RESUMO
The capacity for long-distance migration of the oligodendrocyte precursor cell, oligodendrocyte-type 2 astrocyte (O-2A), is essential for myelin formation. To study the molecular mechanisms that control this process, we used an in vitro migration assay that uses neurohypophysial explants. We provide evidence that O-2A cells in these preparations express functional N-methyl-D-aspartate (NMDA) receptors, most likely as homomeric complexes of the NR1 subunit. We show that NMDA evokes an increase in cytosolic Ca2+ that can be blocked by the NMDA receptor antagonist AP-5 and by Mg2+. Blocking the activity of these receptors dramatically diminished O-2A cell migration from explants. We also show that NMDA receptor activity is necessary for the expression by O-2A cells of the highly sialylated polysialic acid-neural cell adhesion molecule (PSA-NCAM) that is required for their migration. Thus, glutamate or glutamate receptor ligands may regulate O-2A cell migration by modulating expression of PSA-NCAM. These studies demonstrate how interactions between ionotropic receptors, intracellular signaling, and cell adhesion molecule expression influence cell surface properties, which in turn are critical determinants of cell migration.
Assuntos
Moléculas de Adesão de Célula Nervosa/metabolismo , Oligodendroglia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Ácidos Siálicos/metabolismo , Cálcio/metabolismo , Movimento Celular , Células Cultivadas , Humanos , Oligodendroglia/citologia , Técnicas de Patch-Clamp , Neuro-Hipófise/citologia , RNA/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/metabolismo , Células-Tronco/citologiaRESUMO
Ii, a 31,000 mol. wt polypeptide chain associated with murine and human Ia antigens was investigated for its labeling pattern, carbohydrate content and structural polymorphism. Two-dimensional gel electrophoretic analysis of tunicamycin treated cells from mouse and human lymphocytes shows that Ii contains two N-linked carbohydrate chains. Ii is a methionine rich polypeptide. Tryptic and chymotryptic two dimensional peptide maps of Ii chain associated with I-A and I-E subregion products are identical. This absence of polymorphism holds true when Ii chain is isolated from different mouse haplotypes. Human Ii chains from different HLA-DR types appear also invariant by peptide map analysis. By molecular weight, carbohydrate content, charge and tryptic and chymotryptic maps criteria, Ii of mouse and human are strikingly homologous.
Assuntos
Antígenos de Histocompatibilidade Classe II , Peptídeos , Animais , Carboidratos/análise , Fenômenos Químicos , Química , Quimotripsina , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoeletroforese Bidimensional , Camundongos , Polimorfismo Genético , TripsinaRESUMO
The complete amino acid sequence of the human neurokinin-3 receptor was deduced by DNA sequence analysis of human genomic fragments. Comparison of the predicted primary structure with those for the human neurokinin receptors 1 and 2 shows a highly conserved pattern of seven hydrophobic regions with maximum divergence occurring at the amino- and carboxy-termini. The position of intron-exon junctions are identical to those in other reported neurokinin genes. Using a chimeric genomic-cDNA gene, the human NK-3 receptor was expressed in Xenopus laevis oocytes where it mediates membrane conductance changes in response to its agonist, neurokinin B. More significantly, expression of the gene in mammalian cells resulted in detection of receptor binding as well as neurokinin-stimulated calcium mobilization and arachidonic acid release, all displaying the pharmacological characteristics expected of a neurokinin-3 receptor. By using the polymerase chain reaction we have shown that mRNA for the human neurokinin-3 receptor is expressed predominantly in the central nervous system.
