3,4-Dinitrophenyl N-acetyl-β-d-glucosaminide, a synthetic substrate for direct spectrophotometric assay of N-acetyl-β-d-glucosaminidase or N-acetyl-β-d-hexosaminidase

Yagi, Takeshi; Hisada, Ryuki; Shibata, Hideto

doi:10.1016/0003-2697(89)90474-0

Cited by 19 publications

(12 citation statements)

References 5 publications

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“…MPO activity, reflecting the presence of neutrophils, was determined as described previously [4]. The presence of monocytes/macrophages in synovial fluid was assessed using the N -acetyl-β-D-glucosaminidase (NAG) assay as described elsewhere [30]. Cytokine (TNF-α, IL-1β, IL-6, IL-8) concentration were determined by ELISA using commercially available kits (R&D Systems) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

Fox

Keeble

et al. 2013

J Cellular Molecular Medi

109

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The role of hydrogen sulfide (H2S) in inflammation remains unclear with both pro- and anti-inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow-releasing H2S donor) on lipopolysaccharide (LPS)-evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund's adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS-induced production of nitrite (NO2−), PGE2, TNF-α and IL-6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced LPS-induced NF-κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX-2, iNOS and TNF-α converting enzyme (TACE). In the CFA-treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti-inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAG) activity and decreased TNF-α, IL-1β, IL-6 and IL-8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti-inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro-inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.

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Section: Methodsmentioning

confidence: 99%

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

Fox

Keeble

et al. 2013

J Cellular Molecular Medi

109

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“…NAGase activities were measured using a modification of the method described by Yagi et al. [27], which is based on the release of p ‐nitrophenol from the respective aryl chitosides. Samples of between 5 and 100 μL were added to 0.5 mL of a solution containing 50 m m potassium phosphate buffer, pH 6.7, and 300 μg·mL −1 4‐nitrophenyl N‐ acetyl‐β‐ d‐ glucosaminide, and the volume made up to 600 μL with buffer.…”

Section: Methodsmentioning

confidence: 99%

The β‐N‐acetylglucosaminidases NAG1 and NAG2 are essential for growth of Trichoderma atroviride on chitin

et al. 2009

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The chitinolytic enzyme machinery of fungi consists of chitinases and β‐N‐acetylglucosaminidases. These enzymes are important during the fungal life cycle for degradation of exogenous chitin, which is the second most abundant biopolymer, as well as fungal cell‐wall remodelling. In addition, involvement of chitinolytic enzymes in the lysis of the host cell wall in mycoparasitic Trichoderma spp. has been reported. In view of the fact that fungi have on average 15–20 chitinases, but only two β‐N‐acetylglucosaminidases, the question arises how important the latter enzymes actually are for various aspects of chitin degradation. In this study, the role of two β‐N‐acetylglucosaminidases, NAG1 and NAG2, was analysed in the mycoparasitic fungus Trichoderma atroviride. No β‐N‐acetylglucosaminidase activity was detected in T. atrovirideΔnag1Δnag2 strains, suggesting that NAG1 and NAG2 are the only enzymes in T. atroviride that possess this activity. Δnag1Δnag2 strains were not able to grow on chitin and chitobiose, but the presence of either NAG1 or NAG2 was sufficient to restore growth on chitinous carbon sources in solid media. Our results demonstrated that T. atroviride cannot metabolize chitobiose but only the monomer N‐acetylglucosamine, and that N‐acetylglucosaminidases are therefore essential for the use of chitin as a nutrient source. NAG1 is predominantly secreted into the medium, whereas NAG2 mainly remains attached to the cell wall. No physiological changes or reduction of the mycoparasitic potential of T. atroviride was detected in the double knockout strains, suggesting that the use of chitin as carbon source is only of minor importance for these processes.

