Volume 11, Nº 3 (2017)

Extracellular vesicles secreted by cells represent an almost spherical membrane structures enriched with biological molecules of different types. The number and molecular composition of these structures depend on both the physiological state of an organism and underlying diseases. Despite extracellular vesicles playing an important role in intercellular communication and being potential biomarkers of pathological processes, the mechanisms of their formation, their functions, and their morphological characteristics are poorly studied. Low-voltage scanning electron microscopy is a promising method for studying extracellular vesicles, since it does not need a layer of conductive covering and, consequently, permits morphological details of studied objects to be vizualized at a high resolution in a nanometer range. The results of investigation of the morphology and sizes of objects in blood-plasma fractions by low-voltage scanning electron microscopy are presented in this study.

The present article reports immunofluorescence-based analysis of the distribution of the actin-binding protein myosin-9 in the cytoplasm of human embryonic lung fibroblasts. Electrophoresis and Western blotting were used to demonstrate that myosin-9, actin, and high molecular weight tropomyosin isoforms are incorporated into cytoplasmic protein complexes not bound to cytoskeletal structures. Cross-immunoprecipitation was used to show that these complexes were rapidly disassembled when the cells were exposed to lysophosphatidic acid (LPA). Moreover, LPA induced proteolytic degradation of myosin-9 associated with the structures of the actin cytoskeleton. The results obtained point at the participation of multimolecular cytoplasmic protein complexes that contain myosin-9 and tropomyosin in the regulation of the cellular response to stimulation with LPA.

Nuclear lamins form nuclear lamina located under the inner nuclear membrane. It was believed that the nuclear lamina plays predominantly a structural role. Recently, its involvement in regulatory processes have been described, e.g., chromatin organization and gene transcription. It is known that mutations in the LMNA gene lead to development of laminopathies, primarily affecting tissues of mesenchymal origin. Today, the mechanisms of the lamina regulation of cell differentiation are largely unknown. In the present work, we studied the effect of LMNA gene mutations on the process of muscle differentiation of primary satellite cells and in С2С12 cell line. The genome of satellite and С2С12 cells was modified by cell transduction via lentiviral constructs encoding LMNA G232E associated with the development of Emery–Dreyfus muscular dystrophy and LMNA R571S associated with the development of dilated cardiomyopathy. Cell morphology was assessed with immunofluorescence, and expression of myogenic genes was analyzed by qPCR. We showed that the analyzed mutations reduced the cell ability to differentiate (to fuse and to form myotubes). We proposed that these mutations enhanced expression of early and reduced expression of late markers of myogenesis. Thus, mutations in nuclear lamins can modify the process of muscle differentiation.

Lipophilic derivatives of peptides corresponding to cytoplasmic regions of G protein-coupled receptors (GPCR) can act as intracellular agonists. Our previous work showed that peptides corresponding to residues 562–572 of luteinizing hormone receptor and modified with palmitate or decanoate at the C-terminus activate adenylate cyclase in rat testes. The stimulating effect of peptide 562–572 modified with decanoates at both the N- and the C-termini (peptide IV) reached its maximum at the peptide concentration of 10^–5 M and diminished with further increase in its concentration. It was supposed that this effect was due to peptide IV ability to form micelles. To verify this hypothesis, the relationship between biological activity, hydrophobicity, and ability to form micelles was investigated for peptide IV and other acylated derivatives of peptide 562–572, including those carrying C-terminal decanoate (peptide III) and palmitate (peptide VI) moieties. It was found that the stimulating effect of peptide IV taken in the concentration of 10^–5 M on adenylate cyclase activity in plasma membranes of rat testes and ovaries was only slightly lower than that of peptide VI and higher than the effect of peptide III. At the concentration of 10^–3 M, the effect of peptide IV was 20–27% lower and amounted to only 50–51 and 87–88% of the effects of peptides VI and III, respectively. In spite of its high hydrophobicity, peptide IV was characterized with an abnormally low retention time when eluted from a Nucleosil C8 column during reverse-phase HPLC: it was even lower than the retention time of nonmodified peptide 562–572. However, the retention time of peptide IV, but not of other peptides, increased significantly when the eluent contained a higher proportion of trifluoroacetic acid, which disrupts micellelike structures (0.5 instead of 0.1%). The surface tension of peptide IV solution in water slightly decreased with increasing peptide concentration, but rapidly dropped and reached a plateau at the concentration of 7 × 10^–6 M, which indicates the beginning of micelle formation. Thus, peptide IV in the concentrations above 10^–5 M forms micelles, which prevents it from interacting with the receptor. The ability of GPCR peptides to aggregate and form micelles should be taken into account in the development of their new membrane-active analogs.

The aim of this study was to compare the effects of sulfated fucopolysaccharides isolated from Fucus vesiculosus on HeLa G-63, Hep G2, and Chang liver cells. Native fucoidan F3 and two fractions (F3-0.5 and F3-1) obtained by anion-exchange chromatography were analyzed using chemical methods and IR spectroscopy. It was demonstrated that F3 and F3-1 are characterized by a higher content of sulfates, location of sulfo groups mostly at the C4 atom of fucose residue, and low content of uronic acids inhibited cell proliferation. Human liver carcinoma Hep G2 appeared to be the most sensitive to fucoidan, whereas nonmalignant human Chang liver cells were the least sensitive.