ABSTRACT
An enzyme, beta-D-galactosidase, was covalently coupled to mammalian cells by means of a bifunctional reagent. The coupling procedure did not cause appreciable loss of cell viability (less than 6%) as measured by plating efficiently and membrane integrity. After 24 h in culture, the cells exhibited an average of 2.6 x 10(4) molecules of beta-D-galactosidase per cell. Histological evidence indicated that the enzyme was localized on the cell surface and distributed uniformly among the cell population. Considerations for choosing enzyme-label include sensitivity of assay by enzymatic, immunologic and histochemical methods, and the possibility of isolating labeled membrane components by enzyme-specific affinity chromatography.
Subject(s)
Cell Membrane/analysis , Cytological Techniques , Galactosidases , beta-Galactosidase , Animals , Autoradiography , Cell Membrane/metabolism , Cell Survival , Cells, Cultured , Mice , beta-Galactosidase/metabolismSubject(s)
Osteoclasts/ultrastructure , Animals , Bone and Bones/cytology , Immunologic Techniques , Macrophages/immunology , Macrophages/ultrastructure , Male , Mice , Microscopy, Electron, Scanning/methods , Organ Culture Techniques , Osteoclasts/immunology , Receptors, Complement/analysis , Receptors, Fc/analysis , Rosette FormationABSTRACT
When cells are infected by C-type viruses such as Moloney sarcoma/leukaemia virus, new antigens appear on the cell membrane. Mice and rats will respond immunologically to the antigen(s). It was uncertain whether the antigens were related to the viral structural proteins or to non-virion, tumour-specific surface antigens (TSSA) or both. In an 125I-antiglobulin binding assay, Moloney virus completely blocked the binding of mouse and rat sera to virus shedding target cells, thus suggesting that mice and rats recognise only viral proteins. Mice responded to type-specific and rats mainly to group-specific determinants on the virus. Individual Moloney viral proteins were prepared using the guanidine HCl method and were used to block the binding of the rat anti-Moloney immune serum to Moloney virus shedding target cells. By this method, it was demonstrated that the rat serum contains specificities for the viral proteins gp70 and p30, but it was not possible to detect any antibodies directed towards non-virion or TSSA-like molecules.
Subject(s)
Antigens, Neoplasm , Immune Sera , Moloney murine leukemia virus/immunology , Neoplasms, Experimental/immunology , Viral Proteins/immunology , Animals , Antigens, Neoplasm/analysis , Antigens, Viral/isolation & purification , Binding Sites, Antibody , Cell Line , Chromatography, Thin Layer , Epitopes , Immunoassay , Male , Mice , Mice, Inbred BALB C , Rats , Rats, Inbred WF , Viral Proteins/isolation & purificationABSTRACT
The effects of immunoglobulin (Ig) purified by affinity chromatography from two cultured T cell lines on in vitro immune responses was studied. IgT purified from both EL-4 and WEHI-22 suppressed the thymus-dependent IgM response, but had no effect on the thymus-independent IgM response. In contrast, the IgG responses to both thymus-dependent and independent antigens were augmented at optimal concentrations of IgT. Ig of B cell origin--serum Ig, myeloma Ig, B cell surface Ig -- did not have any of these effects. Because of the similarity of the effects of IgT purified from the lymphomas with the effects of T cells and their IgT, it seems likely that the IgT obtained from EL-4 and WEHI-22 cell lines are analogues of normal IgT and thus should be useful in elucidating some of the chemical and biological properties of IgT.
Subject(s)
Immunoglobulins , T-Lymphocytes/immunology , Animals , Cell Line , Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Immunoglobulins/biosynthesis , Immunosuppression Therapy , Lymphoma , MiceABSTRACT
The enzyme lactoperoxidase (iodide:hydrogen-peroxide oxidoreductase, EC 1.11.1.8) was used to iodinate ((125)I) accessible proteins on membranes of intact virally transformed and untransformed cells. A number of labeled bands of proteins were detected by acrylamide gel electrophoresis. A heavily labeled band with a molecular weight of approximately 250,000 daltons was found in all untransformed cells but was absent from transformed cells. When Coomassie-blue-stained membrane preparations were compared, a band was seen in normal cells which co-migrated with the lactoperoxidase-labeled band. In transformed cell membranes, three discrete bands were present in the same position. Thus, the expression of this protein may be altered when cells are in the transformed state.
Subject(s)
Cell Membrane/analysis , Cell Transformation, Neoplastic , Neoplasm Proteins/analysis , Proteins/analysis , Animals , Autoradiography , Catalase , Cell Line , Electrophoresis, Polyacrylamide Gel , Fibroblasts , Glucose Oxidase , Iodine Radioisotopes , Isotope Labeling , Mice , Mice, Inbred BALB C , Molecular Weight , Moloney murine leukemia virus , Polyomavirus , Simian virus 40Subject(s)
Antigens , Lymphocytes/immunology , Thymus Gland/cytology , Animals , Antibody-Producing Cells , Bone Marrow/immunology , Bone Marrow Cells , Erythrocytes/immunology , Female , Gossypium , Hemolytic Plaque Technique , Immune Adherence Reaction , Immunization , Male , Methods , Mice , Sheep , Spleen/cytologyABSTRACT
The amino acid sequences of the Fd fragments of two human pathological immunoglobulins of the immunoglobulin G1 class are reported. Comparison of the two sequences shows that the heavy-chain variable regions are similar in length to those of the light chains. The existence of heavy chain variable region subgroups is also deduced, from a comparison of these two sequences with those of another gamma 1 chain, Eu, a mu chain, Ou, and the partial sequence of a fourth gamma 1 chain, Ste. Carbohydrate has been found to be linked to an aspartic acid residue in the variable region of one of the gamma 1 chains, Cor.
