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

A putative plastidic glucose translocator is expressed in heterotrophic tissues that do not contain starch, during olive (Olea europea L.) fruit ripening

Metabolite-specific transporters are present in the inner membrane of the plastid envelope allowing transport between the plastid and other cellular compartments. A plastidic glucose translocator (pGlcT) in leaf mesophyll cells transports glucose from chloroplast stroma to the cytosol after amylolytic starch degradation at night. Here we report the cloning of a pGlcT expressed in olive fruits (Olea europea L.). Our results showed high expression of pGlcT in non-green heterotrophic fruit tissues. Expression of pGlcT in olive fruits was somewhat higher compared to leaves, and continued until the black, mature fruit stage. We cloned part of tomato pGlcT and found that it is also expressed throughout fruit development implying a role for pGlcT in heterotrophic tissues. Light and electron microscopic characterization of plastid structural changes during olive fruit ripening revealed the transition of chloroplast-like plastids into starchless, non-green plastids; in mature olive fruits only chromoplasts were present. Together, these findings suggest that olive pGlcT is abundant in chromoplasts during structural changes, and provide evidence that pGlcT may play different physiological roles in ripening fruits and possibly in other non-photosynthetic organs.This work was supported by the Instituto National de Investigaciones Agrarias (INIA) through Project no. CAO99-003, and by a fellowship to R.B. awarded by the Ministry of Education, Culture and Sports as part of Estancias de Cientificos y Tecnologos Extranjeros en España program (2000–2002).Peer reviewe

Distribution of loosely-bound calcium in the vegetative and generative cells of the pollen grains in Chlorophytum elatum

We used chlorotetracycline fluorescence, alizarin staining and potassium pyroantimonate methods, as well as X-ray microanalysis, to demonstrate the differential localization of Ca2+ during pollen maturation. Level of loosely-bound Ca2+ ions was higher in the generative cell than in the vegetative cell of the mature pollen grain which is one of the symptoms of metabolic differentiation of the two sister cells.Peer reviewe

Multi-tasking by the p75 neurotrophin receptor: sortilin things out?

Signalling by the p75 neurotrophin receptor has been implicated in diverse neuronal responses, including increased differentiation or survival, inhibition of regeneration, and initiation of apoptotic cell death. These numerous roles are matched by, but are not yet correlated with, a multiplicity of extracellular ligands and intracellular interactors. Membrane proteins such as sortilin, a member of the Vps10p family of sorting receptors, and the glycosylphosphatidylinositol-linked Nogo receptor (NgR) and the associated adaptor lingo 1 have recently been added to the list of p75-interacting modulators. Other studies have described intramembranal cleavage of p75 and the potential nuclear targeting of cleavage fragments or of the complete receptor after it has been internalized into a putative signalling endosome. These findings suggest that some of the diversity in p75 activities might be due to differential subcellular localization and transport of p75 receptor complexes. We therefore argue that cell-biology-driven approaches are now required to make sense of p75 signalling

Involvement of JIM13- and JIM8-responsive carbohydrate epitopes in early stages of cell wall formation

We studied the possible involvement of JIM13- and JIM8-responsive epitopes in the formation of the cell wall at the surface of regenerating protoplasts of sugar beet (Beta vu/garis L.). The spatial and temporal expression of both antigens was studied in suspension cells used as the source-tie for protoplast isolation, in suspension- and mesophyll-derived protoplasts, and in cells which developed from both types of protoplast. lmmunofluorescence and immunocytochemical-electron microscopic methods revealed that labeling was present in the cell walls of most suspension cells and also in the incipients of cell walls synthesized around the protoplasts. This signal became much more intense as rebuilding of the cell wall progressed during culture. Relatively weaker labeling was observed in the cytoplasm, where it was frequently associated with the vacuolar compartment. Signal intensity varied between individual cells of the Same population and in successive stages of development, but was always stronger with JIM13 than with JIM8. The role of JIM13-responsive epitope in the development of suspension-derived protoplasts was further studied by its ability to bind antibody added to cultures of dierent ages. Both JIM8- and JIM13-responsive epitopes were widespread in sugar beet cells of dierent origin and stage of cell wall synthesis. These epitopes may play an important role in cell wall formation and growth under in vh conditions.This study was supported by the Polish Committee for Scientific Research (project no. 6 PWC 087 12 and a Bilateral Action Poland-Spain).Peer reviewe

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

Marcadores moleculares que se expresan en el fruto del olivo

13 páginas, 17 figuras, 1 tabla.[EN] With the aim of setting new molecular markers of certain function in the olive, several genes expressed during olive fruit ripening have been isolated and characterized, also analyzing temporal an spatial expression of such genes. Among other, the following marker genes have been analyzed: Δ9- stearoyl-ACP-desaturase, a key enzyme for fatty acid biosynthesis; a putative plastidic glucose translocator (pGlcT) expressed in non-photosynthetic tissues like the mature olive fruit; and finally, polyubiquitin and cullin, components of the ubiquitin-mediated protein degradation system. In a parallel way, protein extracts have been isolated from olive reach, in order to analyze their protein profiles by SDS-PAGE. The major protein component of such extracts corresponds to a family of storage proteins of the 11Stype. After raising a rabbit antiserum to these proteins, a spatial and temporal analysis of them was performed during seed development and germination. Finally, oleosin purification from olive seeds has been accomplished. The biological meaning attributed to each of these markers, forecast their future usage in olive improvement programmes. However, more information particularly concerning the knowledge of these genes and their expression is needed, and therefore, basic research on olive should be carried through[ES] Con objeto de establecer nuevos marcadores moleculares en el olivo con función determinada, se han aislado y caracterizado varios genes que se expresan durante la maduración del fruto del olivo, y se ha realizado un análisis temporal y espacial de la expresión de estos genes. Entre otros, se han analizado los siguientes marcadores: la Δ9- estearoyl-ACP- desaturasa, enzima clave en la biosíntesis de ácidos grasos; un posible translocador plastídico de glucosa (pGlcT), que se expresa en tejidos no fotosintéticos como es el fruto maduro del olivo; y finalmente, también se han identificado y caracterizado poliubiquitina y culina, componentes del sistema de degradación proteica mediado por ubiquitina. Paralelamente, se han preparado extractos crudos a partir de semillas de olivo con objeto de analizar sus perfiles proteicos mediante SDS-PAGE,. Los componentes mayoritarios de estos extractos corresponden a una familia de proteínas de almacenamiento tipo 11S. Después de obtener un suero específico para este tipo de proteínas, se hizo un seguimiento espacial y temporal de las mismas durante el desarrollo y germinación de las semillas. Por último, también se ha llevado a cabo la purificación de oleosinas a partir de semillas. El significado biológico atribuido a cada uno de estos marcadores, hace preveer que en un futuro puedan ser utilizados en programas de mejora del olivo. Pero antes se requiere una mayor información y profundizar en el conocimiento de estos genes y su expresión, por lo que es necesario continuar las investigaciones a nivel básico en el olivo.Este trabajo fue financiado por el proyecto INIA-CAO99-003.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