Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion

The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Many studies have shown that fusion involves the cooperative action of a large number of these glycoproteins, but the underlying mechanisms are unknown. We used electron microscopy and tomography to study the low pH-induced fusion reaction catalyzed by vesicular stomatitis virus glycoprotein (G). Pre- and post-fusion crystal structures were observed on virions at high and low pH, respectively. Individual fusion events with liposomes were also visualized. Fusion appears to be driven by two successive structural rearrangements of G at different sites on the virion. Fusion is initiated at the flat base of the particle. Glycoproteins located outside the contact zone between virions and liposomes then reorganize into regular arrays. We suggest that the formation of these arrays, which have been shown to be an intrinsic property of the G ectodomain, induces membrane constraints, achieving the fusion reaction.     

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm² on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10^-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.     

A Bioengineered Nisin Derivative to Control Biofilms of Staphylococcus pseudintermedius

Antibiotic resistance and the shortage of novel antimicrobials are among the biggest challenges facing society. One of the major factors contributing to resistance is the use of frontline clinical antibiotics in veterinary practice. In order to properly manage dwindling antibiotic resources, we must identify antimicrobials that are specifically targeted to veterinary applications. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many gram-positive bacteria, including human and animal pathogens such as Staphylococcus, Bacillus, Listeria, and Clostridium. Although not currently used in human medicine, nisin is already employed commercially as an anti-mastitis product in the veterinary field. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and also against staphylococci and streptococci associated with bovine mastitis. However, newly emerging pathogens such as methicillin resistant Staphylococcus pseudintermedius (MRSP) pose a significant threat in terms of veterinary health and as a reservoir for antibiotic resistance determinants. In this study we created a nisin derivative with enhanced antimicrobial activity against S. pseudintermedius. In addition, the novel nisin derivative exhibits an enhanced ability to impair biofilm formation and to reduce the density of established biofilms. The activities of this peptide represent a significant improvement over that of the wild-type nisin peptide and merit further investigation with a view to their use to treat S. pseudintermedius infections.     

Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis.

In plants, recent studies have demonstrated links between the regulation of developmental processes and chromatin dynamics and organisation. Analysis of new mutations affecting overall plant architecture, leaf development and flowering time in Arabidopsis has allowed us to clone and characterise LHP1, the Drosophila heterochromatin protein 1 (HP1) homologue. LHP1 has the chromo and chromo shadow domains central to the function of animal proteins. Yeast two hybrid studies and in planta deletion experiments suggest similar modes of action in plants and animals via homodimer formation. In vivo localisation experiments revealed a specific subnuclear protein distribution in foci throughout the nucleus. Our data suggest that LHP1 may act as a main regulator of gene expression in plants, through formation of heterochromatin-like repressive complexes, to control developmental pathways involved in organ and cell size, and the vegetative to reproductive phase transition.     

Vesicular stomatitis virus M protein may be inside the ribonucleocapsid coil.

Vesicular stomatitis virus is an enveloped virus with an external glycoprotein G and a nucleocapsid that form, together with the M protein, a tight helically coiled structure: the skeleton. Negative staining and immunoelectron microscopy studies on skeleton preparations were performed to determine the localization of the M protein. These studies have resulted in a new model for the structure of rhabdoviruses in which the nucleocapsid is wound around a core containing the M protein. This model predicts contact between M and lipid only at the extreme ends of the skeleton, which is confirmed by skeleton-liposome binding studies.     

Reversible conformational changes and fusion activity of rabies virus glycoprotein.

In an attempt to understand the implication of the rabies virus glycoprotein (G) in the first steps of the viral cycle, we studied the pH dependence of virus-induced fusion and hemagglutination, as well as modifications of the structure and properties of the viral glycoprotein following pH acidification. Our results suggest that the G protein adopts at least three distinct configurations, each associated with different properties. At neutral pH, G did not fuse membranes or hemagglutinate erythrocytes. It was insensitive to digestion with bromelain and trypsin. At pH 6.4, the glycoprotein became sensitive to proteases. Hemagglutination was at its maximum and then sharply decreased with the pH. No fusion was detected. Aggregation of virus was also observed. The third configuration, at below pH 6.1, was associated with the appearance of fusion. Some neutralizing monoclonal antibodies were able to differentiate these three configurations. Preincubation of the virus at below pH 6 inhibited fusion, but this inhibition, like the structural modifications of the glycoprotein, was reversible when G was reincubated at neutral pH.     

Prédation exercée par le chabot (Cottus Gobio L.) sur l'alevin de truite commune (Salmo trutta L.): taille maximale de capture des alevins par les chabots

In laboratory conditions, where predation by sculpins (Cottus gobio L.) upon brown trout fry (Salmo trutta L.) was strong, predation was shown to be closely linked to total length of prey and predator. The limit of this predation can be defined by the equation: Y = 0.484 X + 5.8 (Y = total length of the trout fry in mm and X = total length of the sculpin in mm).

     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.