Superoxide dismutase from bovine erythrocytes

The pCW-3A4-BMR plasmid containing the gene of human P450 3A4 fused to the reductase of cytochrome P450 BM3 from B. megaterium (BMR) linked via a Pro-Ser-Arg linker was available in our laboratory (Dodhia et al., 2006 (link)). GenElute plasmid miniprep kit was obtained from Sigma (Italy). PCR purification kit and gel extraction kit were purchased from Macherey-Nagel (Germany).
The primers for PCR and oligonucleotides for the linker region between P450 3A4 and BMR reductase were prepared by MWG (Germany). DNA Ladder, restriction endonucleases enzymes Avr II and Hind III, Vent polymerase, T4 DNA ligase enzyme and buffers used in sub-cloning were from New England Biolabs (UK) and Promega (Italy). Horse heart cytochrome c, erythromycin, testosterone, 6β-hydroxytestosterone, horseradish peroxidase type X, superoxide dismutase from bovine erythrocytes, catalase, N,N-dimethylaniline, and 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one were purchased from Sigma (Italy). NADPH (tetrasodium salt) was acquired from Calbiochem (Germany). Human CPR was purchased from Life Technologies. All other chemicals, biochemicals used in this study were purchased from Sigma (Italy) at the highest available grade and used as recommended by the manufacturer.

The lytic polysaccharide monooxygenase LPMO9C from the ascomycete N. crassa was recombinantly expressed in Pichia pastoris X-33 as reported previously (39 (link)). Production in a 5-liter laboratory fermenter and column purification were done according to a published protocol (29 (link)). The purity of the enzyme was checked by SDS-PAGE. Superoxide dismutase from bovine erythrocytes, peroxidase from horseradish, and catalase from C. glutamicum were purchased from Sigma-Aldrich. Activity of catalase was determined by measuring the decrease of H₂O₂ at 240 nm (ϵ₂₄₀ = 46,900 mm⁻¹ cm⁻¹) (40 (link)) in 100 mm sodium phosphate buffer, pH 6.0. Reactions were monitored in an Agilent 8453 UV-visible spectrometer featuring a photodiode array detector and a temperature-controlled 8-cell changer. One unit of catalase activity is defined as the amount of enzyme consuming 1 μmol of H₂O₂/min under the specified assay conditions. Blank runs in the absence of catalase were recorded for all conditions and subtracted from the catalase reactions. Catalase concentrations were determined with the Bradford protein assay (Bio-Rad) using BSA as a calibration standard.

T cells were stimulated with immobilized CD3×CD28 mAbs as previously described [25 (link)]. Briefly, SuperAvidin™-coated polystyrene microspheres (Bangs laboratories, Inc., Ø ~ 10 μm, binding capacity: 0,02-0,04 μg biotin/mg) were coated with biotinylated CD3 (clone UCHT1) and CD28 (clone CD28.2) mAbs (10 μg/ml each, BioLegend) for 30 min at 37°C in PBS Dulbecco (Biochrom AG). Antibody-coated microspheres were washed three times, resuspended in PBS at 10⁸ beads/ml and incubated with T cells in a 1 bead per cell ratio. Microspheres coated with 20 μg/ml of biotinylated mouse IgG1κ (eBioscience) were used as a control.
Stimulations in the presence of 100 U/ml superoxide dismutase from bovine erythrocytes, 1000 U/ml catalase from Corynebacterium glutamicum and 100 μM L-ascorbic acid (all from Sigma-Aldrich) were performed by pre-incubating T cells for 30 min with the compounds before stimulation.
Mouse T cells were stimulated with CD3 (clone 145-2C11) and CD28 (clone 37.51) mAbs (10 μg/ml each, both from BD Pharmingen) immobilized on microspheres as described above. Microspheres coated with biotinylated hamster IgG1κ and IgG2λ1 (10 μg/ml each, both from BD Pharmingen) were used as a control.

5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Alexis Biochemicals (Lausen, Switzerland). Ciprofloxacin (CPFX), norfloxacin (NRFX), pazfloxacin (PZFX), levofloxacin (LVFX), lomefloxacin (LFLX) and 1, 3-dimethylthiourea (DMTU) were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Diethylenetriaminepentaacetic acid (DTPA), NaNO₂, NaNO₃ and dextran were purchased from Nacalai Tesque (Kyoto, Japan). Ficoll-Paque was purchased from GE Healthcare (Tokyo, Japan), and phorbol 12-myristate 13-acetate (PMA) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Mayer’s hematoxylin and eosin alcohol solutions were purchased from Muto Pure Chemicals (Tokyo, Japan). Superoxide dismutase from bovine erythrocytes was purchased from Sigma (Tokyo, Japan).

Yeast extract, potato dextrose agar, agar, and sucrose were purchased from Biolife (Milan, Italy). Ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) was from Pharmacia Biotech (Uppsala, Sweden), stabilized 3% solution of hydrogen peroxide (H₂O₂) was obtained from Fluka (Buchs, Switzerland), and potassium cyanide was purchased from Sigma Aldrich (Taufkirchen, Germany). Hydrochloric acid as purchased from Merck (Darmstadt, Germany). Trichloroacetic acid (TCA) and ascorbic acid were from Kemika (Zagreb, Croatia), absolute ethanol was from Panreac (Barcelona, Spain), while butylated hydroxytoluene (BHT) and 2-thiobarbituric acid (TBA) was obtained from Acros Organics (Morris Plains, New Jersey, USA). Superoxide dismutase from bovine erythrocytes (3000 U mg ⁻¹ protein) (SOD) and xanthine oxidase from bovine milk (0.4-1.0 U mg ⁻¹ protein) (XOD) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Formic acid was purchased from Fluka (Buchs, Switzerland). Aflatoxin standard mix (B₁, B₂, G₁, G₂) was purchased from Biopure (Tulln, Austria). Acetonitrile and methanol (both HPLC grade) were purchased from J. T. Baker (Radnor, PA, USA).

Poly(D,L-lactide-co-glycolide) (PLGA; Resomer RG 503H) was purchased from Sigma-Aldrich (Darmstadt, Germany) and used as received. Poly(ethylene imine) (PEI), branched, MW 25.000 Da was purchased from Sigma-Aldrich (Darmstadt, Germany) and used as 14 mg/mL stock solution in dimethyl sulphoxide (DMSO). Bovine Serum Albumin (BSA) and Superoxide Dismutase from bovine erythrocytes were purchased from Sigma-Aldrich (Dartmstadt, Germany) and used as received. Atto 488 NHS-ester, Atto 550 NHS-ester and Atto 633 amine were purchased from Atto-TEC GmbH (Siegen, Germany) and used as 10 mg/mL stock solution in DMSO. Fluorescently labelled PLGA was obtained as described elsewhere [16 (link)]. Lysotrack Red DND-99 and CellMask Green Plasma Membrane Stain were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and used as received. All other chemicals were purchased from Sigma Aldrich (Darmstadt, Germany) unless specified and used as received.

L-arginine, apocynin, sodium nitrite, sodium nitrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Bradford reagent, cytochrome c from bovine heart, sodium dodecyl sulfate, superoxide dismutase from bovine erythrocytes, L-Nω-nitro-arginine (L-NNA), phosSTOP, cOmpleteMini, HT, TYR, and HVA were purchased from Sigma Aldrich (Gillingham, UK). Hydroxytyrosol-3-glucuronide (HT-3G), hydroxytyrosol-3-sulfate (HT-3S), tyrosol-glucuronide (TYR-G), and tyrosol-sulfate (TYR-S) were obtained from Toronto Research Chemicals Inc. (North York, ON, Canada).