Biomedical subjects

M Lafleur

Publications and source records attributed to M Lafleur.

At least 37 records · Page 2Linked to original sources

PubMed1993

Effect of cholesterol on the polymorphism of dipalmitoylphosphatidylcholine/melittin complexes: an NMR study.

In order to get insights into the effects of cholesterol on protein activity, the lytic power of melittin on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol mixtures was studied using solid-state deuterium and phosphorus-31 nuclear magnetic resonance spectroscopy (2H and 31P-NMR). After incubation, melittin disrupts pure DPPC vesicles, leading to the formation of small lipid/peptide complexes below the phase transition temperature (Tm), whereas large bilayer assemblies are reformed above Tm; the transition between these two species is thermally reversible. This study reveals that cholesterol modifies this thermal behavior and that this modulation of the lytic power of melittin is indirect, since it is essentially related to the original effect of the sterol on the thermotropism of pure lipid bilayers. It is known that melittin does not lyse gel phase DPPC bilayers spontaneously. Our study shows that the addition of large amounts of sterol (30 mol%) does not promote the spontaneous lysis at 26 degrees C, despite the increased fluidity of the lipid system. The lysis takes place around 32 degrees C, regardless of the cholesterol concentration. This study also shows that high concentrations of cholesterol (> or = 30%) in DPPC bilayer inhibit the lysis. It is proposed that the tight lipid packing due to high cholesterol concentrations prevents the penetration of melittin into the bilayer. When melittin interacts with cholesterol-rich bilayers (30 mol%), the lysis is only partial, and leads to the formation of small cholesterol-depleted particles. Finally, DPPC which bears deuteriated acyl chains was used to determine the influence of melittin on the orientational order of the lipid chains in the large assemblies. The quadrupolar splittings obtained in the presence of melittin are not considerably different than those obtained in the absence of melittin.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1993

Evidence for two pools of cholesterol in the Acholeplasma laidlawii strain B membrane: a deuterium NMR and DSC study.

Recent investigations have indicated that there exists a well-defined range of membrane hydrocarbon order compatible with good growth of the microorganism Acholeplasma laidlawii B [Monck, M., Bloom, M., Lafleur, M., Lewis, R. N. A. H., McElhaney, R. N., & Cullis, P. R. (1992) Biochemistry 31, 10037-10043]. Since cholesterol increases hydrocarbon order in membranes, it was of interest to examine the effect of cholesterol on the hydrocarbon order and growth characteristics of A. laidlawii B. Cholesterol is normally absent from A. laidlawii membranes since it is neither biosynthesized nor required for the growth or survival of the microorganism. However, cholesterol will be incorporated into the membrane if exogenously supplied to the A. laidlawii culture. For membranes prepared from cells grown in the presence of cholesterol, chemical determinations indicated cholesterol represented as much as 40 mol% of the total membrane lipid. However, 2H NMR order parameter measurements and DSC studies of the same membrane preparation suggested that cholesterol was present at significantly lower levels (approximately 10-15 mol%) in the membrane lipid bilayer. Further incorporation of cholesterol into the A. laidlawii lipid bilayer was found to occur with an increase in temperature or by lyophilization and rehydration at high temperatures, suggesting that sterol present in a separate pool in the membrane preparation could then gain access to the bilayer. 2H NMR spectra of A. laidlawii membrane preparations containing deuterium-labeled cholesterol indicate that the bulk of the cholesterol present in this separate pool is in a solid form.

Acholeplasma laidlawii↗

PubMed1993

Comparison between orientational and conformational orders in fluid lipid bilayers.

The orientational order as determined by 2H NMR and the infrared frequencies of the C--H stretching modes of the methylene groups have been measured for several systems (POPC, POPC/cholesterol and POPE), all in the fluid phase, and then were compared; this work reveals an unexpected linear correlation between them. This experimental result shows that both measurements are essentially sensitive to a common motion, most likely trans/gauche isomerisation. This new correlation with those already found in the literature suggest that several measurements related to the hydrophobic core of the fluid bilayer describe different aspects of a universal behavior. The correlation presented here does not extend to the lipid in gel phase where slower motions affect the NMR lineshape.

Biophysical Phenomena↗

PubMed1992

Influence of lipid composition on the orientational order in Acholeplasma laidlawii strain B membranes: a deuterium NMR study.

2H NMR techniques have recently been developed to determine the complete orientational order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid [Lafleur, M., Fine, B., Sternin, E., Cullis, P.R., & Bloom, M. (1989) Biophys. J. 56, 1037-1041]. In this work, these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A. laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuteriated fatty acid of varying length and unsaturation. By variation of the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic, or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter, mean value of S, varies over the range 0.140-0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2H NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.

Acholeplasma laidlawii↗

PubMed1992

Permeability of liposomal membranes to water: results from the magnetic field dependence of T1 of solvent protons in suspensions of vesicles with entrapped paramagnetic ions.

The diffusive permeability to water molecules, Pd, of lipid vesicles with entrapped paramagnetic solute ions can be determined rapidly from analysis of the magnetic field dependence (nuclear magnetic relaxation dispersion, or NMRD profile) of T1 of exterior solvent water protons. Such data yield tau, the mean lifetime of solvent molecules inside the vesicles, from tau = (fT1Para) - T1Ves, where f is the volume fraction of entrapped water, T1Para is the observed T1 corrected for buffer background, and T1Ves is the relaxation time of water protons in the entrapped solution. For small spherical unilamellar vesicles of inner radius R, Pd = R/3 tau, f can be obtained accurately from knowledge of both the concentration of Gd(DTPA)2- in the solution in which the vesicles were formed and the average concentration of ions in the final sample. At low temperatures, in the limit of slow exchange, T1Para becomes independent of field and tau = fT1Para; the observation of a field-independent profile is a control that confirms that no paramagnetic material is external to the vesicles. We have measured T1Para, using a field-cycling relaxometer, for suspensions of POPC (1-palmitoyl-2-oleoyl-lecithin) vesicles with 100-500 mM entrapped Gd(DTPA)2- and membrane concentrations of cholesterol ranging from 0 to 40 mol %. These profiles, which span the field range 0.01-50 MHz proton Larmor frequency, were taken at 5, 15, 25, and 35 degrees C. Concentrations of Gd(DTPA)2- were determined independently by both ICP analyses and NMRD methods. Values for Pd for vesicles with 100 mM Gd(DTPA)2- and outer diameters 100 nm +/- 20%, as determined by quasielastic light scattering, are 63, 47, 24, 16, and 8.7 x 10(-4) cm s-1, at 25 degrees C, for cholesterol concentrations of 0, 10, 20, 30, and 40%, respectively. The corresponding activation enthalpies are 14, 14, 14, 17, and 17 kcal/M. Comparison with 2H NMR studies of deuterated POPC vesicles with no cholesterol at 20 degrees C, and with 10% at 40 degrees C, which yielded the same order parameter for the palmitoyl acyl chains, gives no indication of a correlation between order parameter and permeability.

Contrast Media↗

PubMed1991

Investigation of the interaction between melittin and dipalmitoylphosphatidylglycerol bilayers by vibrational spectroscopy.

Melittin is shown to affect the structure of the charged phospholipid dipalmitoylphosphatidylglycerol (DPPG). In the gel phase, the presence of melittin leads to (i) an increased lipid interchain vibrational coupling, (ii) a shift of the rectangular to hexagonal lipid packing transition toward low temperatures, (iii) a very small conformational disordering effect, (iv) a decrease of the polarity or hydrogen bonding capability of the lipid ester group surrounding, (v) an important decrease of the water content in the complexes where the remaining water has a more disordered structure than bulk water, and (vi) an interlamellar repeat distance of 79 A. All these observations are rationalized by the following model: adjacent bilayers of DPPG are bridged by tetramers of melittin through electrostatic interactions inducing surface charge neutralization and partial dehydration of the complexes. Melittin also affects the thermotropic behavior of DPPG. When a small amount of the toxin is present, its affinity for charged lipids is such that a phase separation occurs, the domains being stable enough to have their own gel to liquid-crystalline phase transition. In the fluid state, a deeper penetration into the lipid matrix is proposed based on the downshift of the phase transition and the low vibrational interchain coupling. This study brings out general features of cationic species/anionic lipid complexes. The charge neutralization leads to stronger interchain coupling, and electrostatic bridging of adjacent bilayers seems to be common. The hydrophobicity of the peptide is a key factor in the modulation of the gel to liquid-crystalline phase transition and in its insertion in the fluid lipid matrix.

Anions↗

PubMed1990

Comparison of the orientational order of lipid chains in the L alpha and HII phases.

The orientational order profile has been determined by using deuterium nuclear magnetic resonance (2H NMR) for POPE in the lamellar liquid-crystalline (L alpha) and the hexagonal (HII) phases and is shown to be sensitive to the symmetry of the lipid phase. In the HII phase, as compared to the L alpha phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. 2H NMR studies of POPE dispersions containing tetradecanol or decane, both of which can induce HII phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the L alpha to HII phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The L alpha to HII phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. This is consistent with a preferential partition of the decane molecules into a disordered environment such as the intercylinder spaces in the HII phase. The presence of decane in the HII phase structure does not modify the order of the lipid chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanes↗

PubMed1990

Modulation of the orientational order profile of the lipid acyl chain in the L alpha phase.

