Vol 166, No 2 (2018)

The molecular mechanism of violation of the contractile function of skeletal muscles caused by oxidative damage to myosin is not fully understood. Using permeabilized fibres from fast (m. psoas) and slow (m. soleus) rabbit muscles, we studied the effect of myosin oxidation on the mechanism of force generation and its calcium regulation. It was found that this treatment simultaneously reduces the maximum force and fibers stiffness without affecting their calcium sensitivity. This suggests that the mechanism of oxidation-related impairment the force-generating ability of fibers consists in suppression of myosin cross-bridges formation and does no affect the characteristics of actin—myosin interaction.

Experiments on rats showed that activation of the peripheral ion channel TRPM8 with menthol and rapid cooling (decrease in core temperature by 3°C) led to 1.5-fold activation of the expression of TRPV3 ion channel gene in the posterior hypothalamus, but had no effect on the expression of this gene in the anterior hypothalamus. Neither stimulation of peripheral TRPМ8, nor acute cooling affected the expression of genes for other thermosensitive ion channels (TRPV1, TRPV2, TRPV4, TRPA1, and TRPМ8) in the hypothalamus. Enhanced expression of Trpv3 gene can indicate increased sensitivity of hypothalamic neurons in the range of TRPV3 ion channel functioning (31-39oC). The relationship between the changes in Trpv3 gene expression and the shift of thermoregulatory reaction thresholds is discussed. Our findings attest to the presence of a functional relationship between TRP ion channels of the peripheral nervous system and TRP channels in the central structures of the brain.

We studied the effects of elastase, cigarette smoke extract, D-galactosamine hydrochloride, and tyrosine kinase inhibitor SU5416 on endothelial progenitor cells and angiogenesis precursors, as well as on Notch-1 expression by immature endothelial cells. Simultaneously with pulmonary emphysema, different damaging factors with diverse mechanisms of action caused pathological changes in the microvascular network of the lungs and destroyed the alveolar endothelium in female C57Bl/6 mice. D-galactosamine hydrochloride disturbed mobilization of endothelial progenitor cells expressing VEGFR (CD45—CD309⁺) and angiogenesis progenitors (CD45^—CD309⁺CD117⁺) and their migration into emphysema expanded lungs. Elastase inhibited VEGFR-expressing endothelial progenitor cells, while cigarette smoke extract inhibited cells with CD45^—CD31⁺CD34⁺ phenotype. In pulmonary emphysema provoked by elastase or D-galactosamine hydrochloride, angiogenesis was provided by endothelial cells with CD45^—CD31⁺CD34⁺ phenotype, whereas in emphysema modeled with SU5416 or cigarette smoke extract, it was provided by the endothelial VEGFR-expressing cells and mature CD31⁺ endothelial cells, respectively. Replenishment of immature endothelial cells damaged by elastase and SU5416 involved Notch-1⁺ angiogenesis precursors and Notch-1⁺ endothelial progenitor cells with VEGFR.

Using DNA comet assay we found that polysaccharides from Tussilago farfara L. reduced the intensity of polychemotherapy-induced apoptosis and DNA damage in bone marrow cells and small intestinal epithelium of C57Bl/6 mice, which attested to genoprotective properties of these polysaccharides.

The genotoxic effects of heterocyclic compounds were evaluated on the basis of genetic and toxicological characteristics of a biological model of Drosophila melanogaster. Analysis of the viability parameters (fertility, progeny mortality) showed that of 6 tested substance, substance No. 3 exhibited minimum toxicity. After application of substances No. 1 and No. 5 in the studied concentrations, the number of survived flies was insufficient for further analysis, which attested to high toxicity of these substances. The intensity of apoptosis was studied in response to substances Nos. 2, 4, and 6. Substance No. 4 proved to be optimal by the parameter toxicity/apoptosis (low toxicity/high apoptosis), while substance No. 3 exhibited low toxicity, which manifested in low apoptosis intensity.

The morphometric analysis of mesenteric lymph nodes was carried out in female Wistar rats with chemically induced breast cancer. In control rats with untreated breast cancer, the volume of the system of sinuses increased in parallel with the appearance of morphological signs of suppression of cell-mediated immunity, inhibition of humoral immunity, and macrophage reaction. Against the background of chemotherapy, we observed a decrease in the volume of paracortex and lymphoid nodules, suppression of proliferative activity of lymphoid cells in paracortical and B-cell zone, and a decrease in macrophage content. After resection of breast cancer followed by chemotherapy course, lymph transport activation, widening of the paracortex, enhanced proliferative activity of cells in the paracortex and B-cell zone, and reduced volumes of lymphoid nodules with and without germinal centers and medullary substance were revealed in comparison with rats subjected neoadjuvant chemotherapy.

