Yield Strength of Human Erythrocyte Membranes to Impulsive Stretching

Deformability while remaining viable is an important mechanical property of cells. Red blood cells (RBCs) deform considerably while flowing through small capillaries. The RBC membrane can withstand a finite strain, beyond which it ruptures. The classical yield areal strain of 2–4% for RBCs is generally accepted for a quasi-static strain. It has been noted previously that this threshold strain may be much larger with shorter exposure duration. Here we employ an impulse-like forcing to quantify this yield strain of RBC membranes. In the experiments, RBCs are stretched within tens of microseconds by a strong shear flow generated from a laser-induced cavitation bubble. The deformation of the cells in the strongly confined geometry is captured with a high-speed camera and viability is successively monitored with fluorescence microscopy. We find that the probability of cell survival is strongly dependent on the maximum strain. Above a critical areal strain of ∼40%, permanent membrane damage is observed for 50% of the cells. Interestingly, many of the cells do not rupture immediately and exhibit ghosting, but slowly obtain a round shape before they burst. This observation is explained with structural membrane damage leading to subnanometer-sized pores. The cells finally lyse from the colloidal osmotic pressure imbalance.     

Cation-impermeable Inside-out and Right-side-out Vesicles from Human Erythrocyte Membranes

The two surfaces of a membrane can be compared by isolating sealed vesicles which bare one side or the other to impermeable probe molecules. For this purpose, inside-out (IO) and right-side-out (RO) vesicles have been generated from human red blood cell membrane ghosts and partially resolved on dextran density gradients¹. We now report further characterization of this system, showing (a) that vesicles can be made which are impermeable to small solutes, including Na⁺ and K⁺, even though the parent ghosts are quite unsealed; (b) that the dextran gradient fractionation serves to separate sealed vesicles (which happened to be IO and RO, respectively, in the original study¹); (c) that impermeable RO vesicles form instead of IO vesicles if the published protocol is modified by the untimely addition of trace divalent cations; and (d) that the vesicle fractions can and must be accurately monitored for sealing and orientation to avoid misinterpretations such as those encountered in a previous sidedness study².     

Use of Steady-State Laurdan Fluorescence to Detect Changes in Liquid Ordered Phases in Human Erythrocyte Membranes

In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains.     

Differential Labeling of Components in Human Erythrocyte Membranes Associated with the Transport of Glucose

The irreversible inhibition of glucose transport by 1-fluoro-2,4-dinitrobenzene (FDNB) has been used to identify membrane proteins possibly associated with glucose transport in human crythrocytes. D-Glucose was shown to enhance significantly the rate of FDNB inhibition of transport when present during the reaction, whereas cytochalasin B (CB) and D-maltose retarded this FDNB inhibition of transport. This modulation of the inhibition reaction formed the basis for a double isotopic differential labeling technique using [¹⁴C]- and [³H]FDNB followed by SDS-polyacrylamide gel electrophoresis to distinguish transport-associated polypeptides from bulk membrane dinitrophenylated proteins.

Reactions in the presence of CB or maltose revealed the presence of a differentially labeled polypeptide(s), with a molecular weight of approximately 60,000-65,000 daltons. This effect could in part be reversed in the presence of D-glucose but not L-glucose. Reactions in the presence of D-glucose resulted in two regions of differential labeling. One region was around 200,000 daltons and the other corresponded to a 90,000-dalton band.

Extraction of membrane proteins with p-chloromercuribenzene sulfonate resulted in no loss of the 60,000-dalton peak, indicating that this labeled polypeptide(s) was firmly anchored in the hydrophobic core of the membrane.

These results indicate that as many as three membrane polypeptides are differentially labeled by FDNB under conditions strongly associated with the inhibition of the glucose transport system and may be involved in the regulation of glucose transport.     

Formation of Two Different Types of Ion Channels by Amphotericin B in Human Erythrocyte Membranes

The polyene antibiotic amphotericin B (AmB) is known to form aqueous pores in lipid membranes and biological membranes. Here, membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. We show that AmB induced a cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (≤1.0 × 10⁻⁶ M), but a sharp reversal of such polarization was observed at concentrations greater than 1.0 × 10⁻⁶ M AmB, an indication that aqueous pores are formed. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K⁺ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. These findings support the conclusion that the two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components. In effect, a membrane lipid reorganization, as induced by incubation of erythrocytes with tetrathionate, a cross-linking agent of the lipid raft–associated protein spectrin, led to differential changes in the activation parameters for the formation of both types of channels, reflecting the different lipid environments in which such structures are formed.     

