Deposition of bacterial cells onto glass and biofilm surfaces

Deposition rates of Pseudomonas putida and Hyphomicrobium ZV620 onto glass and biofilm surfaces were quantified. Both species deposited to glass at a much slower rate than to biofilm. A definite bias by depositing cells for biofilms of their own species was evident in the highest attachment rates observed in this study.     

Probing the silica surfaces by red blood cells

BACKGROUND: The polymorphic forms of silica (silicon dioxide; SiO(2)) interact with the cell membranes of many mammalian cells, including red blood cells (RBCs), causing hemolysis. The electrostatic factor, which is believed to be a major contributor to the silica-cell contact, might have potential interest for the study of cell surface properties. The surface properties of SiO(2) particles are also of interest. METHODS: Washed human RBCs interacted with the particles of highly dispersed fumed silica (Aerosil A-300) and silicas (nine samples) obtained from the initial A-300 by its dehydroxylation at various thermal conditions. Their light scatter (forward and side light scatter) in 0.01% silica solution was measured uninterruptedly within the first 5 min of the reaction by flow cytometry (flow erythrogram). The hemolytic effect of SiO(2) particles was evaluated by photometric measurement of hemoglobin in the supernatant 90 min after the reaction. RESULTS: Light scatter of affected RBCs and the degree of hemolysis revealed that the surface properties of SiO(2) particles had various effects on the RBCs. After thermal reduction of the surface hydroxyl groups, the membranotoxic effect of silica increased and then decreased. CONCLUSIONS: RBCs offer a convenient and informative model for examining the surface properties of silica.     

Micropatterned surfaces for the control of endothelial cell behaviour

Micropatterned materials were synthesised by photoimmobilising the sulphated hyaluronic acid, adequately functionalised with a photoreactive moiety, on glass substrates. Four different patterns (10, 25, 50 and 100 microns) were obtained. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask was well reproduced on the sample surface. Analysis of endothelial cell behaviour on these micropatterned materials was performed in terms of adhesion, locomotion and orientation. Decreasing the stripe dimensions a more fusiform shape of the adhered endothelial cells was observed. At the same time the cell locomotion and orientation were increased. Furthermore, a photoimmobilisation of stripes of HyalS (10 and 100 microns) was performed on a continuous HyalS layer, in turn immobilised on glass substrate. Being excluded a different chemistry between the stripe and the substrate, the influence of topography on the behaviour of endothelia cells was thus envisaged.     

The kinetics of glucose transport in human red blood cells

A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[14C]glucose transport into human red blood cells at temperatures ranging from 0 degrees to 53 degrees C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20 degrees C, but Arrhenius plots of maximum D-[14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0 degrees C of 0.726 +/- 0.0498 s-1 and 12.1 +/- 0.98 s-1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 +/- 3.47 s-1 and 1113 +/- 494 s-1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 +/- 3.10, 127 +/- 4.78, 88.0 +/- 6.17 and 31.7 +/- 5.11 kJ X mol-1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.     

Modeling the kinetics of cell membrane spreading on substrates with ligand density gradient

An analytical model is developed for the effect of surface gradient in ligand density on the adhesion kinetics of a curved elastic membrane with mobile receptors. The displacement and speed of spreading at the edge of adhesion zone as well as the density profile of receptors along the membrane are predicted as a function of time. According to results, in the diffusion-controlled regime, the front edge displacement of adhesion zone and the rate of membrane spreading decreased with increasing the ligand density in a certain direction. Furthermore, the displacement of the edge of the adhesion zone did not scale with the square root of time, as observed on substrates with uniform ligand density.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.     

Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.     

Kinetics of specific and nonspecific adhesion of red blood cells on glass.

Fixed spherical human red blood cells suspended in 17% sucrose were allowed to adhere on either clean glass surfaces or glass surfaces preincubated with antibodies specific to a certain blood group antigen. The adhesion experiments were performed in an impinging jet apparatus, in which the cells are subjected to stagnation point flow. The objective of this study was to compare the efficiencies of nonspecific and specific (antigen-antibody mediated) adhesion of red blood cells on glass surfaces. The efficiency was defined as the ratio of the experimental adhesion rate to that calculated based on numerical solutions of the mass transfer equation, taking into account hydrodynamic interactions as well as colloidal forces. The efficiency for nonspecific adhesion was nearly unity at flow rates lower than 85 microliter/s (corresponding to a wall shear rate, Gw, of 30 s-1 at a radial distance of 110 microns from the stagnation point). The values of efficiency dropped at higher flow rates, due to an increase in the tangential force. The critical deposition concentration is found to occur at 120-150 mM NaCl, which is consistent with the theoretically predicted values. At low salt concentrations, the experimental values are higher than the theoretical ones. Similar discrepancies have been found in many colloidal systems. Introducing steric repulsion by adsorbing a layer of albumin molecules on the glass completely prevents nonspecific adhesion at flow rates below 60 microliter/s (Gw congruent to 15 s-1). The efficiency of specific adhesion depends both on the concentration of antibody molecules on the surface and the flow rate. Normal red cells adhere more readily through antigen-antibody bonds than fixed cells. Fixed spherical cells have a higher adhesion efficiency than fixed biconcave ones.     

