Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells

Cationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations — one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP — with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.     

Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.     

Structural characterization of diC14-amidine, a pH-sensitive cationic lipid used for transfection

The structure of N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC(14)-amidine) cationic vesicles, used for transfection, was investigated at different pH values and ionic strengths, through the analysis of the electron spin resonance (ESR) spectra of spin labels. Phospholipid derivatives, spin labeled at the 5th and 16th C-atoms along the hydrocarbon chain, incorporated in diC(14)-amidine bilayers, show that the bilayer structure is highly sensitive to the pH value of the medium, due to the two titratable groups present in the amphiphile. Compared with samples at higher pH values, the double charged diC(14)-amidine at pH 3 presents a rather non-organized bilayer gel phase, and a much lower gel-fluid temperature transition, in accord with a strong headgroup electrostatic repulsion. In addition, the structure was found to be highly dependent on the ionic strength of the medium. However, pH 3 diC(14)-amidine bilayer, in the fluid phase, was found to be slightly more closely packed than those at pH 7.4 or 9.0, which are less charged. Parallel to that, the larger isotropic hyperfine splitting measured for nitroxides in the center of the pH 3 diC(14)-amidine bilayer suggests a higher membrane polarity for the highly charged low pH sample.     

DNA alters the bilayer structure of cationic lipid diC14-amidine: A spin label study

Cationic lipids-DNA complexes (lipoplexes) have been used for delivery of nucleic acids into cells in vitro and in vivo. Despite the fact that, over the last decade, significant progress in the understanding of the cellular pathways and mechanisms involved in lipoplexes-mediated gene transfection have been achieved, a convincing relationship between the structure of lipoplexes and their in vivo and in vitro transfection activity is still missing. How does DNA affect the lipid packing and what are the consequences for transfection efficiency is the point we want to address here. We investigated the bilayer organization in cationic liposomes by electron spin resonance (ESR). Phospholipids spin labeled at the 5th and 16th carbon atoms were incorporated into the DNA/diC14-amidine complex. Our data demonstrate that electrostatic interactions involved in the formation of DNA-cationic lipid complex modify the packing of the cationic lipid membrane. DNA rigidifies the amidine fluid bilayer and fluidizes the amidine rigid bilayer just below the gel-fluid transition temperature. These effects were not observed with single nucleotides and are clearly related to the repetitive charged motif present in the DNA chain and not to a charge-charge interaction. These modifications of the initial lipid packing of the cationic lipid may reorient its cellular pathway towards different routes. A better knowledge of the cationic lipid packing before and after interaction with DNA may therefore contribute to the design of lipoplexes capable to reach specific cellular targets.     

Structure, stability, and thermodynamics of lamellar DNA-lipid complexes.

We develop a statistical thermodynamic model for the phase evolution of DNA-cationic lipid complexes in aqueous solution, as a function of the ratios of charged to neutral lipid and charged lipid to DNA. The complexes consist of parallel strands of DNA intercalated in the water layers of lamellar stacks of mixed lipid bilayers, as determined by recent synchrotron x-ray measurements. Elastic deformations of the DNA and the lipid bilayers are neglected, but DNA-induced spatial inhomogeneities in the bilayer charge densities are included. The relevant nonlinear Poisson-Boltzmann equation is solved numerically, including self-consistent treatment of the boundary conditions at the polarized membrane surfaces. For a wide range of lipid compositions, the phase evolution is characterized by three regions of lipid to DNA charge ratio, rho: 1) for low rho, the complexes coexist with excess DNA, and the DNA-DNA spacing in the complex, d, is constant; 2) for intermediate rho, including the isoelectric point rho = 1, all of the lipid and DNA in solution is incorporated into the complex, whose inter-DNA distance d increases linearly with rho; and 3) for high rho, the complexes coexist with excess liposomes (whose lipid composition is different from that in the complex), and their spacing d is nearly, but not completely, independent of rho. These results can be understood in terms of a simple charging model that reflects the competition between counterion entropy and inter-DNA (rho < 1) and interbilayer (rho > 1) repulsions. Finally, our approach and conclusions are compared with theoretical work by others, and with relevant experiments.     

Infrared and time-resolved fluorescence spectroscopic studies of the polymorphic phase behavior of phosphatidylethanolamine/diacylglycerol lipid mixtures

Fourier transform infrared (FTIR) and time-resolved fluorescence spectroscopy have been employed to examine the structural dynamics of lipid fatty acyl chains and lipid/water interfacial region of a binary lipid mixture containing unsaturated phosphatidylethanolamine (PE) and diacylglycerol (DG). Infrared vibrational frequencies of the CH2 symmetric stretching and the C = O stretching bands of the lipids were measured at different lipid compositions and temperatures. For 0% DG, the lamellar gel to lamellar liquid crystalline (L beta-L alpha) and the L alpha to inverted hexagonal (L alpha-HII) phase transitions were observed at approximately 15 degrees and 55 degrees C, respectively. As the DG content increased gradually from 0% to 15%, the L alpha-HII phase transition temperature decreased drastically while the L beta-L alpha phase transition temperature decreased only slightly. At 10% DG, a merge of these two phase transitions was noticed at approximately 10 degrees C. For the composition study at 23 degrees C, the L alpha-HII transition occurred at approximately 6-10% DG as indicated by abrupt increases in both the CH2 and C = O stretching frequencies at those DG contents. Using time-resolved fluorescence spectroscopy, abrupt decreases in both the normalized long time residual and the initial slope of the anisotropy decay function of lipid probes, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine, in these PE/DG mixtures were observed at the L alpha-HII phase transition. These changes in the anisotropy decay parameters suggested that the rotational dynamics and orientational packing of the lipids were altered at the composition-induced L alpha-HII transition, and agreed with a previous temperature-induced L alpha-HII transition study on pure unsaturated PE (Cheng (1989) Biophys. J. 55, 1025-1031). The fluorescence lifetime of water soluble probes, 8,1-anilinonapthalenes sulfonate acid, in PE/DG mixtures increased abruptly at the L alpha-HII phase transition, suggesting that the conformation and hydration of the lipid/water interfacial region also undergo significant changes at the L alpha-HII transition.     

The phase behavior of cationic lipid-DNA complexes

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram.     

Kinetics and mechanism of the barotropic lamellar gel/lamellar liquid crystal phase transition in fully hydrated dihexadecylphosphatidylethanolamine: a time-resolved x-ray diffraction study using pressure jump.

The kinetics and mechanism of the barotropic lamellar gel (L beta')/lamellar liquid crystal (L alpha) phase transition in fully hydrated 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) has been studied using time-resolved x-ray diffraction (TRXRD). The phase transition was induced by pressure jumps of varying amplitudes in both the pressurization and depressurization directions at controlled temperature (78 degrees C). Both low- and wide-angle diffracted x rays were recorded simultaneously in live time using an x-ray-sensitive image intensifier coupled to a CCD camera and Super-VHS videotape recorder. Such an arrangement allowed for the direct and quantitative characterization of the long- (lamellar repeat spacing) and short-range order (chain packing) during a kinetic experiment. The image-processed live-time x-ray diffraction data were fitted using a nonlinear least-squares model, and the parameters of the fits were monitored continuously throughout the transition. The pressure-induced transitions from the L alpha to the L beta' phase and from the L beta' to the L alpha phase was two-state (no formation of intermediates apparent during the transition) to within the sensitivity limits of the method. The corresponding transit time (the time during which both phases coexist) associated with the long- and short-range order of the pressurization-induced L alpha-to-L beta' phase transition decreased to a limiting value of approximately 50 ms with increasing pressure jump amplitude. This limiting value was close to the response time of the detector/recording system. Thus, the intrinsic transit time of this transition in fully hydrated DHPE at 78 degrees C was less than or equal to 50 ms. In contrast, the depressurization-induced L beta'-to-L alpha phase transition was slower, taking approximately 1 s to complete, and occurred with no obvious dependence of the transit time on pressure jump amplitude. In the depressurization jump experiment, the lipid responded rapidly to the pressure jump in the L beta' phase up to the rate-determining L beta'-to-L alpha transition. Such behavior was examined carefully, as it could complicate the interpretation of phase transition kinetic measurements.     

