Hydrophobic Head Group Research Articles

Molecular segregation observed in a concentrated alcohol-water solution.

When a simple alcohol such as methanol or ethanol is mixed with water, the entropy of the system increases far less than expected for an ideal solution of randomly mixed molecules. This well-known effect has been attributed to hydrophobic headgroups creating ice-like or clathrate-like structures in the surrounding water, although experimental support for this hypothesis is scarce. In fact, an increasing amount of experimental and theoretical work suggests that the hydrophobic headgroups of alcohol molecules in aqueous solution cluster together. However, a consistent description of the details of this self-association is lacking. Here we use neutron diffraction with isotope substitution to probe the molecular-scale structure of a concentrated alcohol water mixture (7:3 molar ratio). Our data indicate that most of the water molecules exist as small hydrogen-bonded strings and clusters in a 'fluid' of close-packed methyl groups, with water clusters bridging neighbouring methanol hydroxyl groups through hydrogen bonding. This behaviour suggests that the anomalous thermodynamics of water alcohol systems arises from incomplete mixing at the molecular level and from retention of remnants of the three-dimensional hydrogen-bonded network structure of bulk water.

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.

Location of the Polyamine Binding Site in the Vestibule of the Nicotinic Acetylcholine Receptor Ion Channel

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, (125)I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies, we conclude that the site of (125)I-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of (125)I-MR44. The site where the hydrophobic head group of (125)I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with (125)I-MR44. We conclude that the polyamine moiety of (125)I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by (125)I-MR44 should therefore be accessible from the luminal side of the vestibule.

Binding of polyamine-containing toxins in the vestibule of the nicotinic acetylcholine receptor ion channel

Several wasp venoms contain philanthotoxins (PhTXs) that act as noncompetitive inhibitors (NCIs) on cation-selective ion channels including the nicotinic acetylcholine receptor (nAChR). In the search for a ligand with high affinity and specificity for the nAChR we tested a series of newly developed PhTX analogues. Modulation of the structural elements of PhTXs can significantly influence their binding affinities. This approach resulted in the development of the photolabile compound MR44. In photoaffinity labelling studies 125I-MR44 was used to map the ligand-binding site at the Torpedo californica nAChR. Upon UV irradiation of the receptor–ligand complex, 125I-MR44 was mainly incorporated into the receptor α-subunit. Proteolytic mapping and microsequencing identified the site of 125I-MR44 cross-linking within the sequence αHis-186 to αLeu-199 that in its C-terminal region partially overlaps with the agonist-binding site. Since bound agonists had only minor influence on 125I-MR44 photocross-linking, the site where the hydrophobic head group of 125I-MR44 binds must be located outside the zone that is sterically influenced by agonists bound at the nAChR. A possible site of interaction of 125I-MR44 would be the N-terminal region of the labelled sequence, in which aromatic amino-acid residues are accumulated. We suggest that the polyamine moiety of 125I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the ion channel, while the aromatic ring of this compound binds in the vestibule of the nAChR to a hydrophobic region on the α-subunit that is located close to the agonist binding site.

Monosialoganglioside containing cationic liposomes with a cationic cholesterol derivative promote the efficiency of gene transfection in mammalian culture cells.

We have studied the effects of monosialoganglioside (GM1)-containing cationic liposomes with a cationic cholesterol on the liposome-mediated gene transfection into mammalian culture cells. The results showed that both cationic liposomes with either a cationic cholesterol derivative of a hydrophobic amino head group (I) and a hydrophilic amino head group (II) promoted the transfection of luciferase plasmids (pGL3) into HeLa and CHO-K1 cells more than the control cationic liposomes without GM1. In addition, we found that cationic liposomes with a cationic cholesterol derivative (II) were about ten times as effective as that by commercially available cationic liposome Lipofectin. Confocal fluorescence microscopy showed that the liposome/DNA complex was transferred more efficiently into the target cells by the GM1-containing liposomes than by the liposomes without GM1. In proportion to the above results, free antisense DNAs were also more efficiently transferred into the nucleus of the target cells by the GM1-containing liposomes. When there was 100 mM galactose in the transfection medium, the luciferase activity by the GM1-containing liposomes was reduced to the level of the control liposomes. The results suggest that GM1-containing cationic liposomes with a cationic cholesterol derivative of a hydrophobic amino head group or a hydrophilic amino head group should significantly increase the transfection efficiency of plasmid DNAs and antisense DNAs by galactose receptor-mediated endocytosis. This means that the GM1-containing liposomes described here should be very promising for gene transfection in vitro.

