Development of heart cells in culture: studies using an affinity purified antibody to a myosin light chain

Cultured neonatal rat heart cells can be used to study the factors that regulate cardiac contractility and myocyte development in vitro. An antibody to the 26,000 dalton light chain of myosin (MLC1), has been produced and purified on a Sepharose 4B affinity column prepared with rat heart myosin. When primary cultures of myocytes are studied by indirect immunofluorescence using this antibody a predictable pattern of myofibrillar structure is observed to develop over 72 h. This myosin cytoskeleton is highly organized and the myosin fibrils exhibit cross striations. The antibody does not stain non-muscle heart cells and there is no evidence for myocyte division in culture. The qualitative immunofluorescent pattern of myosin organization is the same in both spontaneously beating and in non-contracting cells.     

The interaction of murine IgG subclass proteins with human monocyte Fc receptors

The use of murine monoclonal antibodies in the immunotherapy of human disease has prompted interest in the interactions of murine IgG with Fc receptors (FcR) expressed on human effector cells. We examined the heterocytophilic interactions between monomeric murine IgG subclass proteins and the FcR expressed on human monocytic cells (peripheral blood monocytes and interferon (IFN)-gamma-induced U937 cells). All four murine IgG2a antibodies and both murine IgG3 antibodies that were tested bound to human monocyte FcR with high affinity (10(8) to 10(9) M-1). By contrast, the affinities of four murine IgG1 and four IgG2b monomers were 100-fold to 1000-fold lower than the affinity of the human IgG1-FcR interaction. A 68,000 to 72,000 dalton protein was isolated by affinity chromatography from blood monocytes and from IFN-gamma-induced U937 cells on murine IgG2a, IgG3, and human IgG immunoadsorbents. In binding assays with IFN-stimulated U937 cells, murine IgG2a and IgG3 antibodies showed complete cross-blocking with a human IgG1 myeloma protein, indicating that murine and human IgG interact with the same population of Fc-binding proteins. No evidence for heterogeneity of cross-reactive FcR was observed. The ability of murine IgG2a and IgG3 monomers to compete with human IgG1 monomers for binding to human monocyte FcR suggests the potential usefulness of antibodies of these isotypes in the immunotherapy of diseases in which monocyte- or macrophage-mediated, antibody-dependent cellular cytotoxicity may play a role in the modification or remission of disease.     

Activation requirements for antigen- and mitogen-induced interferon-gamma release from cytotoxic T lymphocytes

We have studied the activation signals that regulate interferon-gamma (IFN-gamma) secretion from murine cytotoxic T lymphocytes (CTL) upon binding mitogen or antigen. CTL clones were found to require at least 1 hr of stimulation with concanavalin A (Con A) in order to produce detectable levels of IFN-gamma. Full activation of IFN-gamma synthesis in CTL clones occurred after stimulation for 2 hr or more, and in those cultures CTL continued to produce high levels of IFN-gamma even after the effects of Con A had been neutralized. Splenic T cells and uncloned long-term CTL lines required a longer period of stimulation than cloned CTL for Con A-induced IFN-gamma secretion. The relationship between IFN-gamma secretion and cytotoxic activity was studied in an antigen-specific system. These studies reveal marked differences in the types of effector responses generated by CTL upon contact with antigen, demonstrating that some antigen-bearing cells promote high levels of IFN-gamma secretion and are poorly lysed by CTL, whereas other cell lines are lysed with high efficiency by CTL but induce low levels of IFN-gamma secretion.     