Assuntos
Neurocinina A/metabolismo , Receptores de Neurotransmissores/genética , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , DNA , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores da Neurocinina-2 , Receptores de Neurotransmissores/metabolismo , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Xenopus laevisRESUMO
Selective death of magnocellular vasopressinergic neurons in the hypothalamus has been reported in cases of hereditary and idiopathic diabetes insipidus and after experimental lesions of the hypothalamo-neurohypophyseal pathway. To identify trophic factors that promote survival of these neurons, an in vitro model system was established in which organotypic cultures of the rat hypothalamic paraventricular nucleus were maintained in chemically-defined medium. We observe that the majority of magnocellular vasopressinergic neurons die in these cultures, while other cell populations such as corticotrophin-releasing factor producing parvicellular and oxytocin producing magnocellular cells retain a well preserved cytoarchitectonic organization. Degenerating vasopressinergic cells exhibit morphological signs of apoptosis and stained positively when analysed by the terminal deoxynucleotidyl transferase biotinylated dUTP nick end-labelling assay. Partial survival of vasopressinergic neurons occurred after co-culturing the paraventricular nucleus with neurohypophyseal explants, indicating that target-derived factors may be required for the survival of these neurons. Cell survival is dramatically increased by the administration of ciliary neurotrophic factor and leukemia inhibiting factor, but not by interleukin 6 or the members of the neurotrophin family. Reverse transcription-polymerase chain reaction followed by Southern analysis shows the presence of ciliary neurotrophic factor messenger RNA in the neurohypophysis. Thus, endogenous ciliary neurotrophic factor and leukemia inhibiting factor, produced by neurohypophyseal cells may function as a physiological survival factor for neurosecretory vasopressinergic neurons.
Assuntos
Inibidores do Crescimento/genética , Interleucina-6 , Linfocinas/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Vasopressinas/genética , Animais , Apoptose/fisiologia , Southern Blotting , Química Encefálica/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Ventrículos Cerebrais/citologia , Fator Neurotrófico Ciliar , Expressão Gênica/fisiologia , Inibidores do Crescimento/análise , Marcação In Situ das Extremidades Cortadas , Fator Inibidor de Leucemia , Linfocinas/análise , Microscopia Eletrônica , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/análise , Neurônios/química , Neurônios/ultraestrutura , Sistemas Neurossecretores/química , Sistemas Neurossecretores/fisiologia , Núcleo Hipotalâmico Paraventricular/citologia , Neuro-Hipófise/química , Neuro-Hipófise/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasopressinas/análiseRESUMO
Amongst different intrinsic and extrinsic inputs, cholinergic striatal interneurons receive afferents from the dopaminergic nigrostriatal projection and from local collaterals of striatonigral cells containing substance P. The following study demonstrates that both dopamine D2 and substance P (neurokinin-1) receptors are expressed by a large proportion of cholinergic interneurons. Using in situ hybridization on triplet adjacent sections with radioactive probes specific for choline acetyltransferase, substance P receptor, and D2 receptor long-splicing form messenger RNAs, we show that these interneurons can be divided into four subpopulations in terms of substance P and D2 receptor expression. A majority of these neurons coexpress both receptors (76%), while other minor subpopulations express either one (respectively, 16% and 2%) or none of them (6%). Our results also show that substance P receptor is expressed by striatal neurons that are not cholinergic. These findings are in agreement with the concept that striatal cholinergic interneurons are heterogeneous in terms of input-output connections and the type of receptors expressed. Moreover, the presence of substance P and D2 receptors on a majority of these neurons is relevant to a putative role of cholinergic interneurons in several conditions such as various neurodegenerative disorders or antipsychotic drug administration, where substance P and dopamine inputs are modified.
