designed experiments and wrote and finalized the draft
designed experiments and wrote and finalized the draft. test with BenjaminiCHochberg (BH) correction, 0.01), indicating that the sensitivity to salicin is higher in the ring-tailed lemur than in the ruffed lemur. TAS2R16 of tested lemurs showed Tamibarotene constitutive activity; IP1 production in the absence of ligands was much greater than that for cells expressing mock or human TAS2R16 (electronic supplementary material, physique S1a). Open in a separate window Physique 1. Different -glucosides evoked different responses of TAS2R16 in lemurs. ( 0.001). By contrast, the acceptance rate of arbutin-treated food was not different from that of water-treated pieces (= 0.39). The acceptance rate of the apple pieces treated with a mixture of salicin and arbutin was significantly lower than that of CD133 control water-treated pieces ( 0.05) but significantly greater than Tamibarotene that of salicin-treated apple pieces ( 0.01). This result indicates that arbutin masked the bitterness of salicin at the behavioural level and that TAS2R16 function was directly associated with feeding behaviour. Open in a separate window Physique 2. Arbutin inhibits salicin bitterness in the black lemur. Water-soaked apple pieces (control) and -glucoside-soaked pieces were presented to a subject (= 1) simultaneously. Acceptance ratios for each -glucoside and the mixture were calculated (mean s.e.m.). Behavioural assays (= 8, respectively) were performed using each -glucoside. Significant differences were determined by one-way ANOVA followed by Welch’s assessments with the BH correction (false discovery rate (FDR) = 0.15) and significantly lower than that of control (water)-treated pieces ( 0.05), indicating that arbutin partially masks salicin bitterness. Incomplete masking of salicin bitterness by arbutin may be caused by an insufficient amount of arbutin for the complete inhibition of 5 mM salicin or other taste receptor activation. This result suggests that arbutin can mask bitterness derived from specific agonists of TAS2R16, including -glucosides, which could weaken bitterness in plants containing various -glucosides. Plants known to contain arbutin, including Ericaceae  and Aquifoliaceae [37C39], are distributed worldwide, including in Madagascar . (Aquifoliaceae)distributed in Madagascar and southern Africa, is usually consumed by the brown mouse lemur ((electronic supplementary material, table S6). Polymerase chain reactions (PCRs) were performed using Tks Gflex DNA Polymerase (TaKaRa Bio) as follows: 1 min of initial denaturation; 30C40 cycles of denaturation at 98C for 10 s, annealing at a heat gradient of 50C57C for 15 s and extension at 68C for 30 s; and a final extension at 68C for 5 min. All sequences of PCR products were decided using the BigDye Terminator v. 3.1 Cycle Sequencing Tamibarotene Kit and the ABI 3130(Applied Biosystems, Foster City, CA, USA). (d) Construction of expression vectors for TAS2R16 and point Tamibarotene mutants Amplified TAS2R16 was tagged at the N terminus with the first 45 amino acids of rat somatostatin receptor type 3 for cell-surface targeting and at the C terminus with the last 8 amino acids of bovine rhodopsin as an epitope tag [11,26]. The tagged TAS2R16s were sub-cloned into the EcoRV site of the mammalian expression vector pEAK10 using the In-Fusion HD Cloning Kit (Clontech, Fremont, CA, USA). Point mutant vectors were constructed using QuikChange (Agilent Technologies, Santa Clara, CA, Tamibarotene USA) and the overlap extension PCR method . All mutations were checked by sequencing. (e) Cell culture, transfection and calcium assay Cell culture, transfection and calcium assays were performed as described previously [14,52,53]. The calcium response is expressed as the normalized peak response ((=[values were used for the calculation of doseCresponse associations using the drc package in R . Detailed.