Vol 165, No 1 (2018)

The effects of sympathetic cotransmitter NAD+ (10 μM) on bioelectric activity of the heart under conditions of adrenergic stimulation were studied on isolated spontaneously contracting preparations (without stimulation) of the right atrium from 2-7-day-old rats. Action potentials were recorded in the working myocardium using standard microelectrode technique. Perfusion of the right atrium with norepinephrine solution (1 μM) altered the configuration and significantly lengthened the action potentials. NAD⁺ against the background of norepinephrine stimulation significantly decreased the duration of action potentials, in particular, at 25% repolarization. The effect of purine compounds NAD⁺, ATP, and adenosine on bioelectrical activity of the heart of newborn rats was studied under basal conditions (without norepinephrine stimulation). The effect of NAD⁺ against the background of adrenergic stimulation was more pronounced than under basal conditions and was probably determined by suppression of I_CaL, which can be the main mechanism of NAD⁺ action on rat heart.

We assessed the effect of intake of easily digested carbohydrates for 133 days on quantitative parameters of neuromotorics and cognitive function in Wistar rats and C57Bl/6J mice. Neuromotorics (muscle tone) was assessed in rats and mice by the forelimb muscle force (grip strength) over 4 months. Anxiety was assessed in the elevated plus-maze test and cognitive function (short-term and long-term memory) was evaluated by conditioned passive avoidance response (CPAR) test over 3 months. The mice, in contrast to rats, receiving the diet with easily digested sugars demonstrated suppression of neuromotorics. Anxiety increased with age in female mice, but not in rats, irrespective of the diet. Cognitive function in rats receiving experimental rations did not change significantly in comparison with the control. In mice, consumption of equimolar mixture of fructose and glucose impared short-term, but not long-term memory, in comparison with the group receiving glucose alone. We revealed a small (by 14-17%), but statistically significant increase in the brain weight in mice receiving fructose and sucrose. The study demonstrates sufficient interspecies differences in the influence of carbohydrate rations on neuromotorics and behavioral responses in the in vivo metabolic syndrome model.

JAK/STAT signaling pathway was examined during functional stimulation of mesenchymal progenitor cells with fibroblast growth factor. The differences were observed in the realizations of the proliferation-differentiation potential of CFU-fibroblasts under blockade of JAKs or during selective inactivation of STAT3. The study revealed stimulating influences of JAKs and STAT3 on mitotic activity of progenitor cells and individual roles of these proteins in the control of their maturation. Blockade of JAKs diminished the level of fibroblast colony formation and the score of actively proliferating CFU-fibroblasts at the background increase of the differentiation rate of progenitor cells. In contrast, STAT3 inhibitor resulted in a coordinated decrease of all examined parameters.

The effects of a non-selective blocker of NO synthases LNNA in a dose of 25 mg/kg and nitrates KNO₃, NaNO₃, Mg(NO₃)₂, and Ca(NO₃)₂ in doses of 5 and 50 mg/kg were studied on the model of experimental ischemic stroke induced by bilateral occlusion of the common carotid arteries. Wistar rats were randomized into 40 treatment (n=960) and 8 control (n=192) groups. Treatment group rats received injection of either nitrate, or LNNA, or a combination LNNA+nitrate. All substances were administered intraperitoneally 1 h prior to brain ischemia or 5 sec after occlusion of the common carotid arteries. Control rats received the same volume of 0.9% NaCl at the same terms. The non-selective inhibitor of NO synthases LNNA administered against the background of brain ischemia increased neurological deficit and animal mortality. Nitrate/nitrite reductase system had protective properties depending on the cation of nitrate and concentration of a nitrate during inhibition of NO synthase system under conditions of brain ischemia and hypoxia.

Parameters of cardiac activity after administration of the cholinesterase inhibitor physostigmine were analyzed in newborn rats and on day 16 of postnatal development. The type of cardiovascular response to acetylcholine excess in newborns and 16-day-old rats were similar, but they significantly differed by the magnitude, which suggests that that maturation of cholinergic structures involved in the regulation of cardiac activity is completed during the early postnatal ontogeny.

Antiviral activity of the polyphenol complex from seagrass of the Zosteraceae family against highly pathogenic strain of the tick-borne encephalitis virus was studied on passaged culture of porcine embryo kidney cells. The antiviral effect of the test compound manifested in a decrease in the infectious titer of the virus and depended on the concentration and application schemes. Polyphenol complex in a concentration of 100 μg/ml suppressed accumulation of the pathogen in the cell culture: pretreatment of the virus reduced its titer by 4 log, pretreatment of cells by 1.4 log, and application of the compound 1 h after cell infection by 2.8 log. Antiviral action of the test compound is determined by direct inactivation of the virus and inhibition of virus replication at the early stage, which attests to potential of this compound in the treatment of tick-borne encephalitis.

We studied association of Oprm1 gene polymorphisms with signs of N-(1-phenethyl-4-piperidyl)propionanilide intoxication in rats. It was found that the rate of intoxication in laboratory animals depends on genetic features. A polymorphic variant rs105312806 of Oprm1 gene can be a possible marker of animal sensitivity to opioid receptor agonists. This hypothesis was supported by differences in the rats of intoxication signs such as time to lateral posture and sleep duration in homozygous rats carrying different alleles. In rats with AA genotype, the time to lateral posture was shorter by 1.3 times and sleep duration was longer by 3.5 times than in carriers of GG genotype.

The diagnostic potentialities of complex immunochemical analysis of the serum and daily urine were evaluated in 118 patients with multiple myeloma. In 95 patients, we observed secretion of monoclonal intact immunoglobulins with heavy chains G (N=69), A (N=19), and M (N=4) and biclonal secretion of paraproteins G and A (N=3). Bence-Jones protein was detected in the sera and daily urine of 16 patients and Bence-Jones proteinuria alone was detected in 3 patients. The diagnostic sensitivity of serum immunoelectrophoresis in multiple myeloma is 94.1%. Analysis of paraproteinuria is particularly important in Bence-Jones myeloma, when paraprotein excretion may be not associated with paraproteinemia. Complex study by immunoelectrophoretic and immunoturbidimetric methods in multiple myeloma increases the diagnostic sensitivity to 99.2%.

Changes (or variants) in BRCA1 and BRCA2 gene sequences can have different lengths and clinical significance: from single nucleotide variants (SNV) and short insertions/deletions (<50 bp) to extended deletions and duplications (so-called copy number variations, or CNV). According to their clinical significance, all variants can be divided into pathogenic, likely pathogenic, variants of uncertain significance, likely benign, and benign. Moreover, variants can be germinal (i.e. inherited from parents) and somatic (arising in the process of development of the organism). A specific somatic event is loss of heterozygosity (LOH), i.e. transition of one or many point and short variants from heterozygous to homozygous state. Such an event can be the key to the development of carcinogenesis for cells carrying a pathogenic variant, if we consider it within the framework of the Knudson’s two-hit carcinogenesis theory. We studied the prevalence and nature of LOH in of ovarian cancer samples carrying or not carrying a pathogenic variant. To this end, a full coding sequence of BRCA1/2 genes was determined in 30 pairs of DNA samples isolated from blood cells and paraffinized histological blocks of patients on a MiSeq Illumina instrument. Analyss of the obtained reads revealed 9 pathogenic point and short variants (30% patients): 6 germinal (20%) and 3 somatic (10%), and 8 somatic CNV (3 deletions and 5 duplications of several or all exons of the BRCA1 gene). LOH was detected in 70% patients; among the carriers of pathogenic variants - in 83%. For pathogenic variants, the percentage of reads with the alternative allele increased more often than for benign variants located in another gene, or detected in other patients (67% vs. 44%). However, the difference was statistically insignificant, which can be due to insufficient number of patients. Only in 3 of 21 cases of LOH (14%), it can be attributed to CNV. In other cases, LOH is most likely determined by gene conversion, but further research is needed.

We studied the effect of intramuscular injections of bone marrow mesenchymal stromal cells on microcirculation parameters in an ischemic limb of Wistar rats. It was shown that transplantation of mesenchymal stem cells with or without erythropoietin stimulates angiogenesis in the ischemic zone. An essential role in angiogenesis stimulation is played by cytokines and growth factors.

We evaluated the efficacy of rat olfactory ensheathing cells in the therapy of experimental cysts of the spinal cord. Improvement of the motor function of the hind limbs after transplantation of the olfactory ensheathing cells into the posttraumatic spinal cord cysts rats was found. We also determined the required number of cells for transplantation and demonstrated a neuroprotective effect of this dosage. For further clinical studies, autologous tissue-specific cell preparation of olfactory ensheathing cells has to be created. Cell therapy in combination surgical and pharmacological treatment will substantially improve the quality of life of patients with posttraumatic spinal cord cysts.

