Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception.

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects' genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.

Comprehensive Analysis of Mouse Bitter Taste Receptors Reveals Different Molecular Receptive Ranges for Orthologous Receptors in Mice and Humans.

One key to animal survival is the detection and avoidance of potentially harmful compounds by their bitter taste. Variable numbers of taste 2 receptor genes expressed in the gustatory end organs enable bony vertebrates (Euteleostomi) to recognize numerous bitter chemicals. It is believed that the receptive ranges of bitter taste receptor repertoires match the profiles of bitter chemicals that the species encounter in their diets. Human and mouse genomes contain pairs of orthologous bitter receptor genes that have been conserved throughout evolution. Moreover, expansions in both lineages generated species-specific sets of bitter taste receptor genes. It is assumed that the orthologous bitter taste receptor genes mediate the recognition of bitter toxins relevant for both species, whereas the lineage-specific receptors enable the detection of substances differently encountered by mice and humans. By challenging 34 mouse bitter taste receptors with 128 prototypical bitter substances in a heterologous expression system, we identified cognate compounds for 21 receptors, 19 of which were previously orphan receptors. We have demonstrated that mouse taste 2 receptors, like their human counterparts, vary greatly in their breadth of tuning, ranging from very broadly to extremely narrowly tuned receptors. However, when compared with humans, mice possess fewer broadly tuned receptors and an elevated number of narrowly tuned receptors, supporting the idea that a large receptor repertoire is the basis for the evolution of specialized receptors. Moreover, we have demonstrated that sequence-orthologous bitter taste receptors have distinct agonist profiles. Species-specific gene expansions have enabled further diversification of bitter substance recognition spectra.

Transsynaptic Tracing from Taste Receptor Cells Reveals Local Taste Receptor Gene Expression in Gustatory Ganglia and Brain.

Taste perception begins in the oral cavity by interactions of taste stimuli with specific receptors. Specific subsets of taste receptor cells (TRCs) are activated upon tastant stimulation and transmit taste signals to afferent nerve fibers and ultimately to the brain. How specific TRCs impinge on the innervating nerves and how the activation of a subset of TRCs leads to the discrimination of tastants of different qualities and intensities is incompletely understood. To investigate the organization of taste circuits, we used gene targeting to express the transsynaptic tracer barley lectin (BL) in the gustatory system of mice. Because TRCs are not synaptically connected with the afferent nerve fibers, we first analyzed tracer production and transfer within the taste buds (TBs). Surprisingly, we found that BL is laterally transferred across all cell types in TBs of mice expressing the tracer under control of the endogenous Tas1r1 and Tas2r131 promotor, respectively. Furthermore, although we detected the BL tracer in both ganglia and brain, we also found local low-level Tas1r1 and Tas2r131 gene, and thus tracer expression in these tissues. Finally, we identified the Tas1r1 and Tas2r131-expressing cells in the peripheral and CNS using a binary genetic approach. Together, our data demonstrate that genetic transsynaptic tracing from bitter and umami receptor cells does not selectively label taste-specific neuronal circuits and reveal local taste receptor gene expression in the gustatory ganglia and the brain. SIGNIFICANCE STATEMENT: Previous papers described the organization of taste pathways in mice expressing a transsynaptic tracer from transgenes in bitter or sweet/umami-sensing taste receptor cells. However, reported results differ dramatically regarding the numbers of synapses crossed and the reduction of signal intensity after each transfer step. Nevertheless, all groups claimed this approach appropriate for quality-specific visualization of taste pathways. In the present study, we demonstrate that genetic transsynaptic tracing originating from umami and bitter taste receptor cells does not selectively label taste quality-specific neuronal circuits due to lateral transfer of the tracer in the taste bud and taste receptor expression in sensory ganglia and brain. Moreover, we visualized for the first time taste receptor-expressing cells in the PNS and CNS.

Cre-Mediated Recombination in Tas2r131 Cells-A Unique Way to Explore Bitter Taste Receptor Function Inside and Outside of the Taste System.

