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Sources and regulation of nitric oxide synthesis in uterus smooth muscle cells
H. V. Danylovych, Yu. V. Danylovych, T. V. Bohach,
V. T. Hurska, S. O. Kosterin
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua
Received: 28 February 2019; Accepted: 17 May 2019
It was proved that NO synthesis in isolated mitochondria of rat uterus smooth muscle depended on the entry of exogenous Ca2+ to mitochondria (inhibited by 1-10 mM Mg2+ in the absence of ATP and by 10 μM ruthenium red) and was suppressed by calmodulin antagonists (0.1-10 μM calmidazolium and 1-100 μM trifluoperazine). It was blocked by NG-nitro-L-arginine, a known antagonist of the constitutive NO-synthase, with a half-maximal inhibition effect at about 25 μM. Moderate deholesterinization of the plasma membrane of myocytes after processing with 0.01% digitonin was followed by increased nitric oxide biosynthesis by cells. The data obtained suggested that mitochondria and plasmalemma is a possible source of NO synthesis in uterine myocytes.
Role of the heparin-binding domain in intracellular trafficking of sHB-EGF
O. I. Krynina, K. Yu. Manoilov, D. V. Kolybo, S. V. Komisarenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: olyakrynina@gmail.com
Received: 11 July 2018; Accepted: 17 May 2019
Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor family that was proven as a potent mitogen and chemoattractant. HB-EGF mediated EGFR activation is a key event in the stimulation of gene expression, cell migration and proliferation during both normal and pathogenic physiological processes. The main goal of this research was to reveal the role of the heparin-binding domain of HB-EGF in the ligand-receptor formation and its further internalization to the cytoplasm. We used fluorescently-labeled recombinant derivative of soluble HB-EGF and its truncated form (sHB-EGFΔ84–106) with deletion of the heparin-binding domain. Firstly, the binding kinetics of two forms of sHB-EGF to its cell surface receptors was determined using flow cytometry. To determine how the absence of heparin-binding domain in the structure of HB-EGF affects its internalization, we analyzed the endocytosis process of EGFP-sHB-EGFΔ84–106 and EGFP-sHB-EGF complexes by confocal microscopy. It was found that the full-size form of HB-EGF is characterized by a lower intensity of translocation to the cytoplasm in comparison to HBD-deleted form. Thus, differences in the trafficking of the full-size or truncated forms of sHB-EGF in the cell cytoplasm may reflect the mechanisms of extracellular matrix influence on the biological activity of sHB‑EGF.
p60-S6K1 represents a novel kinase active isoform with the mode of regulation distinct from p70/p85-S6K1 isoforms
I. V. Zaiets, V. V. Holiar, A. S. Sivchenko,
V. V. Smialkovska, V. V. Filonenko
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv;
e-mail: filonenko@imbg.org.ua
Received: 13 March 2019; Accepted: 17 May 2019
The phosphatidylinositol-3-kinase (PI3K)/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway controls plenty of cellular functions regulating phosphorylation one of its mediators ribosomal protein S6 kinase 1 (S6K1). Alternative translation of the common S6K1 transcript can generate three protein kinase isoforms, including p85-S6K1, p70-S6K1 and p60-S6K1. The catalytic activity of S6K1 is modulated by mitogens and growth factors via phosphorylation at three critical sites such as the activation loop (T-loop site), turn motif (TM site), and hydrophobic motif (HM site). Both members of the PI3K/mTORC1 pathway, PDK1 and mTORC1, directly phosphorylate the T-loop site and HM site, respectively. Indeed, most studies aimed at elucidating S6K1 regulation have focused on p70- and p85-S6K1. Meanwhile, however, the activity of p60-S6K1 and its regulation have not been elucidated so far. To test whether p60-S6K1 was an active kinase isoform that was regulated similar to p70/p85-S6K1, we employed previously generated p85–/p70–/p60+HEK-293 cells. First, an in vitro kinase assay confirmed the ability of p60-S6K1 to phosphorylate ribosomal protein S6 (rpS6), a well-known S6K1 substrate. Next, analysis of p60-S6K1 phosphorylation under different cell growth conditions showed that p60-S6K1 does not have detectable levels of phosphorylation at PDK1- and mTORC1-regulated sites, yet this isoform undergoes phosphorylation at the TM site. Finally, we found that activity of p60-S6K1 was not sensitive to mitogenic stimulation and cell treatment by potent inhibitor of the PI3K1/mTORC1-dependent signaling pathway rapamycin suggesting the existence of a PI3K/mTORC1-independent mechanism of p60-S6K1 regulation in HEK-293. The data of the current study suggest that the p60-S6K1 isoform possesses intrinsic kinase activity that is independent of PI3K/mTORC1 signaling regulation in HEK-293 cells. What is more, modulation of p60-S6K1 activity via the PI3K/mTORC1 signaling pathway seems to be cell-type specific, since the p60-S6K1 isoform undergoes PDK1- and mTORC1-mediated phosphorylation in breast cancer cell line MCF-7.