Structure–activity relationship and site of binding of polyamine derivatives at the nicotinic acetylcholine receptor

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues. The stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well-characterized luminal NCIs. To elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross-linked to the nAChR in its closed channel conformation resulting in labeling yields for the two alpha and the beta, gamma and delta subunits of 10.4, 11.1, 4.0 and 7.4%, respectively. Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The hydrophobic head groups most likely bind to the high-affinity NCI site, while the positively charged polyamine chains presumably interact with the negatively charged selectivity filter located deep in the channel lumen.

α-Chymotrypsin Superactivity in Cetyltrialkylammonium Bromide-Rich Media

α-Chymotrypsin (a-CT) activity was tested with N-glutaryl-L-phenylalanine p-nitroanilide in buffered media with added cationic surfactants. The effect of the commercial cetyltrimethylammonium bromide (CTABr) was compared with that of three other surfactants with ethyl (CTEABr), propyl (CTPABr), and butyl (CTBABr) head groups. These were synthesized and purified in this laboratory. Surfactant head groups provided distinct environments that largely modulated the catalytic performance. Larger alkyl head group hydrophobicity led to a marked enhancement of α-CT activity. CTBABr-rich media induced the highest superactivity. Kinetic measurements were performed in Tris-HCl buffer at a surfactant concentration either below or above CMC, and α-CT superactivity occurred in both media. Positive interactions between the enzyme and surfactants happened independently of the supramolecular organization of the medium. The reaction followed the Michaelis-Menten kinetics. The substrate to micelle aggregates binding constant was used to calculate the substrate concentration available for catalysis. The k cat to K m ratio was in CTBABr-rich media always higher than in pure buffer and depended on the surfactant concentration. α-CT superactivity depended on the pH value of buffer solution. Enzyme inactivation followed a single-step mechanism in pure buffer and a series mechanism in the presence of a surfactant. The rate of activity decay obeyed a first-order kinetics.

Surface properties and hemocompatibility of alkyl-siloxane monolayers supported on silicone rubber: effect of alkyl chain length and ionic functionality.

Self-assembled monolayers of alkylsiloxanes supported on poly(dimethylsiloxane) (PDMS) rubber were used as model systems to study the relation between blood compatibility and surface composition. The inner lumen of PDMS tubes were first treated with an oxygen plasma. The resultant oxidized surfaces were post-derivatized by reaction with alkyltrichlorosilanes to form the monolayer films. The alkyl chain lengths used were slightly longer than in a previous study, and this may alter the phase-state of the monolayer from liquid-like to crystalline. The chemical properties of the monolayer were controlled by varying the chemical composition of the alkyltrichlorosilanes used. Terminal functionalities included -CH3, -CF3, -COOH, -SO3H and -(CH2CH2O)4OH. Surface derivatization was verified with static contact angle measurements and X-ray photoelectron spectroscopy. Blood compatibility was evaluated using a canine ex vivo arterio-venous series shunt model. Surfaces grafted with hydrophobic head groups such as -CH3 and -CF3 were significantly less thrombogenic than the surfaces composed of ionic head groups such as -COOH and -SO3H. Surfaces enriched in -(CH2CH2O)4OH had an intermediate thrombogenicity. Silastic pump grade tubing and polyethylene tubing, used as controls, were found to be the least thrombogenic of all the surfaces tested.

Monolayer and epi-fluorescence microscopy studies of amino acid derivatized diacetylene lipids

Monolayers of amino acid derivatized diacetylene lipids on water were studied by Langmuir trough and epi-fluorescence microscopy (EFM). Examination of the amino acid derivatized diacetylene lipids showed a wide variety of surface characteristics with many novel properties. Many of these new properties are advantageous for film-based biosensor development. To characterize this family of molecules, pressure–area (π–A) isotherms were measured that demonstrate hydrophilic headgroup compounds allow for close-packed monomolecular layer formation in the solid state upon continuous compression. The effect of subphase pH and temperature on monolayer compressibility and phase transition was examined. Decrease of pH results in attenuation of LE–LC phase transition in Gln-PDA monolayers due to charge screening, while temperature affects the monolayers through regular thermal agitation, and the effect is opposite to the thermal anomaly observed for the non-derivatized diacetylene lipids. EFM studies reveal that only the lipids derivatized with hydrophilic headgroups exhibit phase transition and domain formation, while those with hydrophobic headgroups do not easily form polymerizable matrices. The shape and growth of the domains were found to be strongly headgroup dependent. Photo-polymerization of diacetylene lipid domains on water using EFM is discussed.

