Biomedical subjects

J Dufourcq

Publications and source records attributed to J Dufourcq.

At least 19 recordsLinked to original sources

PubMed1997

Bending elasticities of model membranes: influences of temperature and sterol content.

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.

Biomechanical Phenomena↗

PubMed1996

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.

Air↗

PubMed1995

Ion channel formation by synthetic analogues of staphylococcal delta-toxin.

Ion channel formation by three analogues of staphylococcal delta-toxin, an amphipathic and alpha-helical channel-forming peptide, has been evaluated by measurement of ionic currents across planar lipid bilayers. Replacement of beta-branched, hydrophobic residues by leucine and movement of a tryptophan residue from the hydrophilic to the hydrophobic face of the helix does not significantly alter ion channel activity. Removal of the N-terminal blocking group combined with the substitution of glycine-10 by leucine changes the single channel properties of delta-toxin, without altering macroscopic conductance/voltage behaviour. Truncation of the N-terminus by three residues results in complete loss of channel-forming activity. These changes in channel-forming properties upon altering the peptide sequence do not mirror changes in haemolytic activity. The results lend support to the proposal that channel formation and haemolysis are distinct events. Channel properties are discussed in the context of a model in which the pore is formed by a bundle of approximately parallel transbilayer helices.

Amino Acid Sequence↗

PubMed1995

Evidence for a two-dimensional molecular lattice in subgel phase DPPC bilayers.

Using a combination of X-ray diffraction data from oriented films and multilamellar liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) in the subgel phase, we have established the presence of a 2D molecular lattice containing two lipid molecules. The proposed 2D lattice is consistent with all the X-ray diffraction data on the subgel phase of DPPC available in the literature. In this phase, the DPPC molecules are ordered in the plane of the bilayer and are also found to be positionally correlated across a single bilayer but not with those in adjacent bilayers. We also present the possible molecular arrangements for the proposed lattice.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Acyl chain length dependence in the stability of melittin-phosphatidylcholine complexes. A light scattering and 31P-NMR study.

Light scattering and 31P-NMR have been used to monitor the effect of the bee-toxin, melittin, on phosphatidylcholine (PC) bilayers of variable acyl chain length (from C16:0 to C20:0). Melittin interacts with all lipids provided the interaction is initiated in the lipid fluid phase. For low-to-moderate amounts of toxin (lipid-peptide molar ratios, Ri > or = 15), the system takes the form of large spheroidal vesicles, in the fluid phase, whose radius increases from 750 A with dipalmitoyl-PC (DPPC) to 1500 A with diarachinoyl-PC (DAPC). These vesicles fragment into small discoids of 100-150 A radius when the system is cooled down below Tc (the gel-to-fluid phase transition temperature). Little chain length dependence is observed for the small objects. Small structures are also detected independently of the physical state of lipids (gel or fluid) when Ri < or = 5 and provided the interaction has been made above Tc. Small discs clearly characterized for DPPC and distearoyl-PC (DSPC) lipids are much less stable with DAPC. However in the long term, all these small structures fuse into large lipid lamellae. Discs are thermodynamically unstable and kinetics of disappearance of the small lipid-toxin complexes increases as the chain length increases in the sense: DAPC >> DSPC > DPPC. Kinetics of fusion of the small discs into extended bilayers is described by a pseudo-first-order law involving a lag time after which fusion starts. Increasing the chain length decreases the lag time and increases the rate of fusion. Formation of both the large vesicles in the fluid phase and the small discs in the gel phase as well as their stability is discussed in terms of relative shapes and dynamics of both lipids and toxin.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin.

An original series of 12- to 22-residue-long peptides was developed, they are only constituted by apolar Leu and charged Lys residues periodically located in the sequence in order to general ideal highly amphipathic alpha-helices. By circular dichroism, the peptides are proven to be mainly alpha-helical in organic and aqueous solvents and in the presence of lipids. The peptides are highly hemolytic, their activity varies according to the peptide length. The 15-, 20-, and 22-residue-long-peptides have LD50 approximately 5 x 10(-8) M for 10(7) erythrocytes, i.e. they are 5-10 times more active than melittin, and are indeed several orders of magnitude more active than magainin or mastoparan.

Amino Acid Sequence↗

PubMed1991

High-performance liquid chromatographic separation of modified and native melittin following transglutaminase-mediated derivatization with a dansyl fluorescent probe.

The 26-amino acid linear, amphiphilic peptide melittin was enzymatically modified with the fluorescent probe monodansylcadaverine using guinea pig liver transglutaminase and a fluorescent derivative of stoichiometry 1:1 was obtained. Reversed-phase and size-exclusion high-performance liquid chromatographic modes were tested in order to resolve the labelled peptide and native species. The influence of several operational variables was analysed and the elution conditions were optimized so that a satisfactory resolution could be achieved in both instances in a rapid, easy manner. Both chromatographic modes offer the possibility of accurate monitoring of the time course of the enzyme-mediated conversion and more interestingly, can be applied to the semi-preparative purification of the labelled peptide.

