RESUMEN
BACKGROUND: The kangaroo pericardium might be considered to be a good candidate material for use in the manufacture of the leaflets of percutaneous heart valves based upon the unique lifestyle. The diet consists of herbs, forbs and strubs. The kangaroo pericardium holds an undulated structure of collagen. MATERIAL AND METHOD: A Red Kangaroo was obtained after a traffic fatality and the pericardium was dissected. Four compasses were cut from four different sites: auricular (AUR), atrial (ATR), sternoperitoneal (SPL) and phrenopericardial (PPL). They were investigated by means of scanning electron microscopy, light microscopy and transmission electron microscopy. RESULTS: All the samples showed dense and wavy collagen bundles without vascularisation from both the epicardium and the parietal pericardium. The AUR and the ATR were 150±25µm thick whereas the SPL and the PPL were thinner at 120±20µm. The surface of the epicardium was smooth and glistening. The filaments of collagen were well individualized without any aggregation, but the banding was poorly defined and somewhat blurry. CONCLUSION: This detailed morphological analysis of the kangaroo pericardium illustrated a surface resistant to thrombosis and physical characteristics resistant to fatigue. The morphological characteristics of the kangaroo pericardium indicate that it represents an outstanding alternative to the current sources e.g., bovine and porcine. However, procurement of tissues from the wild raises supply and sanitary issues. Health concerns based upon sanitary uncertainty and reliability of supply of wild animals remain real problems.
Asunto(s)
Bioprótesis , Prótesis Valvulares Cardíacas , Ligamentos/ultraestructura , Macropodidae/anatomía & histología , Pericardio/ultraestructura , Animales , Australia , Enfermedades de las Válvulas Cardíacas/cirugía , Humanos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de TransmisiónRESUMEN
Heteromultimeric cyclic nucleotide-gated (CNG) channels play a central role in the transduction of odorant signals and subsequent adaptation. The contributions of individual subunits to native channel function in olfactory receptor neurons remain unclear. Here, we show that the targeted deletion of the mouse CNGA4 gene, which encodes a modulatory CNG subunit, results in a defect in odorant-dependent adaptation. Channels in excised membrane patches from the CNGA4 null mouse exhibited slower Ca2+-calmodulin-mediated channel desensitization. Thus, the CNGA4 subunit accelerates the Ca2+-mediated negative feedback in olfactory signaling and allows rapid adaptation in this sensory system.
Asunto(s)
Adaptación Fisiológica , Calcio/metabolismo , Calmodulina/metabolismo , Canales Iónicos/genética , Canales Iónicos/fisiología , Monoterpenos , Odorantes , Neuronas Receptoras Olfatorias/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Señalización del Calcio , AMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Ciclohexanoles/farmacología , Electrofisiología , Eucaliptol , Marcación de Gen , Activación del Canal Iónico , Cinética , Ratones , Ratones Endogámicos C57BL , Bulbo Olfatorio/fisiología , Mucosa Olfatoria/fisiología , Neuronas Receptoras Olfatorias/metabolismo , Subunidades de Proteína , Terpenos/farmacologíaRESUMEN
Mapping of the chromosomal location of genes essential for sweet and bitter taste and identification of the relevant G protein-coupled receptors reveals unanticipated complexity in taste signaling pathways. The distribution of sweet and bitter receptors suggests complete cellular segregation of these taste modalities. Sweet compounds may be distinguished through differential expression of sweet receptors. Novel heterologous expression systems to test bitter and sweet modalities now provide the tools necessary for understanding taste coding.