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“…NAGase activity was measured by a modification of the method of Yagi et al (1989), which is based on the release of p-nitrophenol from the respective arylchitosides. After incubation of the microplates at 25 uC in constant darkness for 30 and 48 h, 20 ml 50 mM potassium phosphate buffer, pH 6?7, containing 300 mg 4-nitrophenyl N-acetylb-D-glucosaminide ml…”

Section: Introductionmentioning

confidence: 99%

A screening system for carbon sources enhancing β-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride)

2006

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To identify carbon sources that trigger b-N-acetylglucosaminidase (NAGase) formation in Hypocrea atroviridis (anamorph Trichoderma atroviride), a screening system was designed that consists of a combination of Biolog Phenotype MicroArray plates, which contain 95 different carbon sources, and specific enzyme activity measurements using a chromogenic substrate. The results revealed growth-dependent kinetics of NAGase formation and it was shown that NAGase activities were enhanced on carbon sources sharing certain structural properties, especially on a-glucans (e.g. glycogen, dextrin and maltotriose) and oligosaccharides containing galactose. Enzyme activities were assessed in the wild-type and a H. atroviridis Dnag1 strain to investigate the influence of the two NAGases, Nag1 and Nag2, on total NAGase activity. Reduction of NAGase levels in the Dnag1 strain in comparison to the wild-type was strongly carbon-source and growth-phase dependent, indicating the distinct physiological roles of the two proteins. The transcript abundance of nag1 and nag2 was increased on carbon sources with elevated NAGase activity, indicating transcriptional regulation of these genes. The screening method for the identification of carbon sources that induce enzymes or a gene of interest, as presented in this paper, can be adapted for other purposes if appropriate enzyme or reporter assays are available. INTRODUCTIONSome species of the soil fungus Hypocrea (anamorph Trichoderma), e.g. Hypocrea atroviridis (Trichoderma atroviride) (Dodd et al., 2003), Hypocrea lixii (Trichoderma harzianum), Hypocrea virens (Trichoderma virens) and Trichoderma asperellum, are potent mycoparasites against several plant-pathogenic fungi, and lysis of the host cell wall has been demonstrated to be an important step in the mycoparasitic attack (Benítez et al., 2004;Chet et al., 1998;Howell, 2003;Kubicek et al., 2001). Consequently, with chitin being a major cell wall component of plant pathogens like Rhizoctonia solani, Botrytis cinerea and Sclerotinia sclerotiorum, several chitinolytic genes, encoding chitinases (EC 3.2.1.14) and b-N-acetylglucosaminidases (NAGases; EC 3.2.1.52), have been cloned from Hypocrea/Trichoderma spp. (Carsolio et al., 1994;Draborg et al., 1995;Garcia et al., 1994;Hayes et al., 1994;Kim et al., 2002;Peterbauer et al., 1996;Seidl et al., 2005;Viterbo et al., 2001Viterbo et al., , 2002 and for some of them the encoded protein also has been characterized (Boer et al., 2004;de la Cruz et al., 1992;Hoell et al., 2005). The regulation of expression of NAGases and chitinases in Hypocrea/Trichoderma has so far, besides Trichoderma-host interaction assays, only been studied with respect to their upregulation during growth on colloidal chitin, chitin degradation products and fungal cell walls (Carsolio et al., 1994; de las Mercedes Dana et al., 2001;Kim et al., 2002;Mach et al., 1999;Ramot et al., 2004). Additionally, the influence of carbon and nitrogen starvation on the expression of chitinolytic genes has been investigated (de las Mercedes D...

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3,4-Dinitrophenyl N-acetyl-β-d-glucosaminide, a synthetic substrate for direct spectrophotometric assay of N-acetyl-β-d-glucosaminidase or N-acetyl-β-d-hexosaminidase

Cited by 19 publications

References 5 publications

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The complex effects of the slow‐releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The β‐N‐acetylglucosaminidases NAG1 and NAG2 are essential for growth of Trichoderma atroviride on chitin

A screening system for carbon sources enhancing β-N-acetylglucosaminidase formation in Hypocrea atroviridis (Trichoderma atroviride)

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