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General Physiology and Biophysics

Volume 35, 2016, No. 2


  Ventricular myocyte injury by high-intensity electric field: Effect of pulse duration
Luiza Prado 1), Jair Goulart, Marcelo Zoccoler, Pedro Oliveira

1)Department of Biomedical Engineering, School of Electrical and Computer Engineering, University of Campinas, São Paulo, Brazil.

Although high-intensity electric fields (HEF) application is currently the only effective therapy available to terminate ventricular fibrillation, it may cause injury to cardiac cells. In this study we determined the relation between HEF pulse length and cardiomyocyte lethal injury. We obtained lethality curves by survival analysis, which were used to determine the value of HEF necessary to kill 50% of cells (E50) and plotted a strength-duration (SxD) curve for lethality with 10 different durations: 0.1, 0.2, 0.5, 1, 3, 5, 10, 20, 35 and 70 ms. For the same durations we also obtained an SxD curve for excitation and established an indicator for stimulatory safeness (stimulation safety factor - SSF) as the ratio between the SxD curve for lethality and one for excitation. We found that the lower the pulse duration, the higher the HEF intensity required to cell death. Contrary to expectations, the highest SSF value does not correspond to the lowest pulse duration but to the one of 0.5 ms. As defibrillation threshold has been described as duration-dependent, our results imply that the use of shorter stimulus duration – instead of the one typically used in the clinic (10 ms) – might increase defibrillation safeness.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 121-130.

  Chronic liquid nutrition feeding affects blood pressure, heart and kidney morphology, and serum lipid profile in Wistar rats
Livia Mikuska 1), Michaela Vrabcova, Lubica Horvathova, Jana Osacka, Ivan Varga, Boris Mravec

1)Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic.

We determined the effect of chronic liquid nutrition (Fresubin) intake in different developmental stages on the cardiovascular and renal system of male Wistar rats. Body weight, water intake and blood pressure were periodically measured. Selected serum and urine biochemical parameters reflecting metabolic and homeostatic changes after Fresubin intake were investigated as well. Heart and kidney weight, diameter of cardiomyocytes, diameter and length of cardiomyocyte nuclei, wall thickness of thoracic aorta, the diameter and the area of renal corpuscles and serum and urine biochemical parameters were assessed at the end of experiment. We showed that Fresubin intake differently affects the investigated morphological and biochemical parameters in rats and this effect was dependent on the developmental stage when Fresubin was provided. Importantly, we have shown that Fresubin-induced elevation of blood pressure is a reversible phenomenon and it is independent of weight gain and subsequent development of obesity.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 131-144.

  The cytoprotective effect of isorhamnetin against oxidative stress is mediated by the upregulation of the Nrf2-dependent HO-1 expression in C2C12 myoblasts through scavenging reactive oxygen species and ERK inactivation
Yung Hyun Choi 1)

1)Department of Biochemistry, Dongeui University College of Korean Medicine, 52-57, Yangjeong-ro, Busanjin, Busan 614-052, Republic of Korea.

This study was designed to confirm the protective effects of isorhamnetin against oxidative stress-induced cellular damage. Our results indicated that isorhamnetin inhibited the hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against the intracellular reactive oxygen species (ROS) in mouse-derived C2C12 myoblasts. Isorhamnetin also significantly attenuated H2O2-induced DNA damage and apoptosis, and increased the levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) and its phosphorylation associated with the induction of heme oxygenase-1 (HO-1). However, the protective effects of isorhamnetin on H2O2-induced ROS and growth inhibition were significantly abolished by an HO-1 competitive inhibitor. Moreover, the potential of isorhamnetin to mediate HO-1 induction and protect against H2O2-mediated growth inhibition was abrogated by transient transfection with Nrf2-specific small interfering RNA. Additionally, isorhamnetin induced the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. However, the specific inhibitor of ERK, but not JNK and p38 MAPK, was able to abolish the HO-1 upregulation and the Nrf2 phosphorylation. Collectively, these results demonstrate that isorhamnetin augments the cellular antioxidant defense capacity by activating the Nrf2/HO-1 pathway involving the activation of the ERK pathway, thus protecting the C2C12 cells from H2O2-induced cytotoxicity.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 145-154.

  Sonodynamic therapy induces apoptosis of human leukemia HL-60 cells in the presence of protoporphyrin IX
Xiaomin Su 1), Xiaobing Wang, Kun Zhang, Shuang Yang, Quanhong Liu, Albert Leung, Chuanshan Xu, Pan Wang

1)Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, China.

