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Biomedical subjects

E A Evans

Publications and source records attributed to E A Evans.

At least 19 recordsLinked to original sources
PubMed2001

Prospective survey of tick paralysis in dogs.

OBJECTIVE: To obtain information on tick paralysis in dogs, including the nature of disease, host signalment, tick-host relationship, treatment, disease progression and recovery, and preventive measures. DESIGN: A prospective survey of 577 dogs affected by tick paralysis was conducted during 1998. Forty-two veterinary clinics along the eastern coast of Australia were instructed to complete survey forms for the first 15 dogs that presented with tick paralysis during September to November. RESULTS: Five percent of dogs died from tick paralysis. Younger dogs were more likely to survive. Long coat length was associated with a greater tick burden but not greater tick size, whereas coat thickness had no bearing on either. Dogs with mild disease recovered more quickly from tick paralysis. Respiratory and gait scores reflected disease severity and were good prognostic indicators. The size of the tick did not reflect the severity of the clinical condition it induced in the host. No method of tick removal or in situ treatment improved recovery time or reduced mortality. However, the time spent in hospital was significantly less for dogs from which the live tick was manually removed. Inspiratory stridor, evident in some dogs with tick paralysis, was not related to tick attachment on the neck. The use of acepromazine maleate or dexamethasone did not reduce recovery time or mortality. Increasing the dose of tick antitoxin serum (TAS) above 0.1 mL/kg had no effect on mortality or recovery time. Dogs with severe disease that received an additional dose of TAS were significantly less likely to survive. Subcutaneous use of TAS at the site of tick attachment was of no benefit in reducing mortality or time to initial clinical improvement. A registered preventative product had not been used on the majority of dogs. Clipping the coat to search for ticks did not reduce mortality. CONCLUSIONS: Therapy needs to address cardiopulmonary dysfunction that may be due directly to the effect of tick toxin and not just respiratory compromise caused by progressive respiratory muscle failure.

Acepromazine↗
PubMed1994

Molecular maps of red cell deformation: hidden elasticity and in situ connectivity.

Fluorescence-imaged micropipette aspiration was used to map redistribution of the proteins and lipids in highly extended human red blood cell membranes. Whereas the fluid bilayer distributed uniformly (+/- 10 percent), the underlying, solidlike cytoskeleton of spectrin, actin, and protein 4.1 exhibited a steep gradient in density along the aspirated projection, which was reversible on release from deformation. Quantitation of the cytoskeletal protein density gradients showed that skeletal elasticity is well represented by a grafted polymer network with a ratio of surface dilation modulus to shear modulus of approximately 2:1. Fractionally mobile integral proteins, such as band 3, and highly mobile receptors, such as CD59 as well as glycophorin C in protein 4.1-deficient cells, appeared to be squeezed out of areas dense in the underlying network and enriched in areas of network dilation. This complementary segregation demonstrates patterning of cell surface components by cytoskeletal dilation.

Actins↗
PubMed1994

Cooperative action between band 3 and glycophorin A in human erythrocytes: immobilization of band 3 induced by antibodies to glycophorin A.

The ability of transmembrane receptor proteins to change their association with the cytoskeleton in response to ligand binding seems to be a key mechanism of signal transduction across membranes. To investigate the molecular features of this mechanism we have used the red cell membrane as a model system to study signal transduction through the integral protein, glycophorin A. In these studies the lateral mobility of integral proteins was measured in situ by fluorescence recovery after photobleaching, and membrane rigidity was characterized by micropipette aspiration technique. We found that binding either a monoclonal antibody or its monovalent Fab to the exoplasmic domain of glycophorin A in normal red cells immobilized the receptor and rigidified the membrane. Further, immobilization and rigidification did not occur when antibodies were bound to Miltenberger V cells containing a mutant form of glycophorin A lacking the cytoplasmic domain. These results imply that the site of the immobilization/rigidification lies within the membrane skeletal structure, not in exofacial receptor crosslinking, and requires the extended cytoplasmic domain of normal glycophorin A. In addition, we found that glycophorin A immobilization and membrane skeletal rigidification were accompanied by immobilization of band 3 receptors. This unexpected result indicates a cooperative coupling between liganded glycophorin A, band 3, and the membrane skeleton. We speculate that cooperation of this type may represent a general mechanism for cytoskeletal linkage and transformation initiated by receptors with short cytoplasmic sequences, such as integrins.

Animals↗
PubMed1993

Osmotic properties of large unilamellar vesicles prepared by extrusion.

We have examined the morphology and osmotic properties of large unilamellar vesicles (LUVs) prepared by extrusion. Contrary to expectations, we observe by cryo-electron microscopy that such vesicles, under isoosmotic conditions, are non-spherical. This morphology appears to be a consequence of vesicle passage through the filter pores during preparation. As a result when such LUVs are placed in a hypoosmotic medium they are able to compensate, at least partially, for the resulting influx of water by "rounding up" and thereby increasing their volume with no change in surface area. The increase in vesicle trapped volume associated with these morphological changes was determined using the slowly membrane-permeable solute [3H]-glucose. This allowed calculation of the actual osmotic gradient experienced by the vesicle membrane for a given applied differential. When LUVs were exposed to osmotic differentials of sufficient magnitude lysis occurred with the extent of solute release being dependent on the size of the osmotic gradient. Surprisingly, lysis was not an all-or-nothing event, but instead a residual osmotic differential remained after lysis. This differential value was comparable in magnitude to the minimum osmotic differential required to trigger lysis. Further, by comparing the release of solutes of differing molecular weights (glucose and dextran) a lower limit of about 12 nm diameter can be set for the bilayer defect created during lysis. Finally, the maximum residual osmotic differentials were compared for LUVs varying in mean diameter from 90 to 340 nm. This comparison confirmed that these systems obey Laplace's Law relating vesicle diameter and lysis pressure. This analysis also yielded a value for the membrane tension at lysis of 40 dyn cm-1 at 23 degrees C, which is in reasonable agreement with previously published values for giant unilamellar vesicles.

Biophysical Phenomena↗
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↗