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The contribution of the Nobel prize laureates to the development of knowledge of vitamin biochemistry: Ch. Eijkman, F. G. Hopkins, A. Szent-Györgyi, W. Haworth, P. Karrer, R. Kuhn, H. Dam, E. A. Doisy, G. Minot, W. Murphy, G. Whipple, D. Hodgkin, R. Woodward

V. M. Danilova, R. P. Vynogradova, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;

In the first half of the 20th century, the experimental research of chemists, biochemists and physiologists in collaboration with doctors led to the discovery of a new class of biologically active compounds – vitamins. Many of these scientists were awarded the Nobel Prizes. Thanks to their efforts, almost all currently known vitamins (B1, B2, B6, B9, B12, C, A, E, K) were identified, their structure and the mechanism of biological action were characteri­zed. Many vitamins were found to serve as coenzymes in important biochemical conversions. This article talks about the history of the discovery of the most familiar vitamins and scientists involved in their research. The contribution made by these distinguished scientists to the development of modern­ biochemical science, in particular, vitaminology, cannot be overestimated.

Kinetics of interaction between polyreactive immunoglobulins and antigen. The theory

S. A. Bobrovnik, M. O. Demchenko, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;

Received: 14 February 2019; Accepted: 17 May 2019

Dynamics of association between polyreactive immunoglobulins (PRIGs) and immobilized antigens is considered on the base of our model of PRIGs-antigen interaction, which was suggested by us earlier. This process of PRIGs binding to an immobilized antigen was described  with a system of differential equations. The solution of this system of differential equations gives mathematical expressions that relate the dynamics of the reactant concentrations and time of the reaction. Using Microsoft Excel program the theoretical curves were calculated and plotted that described the dynamics of “active”, “nonactive” PRIGs in solution as well as PRIGs that were bound to an immobilized antigen. Conclusions drawn by us earlier about very high dependen­ce of reaction PRIGs with an antigen from temperature were confirmed.

Consideration of the contribution of chemical (non-enzymatic) conversion of substrate in the general mechanism of enzyme reaction

S. O. Kosterin, S. O. Karakhim, P. F. Zhuk

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;

Received: 13 September 2018; Accepted: 13 December 2018

When enzyme-catalyzed reactions are studied, it is necessary to take into account the contribution of the chemical (non-enzymatic) conversion of the substrate to the product, which is carried out together with the enzyme-catalyzed conversion of the substrate. It is generally believed that the difference of the product concentration that was formed in the presence of the enzyme and in its absence (during the same time interval) is the concentration of the product that was formed directly in the enzyme-catalyzed reaction, i.e. that there is additivity of the product concentrations at each time point. In this paper, we have analyzed when there is additivity and how to correctly take into account the contribution of chemical (non-catalytic) substrate conversion when the enzyme-catalyzed reactions are investigated. We have shown that the additivity of product­ concentrations and initial rates is observed only for a period when the product concentration increases linear­ly with time. The longer the reaction proceeds the more the deviation from the additivity. Under equilibrium condition, there is no additivity of equilibrium product concentrations but under conditions of detailed balance the equilibrium product concentration of the overall reaction, including the enzyme-catalyzed and chemical (non-enzymatic) conversion of the substrate, is also at the same time the equilibrium concentration of the product of the enzyme-catalyzed conversion of the substrate.

Calculations of supramolecular structures of peptidylboronic acid (bortezomib) with ABO blood system antigen

A. D. Kustovska1, S. V. Prymachenko1, Zh. M. Minchenko2,
T. F. Liubarets2, O. O. Dmytrenko2

1National Aviation University, Kyiv, Ukraine;
2SI “National Research Center for Radiation Medicine of National Academy of Medical Sciences of Ukraine”, Kyiv, Ukraine

Received: 08 April 2019; Accepted: 17 May 2019

Peptidylboronic acids have recently become widespread as effective drugs for cancer treatment. Howe­ver, the use of these drugs is often accompanied by negative side effects associated with the phenotypic affiliation to the ABO system. Therefore, it is important to determine the role of pharmaco-chemical charac­teristics of antigens of ABO system and therapeutic agents (for example, bortezomib) in the selection of individualized therapies for patients with chronic lymphoproliferative neoplasms. The expediency and efficiency of bortezomib use for patients with plasma cell myeloma depen­ding on the phenotype affiliation to the ABO system were evaluated. Efficiency of plasma cell myeloma therapy was analyzed in 104 patients who received treatment according to clinical protocols. Dependence of the duration of remission in performance of standard polychemotherapy was researched. Calculation of energy parameters and geomet­ry of probable supramolecular structures of peptidylboronic (bortezomib) and boric acids with antigens of the ABO blood system was performed in the HyperChem 8.07 software package. It was demonstrated that the ability of antigens to form supramolecular complexes with bortezomib varies in the series: B1 >> 01 > A1, which is in line with the results of clinical researches. It was assumed that in case of patients with В blood group secondary process (interaction of bortezomib with carbohydrate antigen) is energetically more beneficial than the main process (inhibition of proteasome), while for patients with O and A groups, equilibrium is shifted toward the main reaction, which results in the therapeutic effect of the drug.

Carassius auratus as a novel model for the hyperglycemia study

H. I. Falfushynska1, O. I. Horyn1, L. L. Gnatyshyna1,2,
B. B. Buyak1, N. I. Rusnak1, O. O. Fedoruk1, O. B. Stoliar1

1Ternopil Volodymyr Hnatiuk National Pedagogical University, Ukraine;
2I. Horbachevsky Ternopil State Medical University, Ukraine

Received: 21 August 2018; Accepted: 13 December 2018

The aim of the present study was to create a suitable model of the glucose toxicity and elucidate the ability of zinc-binding proteins metallothioneins in the crucian carp Carassius auratus to reflect it. For that, fish was loaded by three waterborne concentrations of glucose (low (5.55 mM, LC), middle (55.5 mM, MC) or high (111 mM, HC)) for 21 days. The level of blood glucose, responses of metallothioneins, oxidative stress, DNA instability in the liver, as well as erythrocytes indices, cholinesterase activity in the brain and morphometric variables were evaluated. An increase in blood glucose levels (up to 3–5 times), glycated hemoglobin (HbA1c, only by the HC, by 55%), methemoglobin (by two times), oxyradicals (16-57%) and TBARS levels (up to 57%), frequency of the micronucleated erythrocytes, DNA fragmentation in hepatocytes, body mass and hepatosomatic indices and a decrease in metallothioneins concentration (40-74%), cholinesterase activity (~70%), total hemoglobin (by 18%) and red blood cells count (only after HC-treatment, by 47%) were detected. The lysosomal membrane stability, evaluated by the neutral red retention time, was affected by all studied concentrations of glucose (decreased by 58%). The most prominent changes were observed after the HC of glucose. CART analysis revealed the significant splitting parameters for studied group differentiation including HbA1c, lysosomal membrane stability and lipid peroxidation. We could consider the crucian carp is a useful model organism to perform DM studies and in the future, this fish model can help in mechanistic investigations and testing therapeutic interventions under glycemic states.

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;

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;

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;

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;

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;

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.