Liposome Model System Research Articles

Liposomes as Target Membranes in the Study of Virus Receptor Interaction and Membrane Fusion

Liposomes represent an excellent model system for the study of virus-receptor interactions and viral membrane fusion processes. The liposomal model system involving lipid-conjugated heparin, described in this chapter, provides a useful tool for the study of virus-receptor interactions. Because many viruses have the capacity to interact with heparan sulfate (HS), the system must be adapted to the study of viruses other than alphaviruses. The assay methods for studying fusion of enveloped viruses with liposomes lacking a specific protein or carbohydrate receptor, described in the chapter, help in examining the role of lipids in the binding and fusion of alphaviruses with target membranes, as well as the pH dependence and the kinetics and extents of those processes. The methods can be readily adapted to the study of fusion of other enveloped viruses, such as flaviviruses. The stringency of the assay methods, including membrane lipid-mixing, as well as content-mixing assays, ensures that the virus-liposome fusion reactions observed represent genuine processes of membrane merging.

Adaptation of alphaviruses to heparan sulfate: interaction of Sindbis and Semliki forest viruses with liposomes containing lipid-conjugated heparin.

Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system involving target liposomes containing lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HS-adapted SIN to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HS-adapted SIN underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at low pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses.

Inhibition of lipid peroxidation and structure-activity-related studies of the dietary constituents anthocyanins, anthocyanidins, and catechins.

The antioxidant activities of a series of commonly consumed and biogenetically related plant phenolics, namely, anthocyanidins, anthocyanins, and catechins, in a liposomal model system have been investigated. The antioxidant efficacies of the compounds were evaluated on their abilities to inhibit the fluorescence intensity decay of an extrinsic probe, 3-[p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid, caused by free radicals generated during metal ion-induced peroxidation. Distinct structure-activity relationships were revealed for the antioxidant abilities of these structurally related compounds. Whereas antioxidant activity increased with an increasing number of hydroxyl substituents present on the B-ring for anthocyanidins, the converse was observed for catechins. However, substitution by methoxyl groups diminished the antioxidant activity of the anthocyanidins. Substitution at position 3 of ring C played a major role in determining the antioxidant activity of these classes of compounds. The anthocyanidins, which possess a hydroxyl group at position 3, demonstrated potent antioxidant activities. For the cyanidins, an increasing number of glycosyl units at position 3 resulted in decreased antioxidant activity. Similarly, the substitution of a galloyl group at position 3 of the flavonoid moiety resulted in significantly decreased antioxidant activity for the catechins. Among catechins, cis-trans isomerism, epimerization, and racemization did not play a role in overall antioxidant activity. The antioxidant activities of test compounds (at 40 microM concentrations) were compared to the commercial antioxidants tert-butylhydroquinone, butylated hydroxytoluene, butylated hydroxyanisole, and vitamin E (all at 10 microM concentrations).

Antioxidant activity of sesame coat

The antioxidant activity of ethanolic extracts of sesame coat (EESC) was investigated. The antioxidant activity (91.4%) of 1.0 mg EESC was equal to 1.0 mg tocopherol (90.5%) but was weaker than 1.0 mg butylated hydroxyanisole (98.6%) on peroxidation of linoleic acid. EESC showed an inhibitory effect against the formation of thiobarbituric acid reactive substances (TBARS) in a liposome model system. EESC at 10.0 mg exhibited a 94.9% scavenging effect on α,α-diphenyl-β-picrylhydrazyl radicals and marked reducing power, indicating that EESC acts as a primary antioxidant. The extracts, at a dose of 1.0 mg, showed a 50.0% scavenging effect on the hydroxyl radical. EESC also exhibited a metal-binding ability. Sesamin and sesamolin, the lignan substances, were found in EESC, by HPLC analysis. In addition, chromatographic analysis demonstrated that phenolic compounds and tetranortriterpenoids, which had positive reactions with β-carotene, indicating antioxidant activity, are present in EESC. According to these results, termination of free radical reaction, metal-binding ability and quenching of reactive oxygen are suggested to be, in part, responsible for the antioxidant activity of EESC.

