Protein-lipid Domains Research Articles

Alteration of Average Thickness of Lipid Bilayer by Membrane-Deforming Inclusions.

Thickness of lipid bilayer membranes is a key physical parameter determining membrane permeability and stability with respect to formation of through pores. Most membrane inclusions or impurities like amphipathic peptides, transmembrane peptides, lipid inclusions of a different molecular shape, lipid domains, and protein-lipid domains, locally deform the membrane. The detailed structure of the locally deformed region of the membrane is a kind of "fingerprint" for the inclusion type. However, most experimental methods allow determining only averaged parameters of membranes with incorporated inclusions, thus preventing the direct obtaining of the characteristics of the inclusion. Here we developed a model that allows the obtaining of characteristic parameters of three types of membrane inclusions (amphipathic peptides, transmembrane peptides, monolayer lipid patches) from experimentally observable dependencies of the average thickness of lipid bilayer on the surface concentration of the inclusions. In the case of amphipathic peptides, the model provided the peptide parameters that were in qualitative agreement with the available experimental data.

Model of lipid diffusion in cytoplasmic membranes

An analytical model of lateral lipid diffusion in heterogeneous native cytoplasmic membranes is presented. The Fourier transform method was used to solve the diffusion equation for the coordinate distribution function of lipids in a periodically inhomogeneous membrane, in which the diffusion coefficient is described by a harmonic function of the coordinates. It is shown that advection and diffusion are present in membrane. The model explains different types of lipid diffusion in membrane observed previously in experiments as a result of structural transitions of periodically located fixed protein-lipid domains associated with the spectrin-actin-ankyrin network. If these domains are the same, then super- and subdiffusion can be seen in experiments, when the mean square displacement of lipids depends non-linearly on time, and their average displacement is zero. Drift during advection was less than the chaotic Brownian displacement of lipids, advection was not observed in the experiment. When not all membrane proteins associated with the spectrin-actin-ankyrin network undergo conformational change in the same way upon ligand binding, two periodic sublattices of inhomogeneities arise in the membrane from fixed protein-lipid domains around membrane proteins associated with the cytoskeleton and nested in one another. In this case, hop diffusion can be found in experiments, when periods of nonlinear diffusion of molecules are replaced by periods of advection-diffusion, in which the average displacement of molecules is not zero. Advection is local in nature and occurs near individual protein-lipid domains. In the presented work, criteria are analytically obtained under which hop diffusion is experimentally observed in a periodically inhomogeneous membrane.

Fifty Years of the Fluid-Mosaic Model of Biomembrane Structure and Organization and Its Importance in Biomedicine with Particular Emphasis on Membrane Lipid Replacement.

The Fluid–Mosaic Model has been the accepted general or basic model for biomembrane structure and organization for the last 50 years. In order to establish a basic model for biomembranes, some general principles had to be established, such as thermodynamic assumptions, various molecular interactions, component dynamics, macromolecular organization and other features. Previous researchers placed most membrane proteins on the exterior and interior surfaces of lipid bilayers to form trimolecular structures or as lipoprotein units arranged as modular sheets. Such membrane models were structurally and thermodynamically unsound and did not allow independent lipid and protein lateral movements. The Fluid–Mosaic Membrane Model was the only model that accounted for these and other characteristics, such as membrane asymmetry, variable lateral movements of membrane components, cis- and transmembrane linkages and dynamic associations of membrane components into multimolecular complexes. The original version of the Fluid–Mosaic Membrane Model was never proposed as the ultimate molecular description of all biomembranes, but it did provide a basic framework for nanometer-scale biomembrane organization and dynamics. Because this model was based on available 1960s-era data, it could not explain all of the properties of various biomembranes discovered in subsequent years. However, the fundamental organizational and dynamic aspects of this model remain relevant to this day. After the first generation of this model was published, additional data on various structures associated with membranes were included, resulting in the addition of membrane-associated cytoskeletal, extracellular matrix and other structures, specialized lipid–lipid and lipid–protein domains, and other configurations that can affect membrane dynamics. The presence of such specialized membrane domains has significantly reduced the extent of the fluid lipid membrane matrix as first proposed, and biomembranes are now considered to be less fluid and more mosaic with some fluid areas, rather than a fluid matrix with predominantly mobile components. However, the fluid–lipid matrix regions remain very important in biomembranes, especially those involved in the binding and release of membrane lipid vesicles and the uptake of various nutrients. Membrane phospholipids can associate spontaneously to form lipid structures and vesicles that can fuse with various cellular membranes to transport lipids and other nutrients into cells and organelles and expel damaged lipids and toxic hydrophobic molecules from cells and tissues. This process and the clinical use of membrane phospholipid supplements has important implications for chronic illnesses and the support of healthy mitochondria, plasma membranes and other cellular membrane structures.

