Visnyk of Dnipropetrovsk University. Biology, medicine

9-16

Authors: S. L. Popel, B. M. Mitckan, S. Z. Krasnopolsky, I. V. Melnik

The purpose of this paper is to study the morphological changes of neurocytes in spinal cord of rats in hypokinesia and subsequent physical loading. Studies were performed on 55 laboratory rats of Wistar line. Materials of the research were the anterior horns of the gray matter of L5-S2 spinal segments. Preparations stained by Nissl and Viktorov were examined histologically. Hypokinesia was modeled following on the author’s technique. It was established that during prolonged hypokinesia in neurocytes of spinal cord of rats morphological changes in cell size and shape of the motor nuclei of all segments under study have been recorded. The number of hypochromic, hyperchromic destructively unchanged and hyperchromic destructively altered neurocytes increase; shadow cells appears, as well as cases of satellitosis and neuronophagia. Decrease in of albumen synthetical neurocyte function has been recorded. Physical loading of the average aerobic capacity leads to normalization of structural and functional state of neurocytes and enhances the reparative processes, as evidenced by a number of positive changes in morphometric parameters: increase in the number of normochromic neurocytes and decreasing the number of hyper- and hypochromic neurocytes with destructive signs, absence of pyknotic forms. Morphological parameters of neurocytes and their nuclei after physical loading of average aerobic capacity do not differ from those in the control group of intact animals. In neurocytes of this group of rats RNA concentration increases by 12.6% compared to animals after prolonged hypokinesia. Neurocytes of spinal cord of rats after prolonged hypokinesia develop significant morphological changes which are characterized by emergence of a significant number of hyperchromic neurocytes with signs of destructive changes and shadow cells, as well as and hypochromic neurocytes with signs of destructive changes, reduction in size and change of shape of perikaryons of neurocytes and their nuclei. Morphological changes of neurocytes after prolonged hypokinesia are accompanied by violations of biosynthetic processes, as evidenced by RNA decrease in the cytoplasm of efferent neurocytes of spinal cord of rats. Physical loading of average aerobic capacity leads to normalization of structural and functional state of neurocytes and promotes the reparative processes suported by positive changes in morphometric parameters.

23-27

Authors: O. V. Kulibaba, S. M. Kozishkurt, O. O. Duzenko, I. L. Vovchuk, S. A. Petrov

The paper presents the results of determining the amount of reduced glutathione, glutathione reductase and glutathione peroxidase activity at transplantation of muscle tissue taken from rats from the same litter. During the work 2 surgical interventions were carried out: transplantation of muscle tissue taken from the same litter of rats, and operation without replanting. Transplantation was performed on nonlinear white male rats weighing 180–300 g. The animals were taken out of the experiment by passing electric current through the medulla oblongata. For donor transplants males from the same litter were used. In adult donor rats abdominal muscle tissue was extracted and transplanted to homologous tissue of the recipient rat. The same procedure was carried out with femoral muscle tissue. Operation without replanting was used to compare the effects of changes in the number of investigated parameters of the surgical exposure. Tissue not subject to any surgery served as a control. Parameters under study were determined on the first, third and seventh day after transplantation. Glutathione levels in tissues were determined byE. Butler, A. Dyubona, B. Kelly. Determination of glutathione reductase is based on the reduction of NADPH registration. Glutathione peroxidase activity was determined by accumulation of oxidized glutathione. Surgery is known to lead to expression of oxidative stress in the organs operated. Special role in antioxidant protection of the body is given to thiols. Expressed hydrophilic properties provide their high content in water fraction of cells and ability to protect biologically important molecules (enzymes, nucleic acids, hemoglobin) from oxidative damage. Property of thiol compounds is known to inhibit both fermented and not fermented free radical oxidation. For the first time it was found that transplantation of muscle tissue taken from animals from the same litter lead to significant reduction of glutathione in the femoral muscle of the recipient and abdominal muscle tissue of the donor and recipient and to significant increase in glutathione reductase activity in the femoral muscle tissue of the donor and in the abdominal muscle tissue of the donor and recipient.

