Vol 167, No 3 (2019)

We studied the possibility of formation of endogenous opioid dependence in rats during periodic intake of 5% ethanol solution. In the control group, both drinking bottles contained water. In the experimental group, the second bottle was filled with 5% ethanol solution for 12 h per day; in the following 12 h, these rats were deprived of food and ethanol. This regimen was maintained over 8 days. The rats were subdivided into alcohol- and water-preferring subgroups. Ethanol deprivation followed by naloxone injection evoked the signs of opiate withdrawal syndrome in both subgroups. These findings suggest that periodic voluntary intake of a weak ethanol solution over 8 days led to the formation of endogenous opioid dependence in rats irrespective of amount of the consumed alcohol.

In experiments on narcotized rats, the electrical potential and impedance of isolated segment of the right femoral and/or carotid artery were simultaneously recorded in situ via two extracellular nonpolarizable Ag/AgCl electrodes mounted along the arteries at the interelectrode distance of 4 mm. The active, passive, and intermediate pulsing modes of arterial segment were determined according to the phase relations between its electrical impedance and BP, which was simultaneously measured in the symmetrical part of the respective left artery and used to assess pressure in the examined segment. The study assessed the effect of amplitude (0.2-2.0 mA) of alternating probe current (100 kHz), which was used to measure the electrical impedance of arterial segment, on its pulsing mode. The pulsing mode determined at the initial minimal probe current of 0.2 mA was passive with out-of-phase pulsatile oscillations of electrical impedance and BP. After elevation of the probe current amplitude to maximal level of 2 mA, these oscillations became in-phase indicating transition of the arterial segment to active pulsing mode. This transition was accompanied by appearance of arterial voltage impulses synchronized with BP upstrokes and an 11-fold median increase in the peak-to-peak value of electrical impedance oscillations with the interdecile range of 7-15 (N=28). Under moderate amplitude of probe current (0.3-0.5 mA), the intermediate mode of arterial pulsing was observed featured by a delayed, weak, and short active constriction during BP front, which was insufficient to resist and counterbalance the dilating effect of rising BP. In this case, the pulsatile oscillations of electrical impedance were smaller than those observed in active or passive pulsing modes indicating a possibility to stabilize the arterial diameter during pulsatile oscillations of BP. The effect of alternating electric current on the mode of arterial pulsation is explained with electrical model of smooth muscle cell membrane reflecting the rectifying features of potassium channels and predicting membrane hyperpolarization in response to external alternating current passing across the cell. The visibilities of therapeutic neurotropic and angiotropic stimulation with alternating electric current are discussed.

Magnetic resonance imaging was employed to examine the morphofunctional status of myocardium in Wistar rats with multifactor cardiovasorenal model of arterial hypertension. In 3 months after the onset of experiment, the rats demonstrated a pronounced hypertrophy in left ventricular myocardium mostly due to thickening of the posterior and lateral walls against the background of relatively stable thickness of ventricular septum. The left ventricular endsystolic volume markedly increased in parallel with moderate increase of the end-diastolic volume. The standard calculated indices were used for precise assessment of the type of remodeling of individual myocardial structures. The study showed that multifactor arterial hypertension model was characterized by domination of hypertrophic mode of the left ventricular remodeling, whereas the concentric variant was observed more rarely by 2.5 times. The greatest alterations were observed in the posterior and lateral walls of the left ventricle, which could result from the hemodynamic effects of hypervolemic arterial hypertension.

The development and manufacturing of serum-free culture media allowing reducing the costs of preparations and standardizing the biotechnological process are important trends in biotechnology. Substitution of protein compounds in the serum-free media with recombinant analogues reduces the risk of contamination with various infectious agents. Human transferrin is a protein component of serum-free media responsible for the transport of Fe³⁺ ions into cells. We generated a producing strain P. pastoris secreting human transferrin to the culture medium. The use of constitutive GAP promoter and maintenance of medium pH at 6.5 allows attaining maximum level of transferrin expression (20 mg/liter).

We studied the effect of streptozotocin-induced diabetes mellitus on the level of glycemia and some other indices of lipid metabolism, including fatty acid metabolism and LPO intensity, during the development of diabetes mellitus in rats. Even at the early terms of diabetes development, hypertriglyceridemia and hypercholesterolemia were accompanied by changes in the blood content of fatty acid (at the expense of ω3 and ω6 fatty acids) that persisted throughout the observation period. Intensification of LPO against the background of suppressed activity of antioxidant enzymes and reduced level of ω3 fatty acids attested to the development of oxidative stress. These data attest to antioxidant property of ω3 fatty acids, which is seen from positive correlations between these fatty acids and activity of antioxidant enzymes.

