Biomedical subjects

A Bienvenue

Publications and source records attributed to A Bienvenue.

At least 19 recordsLinked to original sources

PubMed2003

Rheological properties of concentrated skim milk: importance of soluble minerals in the changes in viscosity during storage.

Properties of condensed milks prior to spray drying dictate to a large extent the functionality of the resulting milk powder. Rheological properties of concentrated skim milk, with total solids content of 45% but different mineral content, were studied as a function of shear rate and storage time at 50 degrees C. These milks are proposed as a model to study the effects of minerals on rheology and age gellation of condensed milk prior to drying. During storage of the concentrated milk, the apparent viscosity, particularly after 4 h, increased markedly at all shear rates studied. The yield stress also increased steeply after 4 h of storage at 50 degrees C. The changes in apparent viscosity of concentrated milk stored for up to 4 h were largely reversible under high shear, but irreversible in samples stored for longer time. The appearance of yield stress suggested the presence of reversible flocculation arising from weak attraction between casein micelles, with a transition from reversible to irreversible aggregation during storage. Particle size analysis confirmed irreversible aggregation and fusion of casein micelles during storage. Gradual reduction of mineral content of concentrated milks resulted in a marked decrease in the apparent viscosity and casein micelle aggregation during storage, while addition of minerals to milk had the opposite effect. The results demonstrated that the soluble mineral content is very important in controlling the storage-induced changes in the rheology of concentrated milks.

Animals↗

PubMed1998

Membrane fusion induced by 11-mer anionic and cationic peptides: a structure-function study.

We recently demonstrated that an amphipathic net-negatively charged peptide consisting of 11 amino acids (WAE 11) strongly promotes fusion of large unilamellar liposomes (LUV) when anchored to a liposomal membrane [Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Biochemistry 36, 3773-3781]. To elucidate a potential relationship between peptide structure and its fusogenic properties and to test the hypothesis that specific structural motifs are a prerequisite for WAE-induced fusion, three 11-mer WAE-peptide analogues (WAK, WAEPro, and WAS) were synthesized and investigated for their structure and fusion activity. Structural analysis of the synthetic peptides by infrared attenuated total reflection spectroscopy reveals a distinct propensity of each peptide toward a helical structure after their anchorage to a liposomal surface, emphasizing the importance of anchorage on conveying a secondary structure, thereby conferring fusogenicity to these peptides. However, whereas WAE and WAK peptides displayed an essentially nonleaky fusion process, WAS- and WAEPro-induced fusion was accompanied by substantial leakage. It appears that peptide helicity as such is not a sufficient condition to convey optimal fusion properties to these 11-mer peptides. Studies of changes in the intrinsic Trp fluorescence and iodide quenching experiments were carried out and revealed the absence of migration of the Trp residue of WAS and WAEPro to a hydrophobic environment, upon their interaction with the target membranes. These results do not support the penetration of both peptides as their mode of membrane interaction and destabilization but rather suggest their folding along the vesicle surface, posing them as surface-seeking helixes. This is in striking contrast to the behavior observed for WAE and WAK, for which at least partial penetration of the Trp residue was demonstrated. These results indicate that subtle differences in the primary sequence of a fusogenic peptide could induce dramatic changes in the way the peptide interacts with a bilayer, culminating in equally drastic changes in their functional properties. The data also reveal a certain degree of sequence specificity in WAE-induced fusion.

Amino Acid Sequence↗

PubMed1990

On the nature of the cytochrome P450scc "ultimate oxidant": characterization of a productive radical intermediate.

The electron paramagnetic (EPR) properties of a transient species detected during a cytochrome P450 (P450)-mediated peroxidative reaction have been compared with those of peroxidases Compound I and model metalloporphyrins. The reaction which was studied with cholesterol-specific P450scc and (20R)-20-hydroperoxycholesterol, occurred without enzyme denaturation. The resulting transient species, which reached its maximum after 50 s reaction, was characterized by a one-line EPR spectrum, g = 2.0035, delta 1/2 = 1.08 mT. The decay of this radical was concomitant with the production of (20R)-20,21-dihydroxycholesterol. The reaction (almost 100% yield) conserved the stereo-specificity of the natural pathway. We suggest this intermediate is a candidate for the in vivo ultimate oxidant in the P450-mediated hydroxylation process. The comparison of the observed EPR spectrum with those of peroxidase Compound I and related synthetic models allows us to propose a FeIV porphyrin II cation radical structure for the intermediate.

