Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry. The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes. The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF-CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes. (C), 2001 Elsevier Science B.V All rights reserved.

Encapsulation of lipopeptides within liposomes: Effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C-8, C-12 and C-16. The C-12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers. (C) 2005 Elsevier B.V. All rights reserved.

Antimicrobial peptides in action

Molecular dynamics simulations of the magainin MG-H2 peptide interacting with a model phospholipid membrane have been used to investigate the mechanism by which antimicrobial peptides act. Multiple copies of the peptide were randomly placed in solution close to the membrane. The peptide readily bound to the membrane, and above a certain concentration, the peptide was observed to cooperatively induce the formation of a nanometer- sized, toroidally shaped pore in the bilayer. In sharp contrast with the commonly accepted model of a toroidal pore, only one peptide was typically found near the center of the pore. The remaining peptides lay close to the edge of the pore, maintaining a predominantly parallel orientation with respect to the membrane.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4degreesC. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period < hydrated in buffer/short equilibration period < hydrated in water/prolonged equilibration period. ISCOM matrices appeared to form over time from samples, which initially contained a high concentration of ring-like micelles suggesting that these colloidal structures may be precursors to ISCOM matrix formation. Helices were also frequently found in samples containing ISCOM matrices as a minor colloidal structure. Equilibration time and presence of buffer salts also promoted the formation of liposomes in systems not containing Quil A. These parameters however, did not appear to significantly affect the occurrence and predominance of other structures present in the pseudo-binary systems containing Quil A. Pseudo-ternary phase diagrams of PC, Chol and Quil A are important to identify combinations which will produce different colloidal structures, particularly ISCOM matrices, by the method of lipid film hydration. Colloidal structures comprising these three components are readily prepared by hydration of dried lipid films and may have application in vaccine delivery where the functionality of ISCOMs has clearly been demonstrated. (C) 2003 Elsevier B.V. All rights reserved.

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg2+-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable K-d, of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 mu M free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.

Stimulating dormancy release and emergence of annual ryegrass (Lolium rigidum) seeds using short-term hydrated storage in darkness

Experiments were performed to determine whether the dormancy release effect of hydrated storage in darkness (dark-stratification) is common amongst annual ryegrass populations and has the potential to occur under field conditions. Dormant seeds from all populations tested (22) became sensitive to light during dark-stratification, enabling them to germinate when subsequently exposed to light. Under controlled temperature (25/15degreesC), light (12-h photoperiod), and hydration (solidified agar-water) conditions, more seeds germinated by 28 days if the first 14 days were in darkness followed by exposure to light for 12 h per day than if they were exposed to light throughout or darkness throughout. Constraint over the conditions imposed during dark-stratification and germination was gradually reduced to investigate whether the dormancy release effect was diminished. Dark-stratification was effective in promoting germination when performed under natural diurnal temperatures, and burial in moist soil provided suitable conditions for dark-stratification to occur. The surface of moist soil, with natural diurnal temperatures and sunlight, was suitable for germination of dark-stratified seeds. Dark-stratification is a quick and effective means to enhance the sensitivity of dormant annual ryegrass seeds to light, enabling the majority of the population to germinate. However, large quantities of light are required to promote germination of dark-stratified seeds, so buried seeds must be moved to the soil surface to allow exposure to adequate light for germination.

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state C-13 NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-C-13]Gly3-[2-C-13]Ala4, [1-C-13]Gly3-[2-C-13]Leu6, [1-C-13]Leu13-[2-C-13]Ala15, [2-C-13]Leu13-[1-C-13]Ala15, and [1-C-13]Leu13-[2-C-13]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-C-13]Gly3-[2-C-13]Ala4 and [1-C-13]Gly3-[2-C-13]Leu6 were consistent with alpha -helical structure in the N-terminus irrespective of environment. The Internuclear distances measured in [1-C-13]Leu13-[2-C-13]Ala15, [2-C-13]Leu13-[1-C-13]Ala15, and [1-C-13]Leu13-[2-C-13]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees) than in lyophilized powder (121 degrees -139 degrees) or crystals (129 degrees). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (similar to 160 degrees) (R. Smith, F. Separovic, T. J. Milne, A. Whittaker, F. M. Bennett, B. A. Cornell, and A. Makriyannis, 1994, J. Mol, Biol 241:456-466). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.

