Presence Of Liposomes Research Articles

A pH-induced dissociation of the dimeric form of a lysine 49-phospholipase A2 abolishes Ca2+-independent membrane damaging activity.

The hydrolysis of phospholipids by class II phospholipase A2 (PLA2) involves a Ca2+ ion cofactor bound to the Asp49 residue in the active site region. In the lysine 49 phospholipase A2 homologues (Lys49-PLA2), the Asp49 residue is substituted by Lys, and consequently the Lys49-PLA2s show no Ca2+ binding and no detectable phospholipid hydrolysis. Nevertheless, the Lys49-PLA2s demonstrate membrane damaging activity by an incompletely understood Ca2+-independent mechanism of action. Using a combination of steady-state and time-resolved fluorescence techniques, we have examined the effect of pH on the monomer-dimer equilibrium of bothropstoxin I (BthTX-I), a Lys49-PLA2 from the venom of Bothrops jararacussu which contains a single Trp77 residue located at the dimer interface. At pH 5.0, we observe a decreased quantum yield, a decreased rotational correlation time, and an increased bimolecular quenching rate constant with iodide. These results are consistent with a pH-induced dissociation of the BthTX-I dimer, with the consequent exposure of the Trp77 residue to aqueous solvent. In the presence of liposomes, membrane damaging activity is observed only under conditions in which the dimeric form of the BthTX-I is favored. These results demonstrate that the dimeric form of the protein is essential for the initiation of the Ca2+-independent membrane damaging activity.

Effect of divalent cations on the ATPase activity of Escherichia coli SecA

It was found that Ca 2+ stimulates the intrinsic SecA ATPase activity in the absence as well as in the presence of liposome. On the other hand, Mg 2+, the general cofactor for ATPase, did not affect the intrinsic SecA ATPase but reduced the portion of ATPase activity enhanced by Ca 2+. The enhancement of SecA ATPase activity correlated well with the increase in 8-anilino-1-naphthalene-sulfonic acid binding of SecA, suggesting that increased exposure of hydrophobic residues stimulates the enzyme activity.

Determination by fluorimetric titration of the ionization constants of ciprofloxacin in solution and in the presence of liposomes.

A fluorescence titration method was applied for the determination of pKa of ciprofloxacin (CPX) in solution. Values of 6.18 +/- 0.05 and 8.76 +/- 0.03 were obtained for pKa1 and pKa2, respectively. The method was used to determine the ionization constants in the presence of liposomes of dipalmitoylphosphatidylcholine (DPPC) and DPPC with 10 mol% of dipalmitoylphosphatidylglycerol. A dependence on the surface charge of liposomes was found which supported the existence of a basic electrostatic interaction between CPX and the phospholipid bilayer. Both pK values for the N-4 butyl-piperazinyl derivative (BCPX) of the parent compound were also determined in solution and in the presence of liposomes. The competition of both drugs for the same binding site as 1-anilino-8-naphtalene sulfonate demonstrate that the interaction is governed by electrostatic forces.

Bioflavonoid rescue of ascorbate at a membrane interface.

In aqueous solution, ascorbate potently prevents bleaching of cytochrome c on exposure to excess H2O2 or t-butyl hydroperoxide. Ascorbate failed to protect cytochrome c in the presence of liposomes of mitochondrial membranelike composition. Like the redox mediator N,N,N,'N'-tetramethyl-p-phenylenediamine (TMPD), however, the bioflavonoids epicatechin and quercetin restored the protection afforded by ascorbate in the presence of liposomes and gave further protection. The quercetin glycoside, rutin, was much less effective, as was the vitamin E analog Trolox. In the presence of liposomes, quercetin alone was relatively ineffective, but cooperated with ascorbate to extend protection synergistically. The results bear specific implications in antioxidant protection of cytochrome c and in moderation of its hydroperoxidase activities in biological membranes. The data also reveal a situation where ascorbate is without effect except in the presence of a bioflavonoid, and substantiate a possibly vital role for certain bioflavonoids in mediating electron transfer from ascorbate into a hydrophobic environment.