Adrenal Cortex↗

PubMed1989

Asymmetric distribution of phospholipids in the membrane of vesicles released during in vitro maturation of guinea pig reticulocytes: evidence precluding a role for "aminophospholipid translocase".

Guinea pig reticulocytes lose their transferrin (Tf) binding activity during maturation, in the form of vesicles (exosomes) released into the extracellular medium. Vesicles were prepared from cultures of reticulocytes to study the possible externalization of a particular membrane-associated activity, i.e., that of "aminophospholipid translocase." Analysis of the peptide composition of these vesicles revealed that the major proteins are the Tf receptor and another peptide (70kDa), which is probably the "clathrin-uncoating ATPase" described by Johnstone et al. (1987). The exosome had a lipid composition similar to erythrocyte membrane, although with a lightly but significantly lower phosphatidylethanolamine content. The aminophospholipid distribution in the vesicle membrane was determined by fluorescamine labeling. The exosomes showed an asymmetric aminophospholipid distribution similar to that of erythrocytes. "Aminophospholipid translocase" activity was absent, as no transverse diffusion of spin-labeled phospholipids occurred over more than 2 hours at 37 degrees C.

Animals↗

PubMed1989

Interactions of human lymphoblasts with targeted vesicles containing Sendai virus envelope proteins.

We have studied the internalization of targeted fusogenic liposome content to leukemic T cells (CEM) in vitro. We describe a method for the covalent coupling of T101 antibody to the surface of liposomes and the incorporation of fusogenic viral protein into the liposome membrane. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted fusogenic liposomes and delivered directly to the cytoplasm of leukemic T cells by fusion between the two membranes. The cytotoxic effect was measured by [3H]thymidine incorporation. We show that CEM are rapidly and specifically killed by the drug encapsulated in the targeted fusogenic liposomes. This effect is due to the binding of the liposome by means of the antibody and then to the fusion of the liposome with the targeted cell membrane, mediated by F protein.

Antibodies↗

PubMed1988

Evidence for bidirectional transverse diffusion of spin-labeled phospholipids in the plasma membrane of guinea pig blood cells.

The distribution and transverse diffusion kinetics of four spin-labeled phospholipid analogues (two with choline heads: phosphatidylcholine (PC) and sphingomyelin (SM); two with amino heads: phosphatidylserine (PS) and phosphatidylethanolamine (PE) were studied in the plasma membrane of guinea pig blood cells: erythrocytes, reticulocytes, and leukemic lymphocytes. Nitroxide reduction by the internal content of the cells was used as an indicator to determine the phospholipids that penetrated the cells. The reduction rates were in the order, PS greater than PE greater than PC greater than SM in all cells. Reoxidation of phospholipids extracted by serum albumin revealed the distribution of the phospholipids at a given time. In all cells, the distribution equilibrium was reached in less than 2 h and the amounts left in the external leaflet were in the following proportional order: PS less than PE less than PC less than SM. In the erythrocytes and especially in the reticulocytes, the shape change induced by adding phospholipids relaxed partially or completely at a lower speed but kept the same proportional order as at equilibrium. All the results were analyzed quantitatively with a simple kinetic model including the rates of transverse diffusion (flip and flop), the exchange between plasma membrane and internal membranes, and the reduction rate of free radicals (determined in either the internal or external membrane leaflet). The calculated rate constants of transverse diffusion varied from 2 x 10(-3) to 1.2 x 10(-1) min-1 for the flip and from 4 x 10(-3) to 1.2 x 10(-1) for the flop, depending on the polar head and the cell type. Possible interpretations of the external phospholipid reduction mechanism and cell deformation are discussed.

Animals↗

PubMed1988

Platelet-activating-factor-induced serum-albumin release from rabbit platelets in the absence of calcium.

Using a radioimmunoassay for rabbit-serum albumin, platelet-activating-factor-induced serum-albumin release by platelets was monitored under non-aggregating conditions. The four main results from this study are as follows. The EC50 of the release was of the same order of magnitude as the aggregation EC50 in the presence of calcium. The release took place within 2 min and was inhibited by BN 52021, which is a very specific inhibitor of the platelet-activating-factor-aggregating effect. Serum-albumin release was much greater than serotonin release.

Animals↗

PubMed1988

Relationship between the transverse distribution of phospholipids in plasma membrane and shape change of human platelets.

