Category Archives: Uncategorized

Specificity and sensitivity of the new test for serological evaluation of tuberculosis using MPT83-MPT63 fusion antigen

A. A. Siromolot1,2, T. O. Chudina2, I. S. Danilova3,
O. M. Rekalova4, D. V. Kolybo1,2, S. V. Komisarenko2

1ESC Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
3NSC Institute of Experimental and Clinical Veterinary Medicine, Kharkiv, Ukraine;
4SI National Institute of Phthisiology and Pulmonology named after F. G. Yanovsky National Academy of Medical Sciences of Ukraine, Kyiv;
e-mail: saa0205@ukr.net

The aim of this work was to characterize the experimental test-system based on MPT83-MPT63 fusion antigen with reference commercial diagnostics and investigate the basic characteristics of enzyme-linked immunosorbent assay (ELISA) tests such as specificity and sensitivity. In addition, we investigated the correlation of biochemical and immunological parameters of blood samples of patients with tuberculosis, which was correctly diagnosed by those test-systems. It was shown that the developed test-system match the existing ones regarding the criteria of reliability and the basic requirements of ELISA kits. Recommendations for the testing of serum samples and indications for the use of the proposed serological diagnostic methods has been suggested.

Plasminogen modulates formation of reactive oxygen species in human platelets

A. A. Tykhomyrov, D. D. Zhernosekov, M. M. Guzyk, V. V. Korsa, T. V. Grinenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: artem_tykhomyrov@ukr.net

Reactive oxygen species (ROS) are considered to be important signalling molecules controlling many platelet functions. ROS production has been shown to be augmented by platelet activation, however, plasminogen (Pg) has not been studied in the context of modulating intraplatelet ROS levels. The aim of this study was to investigate the ability of different Pg forms to affect platelet metabolic activity/survival and intracellular ROS production in resting and activated platelets. Platelets isolated from donor plasma were pre-treated with Glu- or Lys-Pg (1.2 µM) and activated by thrombin (1.0 NIH unit/ml) or collagen (1.25 mg/ml). MTT assay was adapted to estimate total mitochondrial dehydrogenase activity, while intracellular ROS levels were monitored with the use of H2DCF-DA probe by flow cytometry. Lys-Pg was shown to slightly, but significantly, mitigate MTT reduction (P < 0.05 vs. control platelets). Two-fold elevation in metabolic activity of platelets stimulated by thrombin as compared to untreated cells was observed. However, this activation was less exhibi­ted in the case of platelets pre-incubated with either Glu- of Lys-Pg, with a predominant effect of Lys-Pg. Unlike thrombin, collagen treatment dramatically suppressed metabolic activity of platelets by 60% compared to control (P < 0.05). Glu- or Lys-Pg pre-incubation had no effects on the activity of collagen-stimulated platelets. Two subpopulations of platelets were observed with distinct characteristics of intracellular ROS formation. Elevated ROS production was demonstrated in these populations of both thrombin- and collagen-treated platelets. Pg (Lys-form to greater extent) enhanced intracellular ROS generation in thrombin-stimulated platelets. These findings suggest that augmented ROS generation within platelets pre-treated with Pg followed by their stimulation may result in down-regulation of their survival and functional activity. This study adds to our understanding one more possible mechanism of Pg impact on the platelet function.

Haemostasis modulation by calix[4]arene methylenebisphosphonic acid C-145 and its sulfur-containing analogue

V. O. Chernyshenko1, O. V. Savchuk1, S. O. Cherenok2,
O. M. Silenko2, A. O. Negelia3, L. O. Kasatkina1, L. V. Pirogova1,
V. A. Didkivskyi1, O. I. Yusova1, V. I. Kalchenko2, L. V. Garmanchuk3,
T. V. Grinenko1, E. V. Lugovskoy1, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
3ESC Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: bio.cherv@gmail.com

C-145 (octasodium salt of calix[4]arene-tetra-methylenebisphosphonic acid) was previously considered as specific anti-сoagulant agent that affects fibrin polymerization and does not notably influence other parameters of coagulation system. C-145S (octasodium salt of thiacalix[4]arene-tetra-methylenebisphosphonic acid) possessing wider hydrophobic hole was expected to be more effective antithrombotic agent than C-145. The aim of present work was to compare the action of both organic compounds on fibrin polymerization, fibrinolysis, platelets and endothelial cells. The change of turbidity during fibrin clot formation induced by APTT-reagent and digestion induced by tPA was estimated. Turbidity study was used for the estimation of polymeric fibrin hydrolysis by plasmin in the presence of thiacalix[4]arene C-145S and calix[4]arene C-145. Effects of thiacalix[4]arene C-145S and calix[4]arene C-145 on the activation of Glu-plasminogen by streptokinase were studied using chromogenic substrate S2251. Platelet aggregation study was performed using aggregometry. Stimulated Ca2+ efflux from endoplasmic reticulum and cytoplasm were determined using specific Ca2+-sensitive probes targeted to endoplasmic reticulum (Mag-Fluo-4) and cytoplasm (FURA-2) by spectrofluorimetry. Both C-145 and C-145S decreased the final turbidity of clot and prolonged clot lysis time in blood plasma in comparison to control value. C-145 was shown to be the more effective fibrinolysis inhibitor when studied in model system of polymerized fibrin desAB. C-145S but not C-145 induced concentration changes of Ca2+ in cytoplasm of resting platelets and significantly inhibited (up to 30%) Ca2+ efflux from endoplasmic reticulum of platelets activated by ADP. Both C-145 and C-145S stimulated the proliferation of endothelial cells of PAE cell line. The effect of C-145S was more prominent. In conclusion, calix[4]arene C 145S proved to be the more potent inhibitor of fibrin polymerization in comparison to C-145, which suggested earlier as anticoagulant agent. C-145S proved to have much more outlined inhibitory action on Ca2+-signaling in platelets and stimulatory effect on endothelial cells proliferation. Thus C-145 remained the most prospective molecular platform for the development of antithrombotic agent.

Phenylalanine ammonia-lyase activity and content of flavonoid compounds in wheat seedlings at the action of hypothermia and hydrogen sulfide donor

Yu. E. Kolupaev1,2, E. I. Horielova1, T. O. Yastreb1, Yu. V. Popov3, N. I. Ryabchun3

1Dokuchaev Kharkiv National Agrarian University, Ukraine;
e-mail: plant_biology@ukr.net;
2Karazin Kharkiv National University, Ukraine;
3Yuryev Рlant Production Institute, National Academy of Agrarian Sciences of Ukraine, Kharkiv

At present hydrogen sulfide (H2S) is considered as one of the signal mediators in plant cells. However, its role in formation of plant resistance to low temperatures and, in particular, in regulation of secondary metabolism under stress conditions remains poorly understood. The influence of H2S donor sodium hydrosulfide (NaHS) on phenylalanine ammonia-lyase (PAL) activity and content of flavonoids in wheat seedlings at normal temperature (21 °C) and under cold hardening conditions (7 days at 3 °C) was studied. After 2 days of the hardening temperature, a transient increase in PAL activity was noted. Also, activity of the enzyme was increased by treatment of plants with 0.1 or 0.5 mM NaHS under normal temperature conditions and especially at the background of cold hardening. By themselves, the cold hardening and the action of H2S donor caused an increase in total content of flavonoids and amount of anthocyanins. With the combination of hypothermia and treatment of seedlings with NaHS, this effect enlarged and the total content of flavonoids increased by 3.8, and anthocyanins increased by 1.8 times in comparison to the control. Treatment with the H2S donor caused a decrease in content of the lipid peroxidation product malonic dialdehyde in seedlings after the action of hardening temperature, and especially after their freezing at –5 °C. Also, under the influence of NaHS, survival of hardened and unhardened seedlings after cryostress increased. It was concluded that one of the mechanisms of the positive influence of the H2S donor on resistance of wheat seedlings to hypothermia is the PAL-dependent accumulation of flavonoid compounds, which have a high antioxidant activity, and a decrease in effects of secondary oxidative stress.

