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The effects of PDK4 inhibition on AMPK protein levels and PGC-1α gene expression following endurance training in skeletal muscle of Wistar rats
S. Aminizadeh1, Y. Masoumi-Ardakani1, B. Shahouzehi2
1Physiology Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Iran;
e-mail: soheilaminizadeh@gmail.com; ymab125@yahoo.com;
2Cardiovascular Research Center, Institute of Basic and Clinical Physiology Sciences, Kerman University of Medical Sciences, Iran;
e-mail: bshahouzehi@gmail.com
There are regulatory networks in cells which surveil the physiological and environmental states. These cellular regulations are conducted through gene expression modulation. Skeletal muscle is able to adapt shortly and produce ATP at different conditions. AMPK (AMP-activated protein kinase) and PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1alpha) are important regulators of cellular energy homeostasis. We designed this study to examine the effects of interactions between endurance training and PDK4 (pyruvate dehydrogenase kinase 4) inhibition on AMPK and PGC-1α expression in rat skeletal muscle. Thirty-two male Wistar rats were randomly selected and divided into 4 groups (n = 8); Group 1 control which did not receive any treatment, Group 2 received dichloroacetic acid (DCA) (150 mg/kg/day), Group 3 (endurance training group), Group 4 which received DCA and performed endurance training. AMPK protein expression, PDK4 and PGC-1α gene expression were measured by western blotting and real-time PCR, respectively. Our data showed that PDK4 inhibition caused AMPK protein elevation. Endurance training (group 2) and PDK4 inhibition (group 4) induce significant enhancement of PGC-1α gene expression compared to control group. The group which received DCA showed significant elevation of PDK4 gene expression compared to control group (P = 0.001), also other two groups (groups 2 & 3) showed significant elevation of PDK4 gene expression compared to control (P = 0.006). It seems that the combination of endurance training and PDK4 inhibition by up-regulation of PGC-1α expression, effectively improves energy state and performance in skeletal muscle.
Disturbance of the transmembrane phosphatidylserine asymmetry in hepatocytes as an apoptosis marker under the action of xenobiotics on rats
O. A. Nakonechna, L. A. Babijchuk, A. I. Bezrodna
Kharkiv National Medical University, Kharkiv, Ukraine;
e-mail: bezrodnaya.ai@gmail.com
It has been reported that unfavorable chemical environmental factors affect the functional state of liver, activate free radical processes against the background of the reduced antioxidant activity, change physico-chemical properties and membrane phospholipid composition of hepatocytes. The aim of our research was to estimate phosphatidylserine distribution in the phospholipid bilayer of hepatocyte membranes and apoptosis stages in hepatocytes of rats under the influence of surfactants: ethyleneglycol (EG), polyethyleneglycol 400, (PEG-400) and polypropyleneglycol (PPG) at a dose of 1/10 DL50. It was found in the subacute toxicological experiment on rats that the investigated xenobiotics EG, PEG-400 and PPG at a dose of 1/10 DL50 caused phosphatidylserine translocation to the outer membrane in the phospholipid bilayer of hepatocytes. This is a specific signal for macrophages aiming at recognition and elimination of apoptotic cells. Analysis of cell death modes under the influence of the investigated xenobiotics at a dose of 1/10 DL50 revealed that the intake of xenobiotics was associated with an increase in the amount of apoptotic/necrotic hepatocytes.
Influence of C(60) fullerene on the ischemia-reperfusion injury in the skeletal muscle of rat limb: mechanokinetic and biochemical analysis
D. O. Zavodovskyi1, S. Yu. Zay2, T. Yu. Matvienko1, Yu. I. Prylutskyy1,
N. Y. Nurishchenko1, S. S. Paradizova3, L. L. Bezuh3, U. Ritter4, P. Scharff4
1ESC Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Ukraine;
2Lesya Ukrainka Eastern European National University, Lutsk, Ukraine;
3SI The Territorial Medical Association of the Ministry of Internal Affairs of Ukraine in Kyiv;
4Institute of Chemistry and Biotechnology, Technical University of Ilmenau, Ilmenau, Germany;
e-mail: Lab@univ.kiev.ua
Influence of the pristine C60 fullerene aqueous colloidal solution (C60FAS) on the ischemia-reperfusion injury in the skeletal muscle of rat limb was studied. Skeletal muscle damage effects were induced by 3 h lasting vascular ischemia. The impact of C60FAS was studied after its intramuscular injection immediately after 1 h of reperfusion at different doses, namely: 1, 2 and 3 mg/kg of body weight. Changes in the mechanokinetic parameters of ischemic skeletal muscle contraction at different modes of functioning and biochemical parameters of blood were used as markers of ischemic injury, and analyzed in detail under action of C60FAS. The obtained results indicate on great promise of use of C60FAS to reduce the consequences of ischemic muscle trauma.
