Stability and aggregation studies of non-sonicated arsonolipid-containing vesicles

In this study, we investigated non-sonicated arsonolipid-containing liposomes (arsonoliposomes) in terms of the influence of lipid composition on their stability, assessed as membrane integrity and physical stability [size]. Vesicles consisting of plain arsonolipids or mixtures of arsonolipids with cholesterol [Chol] or with distearoyl-phospatidylcholine [DSPC] were studied. Membrane integrity was evaluated by measuring the retention of incorporated 5-(6)carboxyfluorescein [CF] during incubation of the vesicles in Tris buffer, pH 7.4. Photon correlation spectroscopy was used to investigate the time-dependent aggregation of arsonoliposomes in the absence and presence of Ca(2+)ions. Vesicles composed of plain C18 (acyl fatty chain) arsonolipids were found to be unstable, with only 15% of the initially incorporated CF remaining in the vesicles after 24 hours. The addition of Chol to the membrane (1:1 mol/mol) significantly increased the stability of arsonoliposomes, while the addition of DSPC to the lipid bilayer (1:1 mol/mol) increased vesicle stability to a lower extent. The results of particle size analysis showed that non-sonicated arsonoliposomes consisting of plain arsonolipid Ars/Stearic are highly and rapidly aggregated, while calcium-induced aggregation is also significant, but slower. Aggregation could not be always explained on the basis of zeta potential changes, indicating that the process is complex.     

Kinetic studies of protein L aggregation and disaggregation

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.     

Biophysical studies of amorphous protein aggregation and in vivo immunogenicity

Amorphous protein aggregates are oligomers that lack specific, high-order structures. Soluble amorphous aggregates are smaller than ~1 µm. Despite their lack of high-order structure, amorphous protein aggregates exhibit specific biophysical properties such as reversibility of formation, density, conformation, and biochemical stability. Our mutational analysis using a Solubility Controlling Peptide (SCP) tag strongly suggests that amorphous aggregation of small globular proteins can significantly increase in vivo immune response and that the magnitude of enhanced immune responses depends on the aggregates’ biophysical and biochemical properties. We propose that SCP tags might help develop subunit (protein) adjuvant-free (immunostimulant-free) vaccines by controlling the aggregation propensity of target proteins.     

Implication of serotonin in the control of vigilance states as revealed by knockout-mouse studies

Genetic manipulation of the 5-HT system leads to alterations of 5-HT neurotransmission and provides new opportunities to investigate the role of 5-HT in sleep regulations. Indeed, it represents an alternative to the use of pharmacological tools and, to some extent, of localized lesions of the 5-HT system, which have been, from the 1960s until recently, the main approaches to investigate this question. Homologous recombination knocking-out genes encoding various proteins involved in 5-HT neurotransmission in the mouse has recently allowed further assesment of the role of the serotonin transporter (5-HTT), the monoamine oxidase A (MAO-A), and the 5-HT1A, 5-HT1B and 5-HT2A receptors in the regulation of sleep. In 5-HT1A -/- and 5-HT1B -/- knock-out mice, Rapid Eye Movement sleep (REMs) was enhanced. Pharmacological blockade of these receptors had the same effects in wild-types. Thus, both receptor types exert a tonic inhibitory influence on REMs. In addition, 5-HT1A -/- and 5-HT1B -/- mutants were hypersensitive to 5-HT1B and 5-HT1A receptor agonists, respectively, which suggests that adaptive changes at 5-HT neurotransmission develop in knock-out animals. In the same manner, 5-HTT-/- knock-out mice exhibited increased REMs. This may be accounted for by a decrease in 5-HT1A and 5-HT1B receptor-mediated sleep regulations. In contrast, decreased REMs was observed in MAOA -/- knock-outs, a phenomenon that mimics the effect of pharmacological MAO inhibition. Finally, 5-HT2A -/- and 5-HT2C -/- mice exhibited more wakefulness and less slow wave sleep (SWS) than wild-types. These effects could not be reproduced by 5-HT2A or 5-HT2c receptor blockade in wild-types. To conclude, constitutive knock-outs undergo adaptive processes involving other proteins than those encoded by the invalidated gene, which renders interpretation of the corresponding sleep phenotype difficult. Inducible knock-outs will probably help to overcome this difficulty. Finally, combination of genetic manipulations with relevant pharmacological ones should allow further progress in the understanding of sleep mechanisms.     

