Cell size and mutual cell adhesion. II. Evidence for a relation between cell size, long-range electrostatic repulsion and intercellular adhesiveness during density-regulated growth in suspension.

The strength of the long-range electrostatic repulsion forces on HeLa cells is measured by agglutinative titration using low molecular weight polylysine (M.W. 11,000). Repulsion forces, found to be present on the smaller HeLa cells from density-inhibited suspension cultures, are weakened by incubation of the cells in hypotonic NaCl solutions. Repulsion forces, found to be absent on the larger cells from fast growing cultures, can be induced on these cells by incubation in hypertonic NaCl solutions. Both effects of anisotonicity are reversible, and disappear on restoration of the medium to normal tonicity. Induction of repulsion forces on fast growing cells is prevented by previous treatment of the cells with neuraminidase. Neuraminidase also abolishes repulsion on density-inhibited cells. It is proposed that alterations of the cell size, produced by anisotonicity or occurring during growth in isotonic suspension medium, affect mutual cell adhesiveness by modifying the strength of the repulsion forces generated by cell surface sialic acids.     

Colloidal iron binding to the surface of HeLa cells, spreading in monolayer culture.

We have used the colloidal iron (CI) binding technique, adapted for transmission electron microscopy, for semiquantitative evaluation of the negative charge density at the surface of HeLa cells in monolayer culture. The surface area increases when HeLa cells spread on the substrate. This increase brings about a decrease in the thickness of the CI rim, indicating a decrease in negative surface charge density. This phenomenon implicates lowering of the electrostatic repulsion, and explains the formation of intercellular contacts at the level of spread parts of the cell. Because of lack of penetration, CI particles are absent in regions of cose apposition between cells and between cells and substrates. Absence of CI binding in broader intercellular or cell-substrate spaces was explained through masking of the anionic groups.     

A correlation between the effects of calcium on intercellular adhesion and electrostatic repulsion between cells.

Calcium ions enchance the mutual adhesiveness of HeLa cells harvested from suspension cultures in which growth is density inhibited. No significant effect of calcium is observed on the mutual adhesiveness of HeLa cells from fast growing suspension cultures. Agglutinative titration of the cells using poly-L-lysine, mol. wt 15000, shows that calcium ions reduce the strength of the repulsive forces on density inhibited HeLa cells. The agglutination curve of the nonrepulsive fast growing HeLa cells is not significantly modified by the addition of calcium. The results support the conclusion that the effect of calcium on the mutual adhesiveness of density inhibited cells is due to a weakening of the repulsive forces on these cells.     

Colloidal iron binding to the surface of HeLa cells,spreading in monolayer culture

Summary We have used the colloidal iron (CI) binding technique, adapted for transmission electron microscopy, for semiquantitative evaluation of the negative charge density at the surface of HeLa cells in monolayer culture. The surface area increases when HeLa cells spread on the substrate. This increase brings about a decrease in the thickness of the CI rim, indicating a decrease in negative surface charge density. This phenomenon implicates lowering of the electrostatic repulsion, and explains the formation of intercellular contacts at the level of spread parts of the cell. Because of lack of penetration, CI particles are absent in regions of close apposition between cells and between cells and substrates. Absence of CI binding in broader intercellular or cell-substrate spaces was explained through masking of the anionic groups.Supported by a Grant from the Algemene Spaar- en Lijfrentekas Cancer FundThe authors acknowledge the technical assistance of B. Buysse, O. Claeys and J. Roels van Kerckvoorde     

Critical polyelectrolyte adsorption under confinement: planar slit, cylindrical pore, and spherical cavity

We explore the properties of adsorption of flexible polyelectrolyte chains in confined spaces between the oppositely charged surfaces in three basic geometries. A method of approximate uniformly valid solutions for the Green function equation for the eigenfunctions of polymer density distributions is developed to rationalize the critical adsorption conditions. The same approach was implemented in our recent study for the "inverse" problem of polyelectrolyte adsorption onto a planar surface, and on the outer surface of rod-like and spherical obstacles. For the three adsorption geometries investigated, the theory yields simple scaling relations for the minimal surface charge density that triggers the chain adsorption, as a function of the Debye screening length and surface curvature. The encapsulation of polyelectrolytes is governed by interplay of the electrostatic attraction energy toward the adsorbing surface and entropic repulsion of the chain squeezed into a thin slit or small cavities. Under the conditions of surface-mediated confinement, substantially larger polymer linear charge densities are required to adsorb a polyelectrolyte inside a charged spherical cavity, relative to a cylindrical pore and to a planar slit (at the same interfacial surface charge density). Possible biological implications are discussed briefly in the end.     

