Computational Studies of Ligand-Receptor Interactions in Bitter Taste Receptors

Phenylthiocarbamide tastes intensely bitter to some individuals, but others find it completely tasteless. Recently, it was suggested that phenylthiocarbamide elicits bitter taste by interacting with a human G protein-coupled receptor (hTAS2R38) encoded by the PTC gene. The phenylthiocarbamide nontaster trait was linked to three single nucleotide polymorphisms occurring in the PTC gene. Using the crystal structure of bovine rhodopsin as template, we generated the 3D structure of hTAS2R38 bitter taste receptor. We were able to map on the receptor structure the amino acids affected by the genetic polymorphisms and to propose molecular functions for two of them that explained the emergence of the nontaster trait. We used molecular docking simulations to find that phenylthiocarbamide exhibited a higher affinity for the target receptor than the structurally similar molecule 6-n-propylthiouracil, in line with recent experimental studies. A 3D model was constructed for the hTAS2R16 bitter taste receptor as well, by applying the same protocol. We found that the recently published experimental ligand binding affinity data for this receptor correlated well with the binding scores obtained from our molecular docking calculations.     

Pathology of Hematodinium infections in snow crabs (Chionoecetes opilio) from Newfoundland, Canada

Bitter crab disease (BCD) of snow crabs, Chionoecetes opilio, is caused by a parasitic dinoflagellate, Hematodinium sp. The disease has shown an alarming increase in prevalence in the commercial fishery in eastern and northeastern areas of Newfoundland and Labrador since it was first recorded there in the early 1990s. We documented histopathological alterations to the tissues in snow crabs with heavy infections of Hematodinium sp. and during sporulation of the parasite. Pressure necrosis was evident in the spongy connective tissues of the hepatopancreas and the blood vessels in most organs. In heavy infections, little remained of the spongy connective tissues around the hepatopancreas. Damage to the gills varied; in some cases it was severe, particularly during sporulation, involving apparent thinning of the cuticle, loss of epithelial cells, and fusion of the membranous layers of adjacent gill lamellae. Affected lamellae exhibited varying degrees of distention with a loss of trabecular cells, hemocyte infiltrations, and swelling or "clubbing" along the distal margins. Large numbers of zoospores were located along the distal margins of affected lamellae suggesting that sporulation may cause a lysis or bursting of the thin lamellar cuticle, releasing spores. Pressure necrosis, due to the build up of high densities of parasites, was the primary histopathological alteration in most tissues. Hematodinium infections in the snow crab are chronic, long-term infections that end in host death, during sporulation of the parasite.     

Saponins from the traditional medicinal plant Momordica charantia stimulate insulin secretion in vitro

The antidiabetic activity of Momordica charantia (L.), Cucurbitaceae, a widely-used treatment for diabetes in a number of traditional medicine systems, was investigated in vitro. Antidiabetic activity has been reported for certain saponins isolated from M. charantia. In this study insulin secretion was measured in MIN6 β-cells incubated with an ethanol extract, saponin-rich fraction, and five purified saponins and cucurbitane triterpenoids from M. charantia, 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (1), momordicine I (2), momordicine II (3), 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-β-glucopyranoside (4), and kuguaglycoside G (5). Treatments were compared to incubation with high glucose (27 mM) and the insulin secretagogue, glipizide (50 μM). At 125 μg/ml, an LC-ToF-MS characterized saponin-rich fraction stimulated insulin secretion significantly more than the DMSO vehicle, p=0.02. At concentrations 10 and 25 μg/ml, compounds 3 and 5 also significantly stimulated insulin secretion as compared to the vehicle, p≤0.007, and p=0.002, respectively. This is the first report of a saponin-rich fraction, and isolated compounds from M. charantia, stimulating insulin secretion in an in vitro, static incubation assay.     

Reengineering the ligand sensitivity of the broadly tuned human bitter taste receptor TAS2R14

Background

In humans, bitterness perception is mediated by ~25 bitter taste receptors present in the oral cavity. Among these receptors three, TAS2R10, TAS2R14 and TAS2R46, exhibit extraordinary wide agonist profiles and hence contribute disproportionally high to the perception of bitterness. Perhaps the most broadly tuned receptor is the TAS2R14, which may represent, because of its prominent expression in extraoral tissues, a receptor of particular importance for the physiological actions of bitter compounds beyond taste.

Extraction of peroxidase from bitter gourd (Momordica charantia) by three phase partitioning with dimethyl carbonate (DMC) as organic phase

Three phase partitioning (TPP) is most renowned technique used for extraction and purification of natural products. In previous studies of TPP, t-butanol is mainly used as an organic phase. This is the first report that explores ability of dimethyl carbonate (DMC) in the field of TPP as an alternate solvent for t-butanol. In the present study TPP process with t-butanol and DMC as organic phase along with different salts was applied to waste bitter gourd powder to obtained peroxidase enzyme. DMC was found to be compatible with most of salts such as ammonium sulphate and sodium citrate and explored as more efficient solvent than t-butanol. This TPP system provides 4.84 fold purity of peroxidase enzyme at optimum source concentration of 0.15 g/mL, with a system comprising DMC as organic phase, sodium citrate (20%) as salt, agitation speed 120 rpm, pH 7, temperature 30 °C and extraction time of 3 h. Present study has aimed for extraction and separation of peroxidase from bitter gourd waste with TPP technique and ensures the scope of carbonated solvents in extraction and purification of proteins.     