Inhibition of lipid peroxidation and nonlipid oxidative damage by carnosine

AbstractThe antioxidant effects of carnosine on lipids and nonlipids, including liposomes, carbohydrates, and proteins, were investigated. Carnosine exhibited a remarkable antioxidant effect in liposome and deoxyribose model systems. Carnosine at high amounts (50 mg/mL) was effective in protecting protein against oxidation. The correlation coefficients between reducing ability and the inhibition of liposome, albumin, and deoxyribose oxidation were r=0.92, 0.83, and 0.41, respectively. Carnosine exhibited metal‐binding ability and scavenging ability for hydroxyl radicals generated by photolysis of H2O2 with UV light. The correlation coefficients between hydroxyl radical scavenging and the inhibitory effect on deoxyribose, liposome, and albumin oxidative damage were r=0.97, 0.60, and 0.29, respectively. These properties may explain how carnosine protects lipids and non‐lipids against oxidative damage.

Antioxidant Effects of Water Extracts from Barley (Hordeum vulgare L.) Prepared under Different Roasting Temperatures

The antioxidant effects of water extracts of roasted barley (WERB) were investigated under different roasting temperatures and compared with those of the water extracts of unroasted barley (WEUB). It was found that the Maillard reaction products increased upon increasing the roasting temperatures. Both WERB and WEUB exhibited significant antioxidant activities in linoleic acid and liposome model systems. Although WERB and WEUB afforded considerable protection against the damage of deoxyribose and proteins, the antioxidant efficiency of roasted samples was weaker than that of unroasted samples because of the reduction of antioxidant components (catechin, tocopherol, and lutein) with increasing roasting temperature. Unroasted samples were more effective in reducing power, quenching free radical, hydroxyl radical, and chelating iron than the roasted samples. The different antioxidant activity among roasted and unroasted barley samples may be partly attributed to the changes in catechin, tocopherol, and lutein contents.

Nutrient uptake by protocells: a liposome model system.

Over the past decade, several liposome-based models for protocells have been developed. For example, liposome systems composed of polymerase enzymes encapsulated with their substrates have demonstrated that complex compartmentalized reactions can be carried out under conditions in which polymeric products are protected from degradation by hydrolytic enzymes present in the external medium. However, such systems do not have nutrient uptake mechanisms, which would be essential for primitive cells lacking the highly evolved nutrient transport processes present in all contemporary cells. In this report, we explore passive diffusion of solutes across lipid bilayers as one possible uptake mechanism. We have established conditions under which ionic substrates as large as ATP can permeate bilayers at rates capable of supplying an encapsulated template-dependent RNA polymerase. Furthermore, while allowing the permeation of monomer substrates such as ATP, bilayer vesicles selectively retained polymerization products as small as dimers and as large as a transfer RNA. These observations demonstrate that passive diffusion could be used by the earliest forms of cellular life for transport of important nutrients such as amino acids, phosphate, and phosphorylated organic solutes.

Membrane Fusion Activity of Tick-Borne Encephalitis Virus and Recombinant Subviral Particles in a Liposomal Model System

We present a kinetic analysis of the membrane fusion activity of tick-borne encephalitis (TBE) virus and TBE-derived recombinant subviral particles (RSPs) in a liposomal model system. Fusion was monitored using a fluorescence assay involving pyrene-labeled phospholipids. Fusion was strictly dependent on low pH, with the optimum being at pH 5.3–5.5 and the threshold at pH 6.8. Fusion did not require a protein or carbohydrate receptor in the target liposomes. Preexposure to low pH of the virus alone resulted in inactivation of its fusion activity. At the optimum pH for fusion and 37°C, the rate and extent of fusion were very high, with more than 50% of the virus fusing within 2 s and the final extent of fusion being 70%. Lowering of the temperature did not result in a significant decrease in the rate and extent of fusion, suggesting that TBE virus fusion is a facile process with a low activation energy, possibly due to the flat orientation of the E glycoprotein on the viral surface facilitating the establishment of direct intermembrane contact. The fusion characteristics of TBE virus and RSPs were similar, indicating that RSPs provide a reliable and convenient model for further study of the membrane fusion properties of TBE virus.

