Vol 11, No 2 (2017)

SOS-response is an important tool of bacteria intended to protect their genome and thereby allow them to survive under adverse conditions. Recently SOS-response was demonstrated to enhance mutagenesis and thus help bacteria to acquire antibiotic resistance. Due to high significance of this phenomena it seems to be important to investigate processes that allow bacteria to survive after SOS-response activation. In current work the recovery of division process of Escherichia coli cells after division arrest due to expression of SOS-response protein SulA was studied. Data indicate that cells are able to rapidly restore normal division; also nucleoid occlusion seems to be the main septum positioning mechanism during the process. In the course of recovery FtsZ forms helix-like structures, which then transform into Z-rings.

Various organic compounds are applied upon cryopreservation and their adding into cell suspension causes modification of subcellular systems, providing cell survival during freeze–thawing. The aim of the study was to assess the modifying effect of cryoprotectant PEG-1500 and low temperatures on Ca²⁺-ATPase activity in saponin-permeabilized erythrocytes. PEG-1500 was revealed to inhibit erythrocyte Ca²⁺-ATPase activity despite the presence of endogenous effectors able to stimulate the enzyme function. Presumably, the Ca²⁺-ATPase modification was determined by the physicochemical properties of the polymer solution, since the removal of PEG-1500 out of the medium recovered the enzyme activity. Reversibility of Ca²⁺-ATPase inhibition was characteristic of erythrocytes both exposed to cryoprotectant without freezing and frozen–thawed in the PEG-1500 presence. The cell freeze–thawing without cryoprotectant had no effect on Ca²⁺-ATPase, suggesting that membrane form of enzyme is cryoresistent. Although the efficiency of erythrocyte cryopreservation with PEG-1500 depends on the incubation temperature before freezing stage, the functional indices of Ca²⁺-ATPase in erythrocytes exposed to PEG-1500 at 37 and 5–7°C had no significant distinctions if the subsequent ATP hydrolysis was conducted at 37°C. However, the enzyme activity was additionally slowed down when the temperature of enzymatic reaction was decreased to 5–7°C after erythrocyte preincubation with PEG-1500 under the same conditions. The identified changes in Ca²⁺-ATPase activity in erythrocytes in the PEG-1500 presence were most likely determined by a modifying effect of the cryoprotectant on the membrane structure; as a result, the Ca²⁺-ATPase endogenous effectors present in the medium could not overcome the restrictions imposed on the enzyme function by a modified membrane macroenvironment.

We have studied mast-cell (MC) activation in the border area of mechanically damaged muscle in Rana temporaria lymph heart for 1 week after the surgery. Twenty-four hours after surgery, the cytoplasm of resident and circulating MCs contained degranulation channels discharging via compound exocytosis. Rare cases of single membrane-free expelled granules were revealed in the space limited with the plasma membrane of a resident MC and surrounding basal lamina. During the first week after the surgery the ultrastructural changes in many secretory granules of activated MCs concerned the granule swelling, reduced amount of the electron-dense component and pronounced loosing of their matrix. It is noteworthy that unaltered and altered granules are located close to empty degranulation channels frequently penetrated MC cytoplasm from the surface to centrally localized nucleus. Immunocytochemical MC labeling with antibodies to histamine revealed that gold particles were located under both altered and unaltered granules. In altered granules, gold particles were arranged above a preserved electron-dense part, whereas a loosened matrix, as a rule, was not labeled. Ultrastructural signs of secretory granule formation in the MC cytoplasm were disclosed to the end of the first week after surgery. Electron-microscopic autoradiography revealed that replicative and transcriptional activity of an MC in the border area was not prominent during all the periods after the surgery. Mitotic MCs were not observed. Occasionally, MCs dying via necrosis were encountered. These observations show that MCs stimulated by mechanical damage of the lymph heart secrete various preformed mediators, histamine in particular. The early response of MCs in the border area is evidence that activated MCs play a key role in the initiation of inflammation in the damaged cross-striated muscle of the lymph heart.

Cyto- and genotoxic effects of usnic-acid (+)- and (–)-enantiomers have been studied in human peripheral-blood lymphocytes. It has been shown that usnic-acid enantiomers in concentrations of 0.04–0.30 mM have pronounced cytotoxic action. Usnic-acid enantiomers in concentrations of 0.04–0.30 mM have exhibited a genotoxic effect; moreover, the genotoxicity of usnic-acid (–)-enantiomer in concentrations of 0.15 and 0.30 mM was twice as high as that of (+)-enantiomer. The effect of usnic-acid (+)- and (–)-stereoisomers has been accompanied by the formation of atypical DNA comets. Furthermore, (–)-usnic acid has induced 2.5–3.5 times more atypical comets than its (+)-stereoisomer.

The connections between the presence of low molecular weight RSH-antioxidants (N-acetylcysteine, glutathione) in serum-free medium, generation of reactive oxygen species (ROS), and proliferation of SP2/0-SF mouse myeloma cells have been demonstrated. It is shown that the presence of RSH compounds in the medium within the studied range of concentrations changed the contents of ROS in cells and had a dose-dependent effect on cell proliferation. Stimulation of the proliferative activity did not depend on the nature of an RSH compound. The optimal concentration for the both antioxidants was 0.2 mM. A further increase of the concentration led to inhibition of cell proliferation to different degrees for N-acetylcysteine and glutathione.

Exosomes, a special type of microparticles produced by cells, are currently of considerable interest for researchers. The term “exosomes” denotes extracellular vesicles of less than 120 nm in size derived from intracellular multivesicular bodies. Multiple studies that address the distinctive features of exosome structure and biochemical composition in various pathological states imply the possibility of development of novel diagnostic techniques based on the detection of changes in the pool of proteins and nucleic acids transported by exosomes. However, methods for isolation and investigation of exosomes are rather difficult to develop because of a small size of these vesicles. A novel approach for preparative-scale isolation of exosomes based on the phenomenon of binding and aggregation of these particles in the presence of lectins has been put forward in the present study. The method developed is relatively cost-effective, allows for the isolation of exosomes from various biological fluids, and has been validated for the subsequent analysis of the protein composition of the exosomes in view of the possible clinical applications. The validation showed that the sedimentation of lectin-aggregated exosomes is a suitable approach for the isolation of these microvesicles from the complete conditioned culture medium in a research-laboratory setup.