Biomedical subjects

R R Anholt

Publications and source records attributed to R R Anholt.

At least 19 recordsLinked to original sources

PubMed2001

The genetic architecture of odor-guided behavior in Drosophila melanogaster.

Animals↗

PubMed2001

Functional genomics of odor-guided behavior in Drosophila melanogaster.

The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of olfactory behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a co-isogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. Candidate genes for several of these transposon-tagged smi loci implicate genes that mediate odorant recognition, including a novel odorant binding protein; signal propagation, including a voltage-gated sodium channel; and a protein containing multiple leucine rich repeats and PDZ domains likely to be involved in postsynaptic organization in the olfactory pathway. Several novel genes of unknown function have also been implicated, including a novel tyrosine-regulated protein kinase. The discovery and characterization of novel gene products that have major, hitherto unappreciated effects on olfactory behavior will provide new insights in the generation and regulation of odor-guided behavior. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.

Animals↗

PubMed2000

Characterization and differential expression of a human gene family of olfactomedin-related proteins.

Olfactomedin-related proteins are secreted glycoproteins with conserved C-terminal motifs. Olfactomedin was originally identified as the major component of the mucus layer that surrounds the chemosensory dendrites of olfactory neurons. Homologues were subsequently found also in other tissues, including the brain and in species ranging from Caenorhabditis elegans to Homo sapiens. Most importantly, the TIGR/myocilin protein, expressed in the eye and associated with the pathogenesis of glaucoma, is an olfactomedin-related protein. The prevalence of olfactomedin-related proteins among species and their identification in different tissues prompted us to investigate whether a gene family exists within a species, specifically Homo sapiens. A GenBank search indeed revealed an entire human gene family of olfactomedin-related proteins with at least five members, designated hOlfA through hOlfD and the TIGR/myocilin protein. hOlfA corresponds to the rat neuronal AMZ protein. Phylogenetic analyses of 18 olfactomedin-related sequences resolved four distinct subfamilies. Among the human proteins, hOlfA and hOlfC, both expressed in brain, are most closely related. Northern blot analyses of 16 human tissues demonstrated highly specific expression patterns: hOlfA is expressed in brain, hOlfB in pancreas and prostate, hOlfC in cerebellum, hOlfD in colon, small intestine and prostate and TIGR/myocilin in heart and skeletal muscle. The link between TIGR/myocilin and ocular hypertension and the expression of several of these proteins in mucus-lined tissues suggest that they play an important role in regulating physical properties of the extracellular environment. Future studies can now assess whether other members of this gene family, like TIGR/myocilin, are also associated with human disease processes.

Amino Acid Sequence↗

PubMed1999

Differential expression of G proteins in the mouse olfactory system.

Transmembrane signaling events at the dendrites and axons of olfactory receptor neurons mediate distinct functions. Whereas odorant recognition and chemosensory transduction occur at the dendritic membranes of olfactory neurons, signal propagation, axon sorting and target innervation are functions of their axons. The roles of G proteins in transmembrane signaling at the dendrites have been studied extensively, but axonal G proteins have not been investigated in detail. We used immunohistochemistry to visualize expression of alpha subunits of G(o) and G(i2) in the mouse olfactory system. G(o) is expressed ubiquitously on axons of olfactory receptor neurons throughout the olfactory neuroepithelium and in virtually all glomeruli in the main olfactory bulb. In contrast, expression of G(i2) is restricted to a sub-population of olfactory neurons, along the dorsal septum and the dorsal recess of the nasal cavity, which projects primarily to medial regions of the olfactory bulb, with the exception of glomeruli adjacent to the pathway of the vomeronasal nerve. In contrast to the overlapping expression patterns of G(o) and G(i2) in the main olfactory system, neurons expressing G(o) and those expressing G(i2) in the accessory olfactory bulb are more clearly separated, in agreement with previous studies. Vomeronasal axons terminating in glomeruli in the rostral region of the accessory olfactory bulb express G(i2), whereas those projecting to the caudal region express G(o). Characterization of the expression patterns of G(i2) and G(o) in the olfactory projection is essential for future studies aimed at relating transmembrane signaling events to signal propagation, axon sorting and target innervation.