ESR spectroscopy was used to investigate the distribution of spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the presence of human platelets. Three rates were determined: hydrolysis of the ester bond at position 2, reduction of labels by cytoplasm, and internalization of labels situated in the outer leaflet of the plasma membrane. We found that the half-time for transverse diffusion of added phospholipids was shorter for aminophospholipids (40 min and less than 10 min for PE and PS, respectively) than for the choline derivatives (greater than 120 min for PC, not measurable for SM). Addition of any of the phospholipids led to a considerable change in the initial platelet shape (assessed by electron microscopy) from a discoid form to a smaller body with very long pseudopods. When aminophospholipids were used, the platelets quickly returned to the initial shape [half-time of 20 min and less than 5 min for (0.2)PE and (0.2)PS, respectively]. Conversely, there was no relaxation after (0.2)PC or (0.2)SM was added. We conclude that there is a relationship between the excess of phospholipids in the outer leaflet of the plasma membrane and cytoskeletal organization presumably via actin polymerization, which is responsible for platelet shape.

Biological Transport, Active↗

PubMed1987

F protein-F protein interaction within the Sendai virus identified by native bonding or chemical cross-linking.

The spatial arrangement of the F protein spike in the Sendai virus was studied after purifying the protein and reconstituting it in lipid vesicles (Sechoy, O., Philippot, J. R., and Bienvenue, A. (1986) Biochim. Biophys. Acta 857, 1-12). The different components of the F protein spikes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under various conditions of treatment, i.e. at different temperatures and sodium dodecyl sulfate concentrations, using different detergents for F protein solubilization (Triton X-100 and octyl glucoside), by fast protein liquid chromatography analysis, and by chemical cross-linking between subunits with bifunctional agents such as dimethyl adipimidate and dithiobis(succinimidyl propionate). The F protein spike appeared to be a structurally stable complex, composed of a noncovalent association of four homooligomers, each consisting of two peptides, F1 and F2, linked by a disulfide bond. Octyl glucoside and Triton X-100 solubilized the F protein, preserving the tetramer, which is probably the native form. Using chemical cross-linking, a covalent bond was formed between two monomers. We hypothesize that the tetrameric form of the F protein in its native form (spike) consists of two identical dimers that can be chemically cross-linked in a stable complex.

Chromatography, High Pressure Liquid↗

PubMed1987

A new paramagnetic analogue of cholesterol as a tool for studying molecular interactions of genuine cholesterol.

The synthesis of a new paramagnetic (nitroxide) analogue of cholesterol is described. This compound (called CNO) contains a doxyl group in the lateral chain at position 25. Our results show that CNO retains three molecular interactions which characterize authentic cholesterol: It assumes an orientation perpendicular to the phospholipid bilayer with the doxyl group buried in the membrane core, as seen by ESR spectroscopy. It widens the transition temperature of dimyristoylphosphatidylcholine, to the same extent as cholesterol, as measured by Raman and ESR spectroscopies. It interacts with polyene antibiotics, such as amphotericin B and filipin, in the same manner as its model. This was proved on the one hand by the change in fluorescence of self quenched vesicle-entrapped calcein, after dilution in the external medium, provoked by filipin, and on the other hand by fluorescence quenching provoked by cobalt ions entering the vesicles under the influence of amphotericin B. We concluded that CNO, although it has a side chain different from genuine cholesterol, can help to solve many physiologically meaningful questions related to the distribution and movement rate of cholesterol itself.

Amphotericin B↗

PubMed1987

The influence of coupling transferrin to liposomes or minibeads on its uptake and fate in leukemic L2C cells.

Coupling transferrin to liposomes or minibeads did not affect its uptake by L2C lymphocytes via the Tf specific receptors. The uptake kinetics of Tf conjugated with particles about 50 nm in diameter was as rapid as in the case of native Tf, and the receptors were recycled with a similar turnover time (about 15 min). Contrary to the generally accepted scheme, we found some Tf degradation provoked by cellular uptake. The degradation represented about 10% of the amount of ligand taken up by the cells. It occurred when transferrin was coupled to liposomes, but not when coupled to minibeads.

Biological Transport↗

PubMed1986

Interaction between blood components and a spin-labeled analogue of PAF-acether.

The interactions between a spin-labeled analogue of PAF-acether (designated as (0,2)PAF) and different human blood components (platelets, erythrocytes, and serum) have been studied. The rate of spin probe reduction by cytosol provided information about the internalization processes when the hydrolysis rate was also available. Although erythrocyte reactivity is lower than that of platelets, erythrocytes, because of their greater numbers, removed (0,2)PAF from whole blood faster than platelets. Lastly, erythrocytes may be more efficient traps for (0,2)PAF than serum acetylhydrolase. Criteria for extending these results to genuine PAF-acether are also discussed.

Blood Platelets↗

PubMed1986

Internalization of low-density-lipoprotein-specific receptors in leukemic guinea pig lymphocytes.

Leukemic guinea pig lymphocytes (L2C) have ten times as many low-density lipoprotein (LDL) receptors as healthy lymphocytes, but LDL accounts for only 38% of the cholesterol in L2C cells, compared to more than 95% in normal cells. Our data show that LDL fails to regulate cholesterol biosynthesis and that there is a defect in LDL internalization and receptor turnover in L2C cells. We also demonstrate that the degradation of LDL is not a limiting process. By discriminating between binding and internalization, we show that internalization in L2C is much slower than in normal cells and that the decrease in metabolism is related to the slow turnover of the LDL receptors.

Animals↗

PubMed1986

Preparation and characterization of F-protein vesicles isolated from Sendai virus by means of octyl glucoside.

We have demonstrated that Triton X-100 is always present in F-protein vesicles at concentrations that can provoke cell lysis. In order to avoid any misinterpretation of the fusogenic capacity of this protein, we solubilized the Sendai virus using octyl glucoside, which can be totally removed from the F protein preparation in less than 16 h by dialysis in the presence of absorbent beads. F-glycoprotein preparations preserved their ability to lyse erythrocytes in the presence of lectins and to induce cell-vesicle fusion as demonstrated by ESR studies. These vesicles were characterized by electron microscopy and SDS-polyacrylamide gel electrophoresis. Lipid analysis of these preparations by thin-layer chromatography indicated that they had the same proportion of lipids as virus envelopes, with slight variations in the sphingomyelin content and the cholesterol/phospholipid molar ratio. F-protein vesicles of different sizes can be obtained by adding exogenous lipids before detergent removal. The hemolytic activity of the vesicles was retained over a large range of lipid concentrations. We conclude that F-protein vesicles prepared with octyl glucoside are convenient tools for studying the fusogenic mechanism of this protein and improving the fusion process between liposomes and cells.

Animals↗

PubMed1986

Comparison of the internalization efficiency of LDL and transferrin receptors on L2C guinea pig lymphocytes.

We demonstrate that L2C lymphocytes have about 10-times more receptors for transferrin (Tf) than healthy lymphocytes, as has been shown in the case of LDL receptors. The dissociation constant is the same in the two cell types (about 4 X 10(-7) M). In contrast to LDL, Tf enters L2C lymphocytes with very rapid kinetics. It is shown by cross-reaction that each receptor is internalized independently of the other.

Animals↗

PubMed1986

Thiolation of low-density lipoproteins and their interaction with L2C leukemic lymphocytes.

We present here, a new method for coupling sulfhydryl groups (SH) to low-density lipoprotein (LDL) surface. This method uses homocysteine thiolactone (HCTL) which reacts with lysine residues in a very mild manner, and permits the selection of the number of SH bound per LDL. Under our experimental conditions (8 SH/LDL), the affinity of thiolated LDL for the specific receptors and their further internalization by L2C lymphocytes are preserved.

Animals↗

PubMed1985

LDL-mediated targeting of liposomes to leukemic lymphocytes in vitro.

We describe a method for the covalent coupling of low-density lipoproteins (LDL) to the surface of small unilamellar vesicles, and the delivery of the liposome content to leukemic L2C lymphocytes in vitro. We demonstrate the stability of the linkage between LDL and liposomes, the preservation of vesicle integrity and the affinity of the LDL for their specific receptors after the coupling reaction. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted liposomes, and delivered into the cytoplasm of leukemic L2C lymphocytes by the LDL pathway, as demonstrated by the lethal effect on cells measured by 51chromium-release assay.

Animals↗

PubMed1984

The influence of the internal content of negatively charged liposomes on their interaction with high-density lipoprotein.

The release of the internal content of negatively charged phosphatidylcholine/phosphatidylserine vesicles under the influence of high density lipoprotein was studied. Under standard conditions (the same composition outside and inside the compartment) the leakage of negative liposomes increased significantly. However, a high internal concentration of calcein provoked a sealing effect, exhibited both in sucrose and in calcein release. This sealing effect is not related to the size of vesicles, the fluidity of the membrane, the distribution of phosphatidylserine molecules, or the membrane potential. Our data indicate that surface potential influences this effect, probably in addition to a lateral pressure effect such as with cholesterol. The surface potential, as measured by the water-lipid partition coefficient of fatty acids, is strongly affected by internal ionic strength when liposomes contain calcein as well as other polyanions (6-carboxyfluorescein, sodium citrate).

Anions↗