title: Application of 6-nitrocoumarin as a substrate for the fluorescent detection of nitroreductase activity in Sporothrix schenckii.
abstract: BACKGROUND
  Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii.
  
  
  METHODS
  Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light.
  
  
  RESULTS
  All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure.
  
  
  CONCLUSIONS
  The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.
authors.affiliation: Universidade Federal do Rio Grande do Sul, Porto AlegreRS, Brazil.
authors.forename: Cheila Denise Ottonelli
authors.initials: CD
authors.lastname: Stopiglia
authors.forename: Mariana
authors.initials: M
authors.lastname: Carissimi
authors.forename: Tatiane Caroline
authors.initials: TC
authors.lastname: Daboit
authors.forename: Valter
authors.initials: V
authors.lastname: Stefani
authors.forename: Valeriano Antonio
authors.initials: VA
authors.lastname: Corbellini
authors.forename: Maria Lúcia
authors.initials: ML
authors.lastname: Scroferneker
mesh_terms: D002855:Chromatography, Thin Layer; D003374:Coumarins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D009601:Nitroreductases / Q000378:metabolism*; D013175:Sporothrix / Q000201:enzymology*; D013379:Substrate Specificity; D014466:Ultraviolet Rays
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D009601:Nitroreductases; C067496:6-amino-1,2-benzopyrone
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