RESUMEN
Cavitation distribution in a High Intensity Focused Ultrasound sonoreactors (HIFU) has been extensively described in the recent literature, including quantification by an optical method (Sonochemiluminescence SCL). The present paper provides complementary measurements through the study of acoustic streaming generated by the same kind of HIFU transducers. To this end, results of mass transfer measurements (electrodiffusional method) were compared to optical method ones (Particle Image Velocimetry). This last one was used in various configurations: with or without an electrode in the acoustic field in order to have the same perturbation of the wave propagation. Results show that the maximum velocity is not located at the focal but shifted near the transducer, and that this shift is greater for high powers. The two cavitation modes (stationary and moving bubbles) are greatly affect the hydrodynamic behavior of our sonoreactors: acoustic streaming and the fluid generated by bubble motion. The results obtained by electrochemical measurements show the same low hydrodynamic activity in the transducer vicinity, the same shift of the active focal toward the transducer, and the same absence of activity in the post-focal axial zone. The comparison with theoretical Eckart's velocities (acoustic streaming in non-cavitating media) confirms a very high activity at the "sonochemical focal", accounted for by wave distortion, which induced greater absorption coefficients. Moreover, the equivalent liquid velocities are one order of magnitude larger than the ones measured by PIV, confirming the enhancement of mass transfer by bubbles oscillation and collapse close to the surface, rather than from a pure streaming effect.
RESUMEN
Acoustic field distribution was determined in HIFU sonoreactors as well as localization of cavitation activity by crossing different techniques: modeling, hydrophone measurements, laser tomography and SCL measurements. Particular care was taken with quantification of this last technique by pixels or photon counting. Cavitation bubbles generated by HIFU are mainly located on the outer layer of the propagation cone in the post-focal zone. Greatest acoustic activity is not located at the geometrical focal, but corresponds to a high concentration of bubbles zone. On the contrary, the main sonochemical activity shifts slightly toward the transducer, whereas quenching of inertial cavitation is observed directly at the focal. Finally, SCL thresholds have been determined.
RESUMEN
The hypothesis of an early vulnerability of the serotonergic system to prion infection was investigated in a murine model of bovine spongiform encephalopathy (BSE). Behavioral tests targeted to 5-HT functions were performed in the course of infection to evaluate circadian activity, anxiety-like behavior, pain sensitivity and the 5-HT syndrome. The first behavioral change was a decrease in nocturnal activity detected at 30% of incubation time. Further behavioral alterations including nocturnal hyperactivity, reduced anxiety, hyperalgesia and exaggerated 5-HT syndrome were observed at 60%-70% of incubation time, before the onset of clinical signs. The same tests performed in 5-HT-depleted mice and in prion protein-deficient mice revealed behavioral abnormalities similar in many aspects to those of BSE-infected mice. Histological and biochemical analysis showed alterations of the serotonergic system in BSE-infected and prion protein-deficient mice. These results indicate that BSE infection affects the homeostasis of serotonergic neurons and suggest that the disruption of prion protein normal function contributes to the early pathological changes in our mouse model of BSE. A similar process may occur in the human variant Creutzfeldt-Jacob disease, as suggested by the early symptoms of alterations in mood, sleep and pain sensitivity.