Asunto(s)
Edulcorantes , Papilas Gustativas/fisiología , Gusto/fisiología , Animales , HumanosRESUMEN
A computer-aided training program was developed in SuperCard and piloted with professional painters. Taking a modern programmed-instruction/behavioral-education approach, cTRAIN is structured as a series of information sets. Each information set consists of a series of information screens (three to five recommended) followed by quiz screens (one to three recommended) structured as four-response multiple choice questions. Correct quiz responses produce positive feedback and continuation in the series, whereas incorrect responses result in "error" feedback and return the student to the beginning of the information set to repeat the same information screens and the same quiz question. This report demonstrates a specific implementation, respiratory protection requirements, using the flexible cTRAIN system for developing training modules. Fifteen adults completed the respiratory protection program, demonstrating substantial and significant (p < .0001 by paired t test) gains from baseline pretest (19.4 out of 30 questions) to the immediate posttest (28.1). Performance remained elevated (26.4) on a retest taken 1 week later.
Asunto(s)
Conducta/fisiología , Instrucción por Computador , Programas Informáticos , Enseñanza , HumanosRESUMEN
The organization of neuronal systems is often dependent on activity and competition between cells. In olfaction, the X-linked OCNC1 channel subunit is subject to random inactivation and is essential for odorant-evoked activity. Reporter-tagged OCNC1 mutant mice permit the visualization of OCNC1-deficient olfactory neurons and their projections. In heterozygous females, X inactivation creates a mosaic with two populations of genetically distinct neurons. OCNC1-deficient neurons are slowly and specifically depleted from the olfactory epithelium and display unusual patterns of projection to the olfactory bulb. Remarkably, this depletion is dependent on odorant exposure and is reversed by odorant deprivation. This suggests that odorants and the activity they evoke are critical for neuronal survival in a competitive environment and implicate evoked activity in the organization and maintenance of the olfactory system.
Asunto(s)
Compensación de Dosificación (Genética) , Canales Iónicos/genética , Canales Iónicos/metabolismo , Olfato/genética , Factores de Edad , Alelos , Animales , Recuento de Células , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Femenino , Expresión Génica/fisiología , Genes Reporteros , Proteínas Fluorescentes Verdes , Heterocigoto , Indicadores y Reactivos/metabolismo , Operón Lac , Proteínas Luminiscentes/genética , Ratones , Ratones Noqueados , Odorantes , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/fisiologíaRESUMEN
Nuclear factor I (NFI) proteins are DNA-binding transcription factors that participate in the tissue specific expression of various genes. They are encoded by four different genes (NFI-A, B, C, and X) each of which generates multiple isoforms by alternative RNA splicing. NFI-like binding sites have been identified in several genes preferentially expressed in olfactory receptor neurons. Our prior demonstration that NFI binds to these elements led to the hypothesis that NFI is involved in the regulation of these genes. To analyse the role of NFI in the regulation of olfactory neuron gene expression we have performed transient transfection experiments in HEK 293 cells using constructs that place luciferase expression under the control of an olfactory marker protein (OMP)-promoter fragment containing the NFI binding site. In vitro mutagenesis of this site revealed a negative modulation of luciferase expression by endogenous NFI proteins in HEK 293 cells. In addition, we have used in situ hybridization to analyse the tissue and cellular distribution of the four NFI gene transcripts during pre- and postnatal mouse development. We have simultaneously characterized the expression of Pax-6, and O/E-1, transcription factors known to regulate the phenotype of olfactory receptor neurons. We demonstrate that all of these transcription factors vary in specific spatio-temporal patterns during the development of the olfactory system. These data on NFI activity, and on transcription factor expression, provide a basis to understand the role of NFI in regulating gene expression in olfactory receptor neurons.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio , Proteínas del Tejido Nervioso/genética , Mucosa Olfatoria/química , Mucosa Olfatoria/embriología , Neuronas Receptoras Olfatorias/química , Factores de Transcripción , Factores de Edad , Animales , Línea Celular , Proteínas del Ojo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Hipocampo/química , Hipocampo/embriología , Humanos , Hibridación in Situ , Riñón/citología , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis/fisiología , Factores de Transcripción NFI , Proteínas Nucleares , Proteína Marcadora Olfativa , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Fenotipo , Embarazo , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/análisis , Proteínas Represoras , Homología de Secuencia de Aminoácido , Transfección , Órgano Vomeronasal/química , Órgano Vomeronasal/embriología , Proteína 1 de Unión a la Caja YRESUMEN