1,3-Oxazol-4-ylphosphonium salts as new non-peptide inhibitors of furin
T. V. Osadchuk1, V. K. Kibirev1,2, O. V. Shybyryn1, A. V. Semyroz1,
Ye. S. Velihina1, Е. R. Abdurakhmanova1, V. S. Brovarets1
1V.P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: brovarets@bpci.kiev.ua;
2Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv
Received: 22 February 2019; Accepted: 17 May 2019
A series of novel triphenylphosphonium derivatives of 1,3-oxazole containing at C2 and C5-positions electron withdrawing or electron-donating groups were synthesized and characterized by 1H, 31P NMR and IR spectroscopy, element analysis and chromato-mass spectrometry. These compounds were found to be a new class of non-peptide inhibitors of furin. Depending on the chemical structure, they inactivated enzyme at micromolar level by mechanism of competitive, non-competitive or mixed inhibition. Evaluation of the synthesized derivatives as furin inhibitors showed that among the triphenylphosphonium salts studied by us, oxazole 12 containing 2,4-dichlorophenyl- in the C2-position and MeS-group at C5 is the most active (Ki = 1.57 μM) competitive inhibitor of furin. Our results provided evidence that chemical modification of 1,3-oxazole-4-yl-triphenylphosphonium salts may be useful for developing new more potent and selective inhibitors of furin.
Born in Ukraine: Nobel prize Winners Ilya Mechnikov, Selman Waksman, Roald Hoffmann AND Georges Charpak
T. V. Danylova1, S. V. Komisarenko2
1National University of Life and Environmental Sciences of Ukraine, Kyiv;
e-mail: danilova_tv@ukr.net;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: svk@biochem.kiev.ua
Received: 18 February 2019; Accepted: 14 March 2019
Our country has not yet gained recognition from a Nobel Committee, however, some Nobel Prize winners were born in the territory, which belongs to present-day Ukraine. Among them are the father of innate cellular immunity Ilya Mechnikov; the famous microbiologist and biochemist Selman Waksman, whose studies had led to the discovery of streptomycin; the outstanding chemist, poet and playwright Roald Hoffmann, and the prominent physicist Georges Charpak who invented and developed particle detectors, in particular, the multiwire proportional chamber. This paper aims to outline briefly the main stages of their scientific activity.
