The antibacterial properties of secreted phospholipases A2: a major physiological role for the group IIA enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall.

The antibacterial properties of human group IIA secreted phospholipase A(2) against Gram-positive bacteria as a result of membrane hydrolysis have been reported. Using Micrococcus luteus as a model system, we demonstrate the very high specificity of this human enzyme for such hydrolysis compared with the group IB, IIE, IIF, V, and X human secreted phospholipase A(2)s. A unique feature of the group IIA enzyme is its very high pI due to a large excess of cationic residues on the enzyme surface. The importance of this global positive charge in bacterial cell membrane hydrolysis and bacterial killing has been examined using charge reversal mutagenesis. The global positive charge on the enzyme surface allows penetration through the bacterial cell wall, thus allowing access of this enzyme to the cell membrane. Reduced bacterial killing was associated with the loss of positive charge and reduced cell membrane hydrolysis. All mutants were highly effective in hydrolyzing the bacterial membrane of cells in which the cell wall was permeabilized with lysozyme. These same overall characteristics were also seen with suspensions of Staphylococcus aureus and Listeria innocua, where cell membrane hydrolysis and antibacterial activity of human group IIA enzyme was also lost as a result of charge reversal mutagenesis.     

Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.     

Correlation between electrokinetic parameters of isolated hepatocyte nuclei and their functional activity in rats

The results of investigations of the electrokinetic properties of cell nuclei in various functional states were summarized. A positive correlation between the value of the electrostatic charge on the nuclear membrane surface and the functional state of the genetic apparatus upon activation was established. It was shown that the value of the electrokinetic potential reflects differences in the mechanisms of action of biologically active substances, and that this may be related to the peculiarity of their action on both membrane surface and the cell genome. It is concluded that the electrokinetic properties of the nuclear membrane reflect changes occurring in the genome of the eukaryotic cell nucleus, and from the value of surface charge density, the extent of involvement of the genome under various influences can be determined.     

Change in electrophoretic mobility of human erythrocyte as the result of membrane shape change in vitro.

Shape change of human erythrocyte due to the membrane externalization induced by incorporation of excess lysolecithin or by hypertonic treatment in vitro, was accompanied by a decrease in the electrophoretic mobility of the cell. No change in sialic acid content of the membrane, mostly responsible for the cell surface charge, was observed in any of these treatments. Therefore, the membrane externalization seems to cause a redistribution of the surface charge and the resulting alteration of the electrophoretic mobility of the cell. Heating at 48 degrees C for 1 hour induced both the shape change and change in the mobility, but at the same time it reduced the membrane sialic acid content.     

Alterations in membrane surface properties during cell differentiation as measured by partition in aqueous two-polymer phase systems: Rat intestinal epithelial cells

Membrane surface properties of rat intestinal epithelial cells (crypt base to villus tips) were studied by cell partition in a two-polymer aqueous phase system. A higher partition generally reflects higher cell surface charge (or charge-associated properties) which is not necessarily the same as the charge determined by cell electrophoresis since the latter reflects only the charge at the plane of shear while the former gauges it deeper into the membrane [10]. Cells were prepared by the method of Weiser [22] which sequentially yields cell fractions from villus tips to crypt base. The isolated cells were subjected to countercurrent distribution in a dextran-polyethylene glycol aqueous two-phase system. Countercurrent distribution on the first fractions obtained by Weiser's method have a peak to the left and a smaller peak to the right indicating a surface membrane heterogeneity of upper villus cells; last fractions have a peak only to the right. When all fractions are pooled before countercurrent distribution two well-separated peaks are obtained with the right peak sometimes showing additional heterogeneities. Experiments combining isotope labeling of cells with countercurrent distribution lead us to conclude that the membrane charge (or charge-associated properties) of crypt base cells increases during differentiation and that the charge of the villus cells to which they give rise then diminishes during maturation. The charge of the bulk of the upper villus cells is the lowest of any in the intestinal cell population. The basis for the alteration in charge has not been established but the phenomenon of changing membrane surface charge (or charge-associated properties) as a function of cell differentiation, maturation and aging appears to be a general phenomenon having been found and traced in different cell populations [14, 16, 17, 28].     

