José Villalaín

The relationship between the antioxidant and the antibacterial properties of galloylated catechins and the structure of phospholipid model membranes

The effects of four catechins, (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), on the physical properties of phospholipid model membranes and the correlation to their antioxidant and antibacterial capacities have been studied by using differential scanning calorimetry (DSC), fluorescence spectroscopy, infrared spectroscopy (IR), AAPH-induced oxidation, and leakage experiments. DSC data revealed that galloylated catechins, especially ECG, affected the physical properties of both the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bilayers dramatically. Galloylated catechins showed higher phospholipid/water partition coefficients than their homologues and were immersed in the phospholipid palisade intercalating within the hydrocarbon chains, ECG being at the deepest position. In contrast, nongalloylated catechins presented a shallow location close to the phospholipid/water interface. ECG also exhibited the highest antioxidant capacity against lipid peroxidation, which correlated with its strong effect on DPH fluorescence anisotropy (as observed by the increase of the lipid order of fluid PC bilayers) and with the presence of highly cooperative transitions as seen by DSC. We propose that the high antioxidant capacity of some galloylated catechins such as ECG could be partially due to the formation of membrane structures showing resistance to detergent solubilization and in which the phospholipids have tightly packed acyl chains and highly hydrated phosphate groups. Significantly, PE was found to be essential to the promotion of carboxyfluorescein leakage from bacterial model membranes by galloylated catechins, indicating that their bactericidal activity, at least at the membrane level, could be due to the specific effect of these catechins on PE.

Influence of liposome charge and composition on their interaction with human blood serum proteins

Lipid composition and specially their electrostatic properties, were found to greatly influence the stability of liposomes in human blood serum. The amount and type of serum proteins bound to the liposomes were also clearly influenced by lipid composition and charge of liposomes. a good correlation was found between the amount of serum proteins adsorbed to a given type of liposome and its instability as measured by the release of an encapsulated fluorescent probe. Liposomes that bind the highest amount of protein were the least stable, except for the case of liposomes containing gangliosides, which were fairly stable even at a high amount of bound protein. Liposomes with neutral charge containing phosphatidylcholine were the most stable and bound the lowest amount of protein. Liposomes with positive charge behaved similarly to those with neutral charge. However, the stability of negatively charged liposomes was very dependent on their composition. Those liposomes containing only one class of negatively charged phospholipids bound a great amount of protein and were very unstable. However, those liposomes containing also phosphatidylcholine bound less protein and were more stable. The examination of the electrophoresis patterns of serum proteins bound to the different types of liposomes indicated the presence of specific proteins which correlated with liposome instability. (Mol Cell Biochem120: 119–126, 1993)

Apparent pKa of the fatty acids within ordered mixtures of model human stratum corneum lipids.

AbstractPurpose. The apparent pKa of the fatty acids within hydrated (30 % w/w) model human stratum corneum (SC) lipid mixtures should be measured. Methods. The degree of ionisation of the fatty acids was calculated as a function of pH using Fourier transform infra-red spectroscopy. The relative intensity of the stretching bands of the unionized and ionized carboxylic groups was determined and fitted to the relevant expression for ionic equilibrium of a monoprotic acid. The pKa was then calculated for increasing proportion of unsaturated fatty acid in the lipid mixture. Results. Values for pKa in the range 6.2-7.3 were found, increasing with greater proportion of oleic acid. These are some 1.5-3 pH units higher than the pKas of fatty acids in molecular solution. Conclusions. As there exists a pH-gradient across the SC, the degree of ionisation will also vary. In the innermost SC layers, a pH of 7 will produce 90% ionization of the fatty acids and head-group repulsion will be great. At the SC surface, the pH of 5 will cause almost minimal head-group repulsion, tending to increase crystallinity and promote a bilayer structure.

