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R R Reed

Publications and source records attributed to R R Reed.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cloning and functional characterization of Roaz, a zinc finger protein that interacts with O/E-1 to regulate gene expression: implications for olfactory neuronal development.

We have identified a protein, Rat O/E-1-associated zinc finger protein (Roaz), that plays a role in regulating the temporal and spatial pattern of olfactory neuronal-specific gene expression. This protein functions by interacting with the olfactory factor O/E-1 and modulating its transcriptional activity. Roaz, isolated via a yeast two-hybrid screen, encoded a protein containing 29 C2H2 zinc fingers of the TFIIIA type. The Roaz mRNA was found in brain, eye, olfactory epithelium, spleen, and heart. In situ hybridization data indicated that Roaz was expressed in the basal layer, consisting of neural precursor cells and immature sensory neurons of the olfactory epithelium, but not in the mature receptor cells. We showed that the Roaz protein bound specifically to O/E-1 by using the yeast two-hybrid system. The two proteins formed a stable complex in coimmunoprecipitation and in vitro binding assays. Introduction of Roaz and O/E-1 into cells containing an olfactory promoter-driven luciferase reporter demonstrated that Roaz abolished O/E-1-mediated transcriptional activation. We propose that the function of Roaz is to modulate negatively the transactivational activity of O/E-1 and to act as a switch protein in the coordination of olfactory sensory neuron differentiation.

Amino Acid Sequence↗
PubMed1997

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study.

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 microns from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.

Amino Acid Sequence↗
PubMed1997

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study.

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 microns from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.

Amino Acid Sequence↗
PubMed1996

Role of Olf-1 and Pax-6 transcription factors in neurodevelopment.

The Olf-1 transcription factor is expressed in olfactory sensory neurons where it regulates the expression of genes that encode components of the odorant signal transduction cascade and contributes to the terminal phenotype of these sensory neurons. We examined the pattern of expression of Olf-1 protein during mouse embryogenesis and observed Olf-1 expression transiently in a subset of neural precursor cells in the CNS and peripheral nervous system. The expression of Olf-1 protein was enriched in sensory components and coincided with postmitotic cells and the initiation of overt differentiation within the nervous system. The spatial and temporal patterns of Olf-1 expression during development suggest a role in neurogenesis that is common among different neural cell types. In parallel, the expression pattern of Pax-6, a transcription factor that is widely expressed in the developing nervous system, including the visual and olfactory systems, was examined with a C-terminal antibody. In the retina, Pax-6 protein is detected in the lens, the cornea, and the neural and pigmented retinas. In the olfactory epithelium, Pax-6 protein is expressed exclusively in cells of non-neuronal lineage, including sustentacular cells, basal cells, and Bowman's glands. The nonoverlapping, cellular localization patterns of Pax-6 and Olf-1 demarcate distinct cell lineages within the developing olfactory epithelium.

Aging↗
PubMed1996

Studies on the metabolism and biological function of APLP2.

Amyloid precursor proteins (APP) are a member of a larger family of proteins that include the amyloid precursor-like proteins (APLP) APLPI and APLP2. We have examined the expression and metabolism of APLP2 and document that APLP2 is expressed at high levels in the nervous system and in peripheral tissues. Furthermore, several APLP2 isoforms encoded by alternatively spliced transcripts are posttranslationally modified by a chondroitin sulfate glycosaminoglycan (CSGAG) chain. Furthermore, CSGAG modification is regulated by the insertion of sequences encoded by an alternatively spliced exon. Notably, expression of the CSGAG form of APLP2 appears restricted to embryonic neurons and mature neuronal populations that undergo regeneration, such as olfactory sensory neurons. Thus, differences in posttranslational modifications between the APLP2 isoforms and APP are likely to underlie differences in the regulation and function of these homologues. Our present efforts are directed towards using gene targeting strategies to disrupt the expression of the mouse APP/APLP2 genes to define the normative roles of the encoded molecules in development, plasticity, regeneration, and repair.

Alternative Splicing↗
PubMed1996

Elementary events underlying voltage-dependent G-protein inhibition of N-type calcium channels.

