RESUMEN
Despite the widespread use of nitric oxide as a signalling molecule in the central nervous system, the molecular makeup of its receptor, soluble guanylyl cyclase (sGC), therein is poorly understood. Accordingly, RT-PCR and in situ hybridization were used to identify sGC subunits expressed in rat brain. In addition to the expected mRNA for alpha 1 and beta1 subunits, message for the beta 2 subunit was detected in the cerebellum at all developmental stages investigated (1--150 days postnatum). The use of degenerate primers allowed the identification of mRNA coding for the rat alpha 2 subunit, which was also expressed at every age studied. All but beta 2 were detected by in situ hybridization in the brains of both 8-day-old and adult rats. The distribution patterns indicated that in some areas, e.g. caudate-putamen and nucleus accumbens, sGC probably exists mainly as the alpha 1 beta 1 heterodimer. In others, e.g. hippocampus and olfactory bulb, alpha 2 beta 1 is likely to be dominant. In the cerebellum, alpha 1 and beta 1 message was strong in the Purkinje cell layer but was not confined to Purkinje cells: smaller cells, presumed to be the Bergmann glia, were also labelled. In contrast, alpha 2 mRNA was concentrated in cerebellar granule cells. Western blotting indicated an excess of alpha 1 over beta 1 protein in the cerebellum, the reverse of what was found in the lung. It is concluded that, in molecular terms, sGC is likely to be more complex and exhibit more regional variation in the brain than previously thought. The functional consequences of this heterogeneity require investigation.
Asunto(s)
Química Encefálica/genética , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Western Blotting , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Guanilato Ciclasa/química , Hibridación in Situ , Masculino , Estructura Terciaria de Proteína , ARN Mensajero/análisis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SolubilidadRESUMEN
Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types--quiescent human CD34+ hematopoietic progenitor cells and dendritic cells--which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a single virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Simplexvirus/genética , Animales , Antígenos CD34/metabolismo , Encéfalo/metabolismo , Línea Celular , Separación Celular , Células Cultivadas , Cricetinae , Células Dendríticas/citología , Células Dendríticas/metabolismo , Citometría de Flujo , Marcadores Genéticos , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histocitoquímica , Humanos , Indicadores y Reactivos/metabolismo , Operón Lac/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía FluorescenteRESUMEN
Ser55 within the head domain of neurofilament light chain (NF-L) is a target for phosphorylation by protein kinase A. To understand further the physiological role(s) of NF-L Ser55 phosphorylation, we generated transgenic mice with a mutant NF-L transgene in which Ser55 was mutated to Asp so as to mimic permanent phosphorylation. Two lines of NF-L(Asp) mice were created and these animals express the transgene in many neurones of the central and peripheral nervous systems. Both transgenic lines display identical, early onset, and robust pathological changes in the brain. These involve the formation of NF-L(Asp)-containing perikaryal neurofilament inclusion bodies and the development of swollen Purkinje cell axons. Development of these pathologies was rapid and fully established in mice as young as 4 weeks of age. The two transgenic lines show no elevation of NF-L, neurofilament middle chain (NF-M), or neurofilament heavy chain (NF-H), and transgenic NF-L(Asp) represents only a minor proportion of total NF-L protein. Because other published transgenic lines expressing higher levels of wild-type NF-L do not exhibit phenotypic changes that in any way resemble those in the NF-L(Asp) mice and because the two different NF-L(Asp) transgenic lines display identical neuropathological changes, it is likely that the pathological alterations observed in the NF-L(Asp) mice are the result of properties of the mutant NF-L. These results support the notion that phosphorylation of Ser55 is a mechanism for regulating neurofilament organisation in vivo.