Induced flocculation of animal cells in suspension culture

Recycle unit operations for suspension cell cultures may be improved by flocculation of the cells. A flocculant search was conducted, and it was found that where strongly cationic polymers were severly toxic to the cells, neutral and anionic polymers were nontoxic and ineffective at flocculating the cells. A weak and poorly soluble polycation, poly-L-histidine, was capable of flocculating cultures of CHO, HeLa, U-937, and CRL 1606 hybridoma cells with no toxicity to the majority of cells. In addition to the lowered acute toxicity, the polymer treatment left the cells in a growing state for recycle. Flocculation was found to be mediated by precipitates of the polymer. The low toxicity of poly-L-histidine is probably due to its low solubility and charge at physiological pH. Nonelectrostatic interactions may also play a role.     

Electrostatic repulsion among erythrocytes in tube flow, demonstrated by the thickness of marginal cell-free layer

Electrostatic repulsion among erythrocytes in flow was evaluated through measurement of the thickness of the marginal cell-free layer in narrow glass tubes of 20-50 microns in inner diameter. To reduce the electrostatic repulsive force, due mainly to sialic acid of the membrane glycoproteins, human erythrocytes were treated with neuraminidase. The surface negative charge of the erythrocytes, as determined from the electrophoretic mobility using free-flow electrophoresis, was found to be proportional to the sialic acid content. When erythrocytes with decreased sialic acid content flowed through narrow tubes, the thickness of cell-free layer determined using an image processor increased even in the absence of erythrocyte aggregation in the suspension. The effect was more pronounced at acidic pH. The addition of Dextran T-70 (70,400 Mol. Wt.) further increased the cell-free layer thickness due to erythrocyte aggregation. Thus, reducing the negative charge density on the erythrocyte surface by itself accelerates the axial accumulation of erythrocytes in flow due to the decreased electrostatic repulsive force between the cells, even in the absence of erythrocyte aggregation.     

Histochemical study of the distribution of sialic acid on the surface of HeLa cells in culture

Summary The distribution of sialic acid on the surface of HeLa cells is studied using Hale's staining technique. Treatment of the cells with neuraminidase before staining, indicates that the staining technique is specific for the demonstration of sialic acid.HeLa cell monolayers, grown in Leighton tubes, are treated with a solution of E.D.T.A. During separation and rounding up, cells are fixed in calcium-formalin and stained. We found a gradual increase of the Hale's positivity during treatment with E.D.T.A.HeLa cells from suspension cultures are grown in Rose-chambers. They are fixed and stained after various periods of incubation. We found a decrease of Hale's positivy during spreading out of cells and monolayer formation.These findings are discussed in terms of surface charge density and formation of stable cell contacts.The authors thank Mr. C. Dragonetti for technical assistance.     

Repulsion between Oppositely Charged Planar Macroions

The repulsive interaction between oppositely charged macroions is investigated using Grand Canonical Monte Carlo simulations of an unrestricted primitive model, including the effect of inhomogeneous surface charge and its density, the depth of surface charge, the cation size, and the dielectric permittivity of solvent and macroions, and their contrast. The origin of the repulsion is a combination of osmotic pressure and ionic screening resulting from excess salt between the macroions. The excess charge over-reduces the electrostatic attraction between macroions and raises the entropic repulsion. The magnitude of the repulsion increases when the dielectric constant of the solvent is lowered (below that of water) and/or the surface charge density is increased, in good agreement with experiment. Smaller size of surface charge and the cation, their discreteness and mobility are other factors that enhance the repulsion and charge inversion phenomenons.     

