RESUMO
mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.
Assuntos
Moléculas de Adesão Celular Neuronais/análise , Sistema Nervoso Central/crescimento & desenvolvimento , Nervo Olfatório/análise , Condutos Olfatórios/crescimento & desenvolvimento , Lectinas de Plantas , Proteínas de Soja , Amidoidrolases , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Química Encefálica , Configuração de Carboidratos , Imunofluorescência , Immunoblotting , Imuno-Histoquímica , Técnicas de Imunoadsorção , Lectinas , Microscopia de Fluorescência , Peso Molecular , Bulbo Olfatório/análise , Mucosa Olfatória/análise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Rana catesbeiana , Ratos , Ratos Endogâmicos , Medula Espinal/análiseRESUMO
Carnosine (beta-alanyl-L-histidine) is present in mouse olfactory bulbs and nasal olfactory epithelium at concentrations exceeding that previously reported for any brain region of any species. After peripheral deafferentation, carnosine concentrations in the olfactory bulbs decrease to less than 10 percent that of normal, while other amino compounds are unaffected. Carnosine appears to be highly localized to the primary olfactory pathway.
Assuntos
Dipeptídeos/análise , Bulbo Olfatório/análise , Mucosa Olfatória/análise , Aminoácidos/análise , Animais , Cromatografia , Denervação , Eletroforese em Papel , Camundongos , Mucosa Olfatória/inervação , Condutos Olfatórios/análiseRESUMO
Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.
Assuntos
DNA/genética , Mucosa Olfatória/análise , Proteínas de Ligação ao Retinol/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/isolamento & purificação , Epitélio/análise , Peso Molecular , Muco/metabolismo , Hibridização de Ácido Nucleico , Odorantes , Mucosa Olfatória/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rana pipiens , Sistema Respiratório/análise , Proteínas de Ligação ao Retinol/genéticaRESUMO
Biochemical and electrophysiological studies suggest that odorants induce responses in olfactory sensory neurons via an adenylate cyclase cascade mediated by a G protein. An olfactory-specific guanosine triphosphate (GTP)-binding protein alpha subunit has now been characterized and evidence is presented suggesting that this G protein, termed Golf, mediates olfaction. Messenger RNA that encodes Golf alpha is expressed in olfactory neuroephithelium but not in six other tissues tested. Moreover, within the olfactory epithelium, Golf alpha appears to be expressed only by the sensory neurons. Specific antisera were used to localize Golf alpha protein to the sensory apparatus of the receptor neurons. Golf alpha shares extensive amino acid identity (88 percent) with the stimulatory G protein, Gs alpha. The expression of Golf alpha in S49 cyc- kin- cells, a line deficient in endogenous stimulatory G proteins, demonstrates its capacity to stimulate adenylate cyclase in a heterologous system.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Neurônios Aferentes/fisiologia , Odorantes , Mucosa Olfatória/inervação , Transdução de Sinais , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Immunoblotting , Imuno-Histoquímica , Dados de Sequência Molecular , Neurônios Aferentes/análise , Bulbo Olfatório/fisiologia , Mucosa Olfatória/análise , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , TransfecçãoRESUMO
From the preparation obtained by ultrasonic disintegration of frog olfactory epithelium scraping, a fraction capable of sensitizing artificial phospholipid membranes to the action of some chemicals assumed to be odorants in frogs was isolated. In the presence of the active fraction, the membranes respond to the addition of camphor, linalool and musk ambrette by an increased permeability to Na+ and Ca2+. The main component of the active fraction is a nucleo-protein with molecular weight of no less than 100 000.
Assuntos
Lipídeos de Membrana , Membranas Artificiais , Odorantes , Mucosa Olfatória/análise , Olfato/fisiologia , Animais , Anuros , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Nucleoproteínas/análiseRESUMO
Some parameters of the receptor element from the rat olfactory epithelium are evaluated; it is characterized by high affinity for camphor (KD = 1.5. x 10(-9) M). Triton X-100 has no marked effect on the binding of [3H]camphor. Neither RNAase nor phospholipase C affected [3H]camphor-binding activity. Pronase and trypsin abolished [3H]camphor binding activity by 65 and 40%, respectively. Sulfhydryl reagents decrease the binding of [3H]camphor by a factor of 5--8. The isoelectric point of the receptor solubilized with Triton X-100 is 4.8, as determined by isoelectric focusing. The molecular weight of the receptor as determined by gel electrophoresis is about 120 000. It is proposed that the camphor receptor is a membrane protein containing sulfhydryl groups and playing a key role in olfactory reception.
