RESUMEN
Mitochondrial pyruvate dehydrogenase (PDH) regulates the delivery of carbohydrate-derived substrate to the mitochondrial tricarboxylic acid cycle and electron transport chain. PDH activity at rest and its activation during exercise is attenuated following high-fat (HFAT) compared with high-carbohydrate (HCHO) diets. Given the reliance on carbohydrate-derived substrate early in transitions to exercise, this study examined the effects of HFAT and HCHO on phase II pulmonary O2 uptake (VÌo2 p) kinetics during transitions into the moderate-intensity (MOD) exercise domain. Eight active adult men underwent dietary manipulations consisting of 6 days of HFAT (73% fat, 22% protein, 5% carbohydrate) followed immediately by 6 days of HCHO (10% fat, 10% protein, 80% carbohydrate); each dietary phase was preceded by a glycogen depletion protocol. Participants performed three MOD transitions from a 20 W cycling baseline to work rate equivalent to 80% of estimated lactate threshold on days 5 and 6 of each diet. Steady-state VÌo2 p was greater (P < 0.05), and respiratory exchange ratio and carbohydrate oxidation rates were lower (P < 0.05) during HFAT. The phase II VÌo2 p time constant (τVÌo2 p) [HFAT 40 ± 16, HCHO 32 ± 19 s (mean ± SD)] and VÌo2 p gain (HFAT 10.3 ± 0.8, HCHO 9.4 ± 0.7 ml·min(-1·)W(-1)) were greater (P < 0.05) in HFAT. The overall adjustment (effective time constant) of muscle deoxygenation (Δ[HHb]) was not different between diets (HFAT 24 ± 4 s, HCHO 23 ± 4 s), which coupled with a slower τVÌo2 p, indicates a slowed microvascular blood flow response. These results suggest that the slower VÌo2 p kinetics associated with HFAT are consistent with inhibition and slower activation of PDH, a lower rate of pyruvate production, and/or attenuated microvascular blood flow and O2 delivery.
Asunto(s)
Carbohidratos de la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Ejercicio Físico , Consumo de Oxígeno , Complejo Piruvato Deshidrogenasa/metabolismo , Adulto , Metabolismo de los Hidratos de Carbono , Dieta Alta en Grasa , Carbohidratos de la Dieta/metabolismo , Grasas de la Dieta/metabolismo , Voluntarios Sanos , Frecuencia Cardíaca , Humanos , Metabolismo de los Lípidos , Masculino , Mitocondrias Musculares/enzimología , Músculos/irrigación sanguínea , Músculos/metabolismo , Fosforilación Oxidativa , Adulto JovenRESUMEN
Mannitol particles, produced by spray drying (SD), have been used commercially (Aridol) in bronchial provocation test. In this study, we propose an alternative method to produce inhalable mannitol powders. The elongated mannitol particles (number median length 4.0microm, and axial ratio of 3.5) were prepared using a confined liquid impinging jets (CLIJs) followed by jet milling (JM). Spray dried and jet milled raw mannitol particles were compared in an attempt to assess the performance of the particles produced by the new method. Aerosol performance of the three different powders (CLIJ, SD, and JM) was relatively poor (fine particle fraction or FPF(loaded) below 15%) when dispersed by the Rotahaler. Dispersion through the Aeroliser led to better aerosol performance of the CLIJ mannitol (FPF(loaded) 20.3%), which is worse than the JM (FPF(loaded) 30.3%) and SD mannitol particles (FPF(loaded) 45.7%) at 60 L/min, but comparable (FPF(loaded) 40.0%) with those of the JM (FPF(loaded) 40.7%) and SD (FPF(loaded) 45.5%) powders at 100L/min. Hence, the optimum use of these elongated mannitol particles can be achieved at increased air flow with a more efficient inhaler. In addition to crystallinity, morphology, and particle size distribution, the surface energies of these powders were measured to explain the differences in aerosol performance. A major advantage of using the CLIJ method is that it can be scaled up with a good yield as the precipitate can be largely collected and recovered on a filter, compared with spray drying which has a low collection efficiency for fine particles below 2microm.
Asunto(s)
Composición de Medicamentos/métodos , Manitol/administración & dosificación , Manitol/química , Administración por Inhalación , Aerosoles , Cromatografía de Gases , Cristalización , Tamaño de la Partícula , Polvos , Propiedades de Superficie , Difracción de Rayos XRESUMEN
A model to predict fractal dimension from sedimentating fractal aggregates has been successfully developed. This model was developed using the settling rate and size data of fractal aggregates. In order to test the validity of the model, a purpose-built settling rig, equipped with lens with magnification of 1200x, which can capture images of particles/flocs down to 2 microm in diameter was used. The performance and technique of the settling rig were validated by comparing the measured settling rates of 30- and 50.7-microm standard particles with their theoretical settling rates calculated using Stokes' law. The measured settling rates were within 10% agreement with the calculated Stokes' velocities. The settling rates and sizes of the particles/flocs were analyzed using image analysis software called WiT 5.3. The maximum temperature gradient across the settling column was 0.1 degrees C, which effectively eliminated convective currents due to temperature differences in the settling column. A total of 1000 calcium phosphate flocs were analyzed. Calcium phosphate flocs with fractal dimensions varying from 2.3 to 2.8 were generated via orthokinetic aggregation. Measurements of fractal dimensions, using light scattering, were done simultaneously with the settling experiments and they were found to be constant. The fractal dimensions calculated using the model agreed with those obtained by light scattering to within 12%.
RESUMEN
Semaphorin 3C is a secreted member of the semaphorin gene family. To investigate its function in vivo, we have disrupted the semaphorin 3C locus in mice by targeted mutagenesis. semaphorin 3C mutant mice die within hours after birth from congenital cardiovascular defects consisting of interruption of the aortic arch and improper septation of the cardiac outflow tract. This phenotype is similar to that reported following ablation of the cardiac neural crest in chick embryos and resembles congenital heart defects seen in humans. Semaphorin 3C is expressed in the cardiac outflow tract as neural crest cells migrate into it. Their entry is disrupted in semaphorin 3C mutant mice. These data suggest that semaphorin 3C promotes crest cell migration into the proximal cardiac outflow tract.
Asunto(s)
Aorta Torácica/anomalías , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Semaforina-3A , Tronco Arterial/química , Proteínas de Pez Cebra/agonistas , Secuencia de Aminoácidos , Animales , Genotipo , Hibridación in Situ , Integrasas/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Recombinación Genética , Retinal-Deshidrogenasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Proteínas Virales/metabolismoRESUMEN
Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, we use these techniques to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. We demonstrate that PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Cresta Neural/embriología , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Semaforina-3A , Animales , Línea Celular , Movimiento Celular , Células Cultivadas , Galactósidos/metabolismo , Hibridación in Situ , Indoles/metabolismo , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Cresta Neural/citología , Neuropilina-1 , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Factores de Tiempo , Proteínas Virales/metabolismoRESUMEN
The present study explores the role of SEMA3A (collapsin-1) in the temporal and spatial regulation of developing sensory projections in the chick spinal cord. During development, SEMA3A mRNA (SEMA3A) is first expressed throughout the spinal gray matter, but disappears from the dorsal region when small caliber (trkA(+)) sensory axon collaterals first grow into the dorsal horn. In explant cultures of spinal cord segments with attached sensory ganglia, the spatial extent of SEMA3A expression varied in different explants, but in each case the growth of trkA(+) sensory collaterals was largely excluded from areas of SEMA3A expression. To test if SEMA3A had a direct effect on sensory axon growth, we injected recombinant protein into the explants before placing them in culture. Increased levels of SEMA3A substantially reduced the ingrowth of trkA(+) axons, whereas trkC(+) axon collaterals were not affected. Consistent with the insensitivity of trkC(+) collaterals to SEMA3A, these collaterals did not express neuropilin-1, a receptor for SEMA3A. The inhibitory effects of SEMA3A on trkA(+) axons within the spinal cord suggests that the fall in SEMA3A expression in the dorsal horn may contribute to the initiation of growth of these axons into gray matter. In addition, the observation that trkA(+) axons frequently grew close to but rarely over areas of SEMA3A expression suggests that semaphorin may act principally as a short-range guidance cue within the spinal cord.
Asunto(s)
Vías Aferentes/embriología , Axones/metabolismo , Ganglios Espinales/embriología , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/metabolismo , Médula Espinal/embriología , Vías Aferentes/citología , Vías Aferentes/metabolismo , Animales , Axones/efectos de los fármacos , Axones/ultraestructura , Células Cultivadas , Embrión de Pollo , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas del Tejido Nervioso/farmacología , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Células del Asta Posterior/citología , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Receptor trkA/efectos de los fármacos , Receptor trkA/metabolismo , Receptor trkC/metabolismo , Semaforina-3A , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
We set out to isolate inhibitory guidance cues that affect retinal ganglion cell (RGC) axons in vitro and that could potentially be involved in RGC pathfinding decisions. Here we describe the biochemical purification of an RGC growth cone collapsing factor from bovine brain membranes and its identification as Slit2. Recombinant human Slit2 collapses and repels RGC growth cones from all quadrants of the chick retina. In the developing mouse visual system, slit2 is expressed in the eye, in the optic stalk, and in the ventral diencephalon. Slit2 expression is strong in anterior ventral diencephalic structures but is absent from the ventral midline where the optic chiasm forms. The putative receptors for Slits, robo1 and robo2, are expressed in the inner retinal layer in which RGCs are located. A comparison of the expression patterns of Slit2 and retinal axon trajectories suggests that slit2 acts as a short range repellent for retinal ganglion cell axons.
Asunto(s)
Axones/fisiología , Proteínas del Tejido Nervioso/fisiología , Células Ganglionares de la Retina/fisiología , Animales , Axones/efectos de los fármacos , Encéfalo/fisiología , Bovinos , Membrana Celular/fisiología , Embrión de Pollo , Ganglios Espinales/embriología , Glicoproteínas/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Ratones , Fibras Nerviosas/fisiología , Factor de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Técnicas de Cultivo de Órganos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Células Ganglionares de la Retina/efectos de los fármacos , Semaforina-3A , Transcripción GenéticaRESUMEN
The semaphorins are a family of intercellular signaling proteins that has grown to include 19 identified members in higher vertebrates. Several of its members act as axonal guidance molecules. One participates in signaling in the immune system. The majority, however, do not yet have known biological functions. Recent studies have shown that neuropilins and plexins act as receptors for semaphorins. The most important challenge for the future is to define the biological roles of semaphorins in vivo.
Asunto(s)
Axones/metabolismo , Glicoproteínas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Movimiento Celular , Eliminación de Gen , Glicoproteínas/genética , Invertebrados , Ratones , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/citología , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Neuropilina-1 , Semaforina-3A , Transducción de Señal , VertebradosRESUMEN
Sensory axons extend from the chick olfactory epithelium to the telencephalon well before the maturation of their target, the olfactory bulb. During a waiting period of several days, olfactory axons arrive and accumulate outside the CNS while the bulb differentiates beneath them. Semephorin-3A is expressed in the tel-encephalon during this period and has been proposed to prevent their entry into the CNS. We show that the misexpression of a dominant-negative neuropilin-1 that blocks SEMA-3A-mediated signaling in olfactory sensory axons induces many of them to enter the tel-encephalon prematurely and to overshoot the olfactory bulb. These results suggest that chemorepellents can prevent the premature innervation of immature targets.
Asunto(s)
Axones/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/metabolismo , Nervio Olfatorio/embriología , Animales , Axones/ultraestructura , Sistema Nervioso Central/citología , Sistema Nervioso Central/embriología , Embrión de Pollo , Electroporación , Genes Dominantes , Péptidos y Proteínas de Señalización Intercelular , Proteínas del Tejido Nervioso/genética , Neuronas Aferentes/citología , Neuropilina-1 , Nervio Olfatorio/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Semaforina-3A , Telencéfalo/embriología , TransfecciónRESUMEN
Neuropilins have recently been characterized as receptors for secreted semaphorins. Here, we report the generation of a dominant negative form of neuropilin-1 by the deletion of one of its extracellular domains. Expression of this variant in cultured primary sympathetic neurons blocks the paralysis of growth cone motility normally induced by SEMA-3A (collapsin-1, semaphorin III, semaphorin D) and SEMA-3C (collapsin-3, semaphorin E) but not that induced by SEMA-3F (semaphorin IV). A truncated form of neuropilin-1 that is missing its cytoplasmic domain fails to act as a dominant negative receptor component. These results suggest that neuropilin-1 is a necessary component of receptor complexes for some, but not all, secreted semaphorin family members. Overexpression of dominant negative neuropilins should provide a powerful new method of blocking the functions of secreted semaphorins.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Receptores de Superficie Celular/fisiología , Animales , Células COS , Células Cultivadas , Embrión de Pollo , Pollos , Ganglios Simpáticos/fisiología , Humanos , Modelos Moleculares , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuropilina-1 , Reacción en Cadena de la Polimerasa , Conformación Proteica , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Semaforina-3A , Eliminación de Secuencia , TransfecciónRESUMEN
Neuropilin-1 (NRP1) is a receptor for two unrelated ligands with disparate activities, vascular endothelial growth factor-165 (VEGF165), an angiogenesis factor, and semaphorin/collapsins, mediators of neuronal guidance. To determine whether semaphorin/collapsins could interact with NRP1 in nonneuronal cells, the effects of recombinant collapsin-1 on endothelial cells (EC) were examined. Collapsin-1 inhibited the motility of porcine aortic EC (PAEC) expressing NRP1 alone; coexpressing KDR and NRP1 (PAEC/KDR/NRP1), but not parental PAEC; or PAEC expressing KDR alone. The motility of PAEC expressing NRP1 was inhibited by 65-75% and this inhibition was abrogated by anti-NRP1 antibody. In contrast, VEGF165 stimulated the motility of PAEC/KDR/NRP1. When VEGF165 and collapsin-1 were added simultaneously to PAEC/KDR/NRP1, dorsal root ganglia (DRG), and COS-7/NRP1 cells, they competed with each other in EC motility, DRG collapse, and NRP1-binding assays, respectively, suggesting that the two ligands have overlapping NRP1 binding sites. Collapsin-1 rapidly disrupted the formation of lamellipodia and induced depolymerization of F-actin in an NRP1-dependent manner. In an in vitro angiogenesis assay, collapsin-1 inhibited the capillary sprouting of EC from rat aortic ring segments. These results suggest that collapsin-1 can inhibit EC motility as well as axon motility, that these inhibitory effects on motility are mediated by NRP1, and that VEGF165 and collapsin-1 compete for NRP1-binding sites.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Glicoproteínas/farmacología , Linfocinas/farmacología , Proteínas del Tejido Nervioso/metabolismo , Actinas/metabolismo , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/crecimiento & desarrollo , Unión Competitiva , Línea Celular , Citoesqueleto/efectos de los fármacos , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/crecimiento & desarrollo , Endotelio Vascular/metabolismo , Ganglios Espinales/efectos de los fármacos , Glicoproteínas/metabolismo , Humanos , Técnicas In Vitro , Linfocinas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Neuropilina-1 , Seudópodos/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Semaforina-3A , Porcinos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Chick collapsin-1/human semaphorin III/mouse semaphorin D is believed to guide the extension of specific axons by a repellent mechanism. Here we examine its role in the guidance of axons of the ganglion of Remak (Remak) in the developing chick intestine. Early in embryogenesis Remak axons extend parallel to, but do not enter, the intestine when collapsin-1 is expressed in the adjacent rectal wall. Remak axons later penetrate the peripheral portions of the rectal wall when collapsin-1 expression retreats from the outer muscle layer to the more internal submucosal and mucosal layers of the rectum. Extension of Remak neurites is repelled in vitro by rectum explants and also by 293T cells expressing collapsin-1. The rectal chemorepellent activity is blocked by anti-collapsin-1 antibodies. Our results suggest that collapsin-1 may help prevent Remak axons from projecting into the intestinal wall at early developmental times and later restricts Remak axon trajectories to the outer part of the intestinal muscle layer.
Asunto(s)
Axones/metabolismo , Glicoproteínas/fisiología , Intestinos/embriología , Intestinos/inervación , Fosfatasa Alcalina/metabolismo , Animales , Axones/inmunología , Células Cultivadas , Embrión de Pollo , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Glicoproteínas/análisis , Glicoproteínas/inmunología , Inmunohistoquímica , Hibridación in Situ , Intestinos/citología , Laminina/metabolismo , Proteínas del Tejido Nervioso/análisis , Neuropilina-1 , Proteoglicanos/metabolismo , Recto/embriología , Recto/metabolismo , Semaforina-3ARESUMEN
Most axons in the CNS innervate specific subregions or layers of their target regions and form contacts with specific types of target neurons, but the molecular basis of this process is not well understood. To determine whether collapsin-1/semaphorin-III/D, a molecule known to repel specific axons, might guide afferent axons within their cerebellar targets, we characterized its expression by in situ hybridization and observed its effects on mossy and climbing fiber extension and growth cone size in vitro. In newborn mice sema-D is expressed by cerebellar Purkinje cells in parasagittal bands located medially and in some cells of the cerebellar nuclei. Later, sema-D expression in Purkinje cells broadens such that banded expression is no longer prominent, and expression is detected in progressively more lateral regions. By postnatal day 16, expression is observed throughout the cerebellar mediolateral axis. Collapsin-1 protein, the chick ortholog of sema-D, did not inhibit the extension of neurites from explants of inferior olivary nuclei, the source of climbing fibers that innervate Purkinje cells. In contrast, when it was applied to axons extending from basilar pontine explants, a source of mossy fiber afferents of granule cells, collapsin-1 caused most pontine growth cones to collapse, as evidenced by a reduction in growth cone size of up to 59%. Moreover, 63% of pontine growth cones arrested their extension or retracted. Its effects on mossy fiber extension and its distribution suggest that sema-D prevents mossy fibers from innervating inappropriate cerebellar target regions and cell types.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Glicoproteínas/genética , Fibras Nerviosas/metabolismo , Factores de Crecimiento Nervioso/genética , Neuronas/metabolismo , Puente/metabolismo , Animales , Animales Recién Nacidos , Axones/metabolismo , Técnicas de Cultivo , Conos de Crecimiento/metabolismo , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Neuronas/ultraestructura , Núcleo Olivar/crecimiento & desarrollo , Núcleo Olivar/metabolismo , Núcleo Olivar/ultraestructura , Células de Purkinje/metabolismo , Semaforina-3ARESUMEN
Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. We examined whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse was accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases were capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolished CNS myelin-induced collapse and allowed remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1-induced growth cone collapse and promoted neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 were effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse.
