RESUMEN
Cell-to-cell communications and autocrine/paracrine regulations are mediated by an extracellular signaling network involving secretion of a variety of different factors, hormones, neurotransmitters, and other signaling molecules that are recognized in an extracellular medium by multiple molecular receptors operating in the plasma membrane of cells. Most of plasma membrane receptors belong to the superfamily of heptahelical receptors, many of which are coupled by G-proteins to adenylate cyclase responsible for cAMP production in the cell cytoplasm. The canonical role of cAMP in cell physiology is to serve as a second messenger and universal regulator of intracellular processes. Meanwhile, increasing body of evidence leaves little doubts that stimulated cells can release cAMP into intercellular space, where it may serve as signaling molecule in cell-to-cell communications and autocrine regulations. This review considers the basic concept on mechanisms of intracellular and extracellular signaling with cAMP as the second and first messenger.
Asunto(s)
Adenilil Ciclasas/metabolismo , AMP Cíclico/metabolismo , Células Eucariotas/metabolismo , Receptores de Superficie Celular/metabolismo , Sistemas de Mensajero Secundario/genética , Adenilil Ciclasas/genética , Animales , Comunicación Celular , Membrana Celular/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Células Eucariotas/citología , Espacio Extracelular/metabolismo , Regulación de la Expresión Génica , Hormonas/genética , Hormonas/metabolismo , Humanos , Neurotransmisores/genética , Neurotransmisores/metabolismo , Receptores de Superficie Celular/genética , Transducción de SeñalRESUMEN
Gene expression analysis facilitates the study of physiological processes operating at the single-cell level. It allows for the identification of key molecules that are involved in distinct intracellular processes in individual cells. The review focuses on the modern techniques and strategies that are used to establish gene expression profile of a single cell. Particularly, it describes how analysis of gene expression patterns at the single-cell level had provided significant insight into understanding of molecular and cellular mechanisms of odor and taste transduction in mammals.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/fisiología , Animales , Humanos , Olfato/genética , Gusto/genéticaRESUMEN
Peroxiredoxins (Prx) are a family of nonselenium peroxidases that are involved in cell defense against oxidative stress and in redox regulation of intracellular signaling. Mammalian peroxiredoxin 6 (Prx6) belongs to the 1-Cys Prx subfamily. The protein--protein interactions of human Prx6 were studied using a yeast two-hybrid system. Hybrid plasmid pHybLex/Zeo/Prx6, which directed synthesis of a chimeric protein consisting of the DNA-binding domain (BD) of LexA and a Prx6 sequence, was used to screen a two-hybrid cDNA library Hybrid Hunter (Invitrogen). The screening identified two potential interaction partners of Prx6: the calcium-activated cysteine endopeptidase calpain and the p50RhoGAP protein of the family of Sec14-like proteins. The possibility for the interactions observed in the two-hybrid system to occur in oxidative stress in vivo is discussed.
Asunto(s)
Calpaína/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Estrés Oxidativo/fisiología , Peroxiredoxina VI/metabolismo , Calpaína/genética , Proteínas Activadoras de GTPasa/genética , Humanos , Peroxiredoxina VI/genética , Unión Proteica/fisiología , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Peroxiredoxins (Prx) are a family of antioxidant proteins with peroxidase activity. The ability of 1-Cys Prx to self-associate was studied with the use of native PAGE and Western blotting. Two protein bands corresponding to monomeric and dimeric forms were detected in the preparation of the recombinant 1-Cys Prx subjected to native PAGE, with dimers being more abundant. The third band corresponding to the oligomeric form was detected after incubation of the recombinant 1-Cys Prx with DTT, although monomers and dimers were also observed. These results indicate that monomeric, dimeric, and oligomeric states of the protein are likely to be interchangeable. Native PAGE in combination with Western blot analysis revealed that self-association of 1-Cys Prx also occurred at physiologically relevant concentrations in vivo. The native 1-Cys Prx existed in the monomeric and dimeric forms in rat olfactory epithelium, with monomers being more common. The structural sensitivity of the recombinant 1-Cys Prx to imidazole was shown.
Asunto(s)
Peroxidasas/química , Secuencia de Aminoácidos , Animales , ADN Recombinante/química , ADN Recombinante/genética , Inhibidores Enzimáticos/química , Imidazoles/química , Datos de Secuencia Molecular , Peroxidasas/genética , Peroxirredoxinas , Estructura Cuaternaria de Proteína , RatasRESUMEN
The 28 kDa peroxiredoxin from rat exhibited peroxidase activity only in the presence of dithiothreitol. Both organic and nonorganic peroxidases were found to be substrates for the 28-kDa peroxiredoxin activity. Analysis of the protective antioxidant activity of the 28-kDa peroxiredoxin revealed that it is accounted for by its peroxidase activity.
Asunto(s)
Antioxidantes/metabolismo , Peroxidasas/metabolismo , Animales , Ditiotreitol/metabolismo , Peroxirredoxinas , Ratas , Especificidad por SustratoRESUMEN
cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.
