Demonstration of an intact complex of epidermal growth factor and its receptor in the endosomes of A-431 cells

The compartmentalization of the epidermal growth factor (EGF) receptors in A-431 cells was studied using centrifugation of the microsomal fraction of these cells in continuous Percoll gradient. The existence of an intact (non-degraded) EGF receptor in plasma membrane and endosome fraction was demonstrated by electrophoretic analysis of in vitro phosphorylated Percoll fractions. No phosphorylated receptor was revealed in lysosomal fraction by this method. The existence of non dissociated EGF-receptor complexes in intracellular compartments 30 minutes after the start of internalization was proven using a synthesized photoreactive labeled EGF derivative (125I-EGF-SANAH). The removing of pH gradient in organellar membranes by 10 mkM of monensin did not affect dissociation from its receptor. The data obtained proved the existence of non-dissociated and non-degraded EGF-receptor complexes in the endosomal compartment of A-431 cells.     

Endocytosis and intracellular transport of epidermal growth factor after destruction of the actin cytoskeleton by cytochalasin B

Endocytosis of the epidermal growth factor (EGF) was investigated in three cell lines--A431, 3T6 and Swiss 3T3--after their incubation with cytochalasin B (CB). CB was introduced into culture medium (10 mkg/ml) 1.5-2 hours before addition of 125I-EGF (20-40 ng/ml). The label uptake rate was measured after a 35-40 minutes incubation of cells with 125I-EGF. It appeared that disorganization of microfilamentous network caused by CB exerted no influence on the binding of EGF to the surface membrane receptors and its internalization. Nevertheless, the experiments performed on A431 cells using a fluorescent label--rhodamine--bound to EGF (EGF-R) indicate that CB, though not influencing the initial steps of endocytosis, inhibits the next step--the intracellular transport of EGF-receptor complexes from the trans-Golgi region to lysosomes. As was shown elsewhere (Barkan, Nikol'sky, 1986), CB inhibits the mitogenic effect of EGF on resting Swiss 3T3 cells. So, the process of EGF-receptor uptake and delivery to the trans-Golgi region is evidently not enough to stimulate the cell proliferation; next steps of transport and degradation of ligand-receptor complexes are presumably needed.     

Magnetic interaction of manganese with pheophytin anion-radical and chlorophyll cation-radical in reaction centers of the photosystem II

The influence of Mn on saturation curves of ESR spectra of Ph(-) and P(+)(680) at 1-200K in samples with different content of Mn has been studied. An analysis of these data and those on photoinduced changes of fluorescence yield of chlorophyll leads to the conclusion that the Mn-containing centre in Photosystem 2 is a cluster of 4 Mn atoms, two of which can be replaced by Mg(2+) or any other divalent metal. The distances between Mn Na Ph as well as between Mn and P(680) have been estimated.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Role of immune cytolysis and its thymosin stimulation in experimental influenza

In experiments on C57BL and CC57W mice the acute or chronic course of experimental influenza infection has been shown to correlate with the activity of immune cytolysis. At a low level of the cytolytic activity of T-lymphocytes the prolonged persistence of influenza virus develops. The stimulation of cell-mediated immunity with thymosin prevents the development of chronic influenza infection.     

Cyclic nucleotides (cAMP and cGMP) in the dog plasma during extracorporeal hemocarboperfusion

The concentrations of cyclic nucleotides (cAMP and cGMP) were measured in blood plasma of dogs subjected to extracorporeal hemocarboperfusion. In spite of complete and irreversible absorption of cyclic nucleotides by the coal absorbent, SKN-2M, the concentration of cGMP in plasma remained within normal. The concentration of cAMP increased 1.5-2.5-fold. The rise in the cAMP concentration was not connected with injection of sodium thiopental and heparin before hemocarboperfusion. It is assumed that cyclic nucleotides play the role of circulating intercellular regulators in blood plasma. These regulators are necessary for the normal course of metabolic processes.