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1.
We studied the interaction with liposomes and the antioxidant activity of flavonoid (quercetin, catechin, taxifolin) complexes with iron(III). It was found that the lipophilicity of complexes depends on an iron:flavonoid ratio and grows at a ratio of 1 to 1, while complexes in a 2: 1 ratio were the most effective to slow down the lipid peroxidation and restore radical 2,2-diphenyl-1-picrylhydrazyl. Thus, the stoichiometry of complexes formed in aqueous solution, may differ from the stoichiometry of complexes that most effectively protect membranes from peroxidation.  相似文献   

2.
Fusogenic capacities of divalent cations and effect of liposome size   总被引:3,自引:0,他引:3  
J Bentz  N Düzgüne? 《Biochemistry》1985,24(20):5436-5443
The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.  相似文献   

3.
(?)-Epigallocatechin-3-gallate (EGCG) is a flavonoid known for its good antioxidant potential and health benefits. It is one of the most intriguing flavonoids, especially because of its specific interactions with model lipid membranes. It was noticed that EGCG might form EGCG rich domains/rafts at certain compositions of lipid membranes. In this article, we investigate whether EGCG forms EGCG rich domains when incorporated in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes. Our results show that EGCG decreases lipid ordering parameter in ordered membranes and increases it in the case of disordered ones. Also, incorporation of EGCG does not affect the zeta-potential and shape of the liposomes, but it can induce aggregation of liposomes. Our study also demonstrates that liposomes with incorporated EGCG are highly protected against UV-light induced oxidation.  相似文献   

4.
Long-term investigations of ternary complexes: DNA–zwitterionic liposomes–divalent metal cations have revealed many details of their structure; but some questions need additional study. The conditions under which fusion or aggregation of liposomes occurs during such complex formation remain obscure. The DNA structure in the ternary complex is still unclear. In this work, using a freeze-fracture method, author demonstrate the thin structure of a complex (early attempts to observe this structure employing other electron microscopic methods, in particular cryo-TEM, have not met with success). After treatment of ternary complexes with nuclease S1, which is able to digest single-stranded DNA, local DNA unwinding in such complexes was confirmed. Author describe how the curvature of liposomes as the main factor may determine the interaction between liposomes and DNA, especially aggregation or fusion of liposomes during ternary complex formation. Therefore, interaction between lipids of membrane vesicles in cell and chromatin DNA can be the first stage of a nuclear envelope and pore complex assembly.  相似文献   

5.
Differential scanning calorimetry was used to study the influence of the flavonoid taxifolin (dihydroquercetin) on the temperature-dependent phase transition of dimyristoylphosphatidylcholine multilamellar liposomes. Taxifolin was added to organic solution of the lipid during the procedure of liposomes preparation (addition from-within) or to a suspension of prepared liposomes (addition from-without). In the first case, liposomes contained from 2 to 50 mol% of taxifolin added from-within; in the second case, lyposomes were treated with 0.001% or 0.01% taxifolin. In both cases, the effect was similar. When the concentration of taxifolin increased, the temperature of lipid melting decreased while the width of transition considerably enlarged. Freeze-fracture electron microscopy revealed that taxifolin did not rupture multilamellar liposomes, while the formation of ripple-phase was retarded in all bilayers even when the liposomes were treated from without. This suggested the ability of taxifolin to penetrate through numerous bilayers of multilamellar liposomes.  相似文献   

6.
The generation of hydroxyl free radicals in 60Co gamma-irradiation of a dilute aqueous suspension of phosphatidyl choline liposomes, resulted in the rapid accumulation of lipid hydroperoxides (linearly with time), but only small concentrations of malondialdehyde. Incubation of the irradiated liposomes with ferric chloride was found to significantly increase the malondialdehyde, and evidence is presented that this resulted from iron catalysed decomposition of the lipid hydroperoxide. This suggests a role for free iron or iron chelates in the propagation of lipid peroxidation stimulated by other systems.  相似文献   

7.
Low-pH-induced fusion of liposomes with rat liver endoplasmic reticulum was evidenced. Fusion was inactivated by treatment of microsomes with trypsin or EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline), indicating the involvement of a protein. The protein was purified 555-fold by chromatographic steps. The identification and purification to homogeneity was obtained by electroelution from a slab gel, which gave a still active protein of about 50 kDa. The protein promoted the fusion of liposomes; laser light scattering showed an increase of mean radius of vesicles from 60 up to about 340 nm. Fusion was studied as mass action kinetics, describing the overall fusion as a two-step sequence of a second order aggregation followed by a first order fusion of liposomes. For phosphatidylcholine containing liposomes aggregation was not rate-limiting at pH 5.0 and fusion followed first order kinetics with a rate constant of 13 · 10−3 sec−1. For phosphatidylethanolamine/phosphatidic acid liposomes aggregation was rate-limiting; however, the overall fusion was first order process, suggesting that fusogenic protein influences both aggregation and fusion of liposomes. The protein binds to the lipid bilayer of liposomes, independently of pH, probably by a hydrophobic segment. Exposed carboxylic groups might be able to trigger pH-dependent aggregation and fusion. It is proposed that the protein inserted in the lipid bilayer bridges with an adjacent liposome forming a fused doublet. Since at endoplasmic reticulum level proton pumps are operating to generate a low-pH environment, the membrane bound fusogenic protein may be responsible for both aggregation and fusion of neighboring membranes and therefore could operate in the exchange of lipidic material between intracellular membranes. Received: 25 August 1997/Revised: 28 April 1998  相似文献   

8.
Synexin enhances the aggregation rate but not the fusion rate of liposomes   总被引:3,自引:0,他引:3  
The effect of synexin on the calcium-induced fusion of large unilamellar liposomes was studied by using two assays for the mixing of aqueous contents. The results were analyzed in terms of the mass action kinetic model, which describes the overall fusion reaction as a two-step sequence consisting of a second-order process of liposome aggregation followed by a first-order fusion reaction. By using several different lipid compositions and varying the electrolyte composition, it was possible to select the rate-limiting step of the overall fusion process. When aggregation was the rate-limiting step, as in the case of Ca2+-induced fusion of phosphatidylserine (PS), phosphatidate (PA)/phosphatidylethanolamine (PE) (1:3), and PS/PE (1:3) liposomes, synexin increased the overall fusion kinetics by increasing the aggregation rate constant (up to 100-fold). When aggregation was rapid compared to destabilization of apposed membranes, i.e., fusion was rate limiting, synexin either had no effect or reduced the overall fusion kinetics. In one such case involving liposomes composed of PA/PS/PE/phosphatidylcholine (PC) (10:15:65:10), synexin reduced the fusion rate constant by 50%. The effect of calcium-induced synexin polymerization was investigated by preincubation of synexin with calcium prior to addition of liposomes. Prepolymerization by Ca2+ always decreased the activity of synexin such that it was less than the activity of an equal amount of untreated monomers. However, it was found that the activity of synexin monomers polymerized to an average hexameric size was greater than that of one-sixth as many untreated monomers, with respect to the liposome aggregation rate constant. Neither polymers nor monomers increased the fusion rate constant.  相似文献   

9.
Cationic polypeptide-induced fusion of acidic liposomes   总被引:1,自引:0,他引:1  
Fusion of acidic liposomes was induced by Mg2+, Ca2+, polylysine and polymyxin B. The extent of fusion and the concomitant change in liposome permeability induced by divalent cations depended on the concentration of liposomes in the suspension as well as on the cation concentration. In contradistinction, the extent of fusion and the change in permeability induced by the polypeptides depended only on the polycation concentration. The difference in the pattern of interaction, between the liposomes and the various cations, is a result of different binding affinities. The binding of the polypeptides to the liposomes, in contrast to divalent cations, is practically irreversible. The potential of polylysine to induce fusion of acidic phosphatidylethanolamine-devoid liposomes was used to demonstrate that in order to obtain fusion, both membranes involved must be susceptible, at least to a certain degree, to fusion by the proper inducer. When lysophosphatidylcholine substituted for phosphatidylcholine in phosphatidylethanolamine-rich acidic liposomes, extensive polylysine-induced fusion was obtained without concomitant spillage of the liposome contents.  相似文献   

10.
Formation of liposome/polynucleotide complexes (lipoplexes) involves electrostatic interactions, which induce changes in liposome structure. The ability of these complexes to transfer DNA into cells is dependent on the physicochemical attributes of the complexes, therefore characterization of binding-induced changes in liposomes is critical for the development of lipid-based DNA delivery systems. To clarify the apparent lack of correlation between membrane fusion and in vitro transfection previously observed, we performed a multi-step lipid mixing assay to model the sequential steps involved in transfection. The roles of anion charge density, charge ratio and presence of salt on lipid mixing and liposome aggregation were investigated. The resonance-energy transfer method was used to monitor lipid mixing as cationic liposomes (DODAC/DOPE and DODAC/DOPC; 1:1 mole ratio) were combined with plasmid, oligonucleotides or Na(2)HPO(4). Cryo-transmission electron microscopy was performed to assess morphology. As plasmid or oligonucleotide concentration increased, lipid mixing and aggregation increased, but with Na(2)HPO(4) only aggregation occurred. NaCl (150 mM) reduced the extent of lipid mixing. Transfection studies suggest that the presence of salt during complexation had minimal effects on in vitro transfection. These data give new information about the effects of polynucleotide binding to cationic liposomes, illustrating the complicated nature of anion induced changes in liposome morphology and membrane behavior.  相似文献   

11.
The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.  相似文献   

12.
N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) has been proposed to play a critical role in the membrane fusion process. The SNARE complex was suggested to be the minimal fusion machinery. However, there is mounting evidence for a major role of calcium in membrane fusion. Hence, the role of calcium in SNARE-induced membrane fusion was the focus of this study. It revealed that recombinant v-SNARE and t-SNARE, reconstituted into separate liposomes, interact to bring lipid vesicles into close proximity, enabling calcium to drive fusion of opposing bilayers. Exposure to calcium triggered vesicle fusion at both, high potency and efficacy. The half-time for calcium-induced fusion of SNARE-reconstituted vesicles was determined to be approximately 10 s, which is two orders of magnitude faster than in its absence. Calcium acts downstream of SNAREs, since the presence of SNAREs in bilayers increases the potency of calcium-induced vesicle fusion, without significantly influencing its efficacy. Hence, this study suggests that in the physiological state in cells, both SNAREs and calcium operate as the minimal fusion machinery.  相似文献   

13.
The fusogenic activity of plant Golgi membranes was studied in a cell-free system by assaying lipid mixing and content leakages of fluorescence probes. Golgi membranes from mung bean (Vigna radiata L.) hypocotyl cells fused to liposomes in the absence of any cytosolic proteins and nucleotides. It was demonstrated that the fusion was mediated by integral membrane protein(s), and was influenced by divalent cations (mm). Mg(2+), Ca(2+), and Mn(2+) ions enhanced the lipid mixing by reducing repulsive forces between membranes. In the content leakage assay, Mg(2+) ions also showed a stimulative effect. However, other divalent cations were inhibitory. It is suggested that the fusion system of Golgi membranes comprises at least two components: one that mediates the formation of fusion intermediates prior to pore opening, and one that mediates the subsequent processes. The latter must be sensitive to divalent cations at millimolar concentrations. The fusion of Golgi and biological membranes was induced by divalent cations. We speculated about the biological role of the fusion system studied here.  相似文献   

14.
We studied the influence of calcium on lipid mixing mediated by influenza hemagglutinin (HA). Lipid mixing between HA-expressing cells and liposomes containing disialoganglioside, influenza virus receptor, was studied at 37 degrees C and neutral pH after a low-pH pulse at 4 degrees C. With DSPC/cholesterol liposomes, calcium present after raising the temperature significantly promoted lipid mixing only when it was triggered by a short low-pH application. In case of DOPC/cholesterol liposomes, calcium promotion was observed regardless of the duration of the low-pH pulse. Calcium present during a short, but not long, low-pH application to HA-expressing cells with bound DSPC/cholesterol liposomes at 4 degrees C inhibited subsequent lipid mixing. We hypothesize that calcium influences lipid mixing because it binds to a vestigial esterase domain of hemagglutinin or causes expulsion of the fusion peptide from an electronegative cavity. We suggest that calcium promotes the transition from early and reversible conformation(s) of low pH-activated HA towards an irreversible conformation that underlies both HA-mediated lipid mixing and HA inactivation.  相似文献   

15.
The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.  相似文献   

16.
Synthetic mannose-containing glycolipids utilizing the cholesterol nucleus as a lipid anchor, and either the 6-aminohexyl- or the 6-(6-aminohexanamido)hexyl-1-thio-alpha-D-mannopyranosides as the carbohydrate ligands, have been synthesized and incorporated into small unilamellar liposomes. Incorporation of these cholesterol-mannoside derivatives at concentrations up to 14 mol% apparently does not affect the physical characteristics of the liposomes. Addition of concanavalin A to a suspension of liposomes containing the long chain cholesterol-mannose derivative causes an increase in light-scattering at 360 nm. As the increase in absorbance is completely reversed by the addition of alpha-methylmannoside, aggregation rather than fusion of the liposomes appears to be occurring. Liposomes containing 14 mol % of the short chain (6-aminohexyl-) derivative are aggregated by concanavalin A indicating that the lectin can approach to within 10 A of the lipid bilayer. Preliminary results suggest that the aggregation of vesicles containing either the long or short chain derivatives is highly dependent on the density of the sugar in the membrane.  相似文献   

17.
The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.  相似文献   

18.
Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.  相似文献   

19.
Pneumolysin, a major virulence factor of the human pathogen Streptococcus pneumoniae, is a soluble protein that disrupts cholesterol-containing membranes of cells by forming ring-shaped oligomers. Magic angle spinning and wideline static (31)P NMR have been used in combination with freeze-fracture electron microscopy to investigate the effect of pneumolysin on fully hydrated model membranes containing cholesterol and phosphatidylcholine and dicetyl phosphate (10:10:1 molar ratio). NMR spectra show that the interaction of pneumolysin with cholesterol-containing liposomes results in the formation of a nonbilayer phospholipid phase and vesicle aggregation. The amount of the nonbilayer phase increases with increasing protein concentration. Freeze-fracture electron microscopy indicates the coexistence of aggregated vesicles and free ring-shaped structures in the presence of pneumolysin. On the basis of their size and analysis of the NMR spectra it is concluded that the rings are pneumolysin oligomers (containing 30-50 monomers) complexed with lipid (each with 840-1400 lipids). The lifetime of the phospholipid in either bilayer-associated complexes or free pneumolysin-lipid complexes is > 15 ms. It is further concluded that the effect of pneumolysin on lipid membranes is a complex combination of pore formation within the bilayer, extraction of lipid into free oligomeric complexes, aggregation and fusion of liposomes, and the destabilization of membranes leading to formation of small vesicles.  相似文献   

20.
Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.  相似文献   

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