The role of gene GJB2 and connexin 26 in hearing impairment

Asmaa Missoum

Department of Biological and Environmental Sciences, Qatar University, Doha, Qatar;
e-mail: amissoum@live.com

Gap Junction Beta 2 (GJB2) gene mutations are the leading causes of hereditary hearing impairment. This gene encodes various gap junction proteins such as connexin 26 (Cx26), which facilitate K+ homeostasis inside the cochlea in the inner ear. It is as well identified in non-syndromic deafness, which is not accompanied with other abnormalities in the body and contributes to 75% of the cases. The protein connexin 26 is composed of four transmembrane helices and two extracellular loops, in which each has three specific, highly preserved, cysteine residues held by intramolecular disulfide bridges. Moreover, 35delG and Cys169Tyr are the most common mutations of GJB2, where the former results in a shortened Cx26 protein due to the termination of coding sequence, and the latter leads to a destabilized protein structure as one of the three cysteine residuals that are affected. This short review gives further insights on how these two types of mutations lead to hearing loss.

The Nobel Prize in Chemistry 2018

https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2018/

The Nobel Prize in Physiology or Medicine 2018

https://www.nobelprize.org/nobel_prizes/medicine/laureates/2018/

The FEBS3+ Meeting – XIth Parnas Conference – Young Scientists Forum ‘Biochemistry and Molecular Biology for Innovative Medicine’

(September 3-5, 2018, Kyiv, Ukraine)

Founder of molecular immunology in Ukraine, well-known political and public figure

On the 75th birthday of Academician of the NAS of Ukraine S. V. Komisarenko

S. O. Kosterin, V. M. Danilova

July 9 marks the 75th birthday of Serhiy Vasylyovych Komisarenko – the well-known scientist-biochemist, founder of the scientific school in molecular immunology, diplomat, state and public figure, laureate of the State Prize of Ukraine in the Field of Science and Technology (1979), Honored Science and Technology Worker of Ukraine (2008), Winner of the O.V. Palladin Prize (2003) and I.I. Mechnikov Prize (2012) of the NAS of Ukraine, Academician-Secretary of the Division of Biochemistry, Physiology and Molecular Biology of the NAS of Ukraine, director of the O.V. Palladin Institute of Biochemistry of the NAS of Ukraine, Doctor of sciences (biology), Professor, Member of the NAS and NAMS of Ukraine.

Purification procedure and assay for the activity of lysyl oxidase

O. O. Gudkova, N. V. Latyshko, O. V. Zaitseva, S. G. Shandrenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: gudkovahelga@gmail.com

The goal of the present study was to extract and purify lysyl oxidase from rodent’s tissues by a fast, simple, effective and inexpensive method and to develop a sensitive, time-saving lysyl oxidase specific activity assay for routine in vitro experiments. Lysyl oxidase was purified by elaborated purification procedure which relies on negative adsorption principle, that is, an effective decrease in the concentration of ballast components by the polar hydrophilic adsorbent and increasing the concentration of the protein of interest. Peroxide-coupled lysyl oxidase activity quantification methods based on luminol chemiluminescence in the presence of horseradish peroxidase as a catalyst and fluorescent detection using folic acid and Cu(II)  with 1,5-diaminopentane as the substrate, were designed. Lysyl oxidase was partially purified from urea extracts of rodent’s tissues. Used purification procedure ensures the fast release of 93% of ballast proteins as shown by polyacrylamide gel electrophoresis. Lysyl oxidase specific activity after purification was 10-22-fold higher than that of the original extract. The molecular mass of murine lysyl oxidase from lung and heart was estimated to be ~32 kDa. We elaborated two sensitive methods for lysyl oxidase activity quantification and fast inexpensive procedure for partial enzyme purification useful in bulky in vitro experiments.