Vol 167, No 1 (2019)

Administration of 5-HT receptor antagonist to snails trained in conditioned food aversion prior to reminding of the conditioning stimulus caused amnesia. At the early period of amnesia (day 3), injections of protein synthesis inhibitor cycloheximide without reminder or reminder alone were ineffective. At the same time, injections of the inhibitor combined with reminder led to memory recovery; this effect in most animals persisted for at least 10 days. In the rest snails, aversive responses to presentations of the conditioning stimulus persisted for 2 days. Cycloheximide injection and reminder in 10 days after induction of amnesia did not affect its development or caused a transient memory recovery (2 days). We hypothesized that amnesia is an active process unfolding in time. One of mechanism of this process is reminder-induced and protein synthesis-depended reactivation of amnesia. Inhibitor of protein synthesis disturbed this reactivation and led to recovery of the initial memory of conditioned food aversion.

The peculiarities of the histological structure and morphometrical parameters of the dentition in ISIAH rats (inherited stress-induced arterial hypertension) before stress and after modeling of everyday life stress regimen (chronic stress) were compared with those in WAG rats. The area of blood vessels of the periodontium, the thickness of the endothelium in the periodontal and pulp vessels, and the number of fibroblasts were increased, while the level of odontoblasts was reduced in ISIAH rats both before and after exposure to chronic stress, which can reflect more pronounced reaction to stress in hypertensive animals.

The content of pro- and anti-inflammatory cytokines in the hypothalamus, hippocampus, and blood serum of C57Bl/6J mice with depressive-like behavior induced by 20-day social stress was analyzed in 4 h after immune stimulation with LPS (250 μg/kg). These animals are characterized by a tendency to an increase in the blood content of IL-6 and a decrease in the level of IL-10. Changes in cytokine content in the brain of mice with depressive-like state developed under these conditions were observed only in the hippocampus: the levels of IL-1β, IL-6, TNFα, and IL-10 increased and the content of IFNγ decreased in comparison the corresponding parameters in the controls (not exposed to social stress) and aggressive animals. No changes in the levels of IL-2, IL-4, and IL-17 were revealed in the hypothalamus and hippocampus.

Increased concentration of lipoprotein(a) is a risk factor of coronary heart disease. lipoprotein(a) consists of LDL-like and highly polymorphic apolipoprotein(a). Here we studied the effect of lipoprotein(a)-containing sera with different apolipoprotein(a) phenotypes on lipid accumulation by THP-1 monocyte-like cells. Cholesterol concentration in lysates of THP-1 cells was significantly higher after their incubation with lipoprotein(a)-containing serum samples with low-molecular-weight phenotype of apolipoprotein(a) in comparison with samples with a high-molecular-weight apolipoprotein(a) phenotype irrespective of initial cholesterol level as well as serum concentrations of apoB-100, oxidized LDL, and circulating immune complexes. The presence of the most atherogenic small dense LDL subfractions in examined sera in addition to a low-molecular-weight apolipoprotein(a) phenotype resulted in significant elevation of cholesterol accumulation by THP-1 cells. The data obtained explain greater atherogenicity of lipoprotein(a) with low-molecular-weight apolipoprotein(a) phenotype.

We performed a comparative study of the cytotoxic effect of endocannabinoid N-arachidonoyl dopamine (AA-DA) on cultured stromal cells of ectopic and eutopic endometrium. It was found that AA-DA in the concentration range of 1-20 μM produces more selective cytotoxic effect on the stromal cells of the ectopic endometrium due to interaction with cannabinoid type 1 receptor. In concentrations below 1 μM, AA-DA stimulated the proliferation of stromal cells of the eutopic endometrium and did not affect the division of ectopic endometrium cells. This effect was realized due to its interaction with cannabinoid type 2 receptor.

The effect of the anti-inflammatory cytokine IL-10 on the ultrastructural distribution of AMPA receptor GluR1 subunit in CA1 field of cultured hippocampal slices was studied by using immunohistochemical technique. It was found that long-term posttetanic potentiation increased the content of GluR1 in the postsynaptic density of the axo-spinous synapse. Addition of IL-10 in concentrations of 1 and 10 ng/ml to the medium facilitated long-term posttetanic potentiation thereby changing the distribution of GluR1 in the spine: the number of receptors increased in the cytoplasm and decreased in the postsynaptic density. It is assumed that activation of neuronal IL-10 receptors affects the distribution of AMPA receptors in axo-spinous synapses of hippocampal field CA1 through interplay of intracellular signaling pathways, thereby participating in the mechanisms of synaptic plasticity under normal conditions.

