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Biomedical subjects

W T Coakley

Publications and source records attributed to W T Coakley.

At least 19 recordsLinked to original sources
PubMed1991

Contact patterns in concanavalin A agglutinated erythrocytes.

Agglutination of human erythrocytes by the lectin concanavalin A is enhanced when the erythrocytes are pretreated with neuraminidase, which removes sialic acids, or with pronase, which degrades both the glycophorins and band 3 protein. In the present work transmission electron microscopy of the enzymatically pretreated erythrocytes shows a regular pattern of interruption of contact between interacting plasma membranes. The lengths characteristic of the pattern were 0.66 and 0.50 microns for pronase- and neuraminidase-pretreated cells, respectively. Agglutination of normal erythrocytes and of neuraminidase-pretreated erythrocytes can be fully reversed by exposure to the competitive inhibitor methyl alpha-D-mannopyranoside. Complete reversal of contact does not occur with pronase-pretreated cells. The comparatively greater tenacity of contact between cells that were treated with pronase before exposure to lectin argues for an involvement of nonspecific interactions in the agglutination process. The results are compared with previously published studies of spatially periodic contact patterns induced by a range of other polymers.

Binding, Competitive↗
PubMed1991

Transport and harvesting of suspended particles using modulated ultrasound.

Polystyrene particles of 9 microns diameter were acoustically concentrated along the axis of a water-filled cylindrical waveguide containing a 3 MHz standing wave field. Modulation of the acoustic field enabled transport of the concentrated particles in the axial direction. Four modulations were investigated: 1, a fixed frequency difference introduced between two transducers; 2, ramping the transducer frequency; 3, tone burst, i.e. sound that is pulsed on and off, allowing intermittent sedimentation under gravity; and 4, switching the sound off to allow continuous sedimentation. The most efficient transport (leaving the fewest particles in suspension) of clumps to one end of the container was achieved with method 1 above. In this system the maximum speed of transport of the axial clumps was 24 mm s-1. A theory developed here for the transport of particles in a pseudo (i.e. slowly moving) standing wave field predicts an upper limit, which increases with particle size, for the speed of an entrained body. For a single 9 microns diameter particle in a field with a spatial peak pressure amplitude of 0.4 MPa this speed would be 0.5 mm s-1. The higher experimental speeds observed here emphasize the value of acoustically concentrating particles into relatively large clumps prior to initiating transport.

Acoustics↗
PubMed1990

Electroacoustic production of murine hybridomas.

The optimal conditions for the production of murine hybridomas by electroacoustic fusion of cells in sugar solutions and of cells in ionic strength media (up to 115 mM NaCl) have been investigated. In the electroacoustic fusion technique cells were brought into contact in a 3 MHz ultrasonic standing wave field and were fused by application of an electric pulse. Hybridomas of murine splenocytes and X63-Ag8.653 mouse myelomas were successfully produced in low ionic strength mannitol solutions and in a range of salt concentrations up to 115 mM. The yield of hybridomas by electroacoustic fusion of cells in mannitol was at least as good as that obtained when cells were fused following conventional dielectrophoresis induced contact. The electroacoustic fusion yield was also comparable to conventional yields when cells were exposed to a pulse in higher ionic strength media where dielectrophoresis induces heating effects. Hybridomas were produced at similar electric field strengths when cells were suspended in high ionic strength media (up to 115 mM NaCl) or in mannitol. The effective electric field strength for hybridoma production was close to that at which trypan blue tests indicated membrane damage.

Animals↗
PubMed1990

Membrane-membrane interactions: parallel membranes or patterned discrete contacts.

Theoretical and experimental studies of thin liquid films show that, under certain conditions, the film thickness can undergo a sudden transition which gives a stable narrower film or ends in film rupture at spatially periodic points. Theoretical analysis have also indicated that similar transitions might arise in the thin aqueous layer separating interacting membranes. Experiments described here show spatially periodic intermembrane contact points and suggest that spontaneous rapid growth of fluctuations can occur on an intermembrane water layer. Normal and pronase pretreated erythrocytes were exposed to 2% Dextran (450,000 Mr) and the resultant aggregates were examined by light and transmission electron microscopy. Cell electrophoresis measurements were used as an index of pronase modification of the glycocalyx. Erythrocytes exposed to dextran revealed a uniform intercellular separation of parallel membranes. This equilibrium between attractive and repulsive intermembrane forces is consistent with the established Derjaguin, Landau, Verwey, Overbeek (DLVO) model for colloidal particle interaction. In contrast to the above uniform separation a spatial pattern of discrete contact regions was observed in cells coming together in dextran following pronase pretreatment. The lateral contact separation distance was 3.0 microns for mild pronase pretreatment and decreased to 0.85 micron for more extensive pronase pretreatments. The system examined here is seen as a useful experimental model in which to study the principles involved in producing either uniform separation or point contacts between interacting membranes.