Determination of L(alpha)-H(II) phase transition temperature for 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine

The thermodynamic properties of fully-hydrated lipids provide important information about the stability of membranes and the energetic interactions of lipid bilayers with membrane proteins (Nagle and Scott, Physics Today, 2:39, 1978). The lamellar/inverse hexagonal (L(alpha)-H(II)) phase transition of 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) water mixtures is a first-order transition and, therefore, at constant pressure, must have a thermodynamically well-defined equilibrium transition temperature. The observed transition temperature is known to be dependent upon the rate at which the temperature is changed, which accounts for the many different values in the literature. X-ray diffraction was used to study the phase transition of fully-hydrated DOPE to determine the rate-independent transition temperature, T(LH). Samples were heated or cooled for a range of rates, 0.212 < r < 225 degrees C/hr, and the rate-dependent apparent phase transition temperatures, T(A)(r) were determined from the x-ray data. By use of a model-free extrapolation method, the transition temperature was found to be T(LH) = 3.33 +/- 0.16 degrees C. The hysteresis, /T(A)(r) - T(LH)/, was identical for heating and cooling rates, +/-r, and varied as /r/beta for beta approximately 1/4. This unexpected power-law relationship is consistent with a previous study (Tate et al., Biochemistry, 31:1081-1092, 1992) but differs markedly from the exponential behavior of activation barrier kinetics. The methods used in this study are general and provide a simple way to determine the true mesomorphic phase transition temperatures of other lipid and lyotropic systems.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.     

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).     

Computational and analytical modeling of cationic lipid-DNA complexes

We present a theoretical study of the physical properties of cationic lipid-DNA (CL-DNA) complexes--a promising synthetically based nonviral carrier of DNA for gene therapy. The study is based on a coarse-grained molecular model, which is used in Monte Carlo simulations of mesoscopically large systems over timescales long enough to address experimental reality. In the present work, we focus on the statistical-mechanical behavior of lamellar complexes, which in Monte Carlo simulations self-assemble spontaneously from a disordered random initial state. We measure the DNA-interaxial spacing, d(DNA), and the local cationic area charge density, sigma(M), for a wide range of values of the parameter (c) representing the fraction of cationic lipids. For weakly charged complexes (low values of (c)), we find that d(DNA) has a linear dependence on (c)(-1), which is in excellent agreement with x-ray diffraction experimental data. We also observe, in qualitative agreement with previous Poisson-Boltzmann calculations of the system, large fluctuations in the local area charge density with a pronounced minimum of sigma(M) halfway between adjacent DNA molecules. For highly-charged complexes (large (c)), we find moderate charge density fluctuations and observe deviations from linear dependence of d(DNA) on (c)(-1). This last result, together with other findings such as the decrease in the effective stretching modulus of the complex and the increased rate at which pores are formed in the complex membranes, are indicative of the gradual loss of mechanical stability of the complex, which occurs when (c) becomes large. We suggest that this may be the origin of the recently observed enhanced transfection efficiency of lamellar CL-DNA complexes at high charge densities, because the completion of the transfection process requires the disassembly of the complex and the release of the DNA into the cytoplasm. Some of the structural properties of the system are also predicted by a continuum free energy minimization. The analysis, which semiquantitatively agrees with the computational results, shows that that mesoscale physical behavior of CL-DNA complexes is governed by interplay among electrostatic, elastic, and mixing free energies.     

