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General Physiology and Biophysics

Volume 31, 2012, No. 1


  Biologic effects of SMF and paclitaxel on K562 human leukemia cells
Run-Guang Sun 1), Wen-Fang Chen, Hao Qi, Kun Zhang, Ting Bu, Ying Liu, Shu-Rui Wang

1)College of Physics and Information Technology, Shaanxi Normal University, Xi’an, Shaanxi 710062, P. R. China.

In this study, we evaluated the ability of 8.8 mT static magnetic fields (SMF) to enhance the in vitro action of a chemotherapeutic agent, paclitaxel, against K562 human leukemia cells. We analyzed the cell proliferation, cell cycle distribution, DNA damage and alteration of cell surface and cell organelle ultrastructure after K562 cells were exposed to paclitaxel in the presence or absence of 8.8 mT SMF. The results showed that in the presence of SMF, the efficient concentration of paclitaxel on K562 cells was decreased from 50 to 10 ng/ml. Cell cycle analysis indicated that K562 cells treated with SMF plus paclitaxel were arrested at the G2 phase, which was mainly induced by paclitaxel. Through comet assay, we found that the cell cycle arrest effect of paclitaxel with or without SMF on K562 cells was correlated with DNA damage. The results of atomic force microscopy and transmission electron microscopy observation showed that the cell ultrastructure was altered in the group treated with the combination of SMF and paclitaxel, holes and protuberances were observed, and vacuoles in cytoplasm were augmented. Our data indicated that the potency of the combination of SMF and paclitaxel was greater than that of SMF or paclitaxel alone on K562 cells, and these effects were correlated with DNA damage induced by SMF and paclitaxel. Therefore, the alteration of cell membrane permeability may be one important mechanism underlying the effects of SMF and paclitaxel on K562 cells.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 1-10.

  Changes in cellular response to the damage induced in PC-3 prostate cancer cells by proton microbeam irradiation
Ewelina Lipiec 1), Anna Wiecheć, Joanna Dulińska-Litewka, Małgorzata Kubica, Janusz Lekki, Zbigniew Stachura, Joanna Wiltowska-Zuber, Wojciech Kwiatek

1)The Henryk Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland.

The aim of this research was to find out whether the passage number effect may influence on the PC-3 cells (the human prostate cancer line derived from bone metastases) response to proton radiation. 2 MeV horizontally focused proton microbeam was used as a radiation source. The cells were treated with a counted number of H+ ions (50–8000) corresponding to doses of 1.3–209 Gy/cell. For comparison, cell death was also induced by UVC radiation. All cells were stained with Hoechst 33342 and propidium iodide and visualized under a fluorescence microscope. Necrosis was observed at: a) 8000 protons per cell (corresponding to ∼209 Gy/cell) after 2–4 passages, b) 3200 protons per cell (corresponding to ∼84 Gy/cell) for cells after 11–14 passages and c) only 800 protons per cell (corresponding to ∼2 Gy/cell ) after 47–50 passages. Apoptosis was efficiently induced, by protons, only in cells after 50 passages. The results showed that the laboratory conditions affected cellular response of PC-3 cell line to the proton irradiation. The cellular response to the radiation treatment strongly depends on number of passages.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 11-18.

  Electroporation-induced changes in normal immature rat myoblasts (H9C2)
Iwona Kaminska 1), Malgorzata Kotulska, Anna Stecka, Jolanta Saczko, Malgorzata Drag-Zalesinska, Teresa Wysocka, Anna Choromanska, Nina Skolucka, Rafał Nowicki, Jakub Marczak, Julita Kulbacka

1)Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-367 Wroclaw, Poland.

Application of a high electric field causes an electric shock to the heart. This is utilized in defibrillation to reestablish normal contraction rhythms during dangerous arrhythmias or in cardiac arrest. If shock-induced transmembrane potentials are large enough, they can cause tissue destruction due to irreversible electroporation (EP). Also electrochemotherapy of nearby tissues may have an adverse effect on the heart. Herein, we present experimental data on effects of electroporation in culture of cardiac cells (H9C2). The electric field was applied in short pulses of 25–3250 V/cm, 50 µs each. The viability of cells was tested by MTT assay after 24 hours. For detection of DNA fragmentation, associated with apoptosis, alkaline and neutral comet assays were performed after EP. Additionally phase contrast images of cells obtained directly after EP were analyzed. Although cell images indicated disruption of cell membranes after EP with high intensities, only a few percent of apoptotic cells and no necrotic effects in the cell nucleus could be observed in comet assay tests performed 2 hours post EP. MTT viability test showed that pulse intensities above 375 V/cm are destructive for myocytes viability.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 19-25.

  Neither inhalative nor intravenous application of carbon monoxide modifies gastric mucosal oxygenation
Christian Vollmer 1), Ingo Schwartges, Katja Obermiller, Christopher Beck, Olaf Picker

1)Department of Anaesthesiology, University Hospital Duesseldorf, Duesseldorf, Germany.

This study was designed to compare the effects of different ways of administering carbon monoxide (intravenous and inhalative) on gastric mucosal oxygenation in a canine model of hemorrhage. Six chronically instrumented dogs were repeatedly anesthetized and randomized to each of the following protocols: In a first series the animals were ventilated either with 100 ppm carbon monoxide (CO) or without followed by hemorrhage and re-transfusion. In a second series a saturated CO solution was infused, compared to normal saline, again followed by hemorrhage and re-transfusion. In a control series, animals received either CO-saline or saline without any further intervention. Microvascular oxygenation of the gastric mucosa (µHbO2) was assessed continuously by tissue reflectance spectrophotometry. Cardiac output was measured intermittently and oxygen delivery (DO2) was calculated. The application of CO, inhalative and intravenous, increased carboxyhemoglobin levels without effect on µHbO2. Hemorrhage reduced µHbO2 in all groups, paralleled by a reduction in DO2 without any differences between groups related to the application of CO. Neither intravenous nor inhalative application of CO alters µHbO2 during physiological conditions or during hemorrhage. Thus, independent of the application way, low dose CO does not seem to modulate regional mucosal oxygenation in cytoprotective concentrations.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 27-37.

  Characteristic induction of steroidogenic factor 1 (SF-1) and DAX-1 and enhanced expression of glucocorticoid synthesis-related genes in adrenals from spontaneously hypertensive rats
Naoki Harikai 1)

1)School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Hyogo.

To gain insight into the molecular mechanism of hyper-glucocorticoidism in spontaneously hypertensive rats (SHR), this study investigated the expression of genes related to glucocorticoid synthesis, melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc) and 11β-hydroxylase (P450c11), and the transcription factors of steroidogenic factor 1 (SF-1), which stimulates expression of the above gene, and DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene 1), which negatively regulates the transcriptional activity of SF-1, in adrenals from SHR. On quantitative real time RT-PCR analysis, gene expression levels of MC2R, StAR, P450scc and P450c11 in SHR were high compared with those in normotensive Wistar Kyoto rats (WKY). The gene expression level of SF-1 was not different between the two rats. However, the expression level of DAX-1 in SHR was markedly lower than that in WKY. Furthermore, the protein levels of StAR, SF-1 and DAX-1 determined by Western blot analysis coