Genetic signature of differential sensitivity to stevioside in the Italian population

The demand for diet products is continuously increasing, together with that for natural food ingredients. Stevioside and other steviol glycosides extracted from the leaves of the plant Stevia rebaudiana Bertoni are the first natural high-potency sweeteners to be approved for consumption in the United States and the European Union. However, the sweetness of these compounds is generally accompanied by aversive sensations, such as bitter and off-tastes, which may constitute a limit to their consumption. Moreover, consumers’ differences in sensitivity to high-potency sweeteners are well known, as well as difficulties in characterizing their aftertaste. Recently, TAS2R4 and TAS2R14 have been identified as the receptors that mediate the bitter off-taste of steviol glycosides in vitro. In the present study, we demonstrate that TAS2R4 gene polymorphism rs2234001 and TAS2R14 gene polymorphism rs3741843 are functional for stevioside bitterness perception.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0401-y) contains supplementary material, which is available to authorized users.     

Positive Darwinian selection in the singularly large taste receptor gene family of an ‘ancient’ fish,Latimeria chalumnae

Background

Chemical senses are one of the foremost means by which organisms make sense of their environment, among them the olfactory and gustatory sense of vertebrates and arthropods. Both senses use large repertoires of receptors to achieve perception of complex chemosensory stimuli. High evolutionary dynamics of some olfactory and gustatory receptor gene families result in considerable variance of chemosensory perception between species. Interestingly, both ora/v1r genes and the closely related t2r genes constitute small and rather conserved families in teleost fish, but show rapid evolution and large species differences in tetrapods. To understand this transition, chemosensory gene repertoires of earlier diverging members of the tetrapod lineage, i.e. lobe-finned fish such as Latimeria would be of high interest.

Results

We report here the complete T2R repertoire of Latimeria chalumnae, using thorough data mining and extensive phylogenetic analysis. Eighty t2r genes were identified, by far the largest family reported for any species so far. The genomic neighborhood of t2r genes is enriched in repeat elements, which may have facilitated the extensive gene duplication events resulting in such a large family. Examination of non-synonymous vs. synonymous substitution rates (dN/dS) suggests pronounced positive Darwinian selection in Latimeria T2Rs, conceivably ensuring efficient neo-functionalization of newly born t2r genes. Notably, both traits, positive selection and enrichment of repeat elements in the genomic neighborhood, are absent in the twenty v1r genes of Latimeria. Sequence divergence in Latimeria T2Rs and V1Rs is high, reminescent of the corresponding teleost families. Some conserved sequence motifs of Latimeria T2Rs and V1Rs are shared with the respective teleost but not tetrapod genes, consistent with a potential role of such motifs in detection of aquatic chemosensory stimuli.

Conclusions

The singularly large T2R repertoire of Latimeria may have been generated by facilitating local gene duplication via increased density of repeat elements, and efficient neofunctionalization via positive Darwinian selection.The high evolutionary dynamics of tetrapod t2r gene families precedes the emergence of tetrapods, i.e. the water-to-land transition, and thus constitutes a basal feature of the lobe-finned lineage of vertebrates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-650) contains supplementary material, which is available to authorized users.     

The inheritance of volatile phenylpropenes in bitter fennel (Foeniculum vulgare Mill. var. vulgare, Apiaceae) chemotypes and their distribution within the plant

The volatile phenylpropenes estragole and t-anethole are the major constituents of the oleoresin of the aerial parts of bitter fennel (Foeniculum vulgare Mill. var. vulgare, Apiaceae). The levels of estragole and t-anethole varied during plant development, being maximal in flowers and developing mericarps. Still the ratio between estragole and t-anethole remained constant throughout development. Estragole-rich types were hybridized with t-anethole rich types to examine the genetic basis of this polymorphism. A reverse correlation between estragole and t-anethole content was evident and the action of a biallelic gene with partial dominance for high estragole content was inferred. Understanding phenylpropene inheritance might explain chemical polymorphism in wild bitter fennel populations, sheds light on the molecular mechanisms that lead to chemotypes evolution and is crucial for breeding fennel varieties with desired chemical compositions.     

