Biomedical subjects

M Ollivon

Publications and source records attributed to M Ollivon.

29 records · Page 2Linked to original sources

PubMed1997

Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus.

Animals↗

PubMed1995

Partition coefficient of a surfactant between aggregates and solution: application to the micelle-vesicle transition of egg phosphatidylcholine and octyl beta-D-glucopyranoside.

The mechanism of the solubilization of egg phosphatidylcholine containing 10% (M/M) of egg phosphatidic acid unilamellar vesicles by the nonionic detergent, octyl beta-D-glucopyranoside, has been investigated at both molecular and supramolecular levels by using fluorescence and turbidity measurements. In the lamellar region of the transition, the solubilization process has been shown to be first a function of the initial size before reaching an equilibrium aggregation state at the end of this region (the onset of the micellization process). The analysis during the solubilization process of the evolution of both the fluorescence energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rho-PE) and the fluorescence of 6-dodecanoyl-2-dimethylaminoaphtalene (Laurdan) has allowed us to determine the evolution of the detergent partitioning between the aqueous and the lipidic phases, i.e., the evolution of the molar fraction of OG in the aggregates (XOG/Lip) with its monomeric detergent concentration in equilibrium ([OG]H2O), throughout the vesicle-to-micelle transition without isolating the aqueous medium from the aggregates. The curve described by XOG/Lip versus [OG]H2O shows that the partition coefficient of OG is changing throughout the solubilization process. From this curve, which tends to a value of 1/(critical micellar concentration), five different domains have been delimited: two in the lamellar part of the transition (for 0 < [OG]H2O < 15.6 mM), one in the micellization part, and finally two in the pure micellar region (for 16.5 < [OG]H2O < 21 mM). The first domain in the lamellar part of the transition is characterized by a continuous variation of the partition coefficient. In the second domain, a linear relation relates XOG/Lip and [OG]H2O, indicating the existence of a biphasic domain for which the detergent presents a constant partition coefficient of 18.2 M-1. From the onset to the end of the solubilization process (domain 3), the evolution of (XOG/Lip) with [OG]H2O can be fitted by a model corresponding to the coexistence of detergent-saturated lamellar phase with lipid-saturated mixed micelles, both in equilibrium with an aqueous phase, i.e., a three-phase domain. The micellar region is characterized first by a small two-phase domain (domain 4) with a constant partition coefficient of 21 M-1, followed by a one-phase mixed-micellar domain for which XOG/Lip no longer linearly depends on [OG]H2O. The results are discussed in terms of a phase diagram.

Animals↗

PubMed1995

Preparation and characterization of dipalmitoylphosphatidylcholine liposomes containing interleukin-2.

Human recombinant interleukin-2 (IL-2) has been associated or mixed with small unilamellar vesicles prepared from dipalmitoylphosphatidylcholine (DPPC). Whatever the mode of IL-2 introduction, a considerable proportion of the added protein was associated with the liposomes, as determined by gel filtration and ultrafiltration/centrifugation, suggesting that IL-2 can interact with the lipid bilayer as well as being entrapped within the aqueous phase. Moreover, IL-2 prevented the aggregation/fusion of the vesicles at 4 degrees C. Liposome-associated protein was partially protected from digestion by pepsin, especially at the C-terminal, since no fluorescence emission from the tryptophan in this region was detected in the resulting peptides after separation by HPLC. Such systems could constitute a sustained release form of IL-2 for immunotherapy.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1993

Reconstitution of non-ionic monoalkyl amphiphile-cholesterol vesicles by dilution of lipids-octylglucoside mixed micelles.