In type 1 diabetes mellitus, the levels of insulin and C-peptide decrease at the periphery and in CNS. C-peptide potentiates the regulatory effects of insulin. We studied the effects of single and repeated (over 7 days) individual and combined nasal administration of C-peptide (10 μg/day) and insulin (20 μg/day) on activity of Akt kinase and kinase-3β-glycogen synthase (GSK3β), the components of 3-phosphoinositide pathway, in the hypothalamus of intact rats and rats with mild streptozotocin-induced type 1 diabetes mellitus. Phosphorylation of Akt kinase at Thr³⁰⁸ and Ser⁴⁷³ (stimulation) and GSK3β at Ser⁹ (inhibition) was evaluated. In diabetes, phosphorylation of Akt kinase and, to a lesser extent, GSK3β, is reduced. A single injection of insulin or C-peptide and insulin increased this process. Long-term combined treatment with C-peptide and insulin normalized activity of Akt kinase and GSK3β in diabetic rats, treatment with insulin alone produced less pronounced effect; monotherapy with C-peptide was ineffective. Intranasal co-administration of C-peptide and insulin effectively stimulates the insulin system in the hypothalamus that is weakened at diabetes mellitus type 1, which can be used in the treatment of this disease.

We have studied the effect of a combination of three natural muramylpeptides containing a meso-diaminopimelic acid residue (polyramyl) on the subpopulations of circulating T cells, spleen morphology, and leukocyte level in the blood of C57Bl/6 mice with cyclophosphamideinduced immunosuppression. Intraperitoneal injections of cyclophosphamide in a dose of 100 mg/kg on days 1, 3, 5, and 7 of the experiment reduced leukocyte count and the relative number of CD4⁺ T cells in the blood, and also depleted the cellular composition of splenic white pulp on day 10. Subcutaneous injections of polyramyl in a dose of 200 μg/mouse on days 8 and 9 practically completely restored blood leukocytes count and morphology of the splenic white pulp. Moreover, administration of polyramyl induced marked tendency to increase in the relative number of CD4⁺ T cells and CD4/CD8 ratio in mice with cyclophosphamideinduced immunosuppression.

We present the results of analysis of skin epidermis thickness in individuals with recessive mutation c.-23+1G>A in the GJB2 gene in comparison with individuals without this mutation living in Eastern Siberia (Yakut population). We examined 152 individuals with different genotypes by GJB2 gene mutation c.-23+1G>A. Homozygotes and heterozygotes by c.-23+1G>A have thicker epidermal layer (0.245 mm and 0.269 mm, respectively) in comparison with individuals without this mutation (0.193 mm) (p<0.05). The obtained data support the hypothesis about selective advantage of carriers of mutant GJB2 gene alleles and partly explain extremely high carrier frequency (10.3%) of c.-23+1G>A mutation in the GJB2 gene in Yakut population in Eastern Siberia.

We studied the expression of mRNA and the level of CAP1 (adenylate cyclase-associated protein 1) and cofilin proteins in the tissues of patients with non-small cell lung cancer. The expression of mRNA and the level of CAP1 in tumor tissue increased during growth of the primary tumor and its metastasis. It was shown that with the growth of the primary tumor, the content of cofilin in the tumor tissue decreases against the background of increased expression of its mRNA; in regional metastasis, the content of cofilin and expression of the corresponding mRNA increased. It was found that increased content of the studied proteins in the tumor tissue increased the risk of metastasis.

Using mouse model of regeneration of critical size cranial defects, we studied combined effect of 1 and 10 μg of BMP-2 of prokaryotic origin and recombinant erythropoietin (Epostim) injected subcutaneously in the area of bone defect in a total dose of 6000 U/kg. Erythropoietin considerably improved quantitative and qualitative characteristics of the bone tissue in the site of implantation when used in combination with BMP-2 in both concentrations.

Vitronectin, extracellular matrix protein, plays an important role in embryonic development and in organ and tissue reparation. A unique characteristic of vitronectin is specific binding of various biological molecules, including urokinase receptor (uPAR), extracellular matrix components, adhesion receptors, growth factors, thus supporting the modulation of cell behavior. Vitronectin is in fact not found in intact myocardium, while after infarction its level increases significantly, which correlates with accumulation of uPAR⁺ progenitor cardiac cells in the focus. The cells isolated from the heart of wild type mice are characterized by higher adhesion to vitronectin than progenitor cardiac cells from the myocardium of uPAR knockout mice. In addition, inhibition of urokinase receptor with specific antibodies on the surface of the progenitor cardiac cells of wild type mice leads to attenuation of their adhesive activity and flattening on vitronectin matrix, which can be important for their distribution in the postinfarction myocardium and realization of the reparative functions.

Previous data showed that myelin-reactive autoantibodies found in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis recognize and hydrolyze various fragments of myelin basic protein (MBP). Moreover, antibody-mediated cleavage of the encephalithogenic fragment MBP_81-103 flanked with two fluorescent proteins can serve as a new biomarker of multiple sclerosis. Here we describe creation of the next generation of this biomarker based on antibody-dependent degradation of a new chemically synthesized fluorescent substrate with resonance energy transfer that contains fluorophore Cy5 and quencher QXL680 separated by MBP_81-99 protein (Cy5-MBP_81-99-QXL680). This substrate is degraded during incubation with purified antibodies and B cells from patients with multiple sclerosis, but not healthy volunteers.

