Ionic strength dependence of localized contact formation between membranes: nonlinear theory and experiment.
(41/3739)
Erythrocyte membrane surface or suspending phase properties can be experimentally modified to give either spatially periodic local contacts or continuous contact along the seams of interacting membranes. Here, for cells suspended in a solution of the uncharged polysaccharide dextran, the average lateral separation between localized contacts in spatially periodic seams at eight ionic strengths, decreasing from 0.15 to 0.065, increased from 0.65 to 3.4 micrometers. The interacting membranes and intermembrane aqueous layer were modeled as a fluid film, submitted to a disjoining pressure, responding to a displacement perturbation either through wave growth resulting in spatially periodic contacts or in perturbation decay, to give a plane continuous film. Measured changes of lateral contact separations with ionic strength change were quantitatively consistent with analytical predictions of linear theory for an instability mechanism dependent on the membrane bending modulus. Introduction of a nonlinear approach established the consequences of the changing interaction potential experienced by different parts of the membrane as the disturbance grew. Numerical solutions of the full nonlinear governing equations correctly identified the ionic strength at which the bifurcation from continuous seam to a stationary periodic contact pattern occurred and showed a decrease in lateral contact and wave crest separation with increasing ionic strength. The nonlinear approach has the potential to recognize the role of nonspecific interactions in initiating the localized approach of membranes, and then incorporate the contribution of specific molecular interactions, of too short a range to influence the beginning of perturbation growth. This new approach can be applied to other biological processes such as neural cell adhesion, phagocytosis, and the acrosome reaction. (+info)
Actin protofilament orientation at the erythrocyte membrane.
(42/3739)
The short actin filaments in the erythrocyte's membrane skeleton are shown to be largely oriented tangent to the lipid bilayer. Actin "proto"-filaments have previously been described as junctional centers intertriangulated by spectrin; however, the protofilaments may simultaneously serve as pinning centers between the network and the overlying bilayer. The latter function now seems of particular importance because near-normal network assembly has been reported with transgenic mouse sphero-erythrocytes that lack the primary linkage protein Band 3. To assess possible physical constraints on actin protofilaments in intact membranes, fluorescence polarization microscopy (FPM) has been used to study rhodamine phalloidin-labeled red cell ghosts. A basis for interpreting FPM images of cells is provided by FPM applied to isolated actin filaments. These are labeled with the same rhodamine probes and imaged at various orientations with respect to the polarizers, including filament orientations perpendicular to the image plane. High aperture and fluorophore conjugation effects are found to be minimal, enabling development of a simple, semi-empirical model which indicates that protofilaments are generally within approximately 20 degrees of the membrane tangent plane. (+info)
Rifins: a second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum.
(43/3739)
Many pathogens evade the host immune response or adapt to their environment by expressing surface proteins that undergo rapid switching. In the case of Plasmodium falciparum, products of a multigene family known as var are expressed on the surface of infected red cells, where they undergo clonal antigenic variation and contribute to malaria pathogenesis by mediating adhesion to a variety of host endothelial receptors and to uninfected red blood cells by forming rosettes. Herein we show that a second gene family, rif, which is associated with var at subtelomeric sites in the genome, encodes clonally variant proteins (rifins) that are expressed on the infected red cell surface. Their high copy number, sequence variability, and red cell surface location indicate an important role for rifins in malaria host-parasite interaction. (+info)
Phosphatidylcholine makes specific activity of the purified Ca(2+)-ATPase from plasma membranes independent of enzyme concentration.
(44/3739)
Ca(2+)-ATPase of plasma membranes (PMCA) was isolated from either human or pig red cells by calmodulin-affinity chromatography and supplemented with phosphatidylcholine (PC). The specific activity of the purified PMCA diluted in media with detergent (C(12)E(10)) was very low, and increased with the concentration of the enzyme along a curve that reached the maximum at 8 microg/ml with K(0.5)=1.2-2.5 microg/ml. Such behavior has been described and attributed to self-association of the enzyme (D. Kosk-Kosicka and T. Bzdega, J. Biol. Chem. 263 (1988) 18184-18189). After heat-inactivation, the PMCA was as effective an activator as the intact enzyme, increasing, to the maximum, the specific activity of diluted enzyme with K(0. 5)=2.2 microg/ml. The inactivated PMCA failed to increase the activity of concentrated enzyme, suggesting that activation did not depend on interaction of intact with denatured enzyme molecules. When enough PC was added to the reaction medium to make its final concentration 16-33 microg/ml, the specific activity of the PMCA was maximum and independent of enzyme concentration. Under these conditions, activation by calmodulin lowered to 10%. As a function of the concentration of pure PC, maximum specific activity was reached along a curve with K(0.5)=4 microg/ml. This curve was identical to that of activation at increasing enzyme concentration, suggesting that, in the latter case, activation could have depended on PC contributed to the assay medium by the enzyme. The results show that PC made the purified PMCA solubilized in detergent reach maximum activity at any concentration of the enzyme. (+info)
Increased erythrocyte phosphatidylserine exposure in chronic renal failure.
(45/3739)
The appearance of phosphatidylserine, an aminophospholipid normally confined to the inner monolayer, at the outer leaflet of red cell membrane may have several pathophysiologic implications. This study examines erythrocyte phosphatidylserine exposure in chronic renal failure (CRF) patients on conservative treatment or on dialysis, to assess possible alterations to phospholipid asymmetry in a condition associated with a state of deranged red cell function. A significant increase in phosphatidylserine-expressing erythrocytes was found in undialyzed patients with CRF (2.32%) and patients on hemodialysis (3.06%) and on peritoneal dialysis (2.14%) compared with control subjects (0.68%). In undialyzed CRF patients, a strong correlation (r = 0.903) was found between the percentage of phosphatidylserine-expressing red cells and the serum creatinine concentration. The increased exposure of phosphatidylserine in uremic erythrocytes may be due to inhibition of phosphatidylserine transport from the outer to the inner leaflet of plasma membrane and may promote an increased erythrophagocytosis. In reconstitution experiments, normal erythrocytes showed an increase in phosphatidylserine-expressing cells when incubated in uremic plasma (3.2% after 2 h versus 1.1% at beginning of incubation), whereas phosphatidylserine-positive uremic erythrocytes decreased when resuspended in normal plasma (2.03% after 2 h and 1.65% after 8 h versus 2.9% at beginning of incubation). Preliminary characterization of the putative uremic compound(s) indicates a molecular weight between 10,000 and 20,000, as well as heat instability. These findings show an impairment of erythrocyte membrane phospholipid asymmetry in CRF patients, regardless of the dialysis treatment. Such abnormality seems related to the uremic state and could contribute to the red cell pathology present in CRF. (+info)
Heart rate variability and fatty acid content of blood cell membranes: a dose-response study with n-3 fatty acids.
(46/3739)
BACKGROUND: Dietary intake of long-chain n-3 polyunsaturated fatty acids (PUFA) may protect against sudden cardiac death, an event that may be predicted by measurement of heart rate variability (HRV). OBJECTIVE: The objectives of this study were to 1) examine the correlations between the content of fatty acids in blood cell membranes (platelets and granulocytes) and HRV in healthy subjects, and 2) assess the effect on HRV of dietary intervention with n-3 PUFA in different doses. DESIGN: Sixty healthy volunteers (25 women and 35 men) were randomly assigned to 3 treatment groups in a double-blind design. Subjects received a daily supplement of either 6.6 g n-3 PUFA, 2.0 g n-3 PUFA, or placebo (olive oil). A 24-h Holter recording was obtained for each subject before supplementation and after 12 wk of supplementation; the 24-h HRV was then related to the content of fatty acids in granulocytes and platelets. RESULTS: Before supplementation, positive correlations were observed in men between the content of docosahexaenoic acid in cell membranes and HRV indexes (r = 0.50, P < 0.01), whereas such correlations were not found in women. Dietary intervention revealed a dose-dependent effect of n-3 PUFA on HRV in men, whereas no effect was found in women. CONCLUSION: The study showed a beneficial effect of n-3 PUFA on HRV in healthy men, suggesting an antiarrhythmic effect of n-3 PUFA. No such effect was observed in healthy women. (+info)
Oligomerization and structural changes of the pore-forming Pseudomonas aeruginosa cytotoxin.
(47/3739)
Pseudomonas aeruginosa produces a pathogenic factor, the 29-kDa pore-forming protein cytotoxin. Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on intermolecular disulfide bridges. SDS induced tetramer, hexamer and mainly pentamers that were resistant to reducing conditions, indicating an additional oligomerization mechanism. Functional oligomerization after incubation with different membranes resulted in an oligomer of approximately 145 kDa that was identified as the pentamer by comparison with the SDS-induced oligomers. Covalent modification with diethylpyrocarbonate showed that histidine residues are indispensable for functional pentamerization. Pentamer formation was not influenced by the lipid composition of the liposomes tested, indicating that rising membrane fluidity did not increase oligomerization. The secondary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% beta-sheet, 20% beta-turn, 10% alpha-helix and 20% remaining structure. Contact with detergent micellae or liposomes induced a reorganization of beta-structure associations, as observed by attenuated total reflection-Fourier transform infrared spectroscopy. Electron microscopy and principle component analysis of the cytotoxin monomer demonstrated a tapered molecule of 11 nm in length and a maximum width of 3.5 nm. These results classify the cytotoxin as a pore-forming toxin, rich in antiparallel beta-structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureus alpha-toxin. (+info)
Analysis of receptor for Vibrio cholerae El tor hemolysin with a monoclonal antibody that recognizes glycophorin B of human erythrocyte membrane.
(48/3739)
El Tor hemolysin (ETH), a pore-forming toxin secreted by Vibrio cholerae O1 biotype El Tor and most Vibrio cholerae non-O1 isolates, is able to lyse erythrocytes and other mammalian cells. To study the receptor for this toxin or the related molecule(s) on erythrocyte, we first isolated a monoclonal antibody, B1, against human erythrocyte membrane, which not only blocks the binding of ETH to human erythrocyte but also inhibits the hemolytic activity of ETH. Biochemical characterization and immunoblotting revealed that this antibody recognized an epitope on the extracellular domain of glycophorin B, a sialoglycoprotein of erythrocyte membrane. Erythrocytes lacking glycophorin B but not glycophorin A were less sensitive to the toxin than were normal human erythrocytes. These results indicate that glycophorin B is a receptor for ETH or at least an associated molecule of the receptor for ETH on human erythrocytes. (+info)