Differential activity of lytic α-helical peptides on lactobacilli and lactobacilli-derived liposomes.

Eukaryotic antimicrobial peptides (AMPs) interact with plasma membrane of bacteria, fungi and eukaryotic parasites. Noteworthy, Lactobacillus delbrueckii subsp. lactis (CIDCA 133) and L. delbrueckii subsp. bulgaricus (CIDCA 331) show different susceptibility to human beta-defensins (ß-sheet peptides). In the present work we extended the study to α-helical peptides from anuran amphibian (Aurein 1.2, Citropin 1.1 and Maculatin 1.1). We studied the effect on whole bacteria and liposomes formulated with bacterial lipids through growth kinetics, flow cytometry, leakage of liposome content and studies of peptide insertion in lipid monolayers. Growth of strain CIDCA 331 was dramatically inhibited in the presence of all three peptides and minimal inhibitory concentrations were lower than those for strain CIDCA 133. Flow cytometry revealed that AMPs lead to the permeabilization of bacteria. In addition, CIDCA 331-derived liposomes showed high susceptibility, leading to content leakage and structural disruption. Accordingly, peptide insertion in lipid monolayers demonstrated spontaneous interaction of AMPs with CIDCA 331 lipids. In contrast, lipids monolayers from strain CIDCA 133 were less susceptible. Summarizing we demonstrate that the high resistance of the probiotic strain CIDCA 133 to AMPs extends to α helix peptides Aurein, Citropin and Maculatin. This behavior could be ascribed in part to differences in membrane composition. These findings, along with the previously demonstrated resistance to ß defensins from human origin, suggest that strain CIDCA 133 is well adapted to host innate immune effectors from both mammals and amphibians thus indicating conserved mechanisms of interaction with key components of the innate immune system.

Mixed lipid aggregates containing gangliosides impose different 2H-NMR dynamical parameters on water environment depending on their lipid composition.

Water dynamics in samples of ceramide tetrasaccharide (Gg4Cer) vesicles and GM1 ganglioside micelles at 300:1 water/lipid mole ratio were studied by using deuterium nuclear magnetic resonance (2H-NMR). GM1 imposes a different restriction on water dynamics that is insensitive to temperatures either above or below its phase transition temperature or below the freezing point of water. The calculated correlation times are in the range of 10(-10) s, typical of water molecules near to the polar groups. Pure GM1 micelles have two distinct water microenvironments dynamically characterized. Their dynamic parameters remain constant with temperature ranging from -18 to 32 degrees C, but the amount of strongly associated water is modified. By contrast, a mixture of single soluble carbohydrates corresponding to GM1 polar head group does not preserve the dynamic parameters of water hydration when the temperature is varied. Incorporation of cholesterol or lysophosphatidylcholine into GM1 micelles substantially increases the mobility of water molecules compared with that found in pure GM1 micelles. The overall results indicate that both the supramolecular organization and the local surface quality (lipid-lipid interaction) strongly influence the interfacial water mobility and the extent of hydration layers in glycosphingolipid aggregates.

A simple method to obtain a covalent immobilized phospholipase A(2).

In the present work, we obtained an immobilized phospholipase A(2) system through covalent coupling by using an acrylic polymer Eupergit C as support. The immobilized enzyme from cobra venom (Naja naja naja) showed good retention activity and excellent stability. Both properties are of great importance for biomedical applications such as hypercholesterolemia treatments.

Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

Cholesterol-induced alterations of the packing properties of gangliosides: an EPR study.

The effect of cholesterol (Chol) on two kinds of glycolipid assemblies, one composed of monosialogangliosides (GM1a) and the other formed by a natural mixture of bovine brain gangliosides (TBG), has been analysed. The experimental approach involves spin label electron paramagnetic resonance (EPR) in aqueous lipid dispersions. The employment of a hydrosoluble spin label and a 'quencher' of the EPR signal that is not able to permeate lipid interfaces, allowed us to conclude that GM1a/Chol mixtures give rise to vesicles at Chol proportions for which TBG/Chol mixtures form micelles. The use of different liposoluble spin labels reveals that cholesterol produces a straightening of the hydrocarbon chains in both lipid systems. In GM1a/Chol mixtures, this feature is more pronounced and it is coupled with a decrease in polarity at the chain ends.

