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.     

Neutron Diffraction with an Excess-Water Cell

As part of a study of the molecular basis of membrane fusion by enveloped viruses, we have used neutron diffraction to study the lamellar (L_α) to inverse hexagonal (H_II) phase transition in the phospholipid N-methylated dioleoylphosphatidylethanolamine. This lipid was chosen because its phase transitions are particularly sensitive to the presence of agents that have been demonstrated to promote or inhibit membrane fusion. Two different geometries of neutron diffraction were used: small angle scattering (SANS) and a membrane diffractometer. The SANS measurements were carried out on the SWAN instrument at KEK, Japan, using dispersions of multilamellar vesicles (MLVs). The diffractometer measurements used the V1 instrument at BeNSC-HMI, Germany, with a specially-constructed cell that holds a stack of lipid bilayers in an excess-water state. The two approaches are compared and discussed. Although the diffractometer takes considerably longer to collect the data, it records much higher resolution than the SANS instrument. The samples recorded in the excess-water cell were shown to be well aligned, despite the lipids being fully hydrated, allowing for the production of high-resolution data. Trial measurements performed have demonstrated that sample alignment is preserved throughout the L_α to H_II phase transition, thereby opening up possibilities for obtaining high-resolution data from non-lamellar phases.     

Bilayer structural destabilization by low amounts of chlorophyll a

The present study shows that small admixtures of one chlorophyll a (Chla) molecule per several hundred lipid molecules have strong destabilizing effect on lipid bilayers. This effect is clearly displayed in the properties of the L_α-H_II transformations and results from a Chla preference for the H_II relative to the L_α phase. Chla disfavors the lamellar liquid crystalline phase L_α and induces its replacement with inverted hexagonal phase H_II, as is consistently demonstrated by DSC and X-ray diffraction measurements on phosphatidylethanolamine (PE) dispersions. Chla lowers the L_α-H_II transition temperature (42 °C) of the fully hydrated dipalmitoleoyl PE (DPoPE) by ∼ 8 °C and ∼ 17 °C at Chla/DPoPE molar ratios of 1:500 and 1:100, respectively. Similar Chla effect was recorded also for dielaidoyl PE dispersions. The lowering of the transition temperature and the accompanying significant loss of transition cooperativity reflect the Chla repartitioning and preference for the H_II phase. The reduction of the H_II phase lattice constant in the presence of Chla is an indication that Chla favors H_II phase formation by decreasing the radius of spontaneous monolayer curvature, and not by filling up the interstitial spaces between the H_II phase cylinders. The observed Chla preference for H_II phase and the substantial bilayer destabilization in the vicinity of a bilayer-to-nonbilayer phase transformation caused by low Chla concentrations can be of interest as a potential regulatory or membrane-damaging factor.     

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: II. On the fine structure of the phase transitions in hydrated dipalmitoylphosphatidylethanolamine

The phase transitions of dipalmitoylphosphatidylethanolamine (DPPE) in excess water have been examined by low-angle time-resolved x-ray diffraction and calorimetry at low scan rates. The lamellar subgel/lamellar liquid-crystalline (L_c → L_α), lamellar gel/lamellar liquid-crystalline (L_β → L_α), and lamellar liquid-crystalline/lamellar gel (L_α → L_β) phase transitions proceed via coexistence of the initial and final phases with no detectable intermediates at scan rates 0.1 and 0.5°C/min. At constant temperature within the region of the L_β → L_α transition the ratio of the two coexisting phases was found to be stable for over 30 min. The state of stable phase coexistence was preceded by a 150-s relaxation taking place at constant temperature after termination of the heating scan in the transition region. While no intermediate structures were present in the coexistence region, a well reproducible multipeak pattern, with at least four prominent heat capacity peaks separated in temperature by 0.4-0.5°C, has been observed in the cooling transition (L_α → L_β) by calorimetry. The multipeak pattern became distinct with an increase of incubation time in the liquid-crystalline phase. It was also clearly resolved in the x-ray diffraction intensity versus temperature plots recorded at slow cooling rates. These data suggest that the equilibrium state of the L_α phase of hydrated DPPE is represented by a mixture of domains that differ in thermal behavior, but cannot be distinguished structurally by x-ray scattering.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0–70°C. In the low-temperature (L_c) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (L_c) phase to the intermediate-temperature (Lβ) phase at 25°C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L_β) to the high-temperature (L_α) phase at 37°C is a gel-to-liquid-crystalline phase transition analogous to the 41.5°C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the L_c phase. In the L_β and L_α phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C=O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A₂ on the basis of a preferred head group conformation.     