Asunto(s)
Glicoproteínas/farmacología , Conos de Crecimiento/fisiología , Neuronas Motoras/fisiología , Vaina de Mielina/fisiología , Actinas/fisiología , Adenoviridae/genética , Animales , Proteínas de Ciclo Celular/fisiología , Células Cultivadas , Sistema Nervioso Central/embriología , Embrión de Pollo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Vectores Genéticos , Neuronas Motoras/enzimología , Mutación/fisiología , Neuritas/fisiología , Semaforina-3A , Proteína de Unión al GTP cdc42 , Proteínas de Unión al GTP rac , Proteína de Unión al GTP rhoARESUMEN
The semaphorin/collapsin gene family is a large and diverse family encoding both secreted and transmembrane proteins, some of which are thought to act as repulsive axon guidance molecules. However, the function of most semaphorins is still unknown. We have cloned and characterized several semaphorins in the zebrafish in order to assess their in vivo function. Zebrafish semaZ2 is expressed in a dynamic and restricted pattern during the period of axon outgrowth that indicates potential roles in the guidance of several axon pathways. Analysis of mutant zebrafish with reduced semaZ2 expression reveals axon pathfinding errors that implicate SemaZ2 in normal guidance.
Asunto(s)
Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Axones/fisiología , Región Branquial/embriología , Sistema Nervioso Central/embriología , Pollos , Regulación del Desarrollo de la Expresión Génica , Mesencéfalo/embriología , Datos de Secuencia Molecular , Mutación , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Cresta Neural/embriología , Notocorda/embriología , Prosencéfalo/embriología , ARN Mensajero , Rombencéfalo/embriología , Semaforinas , Homología de Secuencia de Aminoácido , Médula Espinal/embriología , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/fisiologíaRESUMEN
Chick collapsin-1, the first identified vertebrate member of the semaphorin family of axon guidance proteins, repels specific growth cones. Like all family members, collapsin-1 contains within its sequence a semaphorin domain that is necessary for specifying activity. Two additional structural domains of collapsin-1, the immunoglobulin (Ig) domain and the basic tail, each potentiate collapsin-1 activity. We identify in this study another structural feature of collapsin-1 that is necessary for its function. Collapsin-1 covalently dimerizes, and dimerization is necessary for collapse activity. This dimerization is mediated through a cysteine at residue 723, between the Ig domain and basic tail. The semaphorin domain alone is not active since it cannot dimerize. The collapsing activity of the semaphorin domain can be reconstituted when made as a chimeric construct with an immunoglobin Fc domain, which promotes dimerization.
Asunto(s)
Glicoproteínas/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Cromatografía Líquida de Alta Presión , Cisteína/metabolismo , Dimerización , Glicoproteínas/genética , Humanos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Semaforina-3ARESUMEN
The semaphorin family contains secreted and transmembrane signaling proteins that function in the nervous, immune, and cardiovascular systems. Chick collapsin-1 is a repellent for specific growth cones. Two other secreted members of the semaphorin family, collapsin-2 and -3, are structurally similar to collapsin-1 but have different biological activities. Semaphorins contain a 500 amino acid family signature semaphorin domain. We show in this study that (1) the semaphorin domain of collapsin-1 is both necessary and sufficient for biological activity, (2) the semaphorin domain contains a 70 amino acid region that specifies the biological activity of the three family members, and (3) the positively charged carboxy terminus potentiates activity without affecting specificity. We propose that semaphorins interact with their receptors through two independent binding sites: one that mediates the biological response and one that potentiates it.