Animals↗

PubMed1998

Evolution of olfactomedin. Structural constraints and conservation of primary sequence motifs.

Olfactomedin is a glycosylated extracellular matrix protein originally identified at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently localized throughout the mammalian central nervous system. Although olfactomedin homologues have been identified in fish, frog, rat, mouse and human, its function is still unknown. As a first step toward elucidating the function of olfactomedin, sequences of teleost, amphibian and human homologues were compared to identify invariant, and hence, potential functionally important motifs. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedin in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homo-polymer formation, such as the CXC motif near the amino terminus, are conserved as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, alignment of a region of 53 amino acids of fish, frog, rat and human olfactomedin reveals seven invariant residues including a negatively charged cluster of aspartic and glutamic acid residues. Molecular evolutionary genetic analysis reveals an accelerated rate of nucleotide substitutions in the mammalian lineage. The evolutionary rate at the protein level, however, is constant, indicating that evolution of olfactomedin is constrained by structural limitations. Whereas considerable evolutionary divergence is evident between fish, frog and mammalian olfactomedins, olfactomedins of rat and human show 98% amino acid sequence identity. It appears that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian and mammalian lineages. The apparent evolutionary pressure toward conservation of primary structure supports the notion that olfactomedin has an important function in the mammalian nervous system.

Amino Acid Sequence↗

PubMed1998

Epistatic interactions between smell-impaired loci in Drosophila melanogaster.

Odor-guided behavior is a polygenic trait determined by the concerted expression of multiple loci. Previously, P-element mutagenesis was used to identify single P[lArB] insertions, in a common isogenic background, with homozygous effects on olfactory behavior. Here, we have crossed 12 lines with these smell impaired (smi) mutations in a half-diallel design (excluding homozygous parental genotypes and reciprocal crosses) to produce all possible 66 doubly heterozygous hybrids with P[lArB] insertions at two distinct locations. The olfactory behavior of the transheterozygous progeny was measured using an assay that quantified the avoidance response to the repellent odorant benzaldehyde. There was significant variation in general combining abilities of avoidance scores among the smi mutants, indicating variation in heterozygous effects. Further, there was significant variation among specific combining abilities of each cross, indicating dependencies of heterozygous effects on the smi locus genotypes, i.e., epistasis. Significant epistatic interactions were identified for nine transheterozygote genotypes, involving 10 of the 12 smi loci. Eight of these loci form an interacting ensemble of genes that modulate expression of the behavioral phenotype. These observations illustrate the power of quantitative genetic analyses to detect subtle phenotypic effects and point to an extensive network of epistatic interactions among genes in the olfactory subgenome.

Animals↗

PubMed1998

Molecular evolution of olfactomedin.

Olfactomedin is a secreted polymeric glycoprotein of unknown function, originally discovered at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently found throughout the mammalian brain. As a first step toward elucidating the function of olfactomedin, its phylogenetic history was examined to identify conserved structural motifs. Such conserved motifs may have functional significance and provide targets for future mutagenesis studies aimed at establishing the function of this protein. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedins in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homopolymer formation such as the CXC motif near the amino terminus are conserved, as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, evolutionary analysis of a region of 53 amino acids of fish, frog, rat, mouse, and human olfactomedins indicates that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian, and mammalian lineages. Indeed, a distant olfactomedin homolog was identified in Caenorhabditis elegans. Although the amino acid sequence of this invertebrate protein is longer and highly divergent compared with its vertebrate homologs, the protein from C. elegans shows remarkable similarities in terms of conserved motifs and posttranslational modification sites. Six universally conserved motifs were identified, and five of these are clustered in the carboxyl terminal half of the protein. Sequence comparisons indicate that evolution of the N-terminal half of the molecule involved extensive insertions and deletions; the C-terminal segment evolved mostly through point mutations, at least during vertebrate evolution. The widespread occurrence of olfactomedin among vertebrates and invertebrates underscores the notion that this protein has a function of universal importance. Furthermore, extensive modification of its N-terminal half and the acquisition of a C-terminal SDEL endoplasmic-reticulum-targeting sequence may have enabled olfactomedin to adopt new functions in the mammalian central nervous system.

