Mechanisms of the release of anterogradely transported neurotrophin-3 from axon terminals

Neurotrophins have profound effects on synaptic function and structure. They can be derived from presynaptic, as well as postsynaptic, sites. To date, it has not been possible to measure the release of neurotrophins from axon terminals in intact tissue. We implemented a novel, extremely sensitive assay for the release and transfer of anterogradely transported neurotrophin-3 (NT-3) from a presynaptic to a postsynaptic location that uses synaptosomal fractionation after introduction of radiolabeled NT-3 into the retinotectal projection of chick embryos. Release of the anterogradely transported NT-3 in intact tissue was assessed by measuring the amount remaining in synaptosomal preparations after treatment of whole tecta with pharmacological agents. Use of this assay reveals that release of NT-3 from axon terminals is increased by depolarization, calcium influx via N-type calcium channels, and cAMP analogs, and release is most profoundly increased by excitation with kainic acid or mobilization of calcium from intracellular stores. NT-3 release depends on extracellular sodium, CaM kinase II activity, and requires intact microtubules and microfilaments. Dantrolene inhibits the high potassium-induced release of NT-3, indicating that release of calcium from intracellular stores is required. Tetanus toxin also inhibits NT-3 release, suggesting that intact synaptobrevin or synaptobrevin-like molecules are required for exocytosis. Ultrastructural autoradiography and immunolabel indicate that NT-3 is packaged in presumptive large dense-core vesicles. These data show that release of NT-3 from axon terminals depends on multiple regulatory proteins and ions, including the mobilization of local calcium. The data provide insight in the mechanisms of anterograde neurotrophins as synaptic modulators

Proteínas ubiquitinadas en el polen y su uso potencial como marcadores de androgénesis

7 páginas, 3 figuras.-- rabajo presentado al XII Simposio de Palinología.[EN]: Ubiquitin is a small, highly conserved protein which is present in all eukaryotes. This protein is found either as a free form, o covanlently linked to substrate proteins, often targeting them for degradation by the proteasoime. Changes in the ubiquitinated species and/or their transcripts, have been related to a number of cellular processes, including stress response and cell cycle control. In olive (Olea europaea L.) pollen, inmunoblot experiments have shown a reduction in the free ubiquitin level taking place in late stages of development. However, this reduction does not coincide with a parallel reduction in the level of the corresponding transcripts, as detected by RT-PCR. In adition, the involvement of the ubiquitin-mediated degradation pathway in pollen cell cycle control is discussed. A model for androgenic induction mediated by ubiquitin is also proposed.[ES]: La ubiquitina es una proteína de pequeño tamaño, muy conservada, y presente en todos los eucariotas. Esta proteína se encuentra en forma libre o unida a proteínas substrato, dirigiendo a éstas hacia su degradación por el proteosoma. Los cambios en los niveles de especies ubiquitinadas, asi como en sus mensajeros, están relacionados con diversos procesos celulares entre los que figuran la respuesta a estrés y el control del ciclo celular. En el polen del olivo (Olea europaea L.), experimentos de inmunoblots han mostrado que existe una dismiución de los niveles de ubiquitina libre en los estadios tardíos del desarrollo del polen. Sin embargo, esta disminución no se corresponde con un descenso en los niveles de transcritos correspondientes, tal y como demuestran los análisis de RT-PCR. Por otra parte. discutimos la posible implicación del sistema degradativo mediado por ubiquitina en el control del ciclo celular del polen y proponemos un modelo de la inducción de androgénesis mediada por dicho sistema.Este trabajo ha sido financiado por la DGES (Proyecto PB95-0080).Peer reviewe

Could ubiquitin and ubiquitinated proteins be used as molecular markers for embryogenesis induction of pollen?

3 páginas.-- Trabajo presentado al 2º Meeting Working Group 4: "Fundamental Aspects of Gametic Embryogenesis" y al Workshop: "Molecular and Cellular Approaches to Gametic E,bryogenesis" celebrados en Granada (España) del 3 al 5 de Julio de 1997.This work has been supported by the Spanish DGES (Project PB95-0080) and the TMR programme of the EC (FMBI-CT95-0470).Peer reviewe

Ubiquitin and ubiquitin-conjugated proteins in the olive ( olea europaea l.) pollen

7 páginas, 4 figuras.A study of free ubiquitin and ubiquitinated proteins has been carried out in olive (Olea europaea L.) during the late stages of pollen development and during in-vitro pollen hydration. As described in relatively few plant species, immunoblot experiments carried out using crude protein extracts have shown a reduction in the free ubiquitin level taking place during the mature pollen stage. This reduction does not coincide with a parallel reduction in the level of the corresponding transcripts, as detected by RT-PCR analysis, suggesting the existence of a post-transcriptional regulatory mechanism. Relatively high levels of free ubiquitin are quickly restored upon in-vitro pollen hydration. TEM immunocytochemical localisation of ubiquitin-conjugated compounds in the mature pollen grain showed the antibodies bound to the cytoplasm, the chromatin and the nucleolus of the vegetative and generative cells. Within the cytoplasm, labelling was concentrated in the cytosol, the endoplasmic reticulum membranes and minuscule vesicles associated to the Golgi apparatus. The fibrillar material adhering to the exine, and probably derived from the tapetum, also showed gold particles. Lipid bodies, aperture regions, mitochondria, the intine and the cell wall were devoid of labelling. The changes observed throughout pollen development, as well as the specific localisation, reflect the involvement of the ubiquitin-mediated degradative pathway in regulation of pollen metabolism.This work was supported by the Spanish DGES (Research project PB95-0080).Peer reviewe

Ubiquitina y proteínas ubiquitinadas en el polen. Un modelo de la implicación del sistema en la inducción de androgénesis

Resumen del trabajo presentado al XII Simposio de Palinología (APLE) celebrado en León (España) en 1998.Trabajo financiado por los Proyectos PB95-0080 y OLI96-2160 (DGES,MEC).Peer reviewe