Voltage-dependent G-protein inhibition of N-type calcium channels reduces presynaptic calcium entry, sharply attenuating neurotransmitter release. Studies in neurons demonstrate that G-proteins have multiple modulatory effects on N-type channels. The observed changes may reflect genuine complexity in G-protein action and/or the intricate interactions of multiple channels and receptors in neurons. Expression of recombinant M2-muscarinic receptors and N-type channels in HEK 293 cells allowed voltage-dependent inhibition to be studied in isolation. In this system, receptor-activated G-proteins had only one effect: a 10-fold increase in the time required for channels to first open following membrane depolarization. There were no changes in gating after the channel first opened, and unitary currents were not detectably altered by modulation. Despite its simplicity, this single change successfully accounts for the complex alterations in whole-cell current observed during G-protein inhibition in neurons.

Calcium Channels↗
PubMed1995

The genetic basis for specific anosmia to isovaleric acid in the mouse.

The detection and discrimination of odorants in mammals is thought to be mediated by a family of 100-1000 seven transmembrane domain receptor proteins, although none of these putative olfactory receptors have been shown to bind individual odorants with high affinity. We have used a genetic approach to identify the genomic regions responsible for the differential ability of two inbred mouse strains to detect a single odorant, isovaleric acid. Results obtained with a behavioral assay were consistent with a limited number of genes conferring the ability to detect isovaleric acid. One genetic location mapped to a 0.3 cM region between D4MIT37 and D4MIT156 on mouse chromosome 4. A second locus mapped to the distal end of mouse chromosome 6. The most likely cause of the behavior difference between the two strains of mice is the loss of the receptor protein or proteins responsible for recognizing isovaleric acid. High resolution genetic mapping provides a novel approach to the identification of genes critical for the detection of particular odorants.

Animals↗
PubMed1995

Identification of functional domains of adenylyl cyclase using in vivo chimeras.

Adenylyl cyclase, the effector molecule of the cAMP signaling pathway, is composed of a family of isoforms that differ in their modes of regulation. Many of these modulatory interactions are dependent upon well characterized molecules from various second messenger pathways; however, very little is known about their mechanisms or sites of action on adenylyl cyclase. Chimeras were produced by a novel in vivo mechanism between two differentially modulated adenylyl cyclases to identify their regulatory domains. The basal activity of the type I adenylyl cyclase (AC1) is activated by calcium/calmodulin, inhibited by G protein beta gamma subunits, and insensitive to protein kinase C regulation. In contrast, type II adenylyl cyclase (AC2) is insensitive to calcium/calmodulin regulation and is activated by G protein beta gamma subunits as well as by activated protein kinase C. Expression and biochemical characterization of chimeras between AC1 and AC2 identified a single specific domain of AC1 responsible for calmodulin binding and a small, well defined region near the C terminus of AC2 required for protein kinase C activation.

Adenylyl Cyclases↗
PubMed1995

The genetics of olfaction.

Our understanding of olfaction has progressed rapidly in recent years as a result of the molecular genetic approaches being used to study this sensory system in a variety of model organisms. Considerable success has been achieved in identifying proteins of the mammalian signaling system that are analogous to those present in other sensory systems. More recently, genetic selection of mutations that cause defects in olfactory function in Drosophila melanogaster and Caenorhabditis elegans has led to the identification of additional proteins that play a role in the detection of odorants. The application of genetic, electrophysiological, and molecular analyses to olfactory function in mammals is also shedding light on the mechanisms that account for sensitivity and specificity in this system.

Animals↗
PubMed1995

The genetics of olfaction.

Our understanding of olfaction has progressed rapidly in recent years as a result of the molecular genetic approaches being used to study this sensory system in a variety of model organisms. Considerable success has been achieved in identifying proteins of the mammalian signaling system that are analogous to those present in other sensory systems. More recently, genetic selection of mutations that cause defects in olfactory function in Drosophila melanogaster and Caenorhabditis elegans has led to the identification of additional proteins that play a role in the detection of odorants. The application of genetic, electrophysiological, and molecular analyses to olfactory function in mammals is also shedding light on the mechanisms that account for sensitivity and specificity in this system.

Animals↗
PubMed1995

Distribution of an APP homolog, APLP2, in the mouse olfactory system: a potential role for APLP2 in axogenesis.

