Stratum corneum lipid matrix: Location of acyl ceramide and cholesterol in the unit cell of the long periodicity phase.

The extracellular lipid matrix in the skin's outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) and involves two lamellar phases: the short periodicity phase (SPP) and the long periodicity phase (LPP). To understand the skin barrier thoroughly, information about the molecular arrangement in the unit cell of these lamellar phases is paramount. Previously we examined the molecular arrangement in the unit cell of the SPP. Furthermore X-ray and neutron diffraction revealed a trilayer arrangement of lipids within the unit cell of the LPP [D. Groen et al., Biophysical Journal, 97, 2242-2249, 2009]. In the present study, we used neutron diffraction to obtain more details about the location of lipid (sub)classes in the unit cell of the LPP. The diffraction pattern revealed at least 8 diffraction orders of the LPP with a repeating unit of 129.6±0.5Å. To determine the location of lipid sub(classes) in the unit cell, samples were examined with either only protiated lipids or selectively deuterated lipids. The diffraction data obtained by means of D2O/H2O contrast variation together with a gradual replacement of one particular CER, the acyl CER, by its partly deuterated counterpart, were used to construct the scattering length density profiles. The acyl chain of the acyl CER subclass is located at a position of ~21.4±0.2Å from the unit cell centre of the LPP. The position and orientation of CHOL in the LPP unit cell were determined using tail and head-group deuterated forms of the sterol. CHOL is located with its head-group positioned ~26±0.2Å from the unit cell centre. This allows the formation of a hydrogen bond with the ester group of the acyl CER located in close proximity. Based on the positions of the deuterated moieties of the acyl CER, CHOL and the previously determined location of two other lipid subclasses [E.H. Mojumdar et al., Biophysical Journal, 108, 2670-2679, 2015], a molecular model is proposed for the unit cell of the LPP. In this model CHOL is located in the two outer layers of the LPP, while CER EOS is linking the two outer layers with the central lipid layers. Finally the two other lipid subclasses are predominantly located in the central layer of the LPP.

Neutron Scattering Studies of the Effects of Formulating Amphotericin B with Cholesteryl Sulfate on the Drug's Interactions with Phospholipid and Phospholipid-Sterol Membranes.

Langmuir surface pressure, small-angle neutron scattering (SANS), and neutron reflectivity (NR) studies have been performed to determine how formulation of the antifungal drug amphotericin B (AmB), with sodium cholesteryl sulfate (SCS)-as in Amphotec-affects its interactions with ergosterol-containing (model fungal cell) and cholesterol-containing (model mammalian cell) membranes. The effects of mixing AmB in 1:1 molar ratio with cholesteryl sulfate (yielding AmB-SCS micelles) are compared against those of free AmB, using monolayers and bilayers formed from palmitoyloleoylphosphatidylcholine (POPC) in the absence and presence of 30 mol % ergosterol or cholesterol, in all cases employing a 1:0.05 molar ratio of lipid:AmB. Analyses of the (bilayer) SANS and (monolayer) NR data indicate that the equilibrium changes in membrane structure induced in sterol-free and sterol-containing membranes are the same for free AmB and AmB-SCS. Stopped-flow SANS experiments, however, reveal that the structural changes to vesicle membranes occur far more rapidly following exposure to AmB-SCS vs free drug, with the kinetics of these changes varying with membrane composition. With POPC vesicles, the structural changes induced by AmB-SCS become apparent only after several minutes, and equilibrium is reached after ∼30 min. The corresponding onset of changes in POPC-ergosterol and POPC-cholesterol vesicles, however, occurs within ∼5 s, with equilibrium reached after 10 and 120 s, respectively. The rate of insertion of AmB into POPC-sterol membranes is thus increased through formulation as AmB-SCS. Moreover, the differences in monolayer surface pressure and SANS structure-change equilibration times suggest significant rearrangement of AmB within these membranes following insertion. The reduced times to equilibrium for the POPC-ergosterol vs POPC-cholesterol systems are consistent with the known differences in affinity of AmB for these two sterols, and the reduced time to equilibrium for AmB-SCS interaction with POPC-ergosterol membranes vs that for free AmB is consistent with the reduced host toxicity of Amphotec.

