Том 10, № 3 (2016)

Stem cell sphere formation is applied as a preconditioning treatment prior to transplantation. Here, stem cells (SC) isolated from human subepicardial adipose tissue were analyzed at different stages of the monolayer–spheres–monolayer cycle by transmission electron microscopy. Spheres obtained on a non-adherent surface or through hanging drops gave similar results. At the first two or three passages (stage 1), isolated SC displayed an embryonal cell-like ultrastructure. With increased passage number (stage 2), SC became larger and more electron-dense with a multilobed nucleus, well-developed rough endoplasmic reticulum (RER), well-developed Golgi apparatus, and numerous vacuoles. At 2 h after sphere induction (stage 3), SC gathered into clusters and formed desmosome-like intercellular contacts. Their nuclei possessed a large loose fibrillo-granular nucleolus, the cytoplasm was tightly packed with disintegrated cisternae of RER, and Golgi apparatus was not identified. Twenty-four hours afterward (stage 4), SC in well-formed spheres exhibited a large dense nucleolus and poorly developed Golgi apparatus and RER. One day after sphere dissociation (stage 5), SC looked like embryonal cells and were morphologically similar to the cells of the first stage except for the presence of a large nucleolus and several Golgi complexes. At 48 h after sphere dissociation (stage 6), SC became electron-dense and resembled SC of the second stage, bearing irregular nuclei and cytoplasm that was rich in RER. We interpret these results as SC senescence with increasing passage number after isolation from the tissue or 1 day after sphere dissociation and rejuvenation of the SC just after sphere dissociation. Further research is needed to determine the genetic, biochemical, and physiological parameters of the SC corresponding to morphologically defined distinct stages in order to provide high quality cellular material for cell therapy.

In development of methods of stimulation of regeneration of nerve tissues and creation of new-generation bioelectronic devices, studying the interaction of nerve cells with specially developed scaffolds with different characteristics of the surface within a nanometer range is a necessary stage. Carbon nanotubes (CNTs), flexible graphene films rolled up into nanosized cylindrical tubes, may represent a promising material for making these scaffolds. CNTs were obtained by chemical vapor deposition. Analysis of PC12 cells cultivated on quartz glasses covered with CNT films using electron and optical microscopy have been performed. It has been demonstrated that CNTs stimulate proliferation and do not inhibit neuronal differentiation of the PC12 cells. The possibility of obtaining neurons differentiated from mouse neural stem cells on quartz glasses covered with the CNT films has been shown. The data obtained indicate the possibility of using CNT films produced by chemical vapor deposition on quartz glasses as an electroconductive substrate for obtaining cells of neural origin and, possibly, mature neurons and studying their functions.

Peripheral blood neutrophils of 123 patients in stage Ia of the endometrial cancer have been assayed. The receptor system and ability of neutrophils to form extracellular traps (NET activity) were assessed by fluorescence microscopy, and spontaneous production of IL-2, IFN-γ, matrix metalloproteinase (MMP), G-CSF was found to exist determined with enzyme-linked immunosorbent assay. Phagocytic and myeloperoxidase activity, the level of cationic proteins, and neutrophil activity were evaluated in an NBT test determining the oxygen-dependent bactericidal action of neutrophils. Topology and rigidity of neutrophil membranes were assessed using scanning probe microscopy. It was established that an increased number of neutrophils was accompanied by changes of their receptor system, aerobic and anaerobic cytotoxicity, enhanced phagocytic activity, and reduced NET activity. The secretory activity of neutrophils changed. An increased level of MMP-1, possibly by enhanced production of reactive oxygen species, reduced IL-2 level, and a drastic increase in G-CSF were observed. The architectonics of neutrophils at stage Ia of endometrial cancer was characterized by altered cell shape and granularity loss. The rigidity of the cell membrane decreased. The changes in neutrophil morphology and persistent hyperactivity suggest the existence of the equilibrium between the immune system and the tumor in stage Ia of endometrial cancer.

