Biomedical subjects

N J Ryba

Publications and source records attributed to N J Ryba.

At least 19 recordsLinked to original sources

PubMed2001

Mammalian sweet taste receptors.

The sense of taste provides animals with valuable information about the quality and nutritional value of food. Previously, we identified a large family of mammalian taste receptors involved in bitter taste perception (the T2Rs). We now report the characterization of mammalian sweet taste receptors. First, transgenic rescue experiments prove that the Sac locus encodes T1R3, a member of the T1R family of candidate taste receptors. Second, using a heterologous expression system, we demonstrate that T1R2 and T1R3 combine to function as a sweet receptor, recognizing sweet-tasting molecules as diverse as sucrose, saccharin, dulcin, and acesulfame-K. Finally, we present a detailed analysis of the patterns of expression of T1Rs and T2Rs, thus providing a view of the representation of sweet and bitter taste at the periphery.

Animals↗

PubMed2001

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↗

PubMed2000

A novel family of mammalian taste receptors.

In mammals, taste perception is a major mode of sensory input. We have identified a novel family of 40-80 human and rodent G protein-coupled receptors expressed in subsets of taste receptor cells of the tongue and palate epithelia. These candidate taste receptors (T2Rs) are organized in the genome in clusters and are genetically linked to loci that influence bitter perception in mice and humans. Notably, a single taste receptor cell expresses a large repertoire of T2Rs, suggesting that each cell may be capable of recognizing multiple tastants. T2Rs are exclusively expressed in taste receptor cells that contain the G protein alpha subunit gustducin, implying that they function as gustducin-linked receptors. In the accompanying paper, we demonstrate that T2Rs couple to gustducin in vitro, and respond to bitter tastants in a functional expression assay.

Animals↗

PubMed2000

T2Rs function as bitter taste receptors.

Bitter taste perception provides animals with critical protection against ingestion of poisonous compounds. In the accompanying paper, we report the characterization of a large family of putative mammalian taste receptors (T2Rs). Here we use a heterologous expression system to show that specific T2Rs function as bitter taste receptors. A mouse T2R (mT2R-5) responds to the bitter tastant cycloheximide, and a human and a mouse receptor (hT2R-4 and mT2R-8) responded to denatonium and 6-n-propyl-2-thiouracil. Mice strains deficient in their ability to detect cycloheximide have amino acid substitutions in the mT2R-5 gene; these changes render the receptor significantly less responsive to cycloheximide. We also expressed mT2R-5 in insect cells and demonstrate specific tastant-dependent activation of gustducin, a G protein implicated in bitter signaling. Since a single taste receptor cell expresses a large repertoire of T2Rs, these findings provide a plausible explanation for the uniform bitter taste that is evoked by many structurally unrelated toxic compounds.

Afferent Pathways↗

PubMed1999

Pheromone reception: A complex map of activation in the brain.

Recent studies of the projection pattern made by sensory neurons involved in mammalian pheromone reception have shown that there is a map of activation in the brain, but this pheromone map appears far more complex than the equivalent map in the main olfactory system responsible for the sense of smell.

Animals↗

PubMed1999

Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity.

Taste represents a major form of sensory input in the animal kingdom. In mammals, taste perception begins with the recognition of tastant molecules by unknown membrane receptors localized on the apical surface of receptor cells of the tongue and palate epithelium. We report the cloning and characterization of two novel seven-transmembrane domain proteins expressed in topographically distinct subpopulations of taste receptor cells and taste buds. These proteins are specifically localized to the taste pore and are members of a new group of G protein-coupled receptors distantly related to putative mammalian pheromone receptors. We propose that these genes encode taste receptors.

Animals↗

PubMed1998

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↗

PubMed1997

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↗

PubMed1997

Analysis and comparison of partial sequences of clones from a taste-bud-enriched cDNA library.

Differential patterns of cellular development and function are determined, at least in part, by the specific gene expression of particular cells. Thus, determination of differential patterns of gene expression between tissues is likely to help elucidate molecular details of tissue-specific processes. Our hypothesis was that cells of the circumvallate papilla involved in taste perception would express genes that are not expressed in the surrounding epithelium and that determination of the nature of these genes could be helpful in our understanding of the molecular details of taste. Using partial sequencing of clones derived from rat circumvallate papillae, we have begun to characterize genes that could be important in taste. We prepared a cDNA library of whole circumvallate papillae and, by means of a novel subtraction procedure, enriched taste-specific clones. Characterization of the libraries showed that subtraction resulted in good enrichment of taste-specific clones. Here we report the partial sequencing and analysis of 410 cDNA clones from the taste-bud-enriched cDNA library. Approximately 25% of the genes were identified on the basis of their high homology to known transcripts. These included the developmentally important molecules Pax-1, esp1, Notch 1, and Notch 3 that may play roles in the continuous turnover of taste receptor cells. A further 20% of the genes had no significant homology to known DNA sequences and were identified as taste-specific by Southern blot analysis.

Animals↗

PubMed1996

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↗

PubMed1996

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↗

PubMed1995

Functional expression of the taste specific G-protein, alpha-gustducin.

The taste-specific G-protein alpha-subunit, alpha-gustducin, was expressed using a baculovirus based system. alpha-Gustducin was demonstrated to be myristoylated and was also palmitoylated in insect larval cells. Recombinant alpha-gustducin was purified to homogeneity. Neither receptors nor effectors that interact with gustducin in taste are known. However, alpha-gustducin has a close structural similarity to the visual G-protein, alpha-transducin. Therefore alpha-gustducin was reconstituted with components of the visual system to determine the degree of its functional similarity with alpha-transducin. Despite the fact that the sequences of alpha-gustducin and alpha-transducin share only 80% identity with each other, the interactions and functions of these two proteins were quantitatively identical. These included the interaction with receptor, bovine rhodopsin, with effector, bovine retinal cyclic GMP-phosphodiesterase, and with bovine brain and retinal G-protein beta gamma-heterodimers; receptor-catalysed GDP-GTP exchange and the intrinsic GTPase activity of alpha-gustducin and alpha-transducin were also identical. Gi alpha which is 70% identical with alpha-transducin interacts with different receptor and effector proteins and has very different guanine-nucleotide binding properties. Therefore, the functional equivalence of alpha-gustducin and alpha-transducin suggest that taste buds are likely to contain receptor and effector proteins that share many properties with their retinal equivalents.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

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↗

PubMed1994

Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development.

Partially purified extracts from newborn calf articular cartilage were found to induce cartilage and bone when subcutaneously implanted in rats. This activity showed characteristics of bone morphogenetic proteins (BMPs). Degenerate oligonucleotide primer sets derived from the highly conserved carboxyl-terminal region of the BMP family were designed and used in reverse transcription-polymerase chain reactions with poly(A)+ RNA from articular cartilage as template to determine which BMPs are produced by chondrocytes. Two novel members of the transforming growth factor-beta (TGF-beta) superfamily were identified and designated cartilage-derived morphogenetic protein-1 (CDMP-1) and -2 (CDMP-2). Their carboxyl-terminal TGF-beta domains are 82% identical, thus defining a novel subfamily most closely related to BMP-5, BMP-6, and osteogenic protein-1. Northern analyses showed that both genes are predominantly expressed in cartilaginous tissues. In situ hybridization and immunostaining of sections from human embryos showed that CDMP-1 was predominantly found at the stage of precartilaginous mesenchymal condensation and throughout the cartilaginous cores of the developing long bones, whereas CDMP-2 expression was restricted to the hypertrophic chondrocytes of ossifying long bone centers. Neither gene was detectable in the axial skeleton during human embryonic development. The cartilage-specific localization pattern of these novel TGF-beta superfamily members, which contrasts with the more ubiquitous presence of other BMP family members, suggests a potential role for these proteins in chondrocyte differentiation and growth of long bones.

