Vol 12, No 6 (2018)

The transmembrane protein endoglin (CD105) is a component of the receptor complex for TGF-β family growth factors. It is expressed primarily in endothelial cells, mesenchymal stromal cells, and early hematopoietic precursors. The density of CD105 on the membrane of endothelial cells increases upon their proliferation. A soluble endoglin form (sEng) is produced by a cleavage of an extracellular fragment from CD105, performed by the matrix metalloproteinase MMP-14. sEng blood level serves as an indicator of angiogenesis during the progression of some tumors. The contribution of tumor cells into the sEng pool remains unknown. We investigated the expression of CD105, production of sEng, as well as the mRNA level of two splice-variants of endoglin and mmp-14 genes using a collection of 43 cultures of tumorous and normal cells of different histological origin. Over half of the tumor cell lines and normal stromal cells contained cell populations expressing the membrane-bound endoglin. Cytoplasmic endoglin with unknown function was found in cells of three tumor lines. In all cell cultures, the mRNA expression of L-endoglin splice-variant prevailed over the expression of S-endoglin. We have investigated, for the first time, the formation of sEng in the stromal and tumor cells, and revealed the endothelial cells as the main source of sEng. Normal stromal cells and the majority of CD105⁺ tumor cells produce only low levels of sEng. Among the tumor cell lines, the highest sEng production was detected in the MeWo melanoma cells, that are characterized by the highest activity of mmp-14 gene.

Using the sectional analysis of two-dimensional electrophoretic gels with liquid chromatography-mass spectrometry, proteoform profiles for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells were obtained. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms that are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy. Using a section analysis of two-dimensional electrophoretic gels with liquid chromatography by mass spectrometry, proteoform profiles were obtained for individual genes expressed in cancer (glioblastoma) and normal (FLEH) cells. Profiles of more than 5000 genes were analyzed. It turned out that many genes encoding potential biomarkers of glioblastoma are characterized by sets of proteoforms, which are different in normal and cancer cells. These proteoforms could be sources of highly specific markers and targets for therapy.

Single intraperitoneal injection of methylnitrosourea (MNU) induces in mice the loss of retinal photoreceptors as a nonlinear dose response (Tronov et al. 2015). DNA repair was the putative mechanism for causing a threshold of DNA alkylation in retina cells. Photoreceptor degeneration can stimulate Müller glial cells to transdifferentiate into photoreceptor-like cells in adult mouse retina treated with MNU (Wan et al., 2008). In this paper, we evaluated Müller cell proliferative response to different doses of MNU and compared the response to DNA damage and repair in suspensions of retinal and Müller cells using a comet assay and BrdU (thymidine analogue) as a marker of proliferation. MNU administration in the dose ≤ 40 mg/kg did not result in the activation of Müller cell proliferation in 3 days after the treatment. By this time point, no DNA damage after this dose was observed. For MNU doses that exceed 50 mg/kg, TUNEL-detected death of retinal photoreceptors increased more than 10-fold in the proliferating pool of Müller cells. DNA breaks (single- and double-strand breaks and AP sites) were observed. The results are discussed within the framework of the concept of misbalance of excision repair that results in formation of cytotoxic intermediates in DNA initiated death of photoreceptors followed by activation of Müller retinal cells.

The purpose of this study was to study the relative distribution of the nucleophosmin (B23) protein and histone H4K20me3 in the granule cells of the rat cerebellar cortex. The material for the study was the samples of the cerebellum of mature male rats of the Wistar line (n = 8). Using the methods of immunocytochemistry and confocal laser microscopy for the first time, the features of the distribution of the nucleophosmin protein in the nuclei of the cerebellar granule cells were studied. In the nucleolus, a region that was characterized by the lack of the B23 protein and, supposedly, a fibrillar center was found. In addition to the nucleolus, nucleophosmin was detected (in a smaller amount) in the nucleoplasm of the cell granules. Using the method of confocal laser microscopy and three-dimensional reconstruction, we demonstrated the presence of zones of colocalization of the B23 protein and histone H4K20me3 within the nucleolus and nucleoplasm of the cell granules. It was shown that histone H4K20me3 is part of the perinucleolus heterochromatin, from which various protrusions often extend into the interior of the nucleolus. Clusters of the extranucleolus nucleophosmin were also often colocalized with heterochromatin blocks. However, nonnucleolus clusters of nucleophosmin that were not associated with heterochromatin blocks were also present. The results obtained make a significant addition to the existing ideas about the structural and functional organization of the nucleus of the cerebellar granule cells.

Differences in the phagocytosis process of opsonized and nonopsonized strains of Staphylococcus aureus 2879 M and Escherichia coli 321 were studied. Differences in the character of pseudopodia during phagocytosis by neutrophil granulocytes (NGs) of opsonized and nonopsonized bacteria were detected, and differences in the nature of pseudopodia in reactions to gram-positive and gram-negative microorganisms were not detected. For the first time in dynamic observations at the late stages of phagocytosis, changes in the volume of nuclei and their movement, variations in the intersegment distance of the nuclei, and a slight increase in the volume of NGs were shown. A decrease in the rigidity of the membrane–cytoskeleton NG complex correlating with the intensity of phagocytosis and opsonization of bacteria was shown for the first time. It was established that opsonization does not affect the oxygen-dependent metabolism of NGs and, at the same time, introduces significant adjustments in the implementation of oxygen-independent bactericidal mechanisms of cells.

It is known that the regulation of muscle contraction is carried out by tropomyosin and calcium-sensitive protein troponin, which form thin filament with the F-actin. The noncanonical glycine residue at position 126 of the central part of the skeletal alpha-tropomyosin destabilizes the structure of this protein. The substitution of glycine residue by arginine residue stabilizes the central region of tropomyosin, displaces tropomyosin to the open position and activates the switching actin monomers on during the ATP hydrolysis cycle. To investigate how Gly126Arg substitution affects the interaction of the myosin head with F-actin in the ATP hydrolysis cycle, the myosin subfragment-1 (S1) was modified with a 1,5-IAEDANS fluorescent probe and AEDANS-S1 was incorporated into the ghost muscle fiber. Multistage changes in the mobility and spatial organization of the myosin head during simulation of different stages of the ATP hydrolysis cycle were studied by polarization fluorescence microscopy. It was shown that, in the regulated thin filaments of the ghost muscle fiber at high concentrations of Ca²⁺, Gly126Arg substitution significantly increases the number of myosin heads strongly associated with F-actin when simulating strong binding of myosin to actin, but reduces the number of such heads when imitating weak binding of myosin. Such changes in the behavior of myosin in the ATP hydrolysis cycle indicate an increase in the efficiency of myosin cross-bridges. A significant increase in the relative amount of myosin strongly bound to actin was also observed at low Са²⁺ concentrations. This indicates an increase in Са²⁺-sensitivity of a thin filament initiated by Gly126Arg substitution. The obtained data suggest that the stabilization effects of the central part of tropomyosin by Gly126Arg substitution are realized through the abnormal behavior of tropomyosin and troponin, which leads to a change in the nature of the interaction of myosin with actin and tropomyosin in the ATP hydrolysis cycle.