Asunto(s)
Encéfalo/metabolismo , Encefalopatía Espongiforme Bovina/metabolismo , Trastornos Mentales/metabolismo , Proteínas PrPC/deficiencia , Proteínas PrPSc/toxicidad , Serotonina/metabolismo , Animales , Trastornos de Ansiedad/genética , Trastornos de Ansiedad/metabolismo , Trastornos de Ansiedad/fisiopatología , Encéfalo/fisiopatología , Tronco Encefálico/citología , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiopatología , Bovinos , Trastornos Cronobiológicos/genética , Trastornos Cronobiológicos/metabolismo , Trastornos Cronobiológicos/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Encefalopatía Espongiforme Bovina/fisiopatología , Femenino , Homeostasis/fisiología , Trastornos Mentales/genética , Trastornos Mentales/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Dolor/genética , Dolor/metabolismo , Dolor/fisiopatología , Proteínas PrPC/genética , Proteínas PrPSc/metabolismo , Núcleos del Rafe/citología , Núcleos del Rafe/metabolismo , Núcleos del Rafe/fisiopatología , Síndrome de la Serotonina/genética , Síndrome de la Serotonina/metabolismo , Síndrome de la Serotonina/fisiopatología , Factores de TiempoRESUMEN
Sonoelectrochemical experiments differ from sonochemical ones by the introduction of electrodes in the sonicated reaction vessel. The aim of the study is to characterize the changes of the ultrasonic activity induced by the presence of an electrode located in front of the transducer. The scope of our investigations concerns two low frequencies vibration modes: 20 and 40 kHz. For this purpose, two laser visualization techniques have been used. The first part of the study, described in the present paper (part I), deals with the laser tomography technique which provides an accurate picture of the reactor actives zones, related to numerous cavitation events. For each frequency, two parameters were studied: the electrical power supplied to the transducer and the electrode transducer distance. At both frequencies, without electrode, we can observe distinct zones corresponding to cavitation production and stationary waves establishment. When increasing the input power, bubble clouds tend to form a unique cloud near the transducer. In presence of the electrode, bubble cavitation clouds are largely influenced by the obstacle. The second part of the paper (part II) will describe the Particle Image Velocimetry (P.I.V.) technique which allows to measure the velocity vector field in the fluid portion between the horn and the electrode.
RESUMEN
Sonoelectrochemical experiments differ from sonochemical ones by the introduction of electrodes in the sonicated reaction vessel. The aim of the study is to characterize the changes in the ultrasonic activity induced by the presence of an electrode located in front of the transducer. The scope of our investigations concerns two low frequency vibration modes: 20 and 40 kHz. For this purpose, two laser visualization techniques have been used. The first part of the study, described in a previous paper (Part I), deals with the laser tomography technique which provides an accurate picture of the reactor active zones, related to numerous cavitation events. The second part of the paper (Part II) will describe the particle image velocimetry (PIV) technique used to measure the velocity vector field in the fluid portion between the horn and the electrode. As for the previous study, two parameters were studied: the electrical power supplied to the transducer and the electrode/transducer distance. The velocity vector fields show a main flow in the reactor axis. This flow seems to correspond to the conical cavitation bubbles structure which is observed on the laser tomography pictures. When an electrode is introduced into the reactor, two additional symmetric transversal flows can be quantified on both sides of the electrode.
RESUMEN
PURPOSE: Robot or computer assisted laparoscopic surgeries have overcome several impediments of conventional laparoscopy in pediatric urology. However, in our practice we faced difficulties while performing specific tasks using the da Vinci Surgical System in small cavities. Thus, we objectively evaluated the performance of robot assisted laparoscopic skills in different sizes of workspace. MATERIALS AND METHODS: Seven assessors performed 5 different drills in 7 different sizes of cubic boxes (edge size ranging from 40 to 150 mm) with the da Vinci Surgical System. The drills were developed based on the McGill Inanimate System for Training and Evaluation of Laparoscopic Skills. Assessor performance was evaluated by 2 reviewers for the drill achievement, and time to completion was recorded. A global score was then calculated for each drill in accordance to 1 assessor and 1 box. RESULTS: There were significant collisions while working with the smaller cubes (edges measuring 40 and 45 mm), preventing the surgeon from performing drills. With difficulty, but without collision, the drills were performed in the 50 and 60 mm size cubes. Drills could be accomplished uniformly with ease in the larger cubes (edge 70 mm and greater). CONCLUSIONS: We found that surgeon ability to perform tasks using the da Vinci Surgical System in a small workspace is restricted. This assessment was confirmed by a statistical analysis of the data collected, demonstrating that with common surgical practice using the da Vinci robot workspace has a major impact on surgeon performance.
Asunto(s)
Laparoscopía , Robótica , Análisis y Desempeño de Tareas , Niño , Humanos , Modelos TeóricosRESUMEN
The pro-apoptotic factor BAX has recently been shown to contribute to Purkinje cell (PC) apoptosis induced by the neurotoxic prion-like protein Doppel (Dpl) in the prion-protein-deficient Ngsk Prnp(0/0) (NP(0/0)) mouse. In view of cellular prion protein (PrP(c)) ability to counteract Dpl neurotoxicity and favor neuronal survival like BCL-2, we investigated the effects of the anti-apoptotic factor BCL-2 on Dpl neurotoxicity by studying the progression of PC death in aging NP(0/0)-Hu-bcl-2 double mutant mice overexpressing human BCL-2 (Hu-bcl-2). Quantitative analysis showed that significantly more PCs survived in NP(0/0)-Hu-bcl-2 double mutants compared with the NP(0/0) mutants. However, number of PCs remained inferior to wild-type levels and to the increased number of PCs observed in Hu-bcl-2 mutants. In the NP(0/0) mutants, Dpl-induced PC death occurred preferentially in the aldolase C-negative parasagittal compartments of the cerebellar cortex. Activation of glial cells exclusively in these compartments, which was abolished by the expression of Hu-bcl-2 in the double mutants, suggested that chronic inflammation is an indirect consequence of Dpl-induced PC death. This partial rescue of NP(0/0) PCs by Hu-bcl-2 expression was similar to that observed in NP(0/0):Bax(-/-) double mutants with bax deletion. Taken together, these data strongly support the involvement of BCL-2 family-dependent apoptotic pathways in Dpl neurotoxicity. The capacity of BCL-2 to compensate PrP(c) deficiency by rescuing PCs from Dpl-induced death suggests that the BCL-2-like property of PrP(c) may impair Dpl-like neurotoxic pathways in wild-type neurons.
Asunto(s)
Apoptosis/efectos de los fármacos , Priones/genética , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Células de Purkinje/efectos de los fármacos , Factores de Edad , Análisis de Varianza , Animales , Recuento de Células , Cerebelo/citología , Fructosa-Bifosfato Aldolasa/metabolismo , Proteínas Ligadas a GPI , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Priones/toxicidadRESUMEN
BACKGROUND: Although patients benefit considerably from minimally invasive surgery, the use of new instruments such as robotic systems is challenging for surgeons, and extensive training is required. METHOD: We developed a computer-based simulator of the da Vinci Surgical System, modelling the robot and designing a new interface. RESULTS: The simulator offers users a two-handed interface to control a realistic model of the da Vinci robot. The simulator can be applied (i) to provide an environment in which to practice simple surgical skills and (ii) to serve as a visualization platform on which to validate port placement and robot pose for operation planning. CONCLUSIONS: Virtual reality is a useful technique for medical training. The simulator is currently in its early stages, but this preliminary work is promising.
Asunto(s)
Instrucción por Computador/métodos , Ecosistema , Robótica/educación , Robótica/métodos , Cirugía Asistida por Computador/educación , Cirugía Asistida por Computador/métodos , Interfaz Usuario-Computador , Instrucción por Computador/instrumentación , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Cuidados Preoperatorios/métodos , Robótica/instrumentación , Cirugía Asistida por Computador/instrumentaciónRESUMEN
Research efforts to deduce the function of the prion protein (PrPc) in knock-out mouse mutants have revealed that large deletions in the PrPc genome result in the ectopic neuronal expression of the prion-like protein Doppel (Dpl). In our analysis of one such line of mutant mice, Ngsk Prnp0/0 (NP0/0), we demonstrate that the ectopic expression of Dpl in brain neurons induces significant levels of cerebellar Purkinje cell (PC) death as early as six months after birth. To investigate the involvement of the mitochondrial proapoptotic factor BAX in the Dpl-induced apoptosis of PCs, we have analyzed the progression of PC death in aging NP0/0:Bax-/- double knockout mutants. Quantitative analysis of cell numbers showed that significantly more PCs survived in NP0/0:Bax-/- double mutants than in the NP0/0:Bax+/+ mutants. However, PC numbers were not restored to wildtype levels or to the increased number of PCs observed in Bax-/- mutants. The partial rescue of NP0/0 PCs suggests that the ectopic expression of Dpl induces both BAX-dependent and BAX-independent pathways of cell death. The activation of glial cells that is shown to be associated topographically with Dpl-induced PC death in the NP0/0:Bax+/+ mutants is abolished by the loss of Bax expression in the double mutant mice, suggesting that chronic inflammation is an indirect consequence of Dpl-induced PC death.
