PubMed Health⌕ Search

Biomedical subjects

E A Evans

Publications and source records attributed to E A Evans.

At least 19 recordsLinked to original sources
PubMed1991

Lipid hydroperoxides permit deformation-dependent leak of monovalent cation from erythrocytes.

Subtle peroxidative perturbation of normal red blood cells (RBC) using t-butylhydroperoxide creates a leak pathway for monovalent cations that is reversibly activated by cell deformation. To determine what factor promotes expression of this unique membrane defect, we have dissected "peroxidation" into components that can be evaluated separately by comparing K leak from suitably modified RBC during elliptical deformation and parallel control incubation. Selective introduction of phospholipid hydroperoxides into normal RBC membranes successfully induces a deformation-dependent leak pathway having the same phenomenology as that previously documented for cells treated with t-butylhydroperoxide itself (fully recoverable; calcium-independent; inhibited at lower pH; K efflux balanced by Na influx). This leak pathway occurs in the absence of detectable secondary peroxidative change and appears to reflect a direct influence of lipid hydroperoxide. Using micropipette examination of vesicular bilayers reconstituted from RBC lipid extracts, we find that lipid from peroxidized RBC exhibits only a slight tendency to be less cohesive than normal lipid, apparently precluding isolated lipid properties as an explanation for altered permeability barrier function. However, addition of a hydrophobic membrane-spanning peptide to these same lipids significantly diminishes bilayer cohesion, an effect that is exacerbated further by the presence of peroxidized lipid. These observations suggest that lipid hydroperoxide is a necessary, but perhaps not sufficient, factor for induction of this unique leak pathway. Our results may be relevant to the abnormal cation homeostasis of sickle RBC in which deformation of an oxidatively perturbed membrane occurs during the sickling phenomenon.

Biophysical Phenomena↗
PubMed1989

Structure and deformation properties of red blood cells: concepts and quantitative methods.

The lamellar configuration of the red cell membrane includes a (liquid) superficial bilayer of amphiphilic molecules supported by a (rigid) subsurface protein meshwork. Because of this composite structure, the red cell membrane exhibits very large resistance to changes in surface density or area with very low resistance to in-plane extension and bending deformations. The primary extrinsic factor in cell deformability is the surface area-to-volume ratio which establishes the minimum-caliber vessel into which a cell can deform (without rupture). Within the restriction provided by surface area and volume, the intrinsic properties of the membrane and cytoplasm determine the deformability characteristics of the red cell. Since the cytoplasm is liquid, the static rigidity of the cell is determined by membrane elastic constants. These include an elastic modulus for area compressibility in the range of 300-600 dyn/cm, an elastic modulus for in-plane extension or shear (at constant area) of 5-7 X 10(-3) dyn/cm, and a curvature or bending elastic modulus on the order of 10(-12) dyn.cm. Even though small, the surface rigidity of the cell membrane is sufficient to return the membrane capsule to a discoid shape after deformation by external forces. Viscous dissipation in the peripheral protein structure (cytoskeleton) dominates the dynamic response of the cell to extensional forces. Based on a time constant for recovery after extensional deformation on the order of 0.1 sec, the coefficient of surface viscosity is on the order of 10(-3) dyn.sec/cm. On the other hand, the dynamic resistance to folding of the cell appears to be limited by viscous dissipation in the cytoplasmic and external fluid phases. Dynamic rigidities for both extensional and folding deformations are important factors in the distribution of flow in the small microvessels. Although the red cell membrane normally behaves as a resilient viscoelastic shell, which recovers its conformation after deformation, structural relaxation and failure lead to break-up and fragmentation of the red cell. The levels of membrane extensional force which is two orders of magnitude less than the level of tension necessary to lyse vesicles by rapid area dilation. Each of the material properties ascribed to the red cell membrane plays an important role in the deformability and survivability of the red cell in the circulation over its several-month life span.

Animals↗
PubMed1988

Prepro-alpha-factor has a cleavable signal sequence.