Low pH-induced Conformational Changes in Vesicular Stomatitis Virus Glycoprotein Involve Dramatic Structure Reorganization

Membrane fusion is the key step in the entry of enveloped animal viruses into their host cells. Fusion of vesicular stomatitis virus with membranes occurs at acidic pH and is mediated by its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the protein from purified virus by incubation with nonionic detergent. At pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of hydrophobic regions of G protein during acidification. Bis-ANS binding was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS binding was observed, probably due to loss of tridimensional structure during the conformational rearrangement. This hypothesis was confirmed by circular dichroism analysis at different pH values, which showed a great decrease in alpha-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure occurs during the fusion reaction. Our results indicate that G protein undergoes dramatic structural changes at acidic pH and acquires a conformational state able to interact with the target membrane.

Application of cationic liposomes containing surfactants to an enhancer in firefly bioluminescent assay of adenosine 5′-triphosphate

The effects of surfactant having single and two alkyl chains as a component of liposomes on the intensity of bioluminescence (BL) from firefly luciferin–luciferase reaction with adenosine 5′-triphosphate (ATP) and on the stability of liposomes were investigated by use of vesicles formed by extrusion technique. Stearyltrimethylammonium chloride (STAC) and distearyldimethylammonium chloride (DSDAC) were used as a surfactant having single and two alkyl chains, respectively. The BL intensity in the presence of liposomes containing DSDAC increased twice compared with those containing STAC. The detection limit for ATP upon using liposomes containing DSDAC was 0.5 pM in aqueous ATP standard solution, and was improved by a factor of 10 compared to that in water alone. Liposomes containing DSDAC were more stable than those containing STAC, and were superior to those containing STAC as an enhancer for the firefly BL assay of ATP.

Phosphatidylserine-mediated phagocytosis of influenza A virus-infected cells by mouse peritoneal macrophages.

Influenza virus induces apoptosis in cultured cell lines as well as in animal tissues. HeLa cells were infected with influenza virus A/Udon/72 (H3N2) under conditions resulting in almost 100% infection. Such cells underwent typical caspase-dependent apoptosis and were efficiently phagocytosed by macrophages prepared from peritoneal fluids of thioglycolate-treated mice. The membrane phospholipid phosphatidylserine appeared on the surfaces of virus-infected cells at around the time efficient phagocytosis became detectable. In fact, the phagocytosis was almost completely inhibited in the presence of liposomes containing phosphatidylserine, which did not influence the antibody-dependent uptake of zymosan particles by the same macrophages. These results indicate that macrophages phagocytose influenza virus-infected HeLa cells in a manner mediated by phosphatidylserine that appears on the surfaces of infected cells during the process of apoptosis.

Characterization of the interaction of IpaB and IpaD, proteins required for entry of Shigella flexneri into epithelial cells, with a lipid membrane.

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the IpaB, IpaC, and IpaD proteins, which are secreted by type III secretion machinery. We report here the purification of IpaB and IpaD and the characterization of their lipid-binding properties as a function of pH. The interaction of IpaB with the membrane was quite independent of the pH whereas that of IpaD took place only at low pH. To support the data obtained with the purified proteins, we designed a system in which protein secretion by live bacteria was induced in the presence of liposomes, thereby allowing interaction of proteins with lipids directly after secretion and bypassing any purification step. In these conditions, both IpaB and IpaC, as well as minor amounts of IpaA and IpgD, were associated with the membrane and the ratio of IpaB to IpaC was modulated by the pH. The relevance of these results with respect to the dual roles of IpaB, IpaC and IpaD in induction of membrane ruffles and lysis of the endosomal membrane is discussed.

GPI-linked proteins do not transfer spontaneously from erythrocytes to liposomes. New aspects of reorganization of the cell membrane.

Exposure of cells to liposomes results in the release of integral membrane proteins. However, it is still controversial whether the release is due to spontaneous protein transfer from cells to liposomes or shed vesicles released from cells. We investigated this issue in an erythrocyte-liposome system by examining the location of acetylcholinesterase (AChE, an integral membrane protein marker), cholesterol (erythrocyte membrane lipid marker), hemoglobin (cytosolic protein marker), and a nonexchangeable lipid marker in liposomes in a sucrose density gradient at high resolution. The density distribution showed that AChE is not transferred to the liposomes but is located on small (about 50 nm) light (10-20 wt % sucrose) or large (about 200 nm) heavy shed vesicles (more than 30 wt % sucrose). AChE in the light shed-vesicle fraction markedly increased even after its level in the heavy fraction reached a plateau. AChE was also released from isolated heavy shed vesicles and accumulated in the small light shed-vesicle fraction in the presence of liposomes. After incubation of spherical erythrocytes (morphological index, 5.0) with liposomes, AChE hardly appeared in the heavy shed-vesicle fraction, and the majority (>99%) appeared in the light shed-vesicle fraction, indicating that AChE is released from both the erythrocytes and heavy shed vesicles to the light shed-vesicle fraction, which becomes rich in AChE. Our results demonstrated for the first time that GPI-linked proteins do not spontaneously transfer from erythrocytes to liposomes. Our study also suggests that in vivo GPI-linked membrane proteins do not spontaneously transfer between cell membranes but that some catalyst is needed.