Amino Acid Sequence↗

PubMed1991

Structure and dynamics of dimyristoylphosphatidic acid/calcium complexes by 2H NMR, infrared, spectroscopies and small-angle x-ray diffraction.

The structural and dynamic properties of complexes of dimyristoylphosphatidic acid (DMPA) and calcium ions have been characterized by 2H NMR, Raman, and infrared spectroscopies and small-angle X-ray diffraction. All techniques used show that these complexes do not undergo a cooperative thermotropic phase transition. Small-angle X-ray diffraction unambiguously demonstrates that the structure of the lipid molecules of the DMPA/Ca2+ complexes remains lamellar even at a temperature as high as 85 degrees C. Raman results indicate that within this temperature range, only a few trans-gauche isomerizations of the C-C bonds of the phospholipid acyl chains arise in this system. The 2H NMR spectra indicate that the DMPA chains are highly motionally restricted up to 65 degrees C and that higher temperatures might activate some low-frequency overall motions of entire lamellar domains. Small-angle X-ray scattering and 2H NMR spectroscopy of 2H2O also show that the interaction of calcium with DMPA promotes an important dehydration of the lipid assembly, even though the latter technique clearly demonstrates that some water molecules remain strongly bond in the DMPA/Ca2+ complexes. The carbonyl stretching mode region of the infrared spectrum of DMPA/Ca2+ complexes suggests that these water molecules are trapped near the interfacial region of the lipid membrane and are hydrogen bonded with the carbonyl groups of the lipid. Finally, comparison of the phosphate stretching mode region of the infrared spectra of complexes of DMPA with calcium ions with those of model compounds provides strong evidence that calcium ions bind to both charges of the phosphate group of DMPA and form bridges between adjacent bilayers.

Glycerophospholipids↗

PubMed1991

Selective labelling of melittin with a fluorescent dansylcadaverine probe using guinea-pig liver transglutaminase.

Melittin, a C-terminal glutamine peptide, incorporated the fluorescent probe monodansylcadaverine (DNC) when catalysed by guinea-pig liver transglutaminase and Ca2+, as determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A 1:1 adduct DNC-melittin was identified in which a single glutamine residue out of two, i.e. Gln25, acts as acyl donor. Incubation of melittin with transglutaminase in the absence of DNC originated high molecular mass complexes indicative that the peptide lysine residue can act as an acyl acceptor. The DNC-melittin was about 3 times more active in the lysis of red cell membranes than native melittin. Fluorescence study of the labelled melittin in the submicromolar range where it is active on cells showed that while totally exposed to solvent in methanol solution, both Trp and dansyl groups are buried in buffer solution. This strongly suggests that DNC-melittin is self-associated and indeed more active than the native melittin in the same conditions.

Amino Acid Sequence↗

PubMed1991

The amphiphilic alpha-helix concept. Consequences on the structure of staphylococcal delta-toxin in solution and bound to lipids.

Staphylococcal delta-toxin, a synthetic analogue and a fragment were studied in order to determine their structure in solution and bound in lipids. In solution, a self-association process is observed. Analytical ultracentrifuge and quasi-elastic light-scattering experiments suggest an isodesmic aggregation in the high concentration domain above 2 microM up to very large asymmetrical species. Decreasing concentrations below 2 microM of delta-toxin and the analogue allows dissociation, probably into monomers. The self-associated species are essentially alpha-helical (70%) with buried and highly immobilized Trp either at position 15 for natural delta-toxin or 16 for the analogue. At the lowest concentration studied, the alpha-helix content severely decreases down to 35% while Trp fluorescence shows that these residues are exposed to buffer. The fragment 11-26 is always monomeric and structureless. From all the data, a structural model of aggregated species is proposed with stacked antiparallel amphipathic rods. When bound to lipids, whatever their initial structure in solution, 26-residue long peptides mainly adopt an alpha-helix conformation (80%) while fragment 11-26 exhibits about 50% alpha-helix. The lipid-peptide interactions were quantitatively analysed. For fragment 11-26, a single-step mechanism fits the spectroscopic changes and defines a single monomeric bound structure. On the other hand, for the 26-residue-long analogue, multiple-step processes must occur. The data were analysed with a partition of tetramers into lipids followed by a partial dissociation. Finally, the affinity of fragment 11-26 severely decreases from micelles to fluid and gel-state bilayers. The partition coefficient of the delta-toxin analogue is higher than those of other more apolar peptides, such as melittin and alamethicin, correlating with Eisenberg's hydrophobic moments. It is therefore proposed that delta-toxin probably lies parallel to the surface, only penetrating weakly in lipids, depending on their packing.