Immunogenicity assay of KatG protein from Mycobacterium tuberculosis in mice: preliminary screening of TB vaccine
P. Purkan1, R. Budiyanto1, R. Akbar1, S. P. A. Wahyuningsih2, W. Retnowati3
1Biochemistry Division, Chemistry Department, Faculty of Sciences and Technology, Airlangga University, Campus C, Jl. Mulyorejo-Surabaya, Indonesia;
2Biology Department, Faculty of Sciences and Technology, Airlangga University, Campus C, Jl. Mulyorejo-Surabaya, Indonesia;
3Microbiology Department, Faculty of Medicine, Airlangga University, Campus C, Jl. Moestopo-Surabaya, Indonesia;
e-mail: purkan@fst.unair.ac.id
The tuberculosis (TB) disease is still widely found even though BCG vaccine given to many people. Ineffectiveness of the BCG vaccine is one of causes that make the difficulties in preventing TB transmission. Objective of the research was to determine the immunogenicity of KatG protein of M. tuberculosis clinical isolate L19 in mice. The KatG protein as antigen was prepared by expression of the katG gene of M. tuberculosis clinical isolate L19 in Escherichia coli BL21 using pColdII-DNA vector. After purification by affinity chromatography, the KatG was vaccinated to mice to detect its immunogenicity. The expression of katG in E. coli BL21 could result in KatG protein with molecular weight 80 kDa in sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The pure KatG protein could significantly stimulate the immune response of mice by triggering the antibodies production of IgG1, IgG2a, IgG2b, IgG2c, IgG3, and IgM. The highest antibody level was obtained when the mice were vaccinated by KatG L19 with the dose of 45 μg/ml. Of the antibodies, the IgG2c isotype was dominantly produced in the blood serum. The KatG protein exhibited a high immunogenicity in mice, so it is possible to develop as a vaccine candidate for TB. A clinical test should be performed in a future to ensure its safety as a therapeutic protein.
Effects of alpha-ketoglutarate on lifespan and functional aging of Drosophila melanogaster flies
M. P. Lylyk1, M. M. Bayliak1, H. V. Shmihel1,
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; bayliak@ukr.net
The effects of an alpha-ketoglutarate-supplemented diet on lifespan and functional senescence were evaluated in the Canton S strain of Drosophila melanogaster. The results suggest that effects of dietary alpha-ketoglutarate (AKG) are dose- and gender-dependent. In males, diets containing 1-10 mM AKG did not affect mean and maximum lifespans, except that an increased maximum lifespan observed at 10 mM AKG. Diet with 20 mM AKG shortened median lifespan and had no effect on maximum lifespan of males. In females, diets with low concentrations of AKG (1 and 5 mM) did not affect lifespan, whereas diets supplemented with 10 and 20 mM AKG increased both median and maximum lifespans. At a lifespan-prolonging concentration (10 mM), AKG decreased fecundity, increased cold resistance and had no effect on climbing activity or resistance to oxidative stress in flies of either gender at middle (24 days) and old (40 days) ages. Moreover, middle-aged AKG-fed females but not males were more resistant to heat stress that was accompanied by higher levels of HSP90 protein as compared with controls. Middle-aged flies on AKG-supplemented diets showed elevated oxidative stress and had higher total protein and triacylglycerol levels as compared with controls. Hence, anti-aging effects of AKG do not seem to be related to preventing oxidative stress development but involve metabolic rearrangement and synthesis of specific protective proteins, which aid to resist destructive processes with age.
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.