Amino Acid Sequence↗

PubMed1997

Pheromone regulated production of inositol-(1, 4, 5)-trisphosphate in the mammalian vomeronasal organ.

Social behaviors of most mammals are profoundly affected by chemical signals, pheromones, exchanged between conspecifics. Pheromones interact with dendritic microvilli of bipolar neurons in the vomeronasal organ (VNO). To investigate vomeronasal signal transduction pathways, microvillar membranes from porcine VNO were prepared. Incubation of such membranes from prepubertal females with boar seminal fluid or urine results in an increase in production of inositol-(1, 4, 5)-trisphosphate (IP3). The dose response for IP3 production is biphasic with a GTP-dependent component at low stimulus concentrations and a nonspecific increase in IP3 at higher stimulus concentrations. The GTP-dependent stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore, the GTP-dependent component of the stimulation of IP3 production is sex specific and tissue dependent. Studies with monospecific antibodies reveal a G alpha(q/11)-related protein in vomeronasal neurons, concentrated at their microvilli. Our observations indicate that pheromones in boar secretions act on vomeronasal neurons in the female VNO via a receptor mediated, G protein-dependent increase in IP3. These observations set the stage for further investigations on the regulation of stimulus-excitation coupling in vomeronasal neurons. The pheromone-induced IP3 response also provides an assay for future purification of mammalian reproductive pheromones.

Animals↗

PubMed1996

Effects of single P-element insertions on olfactory behavior in Drosophila melanogaster.

Single P-element (P[lArB]) insertional mutagenesis of an isogenic strain was used to identify autosomal loci affecting odor-guided behavior of Drosophila melanogaster. The avoidance response to benzaldehyde of 379 homozygous P[lArB] element-containing insert lines was evaluated quantitatively. Fourteen smell impaired (smi) lines were identified in which P[lArB] element insertion caused different degrees of hyposmia in one or both sexes. The smi loci map to different cytological locations and probably are novel olfactory genes. Enhancer trap analysis of the smi lines indicates that expression of at least 10 smi genes is controlled by olfactory tissue-specific promoter/enhancer elements.

Animals↗

PubMed1996

Quantitative genetic variation of odor-guided behavior in a natural population of Drosophila melanogaster.

Quantitative genetic variation in behavioral response to the odorant, benzaldehyde, was assessed among a sample of 43 X and 35 third chromosomes extracted from a natural population and substituted into a common inbred background. Significant genetic variation among chromosome lines was detected. Heritability estimates for olfactory response, however, were low, as is typical for traits under natural selection. Furthermore, the loci affecting naturally occurring variation in olfactory response to benzaldehyde were not the same in males and females, since the genetic correlation between the sexes was low and not significantly different from zero for the chromosome 3 lines. Competitive fitness, viability and fertility of the chromosome 3 lines were estimated using the balancer equilibrium technique. Genetic correlations between fitness and odor-guided behavior were not significantly different from zero, suggesting the number of loci causing variation in olfactory response is small relative to the number of loci causing variation in fitness. Since different genes affect variation in olfactory response in males and females, genetic variation for olfactory response could be maintained by genotype x sex environment interaction. This unusual genetic architecture implies that divergent evolutionary trajectories for olfactory behavior may occur in males and females.

Animals↗

PubMed1994

Signal integration in the nervous system: adenylate cyclases as molecular coincidence detectors.

