based on the ideXlab platform.
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identification of fungal cell wall mutants using susceptibility assays based on calcofluor white and congo red
Nature Protocols, 2006Co-Authors: Arthur F J Ram, Frans M KlisAbstract:The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the fungal cell wall. Here we present a simple approach for detecting these genes. The method is based on the observation that cell wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the cell wall. CFW-based and CR-based susceptibility assays identify cell wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4–6 h (excluding the incubation time required for fungal growth).
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expression of agsa one of five 1 3 α d glucan synthase encoding genes in aspergillus niger is induced in response to cell wall stress
Fungal Genetics and Biology, 2005Co-Authors: Robbert A Damveld, Frans M Klis, Patricia A Vankuyk, Mark Arentshorst, Cees A M J J Van Den Hondel, Arthur F J RamAbstract:1,3-α-d-Glucan is an important component of the cell wall of filamentous fungi. We have identified a family of five 1,3-α-d-glucan synthase-encoding genes in Aspergillus niger. The agsA gene was sequenced and the predicted protein sequence indicated that the overall domain structure of 1,3-α-d-glucan synthases is conserved in fungi. Using RT-PCR and Northern blot analysis, we found that expression of the agsA gene and to a lesser extent also of agsE were induced in the presence of the cell wall stress-inducing compounds such as Calcofluor White (CFW), SDS, and caspofungin. Loss of agsA function did not result in an apparent phenotype under normal growth conditions but rendered the cells more sensitive to CFW. The induction of 1,3-α-d-glucan synthase-encoding genes in response to cell wall stress was not limited to A. niger, but was also observed in Penicillium chrysogenum. We propose that this response to cell wall stress commonly occurs in filamentous fungi. © 2004 Elsevier Inc. All rights reserved.
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large scale identification of genes involved in cell surface biosynthesis and architecture in saccharomyces cerevisiae
Genetics, 1997Co-Authors: Marc Lussier, Arthur F J Ram, Annmarie White, Jane Sheraton, Tiziano Di Paolo, Julie Treadwell, Susan B Southard, Craig I Horenstein, Joan Chenweiner, J C KapteynAbstract:The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.
Raffael Schaffrath - One of the best experts on this subject based on the ideXlab platform.
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molecular analysis of kti12 tot4 a saccharomyces cerevisiae gene required for kluyveromyces lactis zymocin action
Molecular Microbiology, 2002Co-Authors: Lars Fichtner, Frank Frohloff, Karin D Breunig, Konstanze Burkner, Michael Larsen, Raffael SchaffrathAbstract:Summary TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1–7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1–6). Unlike TOT1–3 (ELP1–3) and TOT5–7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.
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saccharomyces cerevisiae elongator mutations confer resistance to the kluyveromyces lactis zymocin
The EMBO Journal, 2001Co-Authors: Frank Frohloff, Lars Fichtner, Daniel Jablonowski, Karin D Breunig, Raffael SchaffrathAbstract:Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G(1) cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with ELONGATOR: Intriguingly, strains lacking non-Elongator HATs (gcn5, hat1, hpa3 and sas3) or non-Elongator transcription elongation factors TFIIS (dst1) and Spt4p (spt4) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin.
Mahmoud A Ghannoum - One of the best experts on this subject based on the ideXlab platform.
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comparison of biofilms formed by candida albicans and candida parapsilosis on bioprosthetic surfaces
Infection and Immunity, 2002Co-Authors: Duncan M Kuhn, Jyotsna Chandra, Pranab K Mukherjee, Mahmoud A GhannoumAbstract:Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P 128 μg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.
Sheldon R Pinnell - One of the best experts on this subject based on the ideXlab platform.