Sonodynamic therapy (SDT) is expected to be a novel therapeutic strategy for tumor. The protoporphyrin IX disodium salt (PpIX), a photosensitizer, can be activated by ultrasound. The present study aims to investigate apoptosis of HL-60 cells induced by PpIX-mediated SDT. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to examine cell toxicity. Apoptosis was detected using Annexin V-PE/7-amino-actinomycin D (7-AAD) double staining. Detection of apoptotic bodies was examined by Hoechst33342 (HO) staining. Western blotting was used to analyze the protein of caspase-3 and poly ADP-ribose polymerase (PARP). Intracellular reactive oxygen species (ROS) was detected by a flow cytometer after exposures. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound were significantly boosted. In addition, as determined by Annexin V-PE/7-AAD staining, SDT significantly induced HL-60 cell apoptosis, the obvious nuclear condensation was also found with HO staining at 4 hours post-SDT treatment. Furthermore, Western blotting showed visible enhancement of caspase-3 and PARP cleavage in this process. Besides, intracellular ROS production was significantly enhanced after SDT. Our findings demonstrate that PpIX-mediated SDT could induce apoptosis on HL-60 cells, suggesting that apoptosis is an important mechanism of cell death induced by PpIX-mediated SDT.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 155-164.

  β-Adrenergic signaling in rat heart is similarly affected by continuous and intermittent normobaric hypoxia
Klara Hahnova 1), Dita Kasparova, Jitka Zurmanova, Jan Neckar, Frantisek Kolar, Jiri Novotny

1)Department of Physiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

Chronic hypoxia may produce a cardioprotective phenotype characterized by increased resistance to ischemia-reperfusion injury. Nevertheless, the molecular basis of cardioprotective effects of hypoxia is still not quite clear. The present study investigated the consequences of a 3-week adaptation to cardioprotective (CNH, continuous normobaric hypoxia) and nonprotective (INH, intermittent normobaric hypoxia; 23 h/day hypoxia followed by 1 h/day reoxygenation) regimen of hypoxia on β-adrenergic signaling in the rat myocardium. Both regimens of hypoxia lowered body weight and led to marked right ventricular (RV) hypertrophy, which was accompanied by 25% loss of β1-adrenergic receptors (β1-ARs) in the RV. No significant changes were found in β-ARs in left ventricular (LV) preparations from animals adapted to hypoxia. Although adenylyl cyclase (AC) activity stimulated through the G proteins was decreased in the RV and increased in the LV after exposure to hypoxia, there were no significant changes in the expression of the dominant myocardial AC 5/6 isoforms and the stimulatory G proteins. These data suggest that chronic normobaric hypoxia may strongly affect myocardial β-adrenergic signaling but adaptation to cardioprotective and nonprotective regimens of hypoxia does not cause notably diverse changes.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 165-173.

  Duration of hexobarbital-induced sleep and monoamine oxidase activities in rat brain: Focus on the behavioral activity and on the free-radical oxidation
Vadim Tseilikman 1), Denis Kozochkin, Eugenia Manukhina, H Downey, Olga Tseilikman, Maria Misharina, Anna Nikitina, Maria Komelkova, Maxim Lapshin, Marina Kondashevskaya, Svetlana Lazuko, Oxana Kusina, Marat Sahabutdinov

1)South-Ural State Medical University, Chelyabinsk, Russia.

The present study is focused on the relationship between monoamine oxidase (MAO) activity and hepatic content of cytochrome P450 (CYP), which reflects the status of microsomal oxidation. For vital integrative evaluation of hepatic microsomal oxidation in rats, the hexobarbital sleep test was used, and content of CYP was measured in hepatic microsomes. Rats with short hexobarbital sleep time (SHST) had higher content of microsomal CYP than rats with long hexobarbital sleep time (LHST). Whole brain MAO-A and MAO-B activities, serotonin and carbonylated protein levels were higher in SHST than in LHST rats. MAO-A and MAO-B activities were higher in brain cortex of SHST rats; MAO-A activity was higher only in hypothalamus and medulla of LHST. The same brain regions of LHST rats had higher concentrations of carbonylated proteins and lipid peroxidation products than in SHST rats. MAO activity was correlated with microsomal oxidation phenotype. Rats with higher hepatic content of CYP had higher activities of MAO-A and MAO-B in the brain and higher plasma serotonin levels than rats with lower microsomal oxidation. In conclusion, data obtained in this study showed a correlation between MAO activity and microsomal oxidation phenotype.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 175-183.