Evaluation of the bitterness of green tea catechins by a cell-based assay with the human bitter taste receptor hTAS2R39

Catechins have a broad range of physiological functions and act as the main taste ingredient of green tea. Although catechins show a strong bitterness, the bitter taste receptor for catechins has not been fully understood. The objective of this study was to identify the receptor for the major green tea catechins such as (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg). By the cell-based assay using cultured cells expressing human bitter taste receptor, a clear response of hTAS2R39-expressing cells was observed to 300 μM of either ECg or EGCg, which elicit a strong bitterness in humans. The response of hTAS2R39-expressing cells to ECg was the strongest among the tested catechins, followed by EGCg. Because the cellular response to EC and EGC is much weaker than those of ECg and EGCg, galloyl groups was strongly supposed to be involved in the bitter intensity. This finding is similar to the observations of taste intensity obtained from a human sensory study. Our results suggest the participation of hTAS2R39 in the detection of catechins in humans, indicating the possibility that bitterness of tea catechins can be evaluated by using cells expressing hTAS2R39.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Somatic fusions between the cultivated potato Solanum tuberosum and the wild species S. circaeifolium subsp. circaeifolium Bitter were produced in order to incorporate desirable traits into the potato gene pool. Selection of the putative hybrids was based on a difference in callus morphology between the hybrids and their parents, with the hybrids showing typical purple-colored cells in otherwise green calli. In all, 17 individual calli regenerated to plants. Of the nine plants that could be transferred to the greenhouse, eight showed a hybrid and one a parental morphology. Restriction fragment length polymorphism (RFLP) analysis confirmed the hybrid character in the former group. Chloroplast counts in stomatal guard cells and flow cytometric determination of nuclear DNA content showed that four hybrid plants were tetraploid (4x), one was mixoploid (5x–8x), and the others were polyploid (6x; 8x). Three out of four tetraploid hybrids were found to be fully resistant to Phytophthora infestans, and all four hybrids were resistant to Globodera pallida pathotypes Pa2 and Pa3. It was further observed that the type and amount of steroidal glycoalkaloids varied among the tubers of the parents and the hybrids. Using the hybrids as female parents in crosses with S. tuberosum, viable seeds could be obtained. This demonstrates the potential of these hybrids in practical plant breeding.     

Genetic variation in bitter taste receptor genes influences the foraging behavior of plateau zokor (Eospalax baileyi)

The ability to detect bitter tastes is important for animals; it can help them to avoid ingesting harmful substances. Bitter taste perception is mainly mediated by bitter taste receptor proteins, which are encoded by members of the Tas2r gene family and vary with the dietary preference of a specific species. Although individuals with different genotypes differ in bitterness recognition capability, little is known about the relationship between genetic variation and food selection tendencies at the intraspecific level. In this study, we examined the relationship between genotypes and diet in plateau zokor (Eospalax baileyi), a subterranean rodent endemic to the Qinghai‐Tibet Plateau that caches food for the winter. We assayed the composition and taste profile of each plant contained in temporary caches and vicinity quadrats, which were representative of selected and available food, respectively. Bitter plant selection indices (E_bitter) were estimated. We also sequenced 26 candidate Tas2r genes from zokors and determined their relationships with the E_bitter of their caches. We identified four key results: (1) zokors varied considerably in both bitter food preference and Tas2r sequences; (2) five genes (zTas2r115, zTas2r119, zTas2r126, zTas2r134, and zTas2r136) exhibited allelic variation that was significantly associated with E_bitter; (3) synonymous SNPs, nonsynonymous SNPs, and pseudogenization are involved in the genotype–phenotype relationship; (4) the minor genotypes of zTas2r115, zTas2r134, and zTas2r136 and the major genotypes of zTas2r119 and zTas2r126 cached more bitter plants. Our results link Tas2r variation with food selection behavior at the population level for the first time.     

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-β-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.     

苦瓜种质遗传多样性的RAPD和ISSR分析

采用RAPD和ISSR分子标记技术对38份苦瓜种质进行遗传多样性分析。结果表明:10个RAPD和10个ISSR引物分别扩增出93条和81条带,多态性比率分别为50.54%和61.29%;RAPD和ISSR标记检测供试材料的遗传相似性系数(GS)范围,分别为0.287~1和0.221~1,ISSR(平均GS值0.672)检测多态性效果高于RAPD(平均GS值0.694)。RAPD标记聚类分析将供试种质分为3个类群6组,分类结果与苦瓜瓜瘤的表型分类比较相似;ISSR标记聚类分析将供试种质分为3个类群7组,ISSR标记划分类群与形态上以颜色分类比较接近。RAPD和ISSR标记的遗传相似性系数呈显著相关(r=0.550)。两个标记整合后聚类分析可检测到更大的遗传变异,结果与苦瓜的农艺性状分类和地理分布有一定的相关性。