Asunto(s)
Apoptosis/fisiología , Priones/fisiología , Células de Purkinje/fisiología , Proteína X Asociada a bcl-2/fisiología , Animales , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI , Genotipo , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/patología , Procesamiento de Imagen Asistido por Computador , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Priones/genética , Priones/metabolismo , Células de Purkinje/metabolismo , Proteína X Asociada a bcl-2/genéticaRESUMEN
Alzheimer's dementia may be considered a synaptic disease of central neurons: the loss of synapses, reflected by early cognitive impairments, precedes the appearance of extra cellular focal deposits of beta-amyloid peptide in the brain of patients. Distinct immunocytochemical patterns of amyloid precursor proteins (APPs) have previously been demonstrated in the synapses by ultrastructural analysis in the cerebellum and hippocampus of adult rats and mice. Now we show that during postnatal development and during aging in these structures, the immunocytochemical expression of APPs increases in the synapses in parallel with the known up-regulation of total APPs brain levels. Interestingly, as shown previously in the adult rodents, the presenilins (PSs) 1 and 2, which intervene in APPs metabolism, exhibit a synaptic distribution pattern similar to that of APPs with parallel quantitative changes throughout life. In the brain tissue, single and double immunocytochemistry at the ultrastructural level shows co-localisation of APPs and PSs in axonal and dendritic synaptic compartments during postnatal synaptogenesis, adulthood and aging. In addition, double-labelling immunocytofluorescence detects these proteins close to synaptophysin at the growth cones of developing cultured neurons. Thusly, the brain expression of APPs and PSs appears to be regulated synchronously during lifespan in the synaptic compartments where the proteins are colocated. This suggests that PS-dependent processing of important synaptic proteins such as APPs could intervene in age-induced adjustments of synaptic relationships between specific types of neurons.
Asunto(s)
Envejecimiento/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Cerebelo/metabolismo , Hipocampo/metabolismo , Proteínas de la Membrana/metabolismo , Sinapsis/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Animales Recién Nacidos , Recuento de Células/métodos , Células Cultivadas , Cerebelo/crecimiento & desarrollo , Cerebelo/ultraestructura , Modelos Animales de Enfermedad , Hipocampo/crecimiento & desarrollo , Hipocampo/ultraestructura , Inmunohistoquímica/métodos , Técnicas In Vitro , Microscopía Electrónica/instrumentación , Microscopía Electrónica/métodos , Presenilina-1 , Presenilina-2 , Ratas , Ratas Long-Evans , Sinapsis/ultraestructura , Factores de TiempoRESUMEN
Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the sigma subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity between Drosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.
Asunto(s)
Dendritas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas/genética , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/metabolismo , Secuencia de Bases , Encéfalo/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Clonación Molecular , Secuencia Conservada , ADN Complementario/genética , Drosophila melanogaster , Células HeLa , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas/química , Ratas , Homología de Secuencia de Aminoácido , Distribución Tisular , Dominios Homologos srcRESUMEN
The cellular prion protein PrP(c) is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrP(c) is converted to infectious protease-resistant isoforms PrP(res) or prions. Prions provoque predictable and distinctive patterns of PrP(res) accumulation and neurodegeneration depending on the prion strain and on regional cell-specific properties modulating PrP(c) affinity for infectious PrP(res) in the host brain. Synaptolysis and synaptic accumulation of PrP(res) during PrP-related diseases suggests that the synapses could be primary sites able to propagate PrP(res) and neurodegeneration in the central nervous system. In the rodent cerebellum, the present light and electron microscopic immuno-cytochemical analysis shows that distinct types of synapses display differential expression of PrP(c), suggesting that synapse-specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrP(c) remain unknown, the concentration of PrP(c) almost exclusively at the Purkinje cell synapses in the cerebellum suggests its critical involvement in the synaptic relationships between cerebellar neurons in agreement with their known vulnerability to PrP deficiencies.