Algal Flocculation with Synthetic Organic Polyelectrolytes

The feasibility of removing algae from water and wastewater by chemical flocculation techniques was investigated. Mixed cultures of algae were obtained from both continuous- and batch-fed laboratory reactors. Representative cationic, anionic, and nonionic synthetic organic polyelectrolytes were used as flocculants. Under the experimental conditions, chemically induced algal flocculation occurred with the addition of cationic polyelectrolyte, but not with anionic or nonionic polymers, although attachment of all polyelectrolyte species to the algal surface is shown. The mechanism of chemically induced algal flocculation is interpreted in terms of bridging phenomena between the discrete algal cells and the linearly extended polymer chains, forming a three-dimensional matrix that is capable of subsiding under quiescent conditions. The degree of flocculation is shown to be a direct function of the extent of polymer coverage of the active sites on the algal surface, although to induce flocculation by this method requires that the algal surface charge must concurrently be reduced to a level at which the extended polymers can bridge the minimal distance of separation imposed by electrostatic repulsion. The influence of pH, algal concentration, and algal growth phase on the requisite cationic flocculant dose is also reported.     

New insights into the mechanism of gelation of alginate and pectin: charge annihilation and reversal mechanism

Studies have been undertaken on the binding of Mn2+ ions to two alginate samples of different mannuronate:guluronate ratios (M:G), a sample of low-ester amidated pectin and poly(acrylic acid) (PAA). The binding of Ca2+ ions has also been included for the latter for comparison. The binding curves showed an initial steep rise at low additions of Mn2+ or Ca2+ indicating that all of the ions were bound to the polymer chains with none remaining in solution. At higher additions, the binding curves showed a plateau region and the maximum amount bound, theta, was found to be 0.2, 0.2, 0.25, and 0.33 mol M(2+)/mol COO- for high M:G alginate, low M:G alginate, pectin, and PAA, respectively. The binding curves for Mn2+ and Ca2+ with PAA were superimposable. In all cases, theta was less than the stoichiometric equivalent and also less than predicted by Manning counterion condensation theory. The linear charge density, xi, for the polymers is 1.49, 1.55, 1.62, and 2.85, and it was found that at maximum binding the effective linear charge density, xi(effective), decreased to a value close to 1 in each case and not 0.5 as predicted from Manning's two-variable theory. The mobility of the PAA chains has been followed by electron spin resonance spectroscopy using nitroxide spin labels covalently attached to the polymer, and the gelation of the pectin and alginate samples has been monitored using small deformation oscillatory experiments. For PAA at maximum binding, it was noted that there was a loss of chain mobility and precipitation. For pectin and alginate, gelation occurred and the stoichiometric ratio for maximum binding corresponded to the stoichiometric ratio for the maximum in G'. Precipitation and gelation are attributed to the formation of polymer-metal complexes involving one or two carboxylate groups resulting in charge reversal or charge annihilation.     

Isolation of a Factor from Apple that Agglutinates Erwinia amylovora

Extracts prepared from apple seeds contain a factor (AF) capable of agglutinating cells of Erwinia amylovora. In drop agglutination tests, AF is more active in agglutinating an avirulent, acapsular strain of E. amylovora than a virulent, capsular strain. AF precipitates in agar plates with a receptor derived from boiled cells of avirulent acapsular strain and, therefore, can be located during fractionation by rocket electrophoresis. AF was heat-stable and had a pH optimum for agglutination near congruent with3.6 pH. The agglutination activity was not affected by the presence of Mg(2+), Ca(2+), or EDTA. AF was separated into two fractions (AF I and AF II) by elution from a Bio-Gel P-100 column. The precipitin and agglutination activities associated with AF II were found to be present in a positively charged molecule which was sensitive to treatment with protease and trypsin and, hence, presumably resides in a protein. The approximate molecular weight of AF II was determined to be 12,600 daltons. Besides precipitating the receptor derived from cells of avirulent acapsular strain, AF II was capable of precipitating extracellular polysaccharide from cultures of virulent capsular strain, sodium polygalacturonate, and carboxymethylcellulose. These three polymers also inhibited the agglutination activity associated with AF II. AF II could be replaced by poly-l-lysines in both the precipitin and agglutination assays. In addition, in antigen absorption experiments, poly-l-lysines were found to remove the receptors for AF II from the boiled extracts of avirulent acapsular strain. Based on these observations, it is proposed that the activity of AF II resides in a highly positively charged protein which causes agglutination of bacterial cells by interacting on a charge-charge basis with negatively charged components on the surface of the bacterial cells.     