Kinetic aspects of the formation of non-ionic surfactant vesicles (NSV) using the mixed micelle dilution procedure are examined. Mixed micelles composed of a mixture of lipids, i.e. diglycerol hexadecylether (C16G2), cholesterol (CHOL), dicetylphosphate (DCP) and detergent, octylglucoside (OG), were diluted with detergent-free buffer added either instantaneously or progressively at different rates ranging from 3.47 x 10(-2) to 6.94 x 10(-4) ml/min. The resulting particles were analysed by quasielastic light scattering (QELS), high performance liquid chromatography (HPLC) on gel exclusion column and cryogenic transmission electron microscopy (cryo-TEM). NSV exhibit mean diameters (MD) varying from 100 to 600 nm depending on the kinetics of OG removal. When the dilution of mixed micelles is instantaneous the vesicles are characterized by a spherical shape and MD values close to 100 nm. They show narrow size distribution and stability for 2 months. NSV recovered with progressive micelle dilution, at fast buffer addition rates (3.47 x 10(-2) and 1.39 x 10(-2) ml/min) exhibit MD values of 170-240 nm, elongated shapes, low polydispersities and 2-month stabilities. When the rate of buffer addition is lowered to 6.94 x 10(-4) ml/min, unstable particles with larger MD values and broad size distributions are obtained. Turbidity monitoring at 350 nm and 25 degrees C was used to characterize the lipids-OG mixed aggregate rearrangements either as a function of time when detergent-free buffer was continously added to the mixed micelles or after equilibrium setting when the micelles were instantaneously diluted. In the latter case the intermediate aggregates were also analysed by QELS. For continuous dilutions, the molecular composition of aggregates, [OG/lip]agg, as well as the OG concentration in the aqueous medium, [OG]bulk, were determined at the break points observed on the plots of optical density (OD) versus total OG concentration ([OG]tot). [OG/lip]agg and [OG]bulk values are independent of the rate of buffer addition, suggesting that the micelle to NSV transition is not mainly limited by the kinetics of the molecular processes involved during detergent removal from the mixed aggregates. The examination of the apparent partition coefficient of OG between the aqueous phase and the lipidic aggregates shows, however, that OG depletion from the bilayered structures is more difficult than its elimination from the mixed micelles. QELS analysis of the intermediate lipids-detergent aggregates, performed with time, demonstrates very slow supramolecular rearrangements during the vesicle closure. These rearrangements explain the significant increase in both size and polydisperity of the final vesicles observed with slow rates of buffer addition.

Cholesterol↗

PubMed1992

Solubilization steps of dark-adapted purple membrane by Triton X-100. A spectroscopic study.

Ultraviolet-visible spectroscopy has been used to follow the solubilization of the dark-adapted purple membrane of Halobacterium halobium by Triton X-100. Turbidity of purple membrane fragments and absorbance of bacteriorhodopsin variations during continuous addition of detergent give solubilization profiles exhibiting several break points corresponding to different equilibrium stages of the solubilization process. The present method allows the determination of the detergent to protein+lipid ratio in mixed aggregates at the corresponding break points. It was concluded that, when performed systematically, this technique is a very convenient and powerful tool for the quantitative study of biomembrane-to-micelle transition.

Bacteriorhodopsins↗

PubMed1991

Size analysis and stability study of lipid vesicles by high-performance gel exclusion chromatography, turbidity, and dynamic light scattering.

Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 micron or by filtration through 0.8-micron cellulosic or 0.22-micron polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 micron, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF "tortuous" ones being less efficient than "straight bores" polycarbonate ones; (ii) mixed EPC/EPA REVs sequentially extruded down through 0.2-micron polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC/EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC/EPA (mole ratio: 91/9) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.

Chromatography, High Pressure Liquid↗

PubMed1989

Temperature dependence of the vesicle-micelle transition of egg phosphatidylcholine and octyl glucoside.

The temperature dependence of octyl glucoside micellization was determined and compared to the phase behavior of the octyl glucoside--egg phosphatidylcholine (PC) mixed system in excess water to help elucidate the process of vesicle formation from mixed surfactant-phospholipid micelles. The critical micelle concentration of octyl glucoside (OG) was determined from the sharp increase of ANS fluorescence at micellization in an NaCl buffer at temperatures ranging from 5 to 40 degrees C. The cmc decreased with increasing temperature from 31 mM at 5 degrees C to 16 mM at 40 degrees C. A similar but less steep temperature dependence is observed for the solubilization of egg PC vesicles by OG as monitored by the surfactant-dependent changes in (1) solution turbidity and (2) the resonance energy transfer between NBD-PE and Rho-PE incorporated in the vesicles. These assays identify two breakpoints, most likely the boundaries of the cylindrical micelle and spheroidal micelle coexistence region. The [OG]aq values at these two breakpoints have similar temperature dependencies. However, the cylindrical mixed micelles at the boundary have nearly identical OG:PC ratios over the temperature range studied, whereas the spheroidal mixed micelles have relatively more OG at the higher temperatures (OG:PC ratio increases from 2.92 to 3.72 between 5 and 35 degrees C). Estimation of the acyl volume to surface area ratio for the compositions observed suggests that this parameter remains constant over temperature. The spheroidal mixed micelles, but not the cylindrical PC-OG micelles, exhibit ideal mixing between the two components at all temperatures (5-35 degrees C). This temperature sensitivity may be utilized to improve the efficacy of membrane protein reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)

