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Alan Cartmell - One of the best experts on this subject based on the ideXlab platform.

  • mobility shift based electrophoresis coupled with fluorescent detection enables real time Enzyme Analysis of carbohydrate sulfatase activity
    Biochemical Journal, 2021
    Co-Authors: Dominic P Byrne, Edwin A. Yates, James A London, Patrick A Eyers, Alan Cartmell
    Abstract:

    Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are Enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation Analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated Enzymes.

  • mobility shift based electrophoresis coupled with fluorescent detection enables real time Enzyme Analysis of carbohydrate sulfatase activity
    bioRxiv, 2020
    Co-Authors: Dominic P Byrne, Edwin A. Yates, James A London, Patrick A Eyers, Alan Cartmell
    Abstract:

    Abstract Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are Enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation Analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated Enzymes. One sentence summary Sulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.

Dominic P Byrne - One of the best experts on this subject based on the ideXlab platform.

  • mobility shift based electrophoresis coupled with fluorescent detection enables real time Enzyme Analysis of carbohydrate sulfatase activity
    Biochemical Journal, 2021
    Co-Authors: Dominic P Byrne, Edwin A. Yates, James A London, Patrick A Eyers, Alan Cartmell
    Abstract:

    Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are Enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation Analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated Enzymes.

  • mobility shift based electrophoresis coupled with fluorescent detection enables real time Enzyme Analysis of carbohydrate sulfatase activity
    bioRxiv, 2020
    Co-Authors: Dominic P Byrne, Edwin A. Yates, James A London, Patrick A Eyers, Alan Cartmell
    Abstract:

    Abstract Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are Enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation Analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated Enzymes. One sentence summary Sulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.

M J Zervos - One of the best experts on this subject based on the ideXlab platform.

  • comparison of restriction Enzyme Analysis versus pulsed field gradient gel electrophoresis as a typing system for torulopsis glabrata and candida species other than c albicans
    Journal of Clinical Microbiology, 1993
    Co-Authors: Jose A Vazquez, A Beckley, Jack D Sobel, Susan M Donabedian, M J Zervos
    Abstract:

    Candida species have recently emerged as important nosocomial pathogens. Because of the lack of a reliable system for detecting differences within the same species, little is known about the epidemiology of infection with Candida species. We describe a typing system for Torulopsis glabrata and the non-C. albicans Candida species that uses contour-clamped homogeneous electric field electrophoresis (CHEF), a version of pulsed-field gradient gel electrophoresis, and compared it with restriction Enzyme Analysis (REA) of genomic DNA. One hundred seventeen clinical isolates from 40 patients were evaluated. CHEF and REA were performed on each of the isolates, and the results of the two procedures were compared. The REA procedure revealed 8 different types of Candida lusitaniae, 20 of Torulopsis glabrata, 5 of Candida tropicalis, 3 of Candida parapsilosis, and 7 of Candida kefyr, whereas the CHEF method revealed 14 different types of C. lusitaniae, 16 of T. glabrata, 10 of C. tropicalis, 10 of C. parapsilosis, and 7 of C. kefyr. The CHEF technique yielded unique patterns of electrophoretic karyotypes that could be used to distinguish intraspecies variations. When compared with REA, CHEF demonstrated greater sensitivity in recognizing subtle strain-to-strain variations in most isolates and will be a useful epidemiologic tool for studying non-C. albicans Candida species and T. glabrata. Images

Robert D. Arbeit - One of the best experts on this subject based on the ideXlab platform.

  • comparison of arbitrarily primed polymerase chain reaction restriction Enzyme Analysis and pulsed field gel electrophoresis for typing clostridium difficile
    Journal of Microbiological Methods, 1996
    Co-Authors: Matthew H Samore, Mar Kristjansson, Lata Venkataraman, Paola C Degirolami, Robert D. Arbeit
    Abstract:

    Abstract Three methods of molecular typing of Clostridium difficile [arbitrarily-primed polymerase chain reaction (AP-PCR), restriction Enzyme Analysis (REA) and pulsed-field gel electrophoresis (PFGE)] were compared using 33 isolates collected during a prospective study of Clostridium difficile transmission. Sixteen isolates (from 13 patients and 3 environmental sites) represented a cluster of C. difficile diarrhea on 2 wards, whereas the other 17 isolates were from sporadic cases of C. difficile diarrhea or asymptomatically colonized patient contacts. Fourteen of the 16 clustered isolates were nontypable by pulsed-field gel electrophoresis because of degradation. All 14 of these isolates were a single strain by REA and AP-PCR; 7 of 17 nonclustered isolates also represented the same strain. The other 12 isolates (2 clustered; 10 nonclustered) were subdivided into 9 subgroups by REA, 10 groups by AP-PCR and 7 subgroups by pulsed-field gel electrophoresis. In one instance, AP-PCR resolved isolates indistinguishable by other methods into different groups. However, the interpretation of AP-PCR patterns was complicated by variable intensity of bands and lack of reproducibility of minor bands. In conclusion, these 3 methods of genotyping yielded comparable results, with AP-PCR showing somewhat greater discriminatory power but lower reproducibility.

James A London - One of the best experts on this subject based on the ideXlab platform.

  • mobility shift based electrophoresis coupled with fluorescent detection enables real time Enzyme Analysis of carbohydrate sulfatase activity
    Biochemical Journal, 2021
    Co-Authors: Dominic P Byrne, Edwin A. Yates, James A London, Patrick A Eyers, Alan Cartmell
    Abstract:

    Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are Enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current 'gold standards' for enzymatic carbohydrate sulfation Analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated Enzymes.

  • mobility shift based electrophoresis coupled with fluorescent detection enables real time Enzyme Analysis of carbohydrate sulfatase activity
    bioRxiv, 2020
    Co-Authors: Dominic P Byrne, Edwin A. Yates, James A London, Patrick A Eyers, Alan Cartmell
    Abstract:

    Abstract Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are Enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation Analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated Enzymes. One sentence summary Sulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.