Ben-Ami Sela

Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells

SummaryA soybean agglutinin was found to agglutinate mouse, rat and human cell lines transformed by viral carcinogens, but not hamster cells transformed by viral or non-viral carcinogens. Normal cells from which the transformed cells were derived were not agglutinated by this agglutinin, but they were rendered agglutinable after short incubation with trypsin or pronase. The transformed hamster cells, on the other hand, became agglutinable only after prolonged treatment with pronase. The agglutination was specifically inhibited by N-acetyl-d-galactosamine, indicating that N-acetyl-d-galactosamine-like saccharides are part of the receptor sites for soybean agglutinin on the surface membrane. Such sites exist in a cryptic form in normal cells; they are exposed in transformed mouse, rat and human cells, but become less accessible in transformed hamster cells. The receptor sites for soybean agglutinin differ from the receptors for two other plant agglutinins (wheat germ agglutinin that interacts with N-acetyl-d-glucosamine-like sites and Concanavalin A that interacts with α-d-glucopyranoside-like sites) which become exposed upon transformation of all lines tested. In normal hamster cells, the receptors for all three agglutinins become exposed after incubation with trypsin, but the exposure of N-acetyl-d-galactosamine-like sites requires the longest enzyme treatment. The results indicate a difference in the location of different carbohydrate-containing sites in the surface membrane. The differences in the exposure of carbohydrate-containing sites in the membrane could not be correlated with the levels of carbohydrate-splitting glycosidases in normal and transformed cells.

Antiganglioside Antibodies Inhibit Neuritic Outgrowth from Regenerating Goldfish Retinal Explants

Abstract: Antibodies elicited in rabbits against bovine brain gangliosides were applied to regenerating retinal explants to examine the role of gangliosides in the trophic effect responsible for the induction of outgrowth. The results indicate that antibodies specific to gangliosides block the neuritic outgrowth from the regenerating retinal explants. It is inferred that gangliosides function as receptors on the cell surface of the retinal explants that interact with the trophic substances inducing the outgrowth. The relevance of gangliosides to the mechanism of regeneration and development induced by trophic factors is discussed.

Quantitation of N-acetyl-d-galactosamine-like sites on the surface membrane of normal and transformed mammalian cells

Measurements of the binding of 125I-labeled soybean agglutinin to cells cultured with fetal calf serum have shown, that there can be a similar number of d-GalNAc-like sites exposed on normal and transformed mouse and rat cells; that there were only 10 % of such sites on transformed hamster cells; and that treatment with pronase can render normal cells agglutinable by soybean agglutinin without increasing the total number of exposed d-GalNAc-like sites.

Compositional aspects of lipid hydration.

The effect of various lipids such as cerebrosides, gangliosides and dipalmitoyl lecithin (DPPC) on the lowering of the melting temperature of water, was determined by differential scanning calorimetry (DSC). The lowering of the melting temperature, and the number of water molecules per lipid molecule which apparently do not undergo melting, increase with the increase of the size and charge of the polar group and with the unsaturation of the hydrocarbon chains. Freezing curves show supercooling to about -20 degrees C. It was found that the number of apparently unfreezable water molecules is about four for glucocerebroside from Gauchers spleen and about eight or nine for galactocerebroside from bovine brain. In gangliosides from bovine brain the following number of water molecules/lipid molecule are apparently unfreezable: 22-30 in GM1, 33-40 in GD1a + GD1b while a fraction of gangliosides containing 75% GQ1b and 25% GT1b affects up to 60 molecules of water/molecule of lipid. A zwitterionic DPPC molecule removes apparently six to seven water molecules from freezing. There is no indication that the apparently unfreezable water molecules are in a distinct state. It is suggested that they freeze at very low temperatures producing a flat tail preceding the transition peak which cannot be discerned from the base line.

Calorimetric studies on various gangliosides and ganglioside-lipid interactions

Differential scanning calorimetry was used to investigate the thermotropic behaviour of various gangliosides differing in size and in the net negative charge. It was found that the number and the position of the negative charges in the headgroup region influence strongly the phase transition profiles. Interaction of GM1 ganglioside with egg phosphatidylcholine or cholesterol was also investigated. GM1 is completely miscible with egg phosphatidylcholine, giving only one transition peak at all ratios of the two components, implying that when gangliosides are in a more fluid lipid environment in biological membranes they will be randomly distributed. Interaction with cholesterol decreases the enthalpy of melting of the ganglioside. The decrease in enthalpy reaches a plateau at about 30 mol% cholesterol, suggesting a lower affinity of cholesterol for gangliosides than for sphingomyelin.

Isolectins from wax bean with differential agglutination of normal and transformed mammalian cells

Abstract Wax bean agglutinin prepared according to Liener was separated by chromatography on hydroxylapatite into three fractions, two of which were biologically active. Both fractions are glycoproteins with essentially identical amino acid composition and molecular weight, each consisting of four subunits, molecular weight 30 000. The isolectins agglutinated transformed cells at a concentration about 100 times lower than that required to agglutinate normal cells. Agglutination was inhibited by fetuin but not by any of the simple sugars tested.

Interaction of the chlorinated hydrocarbon insecticide lindane or DDT with lipids—A differential scanning calorimetry study

The interaction of DDT and lindane with glycosphingolipids and phospholipids was investigated by employing differential scanning calorimetry. The degree of perturbation produced by lindane is stronger than that of DDT and depends also on the lipid.

Regenerative capacity of the goldfish visual system is affected by antibodies specific to gangliosides injected intraocularly

An association between gangliosides and neuronal regeneration in goldfish is demonstrated in the present study. A single intraocular injection of affinity purified anti-GM1 antibodies administered simultaneously with crush injury of the optic nerve, inhibits the regenerating process as expressed by two parameters: protein synthesis in the retina and in vitro sprouting ability from the retina. The retinal level of several gangliosides (such as GD3, GD1a, GD1b and GT1b) is enhanced during regeneration. Although GM1 appears to be a minor retinal ganglioside, antibodies to GM1 exert a marked effect on retinal regenerative process. It is assumed that such antibodies could interact with more abundant retinal gangliosides such as GD1b which shows enhanced biosynthesis during regeneration and which shares a similar disaccharide terminal residue with GM1.

Abnormalities in brain myelin of rabbits with experimental autoimmune multiple sclerosis-like disease induced by immunization to gangliosides

An experimental autoimmune multiple sclerosis‐like disease (EAMSD) was induced in rabbits by immunizing them with bovine brain gangliosides. Forebrain myelin was isolated and fractionated on a discontinuous sucrose gradient into light myelin (LM, buoyant density 0.625 M), and heavy myelin (HM, buoyant density > 0.625 M). No abnormalities in either protein or lipid composition of EAMSD myelin fractions were observed. However, the EAMSD tissue yielded 31% less light and 39% more heavy myelin compared to the control brains. Thus, the HM/LM ratio was two‐fold greater in experimental than in control myelin. This pathological pattern is similar to that which has been observed in myelin obtained from the brains of multiple sclerosis patients and from the optic nerves of rabbits with experimentally‐induced demyelination.

Insulin-like effects of wax bean agglutinin in rat adipocytes

Wax bean agglutinin (WBA) was found to mimic the activities of insulin in mediating glucose oxidation and antilipolysis. In contrast, soybean and peanut agglutinins do not exert any of these activities. Unlike concanavalin A and wheat germ agglutinin that were reported previously to exhibit insulin-like activites, WBA neither enhances nor competes with the [125I]insulin binding at relatively high concentrations. Moreover, mild trypsinization of adipocytes, a treatment which greatly diminishes the binding and bioactivity of insulin in fat cells, only slightly affects glucose oxidation induced by WBA. ED50 values for WBA mediated glucose oxidation and antilipolysis are 9.3 μg and 40.0 μg, respectively, compared with the nearly identical concentrations required for 50% of maximal effect of both glucose oxidation and antilipolysis, mediated by wheat germ agglutinin. The present studies suggest that these two activities may be triggered by WBA via surface glycoproteins that are distinct from the binding site of insulin.