Interaction of amphotericin B with lipid monolayers.

Langmuir isotherm, neutron reflectivity, and Brewster angle microscopy experiments have been performed to study the interaction of amphotericin B (AmB) with monolayers prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and mixtures of this lipid with cholesterol or ergosterol to mimic mammalian and fungal cell membranes, respectively. Isotherm data show that AmB causes a more pronounced change in surface pressure in the POPC/ergosterol system than in the POPC and POPC/cholesterol systems, and its interaction with the POPC/ergosterol monolayer is also more rapid than with the POPC and POPC/cholesterol monolayers. Brewster angle microscopy shows that, in interaction with POPC monolayers, AmB causes the formation of small domains which shrink and disappear within a few minutes. The drug also causes domain formation in the POPC/cholesterol and POPC/ergosterol monolayers; in the former case, these are formed more slowly than is seen with the POPC monolayers and are ultimately much smaller; in the latter case, they are formed rather more quickly and are more heterogeneous in size. Neutron reflectivity data show that the changes in monolayer structure following interaction with AmB are the same for all three systems studied: the data are consistent with the drug inserting into the monolayers with its macrocyclic ring intercalated among the lipid acyl chains and sterol ring systems, with its mycosamine moiety colocalizing with the sterol hydroxyl and POPC head groups. On the basis of these studies, it is concluded that AmB inserts in a similar manner into POPC, POPC/cholesterol, and POPC/ergosterol monolayers but does so with differing kinetics and with the formation of quite different in-plane structures. The more rapid time scale for interaction of the drug with the POPC/ergosterol monolayer, its more pronounced effect on monolayer surface pressure, and its more marked changes as regards domain formation are all consistent with the drug's selectivity for fungal vs mammalian cell membranes.

Localization of cholesterol and fatty acid in a model lipid membrane: a neutron diffraction approach.

The intercellular lipid matrix of the skin's stratum corneum serves to protect the body against desiccation and simultaneously limits the passage of drugs and other xenobiotics into the body. The matrix is made up of ceramides, free fatty acids, and cholesterol, which are organized as two coexisting crystalline lamellar phases. In studies reported here, we sought to use the technique of neutron diffraction, together with the device of isotopic (H/D) substitution, to determine the molecular architecture of the lamellar phase having a repeat distance of 53.9 ± 0.3 Å. Using hydrogenous samples as well as samples incorporating perdeuterated (C24:0) fatty acids and selectively deuterated cholesterol, the diffraction data obtained were used to construct neutron scattering length density profiles. By this means, the locations within the unit cell were determined for the cholesterol and fatty acids. The cholesterol headgroup was found to lie slightly inward from the unit cell boundary and the tail of the molecule located 6.2 ± 0.2 Å from the unit cell center. The fatty acid headgroups were located at the unit cell boundary with their acyl chains straddling the unit cell center. Based on these results, a molecular model is proposed for the arrangement of the lipids within the unit cell.

Changes in ambient temperature differentially alter the thermoregulatory, cardiac and locomotor stimulant effects of 4-methylmethcathinone (mephedrone).

BACKGROUND: The substituted cathinone compound known as mephedrone (4-methylmethcathinone; 4-MMC) has become popular with recreational users of psychomotor-stimulant compounds. Only recently have the first preclinical studies provided information about this drug in the scientific literature; nevertheless, media reports have led to drug control actions in the UK and across several US states. Rodent studies indicate that 4-MMC exhibits neuropharmacological similarity to 3,4-methylenedioxymethamphetamine (MDMA) and prompt investigation of the thermoregulatory, cardiac and locomotor effects of 4-MMC. This study focuses on the role of ambient temperature, which has been shown to shift the effects of MDMA from hyperthermic to hypothermic. METHODS: Male Sprague-Dawley rats were monitored after subcutaneous administration of 4-MMC (1.0-5.6 mg/kg) using an implantable radiotelemetry system under conditions of low (20 °C) and high (30 °C) ambient temperature. RESULTS: A pharmacokinetic study found a T(max) of 0.25 h and a C(max) of 1206 ng/ml after 5.6 mg/kg 4-MMC. A dose-dependent reduction of body temperature was produced by 4-MMC at 20 °C but there was no temperature change at 30 °C. Increased locomotor activity was observed after 4-MMC administration under both ambient temperatures, however, significantly more activity was observed at 30 °C. Heart rate was slowed by 1.0 and 5.6 mg/kg 4-MMC at 20°C, and was slower in the 30 °C vs. 20 °C condition across all treatments. CONCLUSION: These results show that the cathinone analog 4-MMC exhibits in vivo thermoregulatory properties that are distinct from those produced by MDMA.

