Enzymatic deglycosylation of human TYR.

<p><b>A.</b> Deglycosylation of TYR469 with PNGase. <b>B.</b> Deglycosylation of TYR469 with Endo H_f. M, molecular weight marker; C, non-treated protein as control; D, deglycosylated sample.</p

Sequence alignment of human TYR and human TYRP1.

<p>Conserved residues are highlighted in red. Putative TYR glycosylation sites are indicated with a green star. Two different TYR constructs were designed for recombinant expression, TYR456 (50 kDa, residues 19–456) and TYR469 (51.5 kDa, residues 19–469).</p

Characterization of TYR variants by gel filtration and enzyme activity assays.

<p>Overlap of the analytical gel filtration profiles of TYR456 (peak B, in red) and TYR469 (peak A, in blue) on a Superdex 200 10/300 GL column (GE Healthcare). Colorimetric activity assays with the corresponding eluted fractions using L-DOPA as substrate generated a pink or a dark pink pigment product (i.e. quinone-MBTH adduct), indicating that both variants are enzymatically active (10 μL of each elution fraction containing TYR was added in a 80 μL reaction well containing L-DOPA. 10 μL gel filtration buffer solution was used as a negative control). The apparent gradient of light pink to dark pink, and back to light pink in the reaction well indicates different protein concentrations of each elution fraction.</p

Determination of the thermal stability of the TYR variants by thermal shift assays.

<p>Fluorescence scan diagrams based on <i>Sypro Orange</i> (Thermo Fisher Scientific) binding upon thermal unfolding of TYR456 and TYR469 proteins, respectively. The apparent stability midpoint values (or melting temperatures) under the analysed buffer conditions are 72°C and 60°C for TYR456 and TYR469, respectively.</p

Crystallization of TYR469.

<p><b>A.</b> Picture of the initial crystallization hit of TYR469 in 100 mM (NH)₄SO₄, 10 mM MgCl₂, 50 mM MES, pH 5.9, and 18% PEG8000 (w/v). <b>B.</b> Picture of a Cryoloop containing multiple microcrystals suitable for an X-ray mesh scan. <b>C.</b> Heat map resulting from the cryoloop mesh scan. The cross spots indicate diffracting crystals positions. The quality of the diffraction is scored based on a colour gradient (from yellow—poor—to dark red—highest–). <b>D.</b> Picture of TYR469 crystals with 0.5% polyvinylpyrrolidone K15 (w/v) as an additive (HR2-138 condition E2, Hampton Research). <b>E.</b> Picture of TYR469 crystals with 10 mM spermine tetrahydrochloride as an additive (HR2-138 condition D3). <b>F.</b> Representative X-ray diffraction pattern of a crystal from E. Resolution circles are indicated.</p

Main Phenolic Compounds of the Melanin Biosynthesis Pathway in Bruising-Tolerant and Bruising-Sensitive Button Mushroom (Agaricus bisporus) Strains

Browning is one of the most common postharvest changes in button mushrooms, which often results in economic losses. Phenolic compounds, which are associated with browning, were extracted from the nonbruised and bruised skin tissue of various button mushrooms with a sulfite-containing solution and analyzed with UHPLC-PDA-MS. In total, 34 phenolic compounds were detected. Only small differences in the total phenolic content between bruising-tolerant and -sensitive strains were observed. The contents of γ-l-glutaminyl-4-hydroxybenzene (GHB) and γ-l-glutaminyl-3,4-dihydroxybenzene (GDHB) correlated with bruising sensitivity; for example, <i>R</i>² values of 0.85 and 0.98 were found for nonbruised brown strains, respectively. In nonbruised skin tissue of the strains with brown caps, the GHB and GDHB contents in sensitive strains were on average 20 and 15 times higher, respectively, than in tolerant strains. GHB and GDHB likely participate in the formation of brown GHB–melanin, which seemed to be the predominant pathway in bruising-related discoloration of button mushrooms

Main Phenolic Compounds of the Melanin Biosynthesis Pathway in Bruising-Tolerant and Bruising-Sensitive Button Mushroom (Agaricus bisporus) Strains

Browning is one of the most common postharvest changes in button mushrooms, which often results in economic losses. Phenolic compounds, which are associated with browning, were extracted from the nonbruised and bruised skin tissue of various button mushrooms with a sulfite-containing solution and analyzed with UHPLC-PDA-MS. In total, 34 phenolic compounds were detected. Only small differences in the total phenolic content between bruising-tolerant and -sensitive strains were observed. The contents of γ-l-glutaminyl-4-hydroxybenzene (GHB) and γ-l-glutaminyl-3,4-dihydroxybenzene (GDHB) correlated with bruising sensitivity; for example, <i>R</i>² values of 0.85 and 0.98 were found for nonbruised brown strains, respectively. In nonbruised skin tissue of the strains with brown caps, the GHB and GDHB contents in sensitive strains were on average 20 and 15 times higher, respectively, than in tolerant strains. GHB and GDHB likely participate in the formation of brown GHB–melanin, which seemed to be the predominant pathway in bruising-related discoloration of button mushrooms

Challenge dosage DBPCFC test with cashew nut [5].

<p>Challenge dosage DBPCFC test with cashew nut [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151055#pone.0151055.ref005" target="_blank">5</a>].</p

Inclusion and exclusion criteria.

<p>Inclusion and exclusion criteria.</p

Demographic and clinical characteristics.

<p>Demographic and clinical characteristics.</p