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Biomedical subjects

B Geiger

Publications and source records attributed to B Geiger.

At least 109 records · Page 6Linked to original sources
PubMed1979

The use of antibody-coated liposomes as a target cell model for antibody-dependent cell-mediated cytotoxicity.

A simple model system was developed for antibody-dependent cell-mediated cytotoxicity (ADCC) using antibody-coated synthetic membranes (liposomes) as a target cell model. A synthetic hapten, dinitrophenyl-phosphatidylethanolamine (DNP-PE), was incorporated into fluorophore-quencher-loaded liposomes and the latter were coated with pure anti-DNP antibodies. Normal spleen lymphocytes were capable of binding and subsequently lysing these liposomes. This process is dependent upon the presence of an intact (Fc-containing) IgG molecule and independent of exogenous serum complement. The effector lymphocytes are nylon non-adherent, devoid of Thy-1 antigen and present in nude mice, suggesting an identity with K (Fc receptor positive) lymphocytes. These studies indicate that liposomes may be used as a model to study the requirements for the binding and lysis of target cells in this cell-mediated cytotoxic system.

Animals↗
PubMed1979

Normal adult with absent HEX A: immunoreactive HEX A is present.

Fibroblasts from a normal adult with absent hexosaminidase A (HEX A) activity were demonstrated to possess immunoreactive HEX A as measured by GM2 beta-D-N-acetylgalactosaminidase activity which precipitated with specific anti-HEX A antibodies. Possible explanations for the molecular defect are presented.

Antibodies↗
PubMed1978

Biochemical and immunochemical characterization of hexosaminidase P.

Hexasaminidase P, the main isozyme of hexosaminidase in pregnancy serum, was isolated and purified 600--700-fold by a two-step purification procedure--affinity chromatography on Sepharose-bound epsilon-aminocaproyl-N-acetylglucosylamine, followed by ion-exchange chromatography on DEAE-cellulose. The purified enzyme was subjected to biochemical and immunochemical analysis. Its catalytic property, namely, kinetic behavior, is similar to that of the major isozymes of hexosaminidase, A and B. However, it differs from these isozymes in its electrophoretic mobility and in its apparent molecular weight which is around 150 000 compared with 100 000 of the A and B isozymes. Immunochemical analysis indicates that the P isozymes is antigenically cross-reactive with both A and B isozymes, but it does not contain the A-specific antigenic determinants, and exhibits identical antigenic specificity to hexasaminidase B. Two possible structures are suggested that are compatible with the experimental data: (a) a hexosaminidase B like structure with higher extent of glycosylation; (b) a hexameter of beta chain, possibly arranged as three beta2 subunits.

Chemical Phenomena↗
PubMed1978

Antibodies to ganglioside GM1 induce mitogenic stimulation and cap formation in rat thymocytes.

Antibodies towards the ganglioside GM1 [galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyglucosyl ceramide] stimulated DNA synthesis in rat thymocytes. No mitogenic stimulation was observed with the monomeric Fab fragment of anti-GM1, suggesting that cross-linking of the gangliosides or associated components was required for activation by these antibodies. Incubation of thymocytes with anti-GM1 and fluorescein-labeled anti-rabbit IgG at 0 degree C resulted in uniform ring-like or patchy staining that developed into a pronounced cap upon elevation of temperature. The cap had a characteristic uropod form, enriched with intracellular organelles. Sodium azide and cytochalasin B completely inhibited cap formation, while colchicine was without effect. These results imply a possible direct or indirect association between surface gangliosides and submembraneous cytoskeletal assemblies that control modulation of these surface components and may transmit stimuli to the interior of the cell.

Animals↗
PubMed1978

Localization of acetylcholine receptor in excitable membrane from the electric organ of Torpedo: Evidence for exposure of receptor antigenic sites on both sides of the membrane.

Nicotinic acetylcholine receptor was localized in a receptor-rich membrane preparation from the electric organ of Torpedo californica by applying an immunoferritin technique. The membrane preparation was incubated with (Fab')2 fragments derived from specific rabbit antibodies against the purified acetylcholine receptor and subsequently with ferritin-conjugated goat antiserum to rabbit immunoglobulin. More than 50% of the vesicles were found to be labeled with ferritin while the rest remained unlabeled. Ferritin labeling on both sides of the membrane was evident in open membrane vesicles, whereas in closed vescles the labeling was confined to the outer surface due to the inability of the tracer to penetrate the membrane. These data suggest that antigenic sites of the receptor molecule are exposed on both sides of the excitable membrane, and that acetylcholine receptor may be a transmembrane protein.

Animals↗
PubMed1978

Purification, biochemical and immunological characterisation of hexosaminidase A from variant AB of infantile GM2 gangliosidosis.

Variant AB of infantile GM2 gangliosidosis is a fatal disease leading invariably to death within the first few years of life, due to the excessive storage of the glycolipids GM2 and GA2 which occurs in the nervous tissue of the patient. Unlike other variants of this hereditary disease, where a deficiency of hexosaminidase A, the ganglioside-GM2-degrading enzyme, could be demonstrated, the variant AB is characterized by a normal or even elevated level of this enzyme. To examine the possibility of a mutant hexosaminidase A, well capable of hydrolyzing the fluorogenic synthetic substrates but unable to attack the ganglioside, the enzyme was isolated from a patients tissue and characterized biochemically and immunologically in comparison with an enzyme preparation from normal control tissue. No differences between hexosaminidase A from normal and variant AB tissue could be detected indicating that the defect involved in this disease is not at the genetic level of production of either alpha or beta chains of hexosaminidase A.