Tetrazolium method for studying the catalytic properties of oxidoreductases in cellular organelles immobilized on glass surfaces

A new quantitative method allowing the measurement of the activity of oxidoreductases, as well as the study of their catalytic properties, is proposed. The method is based on photometering a smear of cellular organelles in the course of incubation in medium containing the reductase substrate and an artificial electron acceptor, tetrazolium salt. Catalytic properties of succinate:p-nitrotetrazolium violet reductase, as revealed on the smears, are shown to be identical to those of the reductase in mitochondrial suspension. Under similar conditions the maximal oxidation rate of succinate with p-nitrotetrazolium violet is the same as that in the presence of an acceptor of another type, Wurster's blue. The method allows the study of a number of reductases.     

Stopped-flow kinetics of oxygen uptake and release by embryonic red blood cells.

The processes of O2 uptake and release by the three embryonic haemoglobins contained within early mouse embryonic red blood cells have been studied using dual-wavelength stopped-flow kinetic spectroscopy. The rate of O2 uptake in the pseudo-spherical, nucleated, embryonic red blood cells exhibits a greater than first-order dependence on O2 concentration. The time courses for the release from the red blood cells into dithionite-containing solutions tends towards a limiting rate at high dithionite concentrations. The rates of both the uptake and release processes observed in the embryonic cells are compared with those previously seen for adult mouse red blood cells. A new mathematical model is described which accurately simulates both uptake and release experimental data for the nucleated embryonic red blood cells.     

Radial dispersion of red blood cells in blood flowing through glass capillaries: the role of hematocrit and geometry

The flow properties of blood in the microcirculation depend strongly on the hematocrit (Hct), microvessel geometry, and cell properties. Previous in vitro studies have measured the radial displacement of red blood cells (RBCs) at concentrated suspensions using conventional microscopes. However, to measure the RBCs motion they used transparent suspensions of ghost red cells, which may have different physical properties than normal RBCs. The present study introduces a new approach (confocal micro-PTV) to measure the motion of labeled RBCs flowing in concentrated suspensions of normal RBCs. The ability of confocal systems to obtain thin in-focus planes allowed us to measure the radial position of individual RBCs accurately and to consequently measure the interaction between multiple labeled RBCs. All the measurements were performed in the center plane of both 50 and 100 microm glass capillaries at Reynolds numbers (Re) from 0.003 to 0.005 using Hcts from 2% to 35%. To quantify the motion and interaction of multiple RBCs, we used the RBC radial dispersion (D(yy)). Our results clearly demonstrate that D(yy) strongly depends on the Hct. The RBCs exhibited higher D(yy) at radial positions between 0.4 and 0.8R and lower D(yy) at locations adjacent to the wall (0.8-1R) and around the middle of the capillary (0-0.2R). The present work also demonstrates that D(yy) tends to decrease with a decrease in the diameter. The information provided by this study not only complements previous investigations on microhemorheology of both dilute and concentrated suspensions of RBCs, but also shows the influence of both Hct and geometry on the radial dispersion of RBCs. This information is important for a better understanding of blood mass transport mechanisms under both physiological and pathological conditions.     

Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.     

Adsorption of LLCMK2 cell-grown Sendai virus onto human red blood cells and its release from the virus adsorbed cells

An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with [3H]leucine in LLCMK2 cells and of human red blood cells (RBCs). The efficiency of viral release from the virus-bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C. When 7.8 x 10(2) virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C. In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C. Such RBC-bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell-grown Sendai virus (LLCMK2-Sendai) particles, but not released by treatment with hemagglutinin-neuraminidase protein (Sendai-gp) isolated from egg-grown Sendai virus.     

Flow cytometric determination of PMCA-mediated Ca2+-extrusion in individual red blood cells.

BACKGROUND: Differences among red blood cells in the activity of the plasma membrane Ca2+-ATPase (PMCA) can impact cell signaling and survival. However, no method has been reported that measures this activity directly in individual cells. METHODS: We have designed a novel assay for PMCA activity that uses the fluorescent Ca2+-reporter Fluo4 and flow cytometric analysis. The method recognizes the extrusion of Ca2+ from the cell after a short Ca2+-loading pulse, which avoids the problem of ATP depletion and ascertains activity at Vmax capacity. RESULTS: Our assay is responsive to known PMCA inhibitors, and while not intended for quantitative kinetic analysis of Ca2+-pumping, it can be used to determine qualitative differences between red blood cell populations that vary in PMCA activity. Using this assay, we confirmed that a normal red blood cell population shows heterogeneity with respect to the PMCA Vmax. CONCLUSION: We report a novel assay of PMCA activity in red blood cells that can provide qualitative information on PMCA activity in individual cells.     

Bacterial glycosidases for the production of universal red blood cells

Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the alpha-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.