Changes in physico-chemical properties of human large intestine tumour cells membrane

Tumour cells produce and excrete to blood many substances which are present in the cell itself in trace amounts only. Our work has been aimed at the determination of changes in electric charge and in phospholipid composition of large intestine normal mucosa and colorectal cancer cells.Surface charge density of tumour unaffected mucosa and of tissue sections from tumours, was measured by electrophoresis. The measurements were carried out at various pH of solution. Membrane isoelectric point was determined by measuring its electric charge in function of pH as well as total positive charge at low pH and total negative charge at high pH. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC. Four phospholipid classes were identified: PI, PS, PE and PC and their surface concentrations were determined.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.Tumour changes provoke an increase in surface charge density of large intestine membrane, whereas the content of individual phospholipids increased or decreased depending on a patient.     

Surface charge of mammalian neurones as revealed by microelectrophoresis

Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M^–1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.     

Protective effect of green tea on erythrocyte membrane of different age rats intoxicated with ethanol

It is known that aging is characterized by changes in cell metabolism resulting in modification of the structure and function of cell membrane components which is mainly the consequence of reactive oxygen species action. These disturbances are also enhanced by different xenobiotics, e.g. ethanol. Therefore, the aim of this paper is to examine green tea influence on total antioxidant status (TAS) and on composition and electric charge of erythrocyte membrane phospholipids in ethanol intoxicated rats of various ages. Antioxidant abilities of erythrocytes were estimated by measuring TAS. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC, while the extent of erythrocytes lipid peroxidation was estimated by HPLC measurement of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels. Electrophoresis was used to determine the surface charge density of the rat erythrocyte membrane. It was shown that the process of aging was accompanied by a decrease in TAS and in the total amount of phospholipids as well as by enhancement of lipid peroxidation and increase in surface charge density of erythrocyte membrane. Ethanol administration caused, in term, decrease in TAS and increase in the level of all phospholipids and lipid peroxidation products. Ethanol as well significantly enhanced changes in surface charge density of erythrocyte membrane. The ingestion of green tea partially prevented decrease in erythrocyte antioxidant abilities observed during aging and ethanol intoxication. Moreover, long-term drinking of green tea protects the structure of the erythrocytes membrane disturbed during aging process and/or chronic ethanol intoxication.     

光动力过程中线粒体膜电位和细胞存活关系

以1-anilionaphthalene-8-sulfonate(ANS)作荧光探针,通过其荧光光谱研究了苯硫基酞菁锌PcS)、苯硫基铝酞菁(AIPcS)和烷氧基铝酞菁(AIPc)这三种金属酞菁配合物作为光敏剂的光动力作用对癌细胞线粒体膜表面电位的影响．研究表明,光动力作用后线粒体膜表面电位降低,表面电荷数面密度增加．ZnPcS的影响最大,这与酶联免疫检测光动力作用后对癌细胞的杀伤效果相一致,提示细胞线粒体膜可能是金属酞菁配合物在光动力过程中的作用位点。通过比较细胞线粒体膜表面电位以及表面电荷数面密度与细胞存活之间的关系,阐述了光动力作用的物理学机制．同时,由于线粒体膜电位与细胞凋亡的密切关系,金属酞菁配合物对线粒体膜表面电位的影响提供了一个衡量药物疗效的判据。     

药物诱导的玉米根尖凋亡细胞表面电特性研究(英文)

细胞凋亡过程中细胞表面膜的电位很可能会发生改变。本文首次报导：应用细胞电泳技术（ｃｅｌｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ）对细胞毒素类药物放线菌酮（ｃｙｃｌｏｈｅｘｉｍｉｄｅ）、放线菌素 Ｄ（ａｃｔｉｎｏｍｙｃｉｎ Ｄ）和秋水仙碱（ｃｏｌｃｈｉｃｉｎｅ）等诱导的植物凋亡细胞与正常细胞之间电泳迁移率（ＥＰＭ）的差异进行了比较,对引起的膜电位变化进行了定量分析。实验以玉米根尖分生组织为材料,制备原生质体,经过适当剂量的药物处理（Ｆｉｇ．１－Ｂ）,在尽量减少细胞膜被破坏的情况下（Ｆｉｇ．２）,观察到：三种细胞毒素类药物的作用有所不同,被诱导的植物凋亡细胞的膜表面Ｚｅｔａ电位绝对值比正常细胞的高（Ｆｉｇ．１－Ａ）。本研究提示细胞电泳可对凋亡细胞表面膜电位的变化进行定量分析,为细胞凋亡的检测在方法上提供了新思路。     

