Phase separation in bimolecular mixed lipid membranes induced by polylysine

We demonstrate, for the first time, polylysine-induced phase separation in a bimolecular lipid membrane of a lecithin/phosphatidylglycerol-mixture by analysing the single channel current fluctuations of gramicidin. The bimodal conductance histograms are direct evidence for the incorporation of the transport system into the two coexisting phases of different composition.     

Phospholipidosis in the alveolar macrophage induced by cationic amphiphilic drugs

The administration of a number of cationic amphiphilic drugs to certain species of laboratory animals results in a phospholipid storage disorder in the lungs. The alveolar macrophage (AM) shows a pronounced response to drug treatment. The most thorough quantification of this response has occurred after chlorphentermine treatment of rats. There is a striking increase in the accumulation of AMs in the alveolar spaces. The accumulated cells are very heterogeneous in size with many being larger than AMs from untreated rats. Cells are present that have a volume 10 times larger than normal AMs. The hypertrophic AMs show striking ultrastructural changes. The cells become engorged with lamellar inclusions, which may give rise to larger quantities of granular or membranous material. The affected AMs show an increase in total phospholipid content, and there is a good correlation between the size of the AM and its level of phospholipid. The phospholipidosis is reversible after termination of drug treatment; however, the above-mentioned changes do not return to control levels at the same time.     

DNA condensation by an oxidizable cationic detergent. Interactions with lipid vesicles.

Cationic amphiphile-mediated delivery of plasmid DNA is the non-viral gene transfer method most often used. In the present work, we considered a new cysteine-detergent, ornithinyl-cysteinyl-tetradecylamide (C(14)-CO), able to convert itself, via oxidative dimerization, into a cationic cystine-lipid. By using fluorescence techniques, we first characterized the structure of complexes of plasmid DNA with C(14)-CO molecules either kept as monomers, or oxidized into dimers. Both forms are able to condense DNA, with the formation of hydrophobic micelle-like domains along the DNA chain. Domains with a larger molecular order were obtained with dimeric C(14)-CO/DNA complexes. In a second step, the interactions of these complexes with lipid vesicles considered as membrane models were investigated. In the presence of vesicles, we observed a decondensation of the DNA involved in complexes obtained with C(14)-CO monomers. With anionic vesicles, the DNA is released into the bulk solution, while with neutral vesicles, it remains bound to the vesicles via electrostatic interactions with inserted C(14)-CO molecules. In sharp contrast, the complexes with C(14)-CO dimers are unaffected by the addition of either neutral or anionic vesicles and show no interaction with them. These results may partly explain the low transfection efficiency of these complexes at the +/-charge ratios used in this study.     

Functional impairment induced by lipophilic cationic compounds on mitochondria

Lipophilic cations, such as rhodamine 123, have selective anticarcinoma activity both in epithelial-derived tumor cells and in tumor cells injected into mice. The mechanism by which rhodamine 123 and safranin have their effect on mitochondrial function was examined. Rhodamine 123 and safranin inhibit the stimulation of mitochondrial respiration by ADP in a similar concentration range. This inhibition occurs whether the mitochondria are respiring on succinate as a substrate or on ascorbate plus tetramethylphenylenediamine. ATP hydrolysis was stimulated twofold by high lipophilic cation concentration. These results demonstrate that rhodamine 123 and safranin affect oxidative phosphorylation in a similar fashion.     

Potassium ion efflux induced by cationic compounds in yeast

Potassium efflux in yeast induced by several cationic compounds showed different characteristics. All of the observed efflux required glucose as substrate at the concentrations used. For most of them, the phenomenon required binding of the cationic compound to the cell surface and increased with the negative cell surface charge, and for all the compounds tested, it depended on a metabolizable substrate. Efflux induced with terbium chloride appeared more likely due to the function of a K+/H+ antiporter. With DEAE-dextran and dihydrostreptomycin, potassium efflux was dependent on the cell potassium content and was also sensitive to osmotic changes of the medium. DEAE-dextran-provoked efflux was not due to cell disruption. Dihydrostreptomycin seemed to activate a potassium efflux system which could not be studied in isolation, but its inhibition of potassium uptake may also be involved. Except for cells treated with ethidium bromide, no appreciable cell disruption was observed. The potassium efflux observed appears to be a membrane phenomenon reversible after washing with magnesium chloride.     

