g(m1)-ganglioside and Hemolysis

In this work a thorough characterization of the GM1 micelle-Amphotericin B (AmB) interaction was performed. The micelle formation as well as the drug loading occurs spontaneously, although influenced by the physicochemical conditions, pH and temperature. The chromatographic profile of GM1-AmB complexes at different molar ratios shows the existence of two populations. The differential absorbance of GM1, monomeric and aggregate AmB, allowed us to discriminate the presence of all of them in both fractions. Thus, we noted that at higher proportion of AmB in the complex, increases the larger population which is composed mainly of aggregated AmB. The physical behavior of these micelles shows that both GM1- AmB complexes were stable in solution for at least 30 days. However upon freeze-thawing or lyophilization-solubilization cycles, only the smallest population, enriched in monomeric AmB, showed a complete solubilization. In vitro, GM1-AmB micelles were significantly less toxic on cultured cells than other commercial micellar formulations as Fungizone, but had a similar behavior to liposomal formulations as Ambisome. Regarding the antifungal activity of the new formulation, it was very similar to that of other formulations. The characterization of these GM1-AmB complexes is discussed as a potential new formulation able to improve the antifungal therapeutic efficiency of AmB.

Topics: Amphotericin B; Animals; Antifungal Agents; Candida albicans; Cell Survival; Chemistry, Pharmaceutical; Chlorocebus aethiops; Drug Carriers; Freeze Drying; G(M1) Ganglioside; Hemolysis; Humans; Hydrogen-Ion Concentration; Micelles; Microbial Sensitivity Tests; Solubility; Technology, Pharmaceutical; Temperature; Vero Cells

Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Biological Assay; Enterotoxins; Escherichia coli; Escherichia coli Proteins; G(M1) Ganglioside; Gangliosides; Glycosides; Hemolysis; In Vitro Techniques; Molecular Sequence Data; Mutagenesis; Oligonucleotide Probes; Plasmids; Radioimmunoassay; Sulfites; Triterpenes

Rat erythrocytes contained ganglio-series gangliosides, GM1, fucosyl GM1, and GD1a, in a high concentration. The concentrations of GM1, fucosyl GM1, and GD1a in rat erythrocyte ghosts were 889.0 nmol, 470.6 nmol, and 462.0 nmol per g dry weight, respectively, and the molar ratio of lipid-bound sialic acid, cholesterol and lipid-bound phosphorus was 3.1:73.9:100.0. The reactions of fucosyl GM1 and GM1 on rat erythrocytes with rabbit anti-fucosyl GM1 and anti-GM1 antisera were measured by means of haemolysis in the presence of complement and a binding assay of antibodies with a FACS after staining erythrocytes by the indirect membrane immunofluorescence technique. When measured by ELISA, anti-fucosyl GM1 antiserum was found to react almost exclusively with fucosyl GM1 with a slight cross-reaction with GM1, but anti-GM1 antiserum cross-reacted to a significant extent with asialo GM1. Rat erythrocytes were haemolyzed specifically with anti-fucosyl GM1 antiserum, but not with antisera to GM1, asialo GM1, asialo GM2, Forssman and globoside, and the haemolysis was proved to be definitely caused by the specific recognition of fucosyl GM1 on rat erythrocytes by anti-fucosyl GM1 antibody according to the haemolysis inhibition reaction using various glycosphingolipid-containing liposomes as inhibitors. In addition, although the binding of anti-fucosyl GM1 antibody on rat erythrocytes was clearly demonstrated with a FACS, anti-GM1 antibody did not bind. The observations that the haemolysis of rat erythrocytes and the binding of antibody to rat erythrocytes were found only with anti-fucosyl GM1 antiserum, and not with anti-GM1 antiserum, but that nevertheless the titer of anti-GM1 antiserum was higher than that of anti-fucosyl GM1 antiserum and GM1 on rat erythrocytes was more abundant in concentration than fucosyl GM1, seem to be a matter of great importance in assessing the specificity of anti-ganglioside antibody and the surface distribution of gangliosides on the cell.

Topics: Animals; Complement Fixation Tests; Enzyme-Linked Immunosorbent Assay; Erythrocyte Membrane; Fluorescence; G(M1) Ganglioside; Gangliosides; Glycosphingolipids; Goats; Hemolysis; Immune Sera; In Vitro Techniques; Lipids; Mice; Rabbits; Rats; Rats, Inbred Strains

Reaction of cholera toxin with NN'-bis(carboximidomethyl)tartaramide dimethyl ester produced several cross-linked species that had subunit B (which binds to the cell surface) and peptides A1 (which activates adenylate cyclase) and A2 all covalently joined together. This cross-linded material had activity with pigeon erythrocytes that was comparable in all respects with that of native toxin. It activated the adenylate cyclase of whole cells, showing a characteristic lag phase, and this activation was increased if the cells had been preincubated with ganglioside GM1, but abolished if the protein had been preincubated with the ganglioside. It activated the enzyme in lysed cells more strongly and without the lag phase. These results show that the toxin is active even when peptide A1 cannot be released from the rest of the molecule.

Topics: Adenylyl Cyclases; Amides; Animals; Cholera Toxin; Columbidae; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Erythrocytes; G(M1) Ganglioside; Hemolysis; In Vitro Techniques; Indicators and Reagents; Kinetics