Effects of the antibiotic peptide microcin J25 on liposomes: role of acyl chain length and negatively charged phospholipid.
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This paper reports the effects of microcin J25 (MccJ25) on the microviscosity and permeability of phospholipid vesicles of different compositions. The results obtained indicate that MccJ25 interacts with egg L-alpha-phosphatidylcholine (PC) vesicles as demonstrated by peptide intrinsic fluorescence determinations. The interaction depends on the lipid composition of the vesicles. MccJ25 interaction induces a significant fluidity increase of egg PC vesicles. This effect is time and concentration dependent. Both trimethyl ammonium 1,6-diphenyl-1,3,5-hexatriene and 1,6-diphenyl-1, 3,5-hexatriene gave the same results. The microviscosity of L-alpha-phosphatidylcholine dipalmitoyl small unilamellar vesicles (SUVs) was affected while that of L-alpha-phosphatidylcholine dimyristoyl vesicles was not, indicating that the effect was strongly dependent on the chain length of fatty acids. On the other hand, negatively charged L-alpha-phosphatidyl-DL-glycerol (PG) vesicles remarkably inhibited the peptide effect. Nevertheless vesicles composed of L-alpha-phosphatidylethanolamine:PG:cardiolipin (7:2:1), a composition resembling bacterial membrane, were sensitive to the MccJ25 effect. MccJ25 effectively dissipated the valinomycin-induced membrane potential, but induced only a modest leakage (5%) of the trapped Tb(+3)-dipicolinic acid complex. These results indicate that the peptides interact and perturb the bilayer of SUVs. The relationships between this effect and bactericidal action remain to be elucidated. (+info)
Vibrio cholerae cytolysin: assembly and membrane insertion of the oligomeric pore are tightly linked and are not detectably restricted by membrane fluidity.
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Hemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes. Pore formation is inhibited at low temperature and in the absence of cholesterol. We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy. None of these methods yielded any evidence of a non-lytic pre-pore oligomer. The second question was addressed by the use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively. The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization. Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5 degrees C. Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin. The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane. The findings of our study distinguish V. cholerae cytolysin from several previously characterized pore-forming toxins. (+info)
Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers.
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Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature. (+info)
Oxidative and drug-induced alterations in brush border membrane hemileaflet fluidity, functional consequences for glucose transport.
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Oxidation of biological membranes has been suggested as a major pathological process in a variety of disease states including intestinal ischemia and inflammatory bowel disease. Previous studies on the small intestinal brush border membrane have shown that part of the decrease in the activity of the Na(+)-dependent glucose transporter (SGLT1) observed after oxidation could be secondary to the derangement in membrane fluidity that accompanied oxidative damage. The present study examined the relationship between oxidative-induced hemileaflet fluidity alterations and the resultant change in Na(+)-dependent glucose transport activity. To address this issue, in vitro oxidation of guinea pig brush border membrane vesicles was induced by incubation of the vesicles with ferrous sulfate and ascorbate. We found that oxidation decreased the fluidity of both the outer and inner hemileaflets, the decrease being greater in the outer leaflet. Moreover, the preferential alteration in hemileaflet fluidity was accompanied by a decrease in glucose transport. However, when membrane perturbing agents such as hexanol and A(2)C were used to restore membrane fluidity to levels comparable to controls, rates of glucose transport could not be interpreted in terms of variation of bulk membrane fluidity or variation in fluidity of any specific membrane leaflet. On the basis of these experiments, we propose that previous studies that reported coincidental alteration in membrane fluidity and glucose transport cannot be interpreted on the basis of bulk fluidity or hemileaflet fluidity. (+info)
Ethnic differences in erythrocyte membrane fluidity and the association with serum triacylglycerols.
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The objectives of this study were to determine whether there are differences between black and white individuals with regard to the membrane fluidity of isolated erythrocytes, and/or in the relationships between membrane fluidity, gender and circulating lipids. Fluorescent polarization anisotropy, as an index of membrane fluidity, was determined using the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in 52 black and 52 white individuals, of whom 39 pairs were matched for age, sex and blood pressure. In the 39 matched pairs, the TMA-DPH anisotropy was significantly higher in the black (0.262+/-0.007) compared with the white (0.258+/-0.005) subjects (P<0.005). There was also a significant difference in serum lipids. Gender differences in TMA-DPH anisotropy were observed in the white but not in the black individuals. The associations between membrane fluidity and serum lipids were examined in the total group, separated according to ethnic group. Although the associations were in the same direction in both groups, the association was only significant in the white subjects (r= - 0.42; P<0.02). The ethnic difference in membrane fluidity was abolished when adjusting for serum triacylglycerols. In conclusion, ethnic differences in erythrocyte membrane fluidity, as determined by the use of TMA-DPH anisotropy, appear to be the result of ethnic differences in the level of serum triacylglycerols. (+info)
Interaction between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neutral helper lipid.
