Yehuda Marikovsky

Use of cationized ferritin as a label of negative charges on cell surfaces.

A polycationic derivative of ferritin for labeling of negative charges on cell surfaces was prepared by coupling horse spleen ferritin with N, N dimethyl-1,3 propanediamine (DMPA) via carbodiimide activation of the protein carboxyl groups. Various derivatives of cationized ferritin prepared in the pH range between 5 and 7 were characterized and tested for their labeling capacity. The DMPA-ferritin prepared at pH 6.5 was chosen as the optimal preparation for the present study. Labeling with cationized ferritin is performed at physiological conditions with or without prefixation with glutaraldehyde. It is effective on a variety of cells, and its geometry permits relatively easy counting on the membrane surface. The difference in surface charge labeling between young, old, and receptor-destroying enzyme (RDE)-treated rabbit red cells is reflected in the number of cationized ferritin particles attached per unit area of membrane in tangentially sectioned red cells. This is in agreement with the values obtained by electrophoresis.

Agglutination by polylysine of young and old red blood cells

Abstract The rate of agglutination by polylysine of young and old human erythrocytes was automatically recorded and correlated with measurements of the electrophoretic mobility of these cells. Old red cells have a lower electrophoretic mobility than young cells, as well as a higher rate of agglutination than young cells. Mild treatment of red cells with receptor destroying enzyme, which reduces their surface charge, results in an increased rate of agglutination. The results indicate that there is correlation between the rate of agglutination and the surface charge of the red cell.

Cholesterol-phospholipid vesicles in human bile: an ultrastructural study

Phospholipid vesicles, a newly described (bile salt independent) mode of cholesterol transport in human bile, were previously characterized by quasi-elastic light scattering and gel filtration. In the present study the ultrastructure of these vesicles was investigated by electron microscopy using freeze-fracture and negative-staining techniques. Vesicles of varying size were found in all 14 hepatic and 3 gallbladder biles examined. The diameter of the vesicles ranged from 25 to 75 nm by electron microscopy after freeze fracture and from 54 to 94 nm by quasi-elastic light scattering. They had a spherical shape and appeared to be unilamellar. The appearance of the vesicles in fresh hepatic and gallbladder biles as well as in chromatographic fractions was similar. Vesicles were dissolved by the addition of exogenous bile salts. Cholesterol is transported in human bile by both vesicles and micelles. The role of the vesicles may be particularly important in preventing cholesterol precipitation in dilute and supersaturated biles.

Distribution of surface charge and concanavalin a-binding sites on normal and malignant transformed cells☆

Abstract Measurement of the rate of agglutination with the positively charged poly- l -lysine of normal lymphocytes, Moloney-virus-transformed lymphoma cells, normal fibroblasts and SV40 transformed fibroblasts, has shown that the normal cells were agglutinated at a higher rate than the transformed cells. The labeling density of cationized ferritin in electron micrographs of sectioned cells, also indicated a higher charge density for the normal lymphocytes and fibroblasts. The normal cells showed a more regular clustered distribution of cationized ferritin than the transformed cells, and pre-fixation of cells with glutaraldehyde before labeling with cationized ferritin resulted in a random distribution in both types of cells. The transformed cells had a higher agglutinability than the normal cells by Concanavalin A (ConA) and this difference was also found after treatment of the cells with neuraminidase. Labeling with ConA-ferritin showed the same distribution on the sectioned normal and transformed cells. The results indicate that there was a difference in the redistribution of surface charge by cationized ferritin in normal and transformed cells and that there was no detectable difference in redistribution of ConA-binding sites with ConA-ferritin.

Phospholipid lamellae are cholesterol carriers in human bile

Cholesterol solubility and precipitation in bile are major factors in the pathogenesis of cholesterol gallstones. At present, mixed micelles and phospholipid vesicles are considered to be the only cholesterol carriers in bile. In this study we present evidence showing that phospholipid lamellae are major cholesterol carriers in human bile. Lamellae are a known aggregational form in pure phospholipid model systems. In the present study, lamellae were demonstrated by electron microscopy after negative staining and by small-angle X-ray diffraction in all human gallbladder bile samples examined. During diffraction experiments, cholesterol was found to crystallize from these lamellae. Cholesterol carriers in bile were separated by high-resolution chromatography and by prolonged ultracentrifugation. Lamellae were shown to solubilize most of the biliary cholesterol; vesicles solubilized a lesser amount; while micelles solubilized only a minor portion. Our data suggest that phospholipid aggregates are the main cholesterol carriers in bile. Bile salts may control the equilibrium between the various aggregational forms of cholesterol-carrying phospholipids.