Integrating multiple incoming messages simultaneously and discriminating 'meaningful' signals from spontaneous neural activity represent central problems to the nervous system. One mechanism by which signal integration and signal-to-noise resolution are achieved is the formation of temporal coincidence circuits by interacting transduction pathways. Signal integration via temporal coincidence detection is exemplified most readily by the way in which neural adenylate cyclases are regulated. This review will discuss the role of adenylate cyclases as coincidence detectors in the nervous system with special focus on adenylate cyclase type III, an isoenzyme that is found in large quantities in olfactory receptor neurons. The notion that olfactory transduction might also utilize an adenylate-cyclase-mediated temporal coincidence circuit strengthens the idea that signal integration via temporal-coincidence pathways is a universal feature of all neural adenylate cyclases.

Adenylyl Cyclases↗

PubMed1993

Molecular cloning of olfactomedin, an extracellular matrix protein specific to olfactory neuroepithelium.

The extracellular mucous matrix of olfactory neuroepithelium is a highly organized structure in intimate contact with chemosensory cilia that house the olfactory transduction machinery. Here we describe the molecular cloning and primary structure of olfactomedin, which is the major component of this extracellular matrix. Olfactomedin is expressed exclusively in olfactory neuroepithelium and its amino acid sequence shows no homologies to any known protein. This olfactory tissue-specific glycoprotein contains cysteines which form disulfide-linked polymers that constitute the primary architecture of the olfactory extracellular matrix. By analogy to other extracellular matrix proteins of the nervous system, olfactomedin may influence the maintenance, growth, or differentiation of chemosensory cilia on the apical dendrites of olfactory neurons.

Amino Acid Sequence↗

PubMed1993

Formation of the extracellular mucous matrix of olfactory neuroepithelium: identification of partially glycosylated and nonglycosylated precursors of olfactomedin.

Olfactomedin is the major glycoprotein of the extracellular mucous matrix of frog olfactory neuroepithelium. It is responsible for the primary architecture of this extracellular matrix by forming via intermolecular disulfide bonds polymers, which are covered with evenly spaced carbohydrate groups. To study glycosylation of olfactomedin, we raised antibodies against the mature protein and antibodies against a region adjacent to an N-linked glycosylation site near its amino terminus. The latter antibodies cannot bind when this site is glycosylated and reveal precursors of olfactomedin in the perinuclear regions of Bowman's glands. In contrast, antiserum against the mature protein stains acinar regions of glands and the ciliary surface. Enzymatic deglycosylation of olfactomedin shows stepwise removal of carbohydrate and reveals a 51-kDa deglycosylated form. Our results indicate that, prior to secretion, most, if not all, of the six potential N-linked glycosylation sites of olfactomedin are glycosylated with carbohydrate moieties of about 8-10 sugar residues.

Amino Acid Sequence↗

PubMed1993

Molecular neurobiology of olfaction.

Odor discrimination is mediated via dendritic cilia of olfactory receptor neurons. Odorants traverse the aqueous mucous interphase that lines the surface of the olfactory neuroepithelium and interact with odorant receptors, which are members of the superfamily of G-protein-linked receptors. These interactions trigger synthesis of second messengers, including cyclic AMP and inositol triphosphate. Cyclic AMP opens a cation channel to elicit the generator current, which depolarizes the cell and, ultimately, leads to action potentials. Inositol triphosphate opens a calcium channel in the ciliary plasma membrane. Calcium entering through both this channel and the cyclic nucleotide-gated channel modulates the response to odorants by amplifying the generation of cyclic AMP after binding to calmodulin. Calcium also is essential for desensitization of olfactory receptor neurons. Differential expression of odorant receptors of diverse ligand specificities by different olfactory neurons ensures that the structures and concentrations of odorants that reach the chemosensory surface are encoded as distinct patterns of neuronal activity, which are relayed to the brain where they take shape as characteristic odor sensations.

