Major taste loss in carnivorous mammals.

Mammalian sweet taste is primarily mediated by the type 1 taste receptor Tas1r2/Tas1r3, whereas Tas1r1/Tas1r3 act as the principal umami taste receptor. Bitter taste is mediated by a different group of G protein-coupled receptors, the Tas2rs, numbering 3 to ∼66, depending on the species. We showed previously that the behavioral indifference of cats toward sweet-tasting compounds can be explained by the pseudogenization of the Tas1r2 gene, which encodes the Tas1r2 receptor. To examine the generality of this finding, we sequenced the entire coding region of Tas1r2 from 12 species in the order Carnivora. Seven of these nonfeline species, all of which are exclusive meat eaters, also have independently pseudogenized Tas1r2 caused by ORF-disrupting mutations. Fittingly, the purifying selection pressure is markedly relaxed in these species with a pseudogenized Tas1r2. In behavioral tests, the Asian otter (defective Tas1r2) showed no preference for sweet compounds, but the spectacled bear (intact Tas1r2) did. In addition to the inactivation of Tas1r2, we found that sea lion Tas1r1 and Tas1r3 are also pseudogenized, consistent with their unique feeding behavior, which entails swallowing food whole without chewing. The extensive loss of Tas1r receptor function is not restricted to the sea lion: the bottlenose dolphin, which evolved independently from the sea lion but displays similar feeding behavior, also has all three Tas1rs inactivated, and may also lack functional bitter receptors. These data provide strong support for the view that loss of taste receptor function in mammals is widespread and directly related to feeding specializations.

Characterization of human fungiform papillae cells in culture.

The ability to maintain human fungiform papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Here we describe a method for enzymatically isolating human cells from fungiform papillae obtained by biopsy and maintaining them in culture for more than 7 passages (7 months) without loss of viability and while retaining many of the functional properties of acutely isolated taste cells. Cells in these cultures exhibited increases in intracellular calcium when stimulated with perceptually appropriate concentrations of several taste stimuli, indicating that at least some of the native signaling pathways were present. This system can provide a useful model for molecular studies of the proliferation, differentiation, and physiological function of human fungiform papillae cells.

Sweet taste receptor gene variation and aspartame taste in primates and other species.

Aspartame is a sweetener added to foods and beverages as a low-calorie sugar replacement. Unlike sugars, which are apparently perceived as sweet and desirable by a range of mammals, the ability to taste aspartame varies, with humans, apes, and Old World monkeys perceiving aspartame as sweet but not other primate species. To investigate whether the ability to perceive the sweetness of aspartame correlates with variations in the DNA sequence of the genes encoding sweet taste receptor proteins, T1R2 and T1R3, we sequenced these genes in 9 aspartame taster and nontaster primate species. We then compared these sequences with sequences of their orthologs in 4 other nontasters species. We identified 9 variant sites in the gene encoding T1R2 and 32 variant sites in the gene encoding T1R3 that distinguish aspartame tasters and nontasters. Molecular docking of aspartame to computer-generated models of the T1R2 + T1R3 receptor dimer suggests that species variation at a secondary, allosteric binding site in the T1R2 protein is the most likely origin of differences in perception of the sweetness of aspartame. These results identified a previously unknown site of aspartame interaction with the sweet receptor and suggest that the ability to taste aspartame might have developed during evolution to exploit a specialized food niche.

Resemblance of tongue anatomy in twins.

This study compared the anatomical features of the tongue in nine pairs of twins - six monozygotic and three dizygotic. The aim of the project was to determine if tongues, like any other anatomical structure, could be used to reliably predict relatedness given that tongue shape, presentation and surface can be influenced by environment. Using the method of forced choice, 30 subjects were asked to match the photographs of tongues from twins. Our data indicate that, based on visual assessment, monozygotic twins have highly similar tongues (60% matches); similarly, dizygotic twins were matched 31% of the time, which is a higher probability than would be expected from random selection. This study should help identify baseline and control data in future behavioral studies of taste, which has a genetic basis.

Analyses of sweet receptor gene (Tas1r2) and preference for sweet stimuli in species of Carnivora.