The olfactory cyclic nucleotide-gated channel subunit 1 (OCNC1) is required for signal transduction in olfactory receptor cells. To further investigate the role of this channel in the olfactory system, the biochemical and morphological consequences of targeted disruption of OCNC1 were investigated in adult mice. Null as compared to wild-type mice had smaller olfactory bulbs, suggesting compromised development of the central target of the receptor cells. Ectopic olfactory marker protein (OMP)-stained fibers localized to the external plexiform layer reflected the relative immaturity of the olfactory bulb in the null mice. The olfactory epithelium of the knock-out mouse was thinner and showed lower expression of olfactory marker protein and growth-associated protein 43, indicating decreases in both generation and maturation of receptor cells. Tyrosine hydroxylase (TH) expression in the olfactory bulb, examined as a reflection of afferent activity, was reduced in the majority of periglomerular neurons but retained in atypical or "necklace" glomeruli localized to posterior aspects of the olfactory bulb. Double label studies demonstrated that the remaining TH-immunostained neurons received their innervation from a subset of receptor cells previously shown to express a phosphodiesterase that differs from that found in most receptor cells. These data indicate that expression of OCNC1 is required for normal development of the olfactory epithelium and olfactory bulb. The robust expression of TH in some periglomerular cells in the OCNC1-null mice suggests that receptor cells innervating these glomeruli may use an alternate signal transduction pathway.
Asunto(s)
Canales Iónicos/fisiología , Bulbo Olfatorio/fisiología , Neuronas Receptoras Olfatorias/fisiología , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Femenino , Proteína GAP-43/genética , Genotipo , Canales Iónicos/deficiencia , Canales Iónicos/genética , Masculino , Ratones , Ratones Noqueados , Fibras Nerviosas/fisiología , Fibras Nerviosas/ultraestructura , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Bulbo Olfatorio/anomalías , Proteína Marcadora Olfativa , Mucosa Olfatoria/anomalías , Mucosa Olfatoria/citología , Mucosa Olfatoria/patología , Mucosa Olfatoria/fisiología , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/patologíaRESUMEN
The processes of neuronal differentiation and survival are key questions in neurobiology. The olfactory system possesses unique regenerative capacity, as its neurons are continually replaced throughout adulthood from a maintained population of precursor cells. Primary cultures of olfactory epithelium enriched in olfactory neurons would provide a useful model to study the processes of neurogenesis, differentiation and senescence. To determine whether immature olfactory neurons could be isolated in primary culture and to investigate the mechanisms underlying these processes, culture conditions which selectively favored the presence of immature olfactory neurons were optimized. Using low plating densities, a population of cells was identified which, by reverse transcription-polymerase chain reaction, demonstrated messages for olfactory neuronal markers, including Golf, olfactory cyclic nucleotide-gated channel and olfactory marker protein, as well as the p75 low-affinity nerve growth factor receptor. Immunocytochemical analysis showed that these putative immature olfactory neurons possessed immunoreactivity to G(olf), neuron-specific tubulin, neural cell adhesion molecule, synaptophysin and neurofilament. These neurons were defined as olfactory receptor neuron-1 cells. Under these conditions, a separate class of rarely occurring cells with different morphology demonstrated immunoreactivity to mature markers, such as adenylyl cyclase III and olfactory marker protein. Electrophysiologically, these cells displayed properties consistent with those of acutely dissociated olfactory receptor neurons. Another class of rarer cells which represented less than 2% of cells in culture demonstrated immunoreactivity to glial fibrillary acidic protein. These cultures can serve as a model for in vitro analysis of olfactory receptor neuronal development and maintenance, and provide a potential substrate for the development of cell lines.