Molecule Specific Imaging of Freeze-Fractured, Frozen-Hydrated Model Membrane Systems Using Mass Spectrometry

Imaging time-of-flight secondary ion mass spectrometry is used to chemically resolve the spatial distribution of lipids in submicrometer sections of phospholipid membranes. The results show that it is possible to unravel dynamical events such as chemical fluctuations associated with domain structure in cellular membranes. In this work, a liposome model system has been used to capture the stages of membrane fusion between two merging bilayer systems. Fracturing criteria for preserving chemical distributions are shown to be much more stringent than morphological electron cryomicroscopy studies. Images of membrane heterogeneity, induced via mixing various liposomes followed by fast freezing, demonstrate the necessary sample preparation groundwork to investigate complex, heterogeneous membrane domains. Clear delineation of membrane structure provides direct evidence that specific domains or “rafts” can exist. Moreover, low concentrations of each phospholipid are distributed throughout newly fused liposomes despite the existence of distinct domains. In the liposome model, membrane structure ranges from specific domains to a fluid mosaic of the phospholipids during the fusion event. The availability of mass spectrometric imaging is proposed to facilitate the discovery of functional rafts or substructure in cell membranes before, during, and after events including cell division, exocytosis, endocytosis, intracellular transport, infection of membrane-bound viruses, and receptor clustering. This technology holds the promise to define the biology of cell membranes at the molecular level.

Canthaxanthin as an Antioxidant in a Liposome Model System and in Minced Patties from Rainbow Trout

ABSTRACTCanthaxanthin (CX) delayed formation of thiobarbituric acid reactive substances (TBARS) in a concentration‐dependent manner in a ferrous chloride (FeCl2)‐initiated liposome system at 37 °C (p < 0.05). CX delayed TBARS formation in minced flesh from rainbow trout (Oncorhynchus mykiss) at 6 d of 4 °C storage in trial 1, and at 0, 2, 4 and 6 d in trial 2 when compared with controls at 4 °C (p < 0.05). Day 0 TBARS values in trial 2 were equivalent to d 6 of trial 1. CX concentration and a* value decreased in minced trout flesh during 6 days of 4 °C storage (p < 0.05). Overall, CX demonstrated antioxidant activity in vitro and in the minced tissue of CX‐supplemented rainbow trout.

Action of Methanolic Extract of Mung Bean Hulls as Inhibitors of Lipid Peroxidation and Non-lipid Oxidative Damage

The antioxidant effects of methanolic extract of mung bean hulls (MEMBH) on lipids and non-lipids, including liposome, carbohydrate, protein and 2′-deoxyguanosine (2′-dG), were investigated. MEMBH exhibited a remarkable antioxidant effect in a liposome model system, indicating that the extract was an inhibitor of lipid peroxidation. The inhibitory effect of MEMBH on deoxyribose damage was amount-dependent and it afforded considerable protection against damage to deoxyribose. In addition, MEMBH at low amounts was more effective in protecting protein oxidation. Furthermore, the oxidation of 2′-dG to 8-hydroxy-2′-deoxyguanosine (8-OH-2′dG) was inhibited by MEMBH. These results show that the extract also was an inhibitor of non-lipid oxidation damage. The extract exhibited metal binding ability and scavenging activity for hydrogen peroxide and hydroxyl radical, which may explain the mechanism of their protecting lipids and non-lipids from oxidative damage.

Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes.

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.

Antioxidant Activity of Water Extract of Harng Jyur (Chrysanthemum morifolium Ramat)

The antioxidant activity of water extracts of Harng Jyur (Chrysanthemum morifolium Ramat) from four varieties, including Huang Harng Jyur (HHJ), Bai Harng Jyur (BHJ), Gan Harng Jyur (GHJ), and Kung Harng Jyur (KHJ), was investigated. The water extracts of the four Harng Jyur varieties showed strong antioxidant activity in linoleic acid and liposome model systems. Each extract at 20 mg exhibited 65.3–71.8% inhibition on malondialdehyde formation of rat liver homogenate in vitro, greater than 20 mg of tocopherol. A correlation established between the total phenolic compounds and antioxidant activity, yielded a coefficient of r2=0.934, 0.997, 0.758 and 0.983 for HHJ, BHJ, GHJ and KHJ respectively. Each extract of the four Harng Jyur varieties had effective activities as radical scavengers, metal chelators, and active oxygen scavengers. These properties may be the major reasons for the inhibition of lipid peroxidation. Of the four varieties, BHJ was the most significant antioxidative material. No mutagenicity in the four varieties was found in Salmonella typhimurium TA 98, either with or without S9 mix.