The effect of phospholipase A(2) immobilization upon calcium interaction: a kinetic study.

In this work we studied the effect of Ca(2+) on the ability of immobilized PLA(2) to hydrolyze phospholipid substrates either in aggregate or monomeric forms. We use a kinetic methodology for the determination of dissociation constants of soluble and immobilized PLA(2)-Ca(2+) complexes. This approach allows us to obtain the values of the dissociation constants of enzyme-Ca(2+) (K(x)) and enzyme-Ca(2+)-substrate (K'(x)) complexes from the kinetic data obtained at different substrate and Ca(2+) concentrations. Results using mixed micelles of phospholipid-Triton X-100 showed that, in most cases, productive complexes were destabilized by immobilization of PLA(2). However, a correct analysis of the interaction must be independent of the classical modes of PLA(2) action toward lipid surfaces. Thus, a substrate in monomeric form was also employed to analyze the effect of immobilization on hydrolysis rate in the absence of interfacial activation. Kinetic data showed that the immobilization affected severely the mode of PLA(2) action. The kinetic data also suggested that immobilization promoted conformational alterations in the Ca(2+)-binding site, destabilizing the productive complex enzyme-Ca(2+)-phospholipid.

A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties.

Although 6-lauroyl-2-(N,N-dimethylamino)naphthalene (LAURDAN) is now widely used as a probe for lipid systems, most studies focus on the effect of the lipid environment on its emission properties but not on the excitation properties. The present study is intended to investigate the excitation properties of LAURDAN in diverse lipid environments. To this end, the fluorescence properties of LAURDAN were studied in synthetic ester and ether phosphatidylcholines and sphingomyelin vesicles below, at and above the corresponding lipid main phase-transition temperature. The excitation spectra of LAURDAN in these environments always show at least two well-resolved bands. In the different lipid vesicles the behavior of the red band in the LAURDAN excitation spectra is sensitive to the lipid chemical environment near the probe fluorescent moiety and to the packing of the different lipid phases (gel and liquid crystalline). We propose that the interaction between the LAURDAN dimethylamino group and the ester linkage of ester phospholipids is responsible for the strong stabilization of LAURDAN's red excitation band in the gel phase of ester phospholipid vesicles. We discuss the consequence of these proposed ground-state interactions on LAURDAN's emission generalized polarization function. In the context of variable excitation wavelengths, information concerning solvent dipolar relaxation through excitation generalized polarization function is also discussed.

Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.

Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.

Extremely high thermal stability of streptavidin and avidin upon biotin binding.

The effect of biotin binding on the thermal stability of streptavidin (STV) and avidin (AVD) was evaluated using differential scanning calorimetry. Biotin binding increases the midpoint of temperature Tm of thermally induced denaturation of STV and AVD in phosphate buffer from 75 and 83 degrees C to 112 and 117 degrees C at full biotin saturation, respectively. This thermostability is the highest reported for proteins coming from either mesophilic or thermophilic organisms. In both proteins, biotin also increases the calorimetric enthalpy and the cooperativity of the unfolding. Thermal stability of STV was also evaluated in the presence of high concentrations of urea or guanidinium hydrochloride (GuHCl). In 6 M GuHCl, STV remains as a tetramer and the Tm of the STV-biotin complex is centered at 108 degrees C, a few degrees below the value obtained in phosphate buffer. On the contrary, STV under fully saturating condition remains mainly in its dimeric form in 8 M urea and the thermogram shows two endotherms. The main endotherm at a lower temperature has been ascribed to the dimeric liganded state with a Tm of 87 degrees C, and the higher temperature endotherm to the tetrameric liganded form with a Tm of 106 degrees C. As the thermostability of unliganded protein in the presence of urea is unchanged upon binding we related the extremely high thermal stability of this protein to both an increase in structural ordering and compactness with the preservation of the tetramer integrity.

Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence.