Rigidity in synkinetic molecular monolayers of functional lipids containing Angstrom Gaps

Abstract

Effect of Counterions and Headgroup Hydrophobicity on Properties of Micelles Formed by Alkylpyridinium Surfactants. 2. Microcalorimetry

We report the influence of counterions and headgroup hydrophobicity on the enthalpies and heat capacities of micelle formation by 1-alkyl-4-n-dodecylpyridinium surfactants in aqueous solution. For micelle-forming 1-alkyl-4-n-dodecylpyridinium iodide surfactants (alkyl = C1−C3), enthalpies of micelle formation show no trend with respect to 1-alkyl chain length. Changes in molecular architecture of the counterion, however, have a large influence on the thermodynamics of micelle formation. It is shown that next to solvation, London dispersion interactions, counterion (substituent)−water interactions, and the microenvironment of counterions adsorbed to micellar surfaces play an important role in determining the exothermicities of micelle formation. Isobaric heat capacities of micelle formation show that the formation of wormlike micelles for salicylate-containing cationic surfactants is due to a favorable microenvironment for salicylate counterions in the Stern layer of micelles.

Cell shrinkage and apoptosis: a role for potassium and sodium ion efflux

In this study we have shown that redistribution of the lipid composition of the external and internal leaflets of the PM during apoptosis results in two main alterations in the cell surface, externalisation of PS, and a looser packing of the lipid hydrophobic head groups in the external leaflet. Significantly, neither of these alterations can be directly implicated in the mechanism of apoptotic cell shrinkage, however they do have functions in other phases of the apoptotic process. Progressional studies involving morphological and flow cytometric evaluation, and DNA gel electrophoresis revealed that apoptotic cell shrinkage is associated with a decrease in [Na+]i and [K+]i which occurs after visualisation of chromatin condensation and internucleosomal DNA fragmentation, and prior to apoptotic body formation. When apoptotic cultures were supplemented with inhibitors of the Na+, K+-ATPase pump or the Ca2+-dependent K+ channel, essentially all of the respective Na+ or K+ efflux during apoptosis can be inhibited, suggesting that essentially all of the Na+ and K+ efflux can be ascribed to active pumping via the Na+, K+-ATPase pump and the Ca2+-dependent K+ channel.

New synthetic, bolaamphiphilic, macrocyclic lipids forming artificial membranes

In order to obtain stable but fluid membranes attached to a solid or gel support, two fundamental problems had to be solved. First, stable coupling of the lipid layer to the surface had to be achieved, and second, it was necessary to prevent the second lipid layer of the membrane from floating on top of the first one. To overcome these limitations we redesigned lipids to mimic the structure and properties of the cyclic tetraether lipids from Archaebacteria. Our new type of artificial lipid is a linear rigid bolaamphiphile consisting of a long hydrophobic core and hydrophilic head groups at both ends. Up to 72-membered macrocyclic tetraesters were prepared in a highly efficient two-step synthesis from maleic anhydride and long-chain linear diols. The lipid building block was modified by attaching various hydrophilic head groups to meet the various biological needs. Lipids were coated on surfaces, studied by Langmuir-Blodgett technology and used to form stable films on patch clamp pipettes.

Microemulsion polymerization: New surfactant systems by counterion variation

Novel surfactant systems that allow the formation of polymerizable microemulsions are required for the synthesis of ultrafine polymer latices. The work reported here extends the ability to formulate new microemulsion “recipes” by means of a new approach: the geometry and polarity of a given surfactant can be adjusted by electrostatic coupling with an appropriate organic counterion, e.g., the generation of a very bulky hydrophobic head group. It is demonstrated that counterion variation of classical surfactants leads to a significant improvement of classical microemulsions.

Phospholipid Vesicles as Organized Media in Spectroluminescence Analysis

The membrane properties of pure dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylserine (DMPS), and dimyristoyl phosphatidylglycerol (DMPG) vesicles were compared using the fluorescence of the probe pyrene in an assessment of the potential utility of phospholipids as organized media in spectroluminescence analysis. The partition coefficients, excimer stability constants, vibronic band ratios, triethylamine quenching constants, and fluorescence lifetime distribution widths of pyrene were used as measures of the solute capacity, solute aggregation promotion, polarity, sequestering efficiency, and heterogeneity of the three media. The results indicate that the head group hydrophobicity and membrane fluidity required to reduce the incidence of excimer formation and to promote solute sequestering in the most hydrophobic part of the aggregate precludes efficient sequestering from cosolubilized quenchers. This implies that significant improvements in dynamic range would be expected in spectroluminescence analyses utilizing DMPG, but improvements of detection limits would be moderate compared to those observed in bile salts, cyclodextrins, or surfactant aggregates.