Characteristic lengths of transmembrane peptides controlling their tilt and lateral distribution between membrane domains.

Lipids and proteins of plasma membranes of eukaryotic cells are supposed to form protein-lipid domains, characterized by a different molecular order, bilayer thickness, and elastic parameters. Several mechanisms of preferable distribution of transmembrane proteins to the ordered or disordered membrane domains have been revealed. The mismatch between the length of the protein transmembrane domain and hydrophobic thickness of the lipid bilayer is considered to be an important driving force of protein lateral sorting. Utilizing the continuum theory of elasticity, we analyzed optimal configurations and preferable membrane domains for single-pass transmembrane peptides of various hydrophobic lengths and effective molecular shapes. We obtained that short transmembrane peptides stand perpendicularly to the membrane plane. The exceedance of a certain characteristic length leads to the tilt of the peptide. This length depends on the bilayer thickness. Thus, in the membrane with coexisting ordered (thicker) and disordered (thinner) phases tilting of the peptide in each phase is governed by its individual characteristic length. The lateral distribution of the peptides between ordered and disordered membrane domains is shown to be described by two additional characteristic lengths. The exceedance of the smaller one drives the peptide towards a more ordered and thicker membrane, while the exceedance of the larger characteristic length switches the preferable membrane domain from ordered and thicker to disordered and thinner. Thus, membrane proteins with long enough transmembrane domains are predicted to accumulate in the thinner disordered membrane as compared to the thicker ordered bilayer. For hourglass-like and barrel-like shaped transmembrane peptides the specific regime of sorting was obtained: the peptides distributed almost equally between the phases in a wide range of peptide lengths. This finding allowed explaining the experimental data on lateral distribution of transmembrane peptide tLAT.

Interaction of Peptides Containing CRAC Motifs with Lipids in Membranes of Various Composition

The lateral distribution of integral and peripheral proteins, as well as lipids in the plasma membranes of mammalian cells is extremely heterogeneous. It is believed that various lipid-protein domains are formed in membranes. Domains enriched in sphingomyelin and cholesterol are called rafts. It is assumed that the distribution of proteins into rafts is largely related to the presence in their primary sequence of a specific amino acid region called the CRAC motif, which is responsible for cholesterol binding. In this work, the interaction of two peptides containing CRAC motifs in their structure with membranes of different compositions was studied by means of molecular dynamics. It has been shown that the average number of lipid molecules in contact with each peptide is proportional to the mole fraction of lipid in the membrane. The predominant interaction of peptides with cholesterol was not observed. In addition, cholesterol did not form long-lived contacts with any amino acid or amino acid sequence. We suppose that in some cases the predominant lateral distribution of peptides and proteins containing CRAC motifs into rafts may be due to amphipathicity of the CRAC motif rather than due to specific strong binding of cholesterol.