We have characterized the fluorescence properties of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN) in pure interfaces formed by sphingomyelin and 10 chemically related glycosphingolipids (GSLs).1 The GSLs contain neutral and anionic carbohydrate residues in their oligosaccharide chain. These systems were studied at temperatures below, at, or above the main phase transition temperature of the pure lipid aggregates. The extent of solvent dipolar relaxation around the excited fluorescence probe in the GSLs series increases with the magnitude of the glycosphingolipid polar headgroup below the transition temperature. This conclusion is based on LAURDAN's excitation generalized polarization (GPex) and fluorescence lifetime values found in the different interfaces. A linear dependence between the LAURDAN GPex and the intermolecular spacing among the lipid molecules was found for both neutral and anionic lipids in the GSLs series. This relationship was also followed by phospholipids. We conclude that LAURDAN in these lipid aggregates resides in sites containing different amounts of water. The dimension of these sites increases with the size of the GSLs polar headgroup. The GP function reports on the concentration and dynamics of water molecules in these sites. Upon addition of cholesterol to Gg4Cer, the fluorescence behavior of LAURDAN was similar to that of pure cerebrosides and sphingomyelin vesicles. This observation was attributed to a change in the interfacial hydration as well as changes in the shape and size of the Gg4Cer aggregates in the presence of cholesterol. After the addition of cholesterol to gangliosides, the changes in the LAURDAN's spectral parameters decrease progressively as the polar headgroup of these lipids becomes more complex. This finding suggests that the dehydration effect of cholesterol depends strongly on the curvature radius and the extent of hydration of these lipid aggregates. In the gel phase of phrenosine, GalCer, Gg3Cer, sulfatide, and sphingomyelin, the excitation red band (410 nm) of LAURDAN was reduced with respect to that of LAURDAN in the gel phase of pure phospholipids. This observation indicates a local environment that interacts differently with the ground state of LAURDAN in GSLs when compared with LAURDAN in phospholipids.

Evidence of a strong interaction of 2,4-dichlorophenoxyacetic acid herbicide with human serum albumin.

The interaction of 2,4-dichlorophenoxyacetic acid herbicide (2,4-D) with human serum albumin (HSA) was studied using fluorescence and differential scanning calorimetry (DSC). Fluorescence displacement of 1-anilino-8-naphtalenesulfonate (ANS) bound to HSA was used to evaluate the binding affinity of 2,4-D to HSA. The binding is associated to a high affinity site of HSA located in the IIIA subdomain. The association constant (Kass) of the herbicide was about 5 microM(-1), several times higher than the affinity found for pharmaceutical compounds. This relatively strong interaction with HSA was evidenced by the increase in HSA protein thermostability induced as consequence of herbicide interaction. 2,4-D induces an increase in the midpoint of thermal denaturation temperature from 60.1 degrees C in herbicide free solution to 75.6 degrees C in full ligand saturating condition. The calorimetric enthalpy and the excess heat capacity also increased upon 2,4-D binding. To investigate the possibility of other/s system/s of 2,4-D transport in blood, besides of HSA, the interaction of the herbicide with lipid monolayers was explored. No interaction was detected with any of the lipids tested. The overall results provided evidence that high affinity 2,4-D-HSA complex exhibits enhanced thermal stability and that HSA is the unique transport system for 2,4-D in blood.

High-density lipoprotein from hypercholesterolemic animals has peroxidized lipids and oligomeric apolipoprotein A-I: its putative role in atherogenesis.

Oxidized lipoproteins have been involved in the pathogenesis of atherosclerosis and atherosclerotic lesions contain oxidized low density lipoprotein. Conversely, the presence of oxidized high density lipoprotein (HDL) in vivo has not been clearly established. Oxidation of HDL in vitro models produces an increase in peroxidized lipids and the appearance of apolipoprotein A-I (apo A-I) oligomers. We investigated the oxidative status of HDL in an in vivo model: the hypercholesterolemic chicken. The HDLs from control and hyperlipemic animals were analyzed for the content of lipid peroxides employing spectroscopic and fluorescence techniques, for the level of apo A-I oligomerization, and for susceptibility to in vitro oxidation. HDL from hypercholesterolemic chickens was more peroxidized (as detected by fluorescence), had higher amount of oligomeric apo A-I, and was oxidized to a greater extent by uv irradiation than that of control animals. We speculate that apo A-I oligomerization could be a key step in the atheroma formation.

