RESUMEN
Oligodendrocyte development is controlled by a number of survival and migratory factors. The present study shows that signaling of CXCR4 receptor by the chemokine CXCL12 regulates survival and migration of neural precursors (NP) as well as oligodendrocyte progenitors (OP). CXCR4 is expressed by E14 striatal NP and OP generated by neurospheres. In CXCR4-defective mice, the number of NP in neurosphere outgrowth was twofold less than in wild-type (WT) mice; NP radial cell migration was also decreased. In contrast, the addition of CXCL12 to WT NP increased radial migration from the sphere in a dose-dependent manner with a maximal response at 200 nM. When oligodendrocytes differentiated in neurosphere outgrowth, CXCR4 was downregulated. OP isolated from newborn brain coexpressed CXCR4 with platelet-derived growth factor receptor-alpha (PDGFR alpha) or chondroitin sulfate proteoglycan; receptor expression also decreased during differentiation in vitro. Neonatal OP showed a peak migratory response to 20 nM of CXCL12 in chemotactic chambers, a migration inhibited by a CXCR4 antagonist and anti-CXCL12 antibody. In the embryonic spinal cord, the number of OP-expressing PDGFR alpha was reduced more than twofold in CXCR4-defective mice compared with WT and the ratio of ventral to dorsal OP was significantly increased. This indicates a defect in OP survival and their dorsal migration from the ventral cord region, probably because CXCR4(-/-) OP are unable to respond to CXCL12 made by vascular endothelia and the pia mater. We propose that CXCR4 signaling regulate survival and outward chemotactic migration of OP during embryonic and postnatal CNS development.
Asunto(s)
Movimiento Celular/fisiología , Neuronas/metabolismo , Oligodendroglía/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismo , Animales , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Sistema Nervioso Central/citología , Sistema Nervioso Central/embriología , Sistema Nervioso Central/crecimiento & desarrollo , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Oligodendroglía/citología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
Stromal cell-derived factor 1 (SDF-1) is an alpha-chemokine that stimulates migration of haematopoietic progenitor cells and development of the immune system. SDF-1 is also abundantly and selectively expressed in the developing and mature CNS, as we show here. At embryonic day 15, SDF-1 transcripts were detected in the germinal periventricular zone and in the deep layer of the forming cerebral cortex. At birth, granule cells in the cerebellum and glial cells of the olfactory bulb outer layer showed an SDF-1 in situ hybridization signal that decreased progressively within the next 2 weeks. In other regions such as cortex, thalamus and hippocampus, SDF-1 transcripts detected at birth progressively increased in abundance during the postnatal period. SDF-1 protein was identified by immunoblot and/or immunocytochemistry in most brain regions where these transcripts were detected. SDF-1 was selectively localized in some thalamic nuclei and neurons of the fifth cortical layer as well as in pontine and brainstem nuclei which relay the nociceptive response. The presence of SDF-1 transcripts in cerebellar granule cells was correlated with their migration from the external to the inner granular layers with disappearance of the signal when migration was completed. In contrast, SDF1 mRNA signal increased during formation of the hippocampal dentate gyrus and stayed high in this region throughout life. The selective and regulated expression of SDF-1 in these regions suggests a role in precursor migration, neurogenesis and, possibly, synaptogenesis. Thus this alpha chemokine may be as essential to nervous system function as it is to the immune system.
Asunto(s)
Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Sistema Nervioso Central/embriología , Sistema Nervioso Central/crecimiento & desarrollo , Quimiocinas CXC/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Animales , Tipificación del Cuerpo/fisiología , Sistema Nervioso Central/metabolismo , Quimiocina CXCL12 , Quimiocinas CXC/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Transcripción Genética/fisiologíaRESUMEN
CXCR4 is the Gi protein-linked seven-transmembrane receptor for the alpha chemokine stromal cell-derived factor 1 (SDF-1), a chemoattractant for lymphocytes. This receptor is highly conserved between human and rodent. CXCR4 is also a coreceptor for entry of human immunodeficiency virus (HIV) in T cells and is expressed in the CNS. To investigate how these CXCR4 ligands influence CNS development and/or function, we have examined the expression and signalling of this chemokine receptor in rat neurons and astrocytes in vitro. CXCR4 transcripts and protein are synthesized by both cell types and in E15 brain neuronal progenitors. In these progenitors, SDF-1, but not gp120 (the HIV glycoprotein), induced activation of extracellular signal regulated kinases (ERKs) 1/2 and a dose-dependent chemotactic response. This chemotaxis was inhibited by Pertussis toxin, which uncouples Gi proteins and the bicyclam AMD3100, a highly selective CXCR4 antagonist, as well as by an inhibitor of the MAP kinase pathway. In differentiated neurons, both SDF-1 and the glycoprotein of HIV, gp120, triggered activation of ERKs with similar kinetics. These effects were significantly inhibited by Pertussis toxin and the CXCR4 antagonist. Rat astrocytes also responded to SDF-1 signalling by phosphorylation of ERKs but, in contrast to cortical neurons, no kinase activation was induced by gp120. Thus neurons and astrocytes can respond differently to signalling by SDF-1 and/or gp120. As SDF-1 triggers directed migration of neuronal progenitors, this alpha chemokine may play a role in cortex development. In differentiated neurons, both natural and viral ligands of CXCR4 activate ERKs and may therefore influence neuronal function.