Cell Adhesion↗
PubMed1990

Depletion flocculation and depletion stabilization of erythrocytes.

At dextran (Mw approximately 500,000) concentrations from 2 to approximately 10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations greater than 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations greater than 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.

Biophysical Phenomena↗
PubMed1990

Spreading of wheat germ agglutinin-induced erythrocyte contact by formation of spatially discrete contacts.

The time dependence of agglutination and cell-cell contact spreading in human erythrocytes exposed to wheat germ agglutinin (WGA) was characterized by light and electron microscopy. Cells (3 x 10(7)/mL) had a threshold lectin concentration in the range of 0.6-2.0 micrograms/mL for initial cell contact. Spreading was essentially completed within 60 and 2 min in undisturbed and gently agitated suspensions, respectively. The cells in large WGA agglutinates retained features of their initial disk form in contrast to the convex outlines of polycation or polyethylene glycol-induced agglutinates. Spreading of contact area was accompanied by development of a pattern of discrete contact regions separated by a distance of the order of 1 micron. Freeze fracture electron microscopy and studies with ferritin-labeled WGA showed no significant aggregation of intramembrane particles or specific lectin receptors under conditions when contact spreading occurred. It is argued that flow stress effects on cells in suspended agglutinates give rise to a situation where opposite membranes, at the leading edge of cell contact, are separated by a thin aqueous layer. When this intercellular water layer exceeds a critical length, it becomes unstable. The layer breaks up by surface wave development to form an array of intracellular water spaces. Formation of the aqueous spaces causes opposite membrane regions to move synchronously toward each other. Lectin molecules crosslink the wave crests to give spatially periodic contact points.

Cell Communication↗
PubMed1990

Microbial content of aerosols produced from suspensions exposed to megahertz frequency ultrasound.

A piezo-electric bowl transducer was used to generate aerosols by focusing ultrasound in the frequency range 1-7 MHz at a liquid/air interface. Atomization at the liquid surface and the production of a fountain contributed to aerosol formation. When the liquid consisted of suspensions of representatives from the viral, bacterial, and yeast groups of micro-organisms (covering a 0.2-11.5 microns size range) living organisms were isolated from the aerosols at all frequencies. The fountains were implicated as a major source of air-borne micro-organisms because significant numbers of isolates were obtained in the presence of fountains but in the absence of obvious atomization, and theoretical predictions make the sizes of droplets arising from atomization at the higher frequencies too small to have carried some of the larger organisms.

Aerosols↗
PubMed1989

Agglutination of Legionella pneumophila by antiserum is accelerated in an ultrasonic standing wave.

The agglutination of Legionella pneumophila (LP) by diluted anti-LP whole rabbit serum has been compared in conventional microwell plates and in capillary containers where the suspension was exposed to a 1 MHz ultrasonic standing wave field. A positive reaction in the standing wave field was detected as a series of cell agglutinates, separated by half an acoustic wavelength (0.75 mm), distributed along the length of the capillary. Agglutination occurred in 60 s or less with ultrasound, while the incubation period for a positive microwell test was often of the order of hours. At a given antiserum concentration, ultrasound-induced agglutination occurred at LP concentrations two-fold lower than those giving a positive result in the microwell plate assays. At cell concentrations near the lower limit for detection of a positive result in the microwell plates a positive reaction was detected in the standing wave field at antiserum concentrations up to 500-fold lower than those forming visible precipitates in the conventional assay.

Agglutination↗
PubMed1989

Membrane-membrane contact: involvement of interfacial instability in the generation of discrete contacts.

The classical approach to understanding the closeness of approach of two membranes has developed from consideration of the net effect of an attractive van der Waals force and a repulsive electrostatic force. The repulsive role of hydration forces and stereorepulsion glycocalyx forces have been recently recognized and an analysis of the effect of crosslinking molecules has been developed. Implicit in these approaches is the idea of an intercellular water layer of uniform thickness which narrows but retains a uniform thickness as the cells move towards an equilibrium separation distance. Most recently an attempt has been made to develop a physical chemical approach to contact which accommodates the widespread occurrence of localized spatially separated point contacts between interacting cells and membranes. It is based on ideas drawn from analysis of the conditions required to destabilize thin liquid films so that thickness fluctuations develop spontaneously and grow as interfacial instabilities to give spatially periodic contact. Examples of plasma membrane behaviour which are consistent with the interfacial instability approach are discussed and experiments involving polycation, polyethylene glycol, dextran and lectin adhesion and agglutination of erythrocytes are reviewed.