A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarará chica) venom with novel kinetic and chromatographic properties.

A new phospholipase A2 isoform, called P-3, isolated from Bothrops neuwiedii (Yarará chica) venom, showed different chromatographic, enzymatic and cytotoxic properties compared to the previously purified isoforms P-1 and P-2 but it had a similar edema-inducing activity. In contrast to previously reported B. neuwiedii phospholipase A2 isoforms, P-3 did not interact with the oligosaccharide matrix of gel filtration columns (Superose, Superdex). Its molecular weight was 15,000 and its N-terminal 14 amino acid sequence was Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an unique histidine, presumably located at the active site, because a full inhibition of enzymatic activity was observed after treatment with p-bromophenacyl bromide. The new isoform also differentiated in its surface pressure activity profile when assayed in lipid monolayers. P-3 had an optimum activity towards dilauroylphosphatidylcholine monolayers of 27 mN/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an optimum of 13 mN/m with a cut-off of 22 mN/m. P-3 retained its edema-inducing activity in the absence of hydrolytic activity, suggesting that the inflammatory activity was not dependent on the enzymatic activity. Neither the enzymatic nor the edema-inducing activity was affected by heparin. The new isoform was not lethal when a single dose of 5 micrograms/g body weight was injected intraperitoneally into mice. All of the isoforms displayed cytotoxic activity in vitro on B16F10 melanoma cells evaluated by direct MTT assay, with an EC50 of 31 micrograms/ml for P-3 and of 15 micrograms/ml for P-1 and P-2. The cytotoxic activity of P-3 was inhibited by p-bromophenacyl bromide treatment of the enzyme (up to 170 micrograms/ml), whereas the same treatment on P-1 and P-2 changed their EC50 to 60 micrograms/ml. The difference observed with inhibited enzymes suggests a different mechanism for the cytotoxic action of P-3 with respect to P-1 and P-2.

Ganglioside hydration study by 2H-NMR: dependence on temperature and water/lipid ratio.

Dynamic properties of 2H2O in samples of ganglioside aggregates hydrated at water/lipid ratios ranging from 25:1 to 8000:1 mole/mole were studied by using deuterium nuclear magnetic resonance (2H-NMR). We present a physical model for the interpretation of the measured spin-spin relaxation times (T2). For all the concentrations studied the model provides evidence for the existence of at least two kinds of water environments: one in which the rotational correlation time is in the range of 10(-9) to 10(-8) s, and a second in which it lies between 10(-11) to 10(-10) s. A detailed study on the temperature dependence was performed for two of the concentrations, one corresponding to the hexagonal phase (100:1 mole/mole) and the other involving a micellar phase (200:1 mole/mole). In the 100:1 2H2O/ganglioside molar ratio sample, most of the water is tightly bound to long cylindrical structures. For the 200: 1 sample, there are on average approximately 30 water molecules tightly bound to the polar head group of each ganglioside molecule. The relative number and dynamics of molecules in this environment are essentially insensitive to temperature variations in the range 220-300K The rest of water molecules are also influenced by the aggregate, having a different mobility from that observed in the free liquid state.

Laurdan properties in glycosphingolipid-phospholipid mixtures: a comparative fluorescence and calorimetric study.