MAT alpha Saccharomyces cerevisiae secrete a small peptide mating pheromone termed alpha-factor. Its precursor, prepro-alpha-factor, is translocated into the endoplasmic reticulum and glycosylated at three sites. The glycosylated form is the major product in a yeast in vitro translation/translocation system. However, there is another translocated, nonglycosylated product that contains a previously unidentified modification. Contrary to previous results suggesting that the signal sequence of prepro-alpha-factor is not cleaved, amino-terminal radiosequencing has identified this product as prepro-alpha-factor without its signal sequence, that is, pro-alpha-factor. The translocated, glycosylated proteins are also processed by signal peptidase. Moreover, we have found that both purified eukaryotic and prokaryotic signal peptidase can process prepro-alpha-factor. Experiments using a yeast secretory mutant (sec 18) blocked in transport from the endoplasmic reticulum to the Golgi indicate that the protein is also cleaved in vivo. Finally, characterization of the Asn-linked oligosaccharide present on pro-alpha-factor in the yeast in vitro system by use of specific glucosidase and mannosidase inhibitors indicates that they have had the three terminal glucoses and probably one mannose removed. Therefore they most likely consist of Man8GlcNAc2 structures, identical to those found in the endoplasmic reticulum in vivo.

Amino Acid Sequence↗
PubMed1987

Membrane-associated sickle hemoglobin: a major determinant of sickle erythrocyte rigidity.

Micropipette aspiration tests on single erythrocytes have previously shown that the static rigidity (membrane shear modulus) of oxygenated sickle cells increased with increasing hemoglobin concentration, whereas the rigidity of normal cells was independent of hemoglobin concentration. Moreover, it was observed that after mechanical extension, sickle cells exhibited persistent deformation more frequently and to a greater extent than normal cells. To ascertain if differences in association of normal and sickle hemoglobin with the membrane could account for these observations, we measured rheologic properties of normal membranes reconstituted with sickle hemoglobin and sickle membranes reconstituted with normal hemoglobin. The static rigidity of normal ghosts reloaded with sickle hemoglobin was higher than those of either normal ghosts reloaded with normal hemoglobin or native normal cells. On the other hand, the increased rigidity of native sickle cells decreased to near-normal values following reconstitution with normal hemoglobin. Furthermore, we observed that normal ghosts reconstituted with sickle hemoglobin exhibited persistent bumps after mechanical extension, but no bumps formed on normal ghosts reconstituted with normal hemoglobin. Moreover residual bumps were not produced on sickle cells reloaded with normal hemoglobin. Since mechanical characteristics peculiar to sickle cells could be induced in normal cells by incorporation of sickle hemoglobin, and since normal characteristics could be restored to sickle cells by incorporation of normal hemoglobin, we suggest that the interaction of sickle hemoglobin with the cell membrane is responsible for augmented static rigidity of oxygenated sickle erythrocytes.

Anemia, Sickle Cell↗
PubMed1986

Thermal-mechanical fluctuations enhance repulsion between bimolecular layers.

Beginning with the free energy potential for long-range interactions between parallel flat sheets and the mechanical energy of bending those sheets, we have derived an upper bound for repulsion enhanced by thermal undulations of sheets in a multilamellar array. Through a self-consistent (mean-field) potential, we cover the full range of layer separations from close-in to far-apart, where the enhanced repulsion approaches the weak steric interaction derived previously by Helfrich [Helfrich, W. (1978) Z. Naturforsch. 33a, 305-315]. We have examined the effect of the fluctuation-enhanced repulsion in experimental studies of osmotic (and mechanical) compression of multilamellar lipid arrays, mechanical compression of bilayers immobilized on mica substrates, and controlled adhesion of giant bilayer membrane vesicles.

Chemical Phenomena↗
PubMed1986

Purification of microsomal signal peptidase as a complex.

