Category Archives: Uncategorized
Kinetic properties of Na(+),K(+)-АТРase of spermatozoa from fertile and infertile men under effect of calix[4]arene C-107
R. V. Fafula, O. I. Meskalo, A. S. Besedina,
Io. A. Nakonechnyi, D. Z. Vorobets, Z. D. Vorobets
Danylo Halytsky Lviv National Medical University, Ukraine;
e-mail: kaf_medicalbiology@meduniv.lviv.ua; roman_fafula@ukr.net
Received: 12 November 2018; Accepted: 14 March 2019
The calix[4]arene C-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxy-calix[4]arene) effects on the kinetic properties of Na+,K+-ATPase in spermatozoa of fertile (normozoospermia) and infertility men (oligozoospermia, and asthenozoospermia) were studied. It was shown that in spermatozoa of healthy men calix[4]arene С-107 inhibited Na+,K+-ATPase activity and decreased the maximum reaction rate of ATP hydrolase reaction without affecting the coefficient of (half-) activation by ATP and Hill coefficient nH. In оligo- and asthenozoospermic samples of spermatozoa almost a 2-fold decrease of cooperativity coefficient nH of ATPase inhibition with calyx[4]aren C-107 was observed. In normozoospermic samples of spermatozoa the KMgCl2 for Na+,K+-ATPase was decreased at calix[4]arene C-107 high concentrations (≥50 nM) in the incubation medium in contrast to oligozoospermic samples of spermatozoa where KMgCl2 was increased only at high calix[4]arene C-107 concentration (100 nM). The increase of the KMgCl2 in the entire range of investigated calix[4]arene concentrations and the decrease of cooperativity coefficient nH of MgCl2 activating effect were detected in asthenozoospermic samples of Na+,K+-ATPase.
Сalix[4]arene chalcone amides effects on myometrium mitochondria
S. G. Shlykov1, A. M. Kushnarova-Vakal1, A. V. Sylenko1,
L. G. Babich1, О. Yu. Chunikhin1, O. A. Yesypenko2,
V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: sshlykov@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv
Received: 19 November 2018; Accepted: 14 March 2019
Mitochondria are a key player in a wide range of the most important functions of the cell. Calixarenes are supramolecular compounds that have been widely used in bioorganic chemistry and biochemistry. The aim of this work was to study the effects of calix[4]arenes with two (С-1012, С-1021), three (С-1023, С-1024) and four (С-1011) chalcone amide groups on the myometrial mitochondria membranes polarization, Ca2+ concentration in the matrix of these organelles ([Ca2+]m ) and on the average hydrodynamic diameter of mitochondria. It was shown that permeabilized myometrium cells incubation with calix[4]arenes containing two or more chalcone amide groups, was accompanied by an increased level of myometrial mitochondria membranes polarization. All studied calix[4]arenes increased [Ca2+]m values in the absence and in the presence of exogenous Ca2+. The values of [Ca2+]m in the absence of exogenous Ca2+ were higher at mitochondria incubation in Mg2+-containing, than in Mg2+,ATP-containing medium. Incubation of isolated mitochondria with the studied calix[4]arenes resulted in changes of mitochondria volume: at incubation with С-1012, С-1021, C-1023 the average hydrodynamic diameter was decreased, while with С-1011 it was increased. Thus, we have shown that a short-term (5 min) incubation of mitochondria in the presence of 10 µM calix[4]arenes, which contain from two to four chalcone amide groups, increased the level of mitochondria membranes polarization, ionized Ca concentration in the matrix and had different effects on the mitochondrial volume.
Adaptive respiratory response of rat pancreatic acinar cells to mitochondrial membrane depolarization
B. O. Manko, O. O. Bilonoha, V. V. Manko
Ivan Franko National University of Lviv, Ukraine;
e-mail: bohdan.manko@lnu.edu.ua
Received: 06 December 2018; Accepted: 14 March 2019
The dependence of uncoupled respiratory capacity of intact pancreatic acini on oxidative substrate supply and functional cell state has not yet been studied in detail. In this study, the respiratory responses of isolated pancreatic acini to FCCP were measured with Clark electrode and mitochondrial membrane potential was assessed with rhodamine123 fluorescence. The response of acini to FCCP was characterized with maximal uncoupled respiration rate, optimal FCCP concentration, respiration acceleration and deceleration. Maximal uncoupled respiration rate substantially increased upon the oxidation of glucose + glutamine (3.03 ± 0.54 r.u.), glucose + glutamine + pyruvate (2.82 ± 0.51 r.u.), glucose + isocitrate (2.71 ± 0.33 r.u.), glucose + malate (2.75 ± 0.38 r.u.), glucose + monomethyl-succinate (2.64 ± 0.42 r.u.) or glucose + dimethyl-α-ketoglutarate (2.36 ± 0.33 r.u.) comparing to glucose alone (1.73–2.02 r.u.) or no substrate (1.76 ± 0.33 r.u.). The optimal FCCP concentration was the highest (1.75 μM) upon glucose + glutamine + pyruvate combination and the lowest (0.5 μM) upon glutamate, combinations of glucose with isocitrate, malate, succinate or α-ketoglutarate. Respiration acceleration after FCCP application was the highest with dimethyl-α-ketoglutarate. Following the peak respiration, time-dependent deceleration was observed. It increased with FCCP concentration and depended on oxidative substrate type. Deceleration was the highest upon malate or isocitrate oxidation but was not observed in case of glutamine or dimethyl-α-ketoglutarate oxidation. Pyruvate alone or in combination with glutamine and glucose significantly decreased the depolarizing effect of FCCP on mitochondrial membrane potential and increased respiration elasticity coefficient with respect to the membrane potential change. Thus, in pancreatic acinar cells, the combination of pyruvate, glutamine and glucose enables the optimal adaptive respiratory response to membrane depolarization.
Kinetics of casein hydrolysis by peptidase from Bacillus thuringiensis var. israelensis
O. V. Sevastyanov1, Yu. A. Shesterenko1, A. A. Ryzhak1,
I. I. Romanovska1, N. A. Dziubliuk2, L. D. Varbanets2
1A. V. Bogatsky Physico-Chemical Institute, National Academy of Sciences of Ukraine, Odesa;
2Danylo Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: romairina@gmail.com
Received: 25 October 2018; Accepted: 14 March 2019
The kinetics of enzyme reaction is generally studied using the Michaelis-Menten equation and various methods of its linearization. Each method has its advantages and drawbacks, so their comparison for determining the kinetics of new enzymes action is topical. The aim of this work was to study the kinetics of casein hydrolysis catalyzed by new peptidase from Bacillus thuringiensis var. israelensis IMB B-7465 using several methods of enzyme activity assessment and Michaelis-Menten equation linearization. The satisfactory agreement between kinetic constants values obtained by the methods of Lineweaver-Burk, Hanes, Eadie-Hofstee, Cornish-Bowden-Eisenthal was established. The Lineweaver-Burk method was shown to be optimal for determining Km and Vmax of casein hydrolysis. Estimation of caseinolytic activity with the use of ortho-phthalic dialdehyde allowed more accurate Vmax determination compared to the use of Anson and Kunitz methods.
Glutathione influence on energy metabolism in rat liver mitochondria under experimental nephropathy
Ye. O. Ferenchuk, I. V. Gerush
Higher State Educational Establishment of Ukraine “Bukovinian State Medical University”, Chernivtsi;
e-mail: yelena_f@ukr.net
Received: 17 October 2018; Accepted: 14 March 2019
Mitochondrial oxidative damage and disorders of energy metabolism contribute to a wide range of pathologies and disease progression. In our work, the effect of glutathione on the activity of respiratory chain enzymes and the content of free SH-groups in rat liver mitochondria was examined with the use of folic acid-induced nephropathy model. Mitochondria were isolated by differential centrifugation, NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and H+-ATPase activity were determined. The activity of these enzymes and the content of the free SH-groups in the liver were shown to be decreased under conditions of nephropathy, evidently due to the intensification of the free radical processes. The introduction of glutathione increased the content of SH-groups and the activity of the Complexes II and V enzymes of mitochondrial respiratory chain but did not change the activity of cytochrome oxidase in mitochondria isolated from the liver of rats under experimental nephropathy. The results obtained demonstrate a positive effect of glutathione on mitochondrial succinate dehydrogenase and H+-ATPase activity normalization in the liver of rats with nephropathy. These findings may help to extend the understanding of mitochondrial energy metabolism under development of kidney diseases.
Phenolic compounds in plants: biogenesis and functions
L. M. Babenko1, O. E. Smirnov2, K. O. Romanenko1,
O. K. Trunova3, I. V. Kosakіvskа1
1M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kyiv;
2Educational and Scientific Center “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv, Ukraine;
3V.I. Vernadsky Institute of General and Inorganic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: lilia.babenko@gmail.com
Received: 05 November 2018; Accepted: 14 March 2019
Phenolic compounds (PCs) in plants play an important role in growth control and have antioxidant, structural, attractant, signaling and protective functions. Information on the discovery, study and identification of phenolic compounds in plants, their role in the complex system of secondary metabolites has been analyzed and summarized. The functions of PCs at the macromolecular, cellular as well as organism and population levels are described. The pathways of PCs formation, enzymes responsible for their synthesis and the plasticity of the synthesis in a plant cell are highlighted. The involvement of PCs in the plant breathing, photosynthesis, oxidation-reduction processes and regulation of the plants physiological state are discussed.
Young scientists conference Modern Aspects of Biochemistry and Biotechnology – 2019
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine
21-22 March, 2019, Kyiv, Ukraine
On March 21-22, 2019 the regular annual young scientists Conference “Modern Aspects of Biochemistry and Biotechnology” was successfully held in Palladin Institute of Biochemistry. The Conference was organized by the Young Scientists Council of Palladin Institute of Biochemistry with the support of ALT Ukraine Ltd –advanced laboratory technologies Company. Young scientists from Kyiv, Dnipro, Kharkiv, Chernivtsi, Ternopil, Poltava took part in the Conference as oral speakers. The scientific program of the Conference included the following sections: Translational Studies; Biochemistry; Biotechnology; Molecular Biology; Medical Biochemistry; Biochemical mechanisms of resistance to adverse environmental conditions. The workshops devoted to computer analysis of biological images; methods of biological experimental data statistical analysis; quantitative polymerase chain reaction for gene expression estimation; spectrofluorometry as a rainbow force for biochemists service were organized to broaden the research skills of young scientists.
Conference was opened by the new section ‘Translational Studies’. So many reports were focused on the efforts to build on basic scientific research to create new therapies, medical procedures, or diagnostics. The members of Scientific Committee specially acknowledged those young scientists who presented data about long way from idea, it’s in vitro approval to the in vivo testing and application.
The honorary awards for the best oral presentation were given to Anna Myronova (“CRISPRa-mediated direct cardiac reprogramming of embryonic rat fibroblasts”), Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv; Yevgen Stohnii (“Epitope determination of novel fibrinogen-specific antibody by limited proteolysis”), Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv; Anton Tkachenko (“Fascin is upregulated in nasal mucosa in chronic rhinosinusitis with nasal polyps”), Kharkiv National Medical University, Kharkiv.
The honourable mention prizes for interesting scientific reports were presented to Vira Borshchovetska (Yuriy Fedkovych Chernivtsi National University, Chernivtsi), Olga Revka and Valerija Zhovannyk (Palladin Institute of Biochemistry, Kyiv), Maxym Skrypnyk (Ukrainian medical stomatological academy, Poltava).
The meeting was held in a creative and friendly atmosphere with constructive and helpful discussions. The abstracts of the oral presentations of participants will be published in the “Ukrainian Biochemical Journal”.
The head of the Conference Competition Commission, D. Sc., prof. Olga Matyshevska
The head of the Young Scientists Council of Palladin Institute of Biochemistry, PhD Tetjana Jatsenko
Blood coagulation parameters in rats with acute radiation syndrome receiving activated carbon as a preventive remedy
V. Chernyshenko1, E. Snezhkova2, M. Mazur2, T. Chernyshenko1,
T. Platonova1, O. Sydorenko2, E. Lugovskoy1, V. Nikolaev2
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: bio.cherv@gmail.com;
2RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv
Received: 13 December 2018; Accepted: 20 March 2019
Radiation-induced coagulopathy (RIC) is one of the major causes of death during acute radiation syndrome (ARS). The aim of this study was to characterize the responses of the hemostasis system to ARS of a moderate level on the 1st and 9th days after irradiation. We aimed to identify molecular markers of the blood coagulation system that are most affected by ARS and to estimate the enterosorption effect on the development of irradiation-induced changes. Platelet aggregation rate, activated partial thromboplastin time (APTT) and fibrinogen concentration were determined by standard methods. Level of protein C (PC) was measured using chromogenic substrate S2366 (p-Glu-Pro-Arg-pNa) and Agkistrodon halys halys snake venom activating enzyme. Functionally inactive forms of prothrombin (FIFPs) were determined using two activators in parallel – thromboplastin or prothrombin activator from Echis multisqumatis venom. Rats of both irradiated groups had a higher risk of intravascular clotting in comparison to both control groups. Statistically significant shortening of clotting time in the APTT test (24 ± 4 s vs. 33 ± 5 s) and increased fibrinogen concentration (4.2 ± 0.6 mg/ml vs. 3.2 ± 0.3 mg/ml) were detected. Both parameters were normalized on the 9th day after irradiation. However the platelet count was decreased (0.3∙106 ± 0.05∙106 1/μl vs. 0.145∙106 ± 0.04∙106 1/μl) due to the impaired megakaryocytic function. The level of PC was decreased after X-ray irradiation (70 ± 10%) and partly restored on the 9th day after irradiation (87 ± 10%). Administration of activated carbon (AC) inhibited the drop in the PC concentration after X-ray irradiation (86 ± 15%) and accelerated its restoration on the 9th day (103 ± 14%). The statistically significant accumulation of FIFPs was detected in blood plasma of irradiated rats at the 1st and 9th days after irradiation. No FIFPs were found in any irradiated rat treated with AC. Characterization of the hemostasis system of rats that were exposed to a semilethal dose of X-rays allowed us to select parameters that can be used for monitoring of ARS development. Apart of from basic coagulation tests (APTT) and the measurement of platelet aggregation, fibrinogen and protein C level we can recommend the determination of FIFPs as a useful tool for estimation of the hemostasis response after irradiation with X-rays. This test indicates the intravascular thrombin generation and can help predict thrombotic complication or disseminated intravascular coagulation. Determination of FIFPs in blood plasma of irradiated rats allowed us to study the enterosorption effect on the development of irradiation-induced changes. It was shown that enterosorption with AC prevented accumulation of FIFPs which appears to be a newly discovered anti-thrombotic effect of therapy with AC. ARS influenced hemostasis by inducing thrombin generation (indicated by FIFPs generation), low-grade inflammation (indicated by PC concentration decrease) and thrombocytopenia. Enterosorption with AC minimizes inflammation and pro-coagulant processes caused by a moderate dose of X-ray irradiation. Accumulation of FIFPs can be assumed to be one of the most sensitive markers of the blood coagulation response to X-ray irradiation.
Interaction of 4 allotropic modifications of carbon nanoparticles with living tissues
S. Ya. Paryzhak1, T. I. Dumych1, S. M. Peshkova1,2,
E. E. Bila2, A. D. Lutsyk1, A. Barras3,
R. Boukherroub3, S. Szunerits3, R. O. Bilyy1
1Danylo Halytsky Lviv National Medical University, Ukraine;
2Ivan Franko Lviv National University, Ukraine;
3Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, France;
e-mail: r.bilyy@gmail.com
Received: 19 January 2019; Accepted: 20 March 2019
Environmental pollution and technological progress lead to carbon nanoparticles that pose a serious health risk. They are present in soot, dust, and printing toner and can also be formed during grinding and cutting. Human neutrophils are able to sequester foreign material by formation of neutrophil extracellular traps (NETs), a process that can cause a strong inflammatory response. In the current work we compared proinflammatory properties of different carbon-based nanostructures: nanodiamonds, graphene oxide, fullerenes C60 and carbon dots. We tested adjuvant properties of carbon nanoparticles in a murine immunization model by investigating humoral (specific IgG and IgM antibodies) and cellular (delayed type hypersensitivity) immune responses. The ability of NETs to sequester nanoparticles was analyzed in a mouse air pouch model and neutrophil activation was verified by in vivo tracking of near-infrared labeled nanodiamonds and ex vivo fluorescent assays using human blood-derived neutrophils. All carbon nanoparticles exhibited proinflammatory adjuvant-like properties by stimulating production of specific IgG but not IgM antibodies (humoral immune response). The adjuvant-like response decreased in this order: from nanodiamonds, graphene oxide, fullerenes C60 to carbon dots. None of the studied carbon nanoparticles triggered a delayed type hypersensitivity reaction (cellular immune response). Nanodiamonds and fullerenes C60 were sequestrated in the body by NETs, as confirmed in the air pouch model and by in vivo fluorescent tracking of near-infrared labeled nanodiamonds.
Apoptosis induction in human leukemia cells by novel 2-amino-5-benzylthiazole derivatives
N. S. Finiuk1,2, I. I. Ivasechko1, O. Yu. Klyuchivska1,
Yu. V. Ostapiuk3, V. P. Hreniukh2, Ya. R. Shalai2,
V. S. Matiychuk3, M. D. Obushak3,
A. M. Babsky2, R. S. Stoika1
1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
2Ivan Franko National University of Lviv, Biology Faculty, Lviv, Ukraine;
3Ivan Franko National University of Lviv, Chemistry Faculty, Lviv, Ukraine;
e-mail: stoika@cellbiol.lviv.ua
Received: 21 December 2018; Accepted: 20 March 2019
Derivatives of 2-amino-5-benzylthiazole are heterocyclic pharmacophores that exhibit different pharmacological activities including anticancer action. The mechanisms of such action of these compounds are not clear. The aim of the present study was to investigate apoptosis induction, particularly DNA damage in human leukemia cells, by the novel synthesized thiazole derivatives ‒ 2,8-dimethyl-7-(3-trifluoromethyl-benzyl)pyrazolo[4,3-e]thiazolo[3,2-a]pyrimidin-4(2H)-one (compound 1) and 7-benzyl-8-methyl-2-propylpyrazolo[4,3-e]thiazolo[3,2-a]pyrimidin-4(2H)-one (compound 2). Western-blot analysis, DNA comet assay in alkaline conditions, diphenylamine DNA fragmentation assay, agarose gel retardation, and methyl green DNA intercalation assays were used to study the effects of the studied compounds in human leukemia cells. These compounds induced PARP1 and caspase 3 cleavage in the leukemia cells, also increased the level of pro-apoptotic Bim protein and the mitochondrion-specific EndoG nuclease, and decreased the level of the anti-apoptotic Bcl-2 protein. They caused DNA single-strand breaks and DNA fragmentation in the leukemia cells without direct DNA binding or DNA intercalation. Thus, novel 2-amino-5-benzylthiazole derivatives may be promising agents for apoptosis induction in the targeted human leukemia cells.