Category Archives: Uncategorized
Curcuma longa aqueous extract prevents myocardial injury in hypercholesterolaemic albino rat
I. B. Ekeigwe, I. C. Ikegwuonu, I. K. Uchendu,
C. A. Uchenna, U. C. Okongwu
Department of Medical Laboratory Science, University of Nigeria, Enugu;
email: ikenna.uchendu@unn.edu.ng
Received: 01 March 2019; Accepted: 17 May 2019
Coronary atherosclerosis is known to be associated with cardiomyopathy in patients worldwide. Presently there is a high prevalence of cardiovascular risk across the globe and the cost of heart disease treating is very high. Therefore, prevention of heart diseases has become the better or easier option. The aim of the present study was to evaluate the cardioprotective effects of Curcuma longa aqueous extract (AECL) in hypercholesterolaemic rats. To achieve this 40 rats were randomly devided into four groups with 10 rats per group. Rats from the group I (normal control) received no treatment. High cholesterol diet (2 g/kg, oral) and high dose of anti-thyroid hormone drug carbimazole (60 mg/kg, oral) were administered once daily for 8 weeks to induce hypercholesterolaemia in rats of II-IV groups. Group II served as toxic (negative) control and group III (positive control) was treated with a standard lipid-lowering drug atorvastatin (20 mg/kg, oral). Group IV received AECL (200 mg/kg) once daily for 8 weeks. For lipid profile analysis the total cholesterol, high density lipoprotein cholesterol, triglycerides levels were estimated and creatine kinase, lactate dehydrogenase and aspartate transminase activities as cardiac biomarkers were assayed in blood serum using standard methods. The histopathological analysis of the heart tissue was carried out. It was shown that AECL significantly reduced hyperlipidaemia, stabilized cardiac biochemical markers and protected the cardiac muscle fibers from injury. The results of the present study suggest that the Curcuma longa extract has cardioprotective potential, and can be used to prevent hypercholesterolaemia-induced myocardial injury.
High thiamine dose restores levels of specific astroglial proteins in rat brain astrocytes affected by chronic ethanol consumption
O. S. Pavlova, A. A. Tykhomyrov, O. A. Mejenskaya,
S. P. Stepanenko, L. I. Chehivska, Yu. M. Parkhomenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: aspavlova92@gmail.com
Received: 23 January 2019; Accepted: 17 May 2019
Long-term ethyl alcohol consumption induces a deficiency of essential nutrient thiamine (vitamin B1 ) and profoundly impairs metabolic processes in nervous tissue, resulting in structural and functional alterations in the central nervous system (CNS). This study was performed to evaluate protective effects of thiamine acute dose on the level of glial fibrillary acidic protein (GFAP), a sensitive marker of astroglia, and B1-related enzyme thiamine pyrophosphokinase (TPK) activity in brain of rats chronically exposed to ethanol. The rats were divided into three groups as follows: i) control group; ii) rats given 15% ethanol solution as drinking water for 9 months (EtOH group), iii) EtOH rats given thiamine per os in a dose of 2.0 mg/kg one day before experiment termination (n = 4 in each group). GFAP levels were analyzed in cerebellum, brain cortex and hippocampus by western blot and immunohistochemistry. Brain TPK activity was measured with the use of the yeast apopyruvate decarboxylase apoenzyme (apoPDC). Thiamine concentration in liver was estimated with the use of thiochrome method. It was demonstrated that GFAP content was dramatically reduced in all studied brain regions of EtOH-exposed rats (approximately by 60%, P < 0.05) compared with control rats indicating profound astroglial dysfunction. Thiamine treatment was shown to recover GFAP levels up to 80% vs. control value in the brain of EtOH-exposed rats (P < 0.05). Ethanol consumption resulted in 3.7-fold decrease in liver thiamine content and 1.4-fold decrease in brain TPK activity, as compared with control (P < 0.05). Thiamine treatment of EtOH-exposed rats significantly elevated B1 liver level, however, had no effect on brain TPK activity. Our data suggest that thiamine deficit can play an important role in alcohol-induced damage to brain astroglia. It is emerged that high-dose thiamine administration can represent effective treatment option against chronic effects of ethanol impact on brain structures.
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.