We report here the purification to near homogeneity of signal peptidase from canine pancreatic microsomes. Purification was monitored using an improved post-translational assay. A 42-fold enrichment over starting membranes was achieved by selective solubilization in nonionic detergent/high-salt buffer followed by gradient sievorptive anion and cation exchange chromatography, hydroxylapatite chromatography, gel filtration, and sucrose gradient velocity sedimentation. When examined by NaDodSO4/PAGE, the purified enzyme consisted of a complex of six polypeptides with apparent molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa subunits were shown to be glycoproteins based on their sensitivity to endoglycosidase H and their ability to bind concanavalin A. We suggest that only one subunit of this complex carries out signal peptide cleavage. The structural association of the other subunits in stoichiometric amounts may reflect their requirement in chain translocation across the microsomal membrane.

Animals↗
PubMed1985

Detailed mechanics of membrane-membrane adhesion and separation. I. Continuum of molecular cross-bridges.

The mechanics of membrane-membrane adhesion are developed for the approximation that the molecular cross-bridging forces are continuously distributed as a normal stress (force per unit area). The significance of the analysis is that the finite range of the cross-bridging forces and the microscopic contact angle are not assumed negligible. Since the cross-bridging and adhesion forces are finite range interactions, there are two membrane regions: a free zone where the membranes are not subject to attractive forces; and an adherent zone where the membranes are held together by attractive stresses. The membrane is treated as an elastic continuum. The approach is to analyze the mechanics for each zone separately and then to require continuity of the solutions at the interface between the zones. Final solution yields the membrane contour and stresses proximal to and within the contact zone as well as the microscopic contact angle at the edge of the contact zone. It is demonstrated that the classical Young equation is consistent with this model. The results show that the microscopic contact angle becomes appreciable when the strength of adhesion is large or the length of the cross-bridge is large; however, the microscopic contact angle approaches zero as the membrane elastic stiffness increases. The solution predicts the width of the contact zone over which molecular bonds are stretched. It is this boundary region where increased biochemical activity is expected. In the classical model presented here, the level of tension necessary to oppose spreading of the contact is equal to the minimal level of tension required to separate the adherent membranes. This behavior is in contrast with that derived for the case of discrete molecular cross-bridges where the possibility of different levels of tension associated with adhesion and separation is introduced. The discrete cross-bridge case is the subject of a companion paper.

Cell Adhesion↗
PubMed1985

Detailed mechanics of membrane-membrane adhesion and separation. II. Discrete kinetically trapped molecular cross-bridges.

In general, membrane-membrane adhesion involves specific molecular binding and cross-bridging reactions. The ideal, classical view is that near equilibrium the forces required to separate adhesive contacts are essentially equal to those induced in the membrane when the contact is formed. In contrast to the classical view, experimental observations often show that negligible levels of tension are induced by the adhesive contact even though the tension required to separate the contact is large enough to rupture the membrane. The deviation in tension levels associated with contact formation and separation appears to be due to the sparse distribution of strong molecular cross-bridges. Here, the mechanics of membrane-membrane adhesion and separation is developed for the case of discrete, kinetically trapped cross-bridges. The solution is obtained by numerical computation of the membrane contour that minimizes the total free energy (membrane elastic energy of deformation plus cross-bridge energies) in the contact zone. This solution is matched with the analytical solution for membrane stresses and geometry derived for the adjacent, unbridged zone. The results yield specific values of the macroscopic tension applied to the membrane in the plane region away from the contact zone and the microscopic angle at the edge of the contact zone. Two disparate values of the macroscopic tension are found: (a) the minimum tension required to separate the adherent membranes; and (b) the maximum tension induced in the membranes when the contact is formed (i.e., the level of tension at which the contact will just begin to spread). The results show that the deviation between these two tensions can be very large and depends strongly on the surface density of cross-bridges. In addition, the results provide an estimate of the restraining forces that anchor receptors within the plane of the membrane.

Cell Adhesion↗
PubMed1983

Bending elastic modulus of red blood cell membrane derived from buckling instability in micropipet aspiration tests.

