Melanosomal targeting via caveolin-1 dependent endocytosis mediates ZN(II) phthalocyanine phototoxic action in melanoma cells.

Melanosomes have been considered crucial targets in melanoma treatments. In this study we explored the role of melanosomes in photodynamic therapy (PDT), employing the synthetic Zn(II) phthalocyanine Pc13, a potent photosensitizer that promotes melanoma cell death after irradiation. Phototoxic action is mediated by reactive oxygen species increase. The internalization mechanism of Pc13 and its consequent subcellular localization were evaluated in melanotic B16-F0 cells. Pharmacological inhibitors of dynamin or caveolae, but not of clathrin, decreased Pc13 cellular uptake and phototoxicity. Similar results were obtained when cells over-expressed dominant negative mutants of dynamin-2 and caveolin-1, indicating that Pc13 is internalized by caveolae-mediated endocytosis. Confocal microscopy analysis revealed that Pc13 targets melanosomes and damage of these structures after irradiation was demonstrated by transmission electron microscopy. Treatment of pigmented B16-F0 and WM35 melanoma cells with the melanin synthesis inhibitor phenylthiourea for 48 h led to cell depigmentation and enhanced cell death after irradiation, whereas a 3-h period of inhibition did not modify melanin content but produced a marked reduction of Pc13 phototoxicity, together with a decrease of oxidative melanin synthesis intermediates. In contrast, the effect of Pc13 in amelanotic A375 cells was not altered by phenylthiourea treatment. These results provide evidence that melanosomes have a dual role in the efficacy of PDT. While melanin antagonizes the phototoxic action of Pc13, the release of cytotoxic synthetic intermediates to cytosol after irradiation and melanosome damage is conducive to the phototoxic response. Based on these findings, we demonstrate that melanosome-targeted PDT could be an effective approach for melanoma treatment.

Further Insights into the Oxidative Pathway of Thiocarbonyl-Type Antitubercular Prodrugs: Ethionamide, Thioacetazone, and Isoxyl.

A chemical activation study of the thiocarbonyl-type antitubercular prodrugs, ethionamide (ETH), thioacetazone (TAZ), and isoxyl (ISO), was performed. Biomimetic oxidation of ethionamide using H2O2 (1 equiv) led to ETH-SO as the only stable S-oxide compound, which was found to occur in solution in the preferential form of a sulfine (ETH═S═O vs the sulfenic acid tautomer ETH-S-OH), as previously observed in the crystal state. It was also demonstrated that ETH-SO is capable of reacting with amines, as the putative sulfinic derivative (ETH-SO2H) was supposed to do. Unlike ETH, oxidation of TAZ did not allow observation of the mono-oxygenated species (TAZ-SO), leading directly to the more stable sulfinic acid derivative (TAZ-SO2H), which can then lose a SOxH group after further oxidation or when placed in a basic medium. It was also noticed that the unstable TAZ-SO intermediate can lead to the carbodiimide derivative as another electrophilic species. It is suggested that TAZ-SOH, TAZ-SO2H, and the carbodiimide compound can also react with NH2-containing nucleophilic species, and therefore be involved in toxic effects. Finally, ISO showed a very complex reactivity, here assigned to the coexistence of two mono-oxygenated structures, the sulfine and sulfenic acid tautomers. The mono- and dioxygenated derivatives of ISO are also highly unstable, leading to a panel of multiple metabolites, which are still reactive and likely contribute to the toxicity of this prodrug.

Using the Integrated Genome Viewer to reveal amplicon-derived polymorphism enriched at the phenylthiocarbamide locus in the teaching lab.

Due to its distinct phenotype and relatively simple inheritance pattern, the phenylthiocarbamide (PTC) loci is frequently utilized in teaching laboratories to demonstrate genetic concepts such as Mendelian inheritance and population genetics. We have developed a next-generation sequencing and bioinformatics approach to analyze the PTC gene locus to reveal single nucleotide polymorphism (SNP) variation at nucleotide position 785 that predicts tasting ability in humans. Here students purify DNA from their own cheek cells, perform polymerase chain reaction (PCR) amplification of the PTC gene followed by cleaved amplified polymorphic sequence (CAPS) testing. Students perform a second PCR on the PTC loci using high-fidelity Taq to create bar-coded amplicons for next-generation sequencing on the Ion Torrent Personal Genome Machine. Bioinformatic verification reveals polymorphic variation by aligning the entire class PTC PCR fragment sequence to the human gene using Bowtie2 and visualizing the results in the Integrated Genome Viewer. This exercise presents a learning opportunity for students to use next-generation sequencing to predict their own PTC taste sensitivity phenotype coupled with the standard CAPS method. This approach brings the PTC teaching method into the genomics era.

Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase.

Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because these bacteria have acquired various mechanisms for antibiotic resistance, which creates the need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival. For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP tyrosinase with high micromolar potency. In this report, we explored structure-activity relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar range (IC50 = 0.57 + 0.05 µM). Binding could be rationalized by molecular docking simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial growth in P. aeruginosa PA01 cultures.

Evolution of the bitter taste receptor TAS2R38 in colobines.

Bitter taste perception enables the detection of potentially toxic molecules and thus evokes avoidance behavior in vertebrates. It is mediated by bitter taste receptors, TAS2Rs. One of the best-studied TAS2R is TAS2R38. Phenylthiocarbamide (PTC) perception and TAS2R38 receptors vary across primate species, and this variation may be related to variation in dietary preferences. In particular, we previously found that the low sensitivity of TAS2R38s in Asian colobines likely evolved as an adaptation to their leaf-eating behavior. However, it remains unclear whether this low PTC sensitivity is a general characteristic of the subfamily Colobinae, a primate group that feeds predominantly on leaves. We performed genetic analyses, functional assays with mutant proteins, and behavioral analyses to evaluate the general characteristics of TAS2R38 in colobines. We found that PTC sensitivity is lower in TAS2R38s of African colobines than in TAS2R38s of omnivorous macaques. Furthermore, two amino acids shared between Asian and African colobines were responsible for low sensitivity to PTC, suggesting that the last common ancestor of extant colobines had this phenotype. We also detected amino acid differences between TAS2R38s in Asian and African colobines, indicating that they evolved independently after the separation of these groups.

Phenylthiourea Binding to Human Tyrosinase-Related Protein 1.

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.

Genotyping Analysis of Bitter-Taste Receptor Genes TAS2R38 and TAS2R46 in Japanese Patients with Gastrointestinal Cancers.

Type-2 bitter-taste receptors (TAS2Rs) are important for the evaluation of food quality and the nutritional control in animals. Mutations in some TAS2Rs including TAS2R38 are known to increase susceptibility to various diseases. However, the involvement of TAS2Rs in cancers has not been well understood. We conducted a pilot study by genotyping two TAS2R genes, TAS2R38 and TAS2R46, in Japanese cancer patients diagnosed with the following types of cancer: biliary tract cancer, hepatocellular carcinoma, pancreatic cancer, colorectal cancer and gastric cancer. We selected the two TAS2Rs because they carry virtually non-functional alleles in human populations. We found that cancer risk is not associated with any TAS2R46 genotypes since there were no significant differences in genotype frequencies between cancer patients and controls. On the other hand, we confirmed that phenylthiocarbamide (PTC) non-tasters homozygous (AVI/AVI) for TAS2R38 were more frequent among Japanese cancer patients than those among controls as suggested in a previous study. The AVI/AVI genotype was therefore considered to increases cancer risk. In contrast, we also found that homozygous (PAV/PAV) PTC tasters are less frequent among cancer patients, suggesting that the PAV/PAV is a cancer resistant genotype that decreases cancer risk. Genotype frequencies for heterozygous AVI/PAV genotype were not significantly different between the two groups. It is suggested that the risk and resistance of cancers is antagonistically controlled by the two TAS2R38 alleles, PAV and AVI, rather than by the AVI allele alone.

