Patrizia Muroni

The Gustin (CA6) Gene Polymorphism, rs2274333 (A/G), as a Mechanistic Link between PROP Tasting and Fungiform Taste Papilla Density and Maintenance

Taste sensitivity to PROP varies greatly among individuals and is associated with polymorphisms in the bitter receptor gene TAS2R38, and with differences in fungiform papilla density on the anterior tongue surface. Recently we showed that the PROP non-taster phenotype is strongly associated with the G variant of polymorphism rs2274333 (A/G) of the gene that controls the salivary trophic factor, gustin. The aims of this study were 1) to investigate the role of gustin gene polymorphism rs2274333 (A/G), in PROP sensitivity and fungiform papilla density and morphology, and 2) to investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity. Sixty-four subjects were genotyped for both genes by PCR techniques, their PROP sensitivity was assessed by scaling and threshold methods, and their fungiform papilla density, diameter and morphology were determined. In vitro experiments examined cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. Gustin and TAS2R38 genotypes were associated with PROP threshold (p=0.0001 and p=0.0042), but bitterness intensity was mostly determined by TAS2R38 genotypes (p<0.000001). Fungiform papillae densities were associated with both genotypes (p<0.014) (with a stronger effect for gustin; p=0.0006), but papilla morphology was a function of gustin alone (p<0.0012). Treatment of isolated cells with saliva from individuals with the AA form of gustin or direct application of the active iso-form of gustin protein increased cell proliferation and metabolic activity (p<0.0135). These novel findings suggest that the rs2274333 polymorphism of the gustin gene affects PROP sensitivity by acting on fungiform papilla development and maintenance, and could provide the first mechanistic explanation for why PROP super-tasters are more responsive to a broad range of oral stimuli.

Associations between Orosensory Perception of Oleic Acid, the Common Single Nucleotide Polymorphisms (rs1761667 and rs1527483) in the CD36 Gene, and 6-n-Propylthiouracil (PROP) Tasting

Orosensory perception of dietary fat varies in individuals, thus influencing nutritional status. Several studies associated fat detection and preference with CD36 or 6-n-propylthiouracil (PROP) sensitivity. Other studies have not confirmed the latter association. We analyzed the relationship between orosensory perception of oleic acid, two CD36 variants, and PROP tasting. Thresholds of oleic acid perception were assessed in 64 subjects using a modification of the three-alternative forced-choice procedure. Subjects were classified for PROP taster status and genotyped for TAS2R38 and CD36 (SNPs: rs1761667 and rs1527483). Subjects homozygous for GG of the rs1761667 polymorphism showed higher sensitivity to oleic acid than AA subjects. The capability to detect oleic acid was directly associated with TAS2R38 or PROP responsiveness. PROP non-tasters had a lower papilla density than tasters, and those with genotype GG of the rs1761667 polymorphism had lower oleic acid thresholds than PROP non-tasters with genotype AA. In conclusion, results showed a direct association between orosensory perception of oleic acid and PROP tasting or rs1761667 polymorphism of CD36, which play a significant role in PROP non-tasters, given their low number of taste papillae. Characterization of individual capability to detect fatty acids may have important nutritional implications by explaining variations in human fat preferences.

NEW INSIGHT INTO THE ANTENNAL CHEMOSENSORY FUNCTION OF OPIUS CONCOLOR (HYMENOPTERA, BRACONIDAE)

Abstract The presence of taste and olfactory chemosensilla is demonstrated by using both morphological and electrophysiological techniques on antennae of Opius concolor, a parasitoid of tephritid larvae. Observation under the Scanning Electron Microscope revealed the presence of large sensilla trichodea with an apical pore, and of flat plates (sensilla placodea) extending almost the entire length of each antennomere. By means of currently used electrophysiological techniques, it was possible to record both the gustatory activity generated by sensilla trichodea following contact with KC1 100 mM and sucrose 100 mM solutions, and olfactory activity probably generated by sensilla placodea in response to volatile stimuli. The analysis of gustatory activity showed that each sensillum trichodeum consisted of at least three chemosensory units. This supports the assumption that in O. concolor single sensilla may, by providing different spike discharge patterns, which depend on the quality and intensity of the stim...