32-35

Authors: O. Y. Galkin, Y. V. Gorshunov, V. F. Solovjova

To address a number of fundamental and applied problems in immunology, molecular and cellular biology and biotechnology it is necessary to obtain Fc-fragments of immunoglobulins. Fc-fragments may be used for studying of the effector functions of antibodies which are mediated by these areas. They are often used as an immunogen to produce anti-specie (based on so-called secondary antibody) conjugate in the development of serological tests for diagnostics (predominantly such conjugate based on monoclonal antibodies). The work is aimed to develop improved methods of obtaining and allocation of Fc-fragments of human IgA. To achieve this objective, optimization of hydrolysis of IgA with subsequent purification of Fс-fragments have been carried out. Improved method of obtaining Fc-fragments of IgA provides: papain hydrolysis of immunoglobulin in the environment of nitrogen for 4 h, allowing to achieve maximum output of Fc-fragments without their further degradation: isolation and purification of Fc-fragments of human IgA by one-stage gel filtration on sephadex G-100; control of purity of the target product by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and Ouchterlony immunodiffusion. Enzymatic hydrolysis was carried out at the optimal temperature of papain (37 °C). As the oxygen in the air may have inhibitory effect on enzymatic hydrolysis reaction, the reaction mixture was incubated in the nitrogen atmosphere to prevent inactivation of papain. To reduce the incident degradation of immunoglobulin molecules, papain hydrolysis was carried out without using an enzyme activator (cysteine). Usage of the proposed scheme allows obtaining Fc-fragments of human IgA of high purity. Outcome of Fc-fragments after all stages of purification was about 18% of the initial amount of IgA in the preparation. Molecular weight from Fc-fragments of human IgA was equal to approximately 70 kDa.

40-44

Authors: G. І. Zvir, O. М. Moroz, S. O. Hnatush

Objects of the study were sulfate-reducing bacteria Desulfovibrio desulfuricans ІМV К-6, isolated from Yavorivske lakе. This strain is kept in the collection of microorganisms at the Department of Microbiology of Ivan Franko National University. Bacteria were grown in the Kravtsov-Sorokin’s liquid medium with the following composition (g/l): Na2SO4 × 10H2O – 0.5, NaH2PO4 – 0.3, K2HPO4 – 0.5, (NH4)2SO4 – 0.2, MgSO4 × 7H2O – 0.1, C3H5O3Na – 2.0. The bacteria were grown for 10 days at 30 °C under anaerobic conditions. In order to study the sensitivity of the sulfate reducing bacteria to action of Uragan and Raundup herbicides, the cells of D. desulfuricans ІМV К-6 were grown at the concentrations of herbicides as follows: 0,28 mМ, 2,8 mМ (concentration recommended for use) and 5,6 mM. Biomass was determined by photometric method. Concentration of hydrogen sulfide in the culture medium was determined by photo-colorimetric method. Concentration of sulfate-ions in the medium was determined by turbidimetric method. Capacity of sulfate reducing bacteria D. desulfuricans ІМV K-6 to grow, reducing sulfates to hydrogen sulfide upon influence of Uragan and Raundup herbicides was studied. Accumulation of bacterial biomass in the control and upon influence of herbicides was the highest on the fourth-sixth day of cultivation, and after that the stationary growth phase began. It was shown that sulfate reducing bacteria upon influence of herbicides grew more intensively compared with the control. It was discovered that the level of biomass changed depending on the increasing concentration of Uragan or Raundup herbicides in the medium. Sulfate reducing bacteria D. desulfuricans ІМV K-6 could reduce sulfates to hydrogen sulfide in the presence of sulfates and organic compounds in the medium (dissimilatory sulfate reduction). Stimulatory influence of Uragan and Raundup on the dissimilatory sulfate reduction process of D. desulfuricans ІМВ К-6 has been discovered. The formation of hydrogen sulfide correlates with the usage of sulfatе ions. The capacity of sulfate reducing bacteria D. desulfuricans ІМV K-6 to grow, reducing sulfate ions to hydrogen sulfide upon influence of Uragan and Raundup may be caused by presence of inert components (sulfates) in these herbicides that can be used by microorganisms as electron acceptors during sulfate respiration.

