muramidase and Cell-Transformation--Viral

A transformed bovine peritoneal macrophage cell line was developed and characterized. Primary peritoneal macrophages were transformed by calcium-phosphate transfection with SV40 plasmid DNA. The transformed cell line retained the morphology of resident peritoneal macrophages as determined by light microscopy and histochemical analysis showed non-specific esterase activity. In addition, immunohistochemical staining of transformed peritoneal macrophages for lysozyme activity was positive. Transformed cells phagocytized Staphylococcus aureus, lysed chicken red blood cell (RBC) targets with and without opsonization and produced hydrogen peroxide radicals and interleukin-6 upon stimulation with opsonized zymosan and lipopolysaccharide, respectively. Transformed cells were also able to ingest and kill Mycobacterium paratuberculosis, an acid-fast bacillus. These results suggest that this cell line should be useful to study interactions between the bovine and intracellular pathogens.

Topics: Animals; Antibody-Dependent Cell Cytotoxicity; Cattle; Cell Line, Transformed; Cell Transformation, Viral; Cytotoxicity, Immunologic; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Female; Hydrogen Peroxide; Interleukin-6; Macrophages, Peritoneal; Muramidase; Phagocytosis; Plasmids; Simian virus 40; Transfection

In an earlier study we found that different forms of the v-myb oncogene transform myeloid cells which resemble either monoblasts [when v-myb of avian myeloblastosis virus (AMV) was used] or promyelocytes [when a point mutant in v-myb of AMV was used; Introna, M., Golay, J., Frampton J., Nakano, T., Ness, S.A. & Graf, T. (1990). Cell, 63, 1287-1297]. In the present study we have searched for genes expressed in AMV mutant-transformed promyelocytes that are not expressed in AMV-transformed monoblasts using a differential screening approach. Eight different genes were identified among more than 500 differentially expressed clones. The most abundant of these was the previously identified myb-regulated mim-1 gene. The others were found to encode a small calcium-binding (MRP-like) protein; the p20K protein; goose-type lysozyme; a ribonuclease A/angiogenin-related protein; and three non-identified proteins. Although these genes appear to be rather lineage restricted, their expression varied in different subtypes of transformed myelomonocytic cells, and only two of them (goose lysozyme and ribonuclease) showed a similar expression pattern in normal promyelocytes and macrophages, suggesting an aberrant gene regulation in the transformed cells. Co-transfection experiments of a reporter construct containing the promoter of the ribonuclease A-related gene indicated that this promoter is regulated by the v-Myb oncoprotein without the involvement of Myb-specific binding sequences.

Topics: Amino Acid Sequence; Avian Myeloblastosis Virus; Base Sequence; Bone Marrow; Calcium-Binding Proteins; Cell Transformation, Viral; Cloning, Molecular; DNA; Gene Expression; Macrophages; Molecular Sequence Data; Monocytes; Muramidase; Oncogene Proteins v-myb; Oncogenes; Proteins; Retroviridae Proteins, Oncogenic; Ribonuclease, Pancreatic; Ribonucleases; Sequence Alignment

Friend-virus transformed erythroblasts (HFL cells) were incubated in solutions containing up to 3 different proteinaceous compounds at pH 7.2 or 5.5 at 5 degrees C. The specificity of interaction of each compound with the cell surface was determined by comparing the amounts of each compound adsorbed and bound in the presence of 2 or 3 different compounds or after prior binding of another compound to the amounts when the individual compounds alone interacted with the cells. At pH 7.2, ovalbumin and gelatin apparently interacted with cell surface components common to both proteins, as indicated by a decrease (up to 80%) in the amount of each compound adsorbed and bound in the presence, or after the binding, of the other compound. The relative amounts of each compound that interacted were different at pH 5.5 and pH 7.2. Gelatin and poly-L-lysine interacted mainly with different components at pH 7.2, whereas common components appeared to be involved at pH 5.5. Concanavalin A interacted preferentially with components that it shared with lysozyme at both pH values. In addition to variations in interactions with changes in pH and type of compound, variations occurred with changes in concentration of the compounds and with their sequence of addition to the cells. Comparative studies with horse red blood cells showed that the interactions differed markedly with cell type.