Deposition of beta-amyloid (A beta) in senile plaques is a major pathological characteristic of Alzheimer's disease. A beta is generated by proteolytic processing of amyloid precursor proteins (APP). APP is a member of a family of related polypeptides that includes amyloid precursor-like proteins APLP1 and APLP2. To examine the distribution of APLP2 in the nervous system, we generated antibodies specific for APLP2 and used these reagents in immunocytochemical and biochemical studies of the rodent nervous system. In this report, we document that in cortex and hippocampus, APLP2 is enriched in postsynaptic compartments. In the olfactory system, however, APLP2 is abundant in olfactory sensory axons, and axon terminals in glomeruli. Confocal microscopy revealed that APLP2 is present in both pre- and postsynaptic compartments in the olfactory bulb. Notably, mRNA encoding chondroitin sulfate glycosaminoglycan (CS GAG)-modified forms of APLP2 are enriched in the olfactory epithelium, relative to alternatively-spliced mRNA, encoding CS GAG-free forms of APLP2. In addition, we demonstrate that CS-modified APLP2 forms accumulate in the olfactory bulb. CS proteoglycans are known to play an important role in regulating cell migration and neuronal outgrowth. Since sensory neurons in the olfactory epithelium are in a state of continual turnover, axons of newly generated cells must establish synaptic connections with neurons in the olfactory bulb in adult life. The presence of APLP2 in olfactory sensory axons and glomeruli is consistent with the view that this protein may play an important role in axonal pathfinding and/or synaptogenesis.

Amyloid beta-Protein Precursor↗
PubMed1994

The molecular basis of sensitivity and specificity in olfaction.

The signaling pathways for converting odorant stimuli from the environment into a signal that is interpreted by the brain has largely been elucidated. The current and formidable challenges lie in understanding the interactions among the second messenger pathways, determining the specificity of olfactory receptors in recognizing chemically similar odorants, and elucidating the regulatory pathways that confine receptor expression to a small subset of the olfactory neurons. It is the sum total of these contributions that result in the ability of the olfactory system to detect odorant with high sensitivity and specificity.

Animals↗
PubMed1993

Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast.

A novel genetic selection in yeast has been used to isolate a complementary DNA for the transcriptional activator, Olf-1, which binds to the regulatory sequences of several olfactory-specific genes. The Olf-1 protein, expressed exclusively in the olfactory receptor neurons and their precursors, contains a new helix-loop-helix motif and functions as an apparent homodimer. Olf-1 may be the first member of a family of related proteins that may direct cellular differentiation in a variety of neuronal tissues.

Amino Acid Sequence↗
PubMed1993

A new subunit of the cyclic nucleotide-gated cation channel in retinal rods.

Retinal rods respond to light with a membrane hyperpolarization produced by a G-protein-mediated signalling cascade that leads to cyclic GMP hydrolysis and the consequent closure of a cGMP-gated channel that is open in darkness. A protein that forms this channel has recently been purified from bovine retina and molecularly cloned, suggesting that the native cGMP-gated channel might be a homo-oligomer. Here we report the cloning of another protein from human retina which has only about 30% overall identity to the rod channel subunit. This protein, immunocytochemically localized to rod outer segments, does not form functional channels by itself. However, when co-expressed with the cloned human rod channel protein, it introduces rapid flickers to the channel openings that are characteristic of the native channel. The hetero-oligomeric channel is also highly sensitive to the blocker L-cis-diltiazem, like the native channel. This new protein thus seems to be another subunit of the native rod channel. The hetero-oligomeric nature of the rod channel means that it is no exception to a common motif shared by other ligand-gated channels.

Adult↗
PubMed1993

Neuronal inositol 1,4,5-trisphosphate receptor localized to the plasma membrane of olfactory cilia.

Both the cyclic adenosine 3',5'-monophosphate and the phosphoinositide second messenger systems are involved in olfactory signal transduction. The inositol 1,4,5-trisphosphate receptor is one of the principal intracellular calcium channels responsible for mobilizing stored calcium. The precise location of the 1,4,5-trisphosphate receptor (endoplasmic reticulum vs surface) and its role in the events of olfactory signal transduction need to be defined. By light microscopic and confocal immunohistochemistry we show expression of the olfactory-enriched G-protein, G(olf), associated with cyclic AMP responses, and of the inositol 1,4,5-trisphosphate receptor in the dendritic projections and cilia of essentially all olfactory receptor neurons, indicating that individual neurons express components of both second messenger systems. By immunoelectron microscopy, we demonstrate that the inositol 1,4,5-trisphosphate receptor is localized to the surface membrane of rat olfactory cilia. This is the first morphological demonstration of the surface membrane localization of inositol 1,4,5-trisphosphate receptor in olfactory cilia. Our findings, taken in conjunction with electrophysiological data from other workers, are supportive of the inositol 1,4,5-trisphosphate receptor playing a novel role in regulating calcium flux at the ciliary surface membrane.

Animals↗