Assuntos
Corpo Estriado/metabolismo , Neurônios/metabolismo , Sistema Nervoso Parassimpático/metabolismo , RNA Mensageiro/biossíntese , Receptores de Dopamina D2/biossíntese , Receptores de Neurotransmissores/biossíntese , Animais , Southern Blotting , Colina O-Acetiltransferase/biossíntese , Corpo Estriado/citologia , Hibridização In Situ , Técnicas In Vitro , Masculino , Oligonucleotídeos/metabolismo , Sistema Nervoso Parassimpático/citologia , Sondas RNA , Ratos , Ratos Sprague-Dawley , Receptores da Neurocinina-1RESUMO
We have reported previously that axotomy induced a marked increase of vasopressin receptor binding in the adult rat facial nucleus, suggesting an increased number of vasopressin receptors. These receptors were pharmacologically undistinguishable from peripheral V(1a) vasopressin receptors. In the present study, we show, using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR), that axotomy regulates the expression of the vasopressin V(1a) receptor mRNA in the facial nucleus. Results were obtained from adult male rats killed 1 week following crush of the right facial nerve. In situ hybridization was performed with a (35)S-labelled riboprobe. A specific hybridization signal was detected in both left and right facial nuclei, with a significantly higher intensity in the nucleus ipsilateral to the lesion. V(1a) receptor transcripts were found associated with large facial motoneuronal cell bodies, not with other cells present in the nucleus, i.e., glial or epithelial cells. RT-PCR analysis of unlesioned facial tissue revealed the presence of mRNAs encoding vasopressin V(1a), vasopressin V(1b) and oxytocin receptors, whereas only the V(1a) receptor mRNA was found to be increased following axotomy in the lesioned facial tissue. These data suggest that the axotomy-induced expression of vasopressin receptors in the rat facial nucleus is due, at least to a large extent, to an increase of the V(1a) vasopressin receptor mRNA in facial motoneurons.
Assuntos
Encéfalo/metabolismo , Nervo Facial/fisiologia , Fígado/metabolismo , Neurônios/metabolismo , Receptores de Vasopressinas/genética , Transcrição Gênica , Regulação para Cima , Animais , Arginina Vasopressina/metabolismo , Axotomia , Primers do DNA , Traumatismos do Nervo Facial , Masculino , Compressão Nervosa , Ocitocina/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Ocitocina/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vasopressinas/genéticaRESUMO
Mouse normal bone marrow pre-B lymphocytes synthesize only membrane mu chains (micron), as shown by mRNA studies and peptide analysis. The micron chains exist in two forms: free micron chains assembled into dimers, or L chain-bound micron chains present in IgM monomers (in the case of 'late pre-B cells', i.e., after productive L chain gene rearrangement). These two forms of molecules are very different in properties, fate and intracellular pathways. Free but not L chain-bound mu chains are highly susceptible to mild proteolysis, which degrades their entire Cmu 1 and VH domains. Free mu chains are rapidly degraded within the lysosomal compartment, which they reach via the cis, avoiding the trans, part of the Golgi complex. In contrast, as soon as mu chains bind to L chains, they are directed towards the 'trans' Golgi compartment, where they undergo terminal glycosylation, then to the cell surface, where they progressively accumulate. It is suggested that the conformation instability of the Cmu 1 and VH domains of the free mu chains plays a critical role in the intracellular targeting of these molecules, as compared with that of L chain-bound mu chains.
Assuntos
Linfócitos B/imunologia , Medula Óssea/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias mu de Imunoglobulina/biossíntese , Proteínas de Membrana/biossíntese , Animais , Linfócitos B/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Compartimento Celular/efeitos dos fármacos , Células Cultivadas , Cloroquina/farmacologia , Cadeias mu de Imunoglobulina/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , Monensin/farmacologia , Peptídeo Hidrolases , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/genéticaRESUMO
A synthetic gene coding for human somatomedin C (SMC) was inserted into an Escherichia coli plasmid vector that contains the bacteriophage lambda pL promoter. Intracellular accumulation of the gene product after induction of the promoter was found to be low. A 200-fold greater yield was obtained with a similar plasmid containing two translationally fused copies of the SMC gene. A series of such tandem genes truncated at their 3' ends were generated with nuclease Bal 31. These gave intermediate expression levels that correlated with the expected sizes of their gene products. Comparison of RNAs extracted from cells containing either the monomer or tandem SMC gene constructions showed that there was no significant difference in expression at the transcriptional level. Pulse-chase experiments demonstrated that the tandem SMC protein was far more stable than the monomer SMC product.