The orientational order profile along the lipid acyl chain has been characterized under several different conditions of polar headgroup composition, temperature, and cholesterol content. Despite the different nature of these factors, the variation of the order is governed by two common trends. First, the relative change of order induced by the variation of these factors is always more pronounced towards the end of the chain than for the methylene groups near the interface. Second, there is, to a first approximation, a distinct correlation between the magnitude of the order parameters and the shape of the order profile. For example when the chain is highly ordered, the relative width of the order distribution is narrow indicating that the plateau region is longer. These conclusions suggest that the orientational order profile depends on only a small number of parameters and demonstrate clearly that the correlation length for changes in orientational order is much greater than one C-C bond length. Our results also show that the reduced temperature is not related in simple terms to orientational order and probably has little theoretical significance. The orientational order profiles of POPC and POPE bilayers are significantly different even when expressed in terms of reduced temperature. The behavior of POPC/cholesterol systems also indicates that the orientational order of the lipid chain and the gel-to-liquid crystalline phase transition temperature are not related in a straightforward manner.

Cell Membrane↗

PubMed1990

Lipid polymorphism and hydrocarbon order.

The use of 2H nuclear magnetic resonance for the characterization of the polymorphic behavior of lipids is illustrated. Different lipid phase preferences may be expected to influence the orientational order and its variation along the acyl chains. Several results are presented to support that view. An increase of motional freedom and a redistribution of the order along the acyl chains are observed during the lamellar-to-hexagonal phase transition, showing that the order profile is sensitive to the lipid phase symmetry. In addition, if the preferences for nonlamellar phases are not expressed explicitly, the presence of "nonbilayer" lipids constrained in bilayer environment induces increased hydrocarbon order. This suggests that order parameters of the acyl chains and lipid polymorphic tendencies are intimately related.

Chemical Phenomena↗

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

Smoothed orientational order profile of lipid bilayers by 2H-nuclear magnetic resonance.

A new method has been developed to determine the complete orientational order profile of lipid bilayers using 2H-NMR. The profile is obtained from a single powder spectrum of a lipid which has a saturated chain fully deuteriated. The smoothed order profile is determined directly from the normalized dePaked spectrum assuming a monotonic decrease of the order along the acyl chain. The oscillatory variations of the order at the beginning of the chain are not described by this method. However the smoothed order profile reveals in a straightforward way the crucial features of the anisotropic order of the bilayer.

Deuterium↗

PubMed1987

Study of the effect of melittin on the thermotropism of dipalmitoylphosphatidylcholine by Raman spectroscopy.

The effect of amphiphilic toxin melittin (Mel) on the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) has been studied by Raman spectroscopy. The spectra show that for complexes that were incubated above 40 degrees C, melittin does not penetrate DPPC bilayers in the gel state as an intrinsic protein since the conformation of the lipid acyl chains is just slightly perturbed by the toxin. Instead, at the DPPC/Mel molar ratios investigated (Ri = 5 and 15), Raman results suggest the formation of discoidal particles as complexes of apolipoproteins with phosphatidylcholines. These lipid/protein assemblies are characterized by a high conformational order, low intermolecular chain-chain interactions due to the size of the particles, and a low cooperativity of the gel to liquid-crystalline transition. The latter is biphasic for samples studied. It is believed that aggregation of these particles into larger ones occurs when the bilayers become less stable at higher temperature and that melittin is partially embedded into the hydrophobic core of the larger lipid/protein units. The freezing of the dispersion at approximately 0 degrees C also causes a reversible aggregation of the particles that leads to the formation of domains in which the interchain interactions are very similar to that of the pure lipid. The small particles of DPPC/Mel are also metastable, and with time, they form larger aggregates from which melittin is expulsed.

Bee Venoms↗

PubMed1984

A restatement of melittin-induced effects on the thermotropism of zwitterionic phospholipids.

Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and/or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures T greater than or equal to Tm efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and/or non-hydrolysable lipids. Melittin monomer does not change Tm. When melittin tetramer is stabilized, it decreases Tm by 10-15 deg. C. The transition broadens, and is finally abolished for Ri less than or equal to 2. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For T less than or equal to Tm, fluorescence and Raman show that melittin decreases the amount of CH2 groups in 'trans' conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at T greater than or equal to Tm, while from Raman the positional intermolecular order decreases without significant change in the CH2 groups 'trans'/'gauche' ratio.

Bee Venoms↗

PubMed1978

[Ineffective erythropoiesis in myeloid splenomegaly: the 59Fe test, bone marrow histologic and cytologic data].

Eight patients presenting primary myelofibrosis or spent polycythemia were submitted to a ferrokinetic study, a triphine bone marrow biopsy and a bone marrow puncture. In all cases before treatment (splenic radiotherapy) ferrokinetic measurements demonstrated an ineffective erythropoiesis, the intensity of which was precised on bone marrow biopsies, in numbering the erythroblasts per surface unit. Bone marrow biopsies permitted also, through a semiquantitative estimate of the bone marrow cellularity, to evaluate an aplasia component not always expected with ferrokinetic study. Study of bone marrow allowed to precise the site of the erythroblastic abortion; cellular death might occur during the last nitosis of the erythroblastic series (transition from the polychromatophil erythroblasts to the acidophil erythroblasts). In order to delineate the general pattern of erythropoiesis before treatment and when assessing the results of a treatment, besides ferrokinetic measurements, the study of bone marrow biopsy and smears appears relevant.

Adult↗

PubMed1976