Intramolecular disorder and its relation to mesophase structure in lipid/water mixtures

NMR spin-half pair dipolar echo measurements are reported for the lamellar (dispersions and multibilayer stacks) and hexagonal phases of potassium palmitate/²H₂O mixtures. In the lamellar L_β and L_γ (gel) phases the alkyl chains are rigid and perfectly ordered, while in the lamellar L_α and hexagonal phases they are flexible and disordered. In particular, the measurements show that in the fluid lamellar L_α phase the chain is “bent” at the C₉–C₁₀ segment; but is “straight” in the hexagonal phase.     

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.     

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.     

Frequency-resolved intramolecular excimer fluorescence study of lipid bilayer and nonbilayer phases

Frequency-domain fluorescence intensity decays of the intramolecular excimer forming (DipyPE) in a fully hydrated dioleoyl-phosphatidylethanolamine (DOPE) suspension have been measured at the monomer (395 nm) and excimer (475 nm) emissions and at different temperatures (0-30°C). A classical Birks (two-state) and a new three-state kinetics models were used to analyze the frequency-domain data. The three-state model allowed us to resolve various intramolecular dynamics parameters of DipyPE in the host DOPE suspension. Those parameters are the excimer association (K_dm) and dissociation (K_md) rate constants, effective concentration (C), and lateral diffusion rate (f) of the pyrene moieties in the DipyPE. In contrast, only CK_dm and K_md were determined based on the two-state model. We observed that K_dm declined while C increased abruptly at ∼12°C, the known thermotropic lamellar liquid crystalline-to-inverted hexagonal (L_α-H_II) phase transition temperature of DOPE. No abrupt changes in K_md and f were observed at all temperatures. We concluded that the rotation of the lipid acyl chains is hindered and the free volume available for the lipid terminal methyl ends is reduced as the lipid membrane enters the highly curved H_II phase from the planar L_α phase.     

Farnesol and geranylgeraniol modulate the structural properties of phosphatidylethanolamine model membranes

The biological activity of farnesol (FN) and geranylgeraniol (GG) and their isoprenyl groups is related to membrane-associated processes. We have studied the interactions of FN and GG with 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes using DSC and X-ray diffraction. Storage of samples at low temperature for a long time favors a multidomain system formed by a lamellar crystalline (Lc) phase and isoprenoids (ISPs) aggregates. We demonstrate that ISPs alter the thermotropic behavior of DEPE, thereby promoting a H_II growth in a lamellar Lc phase with a reduced degree of hydration. The H_II phase occurs with the same repeat distance (d_HII=5.4 nm) as the Lc phase and upon heating it expands considerably (δd/δT≈0.22 nm/°C). The dimensional stabilization of this H_II phase coincides with the transition temperature of the Lc to L_α phase. Thereafter, the system DEPE/ISP will progress by increasing the nonlamellar-forming propensity and reaching a single H_II phase at high temperature. The cooling scan followed a similar structural path, except that the system went into a stable gel phase L_β with a repeat distance, d_Lβ=6.5 nm, in co-existence with a H_II phase. The formation of ISP microdomains in model PE membranes substantiates the importance of the isoprenyl group in the binding of isoprenylated proteins to membranes and in lipid–lipid interactions through modulation of the membrane structure.     

Morphology of the intermediate stages in the lamellar to hexagonal lipid phase transition

Summary The addition of calcium to suspensions of egg phosphatidylcholine and cardiolipin converts multiwalled liposomes to the hexagonal (H_II) phase (Rand, R.P., Sengupta, S. (1972)Biochim. Biophys. Acta 255:484–492). We have studied this lamellar to hexagonal phase transition by freeze-fracture, thin-section electron microscopy, and X-ray diffraction and have morphologically characterized the intermediate stages. The first step in the transition involves the invagination and fusion of bilayers, marked by the appearance of lipidic intramembrane particles and crater-like indentations, as the large liposomes are converted to smaller flattened and elongated vesicles. The next step is the formation of tightly packed hexagonal arrays of tubules, each tubule being about 11 to 15 nm in diameter. These tubules are filled with fluid and a lipid bilayer forms the wall of each cylinder. Finally this tubular bilayer phase is converted to the hexagonal (H_II) phase, where the distance between tubes is 5.5 to 7.5 nm.     

The Kinetics of Non-Lamellar Phase Formation in DOPE-Me: Relevance to Biomembrane Fusion

The mechanism of the lamellar/inverted cubic (Q_II) phase transition is related to that of membrane fusion in lipid systems. N-Monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) exhibits this transition and is commonly used to investigate the effects of exogenous substances, such as viral fusion peptides, on the mechanism of membrane fusion. We studied DOPE-Me phase behavior as a first step in evaluating the effects of membrane-spanning peptides on inverted phase formation and membrane fusion. These measurements show that: a) the onset temperatures for Q_II and inverted hexagonal (H_II) phase formation both are temperature scan rate-dependent; b) longer pre-incubation times at low temperature and lower temperature scan rates favor formation of the Q_II phase; and c) in temperature-jump experiments between 61 and 65°C, the meta-stable H_II phase forms initially, and disappears slowly while the Q_II phase develops. These observations are rationalized in the context of a mechanism for both the lamellar/non-lamellar phase transition and the related process of membrane fusion. Current address for D.P.S.: Givaudan, Cincinnati, OH 45216 Data Deposition: Relevant transition temperatures in this paper have been deposited in the LIPIDAT ( )     

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.     