Mechanism of Selective Release of Membrane Proteins from Human Erythrocytes in the Presence of Liposomes

Incubation of erythrocytes with liposomes results in the release of shed vesicles rich in glycosyl-phosphatidylinositol (GPI)-anchored proteins but poor in transmembranous proteins. We investigated the mechanisms of membrane protein polarization by examining the effect of the interaction between spectrin and membrane proteins on the release of a transmembranous protein, band 3, and a GPI-anchored protein, acetylcholinesterase (AChE), from erythrocyte ghosts. Polymerization of spectrin resulted in a 30-fold decrease in the released amount of band 3 per constant amount of shed vesicles but did not affect the amount of released AChE per constant amount of shed vesicles. On the other hand, the amount of released band 3 per constant amount of shed vesicles increased by cleaving the cytoplasmic part of band 3. Our results first demonstrated that the diffusibility of membrane proteins determined by steric hindrance between membrane proteins and protein mesh primarily determines the ease of localization of membrane proteins into shed vesicles. Taken together with the recent biophysical studies, we built a “fence selection model” that retrograding spectrin mesh sweeps diffusing band 3 molecules from the tip of the membrane crenated area toward the entry of the crenated area, but not AChE molecules. Our study describes a novel method for isolation of a large number of vesicles containing special and intact membrane proteins from cells not by using detergents or organic solvents, but by utilizing the fence effect between the cytoskeleton and membrane proteins.

Immobilized liposome chromatography for refolding and purification of protein

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield.

Determination of liposomes drug retention capacity using differential pulse polarography: the case of chlorothiazide

A methodology based on differential pulse polarography (DPP) was developed for the quantitative, non-destructive determination of the entrapped, adsorbed and free chlorothiazide (CHT) in the presence of liposomes. Results were compared to those obtained from the application of conventional procedure and found equally reliable. Discrimination between adsorbed and free CHT and the capability for non-destructive determination of the entrapped drug were among the advantages of DPP methodology over the currently used technique. From the application of DPP to a 1 mg ml −1, 4.5 μm diameter liposome solution, it was found that drug adsorption was dependent on whether or not drug was entrapped in liposomes, it being twice as large for the empty liposomes. The amount of encapsulated CHT was found to be linearly dependent on the drug concentration, up to 100 mg l −1, present during the encapsulation procedure and stabilized to a maximum when larger CHT concentrations were used.

Anti-atherosclerotic activity of the calcium antagonist lacidipine in cholesterol-fed hamsters

We have investigated the activity of the calcium antagonist lacidipine in male hamsters fed an atherogenic diet containing 2% cholesterol and 5% butter. Animals were examined at 14, 20 and 24 weeks of treatment. At 14 weeks, in hamsters fed the atherogenic diet and without lacidipine treatment, there were significant increases in serum levels of total cholesterol, triglycerides and lipoproteins; these values were approximately similar at week 24. Lacidipine treatment at 0.3, 1.0 and 3.0 mg/kg/d did not affect levels of serum cholesterol, triglycerides and lipoproteins. At 24 weeks, in hyperlipidemic hamsters fed the atherogenic diet, the area of the fatty streak in the aortic arch covered a mean area of 375 +/- 145 micron2 x 100, which accounted for 2.7% of the total surface area of the aortic arch. In hamsters fed the atherogenic diet and treated with lacidipine at 0.3, 1.0 and 3.0 mg/kg, at 24 weeks, the surface area of the aortic arch lesion was significantly reduced by 41 to 71%. In the thoracic aorta at 24 weeks, in lacidipine-treated animals, both the incidence and degree of severity of the lesions was reduced, the area of the fatty streak being lowered by 78 to 97% in comparison with non-lacidipine-treated control animals. Ultrastructural examination demonstrated that the early changes in the aorta in hamsters fed the atherogenic diet involved the intima and smooth muscle cells; lacidipine treatment reduced the severity of the intimal lesions significantly. With SEM, lacidipine administration was seen to reduce endothelial irregularity and the presence of crater-like lesions. At TEM, treatment with lacidipine reduced the number of foam cells and the presence of liposomes in the subendothelium. This investigation demonstrates that in the hyperlipidemic hamster, lacidipine treatment decreases atheromatous lesions without lowering serum lipids. It is suggested that lacidipine influences the atherogenic process by an unusual mechanism which may be related to a combination of both the long-lasting calcium antagonism of the drug and significant antioxidant activity.