We created an anisotropic material based on collagen sponge and reactive polylactide structured by laser photopolymerization. The combination of collagen with reactive polylactide improves the resistance of the formed matrices to biodegradation in comparison with collagen sponge, while the existence of sites with different mechanical characteristics and cell affinity on the matrix provides directed cell growth during their culturing. It was shown that reinforcement of the collagen sponges 7-fold increased the mean Young’s modulus for the hybrid matrix without affecting its cytotoxicity. The developed matrix provides cell adhesion and proliferation along reinforcement lines and can be used for fabrication of tissue engineering constructs.

Within the framework of the previously proposed model of structural organization of DNA that supplements the Watson—Crick model and is based on a mathematical regulation — Fibonacci sequence, we suppose the existence of nucleotides without nitrogenous base and acting as linkers connecting DNA subunits.

Quantitative analysis of blood vessels in the distal segment of rat sciatic nerve after its ligation for 40 sec and subperineurial administration of mesenchymal stem cells or dissociated cells of rat embryonic spinal cord was carried our by immunohistochemical tracing of von Willebrand factor, a marker of endothelial cells of blood vessels. It was found that the number of blood vessels per unit area of the nerve trunk in 21 days after injury and administration of mesenchymal stem cells increased by more than 1.5 times in comparison with the control (damaged nerve). After administration of dissociated cells of the embryonic spinal cord, this effect was not observed. It is assumed that mesenchymal stem cells stimulate the growth of vessels of the damaged nerve via production of angiogenic factors.

Pilot experiments on kinetic vitrification of human pluripotent stem cells and spermatozoa using a KrioBlast^TM-2 without penetrating cryoprotectants have shown high survival of cells (75-85% in both cases).

Repeated administration of songorine to mice restored mnestic processes impaired by scopolamine treatment, which manifested in improvement of CPAR conditioning and normalization of behavioral activity throughtout the observation period. This thearpeutical effect surpassed that of pyracetam used as the reference drug.

The study examined the effect of calcium salt of N-(5-hydroxynicotinoil)-L-glutamic acid (Ampasse preparation) on neuronal activity in hippocampal CA1 area evoked by stimulation of Schaffer collaterals at a rate of 1 Hz (30 impulses during 30 sec) in the surviving hippocampal slices of Wistar rats. The records of 1st and 30th orthodromic population spikes showed that Ampasse in concentrations of 500 μM, 1, 2, and 10 mM facilitated the synaptic transmission in Schaffer collaterals — hippocampal CA1 pyramidal neurons axis; the maximum effect was observed at 2 mM Ampasse. When used in a concentration of 10 mM, Ampasse provoked epileptiform activity, which could be prevented by MK-801, a specific noncompetitive antagonist of the NMDA-receptor complex.

The efficiency of neomitilan, a polysaccharide isolated from Crenomytilus grayanus mussels, was studied in experimental model of radiation pneumonia (irradiation of the animal lungs with a total dose of 14 and 28 Gy). Histological study showed that an increase in the number of nucleated components in the lungs of animals subjected to neomitilan inhalation prior to irradiation. Proliferation of bronchial epitheliocytes and the formation of additional lymphoid structures were also revealed in these animals. The absence of a tendency to lung tissue recovery in animals irradiated with 28 Gy is due to extreme radiation toxicity.

Light microscopy, immunohistochemistry, and morphometric examinations established that cell death in lung granulomas of BCG-infected mice resulted mainly from activation of receptor-mediated apoptosis, which did not prevent the persistence of the causative agent in macrophages of the granulomas and promoted the formation of pronounced fibrosis in granulomas and pulmonary interstitium.

For stabilization of vaccine preparations, they are lyophilized. The composition of the protective medium is an important parameter affecting the quality of the vaccine after drying. In view of the risk of spreading prion diseases, the use of media containing animal proteins is not recommended. In this study, protective media containing no animal proteins and lyophilization regimen were determined. The optimum lyophilization regimen consisted of three stages: freezing at -70°C, main stage at -35°C, and drying at 24°C. Protective medium containing 4% trehalose or protective medium with 10% sucrose and 5% soy peptone ensured highest stability of the lyophilized vaccine preparation in temperature range of 4-24°C. This can help to overcome possible break in the cold chain, which is important during transporting or storage of vaccine preparations.