Raman and fluorescence imaging of phospholipidosis induced by cationic amphiphilic drugs in endothelial cells

Cationic amphiphilic drugs (CADs) are known from lysosomotropism, drug-induced phospholipidosis (DIPL), activation of autophagy, and decreased cell viability, but the relationship between these events is not clear and little is known about DIPL in the endothelium. In this work, the effects of fluoxetine, amiodarone, clozapine, and risperidone on human microvascular endothelial cells (HMEC-1) were studied using a combined methodology of label-free Raman imaging and fluorescence staining. Raman spectroscopy was applied to characterize biochemical changes in lipid profile and their distribution in the cellular compartments, while fluorescence staining (LysoTracker, LipidTOX, LC3B, and JC-1) was used to analyze lysosome volume expansion, activation of autophagy, lipid accumulation, and mitochondrial membrane depolarization. We demonstrated that fluoxetine, amiodarone, and clozapine, but not risperidone, at non-toxic concentrations induced lipid accumulations in the perinuclear and cytoplasmic regions of endothelial cells. Spectroscopic markers of DIPL included a robust increase in the ratio (lipid/(protein + lipid)), an increase in choline-containing lipid, fatty acids, and the presence of cholesterol esters, while starvation-induced activated autophagy revealed a spectroscopic signature associated with subtle changes in the lipid profile only. Interestingly, lysosomal volume expansion, occurrence of DIPL, and activation of autophagy induced by selected CADs all depended on drug-accumulation in acidic pH of lysosome cellular compartments whereas reduced endothelial viability did not, and was attributed to mitochondrial mechanisms as evidenced by a decreased mitochondrial transmembrane potential. In conclusion, drug-induced phospholipidosis in the endothelium did not reduce endothelial viability per se and can be efficiently assayed by Raman imaging.     

Polymorphism of actin paracrystals induced by polylysine

We describe a method for the induction of different polymorphic forms of actin filament paracrystals. This polymorphism is probably based on differences in the stagger and/or polarity of adjacent filaments in single-layered paracrystals and by superposition of different layers in multilayered paracrystals. The helical parameters defining the filament geometry are indistinguishable for the different polymorphic forms observed and for the four different actins used. Analysis of these paracrystals, some of which are ordered to better than 2.5 nm, should provide a reference structure suitable for alignment and orientation within the actin filament of high resolution models of the actin monomer obtained from crystal data.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Reduction of acid sphingomyelinase activity in human fibroblasts induced by AY-9944 and other cationic amphiphilic drugs

AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride), a cationic amphiphilic drug, caused a rapid, irreversible and dose-dependent reduction of acid sphingomyelinase activity in normal human fibroblasts without changing the activities of other lysosomal hydrolases tested. Examinations of activities against synthetic substrates and of the pH-dependency of sphingomyelinase in the drug-treated cells also suggested that the reduction of activity was specific to acid sphingomyelinase. Such a specific reduction was also found with 12 other cationic amphiphilic drugs, most of which have been shown to be inducers of experimental phospholipidosis in animals and/or cultured cells. These results strongly suggest that acid sphingomyelinase is involved in the process of drug-induced lipidosis. The reduction of acid sphingomyelinase seemed not to be due to direct inhibition by these drugs, a specific loss of the enzyme into the culture medium, the presence of inhibitor in the drug-treated cells, or impaired synthesis of the enzyme. There was no indication that changes in the catalytic properties of the enzyme, or changes in the requirement of detergents for its activity occurred in the cell. These results suggest that AY-9944 and other cationic amphiphilic drugs may cause the reduction of acid sphingomyelinase activity by inducing an increased rate of degradation of the enzyme or by causing an irreversible inactivation via some undetected factor.     

Freeze-fracture studies of cytoplasmic inclusions occurring in experimental lipidosis as induced by amphiphilic cationic drugs.

The ultrastructure of cytoplasmic inclusions, which characterize experimental lipidosis as induced by several amphiphilic cationic drugs, was studied by means of freeze-fracturing and thin-sectioning. Retinal and adrenal tissues of rats chronically treated with high oral doses of chlorphentermine were used. In thin sections the cytoplasmic inclusions, which were previously shown to represent lysosomes overloaded with polar lipids, exhibit lamellated or lattice-like internal patterns. The present freeze-fracture observations are interpreted as to indicate that the lamellated inclusions contain polar lipids in the lamellar phase, whereas those with lattice-like patterns contain polar lipids in a hexagonal phase.     