Observation of cell membrane buckling and cell folding in micropipette aspiration experiments was used to evaluate the bending rigidity of the red blood cell membrane. The suction pressure required to buckle the membrane surface initially was found to be about one-half to two-thirds of the pressure that caused the cell to fold and move up the pipet. A simple analytical model for buckling of a membrane disk supported at inner and outer radii correlates well with the observed buckling pressures vs. pipet radii. The buckling pressure is predicted to increase in inverse proportion to the cube of the pipet radius; also, the buckling pressure depends inversely on the radial distance to the toroidal rim of the cell, normalized by the pipet radius. As such, the pressure required to buckle the membrane with 1 X 10(-4) cm diam pipet would be about four times greater than with a 2 X 10(-4) cm pipet. This is the behavior observed experimentally. Based on analysis of the observed buckling data, the membrane bending or curvature elastic modulus is calculated to be 1.8 X 10(-12) dyn-cm.

Elasticity↗
PubMed1983

Force relaxation and permanent deformation of erythrocyte membrane.

Force relaxation and permanent deformation processes in erythrocyte membrane were investigated with two techniques: micropipette aspiration of a portion of a flaccid cell, and extension of a whole cell between two micropipettes. In both experiments, at surface extension ratios less than 3:1, the extent of residual membrane deformation is negligible when the time of extension is less than several minutes. However, extensions maintained longer result in significant force relaxation and permanent deformation. The magnitude of the permanent deformation is proportional to the total time period of extension and the level of the applied force. Based on these observations, a nonlinear constitutive relation for surface deformation is postulated that serially couples a hyperelastic membrane component to a linear viscous process. In contrast with the viscous dissipation of energy as heat that occurs in rapid extension of a viscoelastic solid, or in plastic flow of a material above yield, the viscous process in this case represents dissipation produced by permanent molecular reorganization through relaxation of structural membrane components. Data from these experiments determine a characteristic time constant for force relaxation, tau, which is the ratio of a surface viscosity, eta to the elastic shear modulus, mu. Because it was found that the concentration of albumin in the cell suspension strongly mediates the rate of force relaxation, values for tau of 10.1, 40.0, 62.8, and 120.7 min are measured at albumin concentrations of 0.0, 0.01, 0.1, and 1.% by weight in grams, respectively. The surface viscosity, eta, is calculated from the product of tau and mu. For albumin concentrations of 0.0, 0.01, 0.1, and 1% by weight in grams, eta is equal to 3.6, 14.8, 25.6, and 51.9 dyn s/cm, respectively.

Erythrocyte Membrane↗
PubMed1982

Extensional flow of erythrocyte membrane from cell body to elastic tether. I. Analysis.

This is the first of two papers on an analytical and experimental study of the flow of the erythrocyte membrane. In the experiment to be discussed in detail in the second paper, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or "tethers" are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnished a "reservoir" of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. In this paper we show that an application of the principle of conservation of mass permits the tether radius (approximately 200 A or less) to be measured with the light microscope as the tether is formed and extended at a constant rate. A static analysis of the axisymmetric cell deformation and tether formation process reveals that the tether radius is uniquely determined by the isotropic tension in the membrane and the elastic constitutive (material) behavior of the tether itself. A dynamic analysis of the extensional flow process reveals that the tether radius must decrease as the velocity of the tether is increased and that the decrease depends on both the viscosity of the membrane and the elasticity of the tether. The analysis also shows that these two factors (membrane viscosity and tether elasticity) are readily decomposed and determined separately when flow experiments are performed at different isotropic tensions.

Elasticity↗
PubMed1982

Extensional flow of erythrocyte membrane from cell body to elastic tether. II. Experiment.