Electrostatic cell-surface repulsion initiates lumen formation in developing blood vessels

Blood vessels function in the uptake, transport, and delivery of gases and nutrients within the body. A key question is how the central lumen of blood vessels develops within a cord of vascular endothelial cells. Here, we demonstrate that sialic acids of apical glycoproteins localize to apposing endothelial cell surfaces and generate repelling electrostatic fields within an endothelial cell cord. Both in?vitro and in?vivo experiments show that the negative charge of sialic acids is required for the separation of endothelial cell surfaces and subsequent lumen formation. We also demonstrate that sulfate residues can substitute for sialic acids during lumen initiation. These results therefore reveal a key step in the creation of blood vessels, the most abundant conduits in the vertebrate body. Because negatively charged mucins and proteoglycans are often found on luminal cell surfaces, it is possible that electrostatic repulsion is a general principle also used to initiate lumen formation in other organs.     

Val22对恶臭假单胞菌扁桃酸消旋酶催化活性的影响

恶臭假单胞菌扁桃酸消旋酶的Val22位于20 s环状结构上, 是与底物结合相关的氨基酸之一。其中Val被替换为Arg后酶活性下降了75.9%。除了酶与底物疏水作用减弱以外, 静电排斥作用增强也可能引起活性的下降。利用分子动力学模拟对酶与底物的米氏复合物进行分析, 结果表明: 突变后第22位氨基酸侧链与底物的静电势从0.036 kJ/mol升高至0.124 kJ/mol。这说明氨基酸侧链极性的改变增加了侧链与底物分子之间的静电排斥作用, 因而静电排斥作用也是导致突变体活性下降的原因之一。同时, 突变后系统势能增加了283 kJ/mol, 进一步证实了第22位氨基酸侧链极性和带电性质的改变导致酶与底物结合状态的势能增大, 从而引起活性大幅下降。因此, 将来对酶的结合口袋区域进行理性设计时, 除了考虑空间位阻效应外, 还需考虑疏水作用和静电作用。     

Charge repulsion in the conformational stability of melittin.

Electrostatic repulsion between positively charged groups has been suggested to be critical in determining the conformation of melittin. To clarify the role of repulsive forces, we prepared a series of succinylated melittins, an acetylated melittin, and a synthetic melittin mutant, with various degrees of charge repulsion. The conformation of the melittin derivatives was examined by far-UV circular dichroism under various conditions of pH and salt at 20 degrees C. The stability of the tetrameric helical state was found to be dependent on the net charge of the peptides. The charge repulsive forces destabilized the helical state of intact melittin by 600 cal/(charge.mol of tetramer). This value was close to the corresponding one (450 cal/(charge.mol)) obtained for the acidic molten globule of horse cytochrome c [Goto, Y., & Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686], which has a molecular weight and a net charge comparable to those of the tetrameric melittin. Small-angle X-ray scattering of the tetrameric melittin and the molten globule of cytochrome c showed that the two states are also comparable to each other in the radius of gyration. These results suggest that the contribution of electrostatic repulsion to the conformational stability of melittin is similar to that of the molten globule.     

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.     

Monolayer characterstics and thermal behaviour of phosphatidic acids

The monolayer and thermal behaviour of different phosphatidic acids are presented. At neutral pH and 22°C dilauroylphosphatidic acid and unsaturated phosphatidic acids form liquid-expanded monolayers, while dipalmitoyl- and distearoylphosphatidic acid form condensed monolayers. Dimyristoylphosphatidic acid undergoes a transition from the liquid-expanded to the condensed state. With long-chain saturated and unsaturated phosphatidic acids little change in molecular area is observed between pH 2 and 7. In contrast, the short chain saturated phosphatidic acids, dilauroyl- and dimyristoylphosphatidic acids, undergo a condensation in the pH range 2 to 7. This is so in spite of the fact that the phosphoric acid group dissociates and the phosphatidic acid molecule attains one negative charge over this pH range. This finding is interpreted to indicate that the electrostatic repulsion between negatively charged phosphatidic acid molecules is compensated for or even outweighed by other intermolecular forces. Hydrogen bonding at the lipid/water interface is supposed to play a major role. All phosphatidates studied exhibit a significant expansion in the pH range 7 to 12. The second apparent pK of the primary phosphate group of phosphatidic acids is 8.6 and the expansion observed in this pH range is therefore due to electrostatic repulsion. At neutral pH the ether analogues of saturated phosphatidic acids have monolayer properties similar to those of the ester compounds. Considering the total pH range of 2 to 12 studied the force-area curves of the ether analogues are more condensed compared to the ester compounds. Synthetic phosphatidates and their ether analogues give reversible sharp crystal(gel)-to-liquid crystal transitions while the naturally occurring egg phosphatidate gives a broad, asymmetric one. The transition temperature T_m of saturated phosphatidates increases with increasing hydrocarbon chain length and at a given chain length T_m decreases markedly with unsaturation. The T_m values of the ether analogues are about 10°C higher and the ΔH values are 10–15% lower than those of the corresponding esters.     

