Thymidine

A 10 ml culture of E. coli BL21(DE3) containing pET-topAI plasmid was grown at 37°C in M9 medium supplemented with glucose. When the O.D.₆₀₀ of the culture reached 0.3, 1.5 ml of the culture was transferred to a tube containing 20 μl [³H]thymidine (Perkin Elmer) and 80 μl cold thymidine (1 mg/ml) for DNA synthesis or 20 μl [³H]uridine (Perkin Elmer) and 80 μl cold uridine (1 mg/ml) for RNA synthesis, respectively. A 50-μl aliquot of the culture was spotted on a 3MM filter paper (Whatman 3 mm, 2.3 cm diameter) at indicated times and the filter paper was soaked in 10% TCA, which was then incubated for 1 h at room temperature. Then the filter papers were washed three times with 10% TCA solution. The filter papers were analyzed using a scintillation counter. Protein synthesis was analyzed as described previously (30 (link)). Data are representative of three independent experiments.

Cells were seeded in 24-well plates and 24 h later washed once with fresh medium only and then treated with fresh medium containing the appropriate drug concentrations. Cells were incubated at 37 °C, 5% (v/v) CO₂ for a further 24 h, with 5 μM thymidine containing 0.5 μCi [³H-methyl]thymidine (Perkin Elmer, Buckinghamshire, UK) added for the final 6 h. Medium was aspirated and cells, which were subconfluent, fixed in 5% (w/v) trichloroacetic acid (TCA) at 4 °C overnight. Fixed cells were then washed with water, solubilized with 0.5 mL 0.1 M NaOH, transferred to scintillation vials containing 4 mL scintillation fluid (0.4% (w/v) TCA; Perkin Elmer, Buckinghamshire, UK), and counted on a Packard Tricarb 4000 series scintillation counter (Perkin Elmer, Buckinghamshire, UK). Each independent experiment was performed with cell culture triplicates.

BP was measured using the ³H-thymidine incorporation technique as described in Berglund et al. [12 (link)]. One millilitre of seawater was added to three Eppendorf tubes, one control and duplicate samples. Bacteria in the control were pre-killed by adding 100 μl ice-cold 50 % TCA and incubating at −20 ^oC for 5 min. Next, 2 μl [³H]-thymidine (84 Ci mmol⁻¹; PerkinElmer, Massachusetts, USA) was added to each tube to a final concentration of 24 nM. The incorporated thymidine was converted to cell production using the conversion factor of 1.4 × 10¹⁸ cells mol⁻¹ [60 (link)]. Carbon biomass production was estimated from cell production and average cell carbon biomass as described in Eriksson-Wiklund et al. [22 (link)]. Primary production (PP) was measured in situ using the ¹⁴C technique: 5 ml seawater was added to three 20 ml transparent polycarbonate tubes with one dark tube as a control. Next, 7.2 μl ¹⁴C was added to each tube (¹⁴C Centralen Denmark, activity 100 μCi/ml) and were incubated at 1 m depth for ∼3 h. The samples were analysed in a Beckman 6500 scintillation counter. Daily PP was calculated as described in Andersson et al. [3 (link)].

[³H]thymidine incorporation assay was performed in the cells cultured as reported above; the cells were incubated for the last 2 h in the presence of methyl [³H]thymidine (1–2 µCi/ml) (Perkin Elmer, Monza, Italy) and then processed using trichloroacetic acid precipitation as reported previously [41] (link). The recovered radioactivity was measured in a beta counter (Beckman Coulter s.r.l., Milano, Italy).

LN and spleen cells were cultured as described above for 72 h in triplicate wells with a range of MOG_35–55 concentrations. We then added [³H] thymidine (Perkin Elmer) for the last 18–24 h of culture and assessed thymidine incorporation using a β-plate scintillation counter.