RESUMEN
First described in the developing nervous system, Semaphorin III/Neuropilin, Ephrin/Eph, and Delta/Notch signaling relays have now been implicated in the elaboration of the blood vessel network during embryogenesis.
Asunto(s)
Neovascularización Fisiológica/fisiología , Animales , Glicoproteínas/fisiología , Humanos , Proteínas de la Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/embriología , Neuropilina-1 , Receptores Notch , Semaforina-3A , Transducción de SeñalRESUMEN
Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.
Asunto(s)
Vectores Genéticos , Liposomas , Péptidos , Fosfatidiletanolaminas , Receptores de Fibronectina/metabolismo , Transfección/métodos , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , Córnea , Portadores de Fármacos , Humanos , Ligandos , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Péptidos/síntesis química , Péptidos/metabolismo , Compuestos de Amonio Cuaternario , Conejos , Proteínas Recombinantes de FusiónRESUMEN
The programmed cell death (apoptosis) of a proportion of the neurons which form plays a critical role in the development of the nervous system and ensures that the correct number of mature neurons are ultimately present. We show that the prior exposure of neuronal cells to an elevated temperature sufficient to induce the heat-shock response partially protects the cells from apoptotic cell death following subsequent transfer to serum-free medium. The degree of protection observed in experiments using different heat-shock or recovery times correlates with the extent of heat-shock protein synthesis. Similarly activation of heat-shock protein synthesis by inducers other than elevated temperature also results in protection from apoptosis. The mechanism by which the heat-shock proteins may protect neuronal cells from apoptosis is discussed.
Asunto(s)
Apoptosis , Proteínas de Choque Térmico/fisiología , Calor , Neuronas/citología , Animales , Medio de Cultivo Libre de Suero , Ganglios Espinales , Regulación de la Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Células Híbridas , Neuroblastoma , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
A prior mild thermal stress (heat shock) can protect neuronal cells against a subsequent exposure to either severe thermal stress or the induction of programmed cell death (apoptosis). By micro-injecting trigeminal ganglion neurons with expression constructs we show that over-expression of the individual heat shock proteins hsp70 and hsp90 can protect these cells against severe thermal stress but not against apoptosis. However, the protective effect of prior heat shock against subsequent apoptosis is dependent upon its ability to induce heat shock protein (hsp) synthesis rather than, for example, the inhibition of other protein synthesis associated with heat shock. The significance of these effects is discussed in terms of the role of different hsps in protecting neuronal cells from distinct stresses.
Asunto(s)
Muerte Celular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/farmacología , Proteínas HSP90 de Choque Térmico/farmacología , Factores de Crecimiento Nervioso/farmacología , Nervio Trigémino/efectos de los fármacos , Animales , Células Cultivadas , Ratones , Ratones EndogámicosRESUMEN
The extent of programmed cell death (apoptosis) which occurs upon transfer of a neuronal cell line (ND7) to serum-free medium can be greatly increased by addition of retinoic acid (RA) to the medium. Here we show that the degree of apoptosis can also be enhanced by agents which activate protein kinase C (PKC) and that such agents synergize with RA in inducing apoptosis. In contrast chronic down regulation of PKC dramatically reduces the ability of RA to induce apoptosis whilst ND7 cell lines selected for resistance to RA-induced apoptosis are also resistant to apoptosis induced by PKC activation. This indicates that a common death pathway mediates the induction of apoptosis by PKC activators and RA. The potential nature of this pathway and the role of PKC in neuronal cell apoptosis is discussed.
Asunto(s)
Apoptosis , Neuronas/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacología , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Activación Enzimática , Células Híbridas , Ratones , Concentración Osmolar , Proteína Quinasa C/metabolismo , RatasRESUMEN
Prior exposure to a mild thermal stress can protect neuronal cells from a subsequent more severe stress including high temperature, ischemia, glutamate toxicity, or stimuli inducing apoptosis. Although the protective effect of thermal stress correlates with the elevated expression of the heat shock proteins (hsps), the protective effect of individual hsps has never been directly demonstrated in neuronal cells. Here we show that the constitutive overexpression of either of the major hsps, hsp90 or hsp70, can protect neuronal cells from thermal stress but not from stimuli that induce apoptosis. The possible mechanisms by which thermal stress can protect neuronal cells from apoptosis are discussed.
Asunto(s)
Apoptosis , Proteínas HSP70 de Choque Térmico/farmacología , Proteínas HSP90 de Choque Térmico/farmacología , Calor , Neuronas/citología , Neuronas/fisiología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/fisiología , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transfección , Células Tumorales CultivadasRESUMEN
Delta-Notch signalling regulates cell-fate choices in a variety of tissues during development. We report the expression of Delta4 (D14) in arterial endothelium during mouse embryogenesis and in the endothelium of tumor blood vessels. The expression of D14 in the mouse begins at 8 dpc in the dorsal aortae, umbilical artery and the heart. Subsequent expression is restricted to smaller vessels and capillaries and is reduced in most adult tissues. However, it is high in the vasculature of xenograft human tumors in the mouse, in endogenous human tumors and is regulated by hypoxia. These data implicate D14 and the Notch signalling pathway in angiogenesis and suggest possible new targets for antiangiogenic tumor therapy.