Asunto(s)
Cerebelo/metabolismo , Proteínas PrPC/análisis , Sinapsis/metabolismo , Animales , Anticuerpos Monoclonales , Cerebelo/ultraestructura , Cricetinae , Fijadores , Inmunohistoquímica/métodos , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Proteínas PrPC/deficiencia , Enfermedades por Prión/metabolismo , Isoformas de Proteínas/análisis , Sinapsis/ultraestructuraRESUMEN
The expression of the cellular form of the prion protein (PrP(c)) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrP(c) is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrP(c), a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrP(c) at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.
Asunto(s)
Proteínas Luminiscentes/genética , Proteínas PrPC/genética , Priones/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Bovinos , Corteza Cerebelosa/metabolismo , Cerebelo/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Queratinocitos/metabolismo , Proteínas Luminiscentes/análisis , Linfocitos/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Células de Purkinje/metabolismoRESUMEN
Elimination of cerebellar granule cells early during postnatal development produces abnormal neural organization that retains immature characteristics in the adult, including innervation of each Purkinje cell by multiple climbing fibers from the inferior olive. To elucidate mechanisms underlying development of the olivocerebellar projection, we studied light-microscopic morphology of single olivocerebellar axons labeled with biotinylated dextran amine in adult rats rendered agranular by a single postnatal X-irradiation. Each reconstructed olivocerebellar axon gave off approximately 12 climbing fibers, approximately twice as many as in normal rats. Terminal arborizations of climbing fibers made irregular tufts in most areas, whereas they were arranged vertically in a few mildly affected areas. Each climbing fiber terminal arborization innervated only part of the dendritic arbor of a Purkinje cell, and multiple climbing fibers innervated a single Purkinje cell. These climbing fibers originated either from the same olivocerebellar axon (pseudomultiple innervation) or from distinct axons (true multiple innervation). Abundant non-climbing fiber thin collaterals projected to all cortical layers. Although the longitudinal pattern of the zonal olivocerebellar projection was generally observed, lateral branching, including bilateral projections, was relatively frequent. These results suggest that the granule cell-parallel fiber system induces several important features of olivocerebellar projection: (1) organization of the climbing fiber terminal arborization tightly surrounding Purkinje cell dendrites, (2) elimination of pseudo- and true multiple innervations establishing one-to-one innervation, (3) retraction of non-climbing fiber thin collaterals from the molecular layer, and (4) probable refinement of the longitudinal projection domains by removing aberrant transverse branches.
Asunto(s)
Axones/efectos de la radiación , Núcleo Olivar/citología , Células de Purkinje/efectos de la radiación , Células de Purkinje/ultraestructura , Factores de Edad , Animales , Axones/fisiología , Biotina/análogos & derivados , Tamaño de la Célula/efectos de la radiación , Dendritas/fisiología , Dendritas/efectos de la radiación , Dextranos , Colorantes Fluorescentes , Vías Nerviosas/efectos de la radiación , Núcleo Olivar/efectos de la radiación , Ratas , Ratas Wistar , Sinapsis/efectos de la radiaciónRESUMEN
We previously generated a mouse model with a mutation in the murine Atm gene that recapitulates many aspects of the childhood neurodegenerative disease ataxia-telangiectasia. Atm-deficient (Atm-/-) mice show neurological defects detected by motor function tests including the rota-rod, open-field tests and hind-paw footprint analysis. However, no gross histological abnormalities have been observed consistently in the cerebellum of any line of Atm-/- mice analyzed in most laboratories. Therefore, it may be that the neurologic dysfunction found in these animals is associated with predegenerative lesions. We performed a detailed analysis of the cerebellar morphology in two independently generated lines of Atm-/- mice to determine whether there was evidence of neuronal abnormality. We found a significant increase in the number of lysosomes in Atm-/- mice in the absence of any detectable signs of neuronal degeneration or other ultrastructural anomalies. In addition, we found that the ATM protein is predominantly cytoplasmic in Purkinje cells and other neurons, in contrast to the nuclear localization of ATM protein observed in cultured cells. The cytoplasmic localization of ATM in Purkinje cells is similar to that found in human cerebellum. These findings suggest that ATM may be important as a cytoplasmic protein in neurons and that its absence leads to abnormalities of cytoplasmic organelles reflected as an increase in lysosomal numbers.
Asunto(s)
Cerebelo/metabolismo , Lisosomas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Recuento de Células , Proteínas de Ciclo Celular , Cerebelo/química , Cerebelo/ultraestructura , Citoplasma/química , Proteínas de Unión al ADN , Femenino , Ganglios Espinales/química , Ganglios Espinales/citología , Inmunohistoquímica , Lisosomas/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones Mutantes , Microscopía Electrónica , Neuronas/química , Proteínas Serina-Treonina Quinasas/análisis , Células de Purkinje/química , Células de Purkinje/citología , Células de Purkinje/ultraestructura , Proteínas Supresoras de TumorRESUMEN
Healthy brain neurons co-express Alzheimer's disease (AD) related proteins presenilins (PS) and beta-amyloid precursor protein (beta-APP). Deposition of beta-amyloid and PS in the senile plaques of AD brain and their ability to interact in vitro suggest that AD pathology could arise from a defect in the physiological interactions between beta-APP and PS within and/or between neurons. The present study compares the immunocytochemical distribution of PS (1 and 2) and beta-APP major isoforms (695 and 751/770) in the synapses of the cerebellum and hippocampus of the adult rat and mouse. In the cerebellar cortex of both species, the four molecules are immunodetected in the presynaptic or the postsynaptic compartments of synapses, suggesting that they are involved in interneuronal relationships. In contrast, PS and beta-APP are postsynaptic in almost all the immunoreactive synapses of the hippocampus. The different distribution patterns of these proteins in cerebellar and hippocampal synapses may reflect specific physiological differences, responsible for differential vulnerability of neurons to AD synaptic pathology. Defective interactions between beta-APP and PS at the synapses could impede the synaptic functions of beta-APP, inducing the selective loss of synapses that accounts for cognitive impairment in AD.
Asunto(s)
Precursor de Proteína beta-Amiloide/análisis , Cerebelo/citología , Hipocampo/citología , Proteínas de la Membrana/análisis , Sinapsis/ultraestructura , Animales , Cerebelo/ultraestructura , Hipocampo/ultraestructura , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Isoformas de Proteínas/análisis , Ratas , Ratas Long-EvansRESUMEN
ClC-K channels are Cl- channels specifically expressed in vertebrate kidneys. Although their heterologous functional expression is still controversial, indirect evidence points to them as major factors involved in Cl- reabsorption in the nephron. We cloned xClC-K, an amphibian (Xenopus) homologue of mammalian ClC-K. The cDNA encodes a 77 kDa protein presenting 62% similarity with human ClC-Kb. The protein is monoglycosylated and is expressed primarily in the Xenopus kidney. It is localized in the basolateral membranes of proximal convoluted tubules of the nephron and in the apical region of the diluting segments. Heterologous expression of xClC-K in HEK-293 cells showed that the full-length protein is glycosylated and targeted to the cell membrane, but no associated Cl- current could be observed with the patch-clamp recording technique. N-glycosylation of both the native kidney channel and the recombinant protein expressed in HEK-293 conferred on them anomalous behaviour in denaturing PAGE, which is indicative of strong interactions at the extracellular side of the plasma membrane. The expression of ClC-K channels in both mesonephric and metanephric kidneys will permit further comparative physiological studies of Cl- permeabilities at the molecular level.
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Membrana Celular/metabolismo , Canales de Cloruro/genética , Túbulos Renales Proximales/metabolismo , Proteínas de Xenopus , Xenopus laevis/metabolismo , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Canales de Cloruro/química , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Clonación Molecular , Femenino , Glicosilación , Humanos , Túbulos Renales Proximales/citología , Datos de Secuencia Molecular , Especificidad de Órganos , Técnicas de Placa-Clamp , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transfección , Xenopus laevis/genéticaRESUMEN
Studies of postnatal neurogenesis have benefited from the use of a relatively non-invasive method for chronic delivery of bioactive substances to a restricted area of cortex. This method consists of the implantation of an Elvax polymer microsource of active substances close to the targeted brain surface. Receptor ligands, as well as macromolecules such as proteins, peptides and enzymes have been shown to be released by the implants in a sustained manner over weeks. Here we describe the kinetics and immunoreactivity of different immunoglobulins released in vitro and in vivo by Elvax polymer. In vitro, the immunoglobulins first diffuse during a burst phase from the pore network of the polymer matrix. Release continues during a slow phase depending on loading, porosity and volume of the matrix but also on intrinsic properties of immunoglobulins. Elvax microsources loaded either with anti-TAG-1 or with anti-HNK-1 antibodies according to the release data in vitro, are implanted on the posterior cerebellar cortex of postnatal rats during the period when the targeted antigens are expressed by the differentiating cells. After several days, the released immunoreactive antibodies are located at the antigenic sites within the cerebellar cortex close to the implants. The sustained local delivery of immunoglobulins using the Elvax implant method allows access to cell surface and matrix molecules and thereby to the mechanisms they control during postnatal neurogenesis.
Asunto(s)
Antígenos CD57/análisis , Moléculas de Adhesión Celular Neuronal , Corteza Cerebelosa/crecimiento & desarrollo , Inmunoglobulina G/administración & dosificación , Inmunoglobulina M/administración & dosificación , Glicoproteínas de Membrana/análisis , Envejecimiento , Animales , Antígenos CD57/biosíntesis , Antígenos CD57/inmunología , Corteza Cerebelosa/citología , Contactina 2 , Preparaciones de Acción Retardada , Portadores de Fármacos , Implantes de Medicamentos , Inmunohistoquímica/métodos , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/inmunología , Polivinilos , Ratas , Ratas WistarRESUMEN
During postnatal development of the rodent cerebellum, a transient enzyme activity of ecto-5'-nucleotidase has been shown in the asymmetrical synapses of Purkinje cells. The alterations of the afferent circuitry and microenvironment of the ectopic Purkinje cells present in the cerebellum of the reeler mutant mouse could enlighten parameters that influence the synaptic 5'-nucleotidase activity of these cells. Ecto-enzyme cytochemistry reveals intense 5'-nucleotidase activity in 43% of synapses of the Purkinje cells throughout the cortex and the core of the reeler cerebellar vermis, although the molecular layer displays large areas with less than 1% of labelled synapses. However, enzymatic labelling is found in considerably more Purkinje cells synapses (73%) throughout the granular layer and the subcortical mass. Climbing fiber synapses of monoinnervated Purkinje cells are labelled by 5'-nucleotidase activity in the molecular layer, as well as asymmetrical synapses made on the subjacent ectopic Purkinje cells by the multiple climbing fibers and by the heterologous afferences. The non-innervated dendritic spines of these cells are also labelled, suggesting that 5'-nucleotidase activity at postsynaptic sites of reeler Purkinje cells does not depend on the presynaptic innervation. Rather, 5'-nucleotidase enzyme activity is enhanced at theses sites when the Purkinje cells have not achieved chemodifferentiation but have conserved immature wiring, i.e., low parallel fiber and multiple climbing fiber inputs.
Asunto(s)
5'-Nucleotidasa/metabolismo , Cerebelo/enzimología , Ratones Mutantes Neurológicos/metabolismo , Células de Purkinje/enzimología , Sinapsis/enzimología , Animales , Cerebelo/ultraestructura , Histocitoquímica , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Distribución TisularRESUMEN
The precursor proteins of Alzheimer's disease beta-amyloid peptide, the beta-amyloid precursor protein isoforms, comprise a family of neuronal proteins with synaptic localization whose physiological roles in brain are poorly understood. One possible role for synaptic proteins is involvement in neuronal plasticity. After exposure to an enriched environment compared to impoverished conditions, rats exhibited superior cognitive capacity. Up to approximately four-fold increased overall levels of beta-amyloid precursor proteins were found in cortical/subcortical tissue of the enriched animals displaying significantly more synapses immunoreactive for the different beta-amyloid precursor protein isoforms (beta-amyloid precursor protein695- and beta-amyloid precursor protein751/770) in hippocampus and adjacent occipital cortex. This correlation thus provides in vivo evidence for an association of beta-amyloid precursor proteins with plastic changes induced by complex environment with consequences for cognitive functions and suggests that impaired beta-amyloid precursor protein metabolism at synapses might contribute to brain dysfunction in Alzheimer's disease.