Solubilization of stratum corneum lipid liposomes by C 14-betaine/sodium dodecyl sulfate mixtures. Influence of the level of ceramides in the solubilization process

The solubilization of liposomes modeling the stratum corneum (SC) lipids by tetradecylbetaine (C 14-Bet)/sodium dodecyl sulfate (SDS) mixtures and the role played by the content of ceramide in this process were studied. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the changes in the static light scattering of the system during solubilization. The fact that the free surfactant concentration was always similar to its CMC indicates that the liposome solubilization was mainly ruled by the formation of mixed micelles. The mole fraction of the zwitterionic component ( X zwitter), 0.4, showed the lowest ability to saturate or solubilize liposomes, whereas that of X zwitter=0.6 exhibited the highest degree of partitioning into liposomes. The presence in liposomes of higher and lower proportion of ceramides than that existing in the SC led to a fall and to a rise in both the activity and the partitioning of a specific surfactant mixture ( X zwitter=0.4) in these bilayer structures. This finding could be related to the recently reported dependencies of the level of ceramides in skin function and barrier abnormalities. Comparison of the present Re and K values with those reported for phosphatidylcholine (PC) liposomes shows that although SC liposomes were more resistant to the action of surfactant mixtures their degree of partitioning into SC bilayers was always higher than that reported for PC liposomes.

Oxidative refolding of lysozyme assisted by negatively charged liposomes: Relationship with lysozyme-mediated fusion of liposomes

Oxidative refolding of denatured/reduced lysozyme was examined in the presence of charged liposomes composed of neutral 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol (POPG). Surface charge density of liposomes had a marked effect on the interaction between liposomes and reduced lysozyme which is observed in the early stage of the refolding. It was found that there was a critical level of surface charge density of liposomes (−0.15 C/nm 2) at which the interaction between liposomes and lysozyme drastically changed. At the surface charge density of liposomes ranging from −0.15 to −1.4 C/nm 2, the interaction between liposomes and lysozyme resulted in aggregate formation. In contrast, at the surface charge density ranging from 0 to −0.15 C/nm 2, no aggregate formation was observed if the lysozyme/liposome molar ratio was less than 600. On the basis of the experimental results, a model for the interaction between charged liposomes and lysozyme was proposed, focusing on the mechanism of protein-induced fusion of charged liposomes as well as protein refolding on liposomes. Then, the optimal condition for oxidative refolding of lysozyme was examined in the presence of charged liposomes by controlling the lysozyme-liposome interaction. The reactivation yield of lysozyme was improved up to 85% in the presence of liposomes with a surface charge density of −0.14 C/nm 2.

Oxidative Refolding of Lysozyme Assisted by Negatively Charged Liposomes. Relationship with Lysozyme-Mediated Fusion of Liposomes.

Oxidative refolding of denatured/reduced lysozyme was examined in the presence of charged liposomes composed of neutral 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoglycerol (POPG). Surface charge density of liposomes had a marked effect on the interaction between liposomes and reduced lysozyme which is observed in the early stage of the refolding. It was found that there was a critical level of surface charge density of liposomes (−0.15 C/nm 2 ) at which the interaction between liposomes and lysozyme drastically changed. At the surface charge density of liposomes ranging from −0.15 to −1.4 C/nm 2 , the interaction between liposomes and lysozyme resulted in aggregate formation. In contrast, at the surface charge density ranging from 0 to −0.15 C/nm 2 , no aggregate formation was observed if the lysozyme/liposome molar ratio was less than 600. On the basis of the experimental results, a model for the interaction between charged liposomes and lysozyme was proposed, focusing on the mechanism of protein-induced fusion of charged liposomes as well as protein refolding on liposomes. Then, the optimal condition for oxidative refolding of lysozyme was examined in the presence of charged liposomes by controlling the lysozyme-liposome interaction. The reactivation yield of lysozyme was improved up to 85% in the presence of liposomes with a surface charge density of −0.14 C/nm 2 .