We propose a modification of Francis agar used for identification of the causative agent of histoplasmosis by in vitro conversion of the mycelial culture to the yeast-like growth phase. For improving of the growth characteristics of the medium, we used FT-agar with glucose-vitamin additives developed for culturing of the tularemia causative agent. The modified Francis medium is characterized by significantly higher growth properties and allowed 10-fold increasing the number of CFU of yeast-like cells of both American and African histoplasmosis causative agent.

Biomarkers of activity of rheumatoid arthritis were analyzed in the blood serum and synovial fluid of affected joints in 84 patients. Significant differences between the serum and synovial fluid levels were revealed for 10 of 17 analyzed biomarkers; the levels of IgM and proinflammatory cytokines TNFα, IL-1β, IFNγ, and IL-6 were higher in the synovial fluid. The concentration of IL-10 in the synovial fluid was also elevated. In the peripheral blood, the content of antinuclear antibodies, circulating immune complexes, and cytokines IL-4 and TGF-β was elevated. These findings attest to the development of local Th1 type immune response in affected joints paralleled by compensatory elevation of immunosuppressive cytokine IL-10 and systemic Th2/Th3 type immune response (judging from peripheral blood parameters) in patients with rheumatoid arthritis.

We studied the effect of lithium carbonate on hepatocellular carcinoma-29 cells in CBA male mice after injection in a dose of 20 mM along the tumor periphery. Transmission electron microscopy revealed a decrease in the volume density of the granular endoplasmic reticulum in the cell cytoplasm and an increase in the total numerical and volume density of autophagosomes and autolysosomes and zones of destruction of intracellular organelles. The ability of lithium carbonate to activate intracellular degradation processes in tumor cells and to stimulate cell death can be used to develop new combined strategies in the chemotherapy for hepatocellular carcinoma.

We propose an original method for controlling BP by administration of Si~ODN nanocomposites containing antisense oligonucleotides fixed on silicon-organic nanoparticles. ODN in nanocomposites are targeted to mRNA of the genes encoding angiotensin-converting enzyme (ACE1) and type 1 angiotensin-II receptor (AT1A). The experiments were performed on hypertensive ISIAH rats, a genetic model of hypertension. Single inhalation or intraperitoneal administration of the nanocomposites targeted to ACE1 mRNA or ATA1 mRNA, respectively, led to a pronounced decrease (by ~30 mm Hg) in systolic BP in ISIAH rats over a week. The use of scrambled ODN in the nanocomposites had no effect. A decrease in the expression of ACE1 and AT1A genes under the effect of the corresponding antisense ODN was demonstrated, which attested to directed effect of the test preparations.

HBL-100 breast epithelial cells were cultured with a blocker (N-ethylmaleimide) and protector (1,4-dithioerythritol) of SH groups. The study assessed changes in redox potential of glutathione and thioredoxin systems, intensity of oxidative modification of proteins, ROS production, and cell proliferation. The roles of thioredoxin system and protein oxidative modification in HBL-100 cell proliferation under redox status modulation were established. The role of carbonylated thioredoxin in arrest of the cell cycle in S-phase was demonstrated, which could be used for targeted therapy of the diseases accompanied by oxidative stress and disturbed redox status.

Voltage clamp and concentration-jump methods were employed to examine the effects of endogenous neuropeptide cycloprolylglycine on GABA-activated ionic currents in isolated cerebellar Purkinje cells. In the concentration range of 0.1-10.0 μM, short-term (600 msec) external application of cycloprolylglycine against the background of GABA-evoked current produced no effect on its amplitude. In contrast, application of 1 μM cycloprolylglycine increased current up to 177±15% control level. The development of potentiating effect and return to the control level of ionic current were slow, which was indicative of possible implication of second messenger systems in these processes. Functional augmentation of GABA_A receptors under the action of cycloprolylglycine can underlie the established neuroprotective and anxiolytic effects of this endogenous dipeptide.

Dose-dependent protective effects of lanthanum nitrate solution and gel were shown on the model of experimental infection caused by a virulent strain of Shigella flexneri 2a or opportunistic bacteria Klebsiella pneumoniae in outbred and DBA mice.

The study included pregnant women aged 23-41 years with preeclampsia and gestation-associated arterial hypertension at weeks 27-40 and patients with essential arterial hypertension developing under conditions of the metabolic syndrome and without it. Frequency analysis of polymorphisms of the renin—angiotensin system genes (ACE, AGT, and AGTR1), ITGB3, FTO and their associations confirmed the syndrome nature of hypertensive disorders in pregnancy. The presence allele T of AGT gene and/or allele C of AGTR1 gene in the genotype of patients with preeclampsia was associated with higher BP and pressure load over 24 h. Allele D of ACE gene was also essential for BP parameters (pressure load) in patients with preeclampsia and gestation-associated arterial hypertension. Due to high genetic heterogeneity of the preeclampsia syndrome and genetic differences in the incidence of the studied gene polymorphisms in preeclampsia and gestation-associated arterial hypertension, no direct associations between these gestation disorders and polymorphic markers of the renin—angiotensin system genes can be established. However, polymorphisms of the renin—angiotensin system genes are essential for the 24-h dynamics of BP and pressure load under conditions of hypertensive disorders in pregnancy.