Defective in vitro T cell colony formation in the acquired immunodeficiency syndrome

Depressed T cell immunity is a universal characteristic of the acquired immunodeficiency syndrome (AIDS). In the present study, 25 patients with AIDS and opportunistic infections, 22 individuals with AIDS-related complex (ARC, or chronic lymphadenopathy syndrome), and 20 healthy homosexuals were evaluated by means of the T cell colony assay. Forty-seven healthy heterosexual controls showed an average of 3964 +/- 319 colonies/7.5 X 10(5) cells, with a range of 880 to 9340. The mean in the 20 healthy homosexuals (3173 +/- 483) did not differ significantly from the controls; in this group, only three patients had values less than 1000 colonies/plate. By contrast, all AIDS patients and 14 ARC patients had colony counts less than 1000. The mean value for the AIDS patients was only 24 +/- 15 (p less than 0.0005 compared with either controls or healthy homosexuals); values in the ARC group were intermediate (1180 +/- 360). The addition of interleukin 2 to the plates promoted correction of the proliferative abnormality in ARC patients. This interleukin increased colony scores in the AIDS group, but the mean value was still significantly less than controls. Comparison indicated that the colony assay is a more sensitive indicator for detecting proliferative abnormalities than responses to PHA, Con A, or pokeweed mitogen in suspension cultures.     

Accumulation of heat shock proteins in field-grown cotton

Cotton (Gossypium hirsutum L.) plants grown under field water deficits exhibited an 80 to 85% reduction in leaf area index, plant height, and dry matter accumulation compared with irrigated controls. Midday photosynthetic rates of dryland plants decreased 2-fold, and canopy temperatures increased to 40°C at 80 days after planting compared with canopy temperatures of 30°C for irrigated plants. Leaves from dryland plants which had exhibited canopy temperatures of 40°C for several weeks accumulated stainable levels of polypeptides with apparent molecular weights of 100, 94, 89, 75, 60, 58, 37, and 21 kilodaltons. These polypeptides did not accumulate in leaves from irrigated plants.

Addition of [³⁵S]methionine to leaves of growth chamber-grown cotton plants and subsequent incubation at 40°C for 3 hours radiolabeled polypeptides with molecular weights similar to those that accumulate in dryland cotton leaves. These data suggest that the proteins which accumulate in water-stressed cotton leaves at elevated temperatures (40°C) are heat shock proteins and that these proteins can accumulate to substantial levels in field-stressed plants.

     

Transformation by SV 40 virus sensitizes fibroblasts of human skin to the lytic action of H-1 parvovirus

Human skin fibroblasts which are naturally resistant to Parvovirus H-1 can be lysed by this virus after SV40 transformation. This observation raises the possibility that oncosuppression by Parvovirus involves a direct oncolytic effect.     

Purification and characterization of a saliva-interacting cell-wall protein from Streptococcus mutans serotype f by using monoclonal-antibody immunoaffinity chromatography.

A rat monoclonal antibody, LO SM2, of the immunoglobulin M class, specific for a saliva receptor (SR) from Streptococcus mutans serotype f, was able to precipitate the SR from crude cell-wall-associated antigens (WEA) of this bacteria in presence of a detergent mixture. We have then used the technique of monoclonal-antibody immunoaffinity chromatography to purify the S. mutans SR. Pure SR was obtained from a crude WEA fraction with a single chromatographic step. The active SR could be eluted from the column in a highly purified form with 0.2 M-glycine/HC1, pH 2.8. The final yield was about 32% in terms of binding activity. Characterization of the SR by crossed immunoelectrophoresis, sodium dodecyl sulphate- or 4-30%-native-gradient-polyacrylamide-gel electrophoresis showed that the receptor is a single polypeptide chain of Mr approx. 74000. Native or denaturated forms of the SR adsorbed on to a solid support, such as nitrocellulose, are recognized by monoclonal antibody LO SM2, and both forms are still able to bind the ligand, saliva.     

Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue

Summary The deffects of a purified homologue of tunicamycin (B₂-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6–8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B₂-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B₂-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B₂-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - MF mouse fibroblasts from Balb/c mouse embryos - 3T3 Balb/3T3 mouse fibroblastic line - SV40 Simian virus 40 - PY polyoma virus - TLC thin layer chromatography