Regulation of Aurora Kinases and Their Activity

The three mitotic protein kinases Aurora-A, B and C are complementary enzymes that regulate multiple mitotic events. To do so, the different kinases must be locally activated, and the control of their activity is tightly regulated in time and space during mitosis. For instance, Aurora-A is first active at the centrosomes, then on microtubules at the spindle pole, Aurora-B is active in the nucleus, then at chromosome kinetochores and later one at the midbody. Aurora kinase activity is regulated in space and time by locally binding to regulators. Aurora kinases must bind to protein partners to be activated. Aurora-A for instance binds to targeting protein for Xenopus kinesin-like protein 2 (TPX2) and is activated at the spindle pole, Aurora-B and Aurora-C to INner CENtromer Protein (INCENP) and is activated on the chromosomes. These activations go through an autophosphorylation of a threonine residue in the T-loop of the kinase. Other protein partners are using different mechanisms to activate Auroras. These allow activation of the kinase at different time and location in the cell. This review is an up-to-date list of regulators of Aurora kinases. The subcellular localization of these regulators explains the presence of an active Aurora kinase. It also explains the changes in the localizations of the Aurora kinases activity observed during cell cycle progression in mitosis. Aurora kinases have been recently reported to be involved in nonmitotic events, and the identity of their activators in these events must be searched

Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A

Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species

Sparse denoising and adaptive estimation enhances the resolution and contrast of fluorescence emission difference microscopy based on array detector

International audienceArray detector allows a resolution gain for confocal microscopy by combining images sensed by a set of photomultipliers tubes (or sub-detectors). Several methods have been proposed to reconstruct a high-resolution image by linearly combining sub-detector images, especially the fluorescence emission difference (FED) technique. To improve the resolution and contrast of FED microscopy based on array detector, we propose to asso-ciate sparse denoising with spatial adaptive estimation. We show on both calibration slides and real data that our approach applied to the full stack of spatially reassigned detector signals, enables to achieve a higher reconstruction performance in terms of resolution, image contrast, and noise reduction

Neutralising Antibodies against Ricin Toxin

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC50 of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD50). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention

Modelling and analysis of protein aggregation - Competing pathways in prion (PrP) polymerisation

Protein aggregation leading to the formation of amyloid fibrils is involved in several neurodegenerative diseases such as prion diseases. To clarify how these fibrils are able to incorporate additional units, prion fibril aggregation and disaggregation kinetics were experimentally studied using Static Light Scattering (SLS). Values that are functions of ∑i ≥ 1i2 Ci, with ci being the concentration of fibrils of size i, were then measured as a function of time. An initial model, adapted from the Becker-Döring system that considers all fibrils to react similarly is not able to reproduce the observed in vitro behaviour. Our second model involves an additional compartment of fibrils unable to incorporate more prion units. This model leads to kinetic coefficients which are biologically plausible and correctly simulates the first experimental steps for prion aggregation

Modelling and analysis of protein aggregation - Competing pathways in prion (PrP) polymerisation*

Protein aggregation leading to the formation of amyloid fibrils is involved in several neurodegenerative diseases such as prion diseases. To clarify how these fibrils are able to incorporate additional units, prion fibril aggregation and disaggregation kinetics were experimentally studied using Static Light Scattering (SLS). Values that are functions of ∑i ≥ 1i2 Ci, with ci being the concentration of fibrils of size i, were then measured as a function of time. An initial model, adapted from the Becker-Döring system that considers all fibrils to react similarly is not able to reproduce the observed in vitro behaviour. Our second model involves an additional compartment of fibrils unable to incorporate more prion units. This model leads to kinetic coefficients which are biologically plausible and correctly simulates the first experimental steps for prion aggregation