Colloids↗

PubMed1988

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside.

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescent Dyes↗

PubMed1986

Sizing and separation of liposomes, biological vesicles, and viruses by high-performance liquid chromatography.

The ability of an HPLC gel exclusion column (TSK G6000PW) to separate lipid vesicles, viruses, and biological vesicles according to size was tested and compared with separations on Sephacryl S1000. The columns were calibrated using vesicular Stokes radii determined by quasielastic light scattering. The vesicles separated according to size on both types of column and remained intact during elution. Viruses of known diameters and clathrin-coated vesicles were also eluted as a function of size. The TSK G6000PW column was able to separate larger particles (greater than 500 nm) than the Sephacryl S1000, and, when used in combination with the TSK G5000PW column, gave more discrete separations of smaller particles (10 to 30 nm diameter). Moreover, the HPLC columns can be run significantly faster (10-20 min vs several hours) and give more precise results than Sephacryl S1000. Therefore, HPLC using a G6000PW column alone, or in combination with a G5000PW column, provides a rapid and accurate means of sizing and selecting specifically sized biological and artificial vesicles.

Acrylic Resins↗

PubMed1985

Lipid solvation of the aqueous form of the myelin proteolipid apoprotein: evidence and characterization of two lipid populations by fluorescence polarization, differential calorimetry, and sucrose gradient centrifugation.

The interaction between dipalmitoylphosphatidylcholine (DPPC) and the aqueous form of the myelin proteolipid apoprotein (PLA) has been investigated. Lyophilization was found to be an efficient and nonperturbing method for membrane reconstitution. Mixtures of different lipid/protein ratios were analyzed by means of differential calorimetry, fluorescence polarization, and sucrose gradient centrifugation. The presence of two coexisting lipid populations, termed "bulk" and "interacting" lipids, was demonstrated by these three techniques. By differential calorimetry, 23 DPPC molecules per molecule of protein (30 kDa) were shown to be excluded from the lipid phase transition. By fluorescence polarization, we detected above the phase-transition temperature a large perturbation of the lipid acyl chain dynamics induced by the aqueous form of PLA. Increasing the protein content above 35% by weight within the recombinants caused drastic changes in both delta H values and the fluorescence anisotropy parameter, which could stem from protein aggregation.

Animals↗

PubMedDate not supplied

Self-evolving microstructured systems upon enzymatic catalysis.

The consequences of cell microstructuration on enzyme functions is discussed in the framework of self-evolving microstructured systems. Molecular assemblies of amphiphiles or lipids are spontaneously formed by self-organisation. Among these different structures, reversed micelles, liquid crystalline mesophases and vesicles are hosts for enzymatic reaction studies. Inside a living cell, phospholipid metabolism is responsible for membrane structural modifications; the catalytic behaviour of lipolytic enzymes, mainly phospholipase (PL) A2, is described in relation with structural aspects of biological membranes. The implication in cellular regulation events of PLC and PLD is discussed in relation with the role of their reaction products as second messengers in membrane fusion processes. The in vitro synthesis of dialkyl phosphatidylcholines, via the enzymatic 'salvage pathway' which leads to the formation of vesicles upon phospholipid formation, is considered in relation with autopoiesis. More recent studies on self-evolving systems based on enzyme-surfactants reactions are detailed. The interactions between amphiphilic aggregates and enzymes allow to explore the OG/octanol/water phase diagram. Enzymatic formation of dipalmitoylphosphatidylcholine (DPPC) liposomes and non-ionic surfactant vesicles (NSV), starting from mixed micelles or open structures, finally sets an example of a biomimetic self-evolving system.

Cholesterol↗