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A study was made of an association of small GTPase Rab7, commonly considered as a marker of late endosomes, with endosomal compartments of cells expressing EGF receptor with different ability to be sorted for degradative pathway. It was found that in cells HER14, expressing normal EGF receptor, Rab7 was associated with both early and late endosomes and the extent of association correlated with the number of EGF-receptor complexes in the specific endosomal fraction. Cels with a receptor, lacking major sites of autophosphorylation by deletion of 126 C-terminal residues (CD126), demonstrated a low efficiency of EGF-receptor sorting to late endosomes and decreased association dynamics with Rab7. Interaction of Rab7 with endosomes of cells expressing kinase negative receptors (K721) was found to be minimal. At the same time, in cells Cd126 and K721 with a low sorting efficiency Rab7 was mainly associated with early endosomes. These data favor Rab7 involvement in mediating early-to-late endosomal transition.

During endocytosis EGF-receptor complexes are transported from early peripheral endosomes to late juxtranuclear-located endosomes to be then degraded in lysosomes. It is suggested that such a spatial organization of endosomal compartments is maintained by microtubule system and is necessary for lysosomal degradation of endocytosed cargo. In the present work, a study was made of the influence of Nocodazole, a microtubule depolymerizing agent, on endocytosis of fluid phase marker HRP and EGF entering the cell via receptor-mediated endocytosis. By subcellular fractionation in Percoll gradient it was shown that Nocodazole did not affect HRP internalization but stimulated its accumulation in a fraction sedimented together with late endosomes, thus preventing HRP delivery to lysosomes. On the contrary, Nocodazole exerted no influence on dynamics of compartmentalization and lysosomal degradation of EGF-receptor complexes. Moreover, no alterations were found in the functioning of a so well-known EGF-stimulated signal transduction pathway as MAP-kinase cascade. At the same time microtubule depolymerization dramatically changed the morphology of endosomal compartments abolishing juxtranuclear localization of late endosomes. Our data suggest that translocation of EGF-receptor complexes is not necessary for their normal lysosomal processing. Rab7, traditionally considered as a marker of late endosomes, has been found to demonstrate in Nocodazole-treated cells, in contrast to the control, a low extent of co-localization with endosomal structures. It could be supposed that the role of Rab7 is not so much to mediate early-to-late endosome transition as to maintain spatial organization of endosomal apparatus by mediating endosome-cytoskeleton interactions.

The transcription factor STAT3 (signal transducers and activators of transcription) takes part in cell signaling. Here we show that STAT3 is associated with the cytoskeleton in A-431 cells. The protein is determined in cytoskeletal (detergent insoluble) fraction of control cells and of cells after RGF treatment. STAT3 is diffusely distributed in the cytoplasm of control cells to be seen relocalized to the nuclei after EGF treatment. Besides, the protein is observed in cell membrane protrusions. Using double immunofluorescence, co-localization of action and STAT3 was shown in these structures.

The endocytosis of 125I-EGF was studied in transfectant NIH3T3 cells expressing a normal full-length EGF receptor (HER14) and a mutant receptor lacking 4 major autophosphorylation sites by deletion of 126 amino acids from the carboxyl terminus (CD126). The rates of 125I-EGF internalization and degradation in CD 126 were found to be slightly lower compared to HER14. Analysis of 125I-EGF-complexes compartmentalization during endocytosis, made by subcellular fractionation in 17% Percoll gradient, revealed different behavior of full-length and truncated receptors. In the former case the major portion of internalized 125I-EGF was found to transit rapidly from light (early) to heavy (or late) endosomes, while in CD126 cells the efficiency of this transition, which reflects sorting of EGF-receptor complexes for degradative pathway, was significantly lower. At the same time, cells with the truncated receptor showed an increased level of 125I-EGF recycling compared to HER14. The results obtained suggest that in regulation of EGF receptor sorting for degradative pathway a protein, containing so called SH2-domain, is involved. Failure of the truncated receptor to be sorted efficiently may indicate a direct interaction of this protein with phosphorylated C-terminal tyrosines of the receptor.

Two subpopulations of epidermal growth factor (EGF) receptor with different affinity to EGF have been demonstrated for many cell types. There are reasons to assume a key role of high-affinity receptors in stimulation of cell response to EGF. Nevertheless, characteristics of its action remain obscure. In the present work an attempt has been made to study internalization and intracellular compartmentalization of high-affinity receptors. For this purpose endocytosis of 125I-EGF in A431 cells was stimulated by low (less than 1 nM) EGF concentrations as well as after blocking of low-affinity binding sites with specific monoclonal antibodies 2E9. By subcellular fractionation in 17% Percoll gradient it was demonstrated that in both cases internalized 125I-EGF was found first in light, and then in heavy endosomes staying there for a long time. Effectiveness of 125I-EGF delivery to prelysosomal heavy endosomes as well as initial internalization rate is maximal at low EGF concentrations and is reduced dramatically with increasing of receptor occupancy. Monoclonal antibody Mab108 specifically recognizing high-affinity receptors were capable of stimulation of receptor internalization with initial rate higher than that of high EGF concentrations, but Mab108-receptor complexes were localized in light endosomes. Preincubation of the cells with low concentrations of EGF led to redistribution of considerable portion of 125I-Mab108 into heavy endosomes, which may be a result of high-affinity receptor tyrosine kinase activation. Our data confirm a hypothesis of TK involvement in regulation of both internalization and sorting of EGF-receptor complexes. Structural organization of high-affinity receptors is not sufficient for receptor targeting to degradation pathway.

In chronic experiments (4 months) in rabbits, rats, and mice biological effects were investigated from 7 exposure regimen of electromagnetic fields with a frequency of 24 MHz at field strengths of the electric field component of 125, 250, 500, and 1,000 V/m, respectively, and an exposure time of 0.25, 1, and 4 hrs. respectively. The effects on the CNS, the immune and hormone systems, the peripheral blood and on the spermato and embryo genesis were estimated. The results delivered the basis for the introduction of an index. In point of time different limit values are to be determined according to the energetic load. A concrete value for the energetic load 7,200 (V/m)2.h is proposed as maximum allowable field strength of the electric component 300 V/m.

Mice with transplantable Harding-Passey melanoma were treated with polysaccharide mannan (50 mg/kg), cyclophosphamide (20 and 100 mg/kg) and their combinations. Mannan proved to possess considerable antitumor properties. It potentiated the therapeutic effect of cyclophosphamide and diminished its toxicity. Mannan treatment resulted in two-phase dynamics of cyclic nucleotide system in mouse liver and melanoma.