Assuntos
Escherichia coli/genética , Regulação da Expressão Gênica , Genes Sintéticos , Fator de Crescimento Insulin-Like I/genética , Somatomedinas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Meia-Vida , Hibridização de Ácido Nucleico , Plasmídeos , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Transcrição GênicaRESUMO
Double-stranded DNA encoding the human hormone somatomedin-C (SMC) has been synthesized. This synthetic gene has been inserted into a plasmid bearing the strong leftward promoter (PL) of bacteriophage lambda and expressed in E. coli. Codons for the N-terminal region of SMC which maximized the hormone's synthesis were chosen in an SMC-lac z fusion assay. The amounts of SMC accumulated in E. coli were influenced by mutations at two chromosomal loci, lon and htpR.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Protease La , Serina Endopeptidases , Somatomedinas/genética , Proteases Dependentes de ATP , Clonagem Molecular , DNA Recombinante , Endopeptidases/genética , Regulação da Expressão Gênica , Genes , Genes Bacterianos , Proteínas de Choque Térmico/genética , Fator de Crescimento Insulin-Like I , Mutação , Conformação de Ácido Nucleico , Regiões Promotoras GenéticasRESUMO
Neurokinins are a family of neuropeptides with widespread distribution mediating a broad spectrum of physiological actions through three distinct receptor subtypes: NK-1, NK-2, and NK-3. We investigated some of the second messenger and cellular processes under control by the recombinant bovine NK-2 receptor stably expressed in Chinese hamster ovary cells. In this system the NK-2 receptor displays its expected pharmacological characteristics, and the physiological agonist neurokinin A stimulates several cellular responses. These include 1) transient inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization, 2) increased out put of arachidonic acid and prostaglandin E2 (PGE2), 3) enhanced cyclic AMP (cAMP) generation, 4) increased de novo DNA synthesis, and 5) an induction of the "immediate early" genes c-fos and c-jun. Although NK-2 receptor-mediated IP3 formation involves activation of a pertussis toxin-insensitive G-protein, increased cAMP production is largely a secondary response and can be at least partially attributed to autocrine stimulation by endogenously generated eicosanoids, particularly PGE2. This is the first demonstration that a single recombinant neurokinin receptor subtype can regulate, either directly or indirectly, multiple signal transduction pathways and suggests several potential important mediators of neurokinin actions under physiological conditions.
Assuntos
Neurocinina A/metabolismo , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais/fisiologia , Animais , Ácido Araquidônico/metabolismo , Células CHO/metabolismo , Cálcio/metabolismo , Bovinos , Cricetinae , AMP Cíclico/biossíntese , DNA/biossíntese , Dinoprostona/biossíntese , Receptores da Neurocinina-2 , Receptores de Neurotransmissores/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
We have synthesized 175-nucleotide-long probes for the DNA of human histocompatibility antigens HLA-DR alpha and beta by extending on poly(A)+ mRNA from B-cell lines with short synthetic deoxyribonucleotide primers complementary to the predicted nucleotide sequence of the NH2 terminus of both polypeptides. The synthesis of the probe for the alpha-chain DNA was a two-step process starting with 11-mers which were extended by dideoxynucleotide chain termination experiments to a 20-mer of predicted sequence. The synthesized 20-mer was then used to generate a 175-nucleotide cDNA probe which was shown to encode the appropriate amino acids for the alpha chain and was used to select a human genomic DNA clone containing the coding sequences for HLA-DR alpha. For the beta polypeptide an 18-mer homologous to the NH2-terminal sequence of a cDNA clone from another B-cell line was used to extend on poly(A)+ mRNA isolated from a B-cell line. Preliminary sequence analysis of a 175-base-long extension product indicates a match of the cDNA sequence to the published sequence of a clone for HLA-DR beta. Information from these extension experiments helps to establish the sensitivity and specificity of the primer extension method.