Sleeping and vigilance in Black-tailed Godwit

I studied the influence of position within the flock of the bird on the sleep-vigilance trade-off in individual Black-tailed Godwits Limosa limosa roosting at high tide. The peeking rate, the percentage of time during which its eyes were open and mean duration of peeks were highest in peripheral birds. The duration of the sleep posture, defined as the amount of time an individual sits or stands with its bill tucked beneath its scapulars, did not differ significantly between central and peripheral birds. The rate of agonistic encounters was minimal, in both peripheral and central birds. These observations suggest different benefits for both positions in the flock, mainly anti-predatory in the case of central birds and thermoregulatory in the case of peripheral birds. Electronic Publication     

Mechanistic studies on aggregation of polyethylenimine-DNA complexes and its prevention

Aggregation of polyethylenimine (PEI)-DNA complexes severely undermines their utility for gene delivery into mammalian cells. Herein we undertook to elucidate the mechanism of this deleterious phenomenon and to develop rational strategies for its prevention. The effect of temperature, surfactants, complex concentration, ionic strength, viscosity, and pH on the time course of this aggregation was systematically examined. The aggregation process was completely inhibited by 2.5% polyoxyethylene (100) stearate (POES) and to a lesser degree by other nonionic surfactants. Importantly, POES preserved the transfection efficiency of the complexes without inducing toxicity. The aggregation was also reduced by lowering the temperature and pH, diluting the complexes, and increasing the solution viscosity. It is concluded that PEI-DNA complexes aggregate primarily due to hydrophobic interactions, while electrostatic attractions play little role.     

Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution.

An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.     

Switching to a feeding method that obstructs vision increases head-up vigilance in dabbling ducks

A major assumption in most models of foraging is that feeding and vigilance are mutually exclusive. A recent experimental study challenged this hypothesis and demonstrated that birds are able to detect predators while pecking seeds on the ground (head-down vigilance). Experimental obstruction of head-down vigilance makes birds increase head-up vigilance (i.e. the classical overt vigilance posture). For many foragers in the wild, visibility varies between habitats and foraging methods. We compared the vigilance of Teal Anas crecca and Shoveler Anas clypeata when foraging with their eyes above the water surface (shallow feeding, only the bill submerged) and when foraging with their eyes underwater (deep feeding, head and neck underwater, or upending), at three wintering sites in western France. Birds of both species spent less time in head-up vigilance during shallow foraging than during deep foraging, with no significant difference between sites, which suggests that they are capable of some vigilance during shallow foraging. During deep foraging, the time spent vigilant increased because the frequency of scans was much higher, while scan length decreased. However, these differences could have resulted from variations in the availability of food at different depths. In an experiment where the food availability was constant, we observed the same pattern, with a higher frequency of scans during deep foraging. This study therefore provides strong support for the idea that vigilance and foraging are not always mutually exclusive and shows that switching between searching methods can cause vigilance time – and, as a consequence, loss of feeding time – to vary. This should be taken into account in future field and experimental studies of the trade-off animals make between vigilance and feeding.     

Electron microscopic studies of aggregation and pellicle formation inAzospirillum spp.

Microscopic studies on aggregation ofAzospirillum brasilense Cd and the local isolate Cd-1 revealed the presence of an extracellular layer (ECL) on the outer surface of cells taken from the early exponential phase. Incubation of the bacteria with cationized ferritin (CF) was followed by labelling of the ECL of both Cd and Cd-1 taken from the stationary phase. Concanavalin-A-ferritin (Con-A-F), a lectin conjugated with ferritin, was bound to the ECL which was developed during the stationary phase, but not the exponential phase, in both isolates. In static liquid cultures,Azospirillum brasilense Cd formed lateral flagella and fimbriae-like structures which were not observed in shaking liquid cultures. It is suggested that these filaments may play a role in cell-to-cell adhesion of Azospirillum that results in pellicle formation observed in static cultures.     

Kinetic studies on the aggregation of Aspergillus niger conidia

Morphology has a crucial effect on productivity and the supply of substrate for cultures of filamentous fungi. However, cultivation parameters leading to the desired morphology are often chosen empirically as the mechanisms governing the processes involved are usually unknown. For coagulating microorganisms like Aspergillus niger the morphological development is considered to start with the aggregation of conidia right after inoculation. To elucidate the mechanism of this process, kinetic studies were carried out using an in-line particle size analyzer. Based on the data obtained from these experiments a model for conidial aggregation is proposed in this article. It consists of two separate aggregation steps. The first one takes place immediately after inoculation, but only leads to a small decrease of total particle concentration. Most suspended conidia aggregate after a second aggregation step triggered by germination and hyphal growth. Aggregation velocity of this second phase is linearly dependent on the particle growth rate.     

Molecular dynamics studies on the aggregation of Y-shaped fluoroalkanes

Molecular dynamics (MD) calculations have been performed on the aggregation of clusters with up to 128 Y-shaped perfluoroalkylated molecules of the type C10F20[C7H15]2 (Y-A/128) and C10H20[C7F15]2 (Y-B/128) as well as mixed clusters (Y-A/64+Y-B/64) using the AMBER 5 program. The effect of the segregation tendency of the chemically different parts and the influence of the steric repulsion due to the wedge shape of the molecules on the structure formation have been studied. The results have been analyzed by snapshots, radial atom pair distribution functions, orientational correlation functions as well as diffusion coefficients and are compared with the corresponding findings on clusters of alkanes and perfluoroalkanes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s008940020092y.