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Pre-peptide regions of secreted proteins display wide sequence variability, even among highly homologous proteins such as coagulation factors, and are intracellularly removed, thus potentially favoring secretion of wild-type proteins upon suppression of nonsense mutations (translational readthrough). As models we selected F9 nonsense mutations with readthrough-favorable features affecting the pre-peptide and pro-peptide regions of coagulation factor IX (FIX), which cause hemophilia B (HB). Only the p.Gly21Ter (c.61G > T) in the variable pre-peptide hydrophobic core significantly responded (secretion, 4.1 ± 0.5% of wild-type; coagulant activity, 4.0 ± 0.3%) to the readthrough-inducer geneticin. Strikingly, for the p.Gly21Ter mutation, the resulting specific coagulant activity (0.96 ± 0.11) was compatible with normal function, thus suggesting secretion of FIX with wild-type features upon readthrough and removal of pre-peptide. Expression of the predicted readthrough-deriving missense variants (Gly21Trp/Cys/Arg) revealed a preserved specific activity (ranging from 0.84 to 0.98), thus supporting our observation. Conversely, rescue of the p.Cys28Ter (c.84T > A) and p.Lys45Ter (c.133A > T) was prevented by constraints of adjacent cleavage sites, a finding consistent with the association of most missense mutations affecting these regions with severe or moderate HB. Overall, our data indicate that suppression of nonsense mutations in the pre-peptide core preserves mature protein features, thus making this class of mutations preferred candidates for therapeutic readthrough.

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In a randomized cross-over study, 15 healthy volunteers consumed 500 mL of green tea (GTFT) with different solid contents (1.4, 1.6, 1.8 and 2.0 g/L) to induce a dose-response effect on plasma antioxidant capacity. Ingestion of GTFT 2.0 g/L significantly increased plasma reducing power (ferric reducing antioxidant power, FRAP) at 1 h (+2.9%; p<0.01), 2 h (+2.5%; p<0.05) and 4 h (+3.6%; p<0.01). GTFT 1.8 g/L showed statistical significance at 1 h (+4.3%; p<0.01) and 2 h (+4.4%; p<0.01), whereas GTFT 1.6 g/L was effective only at 1 h (+2.9%; p<0.01) and GTFT 1.4 g/L did not induce any changes. The maximum peak of increase in plasma FRAP for different GTFTs was clearly correlated with in vitro FRAP (R=0.778). GTFT 2.0 g/L significantly increased plasma antioxidant potential (total radical-trapping antioxidant parameter) at 1 h (+8.4%; p<0.01), 2 h (+4.4%; p<0.05) and 4 h (+5.9%; p<0.01). The effect of GTFT 1.8 g/L was evident at 1 h (+5.2%; p<0.05) and 2 h (+4.6%; p<0.05) but not at 4 h. No changes in plasma total radical-trapping antioxidant parameter were detected for GTFT at 1.6 and 1.4 g/L. An evidence for a linear correlation between GTFT antioxidant content and the extent of the antioxidant effect in vivo has been provided.