This is the second of two papers on an analytical and experimental study of the flow of erythrocyte membrane. In the experiments discussed here, preswollen human erythrocytes are sphered by aspirating a portion of the cell membrane into a small micropipette; and long, thin, membrane filaments or tethers are steadily withdrawn from the cell at a point diametrically opposite to the point of aspiration. The aspirated portion of the membrane furnishes a reservoir of material that replaces the membrane as it flows as a liquid from the nearly spherical cell body to the cylindrical tether. The application of the principle of conservation of mass permits the tether radius Rt to be measured with the light microscope as the tether is formed and extended at a constant rate. The tether behaves as an elastic solid such that the tether radius decreases as the force or axial tension acting on the tether is increased. For the range of values for Rt is these experiments (100 A less than or equal to Rt less than or equal to 200 A), the slope of the tether-force, tether-radius line is -1.32 dyn/cm. The surface viscosity of the membrane as it flows from cell body to tether is 3 x 10(-3) dyn.s/cm. This viscosity is essentially constant for characteristic rates of deformation between 10 and 200 s-1.

Elasticity↗
PubMed1980

Calibration of beam deflection produced by cellular forces in the 10(-9)--10(-6) gram range.

An experimental procedure and method of analysis are presented for calibration of a thin-beam force transducer. The beam transducer can be produced and calibrated with a minimum coefficient of 10 ng (10(-5) dyne) force per micron (10(-4) cm) deflection, i.e., kB approximately 0.1 dyne/cm. Since beam deflections on the order of 0.1 micron can be detected, forces of a few nanograms can be resolved. Such forces are common in mechanical experiments on microscopic bodies, e.g., biological cells, artificial membrane capsules, droplets, etc.

Biomechanical Phenomena↗
PubMed1980

Analysis of adhesion of large vesicles to surfaces.

An experimental procedure that can be used to measure the interfacial free energy density for the adhesion of membranes of large vesicles to other surfaces is outlined and analyzed. The approach can be used for both large phospholipid bilayer vesicles and red blood cells when the membrane force resultants are dominated by isotropic tension. The large vesicle or red cell is aspirated by a micropipet with sufficient suction pressure to form a spherical segment outside the pipet. The vesicle is then brought into close proximity of the surface to be tested and, the suction pressure reduced to permit adhesion, and the new equilibrium configuration is established. The mechanical analysis of the equilibrium shape provides the interfacial free energy density for the surface affinity. With this approach, the measurable range of membrane surface affinity is 10(-4)-3 erg/cm2 for large phospholipid bilayer vesicles and 10(-2)-10 erg/cm2 for red blood cells.

Adhesiveness↗
PubMed1980

Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells.

An experimental procedure is demonstrated which can be used to determine the interfacial free energy density for red cell membrane adhesion and membrane elastic properties. The experiment involves micropipet aspiration of a flaccid red blood cell and manipulation of the cell proximal to a surface where adhesion occurs. A minimum free energy method is developed to model the equilibrium contour of unsupported membrane regions and to evaluate the partial derivatives of the total free energy, which correspond to the micropipet suction force and the interfacial free energy density of adhesion. It is shown that the bending elasticity of the red cell membrane does not contribute significantly to the pressure required to aspirate a flaccid red cell. Based on experimental evidence, the upper bound for the bending or curvature elastic modulus of the red cell membranes is 10-12 ergs (dyn-cm). Analysis of the adhesion experiment shows that interfacial free energy densities for red cell adhesion can be measured from a lower limit of 10-4 ergs/cm2 to an upper limit established by the membrane tension for lysis of 5-10 ergs/cm2.

Cell Adhesion↗
PubMed1980

Temperature dependence of the viscoelastic recovery of red cell membrane.

The time-dependent recovery of an elongated red cell is studied as a function of temperature. Before release, the elongated cell is in static equilibrium where external forces are balanced by surface elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of a viscoelastic solid material undergoing large ("finite") deformation. The recovery process is characterized by a time constant, tc, that decreases from approximately 0.27 s at 6 degrees C to 0.06 s at 37 degrees C. From this measurement of the time constant and an independent measurement of the shear modulus of surface elasticity for red cell membrane, the value for the membrane surface viscosity as a function of temperature can be calculated.

Adult↗