Child, Preschool↗
PubMed1978

Hexosaminidase A in amniotic fluid of Tay-Sachs fetuses.

Hexosaminidase A is present in relatively low concentrations in cell-free amniotic fluids from pregnancies with Tay-Sachs fetuses. This isoenzyme was determined by an immunological procedure, radial immunodiffusion, by which hexosaminidase A can be directly and specifically detected, even in the presence of excess amounts of hexosaminidase B. No hexosaminidase A could be detected by the same procedure in Tay-Sachs fetal tissues, implying that this isoenzyme in the amniotic fluid originates from the mother.

Amniotic Fluid↗
PubMed1978

The mode of interaction with macrophages of two ordered synthetic polypeptides which differ in their thymus dependency.

The mode of interaction with macrophages of two ordered synthetic polypeptides (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys), (T-T-G-G)-A--L, and (Tyr-Glu-Tyr-Glu)-poly(DLAla)--poly(Lys), (T-G-T-G)-A--L, which differ in their requirements for T-B cell co-operation in the process of antibody production, was compared. The binding of the two radiolabelled antigens to the surface of peritoneal adherent cells, their uptake by the cells and the rate of their degradation were investigated. Macrophages were found to be capable of degrading both poly-peptides with the same efficiency. (T-G-T-G)-A--L, the antigen which is less T-dependent, was bound to macrophage surfaces more readily than (T-T-G-G)-A--L, the T-dependent antigen, however, its uptake by the cells was found to be lower. Thus, (T-G-T-G)-A--L remains for a longer period in the form of a membrane bound polyvalent antigen.

Animals↗
PubMed1977

Stabilization of human beta-D-N-acetylhexosaminidase A towards proteolytic inactivation by coupling it to poly(N-vinylpyrrolidone).

Human hexosaminidase A was covalently bound to soluble poly(N-vinylpyrrolidone), and the effect of this binding on the enzyme inactivation by various procedures was investigated. Whereas the polymer-bound hexosaminidase underwent inactivation to the same extent as the free enzyme, when exposed to heat or acidic pH, the conjugation to polymer appeared to protect the enzyme towards proteolysis. Thus, the polymer-bound enzyme exhibited considerably higher resistance to treatment of both pronase and macrophage cathepsins. The clearance rate from rabbit blood, of the polymer-bound enzyme (expressed as enzyme activity), was shown to be significantly slower than that of the free enzyme.

Drug Stability↗
PubMed1977

Immunochemical determination of ganglioside GM2, by inhibition of complement-dependent liposome lysis.

Immunochemical quantitative determination of a lipid antigen, ganglioside GM2, has been developed, based on the inhibition of the immune lysis of liposomes containing the antigen in their lipid bilayer. It has been shown that the full expression of the antigenicity of the competing lipid requires its dispersion in accessory lipids. The assay of inhibition of liposome lysis can be used also for the establishment of the antigenic similarity of structurally related lipid antigens.

Animals↗
PubMed1977

Immunochemical and biochemical investigation of hexosaminidase S.

Hexosaminidase S (HEX S), the residual isozyme found in tissues and body fluids of children with the O variant of GM2 gangliosidosis, was purified from tissues of variant individuals and biochemically and immunochemically characterized. This enzyme has an apparent molecular weight of 103,000 with an isoelectric point of 4.2, is heat labile to the same extent as HEX A, and loses most of its activity following heating for 30 min at 50 degrees C. HEX S reacts immunologically with the antisera against either HEX A or B, but the reaction is considerably stronger with the anti-A serum or with antibody preparations which react exclusively with the A isozyme. Results obtained by a radioimmunoassay using the various antisera indicated that there is no antigenically cross reacting material which lacks enzymatic activity in the variant tissues. These findings are in accord with a suggested molecular structure of two subunits, each composed of two alpha chains (alpha2 alpha2) for HEX S; it also implies that alpha and beta chains have some structural similarity which is manifested in antigenic cross-reactivity.

Antibodies↗
PubMed1976

Chemical characterization and subunit structure of human N-acetylhexosaminidases A and B.

Human hexosaminidases A and B were purified from placentae, using two stages of affinity chromatography, to a high degree of purity. Each enzyme was purified 5000-6000-fold, and isolated in 25-40% yield. Enzyme preparations appeared homogeneous in the analytical ultracentrifuge and by acrylamide gel electrophoresis. Hexosaminidase A contained 1.65 residues of sialic acid per molecule, whereas no sialic acid was present in hexosaminidase B. The molecular weights of the A and B isozymes as determined by gel filtration and sedimentation equilibrium are 100 000 and 108 000, respectively. In 5 M guanidine-HCl each of the enzymes yielded a 50 000-dalton species, which can further be dissociated into 25 000-dalton polypeptide chains by reduction and alkylation. The hexosaminidase B yielded one type of polypeptide chain, denoted beta, whereas the product from hexosaminidase A could be separated by ion-exchange chromatography into two species of chains, denoted alpha and beta, in equal amounts. The amino acid compositions of the separated alpha and beta chains were determined, and were found to correlate well with those of the intact enzymes. These findings enable the construction of a plausible model for the molecular structure of both enzymes. According to this model hexosaminidase A is composed of two subunits alpha2 and beta2, in which the two polypeptide chains are linked by a disulfide bridge. The structure of hexosaminidase B is, in parallel, beta2beta2. The suggested model is discussed in view of the accumulated information about the interrelationships between hexosaminidase A and B and the genetic metabolic disorders with which they are involved.

Amino Acids↗