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R Tirindelli

Publications and source records attributed to R Tirindelli.

At least 19 recordsLinked to original sources

How mammals detect pheromones.

One of the most intriguing discoveries in mammalian pheromone research is the report that a short exposure of women to volatile compounds from sweat can significantly alter their menstrual cycle. This work suggests that specific molecules are produced by women at different stages of the menstrual cycle and that this putative 'pheromonal' blend has effects on the timing of the cycle in women that were briefly exposed to it. What human pheromones are and how they work are not known, however a considerable progress has been made in understanding how other mammals are likely to detect pheromones with the discovery of pheromone receptors. Even though it is proved that pheromones affect human responses, it remains unlikely that similar receptors account for these effects.

Animals↗

Co-expression of putative pheromone receptors in the sensory neurons of the vomeronasal organ.

Two large and divergent families of G-protein-coupled receptors (V1Rs and V2Rs) are expressed in subsets of neurons in the vomeronasal organ. These receptors are likely to mediate pheromone responses, but it appears that many V2R genes may encode expressed pseudogenes rather than functional proteins. Therefore we have raised antibodies to representative V2Rs and show labeling of vomeronasal neurons demonstrating that V2R genes encode expressed receptors. V2R immunoreactivity was detected at the sensory surface of the vomeronasal organ in dendritic terminals, indicating that these receptors are capable of directly interacting with pheromones and mediating physiological responses. Immunohistochemistry confirmed that three V2R receptors are expressed in small subsets of sensory neurons. However, surprisingly we found that a subfamily of V2R genes is broadly expressed in the Goalpha-layer of the vomeronasal organ and are coexpressed in the same cells as other V2Rs. This is in direct contrast to the main olfactory epithelium where sensory neurons express only a single receptor. Thus, our results suggest that different modes of the information processing may occur in the main and accessory olfactory systems.

Animals↗

Proliferation and migration of receptor neurons in the vomeronasal organ of the adult mouse.

Cell proliferation and differentiation in the vomeronasal organ of the adult mouse was studied by bromodeoxyuridine (BrdU) immunohistochemistry coupled to immunostaining for specific markers of the differentiation, such as carnosine, B50-GAP43 (growth-associated protein) and stathmin. The present study shows that three populations of proliferating elements are present in the vomeronasal sensory epithelium that are placed, respectively, in the supporting cell layer, at the boundaries between the sensory epithelium (S-VNO) and the non-sensory (NS-VNO) and in the basal region of the S-VNO. The number of dividing cells at the boundaries of the S-VNO is by far prevailing. Few proliferating cells located adjacent to the basal membrane are, however, present 1 day after BrdU inoculations. Seven days after BrdU treatment immunopositive nuclei were detected in more central regions of the VNO and at longer survival times they were also positive to carnosine, a marker of fully differentiated neurons. In conclusion, the present results suggest that at least two populations of VNO neuronal precursors are responsible for cell replacement throughout life.

Animals↗

Pheromone signalling in the mouse: role of urinary proteins and vomeronasal organ.

The lipocalin protein family is characterized in structure by a conserved hydrophobic pocket which can bind small volatile odorants. The Major Urinary Proteins (MUPs) are a class of lipocalins found in the urine of adult male mice which concentrate in the urine odorants which confer a characteristic odor. The behavioural as well as the endocrine effects of mouse urine and MUPs are briefly reviewed, suggesting a complex role is pheromonal communication. Some recent data on the molecular receptors of the vomeronasal organ further suggest a complex interaction with the MUP system.

Alpha-Globulins↗

Molecular aspects of pheromonal communication via the vomeronasal organ of mammals.

Recently, two large multigene families of putative G-protein-linked receptors that are expressed in distinct subpopulations of neurones in the vomeronasal organ have been identified. These receptors probably mediate pheromone detection. The most surprising aspects of these findings are that there are so many receptors of two very different classes and that the receptors are unrelated to their counterparts in the main olfactory epithelium. This suggests that many active ligands are likely to exert effects through the vomeronasal organ. Parallel experiments addressing the nature of these ligands indicate a role for some proteins, as well as small molecules, as functional mammalian pheromones. In combination, these results begin to suggest a molecular basis for mammalian pheromone signalling.

Animal Communication↗

Expression of a lipocalin in Pichia pastoris: secretion, purification and binding activity of a recombinant mouse major urinary protein.

The proteins of the mouse major urinary protein complex (MUP), members of the lipocalin family, bind volatile pheromones and interact with the vomeronasal neuroepithelium of the olfactory system. We report the expression of a MUP protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. Mature recombinant MUP (rMUP) is secreted at a concentration of 270 mg/l in minimal medium and it is isolated from the culture supernatant by one step ion-exchange chromatography in a nearly pure form. Binding activity, tested with an odorant molecule which displays high affinity for native MUP, indicates that rMUP has a behavior similar to the native one. This finding suggests that the protein, and in particular its hydrophobic binding pocket, is properly folded.