51-56

Authors: I. U. Pismenetskaya, T. D. Butters

Free oligosaccharides (FOS) are unbound structural analogs of glycans of glycoconjugates. There are several sources of them inside the cell: 1) multistep pathways of N-glycosylation, 2) the cell quality control and endoplastic reticulum-associated degradation of mis-glycosylated and/or misfolded glycoproteins, 3) lysosomal degradation of mature glycoconjugates. Some of these FOS are the earliest indicators of potential glycosylation alterations that would be revealed in the course of the cell quality control and the endoplastic reticulum-associated degradation. Ischemia and hypertension cause stress of intracellular organelles leading to disruption of their functions. The main objective of the work was the characterization of free oligosaccharides (FOS) in plasma obtained from patients with cardiovascular diseases compared to those from healthy subjects to evaluate the potential of these compounds for diagnostics. Chromatographic profiles of FOS composed of 4–12 monosaccharides were obtained and analyzed for quantitative and qualitative differences between the samples. After plasma deproteinization and FOS purification the oligosaccharides were labelled with anthranilic acid (2-AA), separated into the neutral and charged with QAE Sephadex (Q25-120) chromatography and analysed using high-performance liquid chromatography (HPLC). Glucose unit values were determined following comparison with a 2-AA-labelled glucose oligomer ladder derived from a partial hydrolysate of dextran as an external standard. The data were collected and processed using Empower software. The charged FOS were digested with the sialidase from Arthrobacter ureafaciens. 2-AA – labelled free oligosaccharides from transferrin were used as an external standard for the structure decoding. The profiles obtained were compared with intracellular free oligosaccharides of known structures and with the glycan structures and their descriptions in the databases GlycoBase and EUROCarbDB. These approaches allowed predicting a range of glycan structures for each of the main peaks of HPLC profiles of plasma free oligosaccharides and managing ways for their future experimental analysis. In the case of cardiovascular disorders, HPLC profiles of FOS revealed a changing pattern of heterogeneity, depending on the severity of the disease. Three main enlarged glycan species in the netral fraction and one peak in the charged fraction distinguished the FOS of the patients from those of the healthy volunteers. It has been revealed that the neutral marker peaks were represented by polimannose glycans with 5–7 mannose residues and 1–2 residues of N-acetylglucosamine, and one of the major peaks of the charged fraction – by two-antennary complex N-glycan with two sialic acid residues. The study of free oligosaccharides of blood plasma is a new field of glycobiology allowing an evaluation of an organism state at the level of the cell organelle functional status and openning up broad prospects for finding early diagnostic and prognostic markers of cardiac insufficiency.

63-67

Authors: O. E. Shaul’ska, L. M. Diachenko, T. P. Nikolayenko-Kamyshova, A. I. Shevtsova

Destruction of malignant cell clones and search for markers of the effectiveness of chemotherapy in patients with hematologic malignancies is an urgent direction of research. The purpose of this paper was to study plasma of patients with various forms of blood tumor, the activity of trypsin-like enzymes, matrix metalloproteinases MMP2 and MMP9, content of α1-proteinase inhibitor and α2-macroglobulin before and after the cytotoxic therapy by anthracycline antibiotics, doxorubicin and daunorubicin. It was established that concentration of inhibitors and activity of trypsin-like enzymes increased in patients with acute myeloid leukemia and multiple myeloma against the backdrop of multidirectional changes of proMMP9. Activity of the latter enzyme was reduced to 0,03±0,01 rel. u. in acute myeloid leukemia, and after chemotherapy it increased 7 times. Reducing level of inhibitors and increasing activity of MMP9 were found in chronic lymphocytic leukemia. The presence of the correlation between the α2-macroglobulin and gelatinase activity in the treatment indicates that the inhibitor is an important mechanism for storing of gelatinоlytic potential. Balance between proteolysis and antiproteolysis depends on the type of proliferating cells and the course of the disease. Indicators of tissue destruction may be an additional criterion for monitoring the stage of blood tumors and efficacy of treatment.