Membrane-Mediated Lateral Interactions Regulate the Lifetime of Gramicidin Channels

The lipid matrix of cellular membranes is an elastic liquid crystalline medium. Its deformations regulate the functionality and interactions of membrane proteins,f membrane-bound peptides, lipid and protein-lipid domains. Gramicidin A (gA) is a peptide, which incorporates into membrane leaflets as a monomer and may form a transmembrane dimer. In both configurations, gA deforms the membrane. The transmembrane dimer of gA is a cation-selective ion channel. Its electrical response strongly depends on the elastic properties of the membrane. The gA monomer and dimer deform the membrane differently; therefore, the elastic energy contributes to the activation barriers of the dimerization and dissociation of the conducting state. It is shown experimentally that channel characteristics alter if gA molecules have been located in the vicinity of the conducting dimer. Here, based on the theory of elasticity of lipid membranes, we developed a quantitative theoretical model which allows explaining experimentally observed phenomena under conditions of high surface density of gA or its analogues, i.e., in the regime of strong lateral interactions of gA molecules, mediated by elastic deformations of the membrane. The model would be useful for the analysis and prediction of the gA electrical response in various experimental conditions. This potentially widens the possible applications of gA as a convenient molecular sensor of membrane elasticity.

Lateral redistribution of transmembrane proteins and liquid-ordered domains in lipid membranes with inhomogeneous curvature

A number of processes in living cells are accompanied by significant changes of the geometric curvature of lipid membranes. In turn, heterogeneity of the lateral curvature can lead to spatial redistribution of membrane components, most important of which are transmembrane proteins and liquid-ordered lipid-protein domains. These components have a so-called hydrophobic mismatch: the length of the transmembrane domain of the protein, or the thickness of the bilayer of the domain differ from the thickness of the surrounding membrane. In this work we consider redistribution of membrane components with hydrophobic mismatch in membranes with non-uniform geometric curvature. Dependence of the components’ energy on the curvature is calculated in terms of theory of elasticity of liquid crystals adapted to lipid membranes. According to the calculations, transmembrane proteins prefer regions of the membrane with zero curvature. Liquid-ordered domains having a size of a few nm distribute mainly into regions of the membrane with small negative curvature appearing in the cell plasma membrane in the process of endocytosis. The distribution of domains of a large radius is determined by a decrease of their perimeter upon bending; these domains distribute into membrane regions with relatively large curvature.

An update on post-ejaculatory remodeling of the sperm surface before mammalian fertilization

The fusion of a sperm with an oocyte to form new life is a highly regulated event. The activation—also termed capacitation—of the sperm cell is one of the key preparative steps required for this process. Ejaculated sperm has to make a journey through the female uterus and oviduct before it can approach the oocyte. The oocyte at that moment also has become prepared to facilitate monospermic fertilization and block immediately thereafter the chance for polyspermic fertilization. Interestingly, ejaculated sperm is not properly capacitated and consequently is not yet able to fertilize the oocyte. During the capacitation process, the formation of competent lipid-protein domains on the sperm head enables sperm-cumulus and zona pellucida interactions. This sperm binding allows the onset for a cascade reaction ultimately resulting in oocyte-sperm fusion. Many different lipids and proteins from the sperm surface are involved in this process. Sperm surface processing already starts when sperm are liberated from the seminiferous tubules and is followed by epididymal maturation where the sperm cell surface is modified and loaded with proteins to ensure it is prepared for its fertilization task. Although cauda epididymal sperm can fertilize the oocyte IVF, they are coated with so-called decapacitation factors during ejaculation. The seminal plasma–induced stabilization of the sperm surface permits the sperm transit through the cervix and uterus but prevents sperm capacitation and thus inhibits fertilization. For IVF purposes, sperm are washed out of seminal plasma and activated to get rid of decapacitation factors. Only after capacitation, the sperm can fertilize the oocyte. In recent years, IVF has become a widely used tool to achieve successful fertilization in both the veterinary field and human medicine. Although IVF procedures are very successful, scientific knowledge is still far from complete when identifying all the molecular players and processes during the first stages the fusion of two gametes into a new life. A concise overview in the current understanding of the process of capacitation and the sperm surface changes is provided. The gaps in knowledge of these prefertilization processes are critically discussed.