Ethanol-induced alterations in human erythrocyte shape and surface properties: Modulatory role of prostaglandin E1

SummaryExposure of human erythrocytes to ethanol (1 to 20% by vol) in Ca2+ and Mg2+-free phosphate-buffered saline, pH 7.4, transformed biconcave discs into spiculated echinocytes within 3 min at 25°C. The effects of ethanol were concentration- and time-dependent, but reversible by washing in the incubation buffer system within 60 min of initial exposure to ethanol. After prolonged ethanol exposure (180 min), washing of cells resulted in the formation of stomatocytes (cup-forms). Ethanol-induced echinocytosis was also accompanied by a 30% enhancement in the agglutinability of erythrocytes by ligands with high affinity for negative surface charge (poly-l-lysine and wheat germ agglutinin, 20 μl/ml) without any alterations in surface charge topography. Concomitant exposure of erythrocytes to prostaglandin E1 (100nm) selectively prevented the enhancement of ligand-mediated agglutinability, but did not modify cell shape. These data indicate that certain erythrocyte surface properties may not be directly influenced by cell shape and suggest a unique modulatory action of prostaglandin E1 on shape-transformed cells.

The cytoskeleton in ATP-depleted erythrocytes: the effect of shape transformation

Erythrocyte aging, accompanied by reduced deformability and low ATP level, is considered to be among the factors limiting the survival of old erythrocytes. The aim of this study was to observe the effect of reversible erythrocyte shape changes of discocyte-echinocyte-discocyte, following ATP-depletion and repletion with adenosine of normal human erythrocytes, on the cytoskeletal network. The effect becomes evident from electron microscopical observations on Triton-treated erythrocyte membranes. The two-dimensional lattice of spectrin-actin junction polygons increases in size in ATP-depleted erythrocytes. This may be due to the spectrin elasticity permitting an expansion of the skeletal lattice. The observations demonstrate alterations in the configuration of the filamentous units visualized in the cytoskeleton during the process of reversible erythrocyte shape changes, a capacity which is limited in aged human erythrocytes.

Rabbit erythrocyte survival following diminished sialic acid and ATP depletion.

The limited deformability and ATP depletion was considered by some authors to be the factor limiting the life span of old red blood cells (RBC) in circulation. Others believed that sialic acid on the RBC surface determines their life span. We compared the life span of 51Cr labelled, neuraminidase treated rabbit RBCs with ATP depleted by incubation at 37 degree C rabbit RBCs. Osmotic fragility, agglutinability, glucose-6-phosphate dehydrogenase (G6PD) and hexokinase activity and ATP levels of these cells were determined. Desyalated RBCs were removed from the circulation within 24 hours. ATP levels, G6d and hexokinase activity and osmotic fragility were normal in these cells. The agglutination by poly(L-lysine) was affected by the loss of surface charge on these cells. Half the ATP depleted RBCs were out of the circulation within three days. Reconstitution of ATP by reincubation with adenosine, elevated the ATP levels to about 80% of their original level, but survival of these cells did not improve. Analysis of sialic acid showed tha 50% of it was removed during the incubation for ATP depletion. The low ATP level and loss of sialic acid fromt he RBC membrane appeared to be conincidental rather than dependent on each other. The latter appears to be a primary factor in red cell survival.

Changes of cell shape and surface charge topography in ATP-depleted human red blood cells

ATP depletion crenates human red blood cells. With ferritin-avidin (FA) and cationized ferritin (CF) cell surface labeling, it is demonstrated that the discocyte----crenated shape transformation alters the two-dimensional topography of negative charge sites. With restoration of ATP levels, cell shape and charge topography return to normal. Concurrent changes in red cell shape and surface charge topography can be explained by associations between membrane integral proteins and the red cell cytoskeleton.

Ribonucleic acid from Escherichia coli: electron microscopical study.

The dimensions of high-molecular weight RNA from E. coli were measured by electron microscopical methods. Filaments 10 to 13 A in diameter, and 2000–4000 A in length, could be observed in preparations obtained from salt-free aqueous solutions of RNA. When ammonium acetate solutions were used, individual fibers became rare, and granules and various forms of aggregates dominated the picture.