Emergence of hydrogen bonds from molecular dynamics simulation of substituted N-phenylthiourea-catechol oxidase complex.

A series of N-phenylthiourea derivatives was built starting from the X-ray structure in the molecular mechanics framework and the interaction profile in the complex with the catechol oxidase was traced using molecular dynamics simulation. The results showed that the geometry and interactions between ligand and receptor were highly related to the position of the substituted side chains of phenyl moiety. At the end of molecular dynamics run, a concentrated multicenter hydrogen bond was created between the substituted ligand and receptor. The conformation of the ligand itself were also restricted in the receptor pocket. Furthermore, the simulation time of 50 ns were found to be long enough to explore the relevant conformational space and the stationary behavior of the molecular dynamic could be observed.

Expression and Functional Activity of the Human Bitter Taste Receptor TAS2R38 in Human Placental Tissues and JEG-3 Cells.

Bitter taste receptors (TAS2Rs) are expressed in mucous epithelial cells of the tongue but also outside the gustatory system in epithelial cells of the colon, stomach and bladder, in the upper respiratory tract, in the cornified squamous epithelium of the skin as well as in airway smooth muscle cells, in the testis and in the brain. In the present work we addressed the question if bitter taste receptors might also be expressed in other epithelial tissues as well. By staining a tissue microarray with 45 tissue spots from healthy human donors with an antibody directed against the best characterized bitter taste receptor TAS2R38, we observed an unexpected strong TAS2R38 expression in the amniotic epithelium, syncytiotrophoblast and decidua cells of the human placenta. To analyze the functionality we first determined the TAS2R38 expression in the placental cell line JEG-3. Stimulation of these cells with diphenidol, a clinically used antiemetic agent that binds TAS2Rs including TAS2R38, demonstrated the functionality of the TAS2Rs by inducing calcium influx. Restriction enzyme based detection of the TAS2R38 gene allele identified JEG-3 cells as PTC (phenylthiocarbamide)-taster cell line. Calcium influx induced by PTC in JEG-3 cells could be inhibited with the recently described TAS2R38 inhibitor probenecid and proved the specificity of the TAS2R38 activation. The expression of TAS2R38 in human placental tissues points to further new functions and hitherto unknown endogenous ligands of TAS2Rs far beyond bitter tasting.

Association of schizophrenia with the phenylthiocarbamide taste receptor haplotype on chromosome 7q.

Phenylthiocarbamide (PTC) taste sensitivity is an inherited trait determined primarily by allelic variation of the taste-receptor gene TAS2R38 on chromosome 7q. Results of prior studies examining the ability to taste PTC in patients with schizophrenia have been mixed because of the difficulties in measuring PTC taste sensitivity behaviorally. In the current study, we examined the TAS2R38 genotypes of schizophrenia patients to determine whether the increased prevalence of nontasters in this patient population was indicative of a specific genetic association. Our a-priori hypothesis was that schizophrenia patients would show an increased prevalence of the nontaster phenotype compared with controls. The genotypes of two nonsynonymous coding single-nucleotide polymorphisms in TAS2R38 were assayed for 176 schizophrenia patients and 229 healthy control individuals, and the two-allele haplotypes were estimated. There was an over-representation of the major PTC nontaster haplotype among patients of European descent, relative to control individuals of similar ancestry. Patients and controls of African ancestry did not differ. The PTC nontaster haplotype is a genetic marker that may be used to identify subsets of schizophrenia patients who potentially harbor vulnerability genes in this region of chromosome 7q.

Effects of known phenoloxidase inhibitors on hemocyanin-derived phenoloxidase from Limulus polyphemus.

Inhibitors of phenoloxidase are used routinely to characterise the structural and functional properties of phenoloxidases. Hemocyanin-derived phenoloxidase activity is also sensitive to standard phenoloxidase inhibitors. In this study, we characterise the effects of a number of phenoloxidase inhibitors on hemocyanin-derived phenoloxidase activity from the chelicerate, Limulus polyphemus. Both inhibition type and K(i) values were similar to those observed for hemocyanin-derived phenoloxidase from another chelicerate, Eurypelma californicum. In addition, substrate inhibition was observed at concentrations above 2mM dopamine. The conformation in which two of the inhibitors, namely tropolone and kojic acid, would bind near the Cu(II) centre of hemocyanin is proposed.

Insights into the binding of Phenyltiocarbamide (PTC) agonist to its target human TAS2R38 bitter receptor.

Humans' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs). Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC) and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38) by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM) helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i) residue N103 participates actively in PTC binding, in line with previous computational studies. (ii) W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii) W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.

Specific alleles of bitter receptor genes influence human sensitivity to the bitterness of aloin and saccharin.

Variation in human taste is a well-known phenomenon. However, little is known about the molecular basis for it. Bitter taste in humans is believed to be mediated by a family of 25 G protein-coupled receptors (hT2Rs, or TAS2Rs). Despite recent progress in the functional expression of hT2Rs in vitro, up until now, hT2R38, a receptor for phenylthiocarbamide (PTC), was the only gene directly linked to variations in human bitter taste. Here we report that polymorphism in two hT2R genes results in different receptor activities and different taste sensitivities to three bitter molecules. The hT2R43 gene allele, which encodes a protein with tryptophan in position 35, makes people very sensitive to the bitterness of the natural plant compounds aloin and aristolochic acid. People who do not possess this allele do not taste these compounds at low concentrations. The same hT2R43 gene allele makes people more sensitive to the bitterness of an artificial sweetener, saccharin. In addition, a closely related gene's (hT2R44's) allele also makes people more sensitive to the bitterness of saccharin. We also demonstrated that some people do not possess certain hT2R genes, contributing to taste variation between individuals. Our findings thus reveal new examples of variations in human taste and provide a molecular basis for them.

Functional variants in TAS2R38 and TAS2R16 influence alcohol consumption in high-risk families of African-American origin.

BACKGROUND: A novel family of G protein-coupled receptors, TAS2Rs, has recently been characterized and linked to sensitivity to bitter taste compounds. We have previously reported that a missense mutation in the TAS2R16 gene reduces the sensitivity of the receptor to bitter-taste stimuli and that it is associated with risk for alcohol dependence. Other family-based studies on the genetic transmittance of taste perception have previously demonstrated a correlation between genetic variation in TAS2R38 and sensitivity to bitter-taste compounds such as phenylthiocarbamide (PTC) and 6-n-propylthiouracil (PROP). Haplotypes resulting from 3 common nonsynonymous coding single-nucleotide polymorphisms in the TAS2R38 gene have been shown to alter receptor functions and taste sensitivity to PTC and PROP. The perceived bitterness of PROP has also been associated with oral sensation and drinking behaviors. METHODS: We used family-based association methods to test for association between TAS2R38 haplotypes and alcohol dependence as well as a measure of alcohol consumption (Maxdrinks) and age of onset of drinking behaviors in a sample of families densely affected with alcoholism. We have also extended our analysis of TAS2R16 to include the Maxdrinks phenotype. RESULTS: A positive correlation was observed between TAS2R38 haplotypes and Maxdrinks in Collaborative Study on the Genetics of Alcoholism (COGA) high-risk women of African-American origin. The common taster haplotype is significantly associated with a lower mean Maxdrinks compared with the other haplotypes. Similarly, the allele of TAS2R16 that is associated with a lower risk for alcohol dependence is also associated with lower mean Maxdrinks scores in African-American families. In contrast to the previously reported significant association between TAS2R16 and alcohol dependence, we found no evidence that TAS2R38 haplotypes influence alcohol dependence in the COGA dataset. CONCLUSION: Functional variants in both TAS2R16 and TAS2R38 correlate with alcohol consumption in African-American families.