Adaptation, Physiological↗

PubMed1991

Olfactomedin: purification, characterization, and localization of a novel olfactory glycoprotein.

We have identified a novel glycoprotein expressed exclusively in frog olfactory neuroepithelium, which we have named "olfactomedin". Olfactomedin is a 57-kDa glycoprotein recognized by seven monoclonal antibodies, previously shown to react solely with proteins of olfactory cilia preparations. It undergoes posttranslational modifications, including dimerization via intermolecular disulfides and attachment of complex carbohydrate moieties that contain N-acetylglucosamine and beta-D-galactoside sugars. Olfactomedin strongly binds to Ricinus communis agglutinin I and has been purified to homogeneity by lectin affinity chromatography. Polyclonal rabbit antiserum raised against purified olfactomedin confirmed that it is expressed only in olfactory tissue. Immunohistochemical studies at the light microscopic and electron microscopic level show that olfactomedin is localized in secretory granules of sustentacular cells, in acinar cells of olfactory glands, and at the mucociliary surface. The massive production of olfactomedin and its striking deposition at the chemosensory surface of the olfactory neuroepithelium suggest a role for this protein in chemoreception.

Animals↗

PubMed1991

Benzodiazepine receptors in the eye.

Central and peripheral benzodiazepine receptors were localized in the rat, monkey, and human eye by in vitro autoradiography. Central benzodiazepine binding sites, visualized with 3H-R015-1788, were enriched in the inner plexiform layer in all three species. Binding sites also were present in the nerve fiber layer, the ganglion cell layer, and in portions of the inner nuclear layer. Peripheral benzodiazepine binding sites, visualized with 3H-PK-11195, were found in the corneal epithelium and endothelium, iris, ciliary epithelium, trabecular meshwork, and throughout the retina. Binding sites for 3H-PK-11195 also were present in the retinal pigment epithelium and choriocapillaris areas and retinal vascular structures.

Aged↗

PubMed1990

Olfactory transduction: cross-talk between second-messenger systems.

Chemosensory cilia of olfactory receptor neurons contain an adenylate cyclase which is stimulated by high concentrations of odorants. Cyclic AMP produced by this enzyme has been proposed to act as second messenger in olfactory transduction. Here we report that olfactory cilia contain calmodulin and that calmodulin potently activates olfactory adenylate cyclase by a mechanism additive to and independent from direct stimulation by odorants. Activation by calmodulin is calcium dependent and enhanced by GTP. Thus, olfactory transduction may involve a second-messenger cascade in which an odorant-induced increase in intracellular calcium concentration leads to activation of adenylate cyclase by calmodulin.

Adenylyl Cyclases↗

PubMed1990

Identification of a group of novel membrane proteins unique to chemosensory cilia of olfactory receptor cells.

We have used a library of monoclonal antibodies (mAbs) against chemosensory cilia of the olfactory epithelium of Rana catesbeiana to identify proteins that are unique to the ciliary membrane. Five different antibodies (mAb 8, 26, 34, 42/45, and 43) identify novel proteins in olfactory cilia that are not detected in olfactory nerve membranes, nonchemosensory cilia from respiratory epithelium, or membranes from brain, heart, liver, kidney, and lung. Deglycosylation of olfactory cilia with endoglycosidase H shows that most of these antibodies (mAb 8, 42/45, 43, and possibly 26) react with antigenic determinants comprised partially or entirely of carbohydrate, while only one (mAb 34) recognizes an 87-kDa protein that is resistant to endoglycosidase H treatment. Furthermore, a 59-kDa glycoprotein visualized by mAb 8 exists as membrane-associated oligomers connected via intermolecular disulfide bonds. These proteins, tagged with distinct high-mannose-containing carbohydrate moieties and found only in chemosensory cilia of olfactory receptor cells, may be involved in odorant recognition and/or olfactory transduction.

Animals↗