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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.

Preparation of highly-concentrated autologous platelet-rich plasma for biomedical use

V. Chernyshenko1, K. Shteinberg2, N. Lugovska1, M. Ryzhykova1,
T. Platonova1, D. Korolova1, E. Lugovskoy1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: bio.cherv@gmail.com;
2‘Dr. Zapolska Clinic’, Kyiv, Ukraine

Received: 21 December 2018; Accepted: 20 March 2019

Cell therapy with platelets is a widely accepted approach for wound healing and tissue regeneration in medicine. However, with most available methods poorly concentrated platelet suspensions (up to 0.3∙106 1/µl) or suspensions of mostly inactivated or lost platelets are obtained. In this study, we aimed to develop a simple and effective method for preparing a suspension of native and resting platelets with over 1∙106 1/µl. Platelet-rich plasma (PRP) was obtained from fresh blood of healthy donors (n = 5) collected using different amounts of heparin as the anticoagulant. Samples of PRP were spun down and re-suspended in auto­logous blood plasma. Count and vitality of platelets in each sample were determined by aggregation study on the Solar AP2110 aggregometer. Platelet shape and cytoplasmic granularity that indicate the nativity of platelets were monitored on the COULTER EPICS XL Flow Cytometer. This study of aggregation of platelets in PRP obtained using various amounts of heparin allowed us to reduce final concentrations to the amount that effectively prevented clotting and did not affect platelet reactivi­ty (5 U/ml). PRP concentrated 5 times with a total concentration of cells of 1∙106 1/µl was able to be activated by adenosine diphosphate (ADP) (aggregation rate 54 ± 7%). The amount of cells with altered shape and granularity in concentrated suspension was not higher than 20%. This finding means that the platelets would still be able to release a number of growth factors and other biologically active compounds after stimulation or injection into tissue during cell therapy. The decrease in heparin concentrations also minimizes haemorrhage in the injection site supporting biomedical use of the suspension. A simple and effective method for preparation of highly-concentrated PRP (1.2∙106 1/µl) for biomedical use was developed. Aggregometry and flow cytometry proved that obtained platelets were resting and able to be activated. Being autologous, the preparation can be widely used for cell therapy without additional precautions.

Targeting of the pro-oxidant-antioxidant balance in vitro and in vivo by 4-thiazolidinone-based chemotherapeutics with anticancer potential

L. Kobylinska1, O. Klyuchivska2, R. Lesyk1, R. Stoika2

1Danylo Halytsky Lviv National Medical University, Ukraine;
2Institute of Cell Biology, Lviv, Ukraine;
e-mail: lesya8@gmail.com

Received: 05 January 2019; Accepted: 20 March 2019

Oxidative stress is one of the main mechanisms by which anticancer chemotherapeutics damage normal tissues and organs. At the same time, it is an important biochemical mechanism of the neoplastic action of such medicines. The aim of the present study was to determine the pro-oxidant-antioxidant balance in vitro and in vivo under the influence of novel 4-thiazolidinone-based chemotherapeutics with anticancer potential. An advantage of using these compounds in vivo is their low general toxicity, compared to doxorubicin (Kobylinska L. et al., 2014, 2015, 2016). The 4-thiazolidinone derivatives (Les-3288, Les-3833, Les-3882) with previously established anti-neoplastic activity in vitro (Kobylinska L. et al., 2016) and antitumor effect in vivo (Kobylinska L. et al., 2018) were synthesized, dissolved in dimethyl sulfoxide, and administered intraperitoneally to Wistar rats daily for 20 days. Doses of the injected drugs equaled 10% of the LD50, namely doxorubicin – 5.5 mg/kg, Les-3882 and Les-3833 – 10.7 mg/kg, and Les-3288 – 24.3 mg/kg. The radical scavenging of 1,1-diphenyl-2-picrylhydrazil (DPPH) activity was measured. Concentrations of thio-barbituric acid-active products were assessed in blood serum, liver, heart and kidney tissues of treated rats. Additionally, the activities of superoxide dismutase, catalase and glutathione peroxidase were measured in blood serum and these tissues. We found that administration for 20 days of Les-3288, Les-3833 and Les-3882 compounds disturbed the pro-oxidant-antioxidant balance in the treated rats. Increased amounts of products of reactions of lipid peroxidation and exhaustion of the enzymatic antioxidant system in liver, heart and kidney tissues were detected. In general, Les-3288, Les-3833 and Les-3882 compounds exhibited less pro-oxidant action, compared with the effect of doxorubicin. According to the results of influencing the pro-oxidant-antioxidant balance in the selected tissues, the studied compounds can be ranked in the following order: doxorubicin >> Les-3833 > Les-3288 >> Les-3882. The results of measuring direct scavenging ability of these compounds observed in 24 h suggests their lower toxic effect compared with the effect of the doxorubicin. The obtained results are in correspondence with the results of our recent experiments demonstrating their antineoplastic effect in vitro (Kobylinska L. et al., 2016) and anticancer action in vivo (Kobylinska L. et al., 2018), as well as their lower general toxicity in vivo compared with doxorubicin (Kobylinska L. et al., 2014, 2015, 2016).