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Recent studies have indicated that there are many barriers to successful systemic gene delivery via cationic lipid vectors using the intravenous route. The purpose of this study was to investigate the effect of binding and interaction between erythrocytes, a major constituent of blood cells, and the complexes, in relation to the role of the helper lipid, on the in vivo gene delivery to the lung following intravenous injection. We used three types of cationic lipid vectors, DNA-DOTMA/Chol liposome complexes, DNA-DOTMA liposome complexes, and DNA-DOTMA/DOPE liposome complexes. Although the three types of vectors bind to murine blood cells in vivo and in vitro, DOTMA/Chol and DOTMA complexes with a higher in vivo transfection activity do not induce fusion between erythrocytes, whereas DOTMA/DOPE complexes, a less efficient vector in vivo, induce fusion between the erythrocytes after a short incubation period. Pre-incubation of DOTMA/DOPE complexes with erythrocytes significantly reduced the transfection efficiency while DOTMA/Chol- and DOTMA complexes were more resistant to such treatment. The differences in the physicochemical and structural properties of these complexes could explain the differences in interaction with erythrocytes and subsequent gene expression. Lipids in DOTMA/Chol and DOTMA complexes have a stable lamellar structure. However, lipids in DOTMA/DOPE complexes have a highly curved structure with high fluidity. These results indicate that the interaction with erythrocytes depends on the properties of the cationic lipid vectors and this is an important factor for intravenous gene delivery using cationic lipid vectors. (+info)
P-glycoprotein does not reduce substrate concentration from the extracellular leaflet of the plasma membrane in living cells.
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P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is an important mediator of multidrug resistance in cancer. Pgp exhibits a very broad specificity for substrates. These substrates share a common feature of being amphipathic and can orient into either leaflet of the membrane bilayer. Current evidence suggests that Pgp recognizes and extracts substrates from the membrane bilayer, but from which leaflet is unresolved. To directly test whether Pgp can decrease substrate concentration in the extracellular leaflet of the plasma membrane in living cells, we used the fluorescent lipid analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH). TMA-DPH in the extracellular solution rapidly partitions into the extracellular leaflet of the plasma membrane and exhibits slow transbilayer flipping into the cytoplasmic leaflet. Because TMA-DPH fluorescence is confined to the extracellular leaflet in early time points after addition but labels intracellular membranes after longer incubation, we can assess the effect of Pgp on TMA-DPH concentration from both extracellular leaflet and intracellular membranes. Transient transfection with a Pgp and the green fluorescence protein (GFP) fusion protein generated cells with heterogeneous expression levels of Pgp-GFP. Compared with nonexpressing cells, cells expressing Pgp-GFP showed decreased accumulation of TMA-DPH in intracellular membranes but similar levels of accumulation in the extracellular leaflet of the plasma membrane. Additionally, in drug-selected MCF7/Adr cells, which constitutively express high levels of Pgp, inhibition of Pgp by cyclosporin A resulted in significantly increased accumulation of TMA-DPH in intracellular membranes but no difference in its accumulation in the extracellular leaflet of the plasma membrane. These data indicate that whereas Pgp can extract TMA-DPH from the cytoplasmic leaflet of the membrane, any activity Pgp may possess in the extracellular leaflet is insufficient to decrease TMA-DPH concentration there and, therefore, does not contribute to lowering the cellular levels. Pgp is the prototype of an increasing number of clinically important ATP-binding cassette transporters of amphipathic drugs and lipids. These results may help decipher a common mechanism of these transporters. (+info)
Modes of membrane interaction of a natural cysteine-rich peptide: viscotoxin A3.
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Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from other members because it is cytotoxic against tumoral cells but weakly hemolytic. We studied the interactions between the most active of these toxins, viscotoxin A3 (VA3), and model membranes made of phosphatidylcholine and phosphatidylserine (PS), the major zwitterionic and acidic phospholipids found in eukaryotic cells. Monolayer studies showed that electrostatic forces are essential for the interaction and are mainly involved in modulating the embedding of the toxin in the PS head group region. This in turn induces membrane stiffening, as shown by fluorescence polarization assays with 1,6-diphenyl-1,3,5-hexatriene and its derivatives. Moreover, vesicle permeabilization analyses showed that there are two modes of interaction, which are directly related to the stiffening effect and depend on the amount of VA3 bound to the surface of the vesicles. We propose an interaction model in which the embedding of VA3 in the membrane induces membrane defects leading to the gradual release of encapsulated dye. When the surfaces of the vesicles are saturated with the viscotoxin, complete vesicle destabilization is induced which leads to bilayer disruption, all-or-none encapsulated dye release and rearrangement of the vesicles. (+info)