Animals↗
PubMed1989

Electroacoustic fusion of millilitre volumes of cells in physiological medium.

A technique is described in which erythrocytes suspended in 1.1 ml of 145 mM NaCl, have been fused by electrofusion. The cells in suspension were brought into close contact by setting up a 3 MHz ultrasonic standing wave in a cylindrical cell container. The aluminium foil base of the container served both to transmit ultrasound and as an electrode for electrofusion. The electric pulse was generated by a capacitor discharge system. The electric field strength required to fuse cells increased as the ionic strength of the cell suspending phase increased. Cells in physiological saline fused at an electric field strength of 7.3 kV/cm with a 50 microseconds pulse.

Cell Fusion↗
PubMed1989

Rapid detection of hepatitis B virus using a haemagglutination assay in an ultrasonic standing wave field.

A haemagglutination test for Hepatitis B virus has been expedited by increasing cell-cell contact through concentrating antibody-coated erythrocytes in an ultrasonic standing wave field. The reactants used were taken from a commercially available Hepatitis B surface antigen screening kit, which is commonly employed in hospital laboratories and blood banks to screen human sera. When performed in microwells, as prescribed in the kit, positive and negative reactions were discernible after 30 min. However, with the ultrasonic technique these reactions can be distinguished within 5 min.

Evaluation Studies as Topic↗
PubMed1988

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers.

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5-2.0% w/v) concentrations of Dextran T500. At concentrations of 5-7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.

Cell Adhesion↗
PubMed1988

Spatially periodic discrete contact regions in polylysine-induced erythrocyte-yeast adhesion.

Cell-cell adhesion occurs when human erythrocytes and yeast cells are suspended together in suprathreshold concentrations of polylysine in saline. The threshold polycation concentration for adhesion depends on cell concentration and decreases with increasing polycation molecular weight. The threshold concentration was similar for erythrocyte-erythrocyte adhesion and for yeast-erythrocyte adhesion. Transmission electron micrographs show that the erythrocytes adhere to yeast as if to engulf the cell. The regions of close contact between the erythrocyte membrane and the yeast cell walls are spatially discrete. The contact separation distance for the asymmetric erythrocyte-yeast adhesion is very similar to that (0.83 micron) observed when polylysine-induced adhesion occurs in the symmetrical erythrocyte-erythrocyte system. The spacing is attributed to the growth of a squeezing wave as an interfacial instability, on the intercellular aqueous layer. Freeze-fracture electron microscopy of cells that were not fixed during preparation for microscopy confirms the discrete nature of contacts between polylysine treated erythrocytes.

Cell Adhesion↗
PubMed1987

The influence of the antiviral drugs amantadine and rimantadine on erythrocyte and platelet membranes and its comparison with that of tetracaine.

The influence of the antivirus drugs amantadine and rimantadine and of the anionic analogue 1-adamantane-carboxylic acid on a range of properties of human erythrocyte membrane and of thrombocytes has been compared with the effect of the local anaesthetic tetracaine. At low antiviral drug concentrations the abilities of the drugs to induce erythrocyte shape change and suppress osmotic haemolysis were quantitatively proportional to their clinical potency (rimantadine more effective than amantadine at the same concentration). Rimantadine was also more effective than amantadine in suppressing influenza virus-erythrocyte fusion and viral induced haemolysis. The antiviral drug effects were qualitatively similar to those induced by tetracaine. At the quantitative level, tetracaine was more efficient than the antiviral drugs in inhibiting osmotic haemolysis, virus membrane fusion and platelet aggregation. In the absence of any specificity of the antiviral drug effects we argue for a lysosomotropic mode of drug action, i.e. that the drugs modify virus-membrane interactions by changing the endosomal or lysosomal pH.

Adamantane↗
PubMed1987

Real time observations of polylysine, dextran and polyethylene glycol induced mutual adhesion of erythrocytes held in suspension in an ultrasonic standing wave field.