Asunto(s)
Astrocitos/fisiología , Quimiocinas CXC/fisiología , Proteína gp120 de Envoltorio del VIH/farmacología , Neuronas/fisiología , Receptores CXCR4/fisiología , Animales , Astrocitos/citología , Células Cultivadas , Corteza Cerebral/fisiología , Quimiocina CXCL12 , Quimiocinas CXC/genética , Quimiocinas CXC/farmacología , Quimiotaxis , Embrión de Mamíferos , Sustancias de Crecimiento/fisiología , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/citología , Células PC12 , Ratas , Ratas Sprague-Dawley , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Madre/citología , Células Madre/fisiología , Transcripción GenéticaRESUMEN
To determine the time and site of origin of the oligodendrocyte lineage in the developing human spinal cord, we have examined tissues from 45 to 83 d postconception (dpc) using sets of probes and antibodies recognizing oligodendrocyte-specific glycolipids, transcripts, and proteins. We found that two clusters of oligodendrocyte precursors appear on or before 45 dpc on each side of the cord ventral ependyma above the floor plate. These precursors express glycolipids recognized by the O4 and Rmab antibodies, platelet-derived growth factor alpha-receptor, myelin basic protein (MBP), and 2', 3'-cyclic nucleotide 3' phosphodiesterase as well as MBP and proteolipid transcripts. Expression of the morphogen sonic hedgehog was detected in the floor plate at 45 dpc and decreased at 58 dpc. During this period, oligodendrocyte precursors emerged in the ventral and lateral region of the forming white matter, a process occurring first in cervical and later in lumbar cord. The majority of O4(+) cells express the proliferating cell nuclear antigen (PCNA), and their pattern of dispersion suggests that these cells progressively populate the lateral and dorsal cord regions. Oligodendrocytes expressing galactocerebroside appeared at 53 dpc and did not express PCNA. Oligodendrocyte precursors were detected in dorsal cord regions at 74 dpc and at 83 dpc when myelination started in the ventral roots. Thus, oligodendrocyte precursors expressing myelin transcripts and proteins emerge in the ventral region of the embryonic cord several weeks before myelination.
Asunto(s)
Oligodendroglía/citología , Línea Celular , Embrión de Mamíferos/citología , Desarrollo Embrionario y Fetal , Expresión Génica , Humanos , Vaina de Mielina/fisiología , Oligodendroglía/metabolismo , Oligodendroglía/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Células Madre/citología , Células Madre/metabolismo , Células Madre/fisiologíaRESUMEN
The nucleotide sequence of the gene encoding in the major capsid protein (MCP) of frog virus 3 (FV3) has been determined and compared to other iridovirus capsid genes. Nucleotide sequence and S1 nuclease analysis showed that the FV3 MCP gene encoded a transcript of 1452 nucleotides containing a 12 nucleotide AU-rich 5' nontranslated region (NTR) and a 50-nucleotide 3' NTR whose terminus was predicted to fold into a hairpin of moderate stability. An open reading frame initiating from the the 5'-most AUG codon encoded a protein of 463 amino acids with a predicted molecular weight of 49,860. Expression of the putative FV3 MCP gene in vitro confirmed that it encoded the major capsid protein of FV3 and supported the suggestion that translation initiated at AUG-1. Pairwise amino acid alignments detected a high degree of sequence identity between the FV3 MCP and other iridoviruses. These results indicate that iridoviruses possess an evolutionarily related major capsid protein and provide information useful not only in studies of viral gene expression, but also in characterizing newly isolated iridoviruses.
Asunto(s)
Proteínas de la Cápside , Cápside/genética , Iridovirus/genética , Secuencia de Aminoácidos , Animales , Anuros , Secuencia de Bases , Cápside/química , Clonación Molecular , ADN Viral/genética , Expresión Génica , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.