Development on knowledge of hormone biochemistry in the works of the Nobel prize laureates of the first half of the 20th century: F. G. Banting, John J. R. Macleod, H. O. Wieland, A. O. Windaus, A. F. Butenandt, L. Ružička, E.Kendall, P. Hench, T. Reichstein
R. P. Vynogradova, V. M. Danilova, S. V. Komisarenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: valdan@biochem.kiev.ua
Received: 18 February 2019; Accepted: 14 March 2019
The first half of the 20th century was marked by significant scientific advances in the study of hormones and vitamins. Among the first researchers working with hormones were F. Banting and J. Macleod, who discovered and characterized the pancreatic hormone insulin. This discovery catalyzed advances in the understanding of the mechanisms regulating biochemical processes – a new topic in the field of biological chemistry. The next important stage in the development of knowledge on biologically active substances was the works of organic chemists G. Wieland, A. Windaus, A. Butenandt and L. Ružička. They almost simultaneously identified and characterized the chemical structures of bile acids, vitamin D as well as female and male sex hormones. They found that all of these compounds are of a steroid nature and identified cholesterol as the starting material for their synthesis in the body. The studies of highly-active substances of steroid nature were continued by E. Kendall, F. Hench and T. Reichstein. They synthesized and investigated the structure and biological effects of corticosteroids, the hormones produced in the adrenal cortex. They were first to develop a method for the commercial manufacturing of cortisone, a hormone which is widely used to treat inflammatory processes. Thus, in the first half of the 20th century, organic chemists gave biochemists knowledge on the structure of essential for the human body substances – steroid compounds.
Lipid profile parameters and oxidative processes intensity in the persons who have been affected by low doses of radiation
V. L. Sokolenko, S. V. Sokolenko
Bohdan Khmelnytsky National University of Cherkasy, Ukraine;
e-mail: sokolenko@ukr.net
Received: 10 October 2018; Accepted: 14 March 2019
The aim of the work was to analyze the relationship between the main parameters of lipid metabolism and the intensity of oxidative processes among the inhabitants of the territories contaminated by radionuclides as a result of the Chornobyl accident. We examined 50 persons from the control group and 50 persons from the territories of strengthened radioecological control (density of soil contamination by isotopes 137Cs 3.7∙104–18.5∙104 Bq/m2, 50 persons). All examined were the students aged 18 to 24, who had no acute illnesses during the study. We determined the parameters of lipid metabolism, oxidative processes and antioxidant system. A positive correlation of all analyzed lipid metabolism parameters (except for HDL-C) with MDA, ceruloplasmin and the index of oxidative stress was discovered. The highest values of correlation coefficients with oxidative stress indices were observed for low-density lipoprotein cholesterol. Under the conditions of additional emotional stress, the correlation coefficients between the main lipid metabolism and the intensity of oxidative processes increased. Persons who lived for a long time in areas contaminated with radionuclides form the risk group for the development and progression of inflammatory processes. The risk increases under the influence of additional factors of a stressful nature.
New monoclonal antibodies to the Chlamydia trachomatis main outer membrane protein and their immunobiological properties
O. Yu. Galkin1,2, O. B. Besarab1, Yu. V. Gorshunov1, O. M. Ivanova3
1National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexfbt@gmail.com;
2Propharma Plant Ltd., Kyiv;
3Xema Ltd., Kyiv
Received: 18 July 2018; Accepted: 14 March 2019
One of the methods that have been widely used in the diagnosis of urogenital chlamydia is an enzyme-linked immunosorbent assay (ELISA), the use of which allows for differential diagnosis. In order to increase the efficiency of ELISA test kits production, for the kits for the diagnosis of urogenital chlamydia, based on the principle of indirect modification, following synthetic positive controls (PCs) can be used: a conjugate of IgM (IgA) normal immunoglobulins and monoclonal antibodies (McAbs) to C. trachomatis major outer membrane protein (MOMP). The goal of this work was to obtain high active and affinity McAbs to the C. trachomatis MOMP as well as the study of its immunobiological properties which are important for future biochemical approaches. The study was conducted using: polyclonal antibodies (PcAbs) to C. trachomatis; recombinant major outer membrane protein (MOMP) (191-354 a.r.; W4-W5); epitope mapping based on phage display technology. The original set from 16 clones of hybridomas, producers of McAbs to the C. trachomatis MOMP has been obtained. More than half of the tested McAbs (8 out of 14) were characterized by a rather high titer (≥1:800), and three of them had a titer of ≥1:1600. In general, the McAbs titer was correlated with the value of the affinity constant: McAbs with higher titles were characterized by a high value of the affinity constant. For McAbs with a titer of <1:800, the average Ka is 5.2×109 M-1, while for McAbs with a titer ≥1:800 – Ka = 10.7×109 M-1. Antigenic determinants of two McAbs 293F4 and 291F8 that actively competed with PcAbs are represented by two linear sequences of 320-325 a.r. and 326-330 a.r., respectively. The epitope, which interacts with McAb 296G2, is represented by a linear sequence of 347-352 a.r. McAb 296G2 did not show active competition with serum PcAbs. The resulting set of data allows selecting McAbs for use in PCs of the ELISA kit for the detection of IgA or IgM antibodies to C. trachomatis.
