Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α → H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α → H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α → H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.     

Influence of the lamellar phase unbinding energy on the relative stability of lamellar and inverted cubic phases

Based on curvature energy considerations, nonbilayer phase-forming phospholipids in excess water should form stable bicontinuous inverted cubic (Q_II) phases at temperatures between the lamellar (L_α) and inverted hexagonal (H_II) phase regions. However, the phosphatidylethanolamines (PEs), which are a common class of biomembrane phospholipids, typically display direct L_α/H_II phase transitions and may form intermediate Q_II phases only after the temperature is cycled repeatedly across the L_α/H_II phase transition temperature, T_H, or when the H_II phases are cooled from T > T_H. This raises the question of whether models of inverted phase stability, which are based on curvature energy alone, accurately predict the relative free energy of these phases. Here we demonstrate the important role of a noncurvature energy contribution, the unbinding energy of the L_α phase bilayers, g_u, that serves to stabilize the L_α phase relative to the nonlamellar phases. The planar L_α phase bilayers must separate for a Q_II phase to form and it turns out that the work of their unbinding can be larger than the curvature energy reduction on formation of Q_II phase from L_α at temperatures near the L_α/Q_II transition temperature (T_Q). Using g_u and elastic constant values typical of unsaturated PEs, we show that g_u is sufficient to make T_Q > T_H for the latter lipids. Such systems would display direct L_α → H_II transitions, and a Q_II phase might only form as a metastable phase upon cooling of the H_II phase. The g_u values for methylated PEs and PE/phosphatidylcholine mixtures are significantly smaller than those for PEs and increase T_Q by only a few degrees, consistent with observations of these systems. This influence of g_u also rationalizes the effect of some aqueous solutes to increase the rate of Q_II formation during temperature cycling of lipid dispersions. Finally, the results are relevant to protocols for determining the Gaussian curvature modulus, which substantially affects the energy of intermediates in membrane fusion and fission. Recently, two such methods were proposed based on measuring T_Q and on measuring Q_II phase unit cell dimensions, respectively. In view of the effect of g_u on T_Q that we describe here, the latter method, which does not depend on the value of g_u, is preferable.     

The nature of lipidic particles and their roles polymorphic transitions

The structural transition between bilayer (L_α), inverted hexagonal (H_II and inverted cubic (C_II) phases in mixtures of unsaturated phosphatidylethanolamines (PE) and phosphatidylcholines (PC) were investigated. Freeze fracture electron micrographs of intermediate stages of phase transitions showed that C_II was a stable intermediate form between the L_α-H_II transition. The electron microscopic observation was supported by X-ray diffraction and ³¹P-NMR results. Detailed morphology revealed that during the L_α-C_II transition, interlamellae attachment points (conical lipidic particles) connect adjacent bilayers to form arrays of entrapped water pockets (inverted micelles). These water-containg spherical units were packed in a cubic lattice. In the C_II to H_II transition, these spherical units were linearly connected to form tubes. During the L_α-H_II transition, a ripple pattern was observed across the otherwise smooth lamellar. The troughs of the ripples were transformed into linear connections between adjacent bilayers, thereby converting multilayer structures into parallel tubes. No lipidic particles were involved in this type of transition. We show that there are different mechanisms involved in the L_α, H_II, C_II polymorphic transitions, and that different types of ‘lipidic particles’ representing different molecular organizations may be involved in each case. Models of these transitions are proposed.     

Fusion Peptides Promote Formation of Bilayer Cubic Phases in Lipid Dispersions. An X-Ray Diffraction Study

Small angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the H_II phase and shifted the L_α–H_II transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, Q_II, which becomes inserted between the L_α and H_II phases on the temperature scale. Peptide-induced Q_II had strongly reduced lattice constants in comparison to the Q_II phases that form in pure lipids. Q_II formation was favored at the expense of both L_α and H_II phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the L_α phase and completely abolished the H_II phase in DOPE-Me/WT-20 50:1 dispersions, converted the Q_II phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative Gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving H_II phase-like intermediates.     

Sugars favour formation of hexagonal (HII) phase at the expense of lamellar liquid-crystalline phase in hydrated phosphatidylethanolamines

The disaccharides, sucrose and trehalose, markedly decreased (up to 17-13C°) the temperature of the lamellar to hexagonal (L_α →H_II) phase transition and simultaneously increase by 2–4 C° the temperature of the lamellar gel to lamellar liquid-crystal (L_β →L_α) phase transition in hydrated dihexadecylphosphatidylethanolamine and distearoylphosphatidylethanolamine. These two transitions merge and convert into a single L_β-H_II phase transition when dispersed in 2.4 M sucrose. These results are inconsistent with recent reports by (8) and (9)) which suggest that trehalose stabilizes the L_α phase relative to the H_II phase and shifts upwards beyond detectability the L_α-H_II transition. The present results are considered as a manifestation of the Hofmeister effect in which the sugars act as kosmotropic reagents stabilizing the structure of bulk water. This tends to decrease the area of contact between the lipid and the aqueous phases and favours the H_II and L_β phases relative to L_α phase. This hypothesis is consistent with the effects of chaotropic reagents on the L_α-H_II phase transition (Yeagle and Sen (1986) Biochemistry 25, 7518–7522) and on the stability of the lamellar phase of dipalmitoylphosphatidylcholine (Oku and MacDonald (1983) J. Biol. Chem. 258, 8733–8738).     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Conformational Dynamics in the Selectivity Filter of KcsA in Response to Potassium Ion Concentration

Conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid-state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the potassium ion concentration is changed from 50 mM to 1 μM. Potassium ion titration studies reveal that the site-specific K_d for K⁺ binding at the key pore residue Val76 is on the order of ∼ 7 μM and that a relatively high sample hydration is necessary to observe the low-K⁺ conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high-K⁺ and low-K⁺ states are in slow exchange on the NMR timescale (k_ex < 500 s^− 1). The slow rate and tight binding for evacuating both inner sites simultaneously differ from prior observations in detergent in solution, but agree well with measurements by electrophysiology and appear to result from our use of a hydrated bilayer environment. These observations strongly support a common assumption that the low-K⁺ state is not involved in ion transmission, and that during transmission one of the two inner sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of the evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.     

The ionic strength effect on the DNA complexation by DOPC - gemini surfactants liposomes

Liposome dispersions obtained from the mixture of gemini surfactants of the type alkane-α,ω-diyl-bis(alkyldimethylammonium bromide) and helper lipid DOPC create complexes with DNA showing a regular inner microstructure, identified by small angle X-ray diffraction as condensed lamellar phase (L_α^c). In addition to the L_α^c phase, a coexisting lamellar phase L_B was also identified in the complexes formed, with periodicities in the range ~ 8.8-5.7 nm, at ionic strengths corresponding to 50-200 mM NaCl. The periodicities of L_B phase did not correspond to those identified in liposome dispersion without DNA using small angle neutron scattering. The observed phase separation is shown to depend on the interplay between the surface charge density of cationic liposomes, ionic strength and method of complex preparation. The effect of ionic strength on complex formation was studied by isothermal titration calorimetry and zeta potential measurements. High ionic strength reduces the fraction of bound DNA in the complexes, and the isoelectric point is attained at a ratio of DNA/gemini surfactant which is lower than the one that can be estimated by calculation based on nominal charges of CLs and DNA.     

Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and β-sitosterol in unilamellar vesicles

The influence of a mammalian sterol cholesterol and a plant sterol β-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n = 14-22 is the even number of acyl chain carbons) was studied at 30 °C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Ku?erka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n = 18-22 similarly. β-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 Å² and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and β-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.     

Synthesis, structure and redox properties of an unexpected trinuclear copper(II) complex with aspartame: [Cu(apm)2Cu(μ-N,O:O′-apm)2(H2O)Cu(apm)2(H2O)] · 5H2O

Structural, magnetic and spectroscopic data of a new trinuclear copper(II) complex with the ligand aspartame (apm) are described. [Cu(apm)₂Cu(μ-N,O:O′-apm)₂(H₂O)Cu(apm)₂(H₂O)] · 5H₂O crystallizes in the triclinic system, space group P1 (#1) with a = 7.3300(1) Å, b = 15.6840(1) Å, c = 21.5280(1) Å, α = 93.02(1)°, β = 93.21(1)°, γ = 92.66(1)° and Z = 1. Aspartame coordinates to Cu(II) through the carboxylate and β-amino groups. The carboxylate groups of the two central ligands act as bidentate bridges in a syn-anti conformation while the carboxylate groups of the four peripheral ligands are monodentate in a syn conformation. The central copper ion is in a distorted square pyramidal geometry with the apical position being occupied by one oxygen atom of the water molecule. The two terminal copper(II) atoms are coordinated to the ligands in the same position but their coordination sphere differs from each other due to the fact that one copper atom has a water molecule in an apical position leading to an octahedral coordination sphere while the other copper atom is exclusively coordinated to aspartame ligands forming a distorted square pyramidal coordination sphere. Thermal analysis is consistent with the X-ray structure. EPR spectra and CV curves indicate a rupture of the trinuclear framework when this complex is dissolved in ethanol or DMF, forming a mononuclear species, with a tetragonal structure.     

N Solid-state NMR spectroscopic studies on phospholamban at its phosphorylated form at Ser-16 in aligned phospholipid bilayers

Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically ¹⁵N-Ala-11- and ¹⁵N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine/2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned ¹⁵N-labeled Ala-11 and Leu-7 WT-PLB samples show ¹⁵N resonance peaks at approximately 71 ppm and 75 ppm, respectively, while the corresponding phosphorylated forms P-PLB show ¹⁵N peaks at 92 ppm and 99 ppm, respectively. These ¹⁵N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter ∼0.1 and 0.2 μm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 °C, this temperature corresponding closely to the heat capacity maxima (T_em) of DNPC MLVs and LUVs (T_em ≈21 °C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T_em. This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans→gauche isomerization.     

Time-resolved OH → EH transition of the aberrant ba3 oxidase from Thermus thermophilus

The kinetics of single-electron injection into the oxidized nonrelaxed state (O_H → E_H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ ∼ 17 μs) reflects oxidation of Cu_A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ ∼ 420 μs) includes reoxidation of both hemes by Cu_B. This is in significant contrast to the O_H → E_H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu_A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O_H forms also in the ba₃ oxidase from T. thermophilus.     

