RESUMEN
The polypyrimidine tract-binding protein-associated splicing factor (PSF), which plays an essential role in mammalian spliceosomes, has been found to be expressed by differentiating neurons in developing mouse brain. The sequence of a fragment of mouse PSF was found to be remarkably similar to that of human PSF. Both the expression of PSF mRNA in cortex and cerebellum and PSF immunoreactivity in all brain areas were high during embryonic and early postnatal life and almost disappeared in adult tissue, except in the hippocampus and olfactory bulb where various neuronal populations remained PSF-immunopositive. Double-labeling experiments with anti-PSF antibody and anti-neurofilaments or anti-glial fibrillary acidic protein antibodies on sections of cortex, hippocampus, and cerebellum indicate that PSF is expressed by differentiating neurons but not by astrocytic cells. In vitro, mouse PSF was found to be expressed by differentiating cortical and cerebellar neurons. Radial glia or astrocyte nuclei were not immunopositive; however, oligodendrocytes differentiating in vitro were found to express PSF. The restricted expression of PSF suggests that this splicing factor could be involved in the control of neuronal-specific splicing events occurring at particular stages of neuronal differentiation and maturation.
Asunto(s)
Neuronas/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular , Cerebelo/citología , Cerebelo/embriología , Cerebelo/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Oligodendroglía/metabolismo , Factor de Empalme Asociado a PTB , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/aislamiento & purificaciónRESUMEN
Using the whole-cell patch-clamp technique, we demonstrate glycine-induced currents in oligosphere-derived oligodendrocyte progenitors cultured from newborn rats. Similar inward currents are also triggered by beta-alanine and taurine, two established glycine receptor agonists. In our recording conditions, glycine-gated currents in oligodendrocyte progenitors reverse about 0 mV and are reversibly inhibited by the glycine competitive antagonist strychnine, the Cl- channel blocker picrotoxinin and the non-competitive antagonist cyanotriphenylborate. The oligodendrocyte progenitors glycine receptor (GlyR) differs from the corresponding neuronal receptor: [3H]strychnine binding data and the strychnine inhibition curve of glycine-induced currents in oligodendrocyte progenitor cultures suggest the existence of two strychnine binding sites on the oligodendroglial GlyR. Using total RNA isolated from oligodendrocyte progenitors cultures, reverse transcription-polymerase chain reaction analysis of glycine receptor subunit expression shows the presence of alpha2 and beta subunits and immunocytochemical stainings confirm that this GlyR contains an alpha subunit which is not alpha1. The molecular structure of the oligodendroglial GlyR could be either homopentameric alpha2 or heteromeric alpha2beta but in both cases, the sequence of the alpha2 or beta subunits have to be different from the known neuronal sequences in order to explain, respectively, the cyanotriphenylborate (alpha2) and picrotoxinin (beta) sensitivities. This work thus demonstrates that GlyR are expressed by oligodendrocytes obtained not only from spinal cord but also from supraspinal structures. The pharmacological properties and presumably the molecular structure of oligodendroglial GlyR are original. The physiological meaning of the presence of such receptors on developing and mature oligodendrocytes remains unknown.
Asunto(s)
Corteza Cerebral/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , Receptores de Glicina/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión , Células Cultivadas , Corteza Cerebral/citología , Canales de Cloruro/fisiología , Glicinérgicos/metabolismo , Inmunohistoquímica , Neuronas/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Oligodendroglía/fisiología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Ratas , Receptores de Glicina/efectos de los fármacos , Receptores de Glicina/fisiología , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/metabolismo , Estricnina/metabolismo , Células Tumorales CultivadasRESUMEN
RT-PCR was used to assay for growth factors and receptors from seven different protein families in cochlea tissues of the juvenile rat. There was a broad representation of the growth factor families in all the cochlea tissues examined, though the organ of Corti and stria vascularis expressed a greater variety than the spiral ganglion. This broad expression suggests that a variety of known growth factors play significant roles in the development, maintenance, and repair of the inner ear. The results of this survey serve as a basis for the design of future in vitro experiments that will address the ability of growth factors to protect hair cells from damage and to evoke a repair-regeneration response by injured hair cells.
