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D Lancet

Publications and source records attributed to D Lancet.

At least 73 records · Page 4Linked to original sources
PubMed1991

Odorant signal termination by olfactory UDP glucuronosyl transferase.

The onset of olfactory transduction has been extensively studied, but considerably less is known about the molecular basis of olfactory signal termination. It has been suggested that the highly active cytochrome P450 monooxygenases of olfactory neuroepithelium are termination enzymes, a notion supported by the identification and molecular cloning of olfactory-specific cytochrome P450s (refs. 13-16). But as reactions catalysed by cytochrome P450 (refs 17, 18) often do not significantly alter volatility, lipophilicity or odour properties, cytochrome P450 may not be solely responsible for olfactory signal termination. In liver and other tissues, drug hydroxylation by cytochrome P450 is frequently followed by phase II biotransformation, for example by UDP glucuronosyl transferase (UGT), resulting in a major change of solubility and chemical properties. We report here the molecular cloning and expression of an olfactory-specific UGT. The olfactory enzyme, but not the one in liver microsomes, shows preference for odorants over standard UGT substrates. Furthermore, glucuronic acid conjugation abolishes the ability of odorants to stimulate olfactory adenylyl cyclase. This, together with the known broad spectrum of drug-detoxification enzymes, supports a role for olfactory UGT in terminating diverse odorant signals.

Amino Acid Sequence↗
PubMed1991

Immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa.

Previously, we described two olfactory-specific cytochromes P-450: rat cytochrome P-450olf1 (IIG1), identified by cDNA cloning, and bovine cytochrome P-450olf2 (IIA), identified by peptide microsequencing of a transmembranal polypeptide (p52). Here we describe the preparation of polyclonal antisera against peptide sequences of these proteins and their use in the immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa. Immunoreactivities related to both enzymes are found in the subepithelial Bowman's glands of olfactory mucosa. Practically no immunoreactivity was found in other rat tissues, including liver, lung, kidney and respiratory mucosa. In addition, double-labeling experiments demonstrated that cytochromes P-450olf1 and P-450olf2 are present in the same population of Bowman's glands. The olfactory-specific localization of cytochromes P-450olf1 and P-450olf2 is consistent with a role for these enzymes in the modification or clearance of odorants from the chemosensory tissue.

Animals↗
PubMed1991

The UDP glucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence.

A nomenclature system for the UDP glucuronosyltransferase superfamily is proposed, based on divergent evolution of the genes. A total of 26 distinct cDNAs in five mammalian species have been sequenced to date. Comparison of the deduced amino acid sequences leads to the definition of two families and a total of three subfamilies. For naming each gene, we propose that the root symbol UGT for human (Ugt for mouse), representing "UDP glucuronosyltransferase," be followed by an Arabic number denoting the family, a letter designating the subfamily, and an Arabic numeral representing the individual gene within the family or subfamily (hyphen before the Arabic number for mouse), e.g., human UGT2B1 and murine Ugt2b-1. Whereas the gene and cDNA should be italicized, the corresponding transcript, protein, and enzyme activity should not be written with lowercase letters or in italics, e.g., human or murine UGT2B1. Recent experimental evidence suggests that several exons of the UGT1 gene might be shared, indicating that distinct UGT1 transcripts and proteins may arise via alternative splicing; the gene and gene product of alternative splicing will be designated with an asterisk, e.g., UGT1*6 and UGT1*6, respectively. When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, we recommend sequential naming of the genes chronologically as they become characterized. We suggest that the human nomenclature system be used for species other than the mouse. We anticipate that this UGT gene nomenclature system will require updating on a regular basis.

Animals↗
PubMed1990

Primary structure of cAMP-gated channel from bovine olfactory epithelium.

The complete amino-acid sequence of the bovine olfactory epithelium adenosine 3',5'-cyclic monophosphate (cAMP)-gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP-gated channel of rod photoreceptors, suggesting that cyclic nucleotide-gated channels fall into a new family of genetically related proteins.

Amino Acid Sequence↗
PubMed1990

Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase.

Two major transmembranal polypeptides of bovine olfactory epithelium were identified by SDS electrophoretic analysis of Triton X-114 solubilized membranes. Both polypeptides were present in large amounts in membranes of the olfactory epithelium but were barely detectable in membranes of the nasal respiratory epithelium. Both polypeptides are enriched in the deciliated epithelium as compared with isolated cilia. One of them is a glycoprotein with an apparent molecular mass of 56 kDa (gp56); the other is an unglycosylated protein with an apparent molecular mass of 52 kDa (p52). Sequence analysis of peptides obtained by CNBr cleavage of purified gp56 indicates that it is highly homologous to UDP-glucuronosyl transferase (UDPGT). Parallel analysis shows that p52 is highly homologous to cytochrome P-450 sequences of the IIA subfamily. This protein is assigned the name P-450olf2. Polyclonal antibodies were raised against synthetic peptides corresponding to gp56 and p52 peptide sequences. Immunoblots with these antibodies reveal the following properties of gp56 and p52: (1) they are enriched in the microsomal fraction of the bovine olfactory epithelium; (2) they are possibly specific to the olfactory epithelium, as we could not detect reactivity in microsomes derived from respiratory epithelium or lung, and only a very small amount of basal reactivity was seen with liver microsomes; (3) cross-reacting proteins exist in microsomes derived from the rat olfactory epithelium. These results are consistent with a mechanism whereby the microsomal enzymes are involved in odorant modification and clearance from the nasal tissue.

Amino Acid Sequence↗
PubMed1989

Olfactory adenylyl cyclase. Identification and purification of a novel enzyme form.

Rat olfactory adenylyl cyclase has been identified by means of a monoclonal antibody BBC-2, which reacts with both Ca2+/calmodulin-sensitive and -insensitive forms of adenylyl cyclase (Mollner, S., and Pfeuffer, T. (1988) Eur. J. Biochem. 171, 265-271). The antibody recognized a 180-kDa polypeptide in olfactory cilia but not in decilitated olfactory epithelial membranes. A protein of the same mobility was observed when olfactory adenylyl cyclase was purified by forskolin-agarose affinity chromatography followed by radioiodination. Its identity was further established by cross-linking to [32P]ADP-ribosylated Gs alpha (GTP-binding protein), to yield a single radiolabeled product of Mr approximately 220. Olfactory adenylyl cyclase has a approximately 3-fold higher turnover number, as assessed from stoichiometric binding of [35S]guanosine 5'-(3-O-thio)triphosphate. Therefore, the considerably higher specific adenylyl cyclase activity in olfactory cilia must be due to a approximately 100-fold higher molar concentration of enzyme in this tissue.

Adenosine Diphosphate Ribose↗
PubMed1989

Bovine olfactory cilia preparation: thiol-modulated odorant-sensitive adenylyl cyclase.

We have characterized the adenylyl cyclase activity in a newly developed preparation of isolated olfactory cilia from the bovine chemosensory neuroepithelium. Like its counterparts from frog and rat, the ciliary enzyme was stimulated by guanine nucleotides, by forskolin, and by a variety of odorants in the presence of GTP. The main difference between the bovine olfactory cilia preparation and the frog and rat olfactory cilia preparation is that odorant stimulation of the bovine olfactory adenylyl cyclase is strongly inhibited by submillimolar concentrations of dithiothreitol. This inhibition is a consequence of a concomitant increase in the GTP-stimulated level and the decrease of the odorant stimulation of the enzyme. Nasal respiratory cilia have a much lower level of adenylyl cyclase activity and show no odorant stimulation. Owing to the large quantities of material available, the bovine olfactory cilia preparation is advantageous for studies of the proteins involved in chemosensory transduction.

Adenylyl Cyclases↗
PubMed1989

Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.

Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.

Adenylyl Cyclases↗
PubMed1989

Olfactory-specific cytochrome P-450. cDNA cloning of a novel neuroepithelial enzyme possibly involved in chemoreception.

We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.

Amino Acid Sequence↗
PubMed1989

Olfactory function following late repair of choanal atresia.

Results of olfactory function tests (threshold determination and odor identification) in three cases of bilateral and one case of unilateral choanal atresia are reported. All four patients underwent successful repair of choanal atresia at relatively advanced ages (8 to 31 years). Test results showed that patients who had suffered from bilateral atresia had permanent olfactory deficits, while the patient who had suffered from unilateral atresia appeared to have normal olfactory acuity. Although these results should be interpreted with caution due to the small number of cases examined, they suggest the possibility that early sensory exposure might be needed for the normal development of central olfactory functions in analogy to the visual system.

Adolescent↗
PubMed1988

Expression of intermediate filaments and desmoplakin in vertebrate olfactory mucosa.

The expression of intermediate filaments (IF) and desmoplakin was investigated in frog, bovine, and human (fetal) olfactory mucosa. IF are tissue-specific molecular cytoskeletal markers; desmoplakin is the major desmosomal protein. Positive immunoreactivity was observed in the epithelium and in the subepithelial Bowman's glands to keratin and to desmoplakin, indicating the epithelial nature of this tissue. Desmin, neurofilaments, and glial fibrillary acidic protein (GFAP) were not detected in the mucosa. The absence of neurofilaments and GFAP in the tissue containing sensory neurons and glia-like supporting cells is a unique feature and may be related to the fact that the chemosensory neurons are situated in a bonafide epithelium and are known to undergo continuous turnover. In view of the controversy regarding the expression of vimentin in the olfactory neurons, three independently derived antibodies to vimentin were used; weak or no labeling was found in the epithelium, whereas mesenchymal cells in the lamina propia were labeled with all three antibodies. Olfactory nerve fascicles in the lamina propia were heterogenously labeled: VIM 13.2 gave very weak labeling; aVimAS showed mild labeling and SBV-21 showed intensive labeling in the nerve fascicle. This heterogenous labeling pattern may suggest that olfactory vimentin is distinct in reacting only with some of the antivimentin antibodies.

Animals↗
PubMed1986

Monoclonal antibodies to ciliary glycoproteins of frog olfactory neurons.

Monoclonal antibodies were produced against isolated frog olfactory cilia, a preparation enriched in dendritic extensions of the chemosensory neurons. Two antibodies, 18.1 and 35.6, were found to react against specific glycoproteins of the sensory organelles. These glycoproteins were identified by their differential binding to the lectins wheat germ agglutinin and Concanavalin A. The antibodies fluorescently labeled isolated olfactory cilia, as well as the ciliary surface layer of olfactory epithelium, whose extent was defined by anti-tubulin and anti-keratin antibodies. Respiratory epithelium (or other tissues) as well as isolated respiratory cilia were not labeled by antibodies 18.1 and 35.6, indicating tissue specificity. The olfactory-specific antibodies can be used as markers of the sensory epithelium and of the sensory regions of olfactory dendritic membranes. Antibody 18.1 recognized gp95, a specific and major integral membrane glycoprotein of frog olfactory cilia. Since gp95 has been suggested as candidate olfactory receptor protein (Chen, Z. and Lancet, D., Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1859-1863), antibody 18.1 could also be useful for functional studies.

Animals↗
PubMed1986

Polypeptide gp95. A unique glycoprotein of olfactory cilia with transmembrane receptor properties.

Polypeptide gp95 is a major glycoprotein present in preparations of isolated ciliary extensions from frog olfactory sensory neurons (Chen, Z., and Lancet, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1859-1863). We report here that gp95 is distinct among the ciliary polypeptides in having several properties that make it a plausible receptor candidate: it is specific to olfactory cilia, it has the appropriate bilayer density, and it is a transmembrane protein. Polypeptide gp95 has a uniquely high content of complex type oligosaccharides compared to other ciliary glycoproteins, a property which is used for its partial purification and can also serve as a probe for functional identification. The present biochemical characterization of frog gp95 and of its putative homologs from other species may open the way to a future assignment of its role in chemosensory reception.

Animals↗