Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.     

The synthesis and SAR of 2-arylsulfanyl-phenyl piperazinyl acetic acids as glyT-1 inhibitors

Elevation of glycine levels and activation of the NMDA receptor by inhibition of the glycine transporter 1 (GlyT-1) is a potential strategy for the treatment of schizophrenia. A novel series of GlyT-1 inhibitors have been identified containing the 2-arylsulfanyl-phenylpiperazine motif. The most prominent member of this series, (R)-4-[5-chloro-2-(4-methoxy-phenylsulfanyl)-phenyl]-2-methyl-piperazin-1-yl-acetic acid (31) is a potent glycine transporter-1 inhibitor (IC(50)=150 nM), which elevated glycine levels in rat ventral hippocampus as measured by microdialysis in vivo at doses of 1.2-4.6 mg/kg s.c.     

Abundance of Zinc Ions in Synaptic Terminals of mocha Mutant Mice: Zinc Transporter 3 Immunohistochemistry and Zinc Sulphide Autometallography

The mocha mouse is an autosomal recessive pigment mutant on mouse chromosome 10 caused by a deletion in the gene for the delta subunit of the adaptor-like complex AP-3. Based on zinc transporter 3 (ZnT3) immunohistochemistry, zinc TSQ fluorescence and a modified Timm method, previous studies found a lack of histochemically-detectable zinc and a substantial reduction in the ZnT3 immunoreactivity. It has, therefore, been suggested that the mocha mouse could serve as a model for studies of the significance of zinc ions in zinc-enriched (ZEN) neurons. We have chosen the mocha-zinc-model in a study of the significance of ZEN neurons in hypoxia-caused damage in mouse brain. In order to establish that the model was either void of zinc ions or had a significantly decreased level of zinc ions in their ZEN terminals, we repeated the studies that had lead to the above assumption, the only methodology difference being that we used the zinc specific Neo-Timm method instead of the Timm method applied in the original study. We found that, although the ZnS autometallography (AMG) technique revealed a reduction in staining intensity as compared to the littermate controls, there were still plenty of zinc ions in the ZEN terminals, in particular visible in telencephalic structures like neocortex and hippocampus. At ultrastructural levels the zinc ions were found in a pool of vesicles of the ZEN terminals as in the control animals, but additionally zinc ions could be traced in ZEN neuronal somata in the neocortex and hippocampus. The mossy fibres in the hippocampus of mocha mice also bind with TSQ, though less than in the controls. We found ZnS AMG grains in ZEN neuronal somata, which were also immunoreactive for ZnT3. Our study confirmed the decreased ZnT3 immunoreactivity in ZEN terminals of the mocha mouse found in the original study. Based on these findings, we suggest that the mocha mouse may not be an ideal model for studies of the histochemically-detectable zinc ion pool of the central nervous system.     

HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.     

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.     

PCR-RFLP is used to investigate relations among species in the entomopathogenic genera Eryniopsis and Entomophaga

The shape and nucleation of primary conidia are important characters in the classification of the Entomophthoraceae (Zygomycetes). The five species in the genus Eryniopsis vary in the shapes of primary conidia, although within most genera in the order Entomophthorales species have the same shapes of primary conidia. Using PCR-RFLP, we investigated two species in Eryniopsis, Ery. caroliniana with oblong-ovoid primary conidia and Ery. ptychopterae with pear-shaped primary conidia, with five species of Entomophaga, all having pear-shaped conidia. Molecular results merged with morphological data indicate that Ery. ptychopterae belongs in the genus Entomophaga while Ery. caroliniana clearly differs from Entomophaga. Ery. ptychopterae and Ery. transitans are transferred to the genus Entomophaga. Our results support the idea that morphology of primary conidia is of major importance in defining entomophthoralean genera. These results also show that such studies can be conducted with species that have not been isolated, if fungal-filled cadavers can be obtained.     

UNC-119 suppresses axon branching in C. elegans

The architecture of the differentiated nervous system is stable but the molecular mechanisms that are required for stabilization are unknown. We characterized the gene unc-119 in the nematode Caenorhabditis elegans and demonstrate that it is required to stabilize the differentiated structure of the nervous system. In unc-119 mutants, motor neuron commissures are excessively branched in adults. However, live imaging demonstrated that growth cone behavior during extension was fairly normal with the exception that the overall rate of migration was reduced. Later, after development was complete, secondary growth cones sprouted from existing motor neuron axons and cell bodies. These new growth cones extended supernumerary branches to the dorsal nerve cord at the same time the previously formed axons retracted. These defects could be suppressed by expressing the UNC-119 protein after embryonic development; thus demonstrating that UNC-119 is required for the maintenance of the nervous system architecture. Finally, UNC-119 is located in neuron cell bodies and axons and acts cell-autonomously to inhibit axon branching.