The extent to which taste receptor specificity correlates with, or even predicts, diet choice is not known. We recently reported that the insensitivity to sweeteners shown by species of Felidae can be explained by their lacking of a functional Tas1r2 gene. To broaden our understanding of the relationship between the structure of the sweet receptors and preference for sugars and artificial sweeteners, we measured responses to 12 sweeteners in 6 species of Carnivora and sequenced the coding regions of Tas1r2 in these same or closely related species. The lion showed no preference for any of the 12 sweet compounds tested, and it possesses the pseudogenized Tas1r2. All other species preferred some of the natural sugars, and their Tas1r2 sequences, having complete open reading frames, predict functional sweet receptors. In addition to preferring natural sugars, the lesser panda also preferred 3 (neotame, sucralose, and aspartame) of the 6 artificial sweeteners. Heretofore, it had been reported that among vertebrates, only Old World simians could taste aspartame. The observation that the lesser panda highly preferred aspartame could be an example of evolutionary convergence in the identification of sweet stimuli.

Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus.

BACKGROUND: The channel catfish, Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers. RESULTS: Both PHA-E and RCA-I almost exclusively labeled an 82-84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82-84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82-84 kDa which may be interpreted as a component of a multimeric receptor/channel complex. CONCLUSIONS: The data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata.

Evidence that taste buds are not present on the human adult uvula.

We conducted a study to investigate whether taste buds are present on the human adult uvula. Our impetus was to determine whether surgical procedures that involve removal of the uvula can affect taste perception. Five human uvulae were removed via a modified carbon dioxide laser-assisted uvulopalatoplasty in an outpatient office setting. The uvulae were serially sectioned and stained with a solution specific for membrane-bound calcium-modulated adenosine triphosphatase, a high concentration of which is found in taste receptors. Examination of the stained sections under light microscopy failed to show that any taste receptors were present in any of the uvulae. This finding suggests that the taste disturbances noted after surgical procedures involving removal of the uvula are not attributable to a loss of taste receptors.

The bamboo-eating giant panda (Ailuropoda melanoleuca) has a sweet tooth: behavioral and molecular responses to compounds that taste sweet to humans.

A growing body of behavioral and genetic information indicates that taste perception and food sources are highly coordinated across many animal species. For example, sweet taste perception is thought to serve to detect and motivate consumption of simple sugars in plants that provide calories. Supporting this is the observation that most plant-eating mammals examined exhibit functional sweet perception, whereas many obligate carnivores have independently lost function of their sweet taste receptors and exhibit no avidity for simple sugars that humans describe as tasting sweet. As part of a larger effort to compare taste structure/function among species, we examined both the behavioral and the molecular nature of sweet taste in a plant-eating animal that does not consume plants with abundant simple sugars, the giant panda (Ailuropoda melanoleuca). We evaluated two competing hypotheses: as plant-eating mammals, they should have a well-developed sweet taste system; however, as animals that do not normally consume plants with simple sugars, they may have lost sweet taste function, as has occurred in strict carnivores. In behavioral tests, giant pandas avidly consumed most natural sugars and some but not all artificial sweeteners. Cell-based assays revealed similar patterns of sweet receptor responses toward many of the sweeteners. Using mixed pairs of human and giant panda sweet taste receptor units (hT1R2+gpT1R3 and gpT1R2+hT1R3) we identified regions of the sweet receptor that may account for behavioral differences in giant pandas versus humans toward various sugars and artificial sweeteners. Thus, despite the fact that the giant panda's main food, bamboo, is very low in simple sugars, the species has a marked preference for several compounds that taste sweet to humans. We consider possible explanations for retained sweet perception in this species, including the potential extra-oral functions of sweet taste receptors that may be required for animals that consume plants.

Biomimetic chemical sensors using nanoelectronic readout of olfactory receptor proteins.

We have designed and implemented a practical nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells make them important pharmaceutical targets. Specifically, we have coupled olfactory receptor proteins (ORs) with carbon nanotube transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress on a path toward a bioelectronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.

Technique to collect fungiform (taste) papillae from human tongue.

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 2008). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Sour ageusia in two individuals implicates ion channels of the ASIC and PKD families in human sour taste perception at the anterior tongue.

BACKGROUND: The perception of sour taste in humans is incompletely understood at the receptor cell level. We report here on two patients with an acquired sour ageusia. Each patient was unresponsive to sour stimuli, but both showed normal responses to bitter, sweet, and salty stimuli. METHODS AND FINDINGS: Lingual fungiform papillae, containing taste cells, were obtained by biopsy from the two patients, and from three sour-normal individuals, and analyzed by RT-PCR. The following transcripts were undetectable in the patients, even after 50 cycles of amplification, but readily detectable in the sour-normal subjects: acid sensing ion channels (ASICs) 1a, 1beta, 2a, 2b, and 3; and polycystic kidney disease (PKD) channels PKD1L3 and PKD2L1. Patients and sour-normals expressed the taste-related phospholipase C-beta2, the delta-subunit of epithelial sodium channel (ENaC) and the bitter receptor T2R14, as well as beta-actin. Genomic analysis of one patient, using buccal tissue, did not show absence of the genes for ASIC1a and PKD2L1. Immunohistochemistry of fungiform papillae from sour-normal subjects revealed labeling of taste bud cells by antibodies to ASICs 1a and 1beta, PKD2L1, phospholipase C-beta2, and delta-ENaC. An antibody to PKD1L3 labeled tissue outside taste bud cells. CONCLUSIONS: These data suggest a role for ASICs and PKDs in human sour perception. This is the first report of sour ageusia in humans, and the very existence of such individuals ("natural knockouts") suggests a cell lineage for sour that is independent of the other taste modalities.

Characterization and long-term maintenance of rat taste cells in culture.

Taste cells have a limited life span and are replaced from a basal cell population, although the specific factors involved in this process are not well known. Short- and long-term cultures of other sensory cells have facilitated efforts to understand the signals involved in proliferation, differentiation, and senescence, yet few studies have reported successful primary culture protocols for taste cells. Furthermore, no studies have demonstrated both proliferation and differentiation in vitro. In this study, we have developed an in vitro culture system to maintain and utilize rat primary taste cells for more than 2 months without losing key molecular and biochemical features. Gustducin, phospholipase C-beta2 (PLC-beta2), T1R3, and T2R5 mRNA were detected in the cultured cells by reverse transcriptase-polymerase chain reaction. Western blot analysis demonstrated gustducin and PLC-beta2 expression in the same samples, which was confirmed by immunocytochemistry. Labeling with bromo-2-deoxyuridine (BrdU) demonstrated proliferation, and a subset of BrdU-labeled cells were also immunoreactive for either gustducin or PLC-beta2, indicating differentiation of newly generated cells in vitro. Cultured cells also exhibited increases in intracellular calcium in response to several taste stimuli. These results indicate that taste cells from adult rats can be generated and maintained under the described conditions for at least 2 months. This system will enable further studies of the processes involved in proliferation, differentiation, and function of mammalian taste receptor cells in an in vitro preparation.

Identification and functional characterization of a voltage-gated chloride channel and its novel splice variant in taste bud cells.

Taste bud cells are epithelial cells with neuronal properties. Voltage-dependent ion channels have been physiologically described in these cells. Here, we report the molecular identification and functional characterization of a voltage-gated chloride channel (ClC-4) and its novel splice variant (ClC-4A) from taste bud cells. ClC-4A skipped an exon near its 5'-end, incurring the loss of 60 amino acids at the N terminus. In situ hybridization and immunohistochemistry localized these two channels' transcripts and proteins to a subset of taste bud cells. Electrophysiological recordings of the heterologously expressed channels in Xenopus oocytes showed that ClC-4 and ClC-4A have opposite sensitivity to pH and unique ion selectivity. The chloride channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid had a slight or no inhibitory effect on the conductance of ClC-4, but both blockers inhibited ClC-4A, suggesting that ClC-4A is a candidate channel for an acid-induced 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive current. Furthermore, these two channels may play a role in bitter-, sweet-, and umami-mediated taste transmission by regulating transmitter uptake into synaptic vesicles.

Sphingolipids influence the sensitivity of lipid bilayers to fungicide, syringomycin E.