The gustin (CA6) gene polymorphism, rs2274333 (A/G), is associated with fungiform papilla density, whereas PROP bitterness is mostly due to TAS2R38 in an ethnically-mixed population.

PROP responsiveness is associated with TAS2R38 haplotypes and fungiform papilla density. Recently, we showed that a polymorphism in the gene coding for the salivary trophic factor, gustin (CA6), affects PROP sensitivity by acting on cell growth and fungiform papillae maintenance, in a genetically homogeneous cohort. Since population homogeneity can lead to over estimation of gene effects, the primary aim of the present work was to confirm gustins role in PROP bitterness intensity and fungiform papillae density in a genetically diverse population. Eighty subjects were genotyped for both genes by PCR techniques. PROP responsiveness was assessed by a filter paper method and fungiform papilla density was determined in each subject. As expected, PROP bitterness ratings were lower in individuals with the AVI/AVI diplotype of TAS2R38 than in individuals with PAV/PAV and PAV/AVI diplotypes. However, no differences in PROP bitterness among genotypes of the gustin gene, and no differences in the density of fungiform papillae related to TAS2R38 diplotype were found. In contrast, the density of fungiform papillae decreased as the number of minor (G) alleles at the gustin locus increased. In addition, the distribution of TAS2R38 genotypes within each gustin genotype group showed that the occurrence of recessive alleles at both loci was infrequent in the present sample compared to other populations. These findings confirm that papillae density is associated with gustin gene polymorphism, rs2274333 (A/G), in an ancestrally heterogeneous population, and suggest that variations in the frequency of allele combinations for these two genes could provide a salient explanation for discrepant findings for gustin gene effects across populations.

Electrophysiological responses of labral apical chemoreceptors to adenine nucleotides in Culex pipiens

Abstract Electrophysiological recordings show that the hair-like sensilla on the tip of the labrum in Culex pipiens L. have neurones that respond to adenine nucleotides. Adenine nucleotides show the following descending order of stimulating effectiveness: ADP > ATP=AMP-PNP=AMP. Sensillar sensitivity to these adenine nucleotides were tested at the concentration range of 10 −5 –10 −3 M. These electrophysiological data are in good accordance with data reported from behavioural studies of this species.

Neuropeptide F peptides act through unique signaling pathways to affect cardiac activity

Elucidating how neuropeptides affect physiology may result in delineating peptidergic mechanisms and identifying antagonists for application in basic and translational science. Human neuropeptide Y (NPY) regulates cardiac activity; frequently invertebrates contain orthologs of vertebrate peptides. We report invertebrate NPY-like neuropeptide F (NPF) arrested the signal frequency of the slow phase of the cardiac cycle (EC50 = 1 pM); however, signal frequency of the fast phase was affected only minimally. Neuropeptide F decreased the duration of the slow phase by ~70% (EC50 = 0.6 pM), but increased the duration of the fast phase by ~57% (EC50 = 10nM). Short NPF-1 (sNPF-1) decreased the signal frequency of the slow phase by ~70% (EC50 = 9 nM); yet, signal frequency of the fast phase was unaffected. Short NPF-1 decreased the duration of the slow phase ~55% (EC50 ~50 nM), but increased the duration of the fast phase ~20% without dose dependency. Neuropeptide F and sNPF-1 increased isoelectric period duration. This novel report demonstrated NPY-like peptides are cardioactive but functionally unique. These data contribute to understanding how invertebrate orthologs affect cardiovascular activity. Dipteran fast and slow phases may be generated from separate pacemakers in the abdominal heart and in the anterior thoracocephalic aorta, respectively. Thus, our research suggests NPF and sNPF-1 act through different mechanisms to regulate cardiac activity. Invertebrate NPY-like peptides act in olfaction and feeding yet mechanisms which are associated with their cardioactive effects remain unknown; our work may provide evidence linking their roles in sensory response and cardiac activity.