Asunto(s)
Encéfalo/patología , Malformaciones del Sistema Nervioso/patología , Proteínas de Neurofilamentos/biosíntesis , Proteínas de Neurofilamentos/genética , Médula Espinal/patología , Sustitución de Aminoácidos , Animales , Ácido Aspártico , Encéfalo/metabolismo , Humanos , Ratones , Ratones Mutantes Neurológicos , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Especificidad de Órganos , Fosforilación , Nervio Ciático/metabolismo , Nervio Ciático/patología , Serina , Médula Espinal/metabolismo , Raíces Nerviosas Espinales/metabolismo , Raíces Nerviosas Espinales/patología , Núcleos del Trigémino/metabolismo , Núcleos del Trigémino/patologíaRESUMEN
In order to investigate the effect on tau of manipulating glycogen synthase kinase (GSK)-3beta activity in the brain, we created transgenic mice harbouring wild-type GSK-3beta genes or a mutant GSK-3beta that is predicted to be more active. Transgene-derived mRNAs were detected in the brains of a number of the transgenic mouse lines and several of these transgenic lines displayed transgenic GSK-3beta activity. Western blot analyses of the two lines with the highest levels of transgenic GSK-3beta activity revealed that the phosphorylation status of tau was elevated at the AT8 epitope. These observations strongly suggest that GSK-3beta is an in vivo tau kinase in the brain. Only low levels of expression of GSK-3beta were obtained and it is possible that high levels of GSK-3beta activity are lethal.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas tau/metabolismo , Animales , Western Blotting , Química Encefálica/efectos de los fármacos , Química Encefálica/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Humanos , Ratones , Ratones Transgénicos , Mutación , Fosforilación , Reacción en Cadena de la Polimerasa , Pruebas de PrecipitinaRESUMEN
Ser55 within the head domain of neurofilament light chain (NF-L) is transiently phosphorylated by protein kinase A, and phosphorylation of this residue is thought to regulate assembly of neurofilaments. To understand how Ser55 phosphorylation influences NF-L assembly, wild-type and mutant NF-L genes in which Ser55 was mutated to alanine, so as to prevent phosphorylation, or to aspartate, so as to mimic permanent phosphorylation, were transfected into mammalian cells that contain or do not contain an endogenous intermediate filament network. Wild-type and mutant NF-Ls localised to the Triton X-100-insoluble fraction, which suggests that phosphorylation of Ser55 does not inhibit assembly of NF-L and NF-L/ vimentin polymers at or below the tetrameric stage. Immunofluorescence microscopy of transfected cells demonstrated that the wild-type and mutant NF-Ls all colocalised with vimentin to produce similar filamentous arrays. However, in cells lacking an endogenous intermediate filament network, the aspartate mutant produced a pattern of staining different from that of the wild-type or alanine mutant. These results suggest that phosphorylation of NF-L Ser55 is not a mechanism that precludes assembly of neurofilaments from monomers into intermediate filament structures but that phosphorylation/dephosphorylation of this residue might confer more subtle characteristics on neurofilament assembly properties and architecture.
Asunto(s)
Filamentos Intermedios/fisiología , Mutación , Proteínas/genética , Proteínas/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Sondas Moleculares/genética , Datos de Secuencia Molecular , Neurofibromina 1 , Fosforilación , TransfecciónRESUMEN
Protein phosphorylation is well established as a regulatory mechanism in higher plants, but only a handful of plant enzymes are known to be regulated in this manner, and relatively few plant protein kinases have been characterized. AMP-activated protein kinase regulates key enzymes of mammalian fatty acid, sterol and isoprenoid metabolism, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. We now show that there is an activity in higher plants which, by functional criteria, is a homologue of the AMP-activated protein kinase, although it is not regulated by AMP. The plant kinase inactivates mammalian HMG-CoA reductase and acetyl-CoA carboxylase, and peptide mapping suggests that it phosphorylates the same sites on these proteins as the mammalian kinase. However, with the target enzymes purified from plant sources, it inactivates HMG-CoA reductase but not acetyl-CoA carboxylase. The kinase is located in the soluble, and not the chloroplast, fraction of leaf cells, consistent with the idea that it regulates HMG-CoA reductase, and hence isoprenoid biosynthesis, in vivo. The plant kinase also appears to be part of a protein kinase cascade which has been highly conserved during evolution, since the kinase is inactivated and reactivated by mammalian protein phosphatases (2A or 2C) and mammalian kinase kinase, respectively. This contrasts with the situation for many other mammalian protein kinases involved in signal transduction, which appear to have no close homologue in higher plants. To our knowledge, this represents the first direct evidence for a protein kinase cascade in higher plants.