  Hepatoprotective potential of zingerone against nonalcoholic fatty liver disease in rats fed with fructose-enriched diet
Jeyabarathy Muniandy Narayanan 1), Victor Jesudoss

1)Faculty of Pharmacy, Asia Metropolitan University, Batu 9, Cheras 43200, Selangor, Malaysia.

Overconsumption of fructose increases the risk of nonalcoholic fatty liver disease (NAFLD), obesity and metabolic syndrome. NAFLD is currently one of the most common etiologies of chronic liver disease worldwide. The aim of the present study is to evaluate the hepatoprotective potential of zingerone against fructose-enriched diet-induced rat model of nonalcoholic fatty liver disease. Male albino Wistar rats were used and randomly divided into four groups: group 1, control rats fed with standard pellet; group 2, rats were fed normal pellet with intragastric intubation of zingerone (100 mg/kg/day); group 3, rats were fed fructose enriched diet alone; group 4, rats were fed fructose enriched diet with intragastric intubation of zingerone (100 mg/kg/day). Body weight, abdominal circumference, blood glucose, lipid profile and hepatic function indicators were increased and HDL reduced in group 3 rats. Liver pathology of group 3 showed marked changes which includes micro- and macrovesicular steatosis, marked inflammatory cell infiltration, sinusoidal fibrosis and with a significant increase in the area percentage of the collagen. Administration of zingerone reversed the fructose enriched diet induced changes especially body weight, abdominal circumference, blood glucose, lipid profile, hepatic function indicators and restored pathological alteration of liver. Taken together these data provide new insights into the preventive approach of zingerone against the development of the NAFLD.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 185-194.

  Role of acid sphingomyelinase in the age-dependent dysregulation of sphingolipids turnover in the tissues of rats
Nataliya Babenko 1), Vladimir Garkavenko, Galina Storozhenko, Olga Timofiychuk

1)Department of Physiology of Ontogenesis, Institute of Biology, Kharkov Karazin National University, 4 Svobody pl., Kharkov, 61022, Ukraine.

Old age-associated pathologies usually coincide with altered sphingolipid metabolism. In the present article, the role of acid sphingomyelinase (ASMase) in the age-dependent changes of sphingomyelin (SM) and ceramide contents in the tissues has been investigated by means of ASMase inhibitors, imipramine and zoledronic acid. It has been determined that ceramide content and ceramide/SM ratio increased, while SM level decreased in the heart, liver, blood serum and skeletal muscles of 24-month old rats in contrast to 3-month old animals. Injections of imipramine or zoledronic acid to 24-month old rats resulted in significant downregulation of ASMase in the liver and skeletal and heart muscles. The both inhibitors decreased the ceramide content and ceramide/SM ratio and increased the SM content in all tissues studied, except the heart, of old rats to the levels close to those observed in the young animals. Long-term treatment of rats by inhibitors, which have different mechanisms of action on ASMase, exerts the similar, but not equal effects on enzyme activity and SM turnover. In summary, the data above strongly suggest that the age-dependent up-regulation of ASMase plays an important role in the modulation of ceramide and SM contents in rat tissues and that imipramine and zoledronic acid are useful tools for SM turnover manipulation at old age.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 195-205.

  Comparative analysis of mesenchymal stromal cells from different tissue sources in respect to articular cartilage tissue engineering
Ľuboš Danišovič 1), Martin Boháč, Radoslav Zamborský, Lenka Oravcová, Zuzana Provazníková, Mária Csöbönyeiová, Ivan Varga

1)Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovak Republic.

The main goal of this study was a comparison of biological properties of mesenchymal stromal cells (MSCs) obtained from bone marrow, adipose tissue and umbilical cord with respect to articular cartilage regeneration. MSCs were isolated and expanded in vitro up to the third passage. The kinetics of proliferation was analyzed by cell analyzer CEDEX XS and expression of selected markers was assessed by flow cytometry. The morphology was analyzed by inverted microscope and TEM. Pellet culture system and chondrogenic medium containing TGF-β1 was used to induce chondrogenic differentiation. Chondrogenesis was analyzed by real-time PCR; the expression of collagen type I and type II was compared. MSCs from all sources showed similar kinetics of proliferation and shared expression of CD73, CD90 and CD105; and were negative for CD14, CD20, CD34 and CD45. Observation under inverted microscope and TEM showed similar morphology of all analyzed MSCs. Cells from all sources underwent chondrogenic differentiation – they expressed collagen type II and acid mucopolysaccharides typical for hyaline cartilage. On the basis of obtained results it should be emphasized that MSCs from bone marrow, adipose tissue and umbilical cord share biological properties. They possess the chondrogenic potential and may be utilized in cartilage tissue engineering.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 207-214.

