Lambda 5

For all experiments (unlabelled and ¹³C-labeled 2,4-D experiments) we estimated 2,4-D (% of initial amount) and bacterial concentrations [Log(CFU).mL⁻¹] after 1, 2, 3, 5, and 10 days of incubation by measuring the optical density in the liquid medium using a Lambda 5 (Perkin-Elmer) spectrophotometer (λ_2,4−D = 282 nm and λ_Bacteria = 600 nm). At each sampling date, 4 replicates of each treatment were destructively sampled for microscopy and carbohydrates measurements (unlabeled 2,4-D experiments) or for Fatty Acid Methyl Ester (FAME) profiling (¹³C-labeled 2,4-D experiments).

The frozen lung samples were homogenized with 10 mM PBS, pH 7.4, sonicated on ice for 1 min., and centrifuged at 100 × g for 10 min. Supernatants were used for SOD measurement. The assay of MnSOD activity was carried out based on SOD-induced inhibition of the conversion of nitro blue tetrazolium (NBT) into formazan mediated by O₂⁻ generated by xanthine-xanthine oxidase. The reaction was performed in sodium carbonate buffer, 50 mM, pH 10.1, containing 0.1 mM EDTA, 25 μM NBT (Sigma-Aldrich, Milan, Italy), 0.1 mM xanthine and 2 nM xanthine oxidase (Sigma-Aldrich) as previously reported [33 (link)]. The rate of reduction in NBT was monitored with a spectrophotometer (Lambda 5; Perkin Elmer, Monza (MB), Italy) set at 560 nm. The amount required to inhibit the rate of reduction in NBT to formazan by 50% was defined as one unit of SOD activity. Specific MnSOD activity was calculated by inhibiting total SOD activity pre-incubating the sample for 30 min. with 2 mM NaCN. Values are mU/mg of protein.

Optical rotations were measured at 20 °C in acetonitrile using an Anton Paar MCP 300 polarimeter in a 100-mm-long 350 μL cell. UV spectra were recorded at 20 °C in acetonitrile or methanol using a PerkinElmer Lambda 5 spectrophotometer. Electronic circular dichroism spectra were acquired at 20 °C in acetonitrile on a JASCO J-810 spectropolarimeter. NMR spectra were recorded on Bruker 300, 500, 600 and 700 MHz spectrometers (Bruker, Rheinstetten, Germany). The chemical shifts (δ) are reported as ppm based on the solvent signal, and coupling constants (J) are in hertz. Preparative HPLC was conducted with a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector and a prep ELSII. All solvents were HPLC grade, purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

Ultraviolet spectra were recorded on a Perkin Elmer Lambda 5 spectrophotometer. For pKa determination, UV spectra of TFX2 were recorded over the range pH 1-13.