Effect of helper lipids on the interaction of DNA with cationic lipid monolayers studied by specular neutron reflection.

The interaction of DNA with monolayers of the cationic lipid dimethyldioctadecylammonium bromide, with/without 50 mol % of a neutral "helper" lipid, either dioleoylphosphatidylethanolamine or cholesterol, has been studied using specular neutron reflection, surface pressure-area isotherms, and Brewster angle microscopy. The amount of DNA bound to the lipid head groups has been comprehensively quantified in the range of 8-39 vol% of DNA with respect to the monolayer composition (monolayers composed of dimethyldioctadecylammonium bromide binding the most DNA and monolayers containing dioleoylphosphatidylethanolamine binding the least) and surface pressure (DNA binding being greatest at highest surface pressures). Surprisingly, regardless of these variables, the thickness of the DNA-containing layer remained approximately constant between 18 and 25 Å. This systematic study is the first direct quantification of the binding of DNA with two different helper-lipid-containing multicomponent monolayers, an important step toward understanding interaction parameters in more realistic models of gene delivery systems.

In-silico studies in Chinese herbal medicines' research: evaluation of in-silico methodologies and phytochemical data sources, and a review of research to date.

The available databases that catalogue information on traditional Chinese medicines are reviewed in terms of their content and utility for in-silico research on Chinese herbal medicines, as too are the various protein database resources, and the software available for use in such studies. The software available for bioinformatics and 'omics studies of Chinese herbal medicines are summarised, and a critical evaluation given of the various in-silico methods applied in screening Chinese herbal medicines, including classification trees, neural networks, support vector machines, docking and inverse docking algorithms. Recommendations are made regarding any future in-silico studies of Chinese herbal medicines.

The effect of neutral helper lipids on the structure of cationic lipid monolayers.

Successful drug delivery via lipid-based systems has often been aided by the incorporation of 'helper lipids'. While these neutral lipids enhance the effectiveness of cationic lipid-based delivery formulations, many questions remain about the nature of their beneficial effects. The structure of monolayers of the cationic lipid dimethyldioctadecylammonium bromide (DODAB) alone, and mixed with a neutral helper lipid, either diolelyphosphatidylethanolamine or cholesterol at a 1 : 1 molar ratio was investigated at the air-water interface using a combination of surface pressure-area isotherms, Brewster angle microscopy (BAM) and specular neutron reflectivity in combination with contrast variation. BAM studies showed that while pure DODAB and DODAB with cholesterol monolayers showed fairly homogeneous surfaces, except in the regions of phase transition, monolayers of DODAB with diolelyphosphatidylethanolamine were, in contrast, inhomogeneous exhibiting irregular bean-shaped domains throughout. Neutron reflectivity data showed that while the thickness of the DODAB monolayer increased from 17 to 24 Å as it was compressed from a surface pressure of 5-40 mN m(-1), the thickness of the helper lipid-containing monolayers, over the same range of surface pressures, was relatively invariant at between 25 and 27 Å. In addition, the monolayers containing diolelyphosphatidylethanolamine were found to be more heavily hydrated than the monolayers of cationic lipid, alone or in combination with cholesterol, with hydration levels of 18 molecules of water per molecule of lipid being recorded for the diolelyphosphatidylethanolamine-containing monolayers at a surface pressure of 30 mN m(-1) compared with only six and eight molecules of water per molecule of lipid for the pure DODAB monolayer and the cholesterol-containing DODAB monolayer, respectively.

Structural studies of the monolayers and bilayers formed by a novel cholesterol-phospholipid chimera.

