A novel solid phase approach to Aia‐containing peptides

A strategy was developed to directly assemble 4‐amino‐1,2,4,5‐tetrahydro‐indolo[2,3‐c]‐azepin‐3‐ones on solid‐phase‐supported peptide sequences. Fmoc‐ and Boc‐based strategies were investigated. The Fmoc‐strategy approach strongly depends on the peptide sequence being synthesized while the Boc‐based synthesis leads to excellent results for all the selected peptide analogs. The method was applied to prepare Aia‐analogs of several bioactive peptides containing one or more Trp‐residues which were shown to be important for biological recognition. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

TOPALi v2: a rich graphical interface for evolutionary analyses of multiple alignments on HPC clusters and multi-core desktops

Summary: TOPALi v2 simplifies and automates the use of severalmethods for the evolutionary analysis of multiple sequence alignments.Jobs are submitted from a Java graphical user interface as TOPALiweb services to either run remotely on high-performance computingclusters or locally (with multiple cores supported). Methodsavailable include model selection and phylogenetic tree estimationusing the Bayesian inference and maximum likelihood (ML) approaches,in addition to recombination detection methods. The optimalsubstitution model can be selected for protein or nucleic acid(standard, or protein-coding using a codon position model) datausing accurate statistical criteria derived from ML co-estimationof the tree and the substitution model. Phylogenetic softwareavailable includes PhyML, RAxML and MrBayes. Availability: Freely downloadable from http://www.topali.orgfor Windows, Mac OS X, Linux and Solaris. Contact: iain.milne{at}scri.ac.uk Associate Editor: Martin Bishop     

Invader–invader interactions in relation to environmental heterogeneity leads to zonation of two invasive amphipods, Dikerogammarus villosus (Sowinsky) and Gammarus tigrinus Sexton: amphipod pilot species project (AMPIS) report 6

As biological invasions continue, interactions occur not only between invaders and natives, but increasingly new invaders come into contact with previous invaders. Whilst this can lead to species replacements, co-existence may occur, but we lack knowledge of processes driving such patterns. Since environmental heterogeneity can determine species richness and co-existence, the present study examines habitat use and its mediation of the predatory interaction between invasive aquatic amphipods, the Ponto-Caspian Dikerogammarus villosus and the N. American Gammarus tigrinus. In the Dutch Lake IJsselmeer, we found broad segregation of D. villosus and G. tigrinus by habitat type, the former predominating in the boulder zone and the latter in the soft sediment. However, the two species co-exist in the boulder zone, both on the short and longer terms. We used an experimental simulation of habitat heterogeneity and show that both species utilize crevices, different sized holes in a plastic grid, non-randomly. These amphipods appear to optimise the use of holes with respect to their ‘C-shape’ body size. When placed together, D. villosus adults preyed on G. tigrinus adults and juveniles, while G. tigrinus adults preyed on D. villosus juveniles. Juveniles were also predators and both species were cannibalistic. However, the impact on G. tigrinus of the superior intraguild predator, D. villosus, was significantly reduced where experimental grids were present as compared to absent. This mitigation of intraguild predation between the two species in complex habitats may explain the co-existence of these two invasive species.     

Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies

Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.     

Characterization of bacterial proteases with a panel of fluorescent peptide substrates

Bacteria produce a range of proteolytic enzymes. In an attempt to detect and identify bacteria on the basis of their protease activity, a panel of protease substrates was investigated. Peptides conjugated to the fluorophore 7-amino-4-methylcoumarin (AMC) are well-established substrates for measuring protease activity. Although peptide-AMC substrates are generally not specific for a single protease, a unique pattern can be achieved for both highly specific enzymes and those with a broader substrate range by comparing different peptide substrates. The panel of 7 peptide-AMC substrates chosen exhibited a unique pattern for nine microbial proteases. The selected peptides were used to determine protease activity in cultured strains of Pseudomonas aeruginosa and Staphylococcus aureus. A signal pattern obtained with peptides with arginine, lysine, and tyrosine in the P1 position characterized the bacterial protease activities in these samples. The kinetic parameters for the three best substrates for the P. aeruginosa sample were calculated. Further information about substrate specificity was gained by the selective use of protease inhibitors. The results presented show that peptide-AMC substrates provide a simple and sensitive tool to characterize protease activity in microbiological samples and that they have the potential to identify and distinguish different bacterial species.     

Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.     

An arginine switch in the species B adenovirus knob determines high-affinity engagement of cellular receptor CD46

Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.