Electromechanical Interactions in Cell Walls of Gram-Positive Cocci

Isolated cell walls of Staphylococcus aureus and Micrococcus lysodeikticus were found to expand and contract in response to changes in environmental pH and ionic strength. These volume changes, which could amount to as much as a doubling of wall dextran-impermeable volume, were related to changes in electrostatic interactions among fixed, ionized groups in wall polymers, including peptidoglycans. S. aureus walls were structurally more compact in the hydrated state and had a higher maximum charge density than M. lysodeikticus walls. However, they were less responsive to changes in electrostatic interactions, apparently because of less mechanical compliance. In media of nearly neutral pH, S. aureus walls had a net positive charge whereas M. lysodeikticus walls had a net negative charge. These charge differences were reflected in Donnan distributions of mobile ions between wall phases and bulk medium phases. Cell walls of unfractionated cocci also could be made to swell and contract, and wall tonus in intact cells appeared to be set partly by electrostatic interactions and partly by mechanical tension in the elastic structures due to cell turgor pressure. The experimental results led to the conclusions that bacterial cell walls have many of the properties of polyelectrolyte gels and that peptidoglycans are flexible polymers. A reasonable mechanical model for peptidoglycan structure might be a sort of three-dimensional rope ladder with relatively rigid, polysaccharide rungs and relatively flexible polypeptide ropes. Thus, the peptidoglycan network surrounding cocci appeared to be predominantly an elastic restraining structure rather than a rigid shell.     

Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls

At 'low' ionic strength, acid phosphatase bound to plant cell walls exhibits an apparent negative co-operativity, whereas it displays classic Michaelis-Menten kinetics in free solution. Conversely, at 'high' ionic strength, the bound enzyme and the soluble enzyme behave identically. This apparent negative co-operativity is explained by the existence of an electrostatic partition of the charged substrate by the fixed negative charges of the cell wall. Raising the ionic strength suppresses these electrostatic repulsion effects. Calcium may be removed from the cell walls by acid treatment and the acid phosphatase is apparently strongly inhibited. This inhibition occurs together with an increased apparent negative co-operativity of the enzyme. Incubating cell wall fragments previously depleted of calcium with CaCl2 restores the initial behaviour of the enzyme. Calcium, which tightly binds to cell wall pectic compounds, has by itself no effect on the enzyme in free solution. It affects the net charge of the cell wall and therefore the amplitude of electrostatic repulsion effects. Non-linear least-square fitting methods make it possible to estimate the density of fixed negative charges as well as the electrostatic partition coefficient, for both the 'native' and 'calcium-deprived' cell wall fragments. It may be shown directly that calcium loading and unloading in the cell wall controls the electrostatic effects, by monitoring proton extrusion from cell wall fragments upon raising the ionic strength. Proton outflux in the bulk phase is considerably enhanced upon removal of calcium from the cell walls. The main conclusion is that loading and unloading of calcium during cell elongation and division may regulate the activity of cell wall enzymes.     

Dielectric properties of native and decoated spores of Bacillus megaterium.

A general model for use in interpreting dielectric data obtained with bacterial endospores is developed and applied to past results for Bacillus cereus spores and new results for Bacillus megaterium spores. The latter were also subjected to a decoating treatment to yield dormant cells with damaged outer membranes that could be germinated with lysozyme. For both spore types, core ions appeared to be completely immobilized, and decoating of B. megaterium spores did not affect this extreme state of electrostasis in the core. The cortex of B. megaterium appeared to contain a high level of mobile ions, in the cortex of B. cereus. The outer membrane-coat complex of B. megaterium acted dielectrically as an insulating layer around the cortex, so that native dormant spores showed a Maxwell-Wagner dispersion over the frequency range from about 1 to 20 MHz. The decoating treatment resulted in a shift in the dispersion to frequencies below the range of observation. Increases in cell conductivity in response to increases in environmental ionic strength indicated that the coats. of B. megaterium could be penetrated by environmental ions and that they had an inherent fixed charge concentration of about 10 to 20 milliequivalents per liter. In contrast, the dispersion for B. cereus spores was very sensitive to changes in environmental ion concentration, and it appeared that some 40% of the spore volume could be penetrated by environmental ions and that these ions traversed a dielectrically effective layer, either the exosporium or the outer membrane. It appears that dormancy is associated with extreme electrostasis of core ions but not necessarily of ions in enveloping structures and that the coat-outer membrane complex is dielectrically effective but not required for maintenance of extreme electrostasis in the core.     

