Category Archives: Uncategorized
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).
Levels of angiogenic regulators and MMP-2, -9 activities in Martorell ulcer: a case report
O. M. Petrenko1, A. A. Tykhomyrov2
1Bogomolets National Medical University, Kyiv, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine;
e-mail: artem_tykhomyrov@ukr.net
Received: 27 July 2018; Accepted: 13 December 2018
Martorell hypertensive ischemic leg ulcers (HYTILU) represent a unique form of lower extremity non-healing ulcers that develop in association with poorly controlled high blood pressure. The present study was performed in order to assess levels of protein regulators of angiogenesis (vascular endothelial growth factor, or VEGF, and angiostatins) and to evaluate activities of matrix metalloproteinases (MMPs) (gelatinases MMP-2 and -9) in wound cutaneous tissue in the case of patient with 2-years HYTILU history. VEGF and angiostatin levels were analyzed by Western blot, MMP activities were evaluated by gelatin zymography. We report here for the first time that wound tissue in HYTILU is characterized with increased levels of VEGF (by 75 folds vs. histologically normal tissue, P < 0.01) and dramatic overproduction of angiostatin levels, which are undetectable in healthy cutaneous tissue. Approximately 10-fold elevation in MMP-2 and -9 activities is observed in wound tissue as compared with uninjured cutaneous tissue. Obtained results indicate that increased production of angiogenic inhibitors, angiostatins, may counteract VEGF-induced pro-angiogenic signaling, and together with MMP overactivation, contributes to failed healing of ischemic ulcer. Further extended studies are needed to clarify how changes of angiogenic profile and imbalance of proteolytic activities in non-healing Martorell ulcers can be considered during their management procedures to improve efficacy of surgery debridement and/or skin grafting.
Evaluation of biochemical indicators in blood plasma of rats with tetracycline-induced hepatosis and their correction by milk phospholipids
V. A. Gryshchenko1, V. V. Musiychuk1, V. O. Chernyshenko2,
O. V. Gornytska2, T. M. Platonova2
1National University of Life and Environmental Sciences of Ukraine, Kyiv;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
е-mail: viktoriya_004@ukr.net
Received: 13 July 2018; Accepted: 13 December 2018
Tetracycline is a drug with direct cytotoxic action on the liver, and therefore it is widely used in pharmaceutical studies of therapeutic effectiveness of hepatoprotective preparations. The aim of the present work was to determine the biochemical indicators in blood plasma of rats with tetracycline-induced hepatosis and correction properties of milk phospholipids under tetracycline-induced hepatosis in rats. To achieve this, Wistar rats were administered 250 mg/kg of 4% tetracycline hydrochloride suspension once a day intragastrically. As the corrective therapy, 1% solution of BAS “FLP-MD” was administered in liposomal form based on milk phospholipids. Under modeled steatohepatitis, significant destructive changes were observed in the cell membranes of hepatocytes in experimental rats. It was confirmed by higher activity of transaminase (in particular, activity of АSТ increased 4 times, that of ALT 1.7 times and the AST/ALT ratio was increased 2.4 times in blood plasma). The synthesis of clotting factors in livers of animals with hepatosis was inhibited. The content of fibrinogen in blood plasma decreased by 21%, factor II (prothrombin) by 27.8%, Xa-factor by 27.9%, and protein C by 40.6%. The animals also had hypochromic anemia, azotemia and bilirubinemia. The calcium-phosphor metabolism and hyperkalemia were observed. The liposomal BAS “FLP-MD” based on milk phospholipids diminished harmful effects of tetracycline, in particular supporting blood coagulation factors’ level restoration, and also by the activity of transaminases. According to the results, it may be used in prophylactics and pharmaceutical correction of steatohepatitis.