Amino Acid Sequence↗

PubMed1990

Coupled changes between lipid order and polypeptide conformation at the membrane surface. A 2H NMR and Raman study of polylysine-phosphatidic acid systems.

Thermotropism and segmental chain order parameters of sn-2-perdeuteriated dimyristoyl-phosphatidic acid (DMPA)-water dispersions, with and without poly(L-lysine) (PLL) of different molecular weights, have been investigated by solid-state deuterium NMR spectroscopy. The segmental chain order parameter profile of this negatively charged lipid is similar to that already found for other lipids. Addition of long PLL (MW = 200,000) increases the temperature, Tc, of the lipid gel-to-fluid phase transition, whereas short PLL (MW = 4000) has practically no effect on Tc. In the fluid phase both varieties of PLL increase the "plateau" character of segmental order parameters up to carbon position 10. At the same reduced temperature, long PLL more significantly increases the segmental ordering, especially at the methyl terminal position. This leads to the conclusion that polar head-group capping and charge neutralization by PLL induce severe changes in lipid chain ordering, even down to the bilayer core. The structure of PLL bound to the lipid bilayer surface was monitored by Raman spectroscopy, following the amide I bands. Results show that the lipid gel-to-fluid phase transition triggers a conformational transition from ordered beta-sheet to random structure of short PLL, while it does not affect the strongly stabilized beta-sheet structure of long PLL. It is concluded that both short and long PLL can efficiently cap and neutralize lipid head groups, whatever their structure, and that peptide length is a key parameter in whether lipids or peptides are the driving force in conformationally coupled changes of both partners in the membrane.

Chemical Phenomena↗

PubMed1990

Delta-haemolysin from Staphylococcus aureus and model membranes. A solid-state 2H-NMR and 31P-NMR study.

Solid-state 2H NMR and 31P NMR of 2H-enriched chains and polar head groups, respectively, of dipalmitoylglycerophosphatidylcholine/water dispersions were undertaken to investigate the action of delta-haemolysin from Staphylococcus aureus on biomembranes. When the lipid/toxin molar ratio, Ri, is greater than or equal to 10, the gel-phase 2H powder patterns and the temperature of the gel-fluid phase transition, tc, are unchanged by the presence of the toxin whereas the 31P powder spectra of polar head groups are perturbed. At t greater than tc, a detailed analysis of methylene ordering indicates that delta-haemolysin orders the lipid chains near tc and disorders them for t much greater than tc. These findings are interpreted in terms of peptide location with regard to the membrane and suggest that the position of the toxin depends on the temperature relative to tc. Experiments carried out at Ri = 4 exhibit sharp, isotropic 2H-NMR lines, at t greater than tc, indicating that delta-haemolysin promotes the appearance of very small objects undergoing fast isotropic reorientation which average to zero the quadrupolar interaction. Below tc, one observes gel-phase powder patterns which indicate that the bacterial toxin is unable to form such small structures with ordered dipalmitoylglycerophosphocholine phospholipids. From comparison of the action of delta-haemolysin with that of melittin on same lipids [Dufourc et al. (1986) Biochemistry 25, 6448-6455] it results that both toxins perturb similarly fluid-phase lipids at elevated temperature, but they behave differently with gel-phase lipids, the former toxin being less efficient in membrane restructuring than the latter.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1990

[Immunosuppressive effect of cyclosporin A incapsulated into liposomes].

A preparation of cyclosporine A (CsA) in small liposomes (300 nm) was tested in a rat model of heterotopic cardiac allograft. At a daily dose of 1,5 mg/kg during 14 days, the graft survival rate was 30.4 +/- 2.8 days with a liposome-CsA solution versus 16 +/- 2.3 days with a Cremophore-CsA solution (p less than 0.01). Animals treated with the liposome-CsA preparation exhibited less weight loss than animals treated with cremophore-CsA solution (p less than 0.01). It is likely that the up-take of CsA by macrophages, when incapsulated into liposomes is dramatically enhanced; these cells have been pointed out at targets for IL2 inhibition by CsA.

Animals↗

PubMed1989

Interaction of staphylococcal delta-toxin and synthetic analogues with erythrocytes and phospholipid vesicles. Biological and physical properties of the amphipathic peptides.

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.

Animals↗

PubMed1989

A further insight into the binding of blood clotting factors to membranes.