Alterations in surface ultrastructure and anionic sites of rat dimethylhydrazine-induced intestinal tumors

The relative roles of cell surface shedding and electronegative charge as determinants of metastatic capacity were studied in experimentally produced intestinal tumors. The ultrastructural organization and distribution of anionic sites on the luminal plasma membrane surface components were examined in small intestinal and colonic tumors induced in male Sprague-Dawley rats with 1,2-dimethylhydrazine. The overall distribution of negatively charged groups was demonstrated with ruthenium red staining. Compared to normal epithelial cells, neoplastic cells revealed evidence of decreased cell surface shedding as manifested by decreased numbers of membrane-bound bodies, and an increased quantity of glycocalyx. Malignant cell surfaces were directly exposed to the intestinal lumen as a result of losing the enteric surface coat covering. The exposed microvilli appeared damaged with shortening and blunting. The glycocalyx and surface coat both reacted strongly with ruthenium red indicating the presence of anionic sites. As a result of surface coat loss, the malignant cell surface components revealed an overall decrease in net negative charge. These alterations in cell surface component ultrastructure and electronegative charge appear to be consistent with the low capacity for chemically induced rat intestinal tumors to metastasize.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Use of amphiphilic spin labels and whole cell isoelectric focusing to assay charge characteristics of sperm surfaces.

The charge characteristics of the surface of bull and rabbit sperm were analyzed using surface-directed spin labels and whole cell isoelectric focusing. Spin label experiments tested charges believed to be localized at the membrane phospholipid-water interface. Charge properties of the glycoprotein calyx were analyzed with isoelectric focusing. Addition of charged detergents altered spin label spectra without changing the isoelectric focusing pH value. Sperm presumed to differ in the amount of adsorbed protein had different isoelectric focusing pH values, but similar spin label spectra. We conclude that these techniques are capable of monitoring charge domains on the sperm surface: one at the polar surface of the phospholipid membrane and one at the interface between the glycocalyx and the suspending fluid. Furthermore, changes in charge density are induced in unique zones of the cell surface during sperm maturation.     

Surface properties of crosslinked erythrocytes as studied by counter-current distribution in aqueous polymer two-phase systems

The bifunctional imidoester dimethyl suberimidate hydrochloride can stabilize rat red blood cells (RBCs) by membrane protein crosslinking, and in that way they can be used as carrier systems for exogenous substances. Counter-current distribution fractionation in charge-sensitive dextran-polyethyleneglycol two-phase systems has been used to detect slight changes in surface charge in stabilized cells. A decrease in the surface charge of crosslinked RBCs and an apparent masking of the age-related cell surface properties have been found to result from the protein crosslinking. Digitonin treatment used to permeabilize crosslinked RBCs produces a significant decrease of the cell surface charge while the age-related surface properties do not seem to be modified by the treatment.     

Recovery of surface membrane in anterior pituitary cells. Variations in traffic detected with anionic and cationic ferritin

Cells dissociated from rat anterior pituitaries were incubated with native or cationized ferritin (CF) to trace the fate of surface membrane. Native ferritin, which did not bind to the cell surface, was taken up in small amounts by bulk-phase endocytosis and was found increasingly (over 1-2 h) concentrated in lysosomes. CF at 100-fold less concentrations bound rapidly to the cell membrane, was taken up by endocytosis in far greater amounts, and within 15-60 min was found increasingly within multiple stacked Golgi cisternae, around forming secretion granules, and within elements of GERL, as well as within lysosomes. The findings demonstrate that the fate of the tracer--and presumably also that of the surface membrane--varies with the same molecule differing only in net charge: vesicles carrying anionic ferritin (net negative charge) fuse only with elements of the lysosomal system whereas those carrying CF (net positive charge) can fuse not only with elements of the lysosomal system, but also with elements along the secretory pathway (Golgi cisternae and condensing granules) as well.     