Asymmetry Determines the Effect of Ceramides on Model Membranes. In Natural Membranes Too?

Ceramides can dramatically influence the lateral organization of biological membranes. In particular, ceramide-induced alterations of protein-lipid domains can be involved in several cellular processes, ranging from senescence to immune response. In this context, an important role is played by the length of the fatty acid bound to the sphingosine moiety. Asymmetric, heterogeneous ceramides, with more than 20 or less than 16 carbon atoms in the fatty acyl chain, in fact exert diverging effects in vivo if compared to their symmetric counterparts. In this work, we investigated the role of ceramide asymmetry and heterogeneity in model membranes showing raft-like phase separation, using a combination of fluorescence imaging, atomic force microscopy, fluorescence correlation spectroscopy and differential scanning calorimetry. We show that ceramide produced enzymatically from natural mixtures of sphingomyelin can dramatically alter the mixing behaviour of proteins and lipids in the membrane, inducing a homogenization of the bilayer. Furthermore, we characterized the physical properties of coexisting lipid phases at equilibrium in membranes with varying ceramide content, emphasizing the differences between symmetric-homogeneous and asymmetric-heterogeneous ceramides. While symmetric ceramides always produce enhanced order, asymmetric ceramides display a more complex behavior similar to that of cholesterol. Our results might help contribute to a more precise understanding of the rearrangements induced by different kinds of ceramide generation in cellular membranes.

Adsorption and Rheological Properties of Myelin Basic Protein and Effects On the Interaction Forces Between Myelin Bilayers

Myelin Basic Protein (MBP) plays an important role in myelin structure, strongly holding the cytoplasmic membrane sides together mainly by electrostatic and hydrophobic interactions [1]. MBP is also known to affect lipid domains, increasing their size by lowering the line tension [2]. In this work, the rheological properties of MBPs between model myelin lipid bilayers and the effect of MBP on interaction forces are studied using the Surface Force Apparatus (SFA) technique. Results show that above a certain critical bulk concentration, Ccrit, MBP adsorbs abruptly on lipid membranes, forming a 3 nm - thick layer in which the viscosity is 2 to 3 times higher than the bulk MBP solution. Also, above this Ccrit, normal myelin membranes show higher adhesion and less repulsion compared to EAE (a model for multiple sclerosis) myelin lipid bilayers while below Ccrit, there was no evidence of MBP adsorption and no significant difference between the force profiles of normal (healthy) and EAE (diseased) myelin bilayers. These results suggest that MBP adsorbs “cooperatively” in clusters/domains to myelin bilayers, and provide new insights in to how lipid-protein domains affect inter-membrane interactions.

Dynamic Structure Formation of Peripheral Membrane Proteins

Using coarse-grained membrane simulations we show here that peripheral membrane proteins can form a multitude of higher-order structures due to membrane-mediated interactions. Peripheral membrane proteins characteristically perturb the lipid bilayer in their vicinity which supports the formation of protein assemblies not only within the same but surprisingly also across opposing leaflets of a bilayer. In addition, we also observed the formation of lipid-protein domains on heteregeneous membranes. The clustering ability of proteins, as quantified via the potential of mean force, is enhanced when radius and hydrophobic penetration depth of the proteins increases. Based on our data, we propose that membrane-mediated cluster formation of peripheral proteins supports protein assembly in vivo and hence may play a pivotal role in the formation of templates for signaling cascades and in the emergence of transport intermediates in the secretory pathway.