Asunto(s)
Cóclea/química , Sustancias de Crecimiento/biosíntesis , Receptores de Factores de Crecimiento/biosíntesis , Animales , Secuencia de Bases , Factor Neurotrófico Ciliar , Cartilla de ADN/química , Factor de Crecimiento Epidérmico/análisis , Factores de Crecimiento de Fibroblastos/análisis , Factor Neurotrófico Derivado de la Línea Celular Glial , Sustancias de Crecimiento/análisis , Células Ciliadas Auditivas/crecimiento & desarrollo , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/análisis , Proteínas del Tejido Nervioso/análisis , Neuronas Aferentes/química , Factor de Crecimiento Derivado de Plaquetas/análisis , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Receptores de Factores de Crecimiento/análisis , Somatomedinas/análisis , Ganglio Espiral de la Cóclea/química , Factor de Células Madre/análisis , Estría Vascular/químicaRESUMEN
Using whole-cell patch-clamp techniques, we show that oligosphere-derived oligodendrocyte progenitor cells (OP) display GABA-, glutamate-, 5-HT-, glycine- and acetylcholine-gated inward currents. When OP differentiate into oligodendrocytes (ODC), the amplitude of peak currents elicited by saturating concentrations of these transmitters decreases except for 5-HT. Intracellular Ca2+ concentration changes induced by microperfusion of glutamate, 5-HT, TRH, met-enkephalin and substance P were monitored using a fluo-3-based calcium imaging system. When OP cells differentiate into ODC, a global decrease of the proportion of responding cells is observed. During type-2 astrocytes commitment, this proportion decreases for 5-HT, TRH- and metenkephalin stimulations whereas it remains constant for substance P and glutamate. These data demonstrate a development regulation of neurotransmitter- and neuropeptide-induced responses within the oligodendroglial lineage.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Neurotransmisores/farmacología , Oligodendroglía/efectos de los fármacos , Acetilcolina/farmacología , Animales , Animales Recién Nacidos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Ácido Glutámico/farmacología , Glicina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Serotonina/farmacología , Células Tumorales Cultivadas , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Various assays of classical PEG-assisted transformation as well as electrotransformation of Streptomyces parvulus IMET41380 and Streptomyces vinaceus NCIB8852 are described. Contrary to the so far reported assays of electrotransforming Streptomyces strains, electroporation in S. parvulus and S. vinaceus was carried out on intact cells, without any lysozyme treatment. In these two strains, the classical PEG-assisted transformation of protoplasts does not work efficiently (10(3) to 10(4) transformants per micrograms of pIJ702 DNA) and electrotransformation gives 10 to 100 times higher yields (10(5) transformants per micrograms of pIJ702 DNA). The electroporation method described here is not applicable to other Streptomyces strains (S. lividans or S. coelicolor).
Asunto(s)
Electroporación , Streptomyces/genética , Transformación Bacteriana , Muramidasa/farmacología , Polietilenglicoles/farmacologíaRESUMEN
We analysed how interactions between protein kinase-dependent intracellular signalling pathways were implicated in the control of the production of tissue-type plasminogen activator (tPA) and the generation of neurite outgrowth by PC12 cells. To that aim, cells were treated with agents that interact with the trk receptor and with protein kinases A and C. Nerve growth factor induced only the formation of large neurites. The release of the protease and the production of short neurite outgrowth were found to be protein-kinase-A-dependent events that could be enhanced by simultaneous activation of protein kinase C with phorbol ester. At high concentration, staurosporine, a nonselective inhibitor of protein kinases, induced the production of short neurites and mimicked the protein-kinase-A-dependent effect on tPA release. Such a response was not observed with K-252a, an analogue of staurosporine devoid of neurite-outgrowth-promoting activity. The responses to protein kinase A stimulation and the addition of staurosporine, although similar, seemed to occur through an activation of distinct, yet interacting, signalling pathways. In conclusion, tPA release and large neurite outgrowth from PC12 cells are controlled by parallel, albeit interacting, pathways, suggesting that these two potentially antagonistic events in PC12 cell differentiation can be modulated in a concerted way or independently of each other, depending on the activity of several protein kinases.
Asunto(s)
Neuritas/efectos de los fármacos , Activadores Plasminogénicos/metabolismo , Proteínas Quinasas/metabolismo , Estaurosporina/farmacología , Animales , Bucladesina/farmacología , Diferenciación Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Factores de Crecimiento Nervioso/farmacología , Neuritas/metabolismo , Neuritas/ultraestructura , Células PC12 , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas , Ratas , Transducción de Señal , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/metabolismoRESUMEN
The alignment of the promoter region of several Streptomyces xylanases shows three conserved sequences which could be involved in gene regulation. By electromobility shift assays these specific sequences, present only in Streptomyces xylanolytic strains, were identified as protein-binding sites. The sequence required for efficient recognition by the retarding protein appeared to be a 4-bp inverted repeat: 5'-CTTT-Nx-AAAG-3'. The DNA-protein affinity was influenced by the culture conditions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Bases , Sitios de Unión/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Genes Bacterianos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/genéticaRESUMEN
Using the p1J702 vector, a xylanase-encoding gene (xin) of Streptomyces sp. EC3 has been cloned by functional complementation of a mutant of Streptomyces lividans TK24, producing xylanase at a very low level. Normal level of xylanase synthesis was restored in at least three clones, containing the same 3802 bp Sstl DNA fragment. In this fragment, several open reading frames (ORFs) have been identified, one of which coded for a xylanase; the products of the other ORFs did not show homology with any of the already known proteins. The complete nucleotide sequence of the 3802 bp Ssti insert has been determined on both strands. Xylanase is very probably synthesized as a 240 amino acid (aa) precursor (25949 Da) including a long (49 aa) signal sequence presenting significant similarity with the signal sequences of other Streptomyces xylanase genes. The xylanase aa sequence showed a clear homology with the aa sequences of other xylanases of the glycanase G family. The xln gene has been introduced into Streptomyces parvulus, a naturally xylanase-negative species. In contrast with its expression in Streptomyces sp. EC3, in S. parvulus, xln was expressed constitutively, a probable consequence of the absence of a regulatory system.