Cellular fatty acid composition of Aeromonas genus – destructor of aromatic xenobiotics
T. V. Gudzenko, O. G. Gorshkova, N. V. Korotaieva,
O. V. Voliuvach, А. М. Ostapchuk, V. O. Іvanytsia
Оdesa I. I. Mechnikov National University, Ukraine;
e-mail: tgudzenko@ukr.net
Received: 17 September 2018; Accepted: 13 December 2018
The aim of this study was a determination of the fatty acid composition of cellular lipids and identification of the strains, isolated from the wastewater of pharmaceutical production, – the destructor of aromatic xenobiotics. The phenotypic characteristics and cellular fatty acid (FA) composition confirmed the strain belonging to the Aeromonas ichthiosmia with the similarity index of library data MIDI Sherlock – 0.564. Analysis of the cellular FA composition of the strain Aeromonas ichthiosmia ONU552 was carried out using the MIDI Sherlock microorganism identification system based on the gas chromatograph Agilent 7890. Chromatographic analysis showed that the fatty acid profile of the strain Aeromonas ichthiosmia ONU552 contains 26 fatty acids with the total number of carbon atoms from 10 to 18. 85.27% of saturated and unsaturated fatty acids had unbranched structure. The total content of unsaturated fatty acids – 16:1 w7c/16:1 w6c, 18:1 w7c, 16:1 w7c alcohol, 17:1 w8c, 17:1 w6c, 16:1 w5c, was 50% of the total fatty acid pool. Less than 1.5% branched fatty acids were predominantly in the iso form: 13:0 iso (0.20%); 15:0 iso (0.97%); 17:1 iso w9c (1.35%), 17:0 iso (1.49%); in the anteiso form, only one acid 17:0 (0.27%) was identified. It was shown that the characteristic of the fatty acid composition of the strain Aeromonas ichthiosmia ONU552 – the destructor of aromatic xenobiotics, was the presence of hydroxyacids 12:0 3OH, 15:0 3OH, 15:0 iso 3OH and dominance of hexadecanoic (16:0) and hexadecenoic (16:1 w7c/16:1 w6c) of fatty acids.
Role of AP-1 transcriptional factor in development of oxidative and nitrosative stress in periodontal tissues during systemic inflammatory response
A. M. Yelins’ka, O. Ye. Akimov, V. O. Kostenko
Ukrainian Medical Stomatological Academy, Poltava, Ukraine;
e-mail: riseofrevan5@gmail.com
Received: 04 August 2018; Accepted: 13 December 2018
Chronic systemic inflammatory response syndrome (SIRS) underlies many diseases (sepsis, atherosclerosis, diabetes mellitus). According to research data of recent years the key role in the development of SIRS is played by the activation of various nuclear transcription factors. The work was aimed at studying the role of such transcription factor as activator protein 1 (AP-1) in the development of oxidative and nitrosative stress in soft periodontal tissues during chronic systemic inflammatory response (SIRS). The experiment was carried out on 24 the Wistar rats. We induced SIRS by bacterial lipopolysaccharide of Salmonella typhi (0.4 μg/kg) intraperitoneal injection. We studied changes in the functioning of the nitric oxide (NO) cycle, the production of superoxide anion radical (O2•-) and the activity of antioxidant enzymes in soft periodontal tissues homogenate. We used SR11302 as an Ap-1 inhibitor (15 mg/kg) for 2 months. We established that during the SIRS modeling, the activity of antioxidant enzymes in soft periodontal tissues decreased with a simultaneous increase in the production of O2•-. SIRS elevated the production of NO by inducible NO-synthase (iNOS) and nitrite reductases. The nonoxidative cleavage of L-arginine under this condition was also increased. The concentration of peroxynitrite (ONOO–) was shown to be elevated more than 2-fold. The inhibition of AP-1 by SR11302 normalized the functional state of the NO cycle, reduced O2•- production and restored the activity of antioxidant enzymes. In this way, under SIRS conditions, “vicious circle” of ONOO– formation is formed. SIRS in soft periodontal tissues poses a threat of oxidative and nitrosative stress development. Usage of AP-1 activation inhibitor SR11302 breaks “vicious circle” of ONOO– formation.
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.