Asunto(s)
Mucosa Olfatoria/química , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas/química , ARN Mensajero/análisis , Ratas , Mapeo Restrictivo , Alineación de SecuenciaRESUMEN
Peroxiredoxins are a novel family of antioxidant proteins that specifically prevent enzymes from metal-catalyzed oxidation. The localization of a member of the mono-cystein subfamily of peroxiredoxins, the 28-kDa protein, in different rat tissues and its antioxidant properties were investigated. By immunoblotting, the 28-kDa peroxiredoxin was found to be most highly concentrated in olfactory epithelium and present in all tissues tested (skin, lung, trachea, kidney, womb, and brain). Immunostaining with rabbit polyclonal antibody raised against the 28-kDa peroxiredoxin revealed the particularly high level of the 28-kDa peroxiredoxin immunoreactivity in air-contacting areas (apical regions and mucus of the olfactory and respiratory epithelium and skin epidermis), which are continually exposed to numerous air-borne reactive oxygen species. In the apical regions of the olfactory and respiratory epithelium, the 28-kDa-peroxiredoxin immunogold labeling outlined microvilli and cilia and was mainly located in sustentacular cells and in respiratory and goblet cells, as electron-microscopic analysis revealed. In skin epidermis, the 28-kDa peroxiredoxin immunoreactivity was confined to the granular layer and specifically concentrated in sebaceous glands of hair follicle. In situ hybridization with 33P-labeled antisense RNA probe revealed the expression of the 28-kDa peroxiredoxin mRNA in tissues with a high level of the 28-kDa peroxiredoxin immunoreactivity. Immunodepletion of the 28-kDa peroxiredoxin profoundly decreased the antioxidant activity of the olfactory tissue extract.
Asunto(s)
Antioxidantes/análisis , Mucosa Olfatoria/química , Peroxidasas/análisis , Animales , Antioxidantes/metabolismo , Dendritas/química , Dendritas/metabolismo , Expresión Génica/fisiología , Hibridación in Situ , Masculino , Microscopía Inmunoelectrónica , Mucosa Olfatoria/metabolismo , Mucosa Olfatoria/ultraestructura , Oxidación-Reducción , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxirredoxinas , ARN Mensajero/análisis , Conejos , Ratas , Ratas WistarRESUMEN
The secretory 28 kD protein, an abundant water-soluble protein from rat olfactory epithelium, belongs to the 1-Cys subfamily of thiol-specific antioxidants (peroxiredoxins). The 28 kD protein contains a single cysteine residue at the position 46 which accounts for the antioxidant activity. Here we studied the effects of N-ethyilmaleimide and t-butyl hydroperoxide on the antioxidant activity of the 28 kD protein and that of the 23 kD protein from rat erythrocyte which is a member of 2-Cys subfamily of peroxiredoxines. N-ethylmaleimide, modifier for cysteine residues, had no effect on antioxidant activity of the dithiothreitol-treated 28 kD protein but irreversibly inhibited activity of the 23 kD protein under reducing conditions. The 28 kD protein was sensitive to treatment with peroxides: t-butyl hydroperoxide at micromolar concentrations was shown to irreversibly inactivate 28 kD protein. In the presence of dithiothreitol, the lower level of peroxide concentrations was required to inhibit 28 kD protein activity. The mechanism of this effect may be mediated through conversion of sulfhydryl group of 46Cys to oxidized states (46Cys-SO2H and 46Cys-SO3H). Antioxidant property of 23 kD protein was impaired by t-butyl hydroperoxide only in the presence of dithiothreitol. The concentrations of t-butyl hydroperoxide needed to affect the 23 kD protein were at least one order of magnitude higher than were required for the 28 kD protein inhibition. The given results suggest the essential differences between catalytic site of 28 kD protein and that of 2-Cys peroxiredoxins.
Asunto(s)
Mucosa Olfatoria/metabolismo , Peroxidasas/metabolismo , Animales , Catálisis , Etilmaleimida/farmacología , Peroxirredoxinas , Ratas , Ratas WistarRESUMEN
Microwave radiation decreased specific camphor binding to a membrane fraction of rat epithelium but not to a Triton X-100 extract of this fraction. Inhibition of the ligand binding did not depend on the modulation frequency of the microwave field in the region 1-100 Hz and was not a linear function of specific absorption rate (SAR). The decreased ligand binding was due to a shedding or release of the specific camphor-binding protein from the membrane into solution. It is highly probable that several other membrane proteins may be shed into solution during microwave exposure.
Asunto(s)
Alcanfor/metabolismo , Microondas , Mucosa Olfatoria/efectos de la radiación , Animales , Femenino , Técnicas In Vitro , Modelos Biológicos , Mucosa Olfatoria/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas EndogámicasRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/aislamiento & purificación , Odorantes , Mucosa Olfatoria/análisis , Células Receptoras Sensoriales/análisis , Aldehídos/metabolismo , Animales , Especificidad de Anticuerpos , Antígenos/inmunología , Alcanfor/metabolismo , Proteínas Portadoras/inmunología , Electroforesis en Gel de Poliacrilamida , Epitelio/análisis , Femenino , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Inmunoglobulina G/inmunología , Masculino , Peso Molecular , Mucosa Olfatoria/inmunología , Ratas , Ratas Endogámicas , Células Receptoras Sensoriales/metabolismo , Especificidad de la EspecieRESUMEN
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.