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide

We have reported here on the structural polymorphism of lipid A, the “endotoxic principle” of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg²⁺] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70°C. These data were correlated with measurements of the β→α phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges—in the absence of Mg²⁺—from 45°C at high to 56°C at low water content, and—at an equimolar content of Mg²⁺—from 52°C at high to 59°C at low water concentrations. In the gel phase—in which the lipid A acyl chains are more disordered than those from saturated phospholipids—cubic phases are adopted at high water content (>60%) and at high [lipid A):[Mg²⁺] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal H_II, state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg²⁺ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.     

Analysis of the hexagonal II phase and its relations to lipidic particles and the lamellar phase A freeze-fracture study

Model systems of phosphatidylethanolamine (PE) and cardiolipin (DPG), as pure components and in binary mixtures with phosphatidylcholine (PC) have been morphologically analysed. The relation between the hexagonal_II (H_II) phase and lipidic particles as well as between the H_II phase and the lamellar phase has been studied. Moreover, the periodicity of the various H_II tubes was determined. (1) The periodicity of the H_II phase of cardiolipin is dependent on the cation involved. DPG-Ca exhibits the smallest tube to tube distance when compared to Mg²⁺ and Mn²⁺. Moreover, the DPG-Ca tubes are quite straight, in contrast to the Mg²⁺ and Mn²⁺ tubes, which appear to be frequently curved. (2) H_II tubes with two distinct diameters have been observed in H_II phase containing lipid mixtures. The thickness of the H_II tube is related to the composition of the tube. In the cardiolipin-lecithin system, structural separation of the pure cardiolipin H_II phase has been suggested with Mg²⁺ and Mn²⁺, but not with Ca²⁺. (3) Models for the H_II to lamellar phase transition and for the H_II phase to the lipidic particles are presented. (4) Lipidic particles are exclusively found in lipid model systems, which contain H_II phase favouring lipids. Morphological evidence is presented which suggests these lipidic particles represent inverted micelles. These observations include: (${}_{\mathrm{<mtext>i</mtext>}}$) there is a strong topological and quantitative relation between H_II tubes and lipidic particles, (${}_{\mathrm{<mtext>ii</mtext>}}$) lipidic particles occur densely packed in conglomerates without the presence of a smooth layer.     

Calcium-induced fusion of didodecylphosphate vesicles: The lamellar to hexagonal II (HII) phase transition

Summary Electron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 Å) to fuse in the presence of Ca²⁺. As revealed by negative staining, Ca²⁺ induces extensive fusion and large vesicles with diameters up to 7000 Å are formed. In a processsecondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with³¹P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (H_II) array and that the amphiphiles are converted from the lamellar to the hexagonal H_II phase.The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal H_II phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.     

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.     

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     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

Seed germination and dormancy in the medicinal woodland herbs Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae)

We used a double germination phenology or “move-along” experiment (sensu Baskin and Baskin, 2003) to characterize seed dormancy in two medicinal woodland herbs, Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae). Imbibed seeds of both species were moved through the following two sequences of simulated thermoperiods: (a) 30/15 °C→20/10 °C→15/6 °C→5 °C→15/6 °C→20/10 °C→30/15 °C, and (b) 5 °C→15/6 °C→20/10 °C→30/15 °C→20/10 °C→15/6 °C→5 °C. In each sequence, seeds of both species germinated to high rates (>85%) at cool temperatures (15/6 and 20/10 °C) only if seeds were previously exposed to cold temperatures (5 °C). Seeds kept at four control thermoperiods (5, 15/6, 20/10, 30/15 °C) for 30 d showed little or no germination. Seeds of both species, therefore, have physiological dormancy that is broken by 12 weeks of cold (5 °C) stratification. Morphological studies indicated that embryos of C. canadensis have “investing” embryos at maturity (morphological dormancy absent), whereas embryos of D. villosa are undeveloped at maturity (morphological dormancy present). Because warm temperatures are required for embryo growth and cold stratification breaks physiological dormancy, D. villosa seeds have non-deep simple morphophysiological dormancy (MPD). Neither species afterripened in a 6-month dry storage treatment. Cold stratification treatments of 4 and 8 weeks alleviated dormancy in both species but C. canadensis seeds germinated at slower speeds and lower rates compared to seeds given 12 weeks of cold stratification. In their natural habitat, both species disperse seeds in mid- to late autumn and germinate in the spring after cold winter temperatures alleviate endogenous dormancy.