Morphological Analysis of the Interaction of Charged Surfactant Vesicles (SVs) with Human Cultured Cells

We analyzed the binding and fusogenic properties of surfactant vesicles (SVs), composed of ionic and nonionic surfactants and cholesterol, with the surface of different human lymphoid cells. The influence of charge on SVs-cell interaction was evaluated by monitoring the presence of fluorescent sodium calcein artificially entrapped in the vesicles using optical fluorescence microscopy and laser scanning confocal microscopy. Our results clearly indicate that only negatively charged vesicles bind and fuse with the plasma membrane of human lymphoid cells, and the number of SVs bound to the cell surface was variable among the positive cells. Thin section electron microscopy illustrated that the fusogenic events of SVs with the cell plasma membrane mostly occurred at smooth and nonvillous regions of the cell surface. Taken together, our results suggest that binding and fusion of SVs with the cell plasma membrane might be dependent on interactions with specific membrane components that preferentially recognize negatively charged SVs.     

Interaction of enkephalin peptides with anionic model membranes

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen²,D-Pen⁵]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Interaction of polycations with cell-surface negative charges of epithelial cells.

The interaction between various polycations and cultured glomerular epithelial cells was studied by cell electrophoresis. It was shown that the glomerular epithelial cell presents a negatively charged surface which imparts a zeta potential of -29.0 +/- 1.5 mV at the peripheral layer of the plasma membrane. The pH at which the GEC charge became 50% reduced (pKa) was determined to be 3.0. A variety of polycations of various sizes and fixed and flexible geometries were tested for their capacity to neutralize the cell charge. All the polycations except cytochrome c and lysozyme were capable of completely neutralizing the cell. Cytochrome c could maximally neutralize only 50% of charge and lysozyme only 72% of charge. However, reduced and 'relaxed' molecules of cytochrome c and lysozyme efficiently neutralized the cell surface, as did larger sized 'flexible' polylysines. On the basis of these findings, we conclude that all polycations are not equal in their capacity to neutralize the cell surface. Flexible molecules in contrast to molecules with rigid structures were more effective in neutralizing the cell. This may likely be due to the exposure and availability of more cationic groups in a flexible molecule which results in stabilization of interaction with cells.     

Surface charge distribution on the endothelial cell of liver sinusoids

The topography of the charged residues on the endothelial cell surface of liver sinusoid capillaries was investigated by using electron microscopic tracers of different size and charge. The tracers used were native ferritin (pl 4.2-4.7) and its cationized (pl 8.4) and anionized (pl 3.7) derivatives, BSA coupled to colloidal gold (pl of the complex 5.1), hemeundecapeptide (pl 4.85), and alcian blue (pl greater than 10). The tracers were either injected in vivo or perfused in situ through the portal vein of the mouse liver. In some experiments, two tracers of opposite charge were sequentially perfused with extensive washing in between. The liver was processed for electron microscopy and the binding pattern of the injected markers was recorded. The electrostatic nature of the tracer binding was assessed by perfusion with high ionic strength solutions, by aldehyde quenching of the plasma membrane basic residues, and by substituting the cell surface acidic moieties with positively charged groups. Results indicate that the endothelial cells of the liver sinusoids expose on their surface both cationic and anionic residues. The density distribution of these charged groups on the cell surface is different. While the negative charge is randomly and patchily scattered all over the membrane, the cationic residues seem to be accumulated in coated pits. The charged groups co-exist in the same coated pit and bind the opposite charged macromolecule. It appears that the fixed positive and negative charges of the coated pit glycocalyx are mainly segregated in space. The layer of basic residues is located at 20-30-nm distance of the membrane, while most of the negative charges lie close to the external leaflet of the plasmalemma.     

Marginal product pricing in the ecosystem.

Surface membrane biosynthesis and turnover is reviewed focusing mainly on the fate of cell surface constituents after they terminated their sojourn as part of a functional cell structure. The different experimental approaches to study this problem are described and original data are presented on the turnover of surface membrane constituents of chicken embryo cells in culture. It is proposed that as a consequence of surface membrane turnover, certain surface macromolecules are continuously shed from cells. The size and charge of these molecules was found to be identical to molecules released from cells by mild trypsin treatment. The term shedding is proposed for this process which is assumed to occur both in vitro and in vivo. Many systems in which shedding of cell surface constituents is clearly demonstrated or can be tentatively suggested are described. The biological significance of cell surface carbohydrate containing macromolecules and the possible role of these shed cellular entities is discussed.