Recognition of lipid-protein rafts in vacuolar membrane

120 The well known Singer–Nicholson model, according to which proteins “float” in a homogeneous lipid sea, has undergone significant changes. Today it is generally accepted that cell membranes are highly heterogeneous: they contain various lipid–protein structures that are involved in many metabolic pro cesses occurring in living cells [1]. According to the model of lipid–protein microdomains (rafts), which is currently most widespread, areas enriched in gly cosphingolipids, sterols, and lipids with saturated fatty acids occur around certain proteins; lipids in these areas are characterized by a denser packing of mole cules compared to the surrounding areas of mem branes [2]. The interest to rafts is determined by the increasing number of experimental data that confirm their involvement in vital cellular processes, such as intrac ellular signaling, endocytosis, apoptosis, sorting of proteins, as well as internalization of toxins, bacteria, viruses, etc. [3, 4]. In addition to specific lipid compo sition, these membrane domains are characterized by specific protein profiles. In plant plasma membrane rafts, P and V type ATPases, protein kinases, mono meric and heteromeric G proteins, 14 3 3 family proteins, aquaporins, etc. were identified [5]. Rafts have been found in the plasma membrane and in endoplasmic reticulum and Golgi membranes. Mitochondrial membranes do not contain rafts [6]. Data on the lipid composition of tonoplasts [7] suggest that plant vacuolar membranes may also contain pro tein–lipid rafts. In this study, we found lipid protein domains (rafts) in the vacuolar membrane. Experiments were performed with isolated vacu oles and membrane vesicles of dormant storage roots of the red beet (Beta vulgaris L.) variety Bordeaux. Vacuoles and tonoplast vesicles from the storage root tissue were isolated and purified as described in [8]. Sterol enriched microdomains (rafts) were detected by fluorescent microscopy using the sterol binding antibiotic filipin [9]. To ensure the binding of filipin to the vacuolar membrane, a suspension of isolated vac uoles in the isolation medium (0.8 M KCl, 20 mM EDTA, and 50 mM Na2HPO4 (pH 8.0)) was supple mented with filipin (0.2% solution in DMSO) to a final concentration of 5 μM. The suspension was incu bated at 20 ± 2°С for 30 min, washed in the same buffer, and analyzed under an Axio observer Z1 fluo rescence microscope (Carl Zeiss, Germany) equipped with an Axio Cam MRm monochrome digital camera and Axio Vision Rel.4.8 software package for captur Recognition of Lipid–Protein Rafts in Vacuolar Membrane

Fundamentals of multilevel mesomechanics of nanostructural transitions in erythrocyte membranes and their destructions in interaction with stress hormones

Stress hormones (cortisol, adrenaline, noradrenaline) are capable for nonspecific binding with erythrocyte membranes thus initiating structural-phase transitions in them. The binding constants for cortisol, adrenaline and noradrenaline are respectively 1.23·106, 6.3·106 and 1.7·106 M−1. The amount of bound hormones varies in the range 10∼10-10∼n mole/mg of protein. The binding involves hydrogen bonds, hydrophobic bonds and electrostatic bonds. Active groups of hormones (amino-, imino-, methylamine-, keto- and hydroxy-groups) interact simultaneously with CO- and NH-groups of both proteins and phospholipids. These processes underlie structural-phase transitions in an erythrocyte membrane in its interaction with stress hormones. The structural-phase transitions result in complex protein-lipid domains of increasing structural order in the membrane. At the same time, water dipoles are displaced from the domains to adjacent regions and promote membrane loosening. The membrane microviscosity thus greatly increases in the regions of both protein-lipid interactions and lipid-lipid interactions. The increase in erythrocyte membrane viscosity hampers the motion of erythrocytes through capillaries, and as a certain critical threshold is approached, this contributes to the development of tissue hypoxia. The mesomechanics of structural-phase transitions and destruction in external fields of biological membranes and nanostructured solids are qualitatively alike.