Laurdan (6-dodecanoyl-2-dimethylamine-naphthalene) is a fluorescent membrane probe of recent characterization. It was shown that this probe discriminates between phase transitions, phase fluctuations and the coexistence of phase domains in phospholipid multilamellar aggregates. We measured the excitation and emission generalized polarization (GP(ex) and GP(em)) of Laurdan in aggregates of complex glycosphingolipids in their pure form and in mixtures with dipalmitoylphosphatidylcholine (DPPC). Our results show that Laurdan detects the broad main phase transition temperature of the neutral ceramide-tetrasaccharide Gg(4)Cer (asialo-G(M1)) and shows a value of GP(ex) in between that of DPPC and that of ganglioside G(M1). In contrast, Laurdan was unable to detect the thermotropic phase transition of G(M1). The probe also appears to be unable to detect phase coexistence in both types of pure glycolipid aggregates. Deconvolution of the excess heat capacity vs. temperature curves of pure Gg(4)Cer and DPPC/Gg(4)Cer mixtures indicates that the thermograms are composed by different transition components. For these cases, Laurdan detects only the high cooperativity component of the transition of the mixture. The peculiar behaviour of Laurdan in aggregates containing complex glycosphingolipids may result from the inherent topological features of the interface that are conferred by the bulky and highly hydrated polar head group of these lipids.

Interaction of biotin with streptavidin. Thermostability and conformational changes upon binding.

The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka approximately 10(15) reproduce the behavior observed, but the estimated values of Tm are 15-20 degrees C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.

Inhibition by gangliosides of Bacillus cereus phospholipase C activity against monolayers, micelles and bilayer vesicles.

The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.

Dual inhibitory effect of gangliosides on phospholipase C-promoted fusion of lipidic vesicles.

The effect of a variety of gangliosides has been tested on the phospholipase C-induced fusion of large unilamellar vesicles. Bilayer composition was phosphatidylcholine:phosphatidylethanolamine: cholesterol (2:1:1 mole ratio) plus the appropriate amounts of glycosphingolipids. Enzyme phosphohydrolase activity, vesicle aggregation, mixing of bilayer lipids and mixing of liposomal aqueous contents were separately assayed. Small amounts ( < 1 mol %) of gangliosides in the lipid bilayer produce a significant inhibition of the above processes. The inhibitory effect of gangliosides increases with the size of the oligosaccharide chain in the polar head group. Inhibition depends in a nonlinear manner on the ganglioside proportion, and is complete at approximately 5 mol %. Inhibition is not due to ganglioside-dependent changes in vesicle curvature or size. Ganglioside inhibition of vesicle fusion is due to two different effects: inhibition of phospholipase C activity and stabilization of the lipid lamellar phase. Enzyme inhibition leads to a parallel decrease of vesicle aggregation and lipid mixing rates. Mixing of aqueous contents, though, is depressed beyond the enzyme inhibition levels. This is explained in terms of the fusion pore requiring a local destabilization of the lipid bilayer, the lamellar structure being stabilized by gangliosides. 31P-NMR and DSC experiments confirm the inhibitory effect of gangliosides in various lamellar-to-nonlamellar transitions.

Interaction of alpha-MSH and substance P with interfaces containing gangliosides.

In the present work we studied the interaction of alpha-MSH and substance P neuropeptides with gangliosides using lipid monolayers, fluorescence spectroscopy, and differential scanning calorimetry techniques. The positively charged weak amphiphilic neuropeptides did not show surface activity in the range of concentrations tested (0.1-0.3 muM), but they were preferentially able to penetrate monolayers formed by acidic lipids, showing the best interaction with the more complex gangliosides. The general order of interaction found for both peptides is GTIh > GDIa = GMI > DLPA > sulphatide. Neither neuropeptide interacted with phosphatidylcholine monolayers above 10 mN.m-1. The binding of alpha-MSH to GMI micelles followed by changes in the fluorescence of its tryptophan residue takes place with an increase in the hydrophobic environment of the neuropeptide. An apparent dissociation constant of 13 muM was estimated for this process. Similar result was found with GMI:DMPC vesicles (1:10 molar ratio). The thermotropic profile of GMI micelles is modified in the presence of the neuropeptides. The calorimetric enthalpy of GMI transition increased 21% and 37% in the presence of alpha-MSH and substance P, respectively. Both neuropeptides induced the same increment in the transition temperature Tm from 19 to 20.5 degrees C. The basic physicochemical studies herein indicated that both positively charged neuropeptides, alpha-MSH and substance P, interact with interfaces containing gangliosides in a mainly electrostatic form, whereas the hydrophobic interaction seems to play a secondary role.