Aqueous solubilization of transmembrane peptide sequences with retention of membrane insertion and function.

We recently reported that the peptide C-K4-M2GlyR mimics the action of chloride channels when incorporated into the apical membrane of cultured renal epithelial monolayers. C-K4-M2GlyR is one of a series of peptides that were prepared by the addition of lysine residues to the N- or C-terminus of the M2 transmembrane sequence of the brain glycine receptor. This study addresses how such modifications affect physical properties such as aqueous solubility, aggregation, and secondary structure, as well as the ability of the modified peptides to form channels in epithelial monolayers. A graded improvement in solubility with a concomitant decrease in aggregation in aqueous media was observed for the M2GlyR transmembrane sequences. Increases in short-circuit current (I(SC)) of epithelial monolayers were observed after treatment with some but not all of the peptides. The bioactivity was higher for the more soluble, less aggregated M2GlyR peptides. As described in our previous communication, sensitivity of channel activity to diphenylamine-2-carboxylate, a chloride channel blocker, and bumetanide, an inhibitor of the Na/K/2Cl cotransporter, was used to assess changes in chloride selectivity for the different assembled channel-forming peptides. The unmodified M2GlyR sequence and the modified peptides with less positive charge are more sensitive to these agents than are the more highly charged forms. This study shows that relatively insoluble transmembrane sequences can be modified such that they are easier to purify and deliver in the absence of organic solvents with retention of membrane association, insertion, and assembly.     

Left handed �� helix models for mammalian prion fibrils

We propose models for in vitro grown mammalian prion protein fibrils based upon left handed beta helices formed both from the N-terminal and C-terminal regions of the proteinase resistant infectious prion core. The C-terminal threading onto a β-helical structure is almost uniquely determined by fixing the cysteine disulfide bond on a helix corner. In comparison to known left handed helical peptides, the resulting model structures have similar stability attributes including relatively low root mean square deviations in all atom molecular dynamics, substantial side-chain-to-side-chain hydrogen bonding, good volume packing fraction, and low hydrophilic/hydrophobic frustration. For the N-terminus, we propose a new threading of slightly more than two turns, which improves upon the above characteristics relative to existing three turn β-helical models. The N-terminal and C-terminal beta helices can be assembled into eight candidate models for the fibril repeat units, held together by large hinge (order 30 residues) domain swapping, with three amenable to fibril promoting domain swapping via a small (five residue) hinge on the N-terminal side. Small concentrations of the metastable C-terminal β helix in vivo might play a significant role in templating the infectious conformation and in enhancing conversion kinetics for inherited forms of the disease and explain resistance (for canines) involving hypothesized coupling to the methionine 129 sulfur known to play a role in human disease.Key words: prion, amyloid fibril, domain swap, beta helix, computational biology     

Optimisation of the P2 pharmacophore in a series of thrombin inhibitors: ion-dipole interactions with lysine 60G.

The optimisation of the P2 pharmacophore in a series of thrombin inhibitors is described. The interaction of a number of piperidine P2 functionalities with lysine 60G of thrombin is explored with reference to the crystal structure of inhibitor enzyme complexes. A primary ion-dipole interaction between the terminal P2 side chain group and lysine 60G is evoked to explain the SAR in this series.     

transAlign: using amino acids to facilitate the multiple alignment of protein-coding DNA sequences

Background  

Alignments of homologous DNA sequences are crucial for comparative genomics and phylogenetic analysis. However, multiple alignment represents a computationally difficult problem. For protein-coding DNA sequences, it is more advantageous in terms of both speed and accuracy to align the amino-acid sequences specified by the DNA sequences rather than the DNA sequences themselves. Many implementations making use of this concept of "translated alignments" are incomplete in the sense that they require the user to manually translate the DNA sequences and to perform the amino-acid alignment. As such, they are not well suited to large-scale automated alignments of large and/or numerous DNA data sets.     

Diel distribution of copepods across a channel of an overwash mangrove island

The distribution of copepod species and their nauplii was studied in a narrow, blind channel on an overwash mangrove island offshore of Belize. Copepodids were sampled with a pump at five stations across the channel during a diel cycle. Diel changes of copepodid stages II – VI were marked by horizontal dispersal of Dioithona oculata, the dominant species, from swarms in the prop roots along the shore during the day to the edge of the prop root habitat at night. Migration of copepodids back to the prop roots appeared to be controlled endogenously because change from a night to a daytime age structure began before first light. Mean copepodid stage at subsurface depths in the channel and prop root edge decreased from 4.2 (with 6.0 = all adults) to 2.9 at predawn to 1.1 during day. The oceanic Oithona nana and O. simplex, and the coastal zone O. fonsecae were evenly distributed with depth and distance from shore during day and night, with avoidance of prop root shoreline during day. These species were much less abundant than Dioithona oculata in the prop roots, but of comparable or greater abundance in the channel. Coastal zone Acartia spinata exhibited evidence of swarming. Nauplii, sampled with a 25m plankton net, were dominated by harpacticoid (50%) and cyclopoid (34%) nauplii, which generally were more abundant at 1m than at the surface and more abundant at night than the day. Lagrangian current measurements indicated velocities at ebb tide twice those of flood tide (1.9 vs. 0.8 cm s^–1) and a minimal residence time of 5 days, which could result in advection of D. oculata nauplii out of the Lair Channel before their recruitment into swarms as copepodid stage II. Previously reported maximum swimming speeds of swarming D. oculata copepodid stages (2.0 cm s^–1) and greater densities in prop roots and near the benthos may help copepodids avoid advection. The swarming behavior and diel horizontal migration (or dispersal) reported for D. oculata appears analogous to that of limnetic zooplankton, which may swarm among macrophytes along shorelines during the day to avoid visual predators and disperse or migrate away from the shoreline at night.     

Assembly of trunk and limb blood vessels involves extensive migration and vasculogenesis of somite-derived angioblasts

Vascular development requires the assembly of precursor cells into blood vessels, but how embryonic vessels are assembled is not well understood. To determine how vascular cells migrate and assemble into vessels of the trunk and limb, marked somite-derived angioblasts were followed in developing embryos. Injection of avian somites with the cell-tracker DiI showed that somite-derived angioblasts in unperturbed embryos migrated extensively and contributed to trunk and limb vessels. Mouse-avian chimeras with mouse presomitic mesoderm grafts had graft-derived endothelial cells in blood vessels at significant distances from the graft, indicating that mouse angioblasts migrated extensively in avian hosts. Mouse graft-derived endothelial cells were consistently found in trunk vessels, such as the perineural vascular plexus, the cardinal vein, and presumptive intersomitic vessels, as well as in vessels of the limb and kidney rudiment. This reproducible pattern of graft colonization suggests that avian vascular patterning cues for trunk and limb vessels are recognized by mammalian somitic angioblasts. Mouse-quail chimeras stained with both the quail vascular marker QH1 and the mouse vascular marker PECAM-1 had finely chimeric vessels, with graft-derived mouse cells interdigitated with quail vascular cells in most vascular beds colonized by graft cells. Thus, diverse trunk and limb blood vessels have endothelial cells that developed from migratory somitic angioblasts, and assembly of these vessels is likely to have a large vasculogenic component.     

Determination of the sequences of protein-derived peptides and peptide mixtures by mass spectrometry

Micro-quantities of protein-derived peptides have been converted into N-acetylated permethyl derivatives, and their sequences determined by low-resolution mass spectrometry without prior knowledge of their amino acid compositions or lengths. A new strategy is suggested for the mass spectrometric sequencing of oligopeptides or proteins, involving gel filtration of protein hydrolysates and subsequent sequence analysis of peptide mixtures. Finally, results are given that demonstrate for the first time the use of mass spectrometry for the analysis of a protein-derived peptide mixture, again without prior knowledge of the protein or components within the mixture.     

Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low‐affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin‐positive (PV) interneurons. Nrg3 and ErbB4 are located pre‐ and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short‐term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4⁺ interneurons and affects short‐term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non‐neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4⁺ interneurons.