Multiple Bilayers Research Articles

Closed state of gramicidin channel detected by X-ray in-plane scattering

An analogue of gramicidin A (gA) was synthesized with the formyl group replaced by a BOC group. The analogue (BOC-gA) exhibited single channel conduction, but the channel is 5-order-of-magnitude destabilized relative to the gA channel. Hydrated mixtures of gramicidin and dilauroyl phosphatidylcholine in the molar ratio of 1:10 were prepared into uniformly aligned multiple bilayers, and X-ray scattering with the momentum transfer in the plane of the membrane was measured. Analysis with the help of computer simulations showed that 70% of BOC-gA are monomers. Thus for the first time it was shown that gramicidin monomers are stable inside the monolayers of a lipid membrane. Furthermore, the monomers have the same β helical conformation as the dimeric channel. The result suggests the possibility that when a gramicidin channel is closed, it dissociates into two monomers floating in opposite monolayers.

Preservation of Permeability Barrier Ontogenesis in the Intrauterine Growth-Retarded Fetal Rat

The epidermal permeability barrier is provided by intercellular lipids forming multiple membrane bilayers in the stratum corneum. In the fetal rat, the barrier to transepidermal water loss forms during the 20th d of gestation and is accompanied by 1) increasing stratum corneum thickness; 2) increasing stratum corneum lipid content, particularly nonpolar ceramide and cholesterol content; and 3) the formation of lamellar unit structures throughout the stratum corneum interstices. In this report, we demonstrate that among pups of 20 d gestational age increasing barrier competence is correlated with increasing fetal weight. It has been previously demonstrated that fetal rats subjected to intrauterine growth retardation (IUGR) exhibit a thinner stratum corneum and decreased content of differentiation-specific epidermal structural proteins. To determine whether IUGR fetal rats also exhibit immaturity of barrier function and the barrier membrane system, maternal rats underwent unilateral uterine vessel ligation on d 17 or 18 of gestation and IUGR and control littermates were harvested on d 20, 21, or 22 of gestation for determination of transepidermal water loss. Despite significant somatic growth retardation and a thinner stratum corneum, barrier function in IUGR fetal rats did not significantly differ from that in control littermates at any gestational age. In both IUGR and control fetal rat epidermis at 21 d gestational age, lipids were deposited in a membrane pattern as visualized by nile red fluorescence microscopy and formed lamellar unit membrane structures throughout the stratum corneum intercellular domains as observed by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content.

The mammalian epidermal permeability barrier is provided by highly hydrophobic lipids forming multiple membrane bilayers within the extracellular domains of the outer, cornified cell layers. To characterize the critical events associated with barrier maturation, we correlated the emergence of a competent barrier to transepidermal water loss with development of the lamellar body secretory system, the organization of stratum corneum membrane bilayers, and the lipid composition of these membranes in the perinatal rat. Whereas pups of 19 d estimated gestational age had no measurable barrier (transepidermal water loss greater than 10 mg/cm2/h), by 21 d the barrier was well established (mean transepidermal water loss 0.41 mg/cm2/h). Development of a functional barrier correlated with increasing thickness of the stratum corneum, as well as with development of a membrane pattern of lipid deposition, visualized with the hydrophobic fluorescent probe nile red. At 19 d estimated gestational age, the stratum corneum intercellular domains exhibited an abundance of secreted lamellar body contents, but they were not organized into basic bilayer unit structures. Lamellar unit structures became evident by 20 d and extended throughout the stratum corneum interstices by 22 d (term). The quantity of lipid in isolated stratum corneum increased significantly between 19 and 20 d (34.08 versus 50.08 mean micrograms lipid/cm2, respectively; p less than 0.02) and still further between 20 and 21 d estimated gestational age (74.49 micrograms lipid/cm2; p less than 0.001). This increase was due to progressive accumulation of neutral lipids, particularly cholesterol, as well as nonpolar ceramides, as shown by thin-layer chromatography/scanning densitometry. These studies imply that in the development of cutaneous barrier function in the fetal rat both the generation of sufficient quantities of hydrophobic lipids and the organization of these lipids into bilayer unit structures are required.

Vehicular influence on transdermal drug penetration

The realization of the composition and structure of the intercellular multiple bilayers of the horny layer lipids has created a basis for understanding the diffusive control of transdermal xenobiotic permeation and the enhancing effects of vehicles and other ingredients added to dermal preparations. The measurement of concentration profiles in the stratum corneum and deeper skin layers yields insight into the transdermal permeation mechanisms. Lipid solubility is a predominant criterion for the transdermal permeation of substances which enter via the lipid route. Applying lipid vehicles, liquid components carrying dissolved drug substances enter the intercellular spaces of the stratum corneum disjunctum by spreading and by capillary action. Drugs and certain vehicle constituents diffuse into the stratum corneum conjunctum and deeper skin layers. There, they lower the diffusion resistance of the horny layer by interfering with the molecular packing of the dermal lipids and by disturbing their order. Furthermore, the dissolving power of the horny layer lipids may be changed.

Phases of egg phosphatidylcholine in an abundance of water

Two methods are described allowing the reversible generation of ‘dark bodies’ in highly swollen egg phosphatidylcholine multilayer systems. The dark bodies are likely to be made of multiple self-connected bilayers. They are stable between roughly 15° and 65°C but disappear at lower and higher temperatures where an optically unresolvable dispersion and the planar multilayer system; respectively, seem to be preferred.