Sphingolipids with long chain bases hydroxylated at the C4 position are a requisite for the yeast, Saccharomyces cerevisia, to be sensitive to the ion channel forming antifungal agent, syringomycin E (SRE). A mutant S. cerevisiae strain, Deltasyr2, having sphingolipids with a sphingoid base devoid of C4-hydroxylation, is resistant to SRE. To explore the mechanism of this resistance, we investigated the channel forming activity of SRE in lipid bilayers of varying composition. We found that the addition of sphingolipid-rich fraction from Deltasyr2 to the membrane-forming solution (DOPS/DOPE/ergosterol) resulted in lipid bilayers with lower sensitivity to SRE compared with those containing sphingolipid fraction from wild-type S. cerevisiae. Other conditions being equal, the rate of increase of bilayer conductance was about 40 times slower, and the number of SRE channels was about 40 times less, with membranes containing Deltasyr2 versus wild-type sphingolipids. Deltasyr2 sphingolipids altered neither SRE single channel conductance nor the gating charge but the ability of SRE channels to open synchronously was diminished. The results suggest that the resistance of the Deltasyr2 mutant to SRE may be partly due to the ability of sphingolipids without the C4 hydroxyl group to decrease the channel forming activity of SRE.

Pseudogenization of a sweet-receptor gene accounts for cats' indifference toward sugar.

Although domestic cats (Felis silvestris catus) possess an otherwise functional sense of taste, they, unlike most mammals, do not prefer and may be unable to detect the sweetness of sugars. One possible explanation for this behavior is that cats lack the sensory system to taste sugars and therefore are indifferent to them. Drawing on work in mice, demonstrating that alleles of sweet-receptor genes predict low sugar intake, we examined the possibility that genes involved in the initial transduction of sweet perception might account for the indifference to sweet-tasting foods by cats. We characterized the sweet-receptor genes of domestic cats as well as those of other members of the Felidae family of obligate carnivores, tiger and cheetah. Because the mammalian sweet-taste receptor is formed by the dimerization of two proteins (T1R2 and T1R3; gene symbols Tas1r2 and Tas1r3), we identified and sequenced both genes in the cat by screening a feline genomic BAC library and by performing PCR with degenerate primers on cat genomic DNA. Gene expression was assessed by RT-PCR of taste tissue, in situ hybridization, and immunohistochemistry. The cat Tas1r3 gene shows high sequence similarity with functional Tas1r3 genes of other species. Message from Tas1r3 was detected by RT-PCR of taste tissue. In situ hybridization and immunohistochemical studies demonstrate that Tas1r3 is expressed, as expected, in taste buds. However, the cat Tas1r2 gene shows a 247-base pair microdeletion in exon 3 and stop codons in exons 4 and 6. There was no evidence of detectable mRNA from cat Tas1r2 by RT-PCR or in situ hybridization, and no evidence of protein expression by immunohistochemistry. Tas1r2 in tiger and cheetah and in six healthy adult domestic cats all show the similar deletion and stop codons. We conclude that cat Tas1r3 is an apparently functional and expressed receptor but that cat Tas1r2 is an unexpressed pseudogene. A functional sweet-taste receptor heteromer cannot form, and thus the cat lacks the receptor likely necessary for detection of sweet stimuli. This molecular change was very likely an important event in the evolution of the cat's carnivorous behavior.

Analysis of a human fungiform papillae cDNA library and identification of taste-related genes.

Various genes related to early events in human gustation have recently been discovered, yet a thorough understanding of taste transduction is hampered by gaps in our knowledge of the signaling chain. As a first step toward gaining additional insight, the expression specificity of genes in human taste tissue needs to be determined. To this end, a fungiform papillae cDNA library has been generated and analyzed. For validation of the library, taste-related gene probes were used to detect known molecules. Subsequently, DNA sequence analysis was performed to identify further candidates. Of 987 clones sequenced, clustering results in 288 contigs. Comparison of these contigs with genomic databases reveals that 207 contigs (71.9%) match known genes, 16 (5.6%) match hypothetical genes, eight (2.8%) match repetitive sequences and 57 (19.8%) have no or low similarity to annotated genes. The results indicate that despite a high level of redundancy, this human fungiform cDNA library contains specific taste markers and is valuable for investigation of both known and novel taste-related genes.