The study was designed to determine genes of microRNAs hypermethylated in malignant ovarian tumors and to select new diagnostic and prognostic markers of the disease and effective system of markers. Using methyl-specific PCR and a representative sample of 54 ovarian cancer specimens, we determined 5 microRNA genes (MIR-34b/c, MIR-9-1, MIR-124-3, MIR-129-2, and MIR-107) hypermethylated in the majority of tumor samples in comparison with paired samples of histologically unchanged tissue (48-57% vs. 4-19%, p<0.001). Using ROC-analysis, we selected an effective system of 4 markers for diagnosis of ovarian cancer (MIR-9-1, MIR-124-3, MIR-129-2, and MIR-107) characterized by high sensitivity and specificity (up to 87-94% at AUC=0.92) relative to the conventional norm (54 paired samples of histologically unchanged tissue) and absolute norm (18 ovarian tissue samples from subjects who died from non-tumor diseases). It was also shown that methylation of MIR-129-2, MIR-9-1, and MIR-34b/c genes is significantly (p<0.01) correlated with the clinical stage or the presence of metastases. The results indicate that epigenetic modifications of the studied microRNA genes are involved in the pathogenesis and progression of ovarian cancer and attest to their diagnostic and prognostic potential.

We studied the influence of magnetite nanoparticles (FeO•Fe₂O₃) and quantum dots (CdSe/ZnS coated with mercaptopropionic acid) on the expression of 5 common reference genes (BA, B2M, PPIA, UBC, and YWHAZ) in peripheral blood cells from 20 volunteers by reverse transcription PCR method. The stability of the expression of reference genes varied depending of the cells type and chemical structure of nanoparticles. The level of YWHAZ mRNA after exposure by nanoparticles demonstrated highest stability in lymphocytes, neutrophils, and monocytes. Stability of YWHAZ expression was confirmed by Western blotting. Our findings suggest that YWHAZ is the most suitable as the reference gene.

We studied immunohistochemical and morphometric characteristics of the spermatogenic epithelium in rats against the background of peroral administration of nanoparticles containing titanium dioxide. Substantial degenerative changes in the spermatogenic epithelium were revealed: thinning, disorganization of layers, and detachment of sperm cells from the basement membrane. Immunohistochemical analysis revealed reduced proliferative activity and differentiation potential of epithelial cells, which was confirmed by changes in the expression of Ki-67 and c-kit markers. Our data attest to unfavorable effects of titanium dioxide nanoparticles on the structural and functional characteristics of the reproductive system in male rats leading to spermatogenesis disturbances.

Based on the data of morphological analysis, we performed histological evaluation of rat tissue reaction to subcutaneous implantation of decellularized matrices of intrathoracic organs and tissues. Cell composition of the inflammatory infiltrate was analyzed, and the dynamics of macrophage and T and B lymphocyte content was assessed on days 7 and 14 of the experiment. It was found that the reaction to implantation depended not only on the quality of decellularization and efficiency of removal of antigen molecules, but also on the original histological structure and quality of preimplantation processing of the transplant.

We presented the results of our study of chronostructure of BP, HR, electrolyte excretion, and locomotor activity under conditions of “free-run rhythm” (light deprivation). In adult male Wistar-Kyoto (normotensive) and SHR (spontaneously hypertensive) rats, BP, biopotentials of the heart (ECG), and locomotor activity were recorded over 24 h by telemetric monitoring and the rate of excretion of electrolytes (Na⁺, K⁺, Ca²⁺, and Mg²⁺) during the nighttime and daytime hours was measured. It was found that under free-run rhythm, 24-h profiles of BP, HR, excretory function of the kidneys, and locomotor activity underwent more considerable changes in normotensive Wistar-Kyoto rats in comparison with hypertensive SHR rats. However, hypertensive rats demonstrated pronounced changes in rhythmic characteristics of HR, which can restrict adaptation reserves of the cardiovascular system.

