Regulation of plasma membrane protein phosphorylation in two mammalian cell types.

The appreciation of protein phosphorylation as a ubiquitous mechanism for the post-translational control of protein function has drawn our attention to the phosphorylation of plasma membrane proteins. We have studied this phenomenon in the human erythrocyte and rat adipocyte, and have observed several features, common to the two systems, which may be of general significance. In examining protein phosphorylation in intact cells incubated with 32Pi, it is evident that the 32P-polypeptides of the plasma membrane are among the most highly labelled species in the cell, despite their minor contribution to overall protein content. The addition of epinephrine (to adipocytes) or cAMP (to erythrocytes) increases the phosphorylation of certain peptides, whereas others are unaffected. The protein kinases mediating these phosphorylations are present in the plasma membrane as isolated, and can be divided into two groups--cAMP dependent and cAMP independent. These two classes of kinase differ markedly in their substrate specificity toward endogenous and exogenous polypeptide substrates. Two classes of protein kinases with similar properties can be detected in the cytoplasm. The relationship between the membrane-bound and cytoplasmic enzymes is uncertain. The potential roles of the plasma membrane cAMP dependent protein kinases are evident from the diverse effects of cAMP on surface properties; however, the prevalence of plasma membrane proteins phosphorylated via cAMP independent pathways is striking. Thus, elucidation of the regulatory properties of the plasma membrane cAMP independent protein kinases may give new insight into the control of a variety of surface phenomena not mediated by cAMP.     

Hybridization frequencies of different mammalian cell types by electrofusion

The efficiency of electrofusion of four types of cells: CHO, HeLa, mouse melanoma cells and human skin fibroblasts has been studied. The frequencies of fusion products were determined 1) directly in a closed flow-through fusion chamber after dielectrophoresis and pulsation; 2) after short-term postfusion cultivation period of 5 to 10 minutes; and 3) in various intervals up to 30 hours after fusion induction. No substantial differences were found in the rates of formation of heterokaryons and synkaryons between the individual cell types, and this confirmed the uniformity of the effects of electric fields on diverse cell membranes. After 5 hours of culture the yield of fusion products reached 15 to 35% in various cell combinations and the frequencies of synkaryons reached up to 7% in almost all the combinations studied 24 to 30 hours after fusion.     

Filament-carrying tubules demonstrated by negative staining in various mammalian cell types

Summary Among other tubular elements obviously representing the endoplasmic reticulum, tubules carrying filaments with a diameter of about 4 m were found in negatively stained specimens of a variety of mammalian cell strains. They have been found in strains of epithelial and fibroblastic, normal and malignant, human and animal origin. So far, it is not possible to identify the filament-carrying tubules with equivalent structures in thin sections.     

Growth in retinal glial cells in vitro is affected differentially by two types of cell contact-mediated interactions

Contact among rabbit retinal glial cells in subconfluent culture was previously shown to stimulate DNA synthesis [J. M. Burke (1983) Exp. Cell Res. 146, 204-206]. In this study nonliving surface membranes and metabolic coupling were investigated as mediators of the contact-dependent phenomenon. To evaluate surface membranes, preparations of fixed glial cells and fixed fibroblasts of several types were added in varying numbers to sparse cultures of glia or fibroblasts. In agreement with published data, fibroblast proliferation was inhibited by the fixed cells in a dose-dependent manner. Growth in glial cells was similarly inhibited. Fixed cells of both types were approximately equally effective in suppressing proliferation in cells of both types. No number of fixed cells was identified which, when added to glial cultures, stimulated glial proliferation. In contrast, metabolic coupling among glial cells was associated with increased DNA synthesis. Coupling was detected radioautographically as a flux of labeled precursor molecules from a prelabeled to a recipient population of glial cells in coculture. The cocultures were secondarily incubated with [3H]thymidine to label the nuclei of S-phase recipient cells. In the cocultures there was a higher rate of nuclear labeling in coupled than in uncoupled recipient glial cells. The results suggest that growth in subconfluent retinal glial cell cultures is modulated differentially by two types of interactions which require cell contact: growth is inhibited by interaction among nonliving cell surfaces but stimulated by metabolic cooperation among living cells.     

The effect of pH and 1,4-dithiothreitol on the adhesion of rumen bacteria

In experiments with ram rumen fluid the pH optimum for adhesion of rumen bacteria to cellulose and starch was found to be near 6. Addition of 1,4-dithiothreitol increased the adhesion logarithmically.     

The presence of ribonucleic acid within the peripheral zones of two types of mammalian cell

An attempt has been made to locate RNA as a structural component of the peripheries of cultured cells derived from a human osteogenic sarcoma, and L1210 mouse leukaemia cells. In the case of cells derived from the osteogenic sarcoma, their detachment from glass was facilitated by incubation with ribonuclease; on removal from glass, they left cellular “footprints” behind, which were visulized in radioautographs of cells previously labeled with tritiated uridine, and removable with ribonuclease. The electrophoretic data show loss of charge by both types of cell following incubation with ribonuclease. These results are interpreted to indicate that RNA is a structural component of the peripheries of these cells. No attempt is made to speculate on the obvious biological improtance of these observations if they are applicable to cells in general.     

Calcium-dependent process in reduction of cell surface charge after x-irradiation.

The electrophoretic mobility (EPM) of rat erythrocytes and cultured melanoma cells decreased with time after X-irradiation in the presence of calcium at concentrations higher than 10 (-5) M. At 37 degrees C, the presence of calcium for the first 20 min of exposure was suffcient to induce the EPM reduction, and Ca 2+ administration subsequent to Ca 2+ -free incubation for 30 min following irradiation had no effect on EPM. At lower temperatures, from 10 down to 20 degrees C however, the effect of calcium on the reduction of EPM decreased drastically. If the cells were kept Ca 2+ -inonophore A23187 also induced to decrease in EPM only in the presence of Ca 2+. These results revealed the transitory existence of membrane condition reactive to extracellular Ca 2+ immediately after X-irradiation, which can be postponed at low temperatures. The reduction of EPM by Ca 2+ -ionophore might suggest that the influx of Ca 2+ is a step in the reduction of EPM after X-irradiation.