Topics: Adsorption; Animals; Cell Line; Cell Transformation, Viral; Concanavalin A; Erythroblasts; Erythrocyte Membrane; Erythrocytes; Friend murine leukemia virus; Gelatin; Horses; Hydrogen-Ion Concentration; Mice; Muramidase; Ovalbumin; Peptides; Polylysine; Sarcosine

Topics: Animals; Cell Aggregation; Cell Transformation, Neoplastic; Cell Transformation, Viral; Concanavalin A; Erythroblasts; Erythrocyte Membrane; Erythrocytes; Friend murine leukemia virus; Gelatin; Mice; Microscopy, Electron, Scanning; Microvilli; Muramidase; Ovalbumin; Peptides; Polylysine; Proteins; Sarcosine; Surface Properties

Hen egg-white lysozyme (HEL)-specific suppressor T cells induced in C57BL/6 mice have been selected by sequential passage over plates coated with goat anti-mouse Ig and HEL. These suppressor T cells, 80% I-J+, were infected in vitro with radiation leukemia virus (RadLV/Nu1) and injected intravenously into sublethally irradiated syngeneic recipients. After 4-6 months, 6 out of 20 injected mice developed thymic lymphomas, which were maintained by transplantation into histocompatible hosts and subsequently established as permanent cell lines. Cells of these six thymomas were screened for the presence of Thy 1.2, Lyt 1, Lyt 2, I-Jb, and Ig cell surface antigens by direct or indirect immunofluorescence. One tumor (thymoma L4) was found to express the expected phenotype of suppressor T cells (Thy 1.2+, Lyt 2+, I-J+). High-speed supernatants of extracts obtained from L4 cells were able to induce HEL-specific suppression in a T cell proliferative assay, demonstrating the presence of an antigen-specific suppressive T cell factor.

Topics: Animals; Antigens; Antigens, Surface; Cell Line; Cell Transformation, Viral; Immune Tolerance; Leukemia Virus, Murine; Leukemia, Experimental; Lymphoma; Mice; Mice, Inbred C57BL; Muramidase; T-Lymphocytes

Equilibrium adsorption and binding isotherms at different pH values and temperatures were used to study the mechanism of interaction of 6 proteinaceous compounds with erythroblasts transformed by Friend virus (HFL cells). The molecular weight of the adsorbate appeared to influence the amounts absorbed: fewer moles interacted with increasing molecular weight. The pI value affected binding of adsorbates of low molecular weight in an almost linear way: more moles bound with increasing pI value. Polylysine and polysarcosine absorbed to labile components on the cell surface. Gelatin, lysozyme, ovalbumin, and polysarcosine interacted with pronase-susceptible, Concanavalin A and polylysine with non-susceptible components.

Topics: Adsorption; Animals; Cell Line; Cell Transformation, Viral; Erythroblasts; Erythrocytes; Friend murine leukemia virus; Gelatin; Hydrogen-Ion Concentration; Mice; Molecular Weight; Muramidase; Ovalbumin; Peptides; Proteins; Temperature

An orang-utan (Pongo pygmaeus) suspension line, CP81, was shown to lack myeloid markers of lysozyme activity an d phagocytosis but to be positive for lymphocytic N-alkaline phosphatase activity, and to release a B-cell-tropic herpesvirus. This herpesvirus, termed Herpesvirus pongo, had 30--40% DNA homology with EBV and was present at 2-3 genome copies per CP-81 cell. Gibbon lymphocytes transformed by H. pongo, Epstein-Barr virus (EBV), and H. papio (of baboon, Papio hamadryas, origin) were found to be virus antigen-positive B cells. Gibbon lymphocytes transformed by H. pongo and EBV and transplanted to nude mice by the intracranial (IC) route (had a 75% and a 45% success rate, respectively), while transplants of similar cells transformed by H. papio were only 10% successful. None of these lines transplanted subcutaneously (SC) nor manifested a high degree of colony formation in 0.33% agarose (less than or equal to 0.5%), Gibbon lymphocytes transformed by H. pongo were hypodiploid while those transformed by EBV or H. papio were diploid. CP-81 cells themselves could be transplanted both IC (100%) and SC (70%) and showed a relatively high degree of colony formation in agarose (6.4-7.6%). B95-8 cells (marmoset, Saguinus oedipus-EBV) could be transplanted IC (66%) but not SC and had a low but significant ability to grow in agarose (1.6%). 594S (baboon, P. hamadryas-H. papio) cells could be transplanted IC (25%) but not SC, and grew to very low levels in agarose (0.1%).

Topics: Alkaline Phosphatase; Animals; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Viral; Fluorescent Antibody Technique; Herpesviridae; Herpesvirus 4, Human; Hominidae; Lymphocyte Activation; Lymphocyte Transfusion; Male; Mice; Mice, Nude; Muramidase; Phagocytosis