Enantioselective Synthesis of a Novel Trans Double Bond Ceramide Analogue via Catalytic Asymmetric Dihydroxylation of an Enyne. The Role of the Trans Double Bond of Ceramide in the Fusion of Semliki Forest Virus with Target Membranes

Intensive interest is currently focused on the role of ceramide (1), a key lipid molecule that functions as a second messenger. The first asymmetric synthesis of a D-erythro-ceramide analogue that contains a C(5)-C(6) trans double bond (3), together with its biological evaluation in a viral-liposome fusion system, is described. Sharpless asymmetric dihydroxylation of 4'-methoxyphenyl trans-5-octadecyn-2-enyl ether (enyne 8a), prepared by the coupling reaction of 1-[(E)-(4'-bromo-2'-butenyl)oxy]-4-methoxybenzene (7) with Lithium tetradecyne, generated yne-diol 9 in 96% yield with the desired stereochemistry at the C(2)and (3) positions and high enantiomeric purity (95% enantiomeric excess). Birch reduction (Li/EtNH2) of yne-diol 9 furnished (2R,3R,5E)-octadecene-1,2,3-triol (10) stereospecifically. The latter was converted to 2 azido derivative 13 in three steps (via a 2-O-triflate-1,3-O-benzylidene intermediate) and 55% overall yield. Reduction of azide 13 and in situ N-acylation with p-nitrophenyl cis-hexadec-4-enoate provided D-erythro-Delta(5)-trans-ceramide (3) in 91% yield. The role of the trans double bond of ceramide in mediating fusion of an alphavirus (Semliki Forest virus) was assessed in a liposomal model system, using target phospholipid/cholesterol vesicles containing either D-erythro-ceramide 1 or 3. The kinetics of virus fusion, as monitored by a change in pyrene excimer fluorescence over a period of 60 s, showed that Delta(5)-trans-ceramide 3 was completely inactive, indicating that there is an absolute requirement for the trans double bond to be located between C(4) and C(5). These data indicate that the molecular determinants on the viral envelope glycoprotein are highly specific for recognition of the unsaturated site in the ceramide molecule.

Antioxidant activity of burdock (Arctium lappa Linné): Its scavenging effect on free‐radical and active oxygen

AbstractThe antioxidant activity and free‐radical and active oxygen‐scavenging activity of burdock extracts were investigated. Of the solvents used for extraction, water yielded the greatest amount of extract that exhibited the strongest antioxidant activity. Water extracts of burdock (WEB) and hot water extracts of burdock (HWEB) exhibited comparable and marked activity on inhibition of linoleic acid peroxidation, indicating that heat treatment did not alter the antioxidant activity of WEB. WEB and HWEB produced significantly lower (P<0.05) malondialdehyde (MDA) in both linoleic acid and liposome model systems than did the control. Moreover, mixtures of tocopherol (Toc), WEB, and HWEB exhibited a remarkable synergistic antioxidant effect in a liposome system; WEB and HWEB thus potentiated the action of Toc. Furthermore, WEB and HWEB displayed a marked inhibitory effect on lipid peroxidation of rat liver homogenate in vitro. WEB and HWEB exhibited an 80% scavenging effect on α,α‐diphenyl‐β‐picrylhydrazyl radical and marked reducing power, indicating that WEB and HWEB act as primary antioxidants. Both extracts at a dose of 1.0 mg exhibited a 60.4–65.0% scavenging effect on superoxide and an 80.5% scavenging effect on hydrogen peroxide. They also showed a marked scavenging effect on the hydroxyl radical. These results revealed that WEB and HWEB are also active as oxygen scavengers and as secondary antioxidants. Based on these results, termination of free‐radical reactions and quenching of reactive oxygen species in burdock extracts are suggested to be, in part, responsible for the antioxidant activity of burdock extracts.

Antioxidative activity of three herbal water extracts

The antioxidative activity of water extracts of three herbs, including the flower of Chrysanthemum morifolium Ramat (FCMR), the calyx of Hibiscus sabdariffa L. (CHSL) and roasted seed of Hordeum vulgare L. (RSHVL), which are commonly called Hang Chu, Lo Shen and Chao Mai in Taiwan, respectively, were investigated. FCMR, CHSL and RSHVL showed marked antioxidative activity, not only in linoleic acid but also in liposome model systems, indicating that the three herbal water extracts may protect the cell from damage by lipid peroxidation. FCMR, CHSL and RSHVL possessed high contents of phenolic compounds and exhibited reducing power, revealing that these herbal extracts may containreductones. The water extracts of the three herbs also showed good hydrogen-donating abilities, indicating that they had effective activities as radical scavengers. No mutagenicity in the water extracts of the three herbs was found in Salmonella typhimurium TA98 and TA100, either with or without S9 mix.