The Sisyphus Effect

Sandor G. Vari

International Research and Innovation in Medicine Program,
Cedars – Sinai Medical Center, Los Angeles, CA 90048-5502, USA;
e-mail: Sandor.Vari@cshs.org

Scientific work for researchers, women and men equally, is an endless challenge. Determining new markers for early diagnosis of diseases, investigating the maintenance of cell and tissue homeostasis, studying metabolic regulation and dysregulation as well as intra-organ and interorgan crosstalk can be tiresome work and can sometimes seem like the punishment Zeus gave to Sisyphus. Sisyphus, the Greek mythological figure, was condemned to roll a boulder up a mountain but near the top of the mountain the boulder roll down; for eternity Sisyphus was forced to repeat this task over and over. Although scientific research can sometimes seem like this, the cleverness of scientists helps to overcome the “Sisyphus Effect”. “I leave Sisyphus at the foot of the mountain. One always finds one’s burden again. But Sisyphus teaches the higher fidelity that negates the gods and raises rocks” [1]. Sisyphus is a symbol of self-overcoming.  Sisyphus was also infamous for his deception though, which had led Zeus to punish him to roll the boulder up a hill that rolled down repeatedly, and Sisyphus had to keep trying again and again for eternity [2].
While the vast majority of scientists are honest and work hard to achieve their research goals, some scientists are egotistical and may resort to trickery and doing things unethically. In Science the role of Sisyphus is played by the community of researchers, and to ensure that the boulder does not roll down the hill again. We researchers have to share our research results as well as repeat the published methods and research protocols again and again and these tasks can seem like “Sisyphean” work. Furthermore, in Science, the research community also plays the role of Zeus and will punish dishonesty so therefore we researchers must roll a boulder up a hill again and again and after we have proved that the published scientific work is sound, the “boulder” and the scien­tist will stay on top of the hill.
In the Association for Regional Cooperation in the Fields of Health, Science and Technology (RECOOP­ HST Association) scientists equally share the burden of Zeus and Sisyphus independently of the status of their X and Y chromosomes, and I know from Albert Camus’ essay that Sisyphus is Happy. Each atom of that stone, each mineral flake of that night-filled mountain, in itself, forms a world. The struggle itself toward the heights is enough to fill a man’s heart. One must imagine Sisyphus happy [1].

The contribution of the Nobel Prize laureates to the development of dynamic biochemistry and bioenergetics. E. Buchner, A. Kossel, R. Willstätter, O. Meyerhof, A. Hill, O. Warburg, A. Szent-Györgyi

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

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiyv;
e-mail: valdan@biochem.kiev.ua

Received: 29 November 2018; Accepted: 13 December 2018

Thanks to the great discoveries of the Nobel laureates of the first half of the 20th century – E. Buchner, A. Kossel, R. Willstätter, O. Meyerhof, A. Hill, O. Warburg, A. Szent-Györgyi, we have gained a deep understanding of the mechanisms of organic­ substances conversion and oxidation in living­ organisms­. This article gives an analysis of the research activity of these distinguished scientists, who, through decoding the main ways of conver­ting carbohydrates and energy in living organisms, laid the foundations of dynamic biochemistry and bioener­getics (one of the branches of biochemical science).