Asunto(s)
Senescencia Celular/fisiología , Neuronas Receptoras Olfatorias/citología , Animales , Biomarcadores , Canales de Calcio/análisis , Canales de Calcio/genética , Diferenciación Celular/fisiología , Células Cultivadas , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Electrofisiología , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Receptores de Inositol 1,4,5-Trifosfato , Canales Iónicos/análisis , Canales Iónicos/genética , Potenciales de la Membrana/fisiología , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/fisiología , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Receptor de Factor de Crecimiento Nervioso/análisis , Receptor de Factor de Crecimiento Nervioso/genética , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/genética , Olfato/fisiologíaRESUMEN
Neuropilin-1 (Npn-1), a receptor for semaphorin III, mediates the guidance of growth cones on extending neurites. The molecular mechanism of Npn-1 signaling remains unclear. We have used a yeast two-hybrid system to isolate a protein that interacts with the cytoplasmic domain of Npn-1. This Npn-1-interacting protein (NIP) contains a central PSD-95/Dlg/ZO-1 (PDZ) domain and a C-terminal acyl carrier protein domain. The physiological interaction of Npn-1 and NIP is supported by co-immunoprecipitation of these two proteins in extracts from a heterologous expression system and from a native tissue. The C-terminal three amino acids of Npn-1 (S-E-A-COOH), which is conserved from Xenopus to human, is responsible for interaction with the PDZ domain-containing C-terminal two-thirds of NIP. NIP as well as Npn-1 are broadly expressed in mice as assayed by Northern and Western analysis. Immunohistochemistry and in situ hybridization experiments revealed that NIP expression overlaps with that of Npn-1. NIP has been independently cloned as RGS-GAIP-interacting protein (GIPC), where it was identified by virtue of its interaction with the C terminus of RGS-GAIP and suggested to participate in clathrin-coated vesicular trafficking. We suggest that NIP and GIPC may participate in regulation of Npn-1-mediated signaling as a molecular adapter that couples Npn-1 to membrane trafficking machinery in the dynamic axon growth cone.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Clonación Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neuropéptidos/genética , Neuropéptidos/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos/genética , Animales , Axones/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Sistema Nervioso Central/metabolismo , Humanos , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Terminaciones Nerviosas/metabolismo , Neuropéptidos/metabolismo , Neuropilina-1 , Vías Olfatorias/metabolismo , Nervios Periféricos/metabolismo , Pruebas de Precipitina , Homología de Secuencia de AminoácidoRESUMEN
Communication with the environment and other animals through chemical cues is an essential process for the survival of many multicellular organisms. Specialized signal transduction pathways are employed in chemodetection and the transformation of information into the electrical signals that elicit behaviors. In organisms as diverse as mice and nematodes, similar molecules are involved in the odorant signaling pathways. Studying the mechanisms of signal transduction in these two systems using biochemical, molecular and genetic approaches has elucidated pathways for odor perception and the roles of specific proteins and second messenger molecules in the signaling cascades.