Ionic interventions that alter the association of troponin C C-domain with the thin filaments of vertebrate striated muscle

The regulatory complex of vertebrate skeletal muscle integrates information about cross-bridge binding, divalent cations and other intracellular ionic conditions to control activation of muscle contraction. Relatively little is known about the role of the troponin C (TnC) C-domain in the absence of Ca2+. Here, we use a standardized condition for measuring isometric tension in rabbit psoas skinned fibers to track TnC attachment and detachment in the absence of Ca2+ under different conditions of ionic strength, pH and MgATP. In the presence of MgATP and Mg2+, TnC detaches more readily and has a 1.5- to 2-fold lower affinity for the intact thin filament at pH 8 and 250 mM K+ than at pH 6 or in 30 mM K+; changes in affinity are fully reversible. The response to ionic strength is lost when Mg2+ and MgATP are absent, whereas the response to pH persists, suggesting that weaker electrostatic TnC-TnI-TnT interactions can be overridden by strongly bound cross-bridges. In solution, titration of a fluorescent C-domain mutant (F154W TnC) with Mg2+ reveals no significant changes in Mg2+ affinity with pH or ionic strength, suggesting that these parameters influence TnC binding by acting directly on electrostatic forces between TnC and TnI rather than by changing Mg2+ binding to C-domain sites III and IV.     

Surface charge measurements on Micrococcus lysodeikticus and the catalytic implications for lysozyme

Electrophoresis measurements on Micrococcus lysodeikticus have shown that the net surface charge density on the cell wall is constant at around -1.5 microC/cm2 for the pH range 4-8. This result has enabled a quantitative analysis to be made of how the electrostatic field associated with the negatively charged cell wall influences the ionic strength and pH dependency of the lytic activity of lysozyme towards M. lysodeikticus. A dominant effect is the creation of a local pH gradient at the cell wall, and at high ionic strengths the lytic activity is found to be controlled by an electrostatic force of attraction between the lysozyme molecule and the cell wall. As the ionic strength of the supporting electrolyte is decreased, however, an electrostatic force of repulsion becomes dominant and is associated with a negative charge carried by the lysozyme molecule, which could possibly be the ionized Asp-52 residue at the active site. This is considered to arise from the fact that at low ionic strengths the fine details of the heterogeneous charge distribution on the cell wall and lysozyme molecule are only partially screened by counter ions.     

Ionic control of acid phosphatase activity in plant cell walls

Abstract. Purified acid phosphatase from sycamore cell walls is not activated by increasing the ionic strength of the reaction mixture. However activation occurs when the enzyme is bound to small cell wall fragments. The apparent activation of the bound enzyme by ions is paralleled by a decline of the substrate concentration C _1/2, that results in half of the maximum rate. Above ionic strengths of about 0.05 the bound and solubilized enzyme forms behave in the same manner. Titration of cell wall fragments at different ionic strengths show that the local pH, inside the cell wall fragments, is lower than the pH in bulk solution. These results are explained in the light of poly-electrolyte theory. The negative charges of the cell walls generate an electrostatic potential that results in the attraction or repulsion of ions. The local concentration of organic phosphate (the substrate of the enzyme) is then lower than its concentration in bulk solution. This concentration difference explains that the value of C _1/2, or of the apparent K_m of the bound enzyme, is greater than the true K_m of the solubilized enzyme. Increasing the ionic strength tends to equalize bulk and local ion concentrations, and therefore apparently activates the bound enzyme.     

Relative Contribution of the Cell Wall, Cytoplasmic Membrane, and Cytoplasm to the Gram-Positive Characteristic of Bacillus megaterium.

Bartholomew, J. W. (University of Southern California, Los Angeles), and Thomas Cromwell. Relative contribution of the cell wall, cytoplasmic membrane, and cytoplasm to the gram-positive characteristic of Bacillus megaterium. J. Bacteriol. 90:643-647. 1965.-A comparison of the roles of the cell wall, cytoplasmic membrane, and cytoplasmic components revealed that the intact cell wall was the dominant contributor to the gram-positive state. Protoplasts of Bacillus megaterium were confirmed as being gram-negative, as reported by Gerhardt et al. The "gram-positive protoplast" report of Amano et al. was shown to be a laboratory-produced artifact, resulting from the comparison of smears made from saline suspensions of Escherichia coli cells with smears made from formalin-sucrose suspensions of B. megaterium protoplasts.     

Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls.

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Unusual electrostatic effects on binding of C1q to anionic liposomes: role of anionic phospholipid domains and their line tension.