The type 2 taste receptors (Tas2rs) comprise a large family of G protein-coupled receptors that recognize compounds bitter to humans and aversive to vertebrates. Tas2rs are expressed in both gustatory and nongustatory tissues, however, identification and functional analyses of T2R-expressing cells have been difficult in most tissues. To overcome these limitations and to be able to manipulate Tas2r-expressing cells in vivo, we used gene-targeting to generate a Tas2r131-specific Cre knock-in mouse strain. We then employed a binary genetic approach to characterize Cre-mediated recombination in these animals and to investigate Tas2r131 expression during postnatal development. We demonstrate that a Cre-activated fluorescent reporter reliably visualizes Tas2r131-cells in gustatory tissue. We show that the onset of Tas2r131 as well as of α-Gustducin expression is initiated at different developmental stages depending on the type of taste bud. Furthermore, the number of Tas2r131- and α-Gustducin-expressing cells increased during postnatal development. Our results demonstrate that the Tas2r131-expressing cells constitute a subpopulation of α-Gustducin positive cells at all stages. We detected Tas2r131-expressing cells in several nongustatory tissues including lung, trachea, ovary, ganglia, and brain. Thus, the Tas2r131-Cre strain will help to dissect the functional role of Tas2r131 cells in both gustatory and nongustatory tissues in the future.

A Binary Genetic Approach to Characterize TRPM5 Cells in Mice.

Transient receptor potential channel subfamily M member 5 (TRPM5) is an important downstream signaling component in a subset of taste receptor cells making it a potential target for taste modulation. Interestingly, TRPM5 has been detected in extra-oral tissues; however, the function of extra-gustatory TRPM5-expressing cells is less well understood. To facilitate visualization and manipulation of TRPM5-expressing cells in mice, we generated a Cre knock-in TRPM5 allele by homologous recombination. We then used the novel TRPM5-IRES-Cre mouse strain to report TRPM5 expression by activating a τGFP transgene. To confirm faithful coexpression of τGFP and TRPM5 we generated and validated a new anti-TRPM5 antiserum enabling us to analyze acute TRPM5 protein expression. τGFP cells were found in taste bud cells of the vallate, foliate, and fungiform papillae as well as in the palate. We also detected TRPM5 expression in several other tissues such as in the septal organ of Masera. Interestingly, in the olfactory epithelium of adult mice acute TRPM5 expression was detected in only one (short microvillar cells) of two cell populations previously reported to express TRPM5. The TRPM5-IC mouse strain described here represents a novel genetic tool and will facilitate the study and tissue-specific manipulation of TRPM5-expressing cells in vivo.

Taste responses in mice lacking taste receptor subunit T1R1.

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1(-/-)) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1(-/-) mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1(-/-) mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1(+/-) mice responded to sweet and umami compounds, whereas those in T1R1(-/-) mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1(-/-) than in T1R1(+/-) mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1(-/-) and T1R1(+/-) mice. Conditioned taste aversion tests demonstrated that both T1R1(-/-) and T1R1(+/-) mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds.

Genetic labeling of Tas1r1 and Tas2r131 taste receptor cells in mice.

Characterization of the peripheral taste system relies on the identification and visualization of the different taste bud cell types. So far, genetic strategies to label taste receptor cells are limited to sweet, sour, and salty detecting cells. To visualize Tas1r1 umami and Tas2r131 bitter sensing cells, we generated animals in which the Tas1r1 and Tas2r131 open reading frames are replaced by expression cassettes containing the fluorescent proteins mCherry or hrGFP, respectively. These animals enabled us to visualize and quantify the entire oral Tas1r1 and Tas2r131 cell populations. Tas1r1-mCherry cells were predominantly detected in fungiform papillae, whereas Tas2r131-hrGFP cells, which are ~4-fold more abundant, were mainly present in foliate and vallate papillae. In the palate, both cell types were similarly distributed. Mice carrying both recombinant alleles demonstrated completely segregated Tas1r1 and Tas2r131 cell populations. Only ~50% of the entire bitter cell population expressed hrGFP, indicating that bitter taste receptor cells express a subset of the bitter receptor repertoire. In extragustatory tissues, mCherry fluorescence was observed in testis and hrGFP fluorescence in testis, thymus, vomeronasal organ, and respiratory epithelium, suggesting that only few extraoral sites express Tas2r131 and Tas1r1 receptors at levels comparable to taste tissue.

Green and efficient synthesis of sulfonamides catalyzed by nano-Ru/Fe(3)O(4).

The environmentally benign synthesis of carbon-nitrogen bonds continues to be an active and challenging field of chemical research. Here, a novel, environmentally benign method for the direct coupling of sulfonamides and alcohols is described. Despite the importance of sulfonamide derivatives as intermediates in drug synthesis, till now such transformations are rarely known. For the first time a domino dehydrogenation-condensation-hydrogenation sequence of alcohols and sulfonamides has been realized in the presence of a nanostructured catalyst. The magnetic property of the catalyst system allows for convenient isolation of the product and efficient recycling of the catalyst. A variety of coupling reactions of benzylic alcohols and sulfonamides including various heterocycles were successfully realized, often with >80% isolated yield. Advantageously, only one equivalent of the primary alcohol is consumed in the process. Mechanistic investigations of the competitive reactions of benzyl alcohol and d(7)-benzyl alcohol with p-toluenesulfonamide revealed a kinetic isotope effect (k(H)/k(D)) of 2.86 (+/-0.109) for the dehydrogenation of benzyl alcohol and 0.74 (+/-0.021) for the hydrogenation of N-benzylidene-p-toluenesulfonamide intermediate, which suggests dehydrogenation of the alcohol to be the rate determining step.

Procedures for the GMP-Compliant Production and Quality Control of [18F]PSMA-1007: A Next Generation Radiofluorinated Tracer for the Detection of Prostate Cancer.

Radiolabeled tracers targeting the prostate-specific membrane antigen (PSMA) have become important radiopharmaceuticals for the PET-imaging of prostate cancer. In this connection, we recently developed the fluorine-18-labelled PSMA-ligand [18F]PSMA-1007 as the next generation radiofluorinated Glu-ureido PSMA inhibitor after [18F]DCFPyL and [18F]DCFBC. Since radiosynthesis so far has been suffering from rather poor yields, novel procedures for the automated radiosyntheses of [18F]PSMA-1007 have been developed. We herein report on both the two-step and the novel one-step procedures, which have been performed on different commonly-used radiosynthesisers. Using the novel one-step procedure, the [18F]PSMA-1007 was produced in good radiochemical yields ranging from 25 to 80% and synthesis times of less than 55 min. Furthermore, upscaling to product activities up to 50 GBq per batch was successfully conducted. All batches passed quality control according to European Pharmacopoeia standards. Therefore, we were able to disclose a new, simple and, at the same time, high yielding production pathway for the next generation PSMA radioligand [18F]PSMA-1007. Actually, it turned out that the radiosynthesis is as easily realised as the well-known [18F]FDG synthesis and, thus, transferable to all currently-available radiosynthesisers. Using the new procedures, the clinical daily routine can be sustainably supported in-house even in larger hospitals by a single production batch.

A novel MCR of isocyanates, aldehydes, and dienophiles.

[reaction: see text] A novel one-pot procedure for the three-component coupling reaction of isocyanates, aldehydes, and dienophiles (IAD reaction) has been developed. Condensation of isocyanates and aldehydes and subsequent Diels-Alder reactions with electron-deficient dienophiles furnishes endo-selective amino-substituted cyclohexenes in good yield.

Headspace solid-phase microextraction with 1-pyrenyldiazomethane on-fibre derivatisation for analysis of fluoroacetic acid in biological samples.

A new and in part automated headspace solid-phase microextraction method for quantitative determination of the highly toxic rodenticide fluoroacetic acid (FAA) in serum and other biological samples has been developed. FAA and deuterated acetic acid (internal standard) were extracted from acidified samples by a StableFlex divinylbenzene-Carboxen on polydimethylsiloxane fibre. The acids were derivatised on the fibre in-situ with 1-pyrenyldiazomethane and detected using gas chromatography-mass spectrometry with electron impact ionisation and selected ion monitoring. The calibration curve for FAA in serum was linear over the range from 0.02 to 5 microg/ml, with limits of detection and quantification of 0.02 and 0.07 microg/ml, respectively. The method was also tested with spiked whole blood, urine, stomach contents and kidney samples. It was sufficiently reliable, reproducible and sensitive for use in routine forensic toxicology applications.

A subset of mouse colonic goblet cells expresses the bitter taste receptor Tas2r131.

The concept that gut nutrient sensing involves taste receptors has been fueled by recent reports associating the expression of taste receptors and taste-associated signaling molecules in the gut and in gut-derived cell lines with physiological responses induced by known taste stimuli. However, for bitter taste receptors (Tas2rs), direct evidence for their functional role in gut physiology is scarce and their cellular expression pattern remained unknown. We therefore investigated Tas2r expression in mice. RT-PCR experiments assessed the presence of mRNA for Tas2rs and taste signaling molecules in the gut. A gene-targeted mouse strain was established to visualize and identify cell types expressing the bitter receptor Tas2r131. Messenger RNA for various Tas2rs and taste signaling molecules were detected by RT-PCR in the gut. Using our knock-in mouse strain we demonstrate that a subset of colonic goblet cells express Tas2r131. Cells that express this receptor are absent in the upper gut and do not correspond to enteroendocrine and brush cells. Expression in colonic goblet cells is consistent with a role of Tas2rs in defense mechanisms against potentially harmful xenobiotics.

In situ generation of chiral N-dienyl lactams in a multicomponent reaction: an efficient and highly selective way to asymmetric amidocyclohexenes.

Chiral N-dienyl lactams are crucial building blocks for the synthesis of complex organic compounds. However, their generation is rather challenging. This paper reports on a highly efficient and diastereoselective multicomponent methodology utilizing chiral amides, aldehydes, and dienophiles (AAD reaction). The three components readily react under in situ generation of chiral N-dienyl lactams which undergo a subsequent Diels-Alder reaction. Different chiral amides have been employed in the standard protocol delivering yields up to 94%, and selectivities up to 90% de. Moreover, DFT calculations were performed to explain the obtained selectivities.

Three-component reactions with (S)-methyl pyroglutamate: an efficient way to diversely substituted asymmetric amidocyclohexenes.

Chiral N-dienyl lactams are crucial building blocks for the synthesis of complex organic compounds. However, their generation is rather challenging. This paper reports the novel one-pot reaction of (S)-methyl pyroglutamate as the amide component with different aldehydes and dienophiles (AAD reaction) to give novel chiral 1-amido-2-cyclohexenes. The corresponding N-dienyl lactams generated in situ undergo subsequent Diels-Alder reactions in good yield and diastereoselectivity. The scope and limitations of the three-component protocol were investigated. X-ray and NMR spectroscopic analysis of the products as well as DFT calculations of the intermediates were also performed to explain the observed stereoselectivity and structural features.

Synthesis and antimicrobial activity of N-analogous corollosporines.

Corollosporine is an antimicrobial metabolite, which was isolated from the marine fungus Corollospora maritima. Owing to its basic 4-hydroxyphthalic acid anhydride structure, it has become an attractive target for a structure/activity relationship modelling of derived compounds with potential antibiotic activity. In this regard we report on the straightforward synthesis of hetero analogous corollosporines, which were easily prepared by a three-step reaction sequence, taking advantage of a novel multicomponent reaction (AAD-reaction) and a subsequent aromatization/Grignard reaction protocol. Furthermore, the obtained products were tested in several biological assays to evaluate their antimicrobial activity.

Synthesis of enantiomerically pure cyclohex-2-en-1-ols: development of novel multicomponent reactions.

Multicomponent reactions of aldehydes, dienophiles, and alcohols or carboxylic acid anhydrides have been developed for the first time. In situ generation of 1-acyloxy- and 1-alkoxy-1,3-butadiene derivatives in toluene in the presence of electron-deficient dienophiles provides selective and efficient access to functionalized cyclohex-2-ene-1-ols in good yields. Subsequent enzyme-catalyzed kinetic resolution gave the corresponding enantiomers with high enantioselectivity.

From a spin-off to the advantageous use in Diels-Alder reactions: a combined synthetic, spectroscopic and computational approach to N-(dienyl)acylamines.

The acid-catalyzed condensation of aldehydes and acetamide has been shown to provide a pool of diverse equilibrating species. For the first time, the synthesis of substituted 1-N-acylamino-1,3-butadienes has been accomplished directly in moderate yields upon telomerization of two molecules of aldehyde with one molecule of carboxamide. Detailed spectroscopic and computational investigations establish the favourable formation of all-trans aminodienes under the reaction conditions. Employment thereof as diene building blocks in Diels-Alder reactions allows for the synthesis of carbocyclic molecules with high stereocontrol.