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calcofluor white combination antifungal treatments for trichophyton rubrum and candida albicans
PLOS ONE, 2012Co-Authors: Joanne M Kingsbury, Joseph Heitman, Sheldon R PinnellAbstract:Superficial mycoses caused by dermatophyte fungi are among the most common infections worldwide, yet treatment is restricted by limited effective drugs available, drug toxicity, and emergence of drug resistance. The stilbene fluorescent brightener calcofluor white (CFW) inhibits fungi by binding chitin in the cell wall, disrupting cell wall integrity, and thus entails a different mechanism of inhibition than currently available antifungal drugs. To identify novel therapeutic options for the treatment of skin infections, we compared the sensitivity of representative strains of the dermatophyte Trichophyton rubrum and Candida albicans to CFW and a panel of fluorescent brighteners and phytoalexin compounds. We identified the structurally related stilbene fluorescent brighteners 71, 85, 113 and 134 as fungicidal to both T. rubrum and C. albicans to a similar degree as CFW, and the stilbene phytoalexins pinosylvan monomethyl ether and pterostilbene inhibited to a lesser degree, allowing us to develop a structure-activity relationship for fungal inhibition. Given the abilities of CFW to absorb UV365 nm and bind specifically to fungal cell walls, we tested whether CFW combined with UV365 nm irradiation would be synergistic to fungi and provide a novel photodynamic treatment option. However, while both treatments individually were cytocidal, UV365 nm irradiation reduced sensitivity to CFW, which we attribute to CFW photoinactivation. We also tested combination treatments of CFW with other fungal inhibitors and identified synergistic interactions between CFW and some ergosterol biosynthesis inhibitors in C. albicans. Therefore, our studies identify novel fungal inhibitors and drug interactions, offering promise for combination topical treatment regimes for superficial mycoses.
Jihad R Albani - One of the best experts on this subject based on the ideXlab platform.
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interaction between carbohydrate residues of α1 acid glycoprotein orosomucoid and progesterone a fluorescence study
Carbohydrate Research, 2002Co-Authors: Melanie De Ceukeleire, Jihad R AlbaniAbstract:Abstract Interaction between progesterone and the carbohydrate residues of α 1 -acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 μM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in α 1 -acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant ( k q ) of 2.23×10 9 M −1 s −1 . This value decreases to 0.19×10 9 M −1 s −1 when calcofluor white is bound to α 1 -acid glycoprotein. Binding of progesterone does not modify the value of k q of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res . 1999 , 322 , 87–94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated α 1 -acid glycoprotein show that binding of progesterone to α 1 -acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.
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effect of binding of calcofluor white on the carbohydrate residues of α1 acid glycoprotein orosomucoid on the structure and dynamics of the protein moiety a fluorescence study
Carbohydrate Research, 2001Co-Authors: Jihad R AlbaniAbstract:Abstract Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of α 1 -acid glycoprotein (orosomucoid) was previously studied at low and high concentrations of Calcofluor compared to that of the protein. α 1 -Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. At equimolar concentrations of Calcofluor and α 1 -acid glycoprotein, the fluorophore displays free motions [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999 , 322 , 87–94], while at high concentration of Calcofluor, its surrounding microenvironment is rigid, inducing the rigidity of the fluorophore itself [Albani, J. R.; Sillen, A.; Plancke, Y. D.; Coddeville, B.; Engelborghs, Y. Carbohydr. Res . 2000 , 327 , 333–340]. In the present work, red-edge excitation spectra and steady-state anisotropy studies performed on Trp residues in the presence of Calcofluor, showed that the apparent dynamics of Trp residues are not modified. However, deconvoluting the emission spectra with two different methods into different components, reveals that the structure of the protein matrix has been disrupted in the presence of high Calcofluor concentrations.
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interaction between carbohydrate residues of α1 acid glycoprotein orosomucoid and saturating concentrations of calcofluor white a fluorescence study
Carbohydrate Research, 2000Co-Authors: Jihad R Albani, Alain Sillen, Yves Plancke, Bernadette Coddeville, Yves EngelborghsAbstract:Abstract Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of α1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193–200]. α1-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to α1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated α1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of α1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of α1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.
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dynamics of carbohydrate residues of α1 acid glycoprotein orosomucoid followed by red edge excitation spectra and emission anisotropy studies of calcofluor white
Carbohydrate Research, 1999Co-Authors: Jihad R Albani, Alain Sillen, Yves Plancke, Bernadette Coddeville, Yves EngelborghsAbstract:Abstract Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated α 1 -acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated α 1 -acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated α 1 -acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.