Asunto(s)
Proteínas Sanguíneas/fisiología , Endotelio Vascular/fisiología , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Arterias/fisiología , Proteínas de Unión al Calcio , Drosophila , Proteínas de Drosophila , Femenino , Humanos , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Receptores Notch , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
We have cloned a Drosophila homologue (D-gsc) of the vertebrate homeobox gene goosecoid (gsc). In the Gsc proteins, the pressure for conservation has been imposed on the homeodomain, the functional domain of the protein: sequence homology is limited to the homeodomain (78% identity) and to a short stretch of 7 aminoacids also found in other homeoproteins such as Engrailed. Despite this weak homology, D-gsc is able to mimic gsc function in a Xenopus assay, as shown by its ability to rescue the axis development of a UV-irradiated embryo. Moreover, our data suggest that the position of insect and vertebrate gsc homologues within a regulatory network has also been conserved: D-gsc expression is controlled by decapentaplegic, orthodenticle, sloppy-paired and tailless whose homologues control gsc expression (for BMP4 and Otx-2), or are expressed at the right time and the right place (for XFKH1/Pintallavis and Tlx) to be interacting with gsc during vertebrate development. However, the pattern of D-gsc expression in ectodermal cells of the nervous system and foregut cannot easily be reconciled with that of vertebrate gsc mesodermal expression, suggesting that its precise developmental function might have diverged. Still, this comparison of domains of expression and functions among Gsc proteins could shed light on a common origin of gut formation and/or on basic cellular processes. The identification of gsc target genes and/or other genes involved in similar developmental processes will allow the definition of the precise phylogenetic relationship among Gsc proteins.
Asunto(s)
Proteínas de Unión al ADN/genética , Drosophila/genética , Genes Homeobox , Genes de Insecto , Proteínas de Homeodominio , Proteínas Represoras , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Drosophila/embriología , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica , Proteína Goosecoide , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Morfogénesis/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Distribución Tisular , XenopusRESUMEN
Transactivation of the herpes simplex virus (HSV) immediate-early (IE) genes is dependent upon the formation of a complex between the viral protein Vmw65 and the cellular transactivation factor Oct-1. Differentiation of the proliferating ND7 neuronal cell to a nondividing phenotype results in a large fall in the amount of Oct-1 to a level characteristic of nondividing neuronal cells but does not dramatically affect the level of IE gene expression following infection or the ability of Vmw65 to transactivate the IE promoter in transfection experiments. This suggests that the low levels of Oct-1 in nonproliferating neuronal cells do not play a key role in the failure of IE gene expression following initial infection of these cells, which is an essential step in the establishment of latent infections with HSV.
Asunto(s)
Proteínas de Unión al ADN/análisis , Simplexvirus/metabolismo , Factores de Transcripción/análisis , Activación Transcripcional , Proteínas Virales/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular , Regulación Viral de la Expresión Génica , Factor C1 de la Célula Huésped , Datos de Secuencia Molecular , Neuronas , Factor 1 de Transcripción de Unión a OctámerosRESUMEN
Goosecoid (Gsc) is a homeodomain protein expressed in the organizer region of vertebrate embryos. Its Drosophila homologue, D-Gsc, has been implicated in the formation of the Stomatogastric Nervous System. Although there are no apparent similarities between the phenotypes of mutations in the gsc gene in flies and mice, all known Gsc proteins can rescue dorsoanterior structures in ventralized Xenopus embryos. We describe how D-Gsc behaves as a transcriptional repressor in Drosophila cells, acting through specific palindromic HD binding sites (P3K). D-Gsc is a 'passive repressor' of activator homeoproteins binding to the same sites and an 'active repressor' of activators binding to distinct sites. In addition, D-Gsc is able to strongly repress transcription activated by Paired-class homeoproteins through P3K, via specific protein-protein interactions in what we define as 'interactive repression'. This form of repression requires the short conserved GEH/eh-1 domain, also present in the Engrailed repressor. Although the GEH/eh-1 domain is necessary for rescue of UV-ventralized Xenopus embryos, it is dispensable for ectopic induction of Xlim-1 expression, demonstrating that this domain is not required for all Gsc functions in vivo. Interactive repression may represent specific interactions among Prd-class homeoproteins, several of which act early during development of invertebrate and vertebrate embryos.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila/genética , Proteínas de Insectos/genética , Proteínas Represoras , Factores de Transcripción , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Secuencia Conservada , ADN/genética , Cartilla de ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Drosophila/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteína Goosecoide , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Fenotipo , Xenopus laevisRESUMEN
Apoptotic cell death plays a critical role in the development of the nervous system. The death of mature nondividing neurons that fail to receive appropriate input from the target field has been extensively studied. However, the mechanisms mediating the extensive cell death occurring in areas of the developing brain where proliferating neuroblasts differentiate into mature nondividing neurons have not been analyzed. We show here that the cell cycle arrest of a proliferating cell of neuronal origin by removal of serum results in either apoptotic cell death or differentiation to a mature nondividing neuronal cell. The proportion of cells undergoing death or differentiation is influenced in opposite directions by treatment of the cells with cyclic AMP and retinoic acid. This suggests that following the withdrawal of signals stimulating neuroblast cell division, neuronal cells either can cease to suppress a constitutive suicide pathway and hence die by apoptosis or, alternatively, can differentiate into a mature neuronal cell. Regulation of the balance between apoptosis and neuronal differentiation could therefore play a critical role in controlling the numbers of mature neurons that form.