The lateral separation of contacts on erythrocytes agglutinated by polylysine

The form of contact seam (whether a continuous parallel seam or membranes in spatially periodic contact) has been characterized for normal and for neuraminidase pretreated human erythrocytes following adhesion in solutions of polylysine in the molecular mass range 10–225 kDa at concentrations from 0.5 to 1.0 mg/mL. The adhesion contact seam was spatially periodic for all normal control cells in polylysine. The lateral separation of contacts decreased from 1.6 to 0.8 μm as the concentration of 225 kDa polylysine was increased threefold from the adhesion threshold value. The separation distance did not change further even at high polymer concentrations that increased the electrophoretic velocity to positive values over twice the modulus of the velocity of control cells. The probability of cell adhesion decreased at these high polymer concentrations. The lateral contact separation increased and cell adhesion decreased for cells pretreated with neuraminidase. Cell adhesion did not occur when neuraminidase reduced the cell electrophoretic velocity modulus by 30%. Following neuraminidase pretreatments that allowed a small amount of adhesion, the cell contact seam was continuous rather than spatially peridic. The results show that a procedure that increases (e.g., polymer concentration increase) or decreases (e.g., enzyme removal of polycation crosslinking site) attraction leads to shorter (to a limiting value) or longer lateral contact separation, respectively.     

Promotion and inhibition of vesicle fusion by polylysine

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.     

Interpretation of particle spectra of electronic counters by microscopical methods

1. Using monocultures or single species dominated natural phytoplankton, cell counts and volume estimations obtained by visual and electronic methods show reasonable agreement. Calibration seems possible (Figs. 1–2).
2. Further examples given (Fig. 3–4) show that microscopical identification of the volume peaks in electronic counter spectra of natural seston is not quite simple. Phytoplankton peaks may not be detected in the Coulter spectrum. Shifts of Coulter peaks to the left side of the visual spectrum may be found when cylindrical, elongated or needle-like phytoplankton dominate the sample.
3. Both visual and electronic methods include potentially large errors. Possibly particle volume is either overestimated by the microscope and/or underestimated by the Coulter counter (Figs. 1a, 2, 3b, 4b, c; Table 2). In grazing studies both methods should be employed. Mutual corrections may be possible, based on the type of the seston present (size and nature of phytoplankton cells and detritus particles). In each case both techniques can yield complementary information about the seston investigated. When performing multitube analysis, screening tests of the samples as described by VANDERPLOEG (1981) are recommended.
4. Detritus, especially the different types of aggregated particles, offers severe problems. In the analysis of detritus-rich samples both methods give unreliable results.
5. In most cases estimates of volume, obtained by microscopical and electronic methods, are used as auxiliary parameters. It is the relation between microscopical or Coulter volume and other parameters (e.g. Coulter volume versus POC and phytoplankton volume versus chlorophyll content) that can give useful ecological information.

     

The lateral separation of contacts on erythrocytes agglutinated by polylysine.

The form of contact seam (whether a continuous parallel seam or membranes in spatially periodic contact) has been characterized for normal and for neuraminidase pretreated human erythrocytes following adhesion in solutions of polylysine in the molecular mass range 10-225 kDa at concentrations from 0.5 to 1.0 mg/mL. The adhesion contact seam was spatially periodic for all normal control cells in polylysine. The lateral separation of contacts decreased from 1.6 to 0.8 microns as the concentration of 225 kDa polylysine was increased threefold from the adhesion threshold value. The separation distance did not change further even at high polymer concentrations that increased the electrophoretic velocity to positive values over twice the modulus of the velocity of control cells. The probability of cell adhesion decreased at these high polymer concentrations. The lateral contact separation increased and cell adhesion decreased for cells pretreated with neuraminidase. Cell adhesion did not occur when neuraminidase reduced the cell electrophoretic velocity modulus by 30%. Following neuraminidase pretreatments that allowed a small amount of adhesion, the cell contact seam was continuous rather than spatially periodic. The results show that a procedure that increases (e.g., polymer concentration increase) or decreases (e.g., enzyme removal of polycation crosslinking site) attraction leads to shorter (to a limiting value) or longer lateral contact separation, respectively.     

Polymorphism of actin paracrystals induced by polylysine

We describe a method for the induction of different polymorphic forms of actin filament paracrystals. This polymorphism is probably based on differences in the stagger and/or polarity of adjacent filaments in single-layered paracrystals and by superposition of different layers in multilayered paracrystals. The helical parameters defining the filament geometry are indistinguishable for the different polymorphic forms observed and for the four different actins used. Analysis of these paracrystals, some of which are ordered to better than 2.5 nm, should provide a reference structure suitable for alignment and orientation within the actin filament of high resolution models of the actin monomer obtained from crystal data.     

Interfacial instability and the agglutination of erythrocytes by polylysine

Human erythrocytes have been exposed to poylysine of molecular weight range 4 to 220 kDa and concentration range 0.5 to 2,000 /ml at 37°C. Threshold concentrations for cell agglutination by the polycation have been determined for the samples of different molecular weight. Light and electron micrographs show that, in the erythrocyte agglutinates, cell-cell contact is generally made only at discrete, spatially periodic, regions which are distributed over a significant part of the cell surface. The average spacing between contact regions is 0.83 m. The cell membrane has a wavy profile between contact regions. Agglutination occurs only in cell samples whose electrophoretic mobility is significantly altered by polylysine and, in agreement with a previous report, occurs even when the electrophoretic mobility reaches high positive values. The electrophoretic mobility data implies that agglutination requires some protrusion of polylysine from the cell glycocalyx. We discuss how a resulting net attractive intercellular force could act to destabilize the aqueous layer between two cells, allowing surface wave growth which results in spatially periodic contact regions. Examples of situations where cell and membrane contact might be explained by the general concept of interfacial instability are discussed.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

The molecular weight cut-off of microcapsules is determined by the reaction between alginate and polylysine

Mammalian cells encapsulated in alginate-polylysine microcapsules are used as artificial organs in cancer research and in biotechnology. These applications require microcapsules with a reproducible mol. wt. cut-off. The high cost of the polycation, polylysine, requires an efficient preparation procedure. This article shows that the overall reported contact time of 5 minutes at ambient conditions should be increased several times in order to reach a maximal binding between the calcium alginate beads and 0.1% (w/v) polylysine solutions. An increase of the polylysine concentration from 0.0125% to 0.8% (w/v) resulted in a faster maximal binding, but the amount of polylysine bound increased also. Immersion of calcium alginate beads with a diameter of 750 mum, prepared from 1 mL alginate, in 30 mL of a 0.8% (w/v) polylysine solution, resulted in a polylysine spill of more than 89%. The time required to reach a maximal binding was related to the reaction temperature. The interaction zone between calcium alginate beads and fluorescein isothiocyanate-labeled polylysine solutions was visualized with a confocal laser scanning microscope as a function of time. Microcapsules, prepared at 40 degrees C with 0.1% (w/v) polylysine solutions with mol. wts. between 12 and 249.2 kD, were permeable for fluorescein isothiocyanate-labeled dextran, mol. wt. 4.7, but not for 40.5 kD. Higher polylysine concentrations resulted in a membrane with a mol. wt. cut-off lower than 4.7 kD. (c) 1993 John Wiley & Sons, Inc.     

The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes

Polypeptide synthesis using either phenylalanine or lysine was initiated on Escherichia coli ribosomes; then the position and conformation of the nascent peptide were monitored by fluorescence techniques. To this end, fluorophores had been attached to the amino terminus of each nascent peptide, and major differences were observed as chain extension occurred. Polyphenylalanine appeared to build up as a hydrophobic mass adjacent to the peptidyl transferase center while polylysine apparently was extended directly from the ribosome into the surrounding solution. An explanation for these differences may be provided by the physical and chemical properties of each polypeptide. These properties may be responsible for the route by which each peptide exits the peptidyl transferase center as demonstrated by the different sensitivity of each to inhibition by erythromycin.