Association of IL6R gene polymorphic variant rs2228145(C>A) with the development of nonalcoholic steatohepatitis in Karelia residents is detected. The risk of nonalcoholic steatohepatitis is more than 2-fold higher in carriers of CC genotype by rs2228145 polymorphic marker than in carriers of other genotypes. Plasma levels of IL-6 and the content of IL6R gene transcripts in the peripheral blood leukocytes are higher in patients with nonalcoholic steatohepatitis than in normal subjects. No relationships between rs2228145 polymorphism and the level of IL-6 and content of IL6 and IL6R mRNA were detected. Gene IL6R polymorphic variant rs2228145(C>A) seems to be involved in genetic predisposition of the population of Karelia to nonalcoholic steatohepatitis. However, biochemical and molecular mechanisms underlying the relationship of rs2228145 with the development of nonalcoholic steatohepatitis are not yet studied.

In patients with endometrial cancer (N=94), endometrial polyps (N=28), endometrial hyperplasia (N=25), and healthy women (N=77), the serum contents of MMP-2, MMP-7, MMP-9, TIMP-1, and TIMP-2 were measured by ELISA. Both carcinoma and benign neoplasms were accompanied by significant elevation of MMP-7 and TIMP-2 in blood serum. The greatest elevation (in comparison with the control) was observed for MMP-7, although serum concentration of this marker was practically identical in patients with carcinoma and benign tumors. In contrast, the levels of MMP-2 and TIMP-1 were lower in cancer patients in comparison with the control; in these patients, the levels of MMP-9 and TIMP-1 were also lower than the corresponding levels in patients with polyps and endometrial hyperplasia. There were no significant correlations between the levels of examined markers with tumor metastasizing, its histological structure, and differentiation degree of endometrial cancer. No differences were observed between examined serological markers in patients with polyps and endometrial hyperplasia of various severities. The examined MMPs and TIMPs cannot be advanced as potential diagnostic markers of endometrial cancer, but they can be used to monitor and prognosticate the disease and to assess effectiveness of the targeted therapy.

Three injections of doxorubicin to rhesus macaques cause severe intoxication, characterized by anemia, cachexia, and degeneration of the viscera. The life span of monkeys injected with the drug and receiving after 24 h mesenchymal stem cell transplantation varied from 96 to 120 days in comparison with 50-74 days in animals receiving stem cells before doxorubicin. Controls received doxorubicin and saline; the lifespan of one monkey was 24 days, of the other — 1 year and 8 months. The increase in activity of proinflammatory cytokine IL-6 was paralleled by an increase in the level of C-reactive protein.

Structural changes in the rat hippocampus in response to chronic cerebrovascular disorders induced by gravity exposure in the caudocranial vector were studied. Qualitative and quantitative morphological analysis detected significant cytoarchitectonic changes in the pyramidal layer: spongiosis, manifest pericellular and perivascular edema, and a drastic increase in the counts of pyramidal neurons with signs of impairment in all hippocampal zones. The density of perikarya in the pyramidal layer decreased. Immunohistochemical study detected high expression of Beclin-1 in CA1 field. High expression of LAMP-2 was detected in CA4 field. Field CA2 was characterized by the maximum counts of damaged cells and high expression of Beclin-1 and LAMP-2.

We studied physiological parameters of rhesus monkeys after administration of anthracycline antibiotic doxorubicin. Intravenous administration of the drug caused intoxication manifested in in an abrupt body weight loss, baldness, vomiting, and exicosis. Intoxication in monkeys determined by the damaging effects of doxorubicin on organs and tissues is also characterized by significant changes in the blood: leukopenia, thrombocytopenia, neutropenia, monocytopenia, lymphocytosis, and a sharp drop of CD20+ B cell content. The total protein and albumin content in the blood significantly decreased. A sharp increase in C-reactive protein was also accompanied by an increase in activity of proinflammatory cytokine IL-6. Transplantation of mesenchymal stem cells in some cases can significantly alleviate doxorubicin-induced damage to organs and maintain normal clinical status of monkeys after two injections of the drug. Late transplantation of stem cells does not have a protective effect and does not protect the animals from the damaging effects of doxorubicin. We found that the protective effect of mesenchymal stem cells depends on the dose of the drug, total number of cells, and the time of their transplantation. It should be noted that human and monkey mesenchymal stem cells produce similar regenerative effects, at least in the doxorubicin toxicity model.

The author name M. V. Edeeva should read M. V. Edeleva.