Study of a factor produced by suspension-cultured Pinus radiata cells which enhances cell growth at low inoculum densities

Pinus radiata cells in suspension culture abruptly lose their growth capacity when diluted below a critical inoculum density. This threshold density can be lowered by adding the supernatant (conditioned medium) from healthy cultures which have been grown separately at high densities. Fresh medium is conditioned rapidly indicating that the factor responsible is either potent or produced rapidly. Activity-response curves increase progressively with concentration indicating that still greater effect may be obtained if the factor can be concentrated following separation from other medium components. The effect is not mimicked by a number of candidate compounds (including auxins, cytokinins, polyamines and vitamins). Partial characterisation studies indicate that the factor is relatively small (<1 000 dalton) and possibly an oligosaccharide. It is considered that the factor is an essential structural component of the walls of expanding cells where it is reversibly-bound.Abbreviations 2,4-D dichlorophenoxyacetic acid - LIGF Low Inoculum Growth Factor - NAA naphthalene acetic acid     

A STUDY ON THE MECHANISM OF INTERCELLULAR ADHESION : Effects of Neuraminidase, Calcium, and Trypsin on the Aggregation of Suspended HeLa Cells

Aggregation of suspended HeLa cells is increased on removal of cell surface sialic acid. Calcium ions promote aggregation whereas magnesium ions have no effect. The calcium effect is abolished by previous treatment of the cells with neuraminidase. Trypsinization of the HeLa cells followed by thorough washing diminishes the rate of mutual cell aggregation. Subsequent incubation with neuraminidase restores the aggregation rate to the original value before trypsin treatment. Cells which had acquired a greater tendency for aggregation after removal of peripheral sialic acid lose this property when subsequently treated with trypsin. Calcium ions have no aggregative effect on trypsinized cells. In contrast to HeLa cells, aggregation of human erythrocytes was not increased after treatment with neuraminidase or on addition of calcium. The results with HeLa cells are interpreted as follows: (a) Trypsin-releasable material confers adhesiveness upon the cells. (b) The adhesive property of this material is counteracted by the presence of cell surface sialic acids. (c) Calcium ions exert their effect by attenuating the adverse effect of sialic acid.     

Modulation of WGA binding sites on marrow sinus endothelium in state of stimulated erythropoiesis: a possible mechanism regulating the rate of cell egress

To study the regulation of cellular and molecular traffic across the marrow-blood barrier, rat marrow endothelial surface was incubated with ferritin-conjugated concanavalin A, wheat germ agglutinin (WGA), recinus communis agglutinin I, and phytohemagglutinin. Normal animals were compared with those after erythropoietic stimulation (phenylhydrazine-induced hemolysis, phlebotomy). A selective and significant reduction in the density of WGA receptors, but not other lectins was noted congruent to the degree of reticulocytosis. Neuraminidase treatment also reduced WGA binding sites and the surface negative charge as detected by polycationic ferritin (PCF). Thus, the reduction in WGA binding sites, may reflect a decrease in the density of membrane sialic acid, rendering the endothelial surface charge less negative and providing an electrostatic attraction for the negatively charged surface of reticulocytes. The findings may also be explained by an increase in the frequency of WGA-excluding fenestrae in the endothelium. These areas, lacking sialic acid, may provide unstable areas in the membrane suitable for the passage of cells and molecules in both directions. It is concluded that, by modulating the density of sialic acid residues, the endothelium may regulate the traffic of cells and molecules across the marrow-blood barrier.