Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.</jats:p

A prime/boost vaccination with HA DNA and Pichia -produced HA protein elicits a strong humoral response in chickens against H5N1

Highly pathogenic avian influenza viruses cause severe disease and huge economic losses in domestic poultry and might pose a serious threat to people because of the high mortality rates in case of an accidental transmission to humans. The main goal of this work was to evaluate the immune responses and hemagglutination inhibition potential elicited by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens in chickens. A plasmid encoding hemagglutinin (HA) from the A/swan/Poland/305-135V08/2006 (H5N1) virus, or the recombinant HA protein produced in Pichia pastoris system, both induced H5 HA–specific humoral immune responses in chickens. In two independent experiments, anti-HA antibodies were detected in sera collected two weeks after the first dose and the response was enhanced by the second dose of a vaccine, regardless of the type of subunit vaccine (DNA or recombinant protein) administered. The serum collected from chickens two weeks after the second dose was characterized by three types of assays: indirect ELISA, hemagglutination inhibition (HI) and a diagnostic test based on H5 antibody competition. Although the indirect ELISA failed to detect superiority of any of the three vaccine regimens, the other two tests clearly indicated that priming of chickens with the DNA vaccine significantly enhanced the protective potential of the recombinant protein vaccine produced in P. pastoris

Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules

Additional file 3: of Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens

Serum humoral response in individual mice and chickens after DNA immunization with and without lipofectin. (PPT 157 kb

Thickness and conductivity determination of thin coatings on ferromagnetic substrates in the case of cylindrical symmetry

An eddy current method allowing the determination of parameters of a thin nonmagnetic conductive coating on a ferromagnetic conductive substrate is reported. At a single operating frequency, two independent quantities can be determined: a permeability-to-conductivity ratio of the substrate and a thickness-conductivity product of the coating. Thus, thickness or conductivity of the coating can be determined independent of variations of the substrate magnetic and electrical parameters. A simple theoretical formula for the normalized electrical impedance of the test coil is obtained using asymptotic expansions of Bessel functions. The method was applied to the evaluation of electrogalvanized wires in the frequency range 100 kHz-1 MHz. A set of low carbon steel wires with diameter around 2.2 mm, coated with zinc layers having thicknesses in the range 2.7-64.6 mu m, was investigated using two long coils. Experimental data of the electrical impedance were compared to those predicted. Agreement between theory and experiment is excellent for coatings thicker than 12 mu m. Despite discrepancies between theory and experiment for very thin layers arising from various imperfections of the coating and interfaces, the method was applied successfully in the thickness range below 12 mu m. To do this, two parameters: an apparent conductivity of the coating and a thickness offset, were introduced. The mathematical inversion of the experimental data with the two-variable Newton-Raphson method and the asymptotic formula is extremely fast. The technique developed has an extremely low sensitivity to variations of the ferromagnetic substrate conductivity and magnetic permeability. A magnetizing field of 0-23 000 A/m, producing large variations in the substrate magnetic permeability, does not significantly influence results of the coating thickness determination. The agreement between measured thickness and that obtained by a chemical method is excellent, typically within 0.5 mu m. An uncertainty of the thickness or conductivity determination better than 1% is obtained. (C) 1997 American Institute of Physics