Surface-active phospholipid in total hip arthroplasty

This is the first report of surface-active phospholipid as the boundary lubricant in total hip arthroplasty. Aspirate and rinsings from the bearing surfaces of 25 revision operations and one primary surgery were analyzed from 3 weeks to 26 years postoperatively. All samples contained substantial amounts of surface-active phospholipids ranging from 14 to 4186 μg. These findings indicate that synoviocytes continue producing the lubricant in significant quantities after arthroplasty surgery independent of the type of joint replacement and its fixation. Surface-active phospholipid was found on all bearing surfaces analyzed including polyethylene, stainless steel, chrome cobalt, alumina, zirconia, and titanium.

A comparison of pseudo-ternary diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by lipid-film hydration and dialysis

Pseudo-ternary diagrams for Quil A, phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and cholesterol were established in order to identify combinations that result in the formation of immune-stimulating complex (ISCOM) matrices and other colloidal structures produced by these three components in aqueous systems following lipid-film hydration or dialysis (methods that can be used to produce ISCOMs). In addition, the effect of equilibration time (1 month at 4degreesC) on the structures formed by the various combinations of the three components was investigated. Depending on the ratio of Quil A, cholesterol and phospholipid, different colloidal particles, including ISCOM matrices, liposomes and ring-like micelles, were found irrespective of the preparation method used. In contrast, worm-like micelles were only observed in systems prepared by lipid-film hydration. For samples prepared by dialysis, ISCOM matrices were predominantly found near the Quil A apex of the pseudo-ternary diagram (> 50% Quil A). On the other hand, for samples prepared by lipid-film hydration, ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram (> 50% phospholipid). The regions in the pseudo-ternary diagrams in which ISCOM matrices were observed increased following an extended equilibration time, particularly for samples prepared by lipid-film hydration. Differences were also observed between pseudoternary diagrams prepared using either PE or PC as phospholipids.

Surface-active phospholipid (surfactant) in equine tendon and tendon sheath fluid

AIM: To investigate the presence of surface-active phospholipid (SAPL, or surfactant) in equine tendon and tendon sheath fluid. METHODS: The left front flexor tendon and sheath were removed from five Thoroughbred horses. Phospholipid was extracted from tendon sheath fluid using Folch reagent and quantified using spectroscopy. Transmission electron microscopy (TEM) was used to observe the tendon surfaces. RESULTS: The presence of phospholipid (90.6 (SD 4.3) mu g/ml) in tendon sheath fluid, plus the appearance of oligolamellar layers and lamellar bodies on the tendon surface were indicative of SAPL. CONCLUSIONS: Evidence of SAPL was found in equine tendon, and may have a similar lubricating function as reported for synovial joints. CLINICAL RELEVANCE: These findings may have important implications for normal tendon function and possible therapeutic adjuncts for tendon and tendon sheath injuries.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Molecular dynamics study of MscL interactions with a curved lipid bilayer

Mechanosensitivity is a ubiquitous sensory mechanism found in living organisms. The simplest known mechanotransducing mechanism is found in bacteria in the form of the mechanosensitive membrane channel of large conductance, MscL. This channel has been studied extensively using a variety of methods at a functional and structural level. The channel is gated by membrane tension in the lipid bilayer alone. It serves as a safety valve protecting bacterial cells against hypoosmotic shock. MscL of Escherichia coli embedded in bilayers composed of asymmetric amounts of single-tailed and double-tailed lipids has been shown to gate spontaneously, even in the absence of membrane tension. To gain insight into the effect of the lipid membrane composition and geometry on MscL structure, a fully solvated, all-atom model of MscL in a stress-free curved bilayer composed of double- and single-tailed lipids was studied using a 9.5-ns molecular dynamics simulation. The bilayer was modeled as a domed structure accommodating the asymmetric composition of the monolayers. During the course of the simulation a spontaneous restructuring of the periplasmic loops occurred, leading to interactions between one of the loops and phospholipid headgroups. Previous experimental studies of the role of the loops agree with the observation that opening starts with a restructuring of the periplasmic loop, suggesting an effect of the curved bilayer. Because of limited resources, only one simulation of the large system was performed. However, the results obtained suggest that through the geometry and composition of the bilayer the protein structure can be affected even on short timescales.