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria.

Yersinia enterocolitica type III secretion-translocation system: channel formation by secreted Yops.

'Type III secretion' allows extracellular adherent bacteria to inject bacterial effector proteins into the cytosol of their animal or plant host cells. In the archetypal Yersinia system the secreted proteins are called Yops. Some of them are intracellular effectors, while YopB and YopD have been shown by genetic analyses to be dedicated to the translocation of these effectors. Here, the secretion of Yops by Y.enterocolitica was induced in the presence of liposomes, and some Yops, including YopB and YopD, were found to be inserted into liposomes. The proteoliposomes were fused to a planar lipid membrane to characterize the putative pore-forming properties of the lipid-bound Yops. Electrophysiological experiments revealed the presence of channels with a 105 pS conductance and no ionic selectivity. Channels with those properties were generated by mutants devoid of the effectors and by lcrG mutants, as well as by wild-type bacteria. In contrast, mutants devoid of YopB did not generate channels and mutants devoid of YopD led to current fluctuations that were different from those observed with wild-type bacteria. The observed channel could be responsible for the translocation of Yop effectors.

Influence of the level of ceramides on the permeability of stratum corneum lipid liposomes caused by a C 14-alkyl betaine/sodium dodecyl sulfate mixture

The role played by the ceramides in the interaction of a mixture of tetradecylbetaine (C 14-Bet)/sodium dodecyl sulfate (mole fraction of the zwitterionic surfactant, 0.6) with liposomes modeling the stratum corneum (SC) lipid composition was studied. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6)-carboxyfluorescein released from the interior of vesicles. The presence in liposomes of higher and lower ceramide proportions than that existing in the SC lipids led to a fall and to a rise in the sublytic activity of the surfactant mixture on these structures, respectively. However, the surfactant mixture partitioning into liposomes (or affinity with these bilayer structures) increased as the proportion of ceramides type III (Cer) increased to achieve the highest value for a Cer proportion similar to that in the SC lipids (about 40%). Thus, at low Cer proportions, the ability of the surfactant mixture to alter the permeability of these bilayer structures was higher than that for liposomes approximating the SC lipid composition, despite their reduced partitioning into liposomes. These findings are in agreement with the recently reported dependencies of the level of ceramides in skin lipids and function barrier abnormalities, and could explain, in part, these dependencies. The fact that the free surfactant concentration needed to achieve the interaction levels investigated was lower than the surfactant mixture critical micellar concentration indicates that permeability alterations were mainly ruled by the action of surfactant monomers, regardless of the liposome lipid composition.

A chemically synthesized version of the insect antibacterial glycopeptide, diptericin, disrupts bacterial membrane integrity.

Insects protect themselves against bacterial infection by secreting a battery of antimicrobial peptides into the hemolymph. Despite recent progress, important mechanistic questions, such as the precise bacterial targets, the nature of any cooperation that occurs between peptides, and the purpose of multiple peptide isoforms, remain largely unanswered. We report herein the chemical synthesis and preliminary mechanistic investigation of diptericin, an 82 residue glycopeptide that contains regions similar to two different types of antibacterial peptides. A revised, highly practical synthesis of the precursor N(alpha)-Fmoc-Thr(Ac(3)-alpha-D-GalNAc) allowed us to produce sufficient quantities of the glycopeptide for mechanistic assays. The synthetic, full-length polypeptide proved to be active in growth inhibition assays with an IC(50) of approximately 250 nM, a concentration similar to that found in the insect hemolymph. Biological analysis of diptericin fragments indicated that the main determinant of antibacterial activity lay in the C-terminal region that is similar to the attacin peptides, although the N-terminal segment, related to the proline-rich family of antibacterial peptides, augmented that activity by 100-fold. In all assays, activity appeared glycosylation independent. Circular dichroism of unglycosylated diptericin indicated that the peptide lacked structure both in plain buffer and in the presence of liposomes. Diptericin increased the permeability of the outer and inner membranes of Escherichia coli D22 cells, suggesting possible mechanisms of action. The ability to access glycopeptides of this type through chemical synthesis will facilitate further mechanistic studies.