Evidence dromyosuppressin acts at posterior and anterior pacemakers to decrease the fast and the slow cardiac activity in the blowfly Protophormia terraenovae

The molecular complexity of the simple blowfly heart makes it an attractive preparation to delineate cardiovascular mechanisms. Blowfly cardiac activity consists of a fast, high-frequency signal phase alternating with a slow, low-frequency signal phase triggered by pacemakers located in the posterior abdominal heart and anterior thoracocephalic aorta, respectively. Mechanisms underlying FMRFamide-related peptides (FaRPs) effects on heart contractions are not well understood. Here, we report antisera generated to a FaRP, dromyosuppressin (DMS, TDVDHVFLRFamide), recognized neuronal processes that innervated the blowfly Protophormia terraenovae heart and aorta. Dromyosuppressin caused a reversible cardiac arrest. High- and low-frequency signals were abolished after which they resumed; however, the concentration-dependent resumption of the fast phase differed from the slow phase. Dromyosuppressin decreased the frequency of cardiac activity in a dose-dependent manner with threshold values between 5 fM and 0.5 fM (fast phase), and 0.5 fM and 0.1 fM (slow phase). Dromyosuppressin structure-activity relationship (SAR) for the decrease of the fast-phase frequency was not the same as the SAR for the decrease of the slow-phase frequency. The alanyl-substituted analog TDVDHVFLAFamide ([Ala9] DMS) was inactive on the fast phase, but active on the slow phase, a novel finding. FaRPs including myosuppressins are reported to require the C-terminal RFamide for activity. Our data are consistent with the conclusions DMS acts on posterior and anterior cardiac tissue to play a role in regulating the fast and slow phases of cardiac activity, respectively, and ligand-receptor binding requirements of the abdominal and thoracocephalic pacemakers are different.

Conserved residues in RF-NH2 receptor models identify predicted contact sites in ligand-receptor binding

Peptides in the RF-NH2 family are grouped together based on an amidated dipeptide C terminus and signal through G-protein coupled receptors (GPCRs) to influence diverse physiological functions. By determining the mechanisms underlying RF-NH2 signaling targets can be identified to modulate physiological activity; yet, how RF-NH2 peptides interact with GPCRs is relatively unexplored. We predicted conserved residues played a role in Drosophila melanogaster RF-NH2 ligand-receptor interactions. In this study D. melanogaster rhodopsin-like family A peptide GPCRs alignments identified eight conserved residues unique to RF-NH2 receptors. Three of these residues were in extra-cellular loops of modeled RF-NH2 receptors and four in transmembrane helices oriented into a ligand binding pocket to allow contact with a peptide. The eighth residue was unavailable for interaction; yet its conservation suggested it played another role. A novel hydrophobic region representative of RF-NH2 receptors was also discovered. The presence of rhodopsin-like family A GPCR structural motifs including a toggle switch indicated RF-NH2s signal classically; however, some features of the DMS receptors were distinct from other RF-NH2 GPCRs. Additionally, differences in RF-NH2 receptor structures which bind the same peptide explained ligand specificity. Our novel results predicted conserved residues as RF-NH2 ligand-receptor contact sites and identified unique and classic structural features. These discoveries will aid antagonist design to modulate RF-NH2 signaling.

Sensitivity to albumin and its terminal amino acids of labellar taste chemoreceptors in Protophormia terraenovae (Diptera: Calliphoridae)

Abstract Stimulation with bovine serum albumin (BSA) evokes spikes discharges from three receptor cells of the labellar chemosensilla in Protophormia , the “sugar” cell being the most sensitive. Confrontation of the spike frequency profiles across all three chemoreceptor cells by the vector space analysis suggests that l -alanine (the C-terminal amino acid of the BSA molecule), but not l -aspartic acid (N-terminal amino acid), may account for the stimulator effectiveness of BSA.

EFFECTS OF ATP ON THE INTERACTION OF ALBUMIN AND ITS C-TERMINAL AMINO-ACID L-ALANINE WITH THE SUGAR RECEPTOR CELL IN PROTOPHORMIA-TERRAENOVAE (DIPTERA, CALLIPHORIDAE)

Abstract Stimulation with bovine serum albumin (BSA) and its C-terminal amino acid l -alanine evokes spike activity from the “sugar” cell of the labellar chemosensilla in Protophormia. The presence of ATP in the BSA solution strongly enhances this effect, but is pH-dependent. On the contrary, addition of ATP to the l -alanine solution inhibits the “sugar” cell response regardless of pH. Reception mechanisms for BSA and l -alanine are discussed.