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.

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.

Genetic analysis of reproduction in the Buryat populations.

A complex anthropological survey based on population-genetic methods and a study of a wide spectrum of genetic systems (43 alleles from 17 independent loci) was undertaken among 450 Buryat women of post-reproductive age. The results obtained showed the influence of particular genetic markers and their complex on the formation of peculiarities in the reproduction structure of the Buryat population.A sharp increase in phenotype GC 2-2 frequency and the corresponding GC*2 allele of the group-specific component (GC) was established for women groups with burdened obstetric records. These groups are characterized also by a considerable decrease in the observed geterozygosity (Ho) as compared to its expected value (He). Samples including women with multiple pregnancies in the recorded obstetric anamnesis are characterized by a significant increase in the frequency of the rare alleles TF*C3 of the transferrin system and those of PI*Z belonging to the proteinase inhibitor system (a1-antitrypsin) as compared to the control group. The results obtained widened current knowledge about the influence of genetic and environmental components on reproduction processes in human populations.

Genetic analysis of a complex trait in the Utah Genetic Reference Project: a major locus for PTC taste ability on chromosome 7q and a secondary locus on chromosome 16p.

The ability to taste phenylthiocarbamide (PTC) shows complex inheritance in humans. We obtained a quantitative measure of PTC tasting ability in 267 members of 26 large three-generation families that were part of a set of CEPH families that had been used for genetic mapping. Significant bimodality was found for the distribution of age and gender adjusted scores (P<0.001), with estimated means of 3.16 (SD=1.80) and 9.26 (SD=1.54). Using the extensive genotyping available in these families from the genetic mapping efforts, we performed a genome scan by using 1324 markers with an average spacing of 4 cM. Analyses were first carried out with a recessive genetic model that has traditionally been assumed for the trait, and a threshold score of 8.0 delineating tasters from non-tasters. In this qualitative analysis, the maximum genome-wide lod score was 4.74 at 246 cM on chromosome 7; 17 families showed segregation of the dichotomous PTC phenotype. No other lod scores were significant; the next highest score was on chromosome 10 (lod=1.64 at 85 cM), followed by chromosome 3 (lod=1.29 at 267 cM). Because PTC taste ability exhibited substantial quantitative variation, the quantitative trait was also analyzed by using a variance components approach in SOLAR. The maximum quantitative genome-wide lod score was 8.85 at 246 cM on chromosome 7. Evidence for other possible quantitative loci was found on chromosomes 1 (lod=2.31 at 344 cM) and 16 (lod=2.01 at 14 cM). A subsequent two-locus whole-genome scan conditional on the chromosome 7 quantitative trait locus identified the chromosome 16 locus (two-locus lod=3.33 at 14 cM).

Photodegradation of diafenthiuron in water.

Diafenthiuron, 1-tert-butyl-3-(2,6-di-isopropyl-4-phenoxyphenyl)thiourea, is an effective insecticide and acaricide. Sunlight degradation of diafenthiuron in various aqueous solutions and pure hexane yielded two major identified products: 1-tert-butyl-3-(2,6-di-isopropyl-4-phenoxyphenyl)-carbodiimide (CGA-140,408) and 1-tert-butyl-3-(2,6-di-isopropyl-4-phenoxy-phenyl)urea (CGA-177,960). CGA-140,408 was further photo-transformed into CGA-177,960 by sunlight. Direct photolysis appeared to be a major photolysis pathway of diafenthiuron in the environment. Photodegradation of CGA-140,408 and CGA-177,960 was enhanced in humic acid water, paddy water and aqueous acetone solutions, and followed first-order kinetics. Isopropanol (a radical quencher) and de-aeration strongly inhibited the photolysis of these chemicals, which suggested oxygen radical-mediated reactions.