Amino Acid Sequence↗

PubMed1993

Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. Identification as a component of pp72 and association with the receptor gamma chain after receptor aggregation.

Protein-tyrosine phosphorylation plays a critical role in the high-affinity IgE receptor (Fc epsilon RI) signaling. Here we investigated the involvement of the tyrosine kinase p72syk in Fc epsilon RI signaling in the rat mast cell line RBL-2H3. Specific antibodies were raised against peptides synthesized on the basis of the deduced peptide sequence of an essentially full-length rat syk cDNA. The expression of p72syk in RBL-2H3 cells was demonstrated with these antibodies. The aggregation of Fc epsilon RI led to the tyrosine phosphorylation of p72syk that was detected after 15 s of stimulation, reached a plateau by 5 min, and was not induced by calcium influx or protein kinase C activation. Association of p72syk with the tyrosine phosphorylated Fc epsilon RI gamma chain was detected only after receptor aggregation. We previously demonstrated that aggregation of the Fc epsilon RI on mast cells results in the tyrosine phosphorylation of a 72-kDa protein (pp72) involved in IgE signaling. The depletion of p72syk from RBL-2H3 cell lysates resulted in only a slight decrease in the amount of pp72. These results demonstrate that pp72 is composed of several phosphoproteins and identify p72syk as one component of pp72. These data, together with recent observations in T cells, indicate that the interaction between p72syk-related tyrosine kinases and zeta-related proteins could play an important role in signal transduction.

Amino Acid Sequence↗

PubMed1993

The molecular cloning of the squid (Loligo forbesi) visual Gq-alpha subunit and its expression in Saccharomyces cerevisiae.

The sequence of the alpha-subunit of the major G-protein from the squid (Loligo forbesi) retina was predicted from its cDNA to be a member of the Gq subclass. The abundance of the squid Gq-alpha in the squid photoreceptor membranes suggests that the protein functions in phototransduction; the sequence of this G-protein is consistent with it mediating the light-dependent activation of a phospholipase C. The squid G-alpha was expressed in the yeast Saccharomyces cerevisiae, where it was unable to replace the function of GPA1, the yeast G-alpha homologue that regulates the mating response, suggesting that Gq-alpha was unable to interact with the endogenous G-beta gamma (STE4-STE18).

Amino Acid Sequence↗

PubMed1993

Rhodopsin mobility, structure, and lipid-protein interaction in squid photoreceptor membranes.

Treatment of outer segment membranes from Loligo forbesi with endoprotease-V8 from Staphylococcus aureus results in cleavage of the C-terminal extension of the squid rhodopsin, with accompanying reduction of the apparent molecular weight from 47,000 to 36,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Negative-stain electron microscopy of the intact membranes shows that small clusters of the rhodopsin C-termini form structures extending from the membrane surface and that these are absent after protease treatment. Fourier transform infrared spectra of the amide I band of the protein indicate that removal of the C-terminal extension increases the relative alpha-helical content of squid rhodopsin to a level comparable to that for bovine rhodopsin in disk membranes, and to an extent which suggests that the alpha-helical structure lies mainly in the M(r) 36,000 (transmembrane) section of the protein. Saturation-transfer electron spin resonance (ESR) spectroscopy of the spin-labeled protein reveals that the rotational diffusion of squid rhodopsin in outer segment membranes that have been extensively washed with urea to remove peripheral proteins is much slower than that of bovine rhodopsin in rod outer segment disk membranes. This reduction in rotational mobility is also found with purified squid rhodopsin reconstituted in egg phosphatidylcholine and in urea-washed outer segment membranes which have been treated with endoprotease-V8 to remove the C-terminal extension of squid rhodopsin. In the latter case, the saturation-transfer ESR spectra are virtually identical to those of the non-proteolyzed membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amides↗

PubMed1993

The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates.

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids.

Animals↗