Water permeability and mechanical strength of polyunsaturated lipid bilayers

Micropipette aspiration was used to test mechanical strength and water permeability of giant-fluid bilayer vesicles composed of polyunsaturated phosphatidylcholine PC lipids. Eight synthetic-diacyl PCs were chosen with 18 carbon chains and degrees of unsaturation that ranged from one double bond (C18:0/1, C18:1/0) to six double bonds per PC molecule (diC18:3). Produced by increasing pipette pressurization, membrane tensions for lysis of single vesicles at 21 degrees C ranged from approximately 9 to 10 mN/m for mono- and dimono-unsaturated PCs (18:0/1, 18:1/0, and diC18:1) but dropped abruptly to approximately 5 mN/m when one or both PC chains contained two cis-double bonds (C18:0/2 and diC18:2) and even lower approximately 3 mN/m for diC18:3. Driven by osmotic filtration following transfer of individual vesicles to a hypertonic environment, the apparent coefficient for water permeability at 21 degrees C varied modestly in a range from approximately 30 to 40 microm/s for mono- and dimono-unsaturated PCs. However, with two or more cis-double bonds in a chain, the apparent permeability rose to approximately 50 microm/s for C18:0/2, then strikingly to approximately 90 microm/s for diC18:2 and approximately 150 microm/s for diC18:3. The measurements of water permeability were found to scale exponentially with the reduced temperatures reported for these lipids in the literature. The correlation supports the concept that increase in free volume acquired in thermal expansion above the main gel-liquid crystal transition of a bilayer is a major factor in water transport. Taken together, the prominent changes in lysis tension and water permeability indicate that major changes occur in chain packing and cohesive interactions when two or more cis-double bonds alternate with saturated bonds along a chain.     

Mechanism of the lamellar/inverse hexagonal phase transition examined by high resolution x-ray diffraction

For the first time the electron density of the lamellar liquid crystalline as well as of the inverted hexagonal phase could be retrieved at the transition temperature. A reliable decomposition of the d-spacings into hydrophobic and hydrophilic structure elements could be performed owing to the presence of a sufficient number of reflections. While the hydrocarbon chain length, d(C), in the lamellar phase with a value of 14.5 A lies within the extreme limits of the estimated chain length of the inverse hexagonal phase 10 A < d(C) < 16 A, the changes in the hydrophilic region vary strongly. During the lamellar-to-inverse hexagonal phase transition the area per lipid molecule reduces by approximately 25%, and the number of water molecules per lipid increases from 14 to 18. On the basis of the analysis of the structural components of each phase, the interface between the coexisting mesophases between 66 and 84 degrees C has been examined in detail, and a model for the formation of the first rods in the matrix of the lamellar phospholipid stack is discussed. Judging from the structural relations between the inverse hexagonal and the lamellar phase, we suggest a cooperative chain reaction of rod formation at the transition midpoint, which is mainly driven by minimizing the interstitial region.     

Search for an endotherm in chloroplast lamellar membranes associated with chilling-inhibition of photosynthesis

The phase transition of chloroplast lamellar membrane lipids has been proposed to be the underlying cause of chilling-induced inhibition of photosynthesis in sensitive plants. Differential scanning calorimetry has been used to search for any endotherms arising from lipid state changes in chloroplast lamellar membranes of the chilling-sensitive plants cantaloupe , kidney bean, domestic tomato, and soybean. For comparison, calorimetric scans of chloroplast lamellar membranes from the chilling-insensitive plants spinach, pea, and wild tomato were made. A large reversible endotherm, extending from below 10 degrees to nearly 40 degrees C, was observed in chloroplast membranes from tomatoes of both chilling-sensitive (Lycopersicon esculentum Mill. cv. Floramerica ) and chilling-insensitive (L. hirsutum LA 1361) species. A much smaller endotherm, approximately 5 to 10% of the area of that seen in the two tomato species, and extending over a similar temperature range, was detected in chloroplasts from chilling-insensitive spinach and peas, and also was generally observed in chloroplasts from chilling-sensitive cantaloupe , kidney bean, and soybean. The enthalpy of these smaller endotherms indicates that, if the endotherm arose entirely from a lipid transition, then it corresponded to the melting of less than about 10% of the total membrane polar lipid. On the basis of these data it is concluded that there is no correlation between chilling sensitivity of photosynthesis and the presence or absence of a phase transition of bulk membrane lipids of the chloroplast lamellar membrane at temperatures above 5 degrees C.     