Calcium alginate–starch hybrid support for both surface immobilization and entrapment of bitter gourd (Momordica charantia) peroxidase

Calcium alginate–starch hybrid gel was employed as an enzyme carrier both for surface immobilization and entrapment of bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase retained 52% of the initial activity while surface immobilized and glutaraldehyde crosslinked enzyme showed 63% activity. A comparative stability of both forms of immobilized bitter gourd peroxidase was investigated against pH, temperature and chaotropic agent; like urea, heavy metals, water-miscible organic solvents, detergent and inhibitors. Entrapped peroxidase was significantly more stable as compared to surface immobilized form of enzyme. The pH and temperature-optima for both immobilized preparations were the same as for soluble bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase showed 75% of the initial activity while the surface immobilized and crosslinked bitter gourd peroxidase retained 69% of the original activity after its seventh repeated use.     

Bitter taste receptor T2R1 is activated by dipeptides and tripeptides

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. In addition, we tested the ability of peptide inhibitors of the blood pressure regulatory protein, angiotensin converting enzyme (ACE) to activate T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium was measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Among the peptides examined, the bitter tri-peptide Phe-Phe-Phe (FFF), is the most potent in activating T2R1 with an EC₅₀ value in the micromolar range. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket.     

Modeling the human PTC bitter-taste receptor interactions with bitter tastants

We employed the first principles computational method MembStruk and homology modeling techniques to predict the 3D structures of the human phenylthiocarbamide (PTC) taste receptor. This protein is a seven-transmembrane-domain G protein-coupled receptor that exists in two main forms worldwide, designated taster and nontaster, which differ from each other at three amino-acid positions. 3D models were generated with and without structural similarity comparison to bovine rhodopsin. We used computational tools (HierDock and ScanBindSite) to generate models of the receptor bound to PTC ligand to estimate binding sites and binding energies. In these models, PTC binds at a site distant from the variant amino acids, and PTC binding energy was equivalent for both the taster and nontaster forms of the protein. These models suggest that the inability of humans to taste PTC is due to a failure of G protein activation rather than decreased binding affinity of the receptor for PTC. Amino-acid substitutions in the sixth and seventh transmembrane domains of the nontaster form of the protein may produce increased steric hindrance between these two α-helices and reduce the motion of the sixth helix required for G protein activation.     

Calcium taste preferences: genetic analysis and genome screen of C57BL/6J x PWK/PhJ hybrid mice

To characterize the genetic basis of voluntary calcium consumption, we tested C57BL/6J mice (B6; with low avidity for calcium), PWK/PhJ mice (PWK; with high avidity for calcium) and their F₁ and F₂ hybrids. All mice received a series of 96-h two-bottle preference tests with a choice between water and the following: 50 m m CaCl₂, 50 m m calcium lactate, 50 m m MgCl₂, 100 m m KCl, 100 m m NH₄Cl, 100 m m NaCl, 5 m m citric acid, 30 μ m quinine hydrochloride and 2 m m saccharin. Most frequency distributions of the parental and F₁ but not F₂ groups were normally distributed, and there were few sex differences. Reciprocal cross analysis showed that B6 × PWK F₁ mice had a non-specific elevation of fluid intake relative to PWK × B6 F₁ mice. In the F₂ mice, trait correlations were clustered among the divalent salts and the monovalent chlorides. A genome screen involving 116 markers showed 30 quantitative trait loci (QTLs), of which six involved consumption of calcium chloride or lactate. The results show pleiotropic controls of calcium and magnesium consumption that are distinct from those controlling consumption of monovalent chlorides or exemplars of the primary taste qualities.     

Taste-active compounds in a traditional Italian food: 'lampascioni'

Nature is a rich source of taste-active compounds, in particular of plant origin, many of which have unusual tastes. Many of these are found in traditional food, where spontaneous plants are used as ingredients. Some taste-active compounds were identified in the bulbs of Muscari comosum, a spontaneous plant belonging to the family of the Liliaceae, very common in the Mediterranean area, and used in traditional gastronomy (called 'lampascioni' in South Italy). The bulbs were extracted with a series of solvents of different polarity. The different fractions were submitted to a preliminary sensory evaluation, and the most interesting ones, characterized by a strong bitter taste and some chemestetic properties, were submitted to further purification and structural analysis. From the ethereal extract, several 3-benzyl-4-chromanones and one stilbene derivative were isolated. Pure compounds were examined for their taste activity by means of sensory evaluation, and proved to be responsible for the characteristic taste of this food. Some of these compounds have been synthesized de novo to confirm their structure.