Identification of the Membrane-Active Regions of the Severe Acute Respiratory Syndrome Coronavirus Spike Membrane Glycoprotein Using a 16/18-Mer Peptide Scan: Implications for the Viral Fusion Mechanism

ABSTRACT We have identified the membrane-active regions of the severe acute respiratory syndrome coronavirus (SARS CoV) spike glycoprotein by determining the effect on model membrane integrity of a 16/18-mer SARS CoV spike glycoprotein peptide library. By monitoring the effect of this peptide library on membrane leakage in model membranes, we have identified three regions on the SARS CoV spike glycoprotein with membrane-interacting capabilities: region 1, located immediately upstream of heptad repeat 1 (HR1) and suggested to be the fusion peptide; region 2, located between HR1 and HR2, which would be analogous to the loop domain of human immunodeficiency virus type 1; and region 3, which would correspond to the pretransmembrane region. The identification of these membrane-active regions, which are capable of modifying the biophysical properties of phospholipid membranes, supports their direct role in SARS CoV-mediated membrane fusion, as well as facilitating the future development of SARS CoV entry inhibitors.

Infrared spectroscopic study of the interaction of diacylglycerol with phosphatidylserine in the presence of calcium

The interaction of 1,2-dipalmitoylglycerol (DPG) with dipalmitoylphosphatidylserine (DPPS) has been studied in aqueous dispersion in the presence and in the absence of Ca2+ by using Fourier transform infrared spectroscopy (FT-IR) and 45Ca(2+)-binding. FT-IR showed that DPG increased the phase transition of DPPS and induced a rigidification of the DPPS/DPG-Ca2+ complex. In the absence of Ca2+, the incorporation of DPG produced an increase in the proportion of dehydrated carbonyl groups in the mixture of DPPS plus DPG whereas, in the presence of Ca2+, DPG suppressed the solid-solid phase transition of phosphatidylserine-Ca2+ complexes. The phosphate band of DPPS was analyzed using a multivariate statistical analysis, indicating that DPG induced a higher dehydration of the PO2- group in the presence of subsaturating Ca2+ concentrations. Even very low concentrations of DPG, such as 2 mol%, already produced a significant effect. In the presence of both DPG and Ca2+, dehydration of DPPS increased, so that full dehydration was reached at a DPPS/Ca2+ molar ratio of 2.94 instead of 2.04 as observed for pure DPPS. However, the stoichiometry of the binding of Ca2+ to DPPS was not significantly altered by the inclusion of DPG as revealed by 45Ca(2+)-binding experiments, indicating that, in this situation, full dehydration of the PO2- groups of DPPS was reached when approx. 2 out of every 3 molecules of DPPS were binding Ca2+. The effects reported here for the interaction of DPG with DPPS may be significant for a number of biological situations where Ca2+, phosphatidylserine and diacylglycerols are involved, such as fusion of membranes or the activation of protein kinase C, where the dehydration effect produced by diacylglycerols may explain, at least in part, their effects.

The interaction of abietic acid with phospholipid membranes

Abietic acid is a major component of the oleoresin synthesized by many conifers and constitutes a major class of environmental toxic compounds with potential health hazard to animal, including human, and plant life. Being an amphipathic molecule, the study of the influence of abietic acid on the structure of membranes would be important to get insight into the mechanism of toxic action of the molecule. The interaction of abietic acid with model membranes of dipalmitoylphosphatidylcholine (DPPC) and dielaidoylphosphatidylethanolamine (DEPE) has been studied by differential scanning calorimetry and 31P-nuclear magnetic resonance spectroscopy. It has been found that abietic acid greatly affects the phase transition of DPPC, shifting the transition temperature to lower values, giving rise to the appearance of two peaks in the thermogram and to the presence of fluid immiscible phases. In a similar way, the phase transition of DEPE, in the presence of abietic acid, was shifted to lower temperatures, and two peaks appeared in the thermograms. The temperature of the lamellar to hexagonal H(II) phase transition was also decreased by the presence of abietic acid, but phase immiscibilities were not detected. The possible implications of these effects on the action of abietic acid on biological membranes are discussed.

Stability of Liposomes on Long Term Storage

Abstract— The effect of the lipid composition of liposomes on their storage for up to one year under different environmental conditions has been examined using 5,6‐carboxyfluorescein as a model drug. When cholesterol and/or α‐tocopherol were included in the liposomes, a significantly greater amount of dye was retained. The presence of α‐tocopherol decreased the breakdown of phosphatidylcholine to lysophosphatidylcholine and also reduced the level of peroxidation. Carboxyfluorescein retention was further enhanced when liposomes were stored at 4°C or at room temperature (20°C) in an O2‐free atmosphere. Lysophosphatidylcholine formation also slowed when the liposomes were kept at 4°C, or in an O2‐free atmosphere. It is concluded that egg yolk lecithin liposomes may be stored for long periods at low temperature in an O2‐free atmosphere or with added stabilizers such as cholesterol and α‐tocopherol.