We identified a group of miRNA genes whose methylation is associated with ovarian cancer metastasis. Based on these data, new markers and the systems of markers predicting tumor dissemination were selected. Using methylation-specific PCR and a representative set of 54 ovarian cancer samples, we identified 10 microRNA genes (MIR-124a-2, MIR-127, MIR-125b-1, MIR-129-2, MIR-137, MIR-193a, MIR-203a, MIR-34b/c, MIR-130b, and MIR-1258) whose methylation is associated with tumor metastasis. The greatest association was established for 4 genes: MIR-137, MIR-193a, MIR-34b/c, and MIR-130b (p<0.01). ROC analysis revealed 3 most optimal marker systems including 4-5 miRNA genes and characterized by high sensitivity (82-94%) and specificity (76-86%) at AUC=0.89-0.92. Methylation of any three genes from these systems is sufficient to predict metastasis with the specified accuracy. Detection of the group of hypermethylated miRNA genes with predictive value for ovarian cancer metastasis is of great importance for personalized treatment of the patients.

We studied dependences of T2 relaxation time on magnetic field and concentration of nanoparticles. It was found that nanocontrast media are effective under the influence of the magnetic fields in the range 0.3-7 T. Data of electron paramagnetic resonance confirm the assumption on aggregation of nanoparticles not coated with proteins in high magnetic fields.

We studied the influence of obesity caused by lethal yellow (A^Y) mutation in the agouti gene, short photoperiod (4/20 h light/darkness), and combination of these factors on depressive-like behavior in the forced swimming test and expression of genes encoding proinflammatory cytokines in the hypothalamus of heterozygous male C57Bl/6-A^Y/a mice and their wild-type littermate controls (C57Bl/6-a/a). It was shown that A^Y mutation as well as short photoperiod increased depressive-like behavior in mice. No effect of the interaction of A^Y mutation and photoperiod on immobility in the forced swimming test was revealed. In wild-type mice, increased depressive-like behavior caused by short photoperiod was accompanied by enhanced expression of Tnf gene. Exposure to short daylight increased the expression of Nos2.

The effects of 15-day restriction diet (from 1 to 1.5 months of age) on some parameters of brain development were studied in rats. The immediate and delayed (15 days after transfer to normal ration) effects were evaluated. The immediate effects included a significant decrease of the absolute weights of the brain and hemispheres. The relative weight of the brain was significantly higher. The thickness of the cortex of the parietal lobe proper and its layer I decreased. The absolute weights of the brain and hemispheres were less than in the control 15 days after the rats were transferred to ad libitum feeding, while the relative weight of the brain was higher than in controls. The thickness of the parietal and anterior parietal cortex and the numerical density of neurons in layers II and V did not differ from the control. In the neurons of layers II and V of the anterior parietal and parietal lobe proper the nuclei were larger, while the nucleoli were enlarged in the neurons of these locations and the hippocampus, the shifts being significant in the anterior parietal layer V. The concentrations of RNA in the parietal, anterior parietal, and hippocampal lobe neurons in different groups were similar directly and 15 days after the diet. Changes in the gravimetric and morphometric parameters of the brain were paralleled by the development of oxidative stress.

Rat glioma cell line C6 expressing human poliovirus receptor (PVR) and susceptible to polioviruses (C6-PVR-BFP) was used to produce a clone with knockout of IFNα/β (Ifnar1) receptor subunit 1 gene (Ifnar1). The sensitivity of C6-PVR-BFP cells to the vaccine strain of poliovirus type 3 (PV3) depended on the signaling pathways of the cell response to type 1 IFN. Using the model of subcutaneous tumor xenografts, we demonstrated oncolytic activity of PV3 against C6-PVR-BFP cells that depended on the expression of PVR and increased considerably upon disturbances in IFN response pathways.