Dynamics of Interaction of Phosphatidylcholine/Octadecylamine Liposomes with Human Erythrocyte Membranes: Electron Microscopic Study

Abstract

The interactions of octadecylamine and of positively charged liposomes (egg phosphatidylcholine/octadecylamine in molar ratio from 7:5 to 7:0.5) with human erythrocyte membrane have been studied by freeze-fracture and thin-section electron microscopy.

For the first time a very fast adsorption of liposomes to the cell membranes (less than 1 sec) is shown, and their intensive incorporation into plasma membrane (probably within the first 2-5 sec) without visible changes in cell morphology.

The prolonged incubation of the cells with liposomes results in certain morphologic changes: the transition of diskocytes to stomatocytes (30-80 sec) accompanied by the formation of isolated membrane vesicles in cell matrix (80-120 sec); the formation of pentalaminar contacts between plasma membrane of spherocytes and the membrane of isolated matrix vesicles (2.5-3 min); and the incorporation of matrix vesicle membranes into the spherocyte membranes (5 min+).

Possible molecular mechanisms underlying the observed structural changes are discussed briefly.     

The Effects of N-Terminal Fragments of Immunophilin on Phospholipid Composition of Rat Brain and Human Erythrocyte Membranes

Rat brain slices and human erythrocyte membranes have been incubated in the presence of water-soluble synthetic peptide fragments corresponding to residues 1–9 and 1–15 of the N-terminus of immunophilin and the effects on the phospholipid composition examined. During a 2 h incubation in the presence of 1 nM, 0.1 M, and 10 M concentrations of the peptides there were observed significant and dose-dependent decreases in the amounts of phosphatidylcholine and phosphatidylethanolamine, as well as increases in the amounts of phosphatidylserine and, to a less extent, phosphatidylinositol, cardiolipin and lysophosphatidylcholine. The overall decrease in the neutral phospholipids of rat brain, and no changes in human erythrocyte membranes with the simultaneous increase in the acidic phospholipids, both in brain and erythrocyte membranes, tended to counteract any changes in the phospholipid composition of the material studied. The results are discussed in terms of the possible effects of immunophilin on modulating phospholipid turnover in brain cell erythrocyte membranes.     

pH-Dependent Effects of Chlorpromazine on Liposomes and Erythrocyte Membranes

Abstract

Chlorpromazine (CPZ) is an amphipathic antipsychotic drug that binds to erythrocytes reaching in this way the central nervous system. CPZ is a basic molecule with pK = 8.6. This paper reports on CPZ-induced lysis of red blood cells and liposomes. Haemolysis was tested under hypotonic conditions, in the pH range 5.0–10.0. Cell sensitivity towards CPZ increased with increasing pH. Increasing pH caused also a decrease in the critical micellar concentrations of CPZ. These results are interpreted in terms of a competition between repulsive electrostatic forces and attractive hydrophobic forces, that would act both in pure CPZ and in mixed CPZ-phospholipid micelles. In order to eliminate possible pH effects mediated by red blood cell proteins, experiments were carried out in which CPZ induced release of a fluorescent dye from liposomes (large unilamellar vesicles). The latter observations confirmed that membrane sensitivity towards CPZ was increased at higher pH.     

马氏蝎毒毒素的分离纯化及其对红细胞膜作用的初步研究

本文用山东产马氏蝎(Buthus martensii kashi)粗毒为材料,经SephadexG-50和Sp-Sephadex C-25二次柱层析,分离纯化获得三个毒峰部分,毒性比粗毒分别提高40—100倍。 纯度鉴定表明三个毒峰的聚丙烯酰胺凝胶电泳和等电聚焦电泳均为一条带,等电点分别为8.7,9.1,9.1,分子量用SDS-不连续聚丙烯酰胺凝胶电泳测定分别为6,600,5,000和8,500。对纯化蝎毒毒素的氨基酸组分也作了分析。 蝎毒毒素对人红细胞膜作用的初步探索结果表明:它使人红细胞膜的Na.K-ATP酶活性和膜脂流动性有所降低。     

The Influence of pH and Ionic Strength on the Electrokinetic Stability of the Human Erythrocyte Membrane