Alpha-Globulins↗

A new multigene family of putative pheromone receptors.

The vomeronasal organ (VNO) mediates detection of pheromones related to social and reproductive behavior in most terrestrial vertebrates. We have identified a new multigene family of G protein-linked receptors (V2Rs) that are specifically expressed in the VNO. V2Rs have no significant homology to other putative pheromone receptors (V1Rs) or to olfactory receptors but are related to the Ca2+-sensing receptor and metabotropic glutamate receptors. V2Rs are expressed at high levels in small subpopulations of VNO neurons. V2Rs are primarily expressed in a different layer of VNO neurons from V1Rs, thus both gene families are likely to encode mammalian pheromone receptors.

Animals↗

Evidence for different chemosensory signal transduction pathways in olfactory and vomeronasal neurons.

Both the olfactory and vomeronasal epithelia mediate chemosensory reception. Here we report that several molecules that are highly expressed in the olfactory epithelium and therefore are likely to be important mediators of olfactory signal transduction (Golfalpha, adenylyl cyclase III and the olfactory cyclic nucleotide gated ion channel) are not present in the vomeronasal epithelium. Therefore it appears that distinct molecules mediate chemosensory signal transduction in the olfactory and vomeronasal epithelia. The genes for Golfalpha, adenylyl cyclase III, the olfactory cyclic nucleotide gated ion channel, Ggamma8 and olfactory marker protein which are all expressed in the olfactory epithelium have consensus Olf-1 binding sites. The transcription factor Olf-1 was found to be highly expressed in the olfactory epithelium and was detected at a similar level in the vomeronasal epithelium. The expression pattern of Olf-1 did not correlate with that of molecules involved in olfactory signaling but was more similar to the expression pattern of Ggamma8 and olfactory marker protein which are found both in olfactory and vomeronasal neurons. Therefore, expression of Olf-1 in the olfactory epithelium and the presence of Olf-1 binding sites in a number of different genes found to be expressed in the olfactory epithelium are not sufficient to explain the observed gene expression patterns.

Adenylyl Cyclases↗

The G-protein gamma-subunit G gamma 8 is expressed in the developing axons of olfactory and vomeronasal neurons.

The tissue localization of the G-protein gamma-subunit, G gamma 8, that is specifically expressed in the olfactory and vomeronasal neurons, was studied in rats at different ages: embryonic day 16, postnatal days 1, 7, 14 and 35, and adult. G8 appears to be a specific marker of the immature olfactory and vomeronasal neurons. Its distribution differs from that of Golf alpha, a G-protein alpha-subunit which is predominantly expressed in mature olfactory neurons. G8 immunoreactivity indicates that an undifferentiated organization of the olfactory epithelium persists up to 3 weeks of age, though neonates possess a functional sense of smell. G gamma 8 accumulates at the highest levels in the axons of the developing olfactory neurons 2 weeks after birth (postnatal day 14). Moreover, up to postnatal day 14, G gamma 8-positive neurons are present in the region of the olfactory and vomeronasal epithelium, where they are not observed in later life. In the olfactory epithelium and in the bulb, G gamma 8 expression becomes weaker and patchy with increasing age, suggesting that the process of continuous regeneration of olfactory neurons occurs in discrete areas. G8-enhanced expression following axotomy indicates that this system is potentially active throughout life. Conversely, in the vomeronasal epithelium G gamma 8 expression persists virtually unmodified in the adult. This indicates that the degree of differentiation may differ between olfactory and vomeronasal neurons.

Aging↗

A novel GTP-binding protein gamma-subunit, G gamma 8, is expressed during neurogenesis in the olfactory and vomeronasal neuroepithelia.

A novel heterotrimeric G-protein gamma-subunit has been cloned, and its function has been confirmed by expression and purification. This gamma-subunit is only detected in the olfactory epithelium, the vomeronasal epithelium and, to a lesser extent, the olfactory bulb. It is absent from all other tissues studied including the nasal respiratory epithelium. During development, expression of G gamma 8 in the olfactory epithelium parallels neurogenesis, peaking shortly after birth and declining in the adult. In situ hybridization studies localize expression of this novel gamma-subunit to the sensory neurons; hybridization is strongest in the region of the epithelium that contains immature neurons. Unlike proteins that are expressed only in mature olfactory neurons (e.g. olfactory marker protein or Golf alpha), expression of G gamma 8 in the olfactory epithelium is relatively unaffected by olfactory bulbectomy. In the vomeronasal epithelium expression of G gamma 8 is also highest in the developing neurons. Taken together, these findings are consistent with a very specific role for G gamma 8 in the development and turnover of olfactory and vomeronasal neurons.

Amino Acid Sequence↗

Crystallization of and preliminary X-ray data for the mouse major urinary protein and rat alpha-2u globulin.

Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution.

Alpha-Globulins↗

Diltiazem at high concentration increases the ionic permeability of biological membranes.

The effects of diltiazem, a drug which inhibits the calcium channels in cardiac muscle as well as the light-sensitive channels in photoreceptor cells, were studied on ionic fluxes in both membrane and intact cell preparations. Diltiazem nonselectively increased the ionic permeability to both anions and cations in photoreceptor rod outer segment and synaptic membrane vesicles as well as in intact erythrocytes. Under our conditions, the estimated threshold for the diltiazem effect varied between 12.5 and 200 microM. In each case the concentration dependence exhibited the sigmoidal shape characteristic of positive cooperativity. The effect of diltiazem on ionic fluxes from phospholipid vesicles were strongly influenced by phospholipid composition and membrane charge. By contrast, diltiazem inhibited the efflux of 86Rb from photoreceptor cells of intact aspartate-isolated retina, an effect opposite to that of diltiazem on ionic permeabilities in photoreceptor membrane vesicle preparations. These data raise serious doubts on the specificity of diltiazem as a calcium channel blocker or as a cGMP channel blocker when used at concentrations higher than 10 microM.

Animals↗

Rhodopsin-detergent micelles aggregate upon activation of cyclic guanosine monophosphate phosphodiesterase.

In the presence of G protein and phosphodiesterase, GTP induces aggregation of phospholipid-free rhodopsin-detergent micelles or rhodopsin reconstituted in phospholipid vesicles. The net electrical charge of the vesicle is not critical to the aggregation process since this phenomenon is not altered by reconstitution with phospholipids with different charge. The aggregation process is observed by monitoring changes in the light-scattering properties of the detergent micelles or vesicle suspension and by phase-contrast microscopy. The lowest light intensity which triggers the aggregation process and concomitant light-scattering changes in a rhodopsin-detergent micellar suspension bleaches 6% rhodopsin. Under these conditions, the signal saturates at 30% rhodopsin bleaching. The aggregation process appears likely to depend on the protein-protein interaction, and the presence of a disk membrane is not necessary for this process.

3',5'-Cyclic-GMP Phosphodiesterases↗

Ligand binding characteristics of homologous rat and mouse urinary proteins and pyrazine-binding protein of calf.

1. The binding affinities of three classes of homologous proteins, alpha 2u protein from rat urine, major urinary protein of mouse and pyrazine-binding protein of calf nasal mucosa have been determined for a panel of ligands. 2. Best ligands were low threshold odorants for man, but chemically unrelated. 3. The binding spectra of the homologous proteins were different.

Animals↗

Complete amino acid sequence of pyrazine-binding protein from cow nasal mucosa.

The sequence is reported of the pyrazine-binding protein from cow olfactory/respiratory mucosa. The protein consists of 159 amino acids and clearly belongs to the retinol-binding protein family. It is most closely related to the urinary proteins from mice and rats and to the odour-binding protein from rat nasal epithelium. It is unique however, in that only one of the otherwise conserved features of the family is still present--namely a single tryptophan. Most surprisingly the protein contains no cysteine and, therefore, does not rely for its structural stability on the disulphide bond(s) present in other members of this group. A model for the protein has been constructed based on the co-ordinates of beta-lactoglobulin. From this, it is possible to identify residues which may line the binding site. The impression gained is of a much larger pocket than occurs with retinol-binding protein or beta-lactoglobulin. The character of the binding pocket remains essentially hydrophobic but with a significant reduction in its aromatic content and an increase in H-bonding side chains.

Amino Acid Sequence↗

The pyrazine-binding protein and olfaction.

1. The present results provide circumstantial evidence, but not a proof, that the Pyrazine-binding Protein is an odorant carrier molecule of fundamental importance. 2. At first sight a role for a secretory protein in olfaction is not obvious. 3. Odorants freely diffuse in air, in water and in lipids, and the use of carrier proteins, would seem superfluous unless a very special combination with the odorant occurs [Gaupp E. (1902) In Anatomie des Frosches, 2nd Edn, pp. 673. Vieweg-Verlag, Braunschweig]. 4. The possibility should be considered that the Pyrazine-binding Protein and the urinary proteins belong to a large family of species-specific secretory molecules which, with the odorant bound, directly stimulate the receptor cell.

Animals↗