Platelets are blood cells without nuclei, which, in conjunction with fibrin, cause bleeding to stop (hemostasis). Cellular microvesicles are microscopic particles released into extracellular space under activation and/or apoptosis of cells of different types. Platelet microvesicles form the main population of blood circulating through microvesicles and play an important role in the reactions of hemostasis, thrombosis, and many other (patho)physiological processes. Despite the large number of studies that have been devoted to the function of platelet microvesicles, the mechanisms of their formation and structural details remain poorly understood. The ultrastructure of the initial platelets and microvesicles formed in vitro from resting cells and platelets activated by arachidonic acid, ADP, thrombin, and calcium ionophore A23187 is investigated in this study. The intracellular origin, stages of formation, structural diversity, and size of microvesicles were analyzed according to the results of transmission electron microscopy of human platelets and isolated microvesicles. It was shown that thrombin, unlike other activators, not only stimulates microvesiculation of the plasma membrane, but also causes decomposition of cells with the formation of subcellular particles that have sizes comparable with the size of the microvesicles from the outer membrane of the cells. Some of these microparticles are cellular organelles surrounded by a thin membrane. The size of isolated microvesicles ranges from 30 to 500 nm, but their size distribution depends on the nature of the activating stimulus. The obtained results contain new data on the formation of platelet microvesicles and their structural diversity, which are important for understanding of their multiple functions in health and disease.

Planar rafts and caveolae are specific membrane clusters that contain high concentrations of cholesterol and lipids consisting of saturated fatty acids. These clusters are resistant to detergents and are known as “detergent-resistant membrane domains” (DRMs). Their morphology and size were studied by atomic force microscopy (AFM). Planar rafts extracted by Lubrol WX from monocytes of healthy donors are 150.6 ± 68.6 nm in diameter and 5.7 ± 2.9 nm in height, while caveolae are 87.3 ± 46.1 nm in diameter and 9.4 ± 5.4 nm in height. Significant differences in size and morphology were found between DRMs isolated from monocytes of healthy donors and patients with myocardial infarction, as well as between DRMs of monocytes and endothelial cells. The morphology dynamics of the isolated planar rafts and caveolae indicates that they quickly aggregate during storage; therefore, in order to assess the actual DRM size and morphology it is necessary to investigate them immediately after isolation.

The ultrastructure and functional parameters of the photosynthetic apparatus in leaves of 14-day-old pea seedlings were studied in conditions of laboratory simulated acid rain (SAR). Pea seedlings were sprayed with an aqueous solution containing NaNO₃ (0.2 mM) and Na₂SO₄ (0.2 mM) (pH 5.6, a control variant), or with the same solution, which was acidified to pH 2.5 (acid variant). Functional characteristics were determined by chlorophyll fluorescence analysis. There was reduction in the efficiency of the photosynthetic electron transport by 25% accompanied by an increase in the quantum yield of thermal dissipation of excess light quanta by 85% without significant change in maximum quantum yield of PSII photochemistry (F_v/F_m). Ultrastructural changes in chloroplasts were revealed by transmission electron microscopy (TEM) 2 days after the SAR treatment of pea leaves. In this case, changes in the structure of the grana and heterogeneity of the thylakoids packing in the granum, namely, an increase in thylakoid intraspace widths and thickness of granal thylakoids compared to the control, were found. It was shown also that carbonic anhydrase activity was significantly inhibited in chloroplast preparations isolated from SAR-treated pea leaves. We hypothesize possible involvement of chloroplast carbonic anhydrase in thylakoid granal structure maintenance. The structural disturbances and the inhibition of photochemical activity of chloroplasts are possible consequences of the carbonic anhydrase inactivation by SAR treatment leading to violation of HCO₃⁻–CO₂ equilibrium. The data obtained suggest that acid rains negatively affect the photosynthetic apparatus by disrupting the membrane system of the chloroplast.