The electrokinetic stability of washed normal human erythrocytes is discussed from the point of view of pH, ionic strength, and composition of the suspending medium. Many of the electrophoretic characteristics at low ionic strengths (sorbitol to maintain the tonicity), such as the isopotential points, are shown to arise principally from adsorption of hemolysate. The concept of electrokinetically stable, metastable, and unstable states for the red cell at various ionic strengths is introduced in preference to the general term "cell injury." In the stable state which exists around pH 7.4 for ionic strengths >0.007, no adsorption of hemolysate occurs, in the metastable state reversible adsorption of hemolysate occurs, and in the unstable state, in which ionic strengths and pH ranges are outside the metastable range, the membrane undergoes irreversible hemolysate adsorption or more general hydrolytic degradation. It is deduced from the equivalent binding of CNS, I, Cl, and F, the pH mobility relationships, and the conformation of the ionic strength data in the stable state to a Langmuir adsorption isotherm, that the membrane of the human erythrocyte behaves as a macropolyanion whose properties are modified by gegen ion association and in some instances by hemolysate adsorption. The experimental results are insufficient to establish conclusively the nature of the ionogenic groupings present in the membrane interphase.     

The Interaction of the Polyene Antibiotic Lucensomycin with Cholesterol in Erythrocyte Membranes and in Model Systems: II. Cooperative Effects in Erythrocyte Membranes

Fluorometric titration curves of erythrocyte membranes with increasing lucensomycin have a sigmoid shape. This behavior, which was not present when colloidal cholesterol suspensions were used, is, however, not peculiar to the membrane structure, being also present in cholesterol-containing phospholipid micelles. Addition of acetic acid induced or increased sigmoidicity. This behavior can either be due to a true cooperativity in binding or to different fluorescence yields of the various lucensomycin-membrane complexes. The latter hypothesis appears to be slightly favored.     

Vitamin E and the Peroxidizability of Erythrocyte Membranes in Neonates

We showed the increased susceptibility of neonatal biomembranes to oxidation by a kinetic analysis using an azo compound as a free-radical initiator and red blood cell (RBC) ghosts as a model membrane. When the RBC ghosts were oxidized, oxygen consumption was suppressed during the induction period in which membrane tocopherol was consumed at a constant rate, while increased oxygen uptake was observed after the tocopherol was exhausted. The total tocopherol content was similar in cord, maternal, and adult RBC ghosts, and there were no differences in the induction period (t/_inh) among the three types of ghosts. While the oxygen uptake rate during the induction period (R_inh) was similar in cord and adult ghosts, the rate in the subsequent phase (R_p) was considerably faster in the cord ghosts. Fatty acid analysis in the membrane lipids showed that the active bisallylic hydrogen (active H) content was greater in cord ghosts than in adult ghosts. The active H content closely correlated with the R_p, but did not with the R_inh. The kinetic chain length (KCL), i.e., the ratio of the rate of propagation to that of initiation, was calculated from R_p and tocopherol consumption rate and KCL values were higher in cord ghosts than in adult ghosts. The faster R_p and the higher KCL of the cord ghosts were attributable to a greater active H content rather than to the tocopherol content.     

Iron-Induced Oxidative Stress in Erythrocyte Membranes of Non-Insulin-Dependent Diabetic Nigerians

The presence of higher level of endogenous free radical reaction products in the erythrocyte ghost membrane (EGM) of Non-insulin-dependent diabetes mellitus (NIDDM) subjects compared with that of normal healthy controls has been demonstrated. The EGMs of NIDDM subjects were also shown to be more susceptible to exogenously generated oxidative stress than those of normal healthy individuals. The decreased level of reactive thiol groups in the EGM of NIDDM individuals supported this observation. We propose that the presence of significant levels of non-heme iron in the EGM of NIDDM subjects is an indication of the potential for iron-catalysed production of hydroxy and other toxic radicals which could cause continuous oxidative stress and tissue damage. Oxygen free radicals could therefore be responsible for most of the erythrocyte abnormalities associated with non-insulin-dependent diabetes and could indeed be intimately involved in the mechanism of tissue damage in diabetic complications.     

若干蝙蝠葛碱衍生物对人红细胞膜Ca~(2+)-Mg~(2+)-ATPase活力的影响

本文测定了数种蝙蝠葛碱衍生物对钙调素(CaM)激活的人红细胞膜Ca~(2+)-Mg~(2+)-ATPase活力的影响。结果表明,这些化合物对该酶都有不同程度的抑制作用,其机制表现为竞争性抑制,过量的CaM能完全逆转这些化合物所引起的抑制。当Ca~(2+)-Mg~(2+)-ATPase被胰蛋白酶(trypsin)限制性酶解完全活化后,其活力不再受CaM激活,但仍被这些化合物所抑制。     

Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte

The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.     

Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte

The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells.