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Diagnostic significance of biochemical indicators of liver fibrogenesis in adolescents with obesity
O. V. Buznytska
Kharkіv Medical Academy of Postgraduate Education,
V. N. Karazin Kharkіv National University, Ukraine
e-mail: ebuznickaa@ukr.net; missbuzelena@gmail.com
Received: 27 September 2018; Accepted: 13 December 2018
Non-alcoholic fatty liver disease occurs in most obese people, the main pathway of which is the process of fibrogenesis. The aim of this work was to determine the potential biomarkers for early diagnosis of liver fibrogenesis in adolescents with obesity. The levels of liver fibrosis markers, such as fibronectin, collagen type IV, N-terminal propeptides and C-terminal telopeptides of type I collagen, were assessed with the use of IFA method in serum of 226 patients with obesity aged 8-18 years. A significant increase in levels of type IV collagen and fibronectin was observed in children with obesity (P < 0.05). As diagnostic criteria for fibrogenesis and fibrolysis, the levels of N-terminal propeptides and C-terminal telopeptides of type I collagen, respectively, were determined. The serum level of N-terminal propeptides of type I collagen significantly exceeds the normal values in all children with obesity compared to the control group (P < 0.05). The biochemical markers (type IV collagen, fibronectin, N-terminal propeptides and C-terminal telopeptides of type I collagen) were proven to have high diagnostic informative value in the early diagnosis of liver fibrogenesis in obese adolescents. It was shown that the signs of fibrosis in non-alcoholic fatty liver disease already occur at the stage of steatosis.
HBD-2 interactions with erythrocyte membranes in vitro
M. V. Makarenko1, D. O. Semeniuk2, I. O. Starenka2, A. P. Pogribna3,
I. V. Sokol4, L. I. Martinova1, D. O. Govsieiev4
1Institute of Postgraduate Education,
Bogomolets National Medical University, Kyiv Ukraine;
2ESC Institute of Biology and Medicine,
Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: dmyt.semenyuk@gmail.com;
3Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv;
4Kyiv City Maternity Hospital No 5
Received: 24 September 2018; Accepted: 13 December 2018
HBD-2 – a member of β-defensin family of antimicrobial peptides – is known to permeabilize cell membranes of susceptible cells, but the mechanism of such interactions is poorly understood. In our study, we have used a hemolytic model to explore the kinetic properties of HBD-2 interactions with membranes of human erythrocytes. We ran hemolytic assays with a wide range of both HBD-2 and erythrocyte concentrations, as well as varying pH values, incubation times, and osmotic strengths; each in the presence or the absence of inhibitory substances such as proteins and salts. The results show that HBD-2 cell membrane permeabilization is both dose- and time-dependent (with plateau effect observed in each case), and inversely dependent on erythrocyte concentration. HBD-2 interactions with cell membranes highly depend on pH value and the presence of inhibitors but are not affected by tested osmotic strength range. Our findings suggest that interactions of HBD-2 with cell membranes are mainly electrostatic in nature and are limited by released cell content. We have developed a speculative model of such interaction based on our results.
Intermittent fasting causes metabolic stress and leucopenia in young mice
O. M. Sorochynska1, M. M. Bayliak1, Y. V. Vasylyk1,
O. V. Kuzniak1, I. Z. Drohomyretska1, A. Ya. Klonovskyi1,
J. M. Storey2, K. B. Storey2, V. I. Lushchak1
1Vasyl Stefanyk Precarpathian National University, Ivano-Frankivsk, Ukraine;
2Institute of Biochemistry, Carleton University, Ottawa, Canada;
e-mail: lushchak@pu.if.ua
Received: 29 October 2018; Accepted: 13 December 2018
Overweight and obesity became the worldwide epidemic resulting from overeating especially when a so-called Western diet rich in carbohydrates and fats is used. It is widely accepted that limitation of food consumption could help to withstand such state of adult organism, but information about younger groups is contradictory. The present study was undertaken to characterize the effects of intermittent fasting, using an every other day (EOD) fasting/feeding protocol, on hematological parameters and biochemical blood plasma indices in young mice from one to two months old. It was shown that intermittently fasted mice were characterized by a reduced body weight, reduced total number of blood leucocytes, lower glucose and lactate levels and higher activity of alanine aminotransferase and aspartate aminotransferase in blood plasma as compared with the age-matched control mice. To gain the same mass EOD animals needed to eat more food than ad libitum fed animals. These differences may probably be explained by a need to expend certain resources to combat stress induced by intermittent fasting. Our data showed that EOD feeding at a young age may negatively influence young mammals.
Influence of sulfite on ATPase activity of coupling factor CF(1) isolated from spinach chloroplasts
O. B. Onoiko, A. P. Khomochkin, O. K. Zolotareva
M. G. Kholodhy Institute of Botany, National Academy of Sciences of Ukraine, Kyiv;
e-mail: membrana@ukr.net
Received: 25 October 2018; Accepted: 13 December 2018
The present study of sulfite participation in the regulation of the chloroplast ATP-synthase complex can help to understand the mechanisms of acid rains toxic effects on the plant photosynthetic apparatus. The aim of the work was to study the effect of sulfite on Ca2+– and Mg2+-dependent ATP hydrolysis catalysed by coupling factor CF1, exposed to preliminary short-term acid treatment. CF1 was isolated from spinach chloroplasts (Spinacia oleracea L.). The latent ATPase activity of CF1 was activated by sodium sulfite addition into incubation media. The rate of ATP hydrolysis was determined by the release of inorganic phosphate. In the presence of 25 mM exogenous sulfite the Mg2+-dependent hydrolytic activity of CF1 was increased 7-fold and Ca2+-dependent activity 3-fold compared to the control. Carbonic anhydrase inhibitors acetazolamide or ethoxyzolamide eliminated sulfite-induced stimulation of ATP hydrolysis. The sulfite stimulating effect on Ca2+– and Mg2+-ATPase activity was increased dramatically after incubation (5 min) of isolated CF1 in a medium with pH 3.5 and its subsequent transition to the alkali medium (pH 8.0). In this case, 1 mM sulfite-induced a 10-fold acceleration of ATP hydrolysis. Carbonic anhydrase specific inhibitors (50 μM) removed the sulfite effect. These data suggest that sulfite was able to replace bicarbonate in the CF1 structure after the release of bound HCO3– during acid treatment.
Spin-orbit coupling effects in O(2) activation by cofactor-independent 2,4-dioxygenase
B. F. Minaev1, R. R. Valiev2
1Bohdan Khmelnytsky National University of Cherkasy, Ukraine;
e-mail: bfmin43@ukr.net;
2Royal Institute of Technology, Stockholm, Sweden;
e-mail: valievrashid@mail.ru
Received: 22 August 2018; Accepted: 13 December 2018
The O2 (dioxygen) is paramagnetic molecule with two non-paired electron spins and triplet ground state (S = 1) while majority of organic molecules are diamagnetic species; they have all electron spins paired and the singlet ground state with the total spin S = 0. Oxygenases catalyze a concerted insertion of the triplet dioxygen into organic (diamagnetic) molecules in a strictly spin-forbidden process and this puzzle is not solved so far in modern enzymology. Many oxidases and oxygenases utilize the π-conjugated organic cofactor (like flavins, pterins) in a singlet ground state and reaction of cofactor with O2 is still spin-forbidden. It is clear that the protein environment in the enzyme active-site “helps” in some way to overcome spin prohibition, but this environment is definitely diamagnetic and the spin-puzzle still exists. Some oxidases and oxygenases use paramagnetic metal ions as a cofactor; in this case the spin prohibition is formally reduced. In recent years, a numbers of oxidative enzymes are discovered which do not contain any cofactor. In the present work, we considered a rather popular cofactor-free bacterial 2,4-dioxygenase and its oxygenolytic reactions with 2-n-alkyl-3-hydroxy-4(1H)-quinolones (AHQ’s). We presented results of quantum-chemical calculations of intermediate diradical proposed recently for direct reaction of dioxygen with AHQ substrate and made conclusion about the mechanism of spin-catalysis.
Different mice inbred strains humoral immune response against human prostate-specific antigen
O. Yu. Galkin1,2, A. G. Komar3, O. B. Besarab1
1National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexfbt@gmail.com;
2Propharma Plant Ltd., Kyiv;
3Ukrainian Medical Center of Certification of Ministry of Health of Ukraine, Kyiv
Received: 21 August 2018; Accepted: 13 December 2018
The aim of the study was an investigation of humoral immune response to prostate-specific antigen (PSA) of different mice inbred strains for further development of recommendations for appropriate immunization schemes for monoclonal antibodies (McAbs) obtaining. The study was conducted using: Balb/c and NZB mice; PSA from human sperm (as immunogen). In case of booster immunization immunogen was previously diluted to the desired concentration (10 or 30 µg per 100 µl) in saline, and injected in the tail vein or intraperitoneally. In case of other immunizations, we prepared emulsion solution of immunogen with adjuvant to final concentration of 10 or 30 µg per 100 µl: PSA was dissolved in saline, the same volume of adjuvant was added, and mixture was thoroughly mixed to form a stable emulsion. When subcutaneous administration, total dose of 100 µl was divided into two equal parts and injected in the hind paw of mice. Intraperitoneal immunization was performed by single administration of 100 µl of emulsion. The level of specific antibodies was determined by titration of blood sera from animals in indirect ELISA. Series of experiments were conducted to determine the level of humoral response of mice of different inbred strains (Balb/c and NZB) for multi-stage immunization with different duration with different amounts of PSA (10 and 30 μg), with different immunoglobulin administration (intraperitoneal and subcutaneously), and with different adjuvants (Freund’s complete and incomplete adjuvants, FCA/FIA). Final booster immunization at a dose of 30 μg was performed either in the same manner as the previous administration of the immunogen, or intravenously in the physiological saline solution. Dependencies of the humoral immune response of Balb/c and NZB mice against PSA on the route of administration of immunogen, the dose and duration of immunization were established. It was shown that intraperitoneal administration provided formation of higher titers of specific antibodies in case of both mice strains. Balb/c mice lines more rapidly responded to PSA, than an NZB mice (for all investigated schemes immunization). It was shown that the most effective immunization scheme was three times intraperitoneal administration with 10 µg of PSA for 8 weeks (the first immunization with FCA, and the rest – with FIA) and booster immunogen intravenous administration in saline solution.
Altered sirtuins 1 and 2 expression in the brain of rats induced by experimental diabetes and the ways of its correction
M. M. Guzyk1, T. M. Tykhonenko1, K. O. Dyakun1,
L. V. Yanitska2, I. B. Pryvrotska3, T. M. Kuchmerovska1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Bogomolets National Medical University, Kyiv, Ukraine;
3I. Horbachevsky Ternopil State Medical University, Ukraine;
e-mail: tkuchmerovska@gmail.com
Received: 03 August 2018; Accepted: 13 December 2018
The molecular pathogenesis of diabetic encephalopathy (DE), one of the serious complications of diabetes mellitus, is complex. In this study, we examined whether expression levels of SIRT1 and SIRT2 were the key for the development of brain dysfunctions and whether PARP-1 inhibitors could affect the expression of these proteins for prevention the development of DE in rats with type 1 diabetes. After 10 weeks of the streptozotocin-induced diabetes mellitus (70 mg/kg), Wistar male rats were treated by i.p. injection with PARP-1 inhibitors, 1.5-isoquinolinediol (ISO) or nicotinamide (NAm) (3 or 100 mg/kg/daily i.p., respectively) for 2 weeks. The rats with blood glucose levels over 19.7 ± 2.1 mmol/l were taken into experiments. Western blots were performed to evaluate effects of PAPR-1 inhibitors on the levels of sirtuins, SIRT1 and SIRT2 expression. Diabetes induced significant reduction of SIRT1 expression and SIRT2 overexpression in brain nuclear extracts of diabetic rats compared to non-diabetic control. In brain, NAm attenuated SIRT2 overexpression in nuclear extracts of diabetic rats and slightly elevated SIRT1 expression, while ISO didn’t affect expression of both sirtuins in diabetic rats. Furthermore, it was observed that in brain of diabetic rats, the ratio of free NAD/NADH couples decreased 3.1-fold compared to non-diabetic control. The administration of ISO increased only slightly the ratio of free NAD/NADH couples in the brain of diabetic rats while NAm increased this parameter 1.7-fold compared to diabetic rats. Therefore, we concluded that alterations in the expression of SIRT1 and SIRT2 in brain cell nuclei of diabetic rats can lead to the development of brain dysfunctions. One of the neuroprotective mechanisms of NAm action can also be realized through inhibition of SIRT2 expression in brain cell nuclei that down-regulate progression of diabetes-induced alterations and can be a therapeutic option for treatment of brain dysfunctions.
Рantoea agglomerans lipopolysaccharides: structure, functional and biological activity
L. D. Varbanets, Т. V. Bulyhina, L. А. Pasichnyk, N. V. Zhytkevich
Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: varbanets_imv@ukr.net
Received: 02 September 2018; Accepted: 13 December 2018
This review analyzed literature data, as well as our own research on lipopolysaccharides (LPS) of gram-negative bacteria, focusing mainly on Pantoea agglomerans, a member of the Enterobacteriaceae family. The unique structures of O-specific polysaccharide chains of LPS from Pantoea agglomerans represented by both linear and branched tetra- and pentasaccharide repeating units were described for the first time. The heterogeneity of the LPS molecule itself and the presence of several LPS in the bacterial cell, which differ in the structure of lipids A, O-specific polysaccharide chains, serological activity, as well as endotoxic properties, such as toxicity and pyrogenicity, were shown. Such heterogeneity represents one of the mechanisms of LPS multifunctionality. Based on the antigenicity of LPS, serotyping of P. agglomerans strains and their assignment to 10 serogroups were carried out for the first time. The high immunomodulatory activity of P. agglomerans LPS suggests the possibility to use their oligosaccharide fragments in the development of conjugated vaccines against diseases caused by gram-negative bacteria.
Nobel Laureates of the early 20th century E. Behring, I. Mechnikov, P. Ehrlich, C. Richet, J. Bordet, K. Landsteiner and their contribution to the development of molecular immunology
V. M. Danilova, R. P. Vynogradova, S. V. Komisarenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: valdan@biochem.kiev.ua
The discoveries of immunologists have often been recognized as the most significant in the field of medicine and physiology, since the immune system is extremely vital for the organism, and the study of the principles of its functioning is of fundamental importance to the prevention (vaccination), diagnosis and therapy of many diseases. This article refers to the scientists of the early twentieth century, who received the most prestigious scientific award – the Nobel Prize in Physiology or Medicine and who built the groundwork for the development of immunology as a science. Thus, in 1901, E. von Behring received the first Nobel Prize “for his work on serum therapy, especially its application against diphtheria, by which he has opened a new road in the domain of medical science and thereby placed in the hands of the physician a victorious weapon against illness and deaths”; in 1908, I. Mechnikov and P. Ehrlich received the Nobel Prize in Physiology or Medicine for the creating of the cellular and humoral theory of immunity; in 1913 – C. Richet – “in recognition of his work on anaphylaxis”; in 1919 – J. Bordet – “for his discoveries relating to immunity (the role of complement, mechanisms of precipitation, agglutination…)”; in 1930 – K. Landsteiner – “for his discovery of human blood groups”. Their works spurred the development of modern molecular immunology – the science of the organization and function of the immune system, as an effective defense barrier in the living organism, which recognize and distinguish between “self” and “non-self”.