Asunto(s)
Olfato/fisiología , Animales , Caenorhabditis elegans/fisiología , Mamíferos , Ratones , Feromonas/fisiología , Transducción de Señal/fisiologíaRESUMEN
Roaz, a rat C2H2 zinc finger protein, plays a role in the regulation of olfactory neuronal differentiation through its interaction with the Olf-1/EBF transcription factor family. An additional role for the Roaz/Olf-1/EBF heterodimeric protein is suggested by its ability to regulate gene activation at a distinct promoter lacking Olf-1/EBF-binding sites. Using an in vitro binding-site selection assay (Selex), we demonstrate that Roaz protein binds to novel inverted perfect or imperfect repeats of GCACCC separated by 2 bp. We show that Roaz is capable of binding to a canonical consensus recognition sequence with high affinity (Kd = 3 nM). Analysis of the structural requirement for protein dimerization and DNA binding by Roaz reveals the role of specific zinc finger motifs in the Roaz protein for homodimerization and heterodimerization with the Olf-1/EBF transcription factor. The DNA-binding domain of Roaz is mapped to the N-terminal 277 amino acids, containing the first seven zinc finger motifs, which confers weak monomeric binding to a single half site and a stronger dimeric binding to the inverted repeat in a binding-site-dependent manner. Full-length protein can form dimers on both the inverted repeat and direct repeat but not on a single half site. These findings support the role of the TFIIIA-type Zn fingers in both protein-protein interaction and protein-DNA interaction and suggest distinct functions for specific motifs in proteins with a large number of zinc finger structures.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/química , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Dimerización , Regulación de la Expresión Génica/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Unión Proteica/fisiología , Ratas , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Factor de Transcripción TFIIIA , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional , Transfección/genéticaRESUMEN
Field potential recording was used to investigate properties of synaptic transmission and long-term potentiation (LTP) at Schaffer collateral-CA1 synapses in both hippocampal slices of mutant mice in which the alpha-subunit of the olfactory cyclic nucleotide-gated channel (alpha3/OCNC)1 was rendered null and also in slices prepared from their wild-type (Wt) littermates. Several measures of basal synaptic transmission were unaltered in the OCNC1 knockout (KO), including maximum field excitatory postsynaptic potential (fEPSP) slope, maximum fEPSP and fiber volley amplitude, and the function relating fiber volley amplitude to fEPSP slope and paired-pulse facilitation. When a high-frequency stimulation protocol was used to induce LTP, similar responses were seen in both groups [KO: 1 min, 299 +/- 50% (mean +/- SE), 60 min, 123 +/- 10%; Wt: 1 min, 287 +/- 63%; 60 min, 132 +/- 19%). However, on theta-burst stimulation, the initial amplitude of LTP was smaller (1 min after induction, 147 +/- 16% of baseline) and the response decayed faster in the OCNC1 KO (60 min, 127 +/- 18%) than in Wt (1 min, 200 +/- 14%; 60 min, 169 +/- 19%). Analysis of waveforms evoked by LTP-inducing tetanic stimuli revealed a similar difference between groups. The development of potentiation throughout the tetanic stimulus was similar in OCNC1 KO and Wt mice when high-frequency stimulation was used, but OCNC1 KO mice showed a significant decrease when compared with Wt mice receiving theta-burst stimulation. These results suggest that activation of cyclic nucleotide-gated channels may contribute to the induction of LTP by weaker, more physiological stimuli, possibly via Ca2+ influx.
Asunto(s)
Hipocampo/fisiología , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Potenciación a Largo Plazo/fisiología , Nucleótidos Cíclicos/fisiología , Olfato/fisiología , Transmisión Sináptica/fisiología , Animales , Estimulación Eléctrica , Electrofisiología , Hibridación in Situ , Ratones , Ratones Transgénicos , ARN Mensajero/biosíntesis , RatasRESUMEN
The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons, as well as defects in dauer formation, a process requiring chemosensory inputs. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and/or neuronal differentiation in both sensory and motor neurons.
