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1.
Science ; 290(5497): 1775-9, 2000 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-11099418

RESUMEN

After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.


Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Encéfalo/citología , Neuronas/citología , Animales , Biomarcadores/análisis , Diferenciación Celular , Tamaño de la Célula , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Neuronas/química , Neuronas/metabolismo , Bulbo Olfatorio/citología , Fenotipo , Fosforilación
2.
J Neurochem ; 75(5): 1889-97, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11032878

RESUMEN

The function of PrP(C), the cellular prion protein (PrP), is still unknown. Like other glycophosphatidylinositol-anchored proteins, PrP resides on Triton-insoluble, cholesterol-rich membranous microdomains, termed rafts. We have recently shown that the activity and subcellular localization of the neuronal isoform of nitric oxide synthase (nNOS) are impaired in adult PrP(0/0) mice as well as in scrapie-infected mice. In this study, we sought to determine whether PrP and nNOS are part of the same functional complex and, if so, to identify additional components of such a complex. To this aim, we looked for proteins that coimmunoprecipitated with PrP in the presence of detergents either that completely dissociate rafts, to identify stronger interactions, or that preserve the raft structure, to identify weaker interactions. Using this detergent-dependent immunoprecipitation protocol we found that PrP interacts strongly with dystroglycan, a transmembrane protein that is the core of the dystrophin-glycoprotein complex (DGC). Additional results suggest that PrP also interacts with additional members of the DGC, including nNOS. PrP coprecipitated only with established presynaptic proteins, consistent with recent findings suggesting that PrP is a presynaptic protein.


Asunto(s)
Encéfalo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas PrPC/metabolismo , Actinas/metabolismo , Animales , Complejo Antígeno-Anticuerpo/química , Fraccionamiento Celular , Cerebelo/metabolismo , Detergentes/química , Distroglicanos , Glicosilfosfatidilinositoles/metabolismo , Sustancias Macromoleculares , Microdominios de Membrana/química , Ratones , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Octoxinol , Polietilenglicoles/química , Pruebas de Precipitina , Sinaptofisina/metabolismo , Tubulina (Proteína)/metabolismo
3.
J Neurochem ; 72(3): 1224-31, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10037495

RESUMEN

PrP(Sc), the only identified component of the scrapie prion, is a conformational isoform of PrPc. The physiological role of PrPc, a glycolipid-anchored glycoprotein, is still unknown. We have shown previously that neuronal nitric oxide synthase (nNOS) activity is impaired in the brains of mice sick with experimental scrapie as well as in scrapie-infected neuroblastoma cells. In this work we investigated the cell localization of nNOS in brains of wild-type and scrapie-infected mice as well as in mice in which the PrP gene was ablated. We now report that whereas in wild-type mice, nNOS, like PrPc, is associated with detergent-insoluble cholesterol-rich membranous microdomains (rafts), this is not the case in brains of scrapie-infected or in those of adult PrP(0/0) mice. Also, adult PrP(0/0), like scrapie-infected mice, show reduced nNOS activity. We suggest that PrPc may play a role in the targeting of nNOS to its proper subcellular localization. The similarities of nNOS properties in PrP(0/0) as compared with scrapie-infected mice suggest that at least this role of PrPc may be impaired in scrapie-infected brains.


Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Priones/genética , Scrapie/enzimología , Animales , Encéfalo/enzimología , Encéfalo/patología , Inmunohistoquímica , Membranas Intracelulares/enzimología , Ratones , Ratones Noqueados , NADPH Deshidrogenasa , Neuronas/ultraestructura , Óxido Nítrico Sintasa de Tipo I , Fracciones Subcelulares/enzimología
4.
J Neurosci ; 16(17): 5405-14, 1996 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-8757253

RESUMEN

This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [3H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3H]GABA and [3H]tiagabine binding. Voltage jumps, Na+ and Cl- concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na+ ions interact with the transporter even in the absence of GABA, and (2) that Cl- facilitates the binding of Na+. Comparison between the charge movements and the transport-associated current shows that this initial Na(+)-transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation--treatment with high-affinity uptake blockers and the W68L mutation-"lock" Na+ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na+ substitutes Li+ and Cs+ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na+ and Cl-, and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Cloruros/farmacología , Electrofisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Células HeLa , Homeostasis , Humanos , Iones , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Mutación , Proteínas del Tejido Nervioso/metabolismo , Inhibidores de la Captación de Neurotransmisores/farmacología , Oocitos/metabolismo , Permeabilidad , Sodio/metabolismo , Xenopus , Ácido gamma-Aminobutírico/metabolismo
5.
Dev Psychobiol ; 29(5): 453-62, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8809495