Capsaicin affects the structure and phase organization of phospholipid membranes.

Capsaicin is a natural compound with pharmacological and toxicological effects, which given its hydrophobicity, can influence the structure of membranes. The interaction of capsaicin with model membranes of dipalmitoylphosphatidylcholine and dielaidoylphosphatidylethanolamine has been studied by using differential scanning calorimetry, fluorescent probe spectroscopy and 31P-nuclear magnetic resonance. Capsaicin remarkably affects the phase transition of dipalmitoylphosphatidylcholine, shifting the transition temperature to lower values, and giving rise, at relatively high capsaicin concentrations, to the appearance of two peaks in the thermogram. These peaks may correspond to separated phases as indicated by the partial phase diagram. Whereas capsaicin did not affect the fluorescence polarization of the probes diphenylhexatriene and trimethylammonium-diphenylhexatriene, it clearly affected that of the probe 2-anthroyloxystearic acid, indicating that the perturbation produced by capsaicin on the membrane would be mainly at the position where this fluorophore is located. On the other hand, capsaicin, at relatively low concentrations, gives rise to immiscible phases in the presence of dielaidoylphosphatidylethanolamine and decrease the temperature of the lamellar to hexagonal HII phase transition. At concentrations of capsaicin higher than 0.3 mol fraction, isotropic phases were detected. The possible implications of the effects of capsaicin on biological membranes are discussed.

Location and orientation of Triclosan in phospholipid model membranes

Triclosan is a hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. Although the molecular mechanism of action of this molecule has been attributed to inhibition of fatty acid biosynthesis, earlier work in our laboratories strongly suggested that the antibacterial action of Triclosan is mediated at least partly through its membranotropic effects. In order to assess its location in phospholipid membranes, high-resolution magic-angle spinning natural abundance 13C NMR of Triclosan embedded within egg yolk lecithin model membranes has been used to obtain 13C spin–lattice relaxation times for both Triclosan and lecithin carbon atoms in the presence of Gd3+ ions. The results indicate that Triclosan is localized in the upper region of the phospholipid membrane, its hydroxyl group residing in the vicinity of the C=O/C2 carbon atoms of the acyl chain of the phospholipid, and the rest of the Triclosan molecule is probably aligned in a nearly perpendicular orientation with respect to the phospholipid molecule. Intercalation of Triclosan into bacterial cell membranes likely compromises the functional integrity of those membranes, thereby accounting for at least some of this compound’s antibacterial effects.

The use of FT-IR for quantitative studies of the apparent pKa of lipid carboxyl groups and the dehydration degree of the phosphate group of phospholipids.

Fourier-transform infrared spectroscopy (FT-IR) has been applied to the quantitative study of the dehydration of the phosphatidylserine phosphate group in the presence of Ca2+ exerted by different molecules, such as diacylglycerol, sphingosine and stearylarnine, by using a partial least-squares statistical procedure. By using this method it was observed that diacylglycerol enhanced the dehydration of this PO2- group produced by Ca2+ whereas the amino-bases sphingosine and stearylamine protected the phosphate group from the dehydration produced by Ca2+ due to the very strong electrostatic interaction established. The apparent pKa of lipid carboxyl groups can also be estimated by using FTIR. The method consisted in quantifying the absorbance intensities due to the protonated and the unprotonated forms of the specific group being studied. The pKa of the carboxyl group of [1-13C]-palmitic acid included in dipalmitoylphosphatidylcholine membranes was found to be 8.7, a value much higher than that estimated from a molecular solution of the fatty acid. It was observed using the same method that the pKa of free fatty acids in model stratum corneum lipid mixtures was in the range 6.2-7.3 increasing with the preponderance of oleic acid over palmitic acid. Finally the pKa of the carboxyl group of phosphatidylserine was shifted from 4.6 in the pure phospholipid to 2.1 and 2.2 in the presence of equimolar sphingosine and stearylamine, respectively, as a consequence of electrostatic interactions.