Langmuir isotherm, neutron reflectivity, and small angle neutron scattering studies have been conducted to characterize the monolayers and vesicular bilayers formed by a novel chimeric phospholipid, ChemPPC, that incorporates a cholesteryl moeity and a C-16 aliphatic chain, each covalently linked via a glycerol backbone to phosphatidylcholine. The structures of the ChemPPC monolayers and bilayers are compared against those formed from pure dipalmitoylphoshatidylcholine (DPPC) and those formed from a 60:40 mol % mixture of DPPC and cholesterol. In accord with previous findings showing that very similar macroscopic properties were exhibited by ChemPPC and 60:40 mol % DPPC/cholesterol vesicles, it is found here that the chimeric lipid and lipid/sterol mixture have very similar monolayer structures (each having a monolayer thickness of ∼26 Å), and they also form vesicles with similar lamellar structure, each having a bilayer thickness of ∼50 Å and exhibiting a repeat spacing of ∼65 Å. The interfacial area of ChemPPC, however, is around 10 Å(2) greater than that of the combined DPPC/cholesterol unit in the mixed lipid monolayer (viz., 57 ± 1 vs 46 ± 1 Å(2), at 35 mN·m(-1)), and this difference in area is attributed to the succinyl linkage which joins the ChemPPC steroid and glyceryl moieties. The larger area of the ChemPPC is reflected in a slightly thicker monolayer solvent distribution width (9.5 vs 9 Å for the DPPC/cholesterol system) and by a marginal increase in the level of lipid headgroup hydration (16 vs 13 H(2)O per lipid, at 35 mN·m(-1)).

Structure of vesicles formed from non-ionic dialkylglycerol poly(oxyethylene) ether surfactants: effect of electrolyte and cholesterol.

Five non-ionic dialkylglycerol poly(oxyethylene) ether surfactants, designated 2C(m)E(n) (where m, the number of carbons in each alkyl chain=16 or 18, and n, the number of oxyethylene units=12, 16 or 17) have been examined for their ability to form vesicles when dispersed in water or in an aqueous solution of 154 mM NaCl, alone or in the presence of 50 mol% cholesterol. Freeze fracture electron microscopy and light scattering showed that regardless of the hydrating fluid, all the non-ionic surfactants, with the exception of 2C(16)E(17) and 2C(18)E(17), formed vesicles in the absence of cholesterol - 2C(16)E(17) and 2C(18)E(17) instead formed micellar aggregates. All surfactants, however, formed vesicles in the presence of 50 mol% cholesterol. Small angle neutron scattering studies of the surfactant vesicles enabled the bilayer thickness and repeat distance (d-spacing) to be determined. The bilayers formed by all the non-ionic surfactants in the absence of cholesterol were surprisingly thin (∼50 Å for the E(12) containing surfactants and ∼64 Å for 2C(18)E(16)) most likely due to the intrusion of oxyethylene groups into the hydrophobic core of the bilayers. In contrast, however, the non-ionic surfactants exhibited a relatively large d-spacing of around ∼130-150 Å. The addition of 50 mol% cholesterol had a dramatic effect on the thickness of the vesicle bilayer, increasing its size by 10-20 Å, most probably because of an extrusion of oxyethylene from the hydrophobic region of the bilayer and/or a reduction in the tilt on the surfactant alkyl chains. Additionally the presence of cholesterol in a vesicle tended to reduce slightly both the d-spacing and the thickness of the water layer separating the bilayers. The presence of NaCl, even at the low concentrations used in the study, did affect the properties of the bilayer such that it reduced the d-spacing and, in the case of cholesterol-containing systems, also reduced bilayer thickness.

Disposition of ceramide in model lipid membranes determined by neutron diffraction.

The lipid matrix present in the uppermost layer of the skin, the stratum corneum, plays a crucial role in the skin barrier function. The lipids are organized into two lamellar phases. To gain more insight into the molecular organization of one of these lamellar phases, we performed neutron diffraction studies. In the diffraction pattern, five diffraction orders were observed attributed to a lamellar phase with a repeat distance of 5.4 nm. Using contrast variation, the scattering length density profile could be calculated showing a typical bilayer arrangement. To obtain information on the arrangement of ceramides in the unit cell, a mixture that included a partly deuterated ceramide was also examined. The scattering length density profile of the 5.4-nm phase containing this deuterated ceramide demonstrated a symmetric arrangement of the ceramides with interdigitating acyl chains in the center of the unit cell.