In vitro inhibition of membrane-mediated calcification by novel phosphonates

The effects of a series of novel phosphonates on the kinetics of mineral development in an ionophore-primed 7:2:1 phosphatidylcholine (PC): dicetylphosphate (DCP): cholesterol (Chol) liposomal model system are reported. When present at 2.5 micromol/liter or 25 micromol/liter concentrations in the solution surrounding the liposomes, the investigated phosphonates did not significantly delay the initial formation of hydroxyapatite-like calcium phosphate salts (HAP) within the liposomes or the penetration of HAP crystals through the enclosing membranes. However, the phosphonates variably retarded the subsequent growth and proliferation of the HAP crystals once they became directly exposed to the phosphonate-containing solution. The effectiveness of phosphonates in inhibiting extraliposomal precipitation strongly depended on their structure. The inhibitory action on active surface growth sites of released intraliposomal crystals was found to be the most effective if the phosphonate molecule contained two phosphonic groups linked to the same C atom. At a phosphonate concentration of 25 micromol/liter, the following general order of effectiveness was established: geminal bisphosphonate >/= geminal tetrakisphosphonate > bisacylphosphonates > monoacylphosphonate > bisalkylphosphonate. Within the bisacylphosphonate family, the highest inhibitory action was observed when four or five -CH2- groups separated the ketophosphonic groups.

Comments of H. Snippe and A.F.M. Verheul

AbstractAbstractGeneral Comments:The manuscripts presented are stimulating in pursuing ongoing investigations and clinical trials to use the liposomal model system in future vaccines. All manuscripts deal with basic studies which show the possibilities for future application but also present the limits of the liposomal model.

Role of the M2 Protein in influenza Virus Membrane Fusion: Effects of Amantadine and Monensin on Fusion Kinetics

We have investigated the effects of the anti-influenza drug amantadine (AMT) and the proton-ionophore monensin on the membrane fusion activity of influenza virus in a liposomal model system, using a kinetic fluorescence lipid mixing assay. Fusion of influenza virus A/turkey/Oregon/71 (H7N3) with liposomes was slowed down in the presence of 2 μ M AMT. The effect of AMT was not observed with an AMT-resistant mutant virus. Fusion inhibition by AMT was reversed by the proton-ionophore monensin. In fact, 1 μ M monensin stimulated fusion of AMT-sensitive or -resistant virus, irrespective of the presence of AMT. The effects of AMT and monensin increased with increasing temperature. They were not observed at 25°, but were very prominent at 45°. Monensin did not influence the fusion rates of reconstituted viral envelopes (virosomes), which lack the nucleocapsid and the M1 protein. These results suggest that intraviral low pH facilitates influenza virus fusion, possibly by weakening interactions of the C-terminus of the viral hemagglutinin with the M1 protein and/or the viral nucleocapsid. The effect of AMT on the fusion capacity of influenza virus may contribute to the anti-influenza action of the drug in the early stages of cellular infection. However, the limited extent of the fusion inhibition suggests that the fusion step is unlikely to be the primary target of AMT.

Role of acidic phospholipids in intermembrane sterol transfer

A liposomal membrane model system was used to examine the effect of acidic phospholipids on spontaneous intermembrane cholesterol transfer. The spontaneous exchange of sterol between small unilamellar vesicles (SUV) containing 35 mol% sterol was monitored with a recently developed assay (Nemecz, G., Fontaine, R.N. and Schroeder, F. (1988) Biochim. Biophys. Acta 943, 511–541), not requiring separation of donor and acceptor membrane vesicles. Acidic phospholipids (2.5–30 mol%) increased the initial rate of spontaneous exchange of sterol by 5–89%, depending on the specific phospholipid. The stimulation of spontaneous sterol transfer by acidic phospholipids was suppressed by high ionic strength, CaCl 2 and low pH. The results suggest that negatively charged phospholipids may fluidize sterol-poor domains in SUV membranes and thereby play an important role in the mechanism whereby sterols desorb from membranes into the aqueous medium.