New anti-candida active nitrogen-containing bisphosphonates as inhibitors of farnesyl pyrophosphate synthase Candida albicans
L. O. Metelytsia, D. M. Hodyna, O. L. Kobzar,
V. V. Kovalishyn, I. V. Semenyuta
V. P. Kukhar Institute of Bioorganic Chemistry and Petrochemistry,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: ivan@bpci.kiev.ua
Received: 05 February 2019; Accepted: 14 March 2019
In our previous work, a number of new nitrogen-containing bisphosphonates (N-BPs) with high predicted and experimental antifungal activity were presented as potential Candida albicans farnesyl pyrophosphate synthase (FPPS) inhibitors. To confirm this hypothesis, a homologous C. albicans FPPS model with high-quality scores has been developed and used in present work to study the molecular mechanism of nitrogen-containing bisphosphonates action as anti-Candida agents. The known FPPS inhibitors ammonium 2-(Pyridin-2-ylamino)ethylidene-1,1-bisphosphonate, risedronate and alendronate were used in molecular docking analysis. The molecular docking analysis of the new N-BPs demonstrated a number of common features of all ligand’s interaction in the active center of FPPS C. albicans. It is established that the ligands phosphonate groups are the key elements in the formation of the stable ligand-protein complexes with binding energy in a range (ΔG) from –6.6 to –7.1 kcal/mol due to a significant number of electrostatic, hydrogen and metal-acceptor bonds. It is confirmed that the new studied N-BPs 1 and 3 with high anti-Candida activity are FPPS inhibitors.
Insulin resistance in obese adolescents and adult men modifies the expression of proliferation related genes
O. H. Minchenko1, Y. M. Viletska1, D. O. Minchenko1,2, V. V. Davydov3
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: ominchenko@yahoo.com;
2Bohomolets National Medical University, Kyiv, Ukraine
3SI “Institute of Children and Adolescent Health Care,
National Academy of Medical Sciences of Ukraine”, Kharkiv
Received: 11 December 2018; Accepted: 14 March 2019
Numerous data demonstrate that key regulatory factors, enzymes and receptors including HSPA5, MEST, SLC1A3, PDGFC, and ADM represent poly-functional, endoplasmic reticulum stress-dependent proteins, which control variable metabolic pathways. The expression level of genes of these proteins in the blood and subcutaneous adipose tissue of obese adolescents and adult men with and without insulin resistance was studied. It was shown that in blood of obese adolescents without insulin resistance the expression level of SLC1A3, HSPA5, MEST, and PDGFC genes was significantly increased, but development of insulin resistance led to down-regulation of these genes expression except HSPA5 gene as compared to the control group as well as to the group of obese adolescents without insulin resistance. At the same time, the expression level of ADM gene did not change significantly in obese adolescents without insulin resistance, but the development of insulin resistance led to down-regulation of this gene expression. In subcutaneous adipose tissue of obese adult men without insulin resistance the level of SLC1A3 gene expression was decreased, although ADM, MEST, and HSPA5 genes – increased. It was also shown that the development of insulin resistance in obese men affected the expression level of ADM and SLC1A3 genes only. Results of this investigation provide evidence that insulin resistance in obese adolescents and adult men is associated with specific changes in the expression of genes, which related to proliferation and development of obesity and insulin resistance as well as to endoplasmic reticulum stress and contribute to the development of obesity complications.