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.     

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.     

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.     

Depletion flocculation and depletion stabilization of erythrocytes

At dextran (Mw ≈ 500,000) concentrations from 2 to ≈10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations > 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 > 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.

     

Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

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.     

Isolation and characterization of bacteriocin‐producing bacteria from the intestinal microbiota of elderly Irish subjects

Aims

To isolate and characterize bacteriocins produced by predominant species of lactic acid bacteria from faeces of elderly subjects.

Methods and Results

Screening over 70 000 colonies, from faecal samples collected from 266 subjects, using the indicator organisms Lactobacillus bulgaricus LMG 6901 and Listeria innocua DPC 3572, identified 55 antimicrobial‐producing bacteria. Genomic fingerprinting following ApaI digestion revealed 15 distinct strains. The antimicrobial activities associated with 13 of the 15 strains were sensitive to protease treatment. The predominant antimicrobial‐producing species were identified as Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus and Enterococcus spp. A number of previously characterized bacteriocins, including ABP‐118 and salivaricin B (from Lact. salivarius), enterocin B (Enterococcus faecium), lactacin B (Lact. acidophilus), gassericin T and a variant of gassericin A (Lact. gasseri), were identified. Interestingly, two antimicrobial‐producing species, not generally associated with intestinally derived microorganisms were also isolated: Lactococcus lactis producing nisin Z and Streptococcus mutans producing mutacin II.

Conclusion

These data suggest that bacteriocin production by intestinal isolates against our chosen targets under the screening conditions used was not frequent (0·08%).

Significance and Impact of the Study

The results presented are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine.     

Ultrasonic deposition of cells on a surface

Bacteria in water have been driven to a glass surface by an ultrasonic standing wave. On an antibody coated surface capture of Bacillus subtilis var niger (BG) spores (6.6 x 10(6) ml(-1)) was increased more than 200-fold over above the efficiency in the absence of ultrasound. In microfluidic (non-turbulent) systems detection of particles by sensors operating at a surface is diffusion limited. This results in very low detection abilities particularly for particles with diameters greater than 1 microm. Ultrasound is used here to drive bacterial spores to a wall and overcome this limitation. The results confirm: (1) pressure nodes can be formed close to the water-glass interface when the glass thickness is near half the ultrasonic wavelength; (2) the antibody used was able to capture spores in the presence of an ultrasonic standing wave.