The binding of 125I-C1q to anionic liposomes was studied as a function of protein concentration, pH, ionic strength, and anionic lipid composition. The maximum amount of protein bound per micromole of lipid was very sensitive to electrostatic factors, increasing strongly with decreased pH and ionic strength or increased anionic lipid content. The apparent association constant was independent of these electrostatic factors, however, in marked contrast to studies on basic peptide binding to anionic lipid vesicles. Microscopic observations of large unilamellar liposomes containing fluorescently labeled C1q or phosphatidylglycerol demonstrated, under conditions causing strong electrostatic interactions, that C1q and anionic lipids colocalized into domains whose radii of curvature were higher than that of the surrounding lipid. These domains were observed to bud and pinch off into brightly fluorescent vesicles. We propose a model for all of these observations in which the line tension or edge energy at the boundary of the domain resists its increase in circumference as the domain grows by electrostatic effects on binding, eventually resulting in vesiculation. We propose that under favorable electrostatic conditions, as larger domains form the edge energy balances the increases in the electrostatic contribution to binding, resulting in a net binding energy independent of electrostatic factors.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.     

Effects of NaCl, pH and Ca2+ on autolysis of isolated tomato fruit cell walls

Cell walls isolated from ripening tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall-bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 m M NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five m M CaCl₂ did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca²⁺ levels and ionic strength of the apoplast.     

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: Computational and experimental evaluation by mutational analysis

Cu, Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu, Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited pro-tonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (>10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu, Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143. © 1994 Wiley-Liss, Inc.     

Permeation of macromolecules into polyelectrolyte microcapsules

Polyelectrolyte microcapsules (PEMCs) have been prepared by coating red blood cells with the polyelectrolytes poly(styrenesulfonate), poly(allylamine hydrochloride), and dextran sulfate applying the layer-by-layer technique with subsequent dissolution of the core. The capsule permeability for human serum albumin (HSA) was studied as a function of the ionic strength and pH by means of confocal microscopy. PEMCs produced with dextran sulfate and poly(allylamine hydrochloride) show a significant increase in permeability for HSA at salt concentrations over 1 mM. For PEMCs prepared with poly(styrenesulfonate) and poly(allylamine hydrochloride) the limiting salt concentration is 5 mM. No pH dependence for permeation was observed. A correlation between the permeation and adsorption of HSA on the PEMC walls was investigated. Finally, a mechanism for the permeability, combining electrostatic interactions, and the presence of pores in the polymer layers is presented confirmed by the considerable increase of permeation of charged molecules in the presence of salt and the permeation of neutral molecules regardless of the ionic strength.     

Aggregation of isolated myofibrils stimulated by their contraction. II. Aggregation mechanism

Aggregation of contracted myofibrils was studied as a function of experimental conditions at myofibril contraction. The aggregation rate increased at higher concentrations of suspensions and at increased ionic strength of the medium to achieve the maximum at 0.1 M KCL in the last case. The aggregate sizes grow with an increase of ionic strength and concentration of MgATP and reduce with addition of F-actin. Aggregation of myofibrils develops only in the case of their complete or significant contraction. It was suggested that aggregation is stimulated by dehydration of myofibril at contraction.     

Membrane interactions in nerve myelin. I. Determination of surface charge from effects of pH and ionic strength on period.

We have used x-ray diffraction to study the interactions between myelin membranes in the sciatic nerve (PNS) and optic nerve (CNS) as a function of pH (2-10) and ionic strength (0-0.18). The period of myelin was found to change in a systematic manner with pH and ionic strength. PNS periods ranged from 165 to 250 A or more, while CNS periods ranged from 150 to 230 A. The native periods were observed only near physiological ionic strength at neutral or alkaline pH. The smallest periods were observed in the pH range 2.5-4 for PNS myelin and pH 2.5-5 for CNS myelin. The minimum period was also observed for PNS myelin after prolonged incubation in distilled water. At pH 4, within these acidic pH ranges, myelin period increased slightly with ionic strength; however, above these ranges, the period increased with pH and decreased with ionic strength. Electron density profiles calculated at different pH and ionic strength showed that the major structural alteration underlying the changes in period was in the width of the aqueous space at the extracellular apposition of membranes; the width of the cytoplasmic space was virtually constant. Assuming that the equilibrium myelin periods are determined by a balance of nonspecific forces/i.e., the electrostatic repulsion force and the van der Walls attractive force, as well as the short-range repulsion force (hydration force, or steric stabilization), then values in the period-dependency curve can be used to define the isoelectric pH and exclusion length of the membrane. The exclusion length, which is related to the minimum period at isoelectric pH, was used to calculate the electrostatic repulsion force given the other forces. The electrostatic repulsion was then used to calculate the surface potential, which in turn was used to calculate the surface charge density (at different pH and ionic strength). We found the negative surface charge increases with pH at constant ionic strength and with ionic strength at constant pH. We suggest that the former is due to deprotonation of the ionizable groups on the surface while the latter is due to ion binding. Interpretation of our data in terms of the chemical composition of myelin is given in the accompanying paper (Inouye and Kirschner, 1988). We also calculated the total potential energy functions for the different equilibrium periods and found that the energy minima became shallower and broader with increasing membrane separation. Finally, it was difficult to account directly for certain structural transitions from a balance of nonspecific forces.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation and characterization of an active lysozyme derivative: Kyn 62-lysozyme.

A novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with hydrochloric acid in frozen solution at -10 degrees C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modification, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active). Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme. Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.     

Skinned muscle fibers of Aplysia: potentiation of contraction by "background" calcium and lack of effect of cyclic AMP

1. Aplysia buccal muscle E1 can be skinned with saponin in a low ionic strength medium. Pulses of calcium, which were ineffective at causing contraction in intact fibers, elicited contraction in skinned fibers. 2. Tension in skinned fibers increased at [Ca2+] greater than 10(-7) M and was maximal at 6 x 10(-7) M. 10(-5) M [Ca2+] caused irreversible damage to the fibers. 3. Fibers did not exhibit "catch", i.e. they relaxed quickly upon removal of calcium. 4. Optimal pH for tension was 7.0. 5. Contractile responses to calcium pulses were increased by raising "background" [Ca2+] to 10(-7) M. 6. Cyclic AMP (10(-4) and 10(-3) M) had no effect on tension.     

Intranasal M Cell Uptake of Nanoparticles Is Independently Influenced by Targeting Ligands and Buffer Ionic Strength

In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to underlying immune cells. Previous studies suggested that high affinity ligands targeting M cells may be used to deliver mucosal vaccines; here, we show that particle composition and dispersion buffer ionic strength can independently influence their uptake in vivo. First, addition of a poloxamer 188 to nanoparticle formulations increased uptake of intranasally administered nanoparticles in vivo, but the effect was dependent on the presence of the M cell-targeting ligand. Second, solvent ionic strength is known to effect electrostatic interactions; accordingly, reduced ionic strength increased the electrostatic potential between the epithelium and the particles. Interestingly, below a critical ionic strength, intranasal particle uptake in vivo significantly was increased even when controlled for osmolarity. Similar results were obtained for uptake of bacterial particles. Surprisingly, at low ionic strength, the specific enhancement effect by the targeting peptide was negligible. Modeling of the electrostatic forces predicted that the enhancing effects of the M cell-targeting ligand only are enabled at high ionic strength, as particle electrostatic forces are reduced through Debye screening. Thus, electrostatic forces can have a dramatic effect on the in vivo M cell particle uptake independent of the action of targeting ligands. Examination of these forces will be helpful to optimizing mucosal vaccine and drug delivery.     

Red blood cells experience electrostatic repulsion but make molecular adhesions with glass.

We have studied the detachment of unfixed red cells from glass coverslips under unit gravity and by centrifugation in buffered isotonic solutions over a range of ionic strengths. Cell-glass contact areas and separation distances were measured by quantitative interference reflection microscopy. Detachment under unit gravity is highly dependent on ionic strength: dilution increases electrostatic repulsion and greatly reduces the proportion of adherent cells. However, even at 1.5 mM some cells stick. Over the range 3-110 mM such adherent cells are progressively removed by increasing centrifugal forces, but in a manner virtually independent of ionic strength. This fact, together with the irreversibility of pre-adherent cells as ionic strength is progressively reduced, as well as the resistance of cells to lateral shearing forces, provide evidence sufficient to reject the notion of secondary minimum adhesion for unfixed cells at any ionic strength down to 1.5 mM. We conclude that all unfixed cells that stick at ionic strengths from 157 to 1.5 mM make molecular contacts with glass. Comparison with long range force calculations suggests that to penetrate the electrostatic repulsion barrier the contact regions are unlikely to have average surface properties. A new method that compares frequency distributions of contact areas with responses to detachment forces shows that detachment forces are not linearly related to contact areas. This lack of relationship is less clearly evident for rigid glutaraldehyde-fixed cells and may therefore depend on the degree of cellular deformability.