Surface and blood‐contacting properties of alkylsiloxane monolayers supported on silicone rubber

Self-assembled monolayers of alkylsiloxanes supported on polydimethyl siloxane (PDMS) rubber were used as model systems to study the relation between blood compatibility and surface chemistry. The inner lumen of PDMS tubes was first treated with an oxygen plasma. The resultant oxidized surfaces were postderivatized by reacting them with alkyltrichlorosilanes to form the monolayer films. The chemical properties of the monolayers were controlled by varying the head-group chemical compositions. Surface derivatization was verified using variable-angle X-ray photoelectron spectroscopy (XPS or ESCA). Blood compatibility was evaluated using a canine ex vivo arteriovenous series shunt model. Surfaces grafted with hydrophobic head-groups as -CH3 and -CF3 had significantly lower platelet and fibrinogen deposition than the surfaces composed of hydrophilic groups such as -CO2CH3, -(CH2CH2O)3COCH3, and -(OCH2CH2)3OH.

Forces between Surfaces Coated with a Polymerizable Surfactant before and after Polymerization

Abstract The forces between layers of a cationic polymerizable surfactant, cetyl p -vinylbenzyl dimethyl ammonium chloride (CVDAC), adsorbed onto muscovite mica have been measured with a surface force technique. The forces were measured both before and after partial UV polymerization of the adsorbed layers, and the stability of adsorbed bilayers after dilution of the adsorption solution was studied for these two cases. Due to their large headgroup, CVDAC molecules do not pack efficiently into monolayers and bilayers. This can be observed by, for instance, the lower surface potential of adsorbed bilayers and the lower pull-off force from monolayer contact, compared to what is found for simpler surfactants like CTAB. It was also found that surface aggregates build up on top of the bilayer, due to the partly hydrophobic headgroup. When the bilayer structure, including the surface aggregates, is polymerized, it is stabilized both with respect to mechanical forces and with respect to dilution of the surrounding solution.

Pentachlorophenol alters the molecular organization of membranes in mammalian cells.

Biophysical measurements of motion parameters for fluorescent and paramagnetic probes partitioned into membranes of mammalian cells have indicated that the lipid bilayers of the membranes become destabilized as a result of treatment with sublethal doses of pentachlorophenol. Decreases in bulk lipid fluidity of up to 50%, which can be attributed to perturbation of the bilayer by the toxicant, were observed for purified plasma membrane and for preparations of total cell membrane. Spin labels that probe the hydrophobic interior and headgroup regions, respectively, of bilayers indicated that pentachlorophenol does not assume an amphipathic orientation in the membrane, but rather it partitions into the hydrophobic interior of the bilayer. Arrhenius plots of motion parameters were continuously linear over temperatures ranging from 20 to 55°C for both treated and control cells, indicating that pentachlorophenol does not induce lipid phase transitions. Treatment with sublethal doses of pentachlorophenol also induced a decline in levels of phospholipid phosphate per cell that can be attributed to a selective action of phospholipase C. Notwithstanding these changes, the cells remained viable judging from their ability to exclude trypan blue and thus the deleterious effects of pentachlorophenol on membranes can be interpreted as sublethal damage.

Ganglioside-modulated protein phosphorylation in myelin.

Gangliosides have profound effects on the phosphorylation of several proteins in myelin. Addition of polysialogangliosides to purified guinea pig brain myelin enhanced the endogenous phosphorylation of a 62-kDa phosphoprotein, but completely inhibited the phosphorylation of myelin basic protein (MBP) (18.5 kDa). The ganglioside-stimulated phosphorylation of the 62-kDa protein was dose-dependent and -specific. Asialo-GM1, ceramide trihexosides, N-acetylneuraminic acid, or colominic acid alone could not mimic this effect, suggesting that the activation process requires both the hydrophobic head group and the anionic character of the gangliosides. Studies on the time course of this reaction revealed that it was a rapid and reversible process and was affected only very slightly by Ca2+. Thus, the stimulatory effect of gangliosides may not involve Ca2+-gangliosides complexes or proteolysis, but may be mediated through an activation of a ganglioside-dependent protein kinase or due to substrate protein-glycolipid interaction. Modulation of the phosphorylation of MBP by gangliosides varies with the states of phosphorylation of this protein. Prior addition of ganglioside to myelin inhibited the phosphorylation of MBP. However, addition of gangliosides to myelin subsequent to maximal phosphorylation of MBP retarded the dephosphorylation of this protein. Phosphorylation of isolated MBP by protein kinase C was stimulated by gangliosides, provided phosphatidylserine was present. In contrast, the glycolipid inhibited the phosphorylation of a unique site catalyzed by cAMP-dependent protein kinase. This site was distinct from those phosphorylated by protein kinase C and was also sensitive to chymotryptic cleavage. Although the exact physiological significance of protein phosphorylation in myelin has yet to be established, gangliosides may play an important role in the modulation of this reversible post-translational modification mechanism.