We studied the effect of Fe²⁺ ions in polymerized hemoglobin (Krunidon blood substitute) and in molecular hemoglobin (Sigma) on OH^• radical initiation in the Fenton system. It was found that polymerized hemoglobin, as a component of Krunidon preparation, in contrast to hemoglobin tetramer, did not intensify OH^• radical generation. The oxidant potential of Krunidon was evaluated in vivo by measuring malondialdehyde level in dog blood plasma after repeated intravenous administration (5 days in a dose of 114 mg/kg) as a biomarker. Administration of the preparation did not significantly increased malondialdehyde content on days 1 and 4 after exposure and did not affect total protein content in blood plasma. Our findings suggest that polymerized hemoglobin in the Krunidon preparation exhibits no pro-oxidant activity and can be used as the basis for the development of non-oxygenic forms of blood substitutes.

We studied functional activity of neutrophilic granulocytes and monocytes isolated from the peripheral blood of women during the follicular and luteal phases of the menstrual cycle. It was shown that phagocytic activity of neutrophilic granulocytes increases, their intracellular oxygen-dependent bactericidal activity decreases, and the number of monocyte extracellular traps increases in women in the luteal phase of the menstrual cycle in comparison with the follicular phase.

We studied the effects of light and non-specific sound stimulation of domestic chick embryos on their filial preference as well as on the expression of two transcriptional factors c-Fos and Egr-1 and neurotrophin BDNF in the embryo brain. Prenatal light stimulation increased preference of the “natural” object, thus producing a priming effect. In the brain of E19 embryos, c-Fos and Egr-1 were expressed at a high basal level and neither light nor sound stimulation affected the number of cells expressing these factors. BDNF mRNA was also present in a number of brain areas of non-stimulated embryos, but light and sound stimulation enhanced the expression of BDNF mRNA in brain structures associated with filial imprinting. These findings suggest that BDNF is probably involved in the effects of prenatal priming on the development of species-specific behavior.

We studied reception of sex steroid hormones in the tissues of thyroid papillary cancer and benign tumor. Enhanced expression of AR and ERβ mRNA reflected malignant tumor growth. Nuclear factors Brn-3α and TRIM16 modulating expression of steroid hormones play an important role in the development of thyroid tumors. It was found that the level of TRIM16 mRNA is associated with the expression of ERβ, which seems to be mediated by its antiestrogen effect.

We studied proliferative activity of multipotent mesenchymal stromal cells on collagen matrices containing platelets stabilized with silver nanoparticles. Dose-dependent stimulation of the growth of stromal cells without their structural damage was observed in the presence of stabilized platelets in proportion of 20-120 mln per 10⁵ multipotent mesenchymal stromal cells. The same doses of non-stabilized platelets produced less pronounced stimulation effect. In higher doses (≥180 mln) stabilized platelets inhibited cell proliferation and induced their damage. Stabilized platelets enhanced migration of multipotent mesenchymal stromal cells; after formation of the monolayer, they actively colonized deep layer of the collagen matrix. The formed monolayer of multipotent mesenchymal stromal cells survived over 14 days without appreciable cell damage.

In experiments on white outbred male rats, a freshly removed (20 experiments) or cryopreserved (10 experiments) testicle from newborn rats (1-2 days after birth) was transplanted under the renal capsule after bilateral orchiectomy. In all experiments with transplantation of freshly removed testicle, it was engrafted. In 3 months, histological examination revealed the formation of mature seminiferous tubules, but spermatogenesis was blocked at the stage of spermatogonia; groups of proliferating Leydig cells in the loose connective tissue between the tubules were also seen. In 6 and 12 months, the status of the seminiferous tubules remained unchanged, but structures typical of the epididymis and developing vas deferens were revealed. The number of proliferating Leydig cells increased. The initially low testosterone concentration in the blood of castrated males increased significantly as soon as in 1 month after transplantation and grew up to 3 months, remaining at a level ~50% of normal. Engraftment of cryopreserved neonatal testicular tissue was observed in 60% cases, however, engrafted tissue, similar to the fresh one, retained the ability for organogenesis with the formation of mature seminiferous tubules, epididymis, and groups of proliferating Leydig cells. The dynamics of blood testosterone concentration in rats with cryopreserved and fresh transplantation was similar. Subcapsular transplantation did not adversely affect the kidneys, which was seen from normal histological structure of the kidneys and creatinine and urea concentrations in the blood.

We propose an in vitro model of chronic oxidative stress based on the use of glucose oxidase. Oxidative stress modeling leads to a significant increase in the number of dead cells in culture. It was shown that the glial cell-derived neurotrophic factor exhibits a pronounced anti-oxidant effect. Preventive application of 1 ng/ml glial cell-derived neurotrophic factor significantly reduced the percentage of dead cells in culture.