Characterization of Membrane Protein Interactions by Isothermal Titration Calorimetry

Understanding the structure, folding, and interaction of membrane proteins requires experimental tools to quantify the association of transmembrane (TM) helices. Here, we introduce isothermal titration calorimetry (ITC) to measure integrin αIIbβ3 TM complex affinity, to study the consequences of helix–helix preorientation in lipid bilayers, and to examine protein-induced lipid reorganization. Phospholipid bicelles served as membrane mimics. The association of αIIbβ3 proceeded with a free energy change of − 4.61 ± 0.04 kcal/mol at bicelle conditions where the sampling of random helix–helix orientations leads to complex formation. At bicelle conditions that approach a true bilayer structure in effect, an entropy saving of > 1 kcal/mol was obtained from helix–helix preorientation. The magnitudes of enthalpy and entropy changes increased distinctly with bicelle dimensions, indicating long-range changes in bicelle lipid properties upon αIIbβ3 TM association. NMR spectroscopy confirmed ITC affinity measurements and revealed αIIbβ3 association and dissociation rates of 4500 ± 100 s^− 1 and 2.1 ± 0.1 s^− 1, respectively. Thus, ITC is able to provide comprehensive insight into the interaction of membrane proteins.     

Structural rearrangement of model membranes by the peptide antibiotic NK-2

We have developed a novel α-helical peptide antibiotic termed NK-2. It efficiently kills bacteria, but not human cells, by membrane destruction. This selectivity could be attributed to the different membrane lipid compositions of the target cells. To understand the mechanisms of selectivity and membrane destruction, we investigated the influence of NK-2 on the supramolecular aggregate structure, the phase transition behavior, the acyl chain fluidity, and the surface charges of phospholipids representative for the bacterial and the human cell cytoplasmic membranes. The cationic NK-2 binds to anionic phosphatidylglycerol liposomes, causing a thinning of the membrane and an increase in the phase transition temperature. However, this interaction is not solely of electrostatic but also of hydrophobic nature, indicated by an overcompensation of the Zeta potential. Whereas NK-2 has no effect on phosphatidylcholine liposomes, it enhances the fluidity of phosphatidylethanolamine acyl chains and lowers the phase transition enthalpy of the gel to liquid cristalline transition. The most dramatic effect, however, was observed for the lamellar/inverted hexagonal transition of phosphatidylethanolamine which was reduced by more than 10 °C. Thus, NK-2 promotes a negative membrane curvature which can lead to the collapse of the phosphatidylethanolamine-rich bacterial cytoplasmic membrane.     

Osmotic parameters of red blood cells from umbilical cord blood

The transfusion of red blood cells from umbilical cord blood (cord RBCs) is gathering significant interest for the treatment of fetal and neonatal anemia, due to its high content of fetal hemoglobin as well as numerous other potential benefits to fetuses and neonates. However, in order to establish a stable supply of cord RBCs for clinical use, a cryopreservation method must be developed. This, in turn, requires knowledge of the osmotic parameters of cord RBCs. Thus, the objective of this study was to characterize the osmotic parameters of cord RBCs: osmotically inactive fraction (b), hydraulic conductivity (L_p), permeability to cryoprotectant glycerol (P_glycerol), and corresponding Arrhenius activation energies (E_a). For L_p and P_glycerol determination, RBCs were analyzed using a stopped-flow system to monitor osmotically-induced RBC volume changes via intrinsic RBC hemoglobin fluorescence. L_p and P_glycerol were characterized at 4 °C, 20 °C, and 35 °C using Jacobs and Stewart equations with the E_a calculated from the Arrhenius plot. Results indicate that cord RBCs have a larger osmotically inactive fraction compared to adult RBCs. Hydraulic conductivity and osmotic permeability to glycerol of cord RBCs differed compared to those of adult RBCs with the differences dependent on experimental conditions, such as temperature and osmolality. Compared to adult RBCs, cord RBCs had a higher E_a for L_p and a lower E_a for P_glycerol. This information regarding osmotic parameters will be used in future work to develop a protocol for cryopreserving cord RBCs.     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using ³¹P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L_β) to liquid-crystalline (L_α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L_α to the inverted hexagonal (H_II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L_α phase compared to those in the presence of H_II phase forming lipids. Our data furthermore imply that the H_II phase is better suited to maintain high VDE activities at lower temperatures.     

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D₂O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (R_i) of 3 the lamellar gel (L or L_c)-to-hexagonal type II (H_II) phase sequence is observed on increasing the temperature. For R_i3 the thermotropic sequence (L or L_c)-L-H_II is detected. On increasing hydration from R_i=3, the H_II phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and H_II phases are R_i 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the H_II phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - T_c lamellar gel-to-lamellar fluid transition temperature - T_h lamellar fluid-to-hexagonal transition temperature     

Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase

The specific volumes of seven 1,2-diacyl-sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 °C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chain CH₂, CH, and terminal CH₃ groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.