Catalase

The selected honey samples No. 1, 3, 6, 7, 8, 11 and 15, at a concentration of 50% (w/w), were treated with catalase (2,000–5,000 U/mg protein; Sigma-Aldrich, UK) at a final concentration ranging from 1,000–2,500 U/ml at ambient temperature for 2 h. catalase-treated honey samples were then used in the antibacterial assay to determine the MIC values against S. aureus.

Whole kidney tissue was homogenized in ice-cold disruption buffer (PBS containing 0.1 mM EDTA, 10% glycerol, protease inhibitor cocktail, and 0.1 mM phenylmethylsulfonyl fluoride [Sigma-Aldrich]), and further lysed as for superoxide. Lysate (50 μg/ml) was added to the assay mixture (25 mM Hepes, pH 7.4, containing 0.12 M NaCl, 3 mM KCl, 1 mM MgCl2, 0.1 mM Amplex red [Invitrogen, CA, USA], and 0.32 U/ml HRP). The reaction was initiated by the addition of 36 μM NADPH. Fluorescence measurements were made using a Biotek Synergy 4 hybrid multimode microplate reader with a 530/25-excitation and a 590/35-emission filter. The reaction was monitored at 25 °C (15 min); the emission increase was linear during this interval. To confirm the H₂O₂ signal, catalase (300 U/ml; Sigma-Aldrich) was added in parallel wells, and the catalase-inhibitable rate of H₂O₂ production was quantified from an H₂O₂ standard curve.

Methanol, gallic acid, cysteine, ascorbic acid and sodium sulphite (as preservative agent) were checked as potential interferences with the colour change of the biosensor response. For all of them, an adequate dilution (1:10) in phosphate buffer 0.1 M pH 7 was the only sample treatment needed. The biosensor in dip stick format was dipped into different solution samples and the colour changes were recorded using the experimental procedure described above. In addition, catalase (C3515 EC 1.11.1.6, 4,000 units/mg from Aspergillus niger, Sigma, ∼10 units of catalase in phosphate buffer) was add to the sample that contained 1% of ethanol.
For application of ethanol detection in coloured solutionss, several colour interference tests have performed, by testing red wine (12% v/v, for red colour), orange (for yellow colour), apple (for brown) and grape juices (purple/dark colour). For all the fruit juices, 1% of ethanol was added to the samples. For all of them, an adequate dilution (1:10) in phosphate buffer 0.1 M pH 7 was needed for sample treatment. The biosensor was dipped in each different colored sample and after the color change, the fluid surrounding the surface of the biosensor was flushed gently with water to remove any colour bound on the surface, then the colour change was visually detected and recorded using the experimental procedure given above.

Mouse melanoma B16, human melanoma Mewo and A375 cells were purchased from the American Type Culture Collection (ATCC) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, CA, USA) containing 5% (for B16 cells) or 10% (for other cells) fetal bovine serum (FBS, Gibco). All cells were in a state of logarithmic growth at 37 °C in a humidified chamber with 5% CO₂. To examine the effects of EGCG, cells were harvested from culture plates and treated with EGCG at the indicated concentrations for the indicated time in DMEM supplemented with 1% FBS including 200 units/mL catalase (Sigma) and 5 units/mL superoxide dismutase (Sigma).
EGCG, catalase, H-89, okadaic acid, dibutyryl-cAMP and the anti-β-actin antibody were purchased from Sigma-Aldrich. Anti-HMGA2 antibody was obtained from Cell Signaling Technology (Beverly MA). Anti-67LR serum was obtained from a rabbit, which had been immunized with synthesized peptides corresponding to residues 161-170 of human 67LR.
Mmu-67LR-siRNA (#4390771), hsa-67LR-siRNA (#4392420) and negative control-siRNA (#4390843) were purchased from Ambion (Austin, TX, USA). Mission microRNA mimic of hsa-let-7b-5p (HMI0007), Mission synthetic microRNA inhibitor of hsa-let-7b-5p (HSTUD0007) and negative control nucleotides (HMC0003 and #199004-00) were purchased from Sigma Aldrich.