Asunto(s)
Adhesinas Bacterianas , Antígenos Bacterianos/genética , Adhesión Bacteriana , Proteínas Bacterianas/genética , Genes Bacterianos , Mycoplasma/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos de Superficie/química , Antígenos de Superficie/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Genes Bacterianos/inmunología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , SolubilidadRESUMEN
Genes encoding enzymes involved in the salvage pathway for nucleosides have been cloned and sequenced from the mollicute Mycoplasma pirum. One of them, encoding deoxyriboaldolase, was functionally identified by complementation of an Escherichia coli mutant. These genes are clustered, suggesting an operon organization, and they are immediately followed by the putative gene for the triose phosphate isomerase, an enzyme used during glycolysis.
Asunto(s)
Genes Bacterianos/genética , Mycoplasma/genética , Nucleósidos/metabolismo , Fosfotransferasas (Fosfomutasas) , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Citidina Desaminasa/genética , Escherichia coli/genética , Isomerasas/genética , Datos de Secuencia Molecular , Mycoplasma/enzimología , Purina-Nucleósido Fosforilasa/genética , Homología de Secuencia de Aminoácido , Timidina Fosforilasa/genética , Triosa-Fosfato Isomerasa/genéticaRESUMEN
Point mutation in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaden their substrate spectrum towards all beta-lactams except cephamicins and imipenem. We describe here hybridization techniques for the detection of point mutations by non-radioactive oligonucleotide probes with plasmid DNA carrying bla T genes immobilized in polystyrene microwells. After hybridization in discriminating conditions with corresponding biotinylated oligonucleotide probes, the hybrids were detected by using a streptavidin-alkaline phosphatase conjugate and a fluorogenic substrate, 4-methylumbelliferyl-phosphate. The adsorption of DNA to microwells used in the present work was found to be independent of Mg2+ and Na+ concentrations. By this method, less than 3 fmols of target DNA were sufficient for the detection of point mutation.
Asunto(s)
Enterobacteriaceae/genética , Fluorometría/métodos , Genes Bacterianos/genética , Mutación/genética , Hibridación de Ácido Nucleico , Adsorción , Secuencia de Bases , ADN Bacteriano/química , Datos de Secuencia Molecular , Sondas de OligonucleótidosRESUMEN
Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The new technology of non-radioactive probes (cold probes) allows the routine use of this method outside of the specialized molecular biology laboratory. Hybridization makes use of a nucleic acid probe to detect a complementary nucleic acid target present in biological fluid or in a biopsy tissue. Hybridization leads to the formation of a double-stranded molecule called hybrid or duplex, which can be detected with a great sensitivity by using high-energy radioisotope. However, the use of radioisotope labeled probes is limited by the short half-life of the isotope, radiolysis of the probe and the radioactive hazards. In order to overcome autoradiography delay and the drawbacks of techniques employing radioisotopes, various non-radioactive labels have been proposed. Labeling and detection systems are currently designed in two ways: The label molecule can be attached directly to the DNA probe (direct labelling) or it can be attached to a molecule which binds either to a modified probe or specifically to the duplex (indirect labeling). Various substances has been used to label directly a nucleic acid probe. The assay detection limit depends largely on the detection limit of the label, therefore, probe assay based on label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). In the indirect labeling, the probe cannot be detected alone; rather, it requires the addition of a detection system.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
ADN/análisis , Hibridación de Ácido Nucleico , Sondas de Ácido Nucleico , ARN/análisis , Humanos , Sensibilidad y EspecificidadRESUMEN
Point mutations in the nucleotide sequence of the structural genes for the TEM-type penicillinases can broaded their substrate spectrum towards all beta-lactams except cephamycins and imipenem. The presence of such variants on self-transferable plasmids accounts for the dissemination of this new type of resistance to numerous species of Enterobacteriaceae in various countries. We have synthetized biotinylated oligonucleotide probes for the detection and the discrimination of parental and mutated nucleotide sequences of TEM enzymes. Seven clinical isolates belonging to four species and harbouring TEM-1, TEM-3 or TEM-6 were studied. The results obtained indicate that detection of TEM-derived broad spectrum beta-lactamases in clinical isolates of Entero-bacteriaceae is possible with biotinylated oligonucleotide probes.