Small-angle neutron scattering studies of the effects of amphotericin B on phospholipid and phospholipid-sterol membrane structure.

Small-angle neutron scattering (SANS) studies have been performed to study the structural changes induced in the membranes of vesicles prepared (by thin film evaporation) from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations and different aggregation states of the anti-fungal drug, amphotericin B (AmB). In the majority of the experiments reported, the lipid vesicles were prepared with the drug added directly to the lipid dispersions dissolved in solvents favouring either AmB monomers or aggregates, and the vesicles then sonicated to a mean size of ~100 nm. Experiments were also performed, however, in which micellar dispersions of the drug were added to pre-formed lipid and lipid-sterol vesicles. The vesicles were prepared using the phospholipid palmitoyloleoylphosphatidylcholine (POPC), or mixtures of this lipid with either 30 mol% cholesterol or 30 mol% ergosterol. Analyses of the SANS data show that irrespective of the AmB concentration or aggregation state, there is an increase in the membrane thickness of both the pure POPC and the mixed POPC-sterol vesicles-in all cases amounting to ~4 Å. The structural changes induced by the drug's insertion into the model fungal cell membranes (as mimicked by POPC-ergosterol vesicles) are thus the same as those resulting from its insertion into the model mammalian cell membranes (as mimicked by POPC-cholesterol vesicles). It is concluded that the specificity of AmB for fungal versus human cells does not arise because of (static) structural differences between lipid-cholesterol-AmB and lipid-ergosterol-AmB membranes, but more likely results from differences in the kinetics of their transmembrane pore formation and/or because of enthalpic differences between the two types of sterol-AmB complexes.

Phytochemical informatics and virtual screening of herbs used in Chinese medicine.

While many experimental and clinical studies of traditional Chinese medicine (TCM) have been reported over recent years, the applications of computational methods to drug discovery from Chinese herbs are still at an early stage. In the light of the spread of TCM to other parts of the world over the last few decades, and the growing number of publications in languages other than Chinese, this article focuses on work published in English and accessible to an international audience. Sources of information in appropriate format are particularly important for informatics, and the growing number of TCM-related databases is discussed. Applications of virtual screening both to the identification of single and multiple target ligands are covered, as are developments in 'target fishing', a novel technique which seeks to identify multiple receptors to which a compound may bind. Finally, the role of informatics in bridging the gulf between the paradigms of TCM and biomedical science is explored, and a discussion presented as to its use in probing the molecular basis of TCM.

Interaction of cationic lipid/DNA complexes with model membranes as determined by neutron reflectivity.

Transfection of cells by DNA for the purposes of gene therapy can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these complexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of these lipoplexes with model membranes composed primarily of zwitterionic lipid 1,2-dimyristoylphosphatidylcholine (DMPC) together with 10 mol % 1,2-dipalmitoylphosphatidylserine (DPPS). Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and complexed with a 1:1 charge ratio of DNA. The interaction of these complexes with the supported phospholipid bilayer was determined. DOPE-containing lipoplexes were found to interact faster with the model cell membrane than those containing cholesterol, and complexes containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the lipoplexes and the model membrane was characterized by an exchange of lipid between the membrane and the lipid/DNA aggregates in solution; the deposition of(additional) lipid on the surface of the model cell membrane was not apparent.

Screening of herbal constituents for aromatase inhibitory activity.

Random Forest screening of the phytochemical constituents of 240 herbs used in traditional Chinese medicine identified a number of compounds as potential inhibitors of the human aromatase enzyme (CYP19). Molecular modelling/docking studies indicated that three of these compounds (myricetin, liquiritigenin and gossypetin) would be likely to form stable complexes with the enzyme. The results of the virtual screening studies were subsequently confirmed experimentally, by in vitro (fluorimetric) assay of the compounds' inhibitory activity. The IC-50s for the flavones, myricetin and gossypetin were determined as 10 and 11 microM, respectively, whilst the flavanone, liquiritigenin, gave an IC-50 of 0.34 microM--showing about a 10-fold increase in potency, therefore, over the first generation aromatase inhibitor, aminoglutethimide.

Enzyme kinetic and molecular modelling studies of sulphur-containing substrates of phenylalanine 4-monooxygenase.

Previous investigations into the binding of substrates/cofactors to the PAH active site have only concentrated on Phe, thienylalanine and BH(4). This is the first reported investigation to model aliphatic thioether amino acid substrates to PAH. The clearance of the thioether substrates (4.82-79.09% of Phe) in the rat and human (1.19-37.41% of Phe) showed species differences. The xenobiotic thioether substrates (SMC and SCMC) were predicted to be poor substrates for PAH by the molecular modelling investigation and this has now been confirmed by the in vitro enzyme kinetic data. However, reaction phenotyping investigations have found that PAH was the major enzyme involved in the metabolism of SCMC in vitro and in vivo.

Effects of surface pressure on the structure of the monolayer formed at the air/water interface by a non-ionic surfactant.

The monolayer formed at an air/water interface by the synthetic non-ionic surfactant, 1,2-di-O-octadecyl-rac-glyceryl-3-(omega-methoxydodecakis (ethylene glycol)) (2C18E12) has been characterized using Langmuir trough measurements, Brewster angle microscopy (BAM), and neutron reflectometry. The BAM and reflectometry studies were performed at four different surface pressures (pi) in the range 15-40 mN/m. The BAM studies (which give information on the in-plane organisation of the surfactant layer) demonstrate that the 2C18E12 molecules are arranged on the water surface to form distinct, approximately circular, 5 microm diameter domains. As the surface pressure is increased these domains retain their size and shape but are made progressively more close-packed, such that the monolayer is made more or less complete at pi=40 mN/m. The neutron reflectometry measurements were made to determine the structure of the interfacial surfactant layer at pi=15, 28, 34 and 40 mN/m, providing information on the thickness of the 2C18E12 alkyl chains', head groups' and associated solvent distributions (measured along the surface normal), along with the separations between these distributions, and the effective interfacial area per molecule. Partial structure factor analyses of the reflectivity data show that the effective interfacial area occupied decreases from 217 A2 per 2C18E12 molecule at pi=15 mN/m down to 102 A2 at pi=40 mN/m. There are concomitant increases in the widths of the surfactant's alkyl chains' and head groups' distributions (modelled as Gaussians), with the former rising from 12 A (at pi=15 mN/m) up to 19 A (at pi=40 mN/m) and the latter rising from 13 A (at pi=15 mN/m) up to 24 A (at pi=40 mN/m). The compression of the monolayer is also shown to give rise to an increased surface roughness, some of which is due to the thermal roughness caused by capillary waves, but with a significant contribution also coming from the intrinsic/structural disorder in the monolayer. At all surface pressures studied, the alkyl chains and head groups of the 2C18E12 are found to exhibit a significant overlap, and this increases with increasing pi. Given the various trends noted on how the structure of the 2C18E12 monolayer changes as a function of pi, we extrapolate to consider the structure of the monolayer at pi>40 mN/m (making comparison with its single chain (CnEm) counterparts) and then relate these findings to the observations recorded on the structure and solute entrapment efficiency of 2C18E12 vesicles.

Memory effects of monolayers and vesicles formed by the non-ionic surfactant, 2C(18)E(12).

The behaviour of monolayers and bilayers formed by the dialkyl chain non-ionic surfactant, 1,2-di-O-octadecyl-rac-glycerol-3-omega-methoxydodecaethylene glycol (2C(18)E(12)) in water at 297 K has been investigated. Using a surface film balance (or Langmuir trough) the compression-expansion cycle of the 2C(18)E(12) monolayer was found to be reversible when compressed to surface pressures (pi) less than 42 mN m(-1). Compression of 2C(18)E(12) monolayer to pi greater than 42 mN m(-1) above this resulted in a considerable hysteresis upon expansion with the pi remaining high relative to that obtained upon compression, suggesting a time/pressure dependent re-arrangement of 2C(18)E(12) molecules in the film. Morphology of the 2C(18)E(12) monolayer, investigated using Brewster angle microscopy, was also found to depend upon monolayer history. Bright, randomly dispersed domains of 2C(18)E(12) of approximately 5 mum in size were observed during compression of the monolayer to pi less than 42 mN m(-1). At pi of 42 mN m(-1) and above, the surfactant film appeared to be almost completely 'solid-like.' Regardless of the extent of compression of the monolayer film, expansion of the film caused formation of chains or 'necklaces' of individual surfactant domains, with the extent of chain formation dependent upon pressure of compression of the monolayer and the length of time held at that pressure. Irreversible effects on 2C(18)E(12) vesicle size were also seen upon temperature cycling the vesicles through their liquid-crystalline phase transition temperature with vesicles shrinking in size and not returning to their original size upon standing at 298 K for periods of more than 24 h. No comparable hysteresis, time, pressure or temperature effects were observed with the monolayer or vesicles formed by the corresponding phospholipid, disteaorylphosphatidylcholine, under identical conditions. The effects observed with 2C(18)E(12) are attributed to the ability of the polyoxyethylene head group to dehydrate and intrude into the hydrophobic chain region of the mono- and bilayers. These studies have important implications for the use of the vesicles formed by 2C(18)E(12) as drug delivery vehicles.

Interaction of cationic lipid vesicles with model cell membranes--as determined by neutron reflectivity.

Transfection of cells by DNA (for the purposes of gene therapy) can be effectively engineered through the use of cationic lipid/DNA "lipoplexes", although the transfection efficiency of these lipoplexes is sensitive to the neutral "helper" lipid included. Here, neutron reflectivity has been used to investigate the role of the helper lipid present during the interaction of cationic lipid vesicles with model cell membranes. Dimethyldioctadecylammonium bromide (DDAB) vesicles were formed with two different helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) and cholesterol, and the interaction of these vesicles with a supported phospholipid bilayer was determined. DOPE-containing vesicles were found to interact faster with the membrane than those containing cholesterol, and vesicles containing either of the neutral helper lipids were found to interact faster than when DDAB alone was present. The interaction between the vesicles and the membrane was characterized by an exchange of lipid between the membrane and the lipid aggregates in solution; the deposition of vesicle bilayers on the surface of the membrane was not apparent.

Assessment of estrogenic activity in some common essential oil constituents.

Estrogenic responses have not only been associated with endocrine function, but also with cognitive function. Several studies have indicated that estrogen replacement therapy has favourable effects on cognition, and may have potential in the prevention and treatment of Alzheimer's disease. Thus, ligands for the estrogen receptor, that have a better efficacy and adverse-effect profile than drugs currently available, require investigation. This study was undertaken to investigate the potential estrogenic activity of a number of essential oil constituents. Initially, estrogenic activity was determined by a sensitive and specific bioassay using recombinant yeast cells expressing the human estrogen receptor. At high concentrations, estrogenic activity was detected for citral (geranial and neral), geraniol, nerol and trans-anethole, while eugenol showed anti-estrogenic activity. Molecular graphics studies were undertaken to identify the possible mechanisms for the interaction of geranial, neral, geraniol, nerol and eugenol with the ligand-binding domain of the estrogen alpha-receptor, using the computer program HyperChem. Citral, geraniol, nerol and eugenol were also able to displace [(3)H]17beta-estradiol from isolated alpha- and beta-human estrogen receptors, but none of these compounds showed estrogenic or anti-estrogenic activity in the estrogen-responsive human cell line Ishikawa Var I at levels below their cytotoxic concentrations, and none showed activity in a yeast screen for androgenic and anti-androgenic activity. The potential in-vivo estrogenic effects of citral and geraniol were examined in ovariectomized mice, but neither compound showed any ability to stimulate the characteristic estrogenic responses of uterine hypertrophy or acute increase in uterine vascular permeability. These results show that very high concentrations of some commonly used essential oil constituents appear to have the potential to interact with estrogen receptors, although the biological significance of this is uncertain.