Asunto(s)
Axones/fisiología , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto , Neuronas/fisiología , Factores de Transcripción/biosíntesis , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Inducción Embrionaria , Exones , Proteínas del Helminto/biosíntesis , Mamíferos , Datos de Secuencia Molecular , Neuronas Motoras/fisiología , Mutagénesis Sitio-Dirigida , Sistema Nervioso/embriología , Mutación Puntual , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Cyclic-nucleotide-activated, nonselective cation channels have a central role in sensory transduction. They are most likely tetramers, composed of two subunits (alpha and beta or 1 and 2), with the former, but not the latter, being able to form homomeric cyclic-nucleotide-activated channels. Identified members of this channel family now include, in vertebrates, the rod and cone channels mediating visual transduction and the channel mediating olfactory transduction, each apparently with distinct alpha- and beta-subunits. Homologous channels have also been identified in Drosophila melanogaster and Caenorhabditis elegans. By co-expressing any combination of two alpha-subunits, or alpha- and beta-subunits, of this channel family in HEK 293 cells, we have found that they can all co-assemble functionally with each other, including those from fly and nematode. This finding suggests that the subunit members so far identified form a remarkably homogeneous and conserved group, functionally and evolutionarily, with no subfamilies yet identified. The ability to cross-assemble allows these subunits to potentially generate a diversity of heteromeric channels, each with properties specifically suited to a particular cellular function.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canales Iónicos/química , Canales Iónicos/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Visión Ocular/fisiología , Animales , Caenorhabditis elegans , Línea Celular , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Drosophila melanogaster , Humanos , Canales Iónicos/biosíntesis , Riñón , Cinética , Sustancias Macromoleculares , Nematodos , Células Fotorreceptoras de Invertebrados/fisiología , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Especificidad de la Especie , Transfección , VertebradosAsunto(s)
Proteínas de Caenorhabditis elegans , Odorantes , Neuronas Receptoras Olfatorias/fisiología , Receptores Odorantes/fisiología , 1-Octanol/farmacología , Adenoviridae/genética , Adenoviridae/fisiología , Animales , Caenorhabditis elegans , Electrofisiología , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas del Helminto/fisiología , Ligandos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Técnicas de Placa-Clamp , Ratas , Receptores Odorantes/genética , Proteínas RecombinantesRESUMEN
Discrimination of odorants is thought to arise from the selective expression of one of a small number of individual receptors in any single olfactory neuron. Receptor genes are expressed in a small subset of neurons throughout a zonally restricted region of the sensory epithelium. We demonstrate that a 6.7 kb region upstream of the M4 olfactory receptor coding region was sufficient to direct expression in olfactory epithelium. Moreover, reporter expression recapitulated the zonal restriction and distributed neuronal expression observed for endogenous olfactory receptors. Transgenic lines were obtained that directed expression in two different receptor zones, one of which was identical to the endogenous M4 receptor. When the reporter was expressed in the same zone as the endogenous M4 receptor, the two expression patterns were, in large part, nonoverlapping. These results suggest a model in which important regulatory elements are located in close proximity to transcription initiation sites of the olfactory receptor genes and receive information defining zonal patterning via long-range processes.
Asunto(s)
Neuronas Receptoras Olfatorias/química , Receptores Odorantes/genética , Transgenes/fisiología , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Epitelio/química , Regulación de la Expresión Génica , Genes Reporteros , Intrones , Ratones , Datos de Secuencia Molecular , Neuronas Receptoras Olfatorias/fisiología , Regiones Promotoras GenéticasRESUMEN
The recognition of odorants by olfactory receptors represents the first stage in odor discrimination. Here, we report the generation of an expression library containing a large and diverse repertoire of mouse olfactory receptor sequences in the transmembrane II-VII region. From this library, 80 chimeric receptors were tested against 26 odorants after transfection into HEK-293 cells. Three receptors were identified to respond to micromolecular concentrations of carvone, (-) citronellal, and limonene, respectively. We also found that the mouse I7 receptor, unlike the rat I7 receptor, prefers heptanal instead of octanal, as a result of a single valine-to-isoleucine substitution. This finding represents the beginning of a molecular understanding of odorant recognition. The identification, on a large scale, of cognate receptor-odorant interactions should provide insight into olfactory coding mechanisms.
Asunto(s)
Clonación Molecular , Monoterpenos , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Acetilcolina/farmacología , Monoterpenos Acíclicos , Aldehídos/metabolismo , Aldehídos/farmacología , Sustitución de Aminoácidos , Animales , Sitios de Unión , Señalización del Calcio , Línea Celular , Membrana Celular/metabolismo , Clonación Molecular/métodos , Regulación hacia Abajo , Alcoholes Grasos/metabolismo , Alcoholes Grasos/farmacología , Isoproterenol/farmacología , Ligandos , Ratones , Datos de Secuencia Molecular , Biblioteca de Péptidos , Ratas , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Receptores Odorantes/química , Proteínas Recombinantes de Fusión/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Terpenos/metabolismo , Terpenos/farmacologíaRESUMEN
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.