Engineered Annexin A5 Variants Have Impaired Cell Entry for Molecular Imaging of Apoptosis Using Pretargeting Strategies

Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell. anxA5 binds PS and crystallizes in a two-dimensional network covering the PS-expressing cell surface. Two-dimensional crystallization is the driving force for anxA5 internalization by PS-expressing cells. Here, we report structure/function analysis of anxA5 internalization. Guided by structural bioinformatics including protein-protein docking, we revealed that the amino acids Arg(63), Lys(70), Lys(101), Glu(138), Asp(139), and Asn(160) engage in intermolecular salt bridges within the anxA5 trimer, which is the basic building block of the two-dimensional network. Disruption of the salt bridges by site-directed mutagenesis does not affect PS binding but inhibits trimer formation and cell entry of surface-bound anxA5. The anxA5 variants with impaired internalization are superior molecular imaging agents in pretargeting strategies as compared with wild-type anxA5.

Interaction Mechanism of Cortisol and Catecholamines with Structural Components of Erythrocyte Membranes

Nonspecific mechanisms of the stress hormones interaction with erythrocyte membranes were studied by means of atomic force microscopy, fluorescence analysis, and IR spectroscopy. It was shown that stress hormones (cortisol, adrenaline, noradrenaline) can bind to erythrocyte membranes with high affinity (K(b) approximately 10(6) M(-1)). The binding mechanism involves hydrogen bonds and hydrophobic and electrostatic interactions. Active groups of the hormones (NH(2), NHCH(3), keto, and hydroxy groups) interact simultaneously with CO and NH groups both of proteins and phospholipids. This leads to the formation of complex protein-lipid domains that distort the surface of the erythrocyte membrane. Water dipoles are displaced from the domains to adjacent regions and facilitate membrane loosening. The interaction of hormones with the membrane is accompanied by structural transitions of disorder --> order (tangle --> alpha-helix, tangle --> beta-structure) in membrane proteins and structural transitions of order --> order in phospholipids. Formation of large domains (clusters) of the lipid-protein and lipid nature leads to distortion of membranes and deteriorates their elasticity and rheological properties.

Investigation on the synergistic effect of a combination of chemical enhancers and modulated iontophoresis for transdermal delivery of insulin

Purpose: The main objective of this study was to assess the flux enhancement of insulin transdermally by utilizing a complex of chemical enhancers in combination with modulated iontophoresis. Methods: The experiments were performed on porcine epidermis model under three different circumstances, namely, (a) 1-hour modulated iontophoresis alone; (b) pretreatment with vehicle and chemical enhancer combinations and (c) combination of (a) and (b). The mechanism of action of the enhancers was studied using infra-red spectra by derivative and curve-fitting techniques and Confocal laser scanning microscopy. The efficacy of the optimized combination was tested in vivo in streptozocin-diabetic Wistar rats. Results: A blend of 1,8 cineole, oleic acid and sodium deoxycholate in propylene glycol : ethanol (7:3) resulted in 45% enhancement, when permeation was performed in combination with iontophoresis as compared to the latter alone. In-depth analysis of infra-red spectra revealed that each of the enhancers acted differentially on lipid-protein domains of the stratum corneum thereby supporting the observed synergism. Movement of fluorescently labeled insulin depicted highlighted follicular regions and paracellular accumulation of the probe after iontophoresis and chemical enhancer treatment respectively. Presence of the fluorescent peptide in these regions 4 hour after treatment with the combination reinforced the results of the permeation studies. Finally the combination of modulated iontophoresis with chemical enhancer blend resulted in lowering of blood glucose for 8 hour in vivo. Conclusions: The study proved the applicability of modulated iontophoresis with chemical pretreatment in delivering insulin transdermally.