Assuntos
Cânfora , Mucosa Olfatória/análise , Células Receptoras Sensoriais/análise , Animais , Fenômenos Químicos , Química , Eletroforese em Gel de Poliacrilamida , Epitélio/análise , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Polietilenoglicóis , Ligação Proteica , Ratos , Reagentes de SulfidrilaRESUMO
Monoclonal antibodies against two olfactory mucosal proteins, one with affinity for anisole-like and the other for benzaldehyde-like compounds, were applied to mouse olfactory epithelium. Responses to three odorants (anisole, benzaldehyde and amyl acetate) were measured. Of 26 antibodies, three (12%) inhibited responses only to the odorant with affinity for the antigen, nine (35%) inhibited responses to all three odorants, and 14 (54%) were without effect. None reduced responses by as much as 50%. The data support the hypothesis that there is a class of related proteins in olfactory neuronal cell membranes that function as receptor molecules and that other mechanisms also mediate odorant stimulation.
Assuntos
Anisóis/fisiologia , Benzaldeídos/fisiologia , Proteínas de Transporte/fisiologia , Mucosa Olfatória/fisiologia , Animais , Anisóis/imunologia , Anisóis/farmacologia , Anticorpos/fisiologia , Benzaldeídos/imunologia , Benzaldeídos/farmacologia , Proteínas de Transporte/imunologia , Cães , Eletrofisiologia , Masculino , Camundongos , Odorantes , Mucosa Olfatória/análise , Mucosa Olfatória/efeitos dos fármacos , Pentanóis/farmacologia , Células Receptoras SensoriaisRESUMO
The specific membrane glycoproteins with high affinity for camphor and decanal were isolated from rat olfactory epithelium. Antibodies to these glycoproteins inhibited both the electroolfactogram and the binding of odorants. The enzyme immunoassay has shown these glycoproteins to be present in the olfactory epithelium of rat, mouse, guinea-pig and hamster but not in that of frog and carp. The molecular mass of the odour-binding glycoproteins from rat olfactory epithelium solubilized by Triton X-100 was approx. 140 kDa. They consisted of two subunits (88 and 55 kDa). The 88 kDa subunit was capable of binding odorants. The data obtained suggest that the glycoproteins isolated have some properties that make them plausible candidates for olfactory receptor molecules.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/isolamento & purificação , Odorantes , Mucosa Olfatória/análise , Células Receptoras Sensoriais/análise , Aldeídos/metabolismo , Animais , Especificidade de Anticorpos , Antígenos/imunologia , Cânfora/metabolismo , Proteínas de Transporte/imunologia , Eletroforese em Gel de Poliacrilamida , Epitélio/análise , Feminino , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Imunoglobulina G/imunologia , Masculino , Peso Molecular , Mucosa Olfatória/imunologia , Ratos , Ratos Endogâmicos , Células Receptoras Sensoriais/metabolismo , Especificidade da EspécieRESUMO
The specific odor-binding glycoproteins have been isolated from rat olfactory epithelium. They consist of two subunits, gp88 and gp55. Subunit gp88 is capable of odorant binding.
Assuntos
Proteínas de Transporte/isolamento & purificação , Glicoproteínas/isolamento & purificação , Mucosa Olfatória/análise , Receptores Odorantes , Células Receptoras Sensoriais/análise , Animais , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese Descontínua , Epitélio/análise , Odorantes , RatosRESUMO
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.
Assuntos
Anticorpos Monoclonais , Glicoproteínas/análise , Neurônios/análise , Mucosa Olfatória/análise , Animais , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Cílios/análise , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Glicoproteínas/imunologia , Mucosa Olfatória/inervação , Condutos Olfatórios/análise , Rana ridibunda , Coloração e Rotulagem/métodosRESUMO
A quantitative method of cross-adaptation was developed to explore the difference in the receptor sites for various amino acids in the carp olfactory receptors. The olfactory responses were measured by recording the stimulant-induced waves from the olfactory bulb. Cross-adaptation was carried out by varying concentrations of amino acids in a wide range. Typical examples of the results obtained are as follows. The response to Thr after Ser was decreased with increasing concentration of Ser applied first and reached the spontaneous level, while that to Thr after Glu was decreased to 77% of the level of the original response with increasing Glu concentration and stayed this constant level with a further increase in Glu concentration. Application of the amino acids in the reverse order gave essentially similar results. Such types of experiments were carried out between various pairs of amino acids. The results obtained suggested that amino acids of very close species (e.g. Thr and Ser, Asp and Glu, Tyr and Phe) stimulate the same respective receptor sites and that amino acids of most other pairs stimulate more or less different receptor sites, although there exist the receptor sites stimulated commonly by both amino acids of one pair. It was concluded that the carp olfactory cells have many different receptor sites stimulated only by one species of amino acid and its close analogues.
Assuntos
Carpas/metabolismo , Cyprinidae/metabolismo , Mucosa Olfatória/análise , Receptores de Superfície Celular/análise , Células Receptoras Sensoriais/análise , Animais , Receptores de AminoácidoRESUMO
Carnosine (beta-alanyl-histidine) is present in the olfactory bulb and olfactory eqithelium of mice and rats at 1-2 nmole/mg tissue. Peripheral deafferentation or central denervation causes a rapid, selective decrease of this depeptide from the reciprocal portion of the primary olfactory pathway. These data demonstrate the localization of carnosine within the primary olfactory chemoreceptor neurons and suggest a possible role for this compound in neural transmission.
Assuntos
Carnosina/análise , Dipeptídeos/análise , Bulbo Olfatório/análise , Mucosa Olfatória/análise , Alanina/análise , Animais , Ácido Aspártico/análise , Cerebelo/análise , Compostos de Dansil/análise , Denervação , Feminino , Glutamatos/análise , Lisina/análise , Masculino , Camundongos , Camundongos Endogâmicos , Ratos , Serina/análise , Especificidade da Espécie , Transmissão Sináptica , Taurina/análise , Ácido gama-Aminobutírico/análiseRESUMO
Spectrophotometric techniques were used to determine the concentrations of Na+, K+ and Ca2+ in the olfactory mucus of frogs. The mean concentrations in mEq/l were: [Na+], 52.7 +/- 4.1; [K+], 10.6 +/- 1.9 and [Ca2+], 10.7 +/- 1.7. Topical application of the odorant cineole was associated with statistically significant increases in [Na+] and [Ca2+]; the secretagogues methacholine and isoproterenol induced transient increases in [Na+]. Cineole and methacholine caused sustained increases in [Na+]/[K+] from the control value of 5:1, while isoproterenol caused a transient increase followed by a decline. The results indicate that the cation concentrations in olfactory mucus samples are more similar to those derived from secretory tissue than to those found in the extracellular fluids surrounding typical neural tissue.
Assuntos
Cálcio/análise , Cicloexanóis , Monoterpenos , Mucosa Olfatória/análise , Potássio/análise , Sódio/análise , Terpenos , Animais , Eucaliptol , Isoproterenol/farmacologia , Mentol/análogos & derivados , Mentol/farmacologia , Compostos de Metacolina/farmacologia , Mucosa Olfatória/efeitos dos fármacos , Rana pipiens , Espectrofotometria AtômicaRESUMO
Substance P has been proposed as a candidate neurotransmitter or neuromodulator in the nociceptive system. Using a light microscopial immunohistochemical peroxidase-anti-peroxidase technique we have detected high substance P-like immunoreactivity (SPLI) in several types of sensory organs of 4 species of gymnotiform teleost fish: olfactory epithelium, vestibular, lateral line and electrosensory organs. The olfactory nerve and its endings within the olfactory bulb and the telencephalon were also strongly labelled. At these sites no SPLI was revealed in other teleosts (Carassius auratus, Gnathonemus petersii). The findings suggest that substance P may be involved in neurotransmission or neuromodulation in these specific sensory systems of these species.
Assuntos
Encéfalo/metabolismo , Sistema Nervoso Central/análise , Peixe Elétrico/metabolismo , Condutos Olfatórios/análise , Órgãos dos Sentidos/análise , Substância P/análise , Animais , Encéfalo/citologia , Carpa Dourada/metabolismo , Imuno-Histoquímica , Bulbo Olfatório/análise , Mucosa Olfatória/análise , Mucosa Olfatória/inervação , Nervo Olfatório/análise , Condutos Olfatórios/citologia , Órgãos dos Sentidos/inervaçãoRESUMO
The immunohistochemical characteristics of the human olfactory system were (OMP). OMP was detected in the olfactory receptor neurons and processes extending from the olfactory neuroepithelium to the olfactory bulb. The olfactory receptor cells located close to the epithelial surface also contained OMP. In severely degenerate regions, only a few OMP-containing cells were observed. Differences in OMP-staining intensity were noted among the olfactory receptor cells. The thick neuroepithelium. Proliferating olfactory neuroepithelium contained OMP reactive and nonreactive olfactory receptor cells. The presence of OMP reactive and nonreactive olfactory neurons indicates the coexistence of two functionally different phases of olfactory neurons. These findings suggest that continuous cell turnover is occurring in human olfactory neuroepithelium.
Assuntos
Proteínas do Tecido Nervoso/análise , Bulbo Olfatório/citologia , Mucosa Olfatória/citologia , Nervo Olfatório/citologia , Adulto , Idoso , Animais , Divisão Celular , Células Epiteliais , Epitélio/análise , Epitélio/embriologia , Feminino , Feto/análise , Feto/citologia , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Neurônios/análise , Bulbo Olfatório/análise , Proteína de Marcador Olfatório , Mucosa Olfatória/análise , Mucosa Olfatória/embriologia , Mucosa Olfatória/inervação , Nervo Olfatório/análise , Ratos , Coloração e RotulagemRESUMO
Acid water-soluble glycoproteins of olfactory mucus in various representatives of the vertebrate have been studied. Olfactory mucus of these representatives is found to be of great similarity in its glycoprotein composition. The universal presence of these glycoprotein components of the mucus is supposed to testify to their similar functional significance in various vertebrates.
Assuntos
Glicoproteínas/análise , Muco/análise , Mucosa Olfatória/análise , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Peixes , Histocitoquímica , Rana temporariaRESUMO
To confirm the diagnosis and determine the cause of the olfactory disturbance, we used an immunohistochemical method to examine biopsy specimens of the olfactory mucosa from a patient who complained of anosmia after head injury. Neuron-specific enolase immunoreactivity was found in the olfactory vesicles and dendrites of the receptor cells in the olfactory epithelium. S-100 protein immunoreactivity was found in the ductal cells of Bowman's gland in the olfactory epithelium and in the acinar cells of Bowman's gland in the lamina propria. This suggests that the olfactory receptor cells and Bowman's gland were normal. The olfactory disturbance in this patient was not caused by nerve transection due to the head injury, but by already existing chronic sinusitis. Immunohistochemical methods are useful for diagnosing olfactory disturbance when used in combination with biopsy of olfactory mucosa.
Assuntos
Mucosa Olfatória/fisiopatologia , Adolescente , Anticorpos/imunologia , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Masculino , Doenças Nasais/diagnóstico , Mucosa Olfatória/análise , Fosfopiruvato Hidratase/imunologia , Proteínas S100/imunologiaRESUMO
Presentation of a 48-y old woman who developed a neuroendocrine tumor of the nasal cavities. This lesion progressed rapidly despite an extensive resection and repeated chemotherapy. The patient refused radiotherapy. Before her death, 28 months later, she exhibited a paraneoplastic Cushing-like syndrome. At autopsy, restricted to the brain, there was a 5 cm diameter tumor invading the frontal area without alteration of the hypothalamus or the pituitary gland. Routine histology and electron microscopy confirmed the neuroendocrine nature of the tumor. Immunohistochemistry revealed the tumor to be positive only for neurone specific enolase, negative for S-100 protein, neurofilament and ACTH. The pituitary gland was positive for most usual hormones (GH, PRL, TSH, LH, FSH) but only few cells were slightly positive for ACTH. Many Crooke cells were observed. These findings suggest that the tumor secreted an ACTH-like substance (not detected actually by immunochemistry) that stimulated the activity of the adrenal cortex but inhibited normal production of ACTH at the pituitary gland level.
Assuntos
Síndrome de ACTH Ectópico/etiologia , Cavidade Nasal/patologia , Tumores Neuroectodérmicos Primitivos Periféricos/metabolismo , Neoplasias Nasais/metabolismo , Mucosa Olfatória/patologia , Síndromes Endócrinas Paraneoplásicas/etiologia , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Tumores Neuroectodérmicos Primitivos Periféricos/análise , Tumores Neuroectodérmicos Primitivos Periféricos/patologia , Neoplasias Nasais/análise , Neoplasias Nasais/patologia , Mucosa Olfatória/análise , Mucosa Olfatória/metabolismo , Hipófise/patologia , Hormônios Adeno-Hipofisários/análise , Sistema Hipófise-Suprarrenal/fisiopatologiaRESUMO
Two forms of Ca(++)-activated protease (calpain I and calpain II) associated with an endogenous inhibitor (calpastatin) were detected in a cytosolic fraction of the olfactory tissue of vertebrates (pig, rat). Using ion exchange chromatography on DEAE-cellulose column, calpain I is divided into 2 peaks (eluting by 0.07-0.15 and 0.22-0.25 M NaCl), and calpain II is eluted by 0.35-0.40 M NaCl. The calpain activity was detected in fractions eluted by 0.1-0.17 M NaCl. The Ca(++)-activated protease was demonstrated also in a fraction of cytoskeleton of olfactory tissue insoluble in a 1% solution of Triton X-100. The activity can be detected by Ca(++)-dependent destruction of exogenous substrate (casein), and by Ca(++)-dependent degradation of cytoskeletal endogenous proteins (16, 18 and 20 kDa), of which one may be calmodulin.