Asunto(s)
Enterobacteriaceae/genética , beta-Lactamasas/genética , Secuencia de Bases , Enterobacteriaceae/enzimología , Genes Bacterianos , Datos de Secuencia Molecular , Mutación , Sondas de OligonucleótidosRESUMEN
The detailed organization of the RNAs transcribed from a region of the FV 3 genome (Sa/I-F fragment and adjacent sequences) has been determined. The information was derived from the cell-free translation of hybrid-selected RNA to locate the genes encoding specific polypeptides, RNA filter hybridization to size the transcripts, and S1 nuclease mapping to locate the 5'- and 3'-ends of the RNAs on the genome. Three genes are contiguous and are transcribed from the same strand: two immediate early genes encoding transcripts of about 1.3 kb that directed the in vitro synthesis of 42K and 46K polypeptides, separated by the late gene encoding the major capsid protein (48K). At an advanced stage in infection, transcripts derived from the immediate early genes are also present. A set of RNAs with different 5'-ends ranging from 1.7 to 0.58 kb is produced from the p46 gene region whereas RNAs, 0.98 and 0.6 kb in size, complementary to the 5'-end of the p42 message, are synthesized. This gene cluster is located between two genes transcribed in the opposite direction from the rightward-reading strand: a late gene whose message is 0.5 kb in size and encodes a 15K polypeptide and a gene transcribed at immediate early and late times of infection which encodes a protein of 70 kDa. The 5'-end of the late RNA maps downstream of the 5'-end of the early one, their sizes being 1.85 and 2 kb, respectively, but both of them can be translated in vitro into a 70K polypeptide. These observations suggest that transcription is not regulated by the organization of the genes; they suggest rather that specific DNA sequences are responsible for the promotion of immediate early and late transcriptions.
Asunto(s)
Genes Virales , Iridoviridae/genética , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , ADN Viral/genética , Regulación de la Expresión Génica , Transcripción GenéticaRESUMEN
To test whether the promoters of two immediate-early genes from frog virus 3 were similar in nucleotide sequence, we have cloned and sequenced an immediate-early gene encoding an infected-cell mRNA of 489 kilodaltons (ICR489) and have shown that the protein product of this gene is approximately 46 kilodaltons. The 5' and 3' ends of the transcripts from this gene, as determined by mung bean nuclease analysis, were microheterogeneous. The promoter region was subcloned upstream from a promoterless chloramphenicol acetyltransferase gene, forming the recombinant plasmid pBS489CAT. As with the previously sequenced frog virus 3 immediate-early gene encoding ICR169, expression of chloramphenicol acetyltransferase in transfected cells required activation by a virion-associated protein. Although the promoter region of the gene encoding ICR489 contained TATA, CAAT, and GC motifs similar to those of typical eucaryotic promoters, it showed no significant homology to the ICR169 promoter, indicating that the concomitant temporal expression of these two genes is not due to similar promoter sequences.
Asunto(s)
Genes Virales , Iridoviridae/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas Virales/genética , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Enzimas de Restricción del ADN , ADN Viral/genética , ADN Viral/ultraestructura , Regulación de la Expresión Génica , Microscopía Electrónica , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Viral/genética , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
A library of cloned fragments representing nearly the entire frog virus 3 (FV 3) genome (99.65%) has been constituted. Individual plasmid recombinants, labeled by nick-translation, were hybridized to Southern blots of genomic FV 3 DNA fragments obtained with XbaI, HindIII, SmaI, and SalI. From these results physical maps were generated and the distribution of restriction sites in the genome was established by double digestion of the fragments. A preliminary translational map was likewise developed. The viral messages were selected by hybridization to the recombinant DNAs immobilized on nitrocellulose filters and were translated in the reticulocyte cell-free system. About 30 polypeptides were detected among the translation products of RNA synthesized in the presence of cycloheximide. It appears that these genes are not clustered but in several cases more than one polypeptide is encoded by a given fragment. The 15 new polypeptide obtained by translation of late mRNAs derive from genes located on one-half of the genome.
Asunto(s)
Virus ADN/genética , Genes Virales , Ranidae/microbiología , Proteínas Virales/genética , Animales , Clonación Molecular , Cicloheximida/farmacología , Enzimas de Restricción del ADN , Peso Molecular , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genéticaRESUMEN
The gene encoding the major capsid polypeptide (MCP 48) of frog virus 3 (FV 3) has been mapped on the viral DNA. Late FV 3 messenger RNA, hybrid-selected by the SalI-F fragment or a subset of these sequences, BamHI-L and -W fragments, directed the synthesis in vitro of a 48 000 mol. wt. (48K) polypeptide. This product was recognized by monospecific antibodies raised against the major capsid polypeptide. The RNA complementary to these DNA sequences was about 1350 nucleotides in size. This transcript, encoding MCP 48, was precisely located; S1 nuclease analysis indicated that its 5' end mapped at 1250 nucleotides to the right and its 3' end at 160 nucleotides to the left of the BamHI site at the junction between the BamHI-W and -L fragments.