Relationship between Mg2+‐Induced Hexagonal Assembly of R‐Form Lipopolysaccharides and Chemical Structure of Their R‐Cores

The relationship between formation of the Mg2(+)-induced hexagonal lattice structure by R-form lipopolysaccharides (LPS) and chemical structure of their R-cores was investigated using different kinds of R-form LPS from a series of mutants of Salmonella minnesota or S. typhimurium. The optimal experimental condition for formation of the hexagonal lattice structure was to suspend LPS preparations, from which cationic material was removed by electrodialysis, in 50 mM tris (hydroxymethyl) aminomethane buffer at pH 8.5 containing 10 mM MgCl2. Under this experimental condition, Rb1 LPS formed the hexagonal lattice structure with the lattice constant of 14.0 +/- 0.2 nm. Ra LPS, which possesses the full length of R-core, also formed the hexagonal lattice structure but its lattice constant was larger (18.1 +/- 0.2 nm) than that of Rb1 LPS (the lattice structure by Ra LPS was looser than that by Rb1 LPS). All the other R-form LPS preparations tested, RcP+, PcP-, Rd1P-, and Re LPS, whose R-cores are shorter than that of Rb1 LPS, did not form the hexagonal lattice structure, but formed membranous structures showing various shapes which consisted of multiple bilayer structures. Failure to form the hexagonal lattice structure was the common feature of these kinds of R-form LPS irrespective of temperature at which the LPS suspensions in 10 mM MgCl2-50 mM Tris buffer were incubated. From the results of the present study it was concluded that capability of R-form LPS to form the hexagonal lattice structure has a close correlation with the chemical structure of their R-cores.

The epidermal permeability barrier.

The permeability barrier of the skin which prevents transcutaneous water loss and penetration of harmful drugs from the environment is localized in the horny layer of the epidermis. Multiple lipid bilayers obstructing the intercellular space of the stratum corneum fulfill this function. In contrast to cellular membranes consisting predominantly of phospholipids, these lamellae contain mostly ceramides, cholesterol and free fatty acids. The lamellae are derived from the contents of lamellar granules (LGs) which are synthesized in the viable epidermal layers by the keratinocytes. LGs display stacks of small disks each of which represents a flattened vesicle or liposome. Prior to terminal differentiation, the disks are exocytosed into the intercellular space and fused to form uninterrupted sheetlike lamellae. The singular lipid composition of LG-disks and of stratum corneum-lamellae reflects the multistage process of barrier formation. It also renders these structures well suited to provide for a barrier function.

Electron microscopy of cholesterol

Negative staining and platinum-carbon shadowing have been used to prepare electron microscope specimens from aqueous colloidal suspensions of cholesterol microscrystals and from crystalline suspensions in methanol and ethanol. Microcrystals prepared by injection of alcoholic solutions of cholesterol into water exhibit angular conformations of varying regularity which contain a number of parallel cholesterol bilayers. The electron optical images of the cholesterol microcrystals, oriented horizontally and ‘on-edge’, obtained by both negative staining and metal shadowing, are in good agreement. Metal shadowing does, however, reveal greater detail within microcrystal clusters than does negative staining, as well as of the bilayer steps at microcrystal edges. The needle-like crystals (from methanol) and plate-like crystals (from ethanol) present considerable difficulties for the negative staining technique, because of their thickness and the consequent depth of the surrounding negative stain. Small crystals are, nevertheless, shown to possess multiple cholesterol bilayers. Platinum-carbon shadowing of cholesterol crystals taken directly from methanol and ethanol provides more satisfactory images than negative staining. The large depth of focus of the transmission electron microscope enables the stacked cholesterol bilayers to be clearly defined at the edges of crystals. The results obtained are discussed in relation to the physicochemical and biological properties of cholesterol, which underlie the fundamental difficulty encountered when fixing and staining cholesterol for thin sectioning, and also the role of cholesterol insolubility in the formation of gallstones.

Preparation of Liposomes from Stratum Corneum Lipids

Mammalian stratum corneum contains multiple intercellular lipid bilayers that constitute the epidermal water barrier. Unlike all other biologic membranes, the epidermal lamellae do not contain phospholipids, as a result of which the ability of the stratum corneum lipid mixture to form bilayers has been questioned. In the present study, a lipid mixture containing only epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%), approximating the composition of stratum corneum lipids, formed stable, unilamellar liposomes when sonicated at 80 degrees C in buffer containing 100 mM NaCl, 5 mM Tris, and 1 mM EDTA at pH 7.5. The size and form of the liposomes were studied by both freeze fracture and negative staining electron microscopy. Lipid mixtures from which either the palmitic acid or the cholesteryl sulfate were omitted were still capable of forming similar liposomes, but a mixture of ceramides and cholesterol, or ceramides alone, were incapable of forming liposomes. The results indicate that lipid mixtures similar to those found in stratum corneum are capable of forming bilayers at physiologic pH.

Magnetic structure of multiple bilayers of thin films of Fe and Ge

A diffraction study of multiple bilayers consisting of thin films of Fe and Ge was performed using both x rays and polarized neutrons. A reduction in the moment of a fraction of the Fe atoms due primarily to interdiffusion at the interface is indicated.

Electric field effects on lipid membrane structure

Multiple bilayers of dimyristoyl phosphatidylcholine and potassium oleate were macroscopically oriented between silver-coated glass slides. These model membranes were subjected to an electric field of up to 10 5V · cm −1. The influence of the field on the molecular structure was monitored by ESR of cholestane and stearic acid spin labels and by NMR of the phosphorus atom in the phosphatidylcholine headgroup. It is concluded that the conformation of the headgroup is greatly affected while no influence on the structure and dynamics of the hydrocarbon chains can be detected. At electric fields above 10 4 V · cm −1, where structural effects are still reversible, spontaneous current fluctuations are observed. At fields above 10 5 V · cm −1, irreversible breakdown of the bilayer structure occurs.