Conformational transition of DNA induced by cationic lipid vesicle in acidic solution: spectroscopy investigation

The conformational transition of DNA induced by the interaction between DNA and a cationic lipid vesicle, didodecyldimethylammonium bromide (DDAB), had been investigated by circular dichroism (CD) and UV spectroscopy methods. We used singular value decomposition least squares method (SVDLS) to analyze the experimental CD spectra. Although pH value influenced the conformation of DNA in solution, the results showed that upon binding to double helical DNA, positively charged liposomes induced a conformational transition of DNA molecules from the native B-form to more compact conformations. At the same time, no obvious conformational changes occurred at single-strand DNA (ssDNA). While the cationic lipid vesicles and double-strand DNA (dsDNA) were mixed at a high molar ratio of DDAB vesicles to dsDNA, the conformation of dsDNA transformed from the B-form to the C-form resulting in an increase in duplex stability (DeltaT(m)=8+/-0.4 degrees C). An increasing in T(m) was also observed while the cationic lipid vesicles interacted with ssDNA.     

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.     

Biophysical mechanisms of endotoxin neutralization by cationic amphiphilic peptides

Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.     

Gene delivery by cationic lipid vectors: overcoming cellular barriers

Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the efficiency of cationic-mediated gene delivery, although often sufficient for cell biological purposes, runs seriously short from a therapeutics point of view, as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement, there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including lipoplex–cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport into the nucleus. Current work indicates that a major obstacle involves the relative inefficient destabilization of membrane-bounded compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices, entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most susceptible to lipoplex-induced destabilization, thereby allowing the most efficient release of DNA, a minimal requirement for optimizing non-viral vector-mediated transfection. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Premature chromosome condensation induced by electrofusion

Premature chromosome condensation of G1, G2, and S-phase chromosomes has been achieved by the use of electrofusion in the fusion of Chinese hamster ovary (CHO) cells and HeLa cells and CHO cells with human leukocytes. Very high yields of heterokaryons, of over 80%, as well as elimination of adverse effects of chemical and viral fusion agents, facilitated induction of premature chromosome condensation of high quality.     

Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins)

Ceragenins are cationic bile salt derivatives having antimicrobial activity. The interactions of several ceragenins with phospholipid bilayers were tested in different systems. The ceragenins are capable of forming specific associations with several phospholipid species that may be involved with their antimicrobial action. Their antimicrobial activity is lower in bacteria that have a high content of phosphatidylethanolamine. Gram negative bacteria with a high content of phosphatidylethanolamine exhibit sensitivity to different ceragenins that corresponds to the extent of interaction of these compounds with phospholipids, including the ability of different ceragenins to induce leakage of aqueous contents from phosphatidylethanolamine-rich liposomes. A second class of bacteria having cell membranes composed largely of anionic lipids and having a low content of phosphatidylethanolamine are very sensitive to the action of the ceragenins but they exhibit similar minimal inhibitory concentrations with most of the ceragenins and for different strains of bacteria. Although Gram negative bacteria generally have a high content of phosphatidylethanolamine, there are a few exceptions. In addition, a mutant strain of Escherichia coli has been made that is essentially devoid of phophatidylethanolamine, although 80% of the lipid of the wild-type strain is phosphatidylethanolamine. Furthermore, certain Gram positive bacteria are also exceptions in that they can have a high content of phosphatidylethanolamine. We find that the antimicrobial action of the ceragenins correlates better with the content of phosphatidylethanolamine in the bacterial membrane than whether or not the bacteria has an outer membrane. Thus, the bacterial lipid composition can be an important factor in determining the sensitivity of bacteria to antimicrobial agents.     

Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins)

Ceragenins are cationic bile salt derivatives having antimicrobial activity. The interactions of several ceragenins with phospholipid bilayers were tested in different systems. The ceragenins are capable of forming specific associations with several phospholipid species that may be involved with their antimicrobial action. Their antimicrobial activity is lower in bacteria that have a high content of phosphatidylethanolamine. Gram negative bacteria with a high content of phosphatidylethanolamine exhibit sensitivity to different ceragenins that corresponds to the extent of interaction of these compounds with phospholipids, including the ability of different ceragenins to induce leakage of aqueous contents from phosphatidylethanolamine-rich liposomes. A second class of bacteria having cell membranes composed largely of anionic lipids and having a low content of phosphatidylethanolamine are very sensitive to the action of the ceragenins but they exhibit similar minimal inhibitory concentrations with most of the ceragenins and for different strains of bacteria. Although Gram negative bacteria generally have a high content of phosphatidylethanolamine, there are a few exceptions. In addition, a mutant strain of Escherichia coli has been made that is essentially devoid of phophatidylethanolamine, although 80% of the lipid of the wild-type strain is phosphatidylethanolamine. Furthermore, certain Gram positive bacteria are also exceptions in that they can have a high content of phosphatidylethanolamine. We find that the antimicrobial action of the ceragenins correlates better with the content of phosphatidylethanolamine in the bacterial membrane than whether or not the bacteria has an outer membrane. Thus, the bacterial lipid composition can be an important factor in determining the sensitivity of bacteria to antimicrobial agents.