  Myocardial connexin-43 and PKC signalling are involved in adaptation of the heart to irradiation-induced injury: Implication of miR-1 and miR-21
Csilla Viczenczova 1), Barbara Szeiffova Bacova, Tamara Egan Benova, Branislav Kura, Chang Yin, Peter Weismann, Rakesh Kukreja, Jan Slezak, Narcis Tribulova

1)Institute for Heart Research, Slovak Academy of Sciences, Bratislava, Slovak Republic.

Intercellular connexin-43 (Cx43) channels are essential for electrical coupling and direct cardiac cell to cell communication to ensure heart function. Expression of Cx43 is altered due to stressful conditions and also affected by the alterations in extracellular matrix. We aimed to explore the effect of chest irradiation on myocardial expression of Cx43 and miR-1 which regulates GJA1 gene transcription for Cx43. Implication of miR-21 that regulates expression of extracellular matrix proteins and PKC signalling that may affect Cx43-mediated coupling was examined as well. Western blot and real-time PCR analyses revealed that six weeks after the exposure of healthy Wistar rats chest to single irradiation of 25 Gy significant myocardial alterations were observed: 1)/ increase of total Cx43 protein expression and its functional phosphorylated forms; 2) suppressed levels of miR-1; 3) enhanced expression of PKCε which phosphorylates Cx43; 4) increase of miR-21 levels; 5) increase of PKCδ expression. These results suggest that irradiation causes post-transcriptional regulation of myocardial Cx43 expression by miR-1 possibly through miR-21 and PKC signalling. We conclude that single dose of irradiation has the potential to enhance myocardial intercellular communication that might be beneficial for the heart that needs to be investigated in details in further studies.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 215-222.

  Photoactivated hypericin is not genotoxic
Jana Feruszová 1), Petronela Imreová, Kristína Bodnárová, Andrea Ševčovičová, Stanislav Kyzek, Ivan Chalupa, Eliška Gálová, Eva Miadoková

1)Department of Genetics, Faculty of Natural Sciences, Comenius University, Mlynská dolina B1, Ilkovičova 6, 842 15 Bratislava, Slovak Republic.

The study was designed to test the potential photogenotoxicity of hypericin (HYP) at three different levels: primary DNA damages, gene mutations and chromosome aberrations. Primary genetic changes were detected using the comet assay. The potential mutagenic activity of HYP was assessed using the Ames/Salmonella typhimurium assay. Finally, the ability of photoactivated HYP to induce chromosome aberrations was evaluated by the in vitro mammalian chromosome aberration test and compared to that of non-photoactivated HYP. The results have shown that photoactivated HYP can only induce primary DNA damages (single-strand DNA breaks), acting in a dose-response manner. This activity depended both on HYP concentrations and an intensity of the light energy needed for its photoactivation. However, mutagenic effect of photoactivated HYP evaluated in the Ames assay using three bacterial strains S. typhimurium (TA97, TA98 and TA100) was not confirmed. Moreover, photoactivated HYP in the range of concentrations (0.005–0.01 µg/ml) was not found to be clastogenic against HepG2 cells. Our findings from both the Ames assay and the chromosome aberrations test provide evidence that photoactivated HYP is not genotoxic, which might be of great importance mainly in terms of its use in the photodynamic therapy.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 223-230.

  Apoptosis and electroretinogram after intravitreal injection of methotrexate in an experimental rabbit model
Eman Aly 1), Amal Ebrahim

1)1. Research Institute of Ophthalmology, Giza, Egypt.

The aim of this study was to explore the changes in electroretinogram of rabbit retina and apoptosis in methotrexate-induced toxicity. Rabbits were divided into 5 groups. Group I served as control in which saline solutions was injected intravitreally. Methotrexate (800 μg, 1.76 μmol) was injected into the vitreous of both eyes of rabbits groups II, III, IV and V by an insulin injector with a 26 gauge needle under general anesthesia. Retinal function was assessed by electroretinogram (ERG) after 2, 4, 10 days and one month then animals were decapitated. The eyes were enucleated and processed for DNA fragmentation studies by gel electrophoresis to retinae and measurement of caspase-3 activities. The results indicated a significant reduction (p ˂ 0.05) in a- and b-wave, a time-dependent appearance of the typical ladder pattern of internucleosomal fragmentation, a characteristic of apoptosis and increase of relative caspase-3 activity after methotrexate intravitreal injection. Methotrexate lead to apoptosis, increase of caspase-3 and affect retinal function.

General Physiology and Biophysics. Volume 35, 2016, No. 2: 231-236.