Intravital microscopy is widely used for in vivo studies of the mechanisms of carcinogenesis and response to antitumor therapy. For visualization of tumor cells in vivo, cell lines expressing fluorescent proteins are needed. Expression of exogenous proteins can affect cell growth rate and their tumorigenic potential. Therefore, comprehensive analysis of the morphofunctional properties of transduced cells is required for creating appropriate models of tumor microenvironment. In the present study, six lines of mouse tumor cells expressing green and red fluorescent proteins were derived. Analysis of cells morphology, growth kinetics, and response to chemotherapy in vitro revealed no significant differences between wild-type and transduced cell lines. Introduction of fluorescent proteins into the genome of 4T1 (murine breast cancer) and B16-F10 (murine melanoma) cells did not affect tumor growth rate after subcutaneous implantation to mice, while both CT26-GFP and CT26-RFP cells (murine colon cancer) were rejected starting from day 8 after implantation. Elucidation of the mechanisms underlying CT26-GFP/RFP rejection is required to modify transduction technique for creating the models of tumor microenvironment accessible for in vivo visualization. Transduced 4T1 and B16-F10 cell lines can be used for intravital microscopic imaging of tumor cells, neoplastic vasculature, and leukocyte subpopulations.

We studied the effect of low-level laser irradiation on proliferative activity of cultured human Wharton’s jelly mesenchymal stromal sells. Cells were irradiated with a solid-state laser emitting at 650 nm; irradiation doses were 0.04, 0.4, or 4 J/cm2. Laser irradiation was performed once at the start of the cell proliferation experiment or daily throughout the experiment. Cells were cultured for 7 days. The number of viable cells was assessed using the MTT test. An increase in cell proliferative activity was detected after daily laser irradiations; the maximum stimulating effect was achieved at a dose of 0.04 J/cm². These results substantiate medical use of lasers for expansion of cells intended for transplantation.

We studied the effect of mesenchymal stem cells from the bone marrow and adipose tissue on the growth rate of melanoma B16 and mammary adenocarcinoma Ca755 tumors after their co-administration with tumor cells to syngeneic mice. Stimulation of tumor growth and formation of melanoma metastases in the lungs was found under the influence of adipose tissue-derived, but not bone marrow-derived stem cells. At delayed terms after irradiation in sublethal doses, the adipose tissue-derived mesenchymal stem cells also stimulated the tumor growth. Stimulation of the tumor growth by adipose tissue-derived mesenchymal stem cells was caused by factors secreted by these cells. Transplantation of mesenchymal stem cells to humans is possible only after accurate exclusion of malignant tumors.

We compared phagocytic activity of macrophages of monocyte origin and Kupffer cells under the influence of M1 and M2 inducers and without activation. Cultures of monocyte-derived macrophages and Kupffer cells were characterized by intensive expression of CD68 that was not affected by activation factors. At the same time, these cultures demonstrated different dynamics of phagocytic activity. Monocyte-derived macrophages initially had more pronounced absorption capacity that gradually increased during the experiment. Kupffer cells were characterized by abrupt fluctuations of phagocytic activity: sharp growth and rapid saturation. Despite these differences, the endosomes produced by monocyte-derived macrophages and Kupffer cells had similar degrees of maturity.

Bone marrow mesenchymal stromal cells are multipotent and can differentiate into cells of various tissues, which determines their high importance for clinical application. We performed an in vitro study of the osteogenic potential of mesenchymal stromal cells cultured on intact polylactide scaffolds or scaffolds modified with collagen I or fibrin. Scanning electron microscopy showed that the cells formed osteogenic nodules or osteogenic nodules on both intact and fibrin-modified polylactide scaffolds. Spectrophotometric detection of alkaline phosphatase activity on days 7 and 11 showed that mesenchymal stromal cell grown on intact polylactide scaffolds and on scaffolds modified with collagen type I or fibrin more intensively synthesized alkaline phosphatase than in the control (culture plastic). This dependence increases in the presence of osteogenic differentiation factors in the medium. After long-term culturing (4 weeks), the presence of calcium deposits detected by alizarin red staining confirmed the osteoinductive properties of intact and protein-modified polylactide scaffolds. These findings suggest that polylactide scaffolds and collagen I increase the osteogenic differentiation potential of mesenchymal stromal cells.

We compared the expression of Aβ42 peptide, τ-protein, and α-synuclein in the substantia nigra and skin fibroblasts of elderly and senile patients with Parkinson’s disease and subjects without neuropathology. Expression of markers in the studied tissues was assessed by immunohistochemical and immunocytochemical methods. The expression of Aβ42 peptide, τ-protein, and α-synuclein in the substantia nigra of elderly and senile patients with Parkinson’s disease was higher by 11-31 times than in subjects without neuropathology. In skin fibroblasts of patients with Parkinson’s disease, the expression of Aβ42 peptide and α-synuclein was 3-14 times higher than in subjects without neuropathology, and expression of τ-protein did not significantly differ in the studied groups. Thus, immunocytochemical analysis of the expression Aβ42 peptide and α-synuclein in skin fibroblasts can be a simple method of early diagnosis of Parkinson’s disease in elderly persons.

The third author’s name should read:

V. V. Matchkov.