The active site of factor Xa, labelled with dansylglutamylglycylarginine (DnsEGR) is sensitive to association with Ca2+, factor Va and phospholipids. When bound to factor Va, DnsEGR-factor-Xa does not change the composition of the binding site of factor Va, as shown by fluorescence energy-transfer experiments between the Trp residues of factor Va and pyrene-labelled phospholipids. Prothrombin was cleaved by alpha-chymotrypsin into two parts: N-terminal residues 1-41 (peptide 1-41) containing the gamma-carboxyglutamic acid residues (Gla), and des-(1-41)-prothrombin; their membrane association was investigated. Peptide 1-41 contains the aromatic residues Tyr and Trp in positions 24 and 41, respectively, and is suitable for fluorescence spectroscopy. The absence of fluorescence energy transfer between these residues suggests that they are more than 2.8 nm apart. Binding of Ca2+ and of phospholipids involves essentially the Tyr residue, while the C-terminal characteristics of the Trp residue remain unchanged. The conformational change which takes place on binding does not shorten the distance between Tyr and Trp beyond 2.8 nm. Our conclusion is that peptide 1-41 has an extended conformation. This result is compatible with the disordered character of the Gla region found in the crystalline structure of fragment 1 of prothrombin. Ca2+ induces a greater fluorescence energy transfer between prothrombin and membranes labelled with pyrene but has no influence on the binding of des-(1-41)-prothrombin. Moreover, the binding curves of des(1-41)-prothrombin are similar to those of prothrombin in the absence of Ca2+. It is concluded that the Ca2+-independent association of prothrombin with membranes involves essentially that part of the prothrombin molecule deleted in the Gla region.

Affinity Labels↗

PubMed1989

Perturbation of binary phospholipid mixtures by melittin: a fluorescence and raman spectroscopy study.

The effect of melittin on different binary mixtures of phospholipids has been studied by polarization of DPH fluorescence in order to determine if melittin can induce phase separation. Since the interaction between lipids and melittin is sensitive to both electrostatic and hydrophobic forces, we have studied the effect of the acyl chain length and of the polar head group of the lipids. In spite of the difference of the chain length between dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), no phase separation occurs in an equimolar mixture of these lipids in presence of melittin. However, when the charged lipid dipalmitoylphosphatidylglycerol (DPPG) is mixed with either DPPC or DSPC, the addition of melittin leads to phase separation. The DSPC/DPPG/melittin system, which shows a very complex thermotropism, has also been studied by Raman spectroscopy using DPPG with deuteriated chains in order to monitor each lipid independently. The results suggest that the higher affinity of melittin for DPPG leads to a partial phase separation. We propose the formation of DPPG-rich domains perturbed by melittin and peptide-free regions enriched in DSPC triggered by the head group charge and chain-length differences.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1989

Membrane structure and dynamics by 2H- and 31P-NMR. Effects of amphipatic peptidic toxins on phospholipid and biological membranes.

The actions of bee venom melittin and delta-lysin from Staphylococcus aureus on membranes have been monitored by solid-state deuterium and phosphorus NMR and shown to differ depending on temperature and on the lipid-to-peptide molar ratio Ri. In the gel phase of phosphatidylcholine model membranes, for lipid-to-peptide ratios Ri greater than 15, melittin induces isotropic lines interpreted as reflecting the presence of small discoidal structures, whereas delta-lysin does not. These small objects are metastable, that is, within a time-scale of hours they return to large lipid bilayers. The kinetics of this process depend on the lecithin chain length. In the fluid phases, at temperatures greater than that of the gel-to-fluid transition Tc, analysis of the quadruplar splittings in terms of chain ordering indicates that both melittin and delta-lysin similarly disorder the membrane. At temperatures above but close to Tc, melittin preferentially orders the center of the bilayer, while delta-lysin promotes ordering throughout the entire bilayer thickness. These effects are interpreted as reflecting different locations of the peptides with respect to the membrane surface. The addition of greater amounts of toxins, Ri = 4, on phosphatidylcholine model membranes induces very small structures irrespective of the temperature in the case of melittin, but only above Tc for delta-lysin. NMR spectral features similar to those characterizing the small fast-tumbling objects with phosphatidylcholine are also observed with egg phosphatidylethanolamine and erythrocyte membranes. The formation of small structures is thus inferred as a general process which reflects membrane supramolecular reorganization.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

Evidence for a Ca2+-independent association between calpain II and phospholipid vesicles.

Possible interactions between calpain II and phospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylcholine were studied using fluorescence and gel filtration techniques. Changes in fluorescence intensity of purified calpain II show that the enzyme strongly interacts with phosphatidylinositol and phosphatidylserine and to a lesser extent with phosphatidylcholine. These results are corroborated by the gel filtration technique which permits the isolation of the enzyme phospholipid complex. Association between calpain II and various phospholipid vesicles can occur in the absence of calcium. Such binding occurs without any observable change of the molecular mass of the two subunits on SDS-polyacrylamide gel electrophoresis.

Animals↗