RESUMEN

The effect of prenatal stress was determined on exploration in situations that induce different levels of fear. Dams (12) were stressed by noise and light thrice weekly on an unpredictable basis throughout pregnancy, and 12 controls were left undisturbed. The time spent by different groups of their adult offspring of both sexes in exploration was assessed during 4 min in a plus maze; large, well-lit open field (1), and open field (2) after prior exposure to a small, dark holebox. Prenatal stress resulted in a significant reduction in the number of arm entries in the plus maze and amount of time spent in the open arms. Locomotion and rearing were also reduced in Open Field 1 and 2, but these activities and hole poking were unchanged in the holebox. It is concluded that prenatal stress renders the animal more fearful to a novel, intimidating environment, which may be expressed as a suppression of exploratory activity.


Asunto(s)
Nivel de Alerta , Conducta Exploratoria , Efectos Tardíos de la Exposición Prenatal , Estrés Psicológico/complicaciones , Animales , Miedo , Femenino , Masculino , Aprendizaje por Laberinto , Actividad Motora , Embarazo , Ratas , Ratas Sprague-Dawley , Medio Social
6.
Pharmacol Biochem Behav ; 54(1): 183-8, 1996 May.
Artículo en Inglés | MEDLINE | ID: mdl-8728556

RESUMEN

The involvement of endogenous opiates in the differentiation of sexual behavior was tested by exposing rat fetuses to continuous naltrexone during the last 9 days of gestation. Time-mated female rats received oral naltrexone, 40 mg/kg/day, via their drinking water, from gestational day 13 until parturition. Early motor development, measured by swimming ability in 7-, 9-, and 11-day-old offspring of the treated dams, was unaffected by prenatal naltrexone. Adult male offspring were given three tests of male sexual behavior, then castrated, primed with ovarian hormones, and given two tests of feminine receptivity (lordosis quotient). Prenatal naltrexone facilitated masculine behavior and suppressed feminine receptivity: latencies to first mount and to ejaculation were shorter, mount rate was higher, and lordosis quotient was lower in naltrexone-treated rats, compared with control animals. These findings implicate endogenous opiates in prenatal organization of sex-specific behavioral dispositions.


Asunto(s)
Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Efectos Tardíos de la Exposición Prenatal , Conducta Sexual Animal/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Eyaculación/efectos de los fármacos , Femenino , Masculino , Actividad Motora/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Postura/fisiología , Embarazo , Ratas , Diferenciación Sexual/efectos de los fármacos , Natación
7.
FEBS Lett ; 371(1): 39-42, 1995 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-7664880

RESUMEN

We have investigated the possible role of selected negatively-charged amino acids of the sodium and chloride-coupled GABA transporter GAT-1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site-directed mutagenesis. The functional consequences were that one of the residues, glutamate-101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate-101 is critical for one or more of the conformational changes of GAT-1 during its transport cycle.


Asunto(s)
Proteínas Portadoras/metabolismo , Ácido Glutámico/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Ácido gamma-Aminobutírico/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Cloruros/fisiología , Conductividad Eléctrica , Proteínas Transportadoras de GABA en la Membrana Plasmática , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Sodio/fisiología
8.
Pharmacol Biochem Behav ; 50(3): 413-9, 1995 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-7617680

RESUMEN

The influence of opioid receptor blockade on the developmental and behavioral effects of prenatal stress was studied. Time-mated dams were implanted with minipumps on day 17 of gestation containing vehicle (V) or naltrexone (NTX, 10 mg/kg/day). Noise and light stress was applied on an unpredictable basis, three times a week throughout gestation to half the dams. Maternal NTX completely prevented the reduction in anogenital distance in prenatally stressed (PS) males and restored the growth rate of both sexes. NTX also decreased the anxiety of PS rats in the plus-maze, increased the opioid component of exploration to control levels, but increased anxiety in control males. NTX did not restore the lower saccharin preference in PS females and decreased it in C females. This suggests that some morphological and behavioral changes induced by prenatal stress could result from excess opioid activity induced by maternal stress.


Asunto(s)
Naltrexona/farmacología , Antagonistas de Narcóticos , Efectos Tardíos de la Exposición Prenatal , Estrés Fisiológico/tratamiento farmacológico , Animales , Conducta de Elección/efectos de los fármacos , Femenino , Bombas de Infusión Implantables , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Sacarina
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