A technique which enables cells to be observed in suspension for times of the order of minutes (employing acoustic radiation forces in a 1 MHz ultrasonic standing wave field) is described. Video recordings of the mutual adhesion of human erythrocytes in suspension have been analysed. Concave-ended cell doublets and linear rouleaux developed in 0.5-1.5% w/v Dextran T500 by a gradual (2.5-17 s) increase in the area of cell contact over the cell cross-section. The concave-ended rouleaux form was not seen in polylysine or in polyethylene glycol. In 5-7% dextran and in 20 micrograms/ml polylysine mutual adhesion was a two stage process. Cells first form a strong local contact which persists (without apparently growing in area) for a number of seconds following which the cell surfaces move suddenly to form a spherical doublet. The average initial contact time and engulfment time for cells in 7% Dextran T500 are 18 and 2.7s, respectively. The corresponding values for cells in 20 micrograms/ml, 14 kDa, polylysine are 2.7 and 0.3s. There was no initial contact delay during spherical doublet formation in 1 mg/ml polylysine. Electron microscopy showed that the intercellular seam for spherical doublets formed with all three agglutinating molecules was bent in a wavy lambda approximately equal to 4 micron) profile. The thickness of the intercellular space varied in a spatially periodic way (lambda approximately equal to 0.8 microns) for cells in polylysine. Examples of periodic intercellular spaces were seen by light microscopy in polyethylene glycol induced clumps. The role of interfacial instability in the adhesion processes is discussed.

Cell Adhesion↗
PubMed1987

Hyperthermia effects on the cytoskeleton and on cell morphology.

Human erythrocyte ghost membranes undergo five thermal transitions at temperatures between 50 and 75 degrees C. Spontaneous fragmentation of whole cells occurs at 50 degrees C, a transition temperature which has been associated with denaturation of the cytoskeletal protein spectrin. Haemolysis occurs at 65 degrees C and microvesiculation of the resulting ghost membrane is seen at temperatures in excess of 70 degrees C. The cell fragmentation develops through spatially periodic growth of surface waves on the erythrocyte membrane. The interfacial instability associated with the surface wave growth arises from thermal impairment of the stabilizing function of spectrin. Interfacial instability is also associated with the beading pattern which arises when long processes drawn mechanically from erythrocytes are heated. Similar beading of cell processes is a feature of many cytoskeleton-weakening agents acting on nonerythroid cells. The complexities of the cytoskeletons of eucaryotic cells including structure, composition and interaction of cytoskeletal microfilaments, microtubules and intermediate filaments, both with each other and with the cell membrane, are outlined. Attention is drawn to the importance of the function of proteins which interact with the cytoskeletal elements and to the influence of calcium concentration on those proteins. Actin monomers are denatured (and are no longer polymerizable) at temperatures a few degrees above the growth temperature of the cell source of the actins. Actin in the filament form requires much higher denaturation temperatures. This greater thermal lability of actin monomers would be expected to result (because of treadmilling in microfilaments) in a gradual depolymerization of the filaments. Depolymerization of microtubules occurs at temperatures close to the cell growth temperature and may be dependent on a thermal effect on microtubule-associated proteins. The response of spread interphase mammalian cells to temperatures around 43 degrees C includes central retraction of membrane, loss of microvilli, concentration of organelles in a juxtanuclear position, rounding up of the cell, retention of contact with the substratum by processes which are sometimes beaded and blebbing of the cell membrane. The morphological effects of heat are compared here with those of cytochalasin, colcemid and a number of morphology modifying agents. Blebbing of membrane is a fairly general response of cells to stress. Proteins in blebs diffuse as if released from a lateral constraint. Moderate heating has been shown to cause cortical microfilament separation from the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗
PubMed1979

Instability development in heated human erthrocytes.

Heated human erythrocytes gradually lose their form-maintaining structure as the temperature is increased to 50 degrees C and can behave in some respects as a viscous fluid. We have developed a technique for heating and stressing these cells that is novel, simple and quantitatively precise. We have applied this technique to heated human erythrocytes and have measured instability development in cells. We have employed instability growth theory to calculate a value for an effective surface tension which, in contrast to other methods of membrane surface tension measurement sought to minimize the effects of membrane supporting structural elements. The value obtained for the surface tension of the heated erythrocyte membrane was 0.9 . 10(-6) N/m with a range of variation from 0.4 . 10(-6)N/m to 1.4 . 10(-6) N/m. The methods described may be useful for determining fundamental physical parameters such as internal viscosity and interfacial tension in other systems.

Erythrocytes↗