Asunto(s)
Comunicación Celular/fisiología , Secuencia Conservada , Glicoproteínas/química , Secuencia de Aminoácidos , Animales , Proteínas Aviares , Células Cultivadas , Embrión de Pollo , Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1 , Plásmidos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Semaforina-3A , Sensibilidad y EspecificidadRESUMEN
Collapsin-1, a member of the semaphorin family, activates receptors on specific growth cones, thereby inhibiting their motility. Neuropilin, a previously cloned transmembrane protein, has recently been identified as a candidate receptor for collapsin-1. We have completed the cloning of chick collapsin-3 and -5 and show that collapsin-1, -2, -3, and -5 bind to overlapping but distinct axon tracts. We infer that in situ, there are distinct receptors with different affinities for collapsin-1, -2, -3, and -5. In contrast, these four collapsins all bind recombinant neuropilin with similar affinities. Strong binding to neuropilin is mediated by the carboxy third of the collapsins, while the semaphorin domain confers their unique binding patterns in situ. We propose that neuropilin is a common component of a semaphorin receptor complex, and that additional differentially expressed receptor components interact with the semaphorin domains to confer binding specificity.
Asunto(s)
Proteínas Aviares , Proteínas Portadoras/genética , Glicoproteínas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Semaforinas , Animales , Células COS/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Pollos , Clonación Molecular , Regulación del Desarrollo de la Expresión Génica/fisiología , Glicoproteínas/química , Péptidos y Proteínas de Señalización Intercelular , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/química , Neuropilina-1 , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Semaforina-3A , Sensibilidad y Especificidad , Homología de Secuencia de Aminoácido , Médula Espinal/química , Médula Espinal/embriología , Colículos Superiores/química , Colículos Superiores/embriologíaRESUMEN
Collapsin-1 is a member of the semaphorin family of signaling molecules that acts as a repellent for growing spinal sensory axons. We have constructed a chimeric collapsin-1/alkaline phosphatase probe to visualize putative collapsin-1 receptors in vitro and in situ. As predicted by the activity profile of collapsin-1, the probe binds spinal sensory tracts, ventral spinal roots, and the sympathetic chain but does not bind retinal axons. In addition, we find that the probe binds sensory axons arising from the olfactory epithelium and some, but not all, cranial sensory nerves. As predicted by these binding studies, in vitro assays demonstrate that primary olfactory sensory, trigeminal, and jugular ganglion growth cones collapse in the presence of soluble collapsin-1. Comparing the expression pattern of collapsin-1 with the trajectories of collapsin-1 responsive axons suggests that in both the spinal cord and the olfactory bulb, collapsin-1 prevents premature entry of sensory axons into their target and helps determine the final location of sensory terminations.
Asunto(s)
Axones/fisiología , Nervios Craneales/embriología , Glicoproteínas/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas Aferentes/fisiología , Bulbo Olfatorio/embriología , Nervio Olfatorio/embriología , Médula Espinal/embriología , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/genética , Animales , Células Cultivadas , Embrión de Pollo , Ganglios Espinales/citología , Ganglios Espinales/embriología , Glicoproteínas/genética , Humanos , Morfogénesis , Proteínas del Tejido Nervioso/genética , Bulbo Olfatorio/citología , Técnicas de Cultivo de Órganos , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Retina/embriología , Semaforina-3A , Raíces Nerviosas Espinales/embriología , Nervio Vago/embriologíaRESUMEN
During embryogenesis, different subclasses of sensory neurons extend central projections to specific locations in the spinal cord. Muscle and cutaneous afferents initially project to the same location in the dorsal cord. Later, specific muscle afferents leave other afferents behind and project into the ventral cord. Previous studies have shown that ventral spinal cord explants secrete a repellent for sensory neurites. We now find that antibodies to collapsin-1 neutralize this repellent activity. Additional data suggest that all afferents respond to collapsin-1 when they are first confined to the dorsal cord, but that ventrally projecting muscle afferents become collapsin-1 insensitive as they project into the ventral cord. Our results suggest that the transient dorsal expression of collapsin-1 prevents all efferents from entering the cord early and sustained ventral expression prevents dorsally terminating afferents from entering the ventral cord later.