Asymmetry determines the effects of natural ceramides on model membranes

Ceramides can dramatically influence the lateral organization of biological membranes. In particular, ceramide-induced alterations of protein-lipid domains can be involved in several cellular processes, ranging from senescence to immune response. In this context, an important role is played by the length of the fatty acid bound to the sphingosine moiety. Asymmetric, heterogeneous ceramides, with more than 20 or less than 16 carbon atoms in the fatty acyl chain, in fact exert diverging effects in vivo if compared to their symmetric counterparts. In this work, we investigated the role of ceramide asymmetry and heterogeneity in model membranes showing raft-like phase separation, using a combination of fluorescence imaging, atomic force microscopy, fluorescence correlation spectroscopy and differential scanning calorimetry. We show that ceramide produced enzymatically from natural mixtures of sphingomyelin can dramatically alter the mixing behaviour of proteins and lipids in the membrane, inducing a homogenization of the bilayer. Furthermore, we characterized the physical properties of coexisting lipid phases at equilibrium in membranes with varying ceramide content, emphasizing the differences between symmetric-homogeneous and asymmetric-heterogeneous ceramides. While symmetric ceramides always produce enhanced order, asymmetric ceramides display a more complex behavior similar to that of cholesterol. Our results might help contribute to a more precise understanding of the rearrangements induced by different kinds of ceramide generation in cellular membranes.

Large Disk Intermediate Precedes Formation of Apolipoprotein A-I−Dimyristoylphosphatidylcholine Small Disks

Small approximately 8.5 nm disks formed spontaneously when dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUVs) were incubated with apolipoprotein A-I (apoA-I) (100:1 molar ratio). However, in a time course study, the transient production of approximately 11 nm large disks was detected and isolated by gel filtration. The intermediate large disks contained three apoA-I molecules and were stable over time; however, when additional apoA-I was added, they formed small disks containing two molecules of apoA-I. The reaction kinetics of apoA-I with DMPC LUVs was monitored by fluorescence resonance energy transfer, and two phases were observed, supporting the presence of the intermediate in the formation of small disks. The lipid dynamics of LUVs and disks were assayed, revealing the presence of sequestered lipid-protein domains upon apoA-I binding to DMPC LUVs. In addition, the lipids in the intermediate large disks were more constrained than those in the small disks. We propose that apoA-I binds with DMPC LUVs to form small lipid-protein domains on the LUV; then the domains are released to form large disks, which can mature in the presence of additional apoA-I to form small disks. Thus, the formation of small apoA-I lipid disks proceeds through the formation of a large disk intermediate.

Effect of DC/mDC iontophoresis and terpenes on transdermal permeation of methotrexate: In vitro study

The systemic toxicity caused by methotrexate limits its use and transdermal delivery would be a possible alternative. Transdermal permeation of methotrexate loaded into polyacrylamide-based hydrogel patch, across mice skin was studied in vitro after pretreatment with terpenes and ethanol, alone or in combination with iontophoresis (DC/mDC). Polyacrylamide patches gave the maximum flux as compared to the copolymers of acrylamide and acrylic acid. Of the terpenes used, pure menthol showed maximum enhancement (38%), whereas pure limonene elicited a minimum of 9.9% enhancement. Binary combination of menthol and ethanol increased the permeation to 54.9%, which was further enhanced to 93.69% and 117% when used in combination with DC and square wave (mDC) iontophoresis, respectively. ATR-FTIR of the stratum corneum treated with terpenes showed a split in the asymmetric C–H stretching vibrations along with decrease in peak heights and areas of asymmetric, symmetric C–H stretching, C O stretching and amide bands. A split in amide II band was observed with iontophoresis. ATR-FTIR studies suggest conformational changes in the lipid–protein domains thereby increasing permeation. Histopathological studies on treated skin samples, gave an insight about the anatomical changes brought by the application of various enhancers. Binary mixture of menthol and ethanol in combination with square wave gave best results.

Expression of the non-compact myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in olfactory bulb ensheathing glia from explant cultures.

The non-compact myelin protein 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) may have a unique role in signaling pathways mediated by lipid-protein domains (Kim and Pfeiffer, 1999). We have tested for CNPase in olfactory bulb ensheathing glia (OBEG) in explant cultures. Migrating bipolar and multipolar cells with OBEG typical morphologies were inimunoreactive for both vimentin- and S100-like proteins. Although apparently devoid of myelin basic protein (MBP)-like immunoreactivity, these cells displayed weak but unambiguous CNPase-like immunoreactivity. Our results suggest a further resemblance to myelinating Schwann cells.