The pharmacokinetics of two fluoxetine capsulated dosage forms and two amitriptyline tablet forms after a single oral intake was studied in dogs and healthy volunteers. High significant correlations were detected between plasma concentrations of fluoxetine (r=0.96, p<0.00001, n=11) and amitriptyline (r=0.78, p<0.0224, n=8) in dogs and volunteers. A correlation of medium strength (though insignificant) was detected between nortriptyline concentrations in the plasma of dogs and volunteers (r=0.69, p<0.199, n=5). The bioavailability parameters of the test drugs in dogs and volunteers did not differ. Similar trends of fluoxetine and amitriptyline pharmacokinetic parameters were revealed in volunteers and animals. Methods for extrapolation of experimental pharmacokinetics parameters of fluoxetine and amitriptyline obtained on dogs for humans are proposed and validated.

We analyzed cytokine profile in the sera of CBA mice in 1, 5, and 24 h after intraperitoneal injection of supernatants of broth cultures of group A Streptococcus types 1M and 3M and Dochez NY5 type 10M strain. The increase of the cytokine content was observed in response to supernatants of all three types, but the highest values were recorded after injection of supernatant of strain Dochez-NY5. The level of IL-2 increased most drastically (by 51 times) and the level of IL-5 increased by 8.9 times in comparison with the control. The level of IL-2 also increased after injection of supernatants of type 1M and type 3M, but to a lesser extent (by 5 and 2.3 times, respectively). The content of proinflammatory cytokines IL-1β, TNFα, and IFNγ in mouse sera increased to a lesser extent than IL-2 after administration of all three supernatants.

The concentration of kidney injury molecule-1 (KIM-1) was measured in blood plasma of 99 patients with clear-cell carcinoma and 14 patients with benign renal tumors using a Human Serum TIM-1/KIM-1/HAVCR Quantikine ELISA kit. The control group consisted of 15 healthy male and 14 healthy female subjects. KIM-1 levels in blood plasma of patients with cancer or benign renal tumors were significantly higher than in the control (p<0.00001 and p<0.01, respectively). In patients with benign tumors, this parameter was significantly lower than in patients with cancer (p<0.0001). KIM-1 level significantly increased with disease stage (p<0.0001), and even in stage I cancer, it was higher than in the control group (p<0.0001) or in patients with benign tumors (p<0.01). The best sensitivity/specificity ratio for stage I renal cancer detection (81 and 83% respectively) was achieved at cut-off level 77 pg/ml, the sensitivity of detection of for stages II-IV being 97%. Plasma level of KIM-1 increased with increasing the size and area of the primary tumor (T). This parameter was higher in patients with metastasis in regional lymph nodes irrespective of their number (N1 or N2) in comparison with patients without regional metastasis (N0). It is also higher in patients with distant metastasis (M+). In patients with grade III-IV cancer, KIM-1 level was 7-fold higher than in patients with grade I-II tumor (p<0.0001). Thus, KIM-1 can be regarded as a highly sensitive marker for early detection of clear-cell carcinoma.

We studied the response of neutrophils, macrophages, and mast cells to local application of silica nanoparticles (10-20 nm). Histological examination of tonsillar postoperative material from 6 patients aged 24-44 years with recurrent tonsillitis was carried out. Irrigation of the tonsillar lacunae was carried out over 5 days before bilateral tonsillectomy: on the right by Polysorb MP suspension (1 g/liter), on the left by saline. The contact of nanoparticles with the mucosa led to a decrease in the number of cells expressing myeloperoxidase (p=0.02) and an increase in the count of CD68+ cells (p=0.04); the count of mast cells remained unchanged. Local use of medical adsorbent based on silica nanoparticles induced changes in cells due to their resorption by the tissue. Positive chemotaxis of CD68+ macrophages revealed in the tonsillar lymphoid tissue attested to stimulation of non-specific immunity and inductive phase of specific immunity. The authors hypothesized that internalization of medical nanoparticles by resident phagocytes of the mucosa could support targeted biodistribution of drugs in the palatine tonsils.

The effects of endocrine disrupters of transcriptional control of morphogenesis are poorly studied. Changes in the expression of transcriptional factor PRH and proliferation of adrenal cortical cells were analyzed in pubertal and postpubertal rats exposed prenatally and postnatally to low doses of endocrine disrupter DDT. In rats exposed to DDT, the expression of PRH and proliferation of adrenal cortical cells differed from those in control rats. Association between these parameters was weakened in the zona glomerulosa and zona reticularis and was absent in the zona fasciculata. These findings suggest that exposure to DDT in pre- and postnatal periods impairs the regulation of proliferative processes by transcriptional factor PRH in all zones of rat adrenal cortex, which can be a mechanism of the disruptive action of DDT.

A stimulating effect of a combination of hepatocyte growth factor (HGF) and glial neurotrophic factor (GDNF) on the growth of neurites in the spinal ganglion model was demonstrated. The mechanism of neurite growth in the spinal ganglion model is associated with transactivation of HGF c-met receptor in the presence of both HGF and GDNF. The combination of HGF and GDNF significantly activated mitogenic signaling cascade mediated by protein kinases ERK1/2, which can be a mechanism for increasing the number of neurites. Our findings can be used for developing effective methods for restoring impaired peripheral nerve function after traumatic and ischemic injury using a combination of GDNF and HGF.