The structure of DNA-DLPC-cationic gemini surfactant aggregates: a small angle synchrotron X-ray diffraction study

The structure of aggregates formed by interaction of DNA with unilamellar dilauroylphosphatidylcholine (DLPC) vesicles (DNA:DLPC=1:1 base/mol) in the presence of gemini surfactant butane-1,4-diyl-bis(dodecyldimethylammonium bromide) (C12GS) was investigated using synchrotron small angle X-ray diffraction. In the concentration range C12GS+:DLPC< or =1 mol/mol, a condensed lamellar Lalphac phase was found with a repeat period of lipid bilayer stacking in the range d approximately 5.70-6.53 nm and the DNA interhelical distance d(DNA) approximately 3.52-3.99 nm, depending on the concentration of C12GS. At molar ratio C12GS+:DLPC> or =0.35:1, the diffractograms have shown the presence of a second lamellar phase with the repeat period d approximately 5.31 nm which slightly decreases with increasing concentration of C12GS+. The increasing fraction of this phase in the aggregates with increasing concentration of C12GS supports the association of this phase with microscopic domains enriched by surfactant molecules. The temperature behaviour of aggregates was investigated in the range 25-60 degrees C and the transversal thermal expansivities of the observed phases were determined.     

Conformational and hydrational properties during the L(beta)- to L(alpha)- and L(alpha)- to H(II)-phase transition in phosphatidylethanolamine

Differential scanning calorimetry (DSC) measurements have been carried out simultaneously with small- and wide-angle X-ray scattering recordings on liposomal dispersions of stearoyl-oleoyl-phosphatidylethanolamine (PE) in a temperature range from 20 to 80 degrees C. The main transition temperature, T(m), was determined at 30.9 degrees C with an enthalpy of 28.5 kJ/mol and the lamellar-to-inverse hexagonal phase transition temperature, T(hex), at 61.6 degrees C with an enthalpy of 3.8 kJ/mol. Additionally highly resolved small angle X-ray diffraction experiments performed at equilibrium conditions allowed a reliable decomposition of the lattice spacings into hydrophobic and hydrophilic structure elements as well as the determination of the lipid interface area of the lamellar gel-phase (L(beta)), the fluid lamellar phase (L(alpha)) and of the inverse hexagonal phase (H(II)). The rearrangement of the lipid matrix and the coincident change of free water per lipid is illustrated for both transitions. Last, possible transition mechanisms are discussed on a molecular level.     

Formation of monolayers and bilayer foam films from lamellar,inverted hexagonal and cubic lipid phases

This study revealed large distinctions between the lamellar and non-lamellar liquid crystalline lipid phases in their spreading at the air/water interface and propensity to form bilayer foam films. Comparative measurements were made for the lamellar L(alpha), the inverted hexagonal H(II) and the bicontinuous cubic Pn3m phases of the phospholipid dipalmitoleoylphosphatidylethanolamine (DPoPE). With regard to monolayer formation, followed as the decrease of surface tension with time, the best spreading (lowest surface tension) was observed for the L(alpha) phase, and poorest spreading (highest surface tension) was recorded for the H(II) phase. The cubic Pn3m phase of DPoPE, induced by temperature cycling, retained an intermediate position between the L(alpha) and H(II) phases. According to their ability to lower surface tension and disintegrate at the air/water interface, the three phases thus order as L(alpha)>Pn3m>H(II). Clearly expressed threshold (minimum) bulk lipid concentrations, C(t), required for formation of stable foam bilayers from these phases, were determined and their values were found to correlate well with the bulk lipid phase behaviour. The C(t) values for L(alpha) and H(II) substantially increase with the temperature. Their Arrhenius plots, ln C(t) versus 1/ T, are linear and intersect at approximately 36-37 degrees C, coinciding with the onset of the bulk L(alpha)-->H(II) phase transition, as determined by differential scanning calorimetry. However, the C(t) value for the Pn3m phase, equal to 30 micro g/mL, was found to be constant over the whole range investigated between 20 degrees C and 50 degrees C. The horizontal C(t) versus T plot for the Pn3m phase crosses the respective plot for the L(alpha) phase at the temperature bounding from below the hysteretic loop of the L(alpha)<-->H(II) transition (approximately 26 degrees C), thus providing a certain insight about the thermodynamic stability of the Pn3m phase relative to the L(alpha) phase. The established strong effect of the particular lipid phase on the formation of monolayers and stable black foam films should be of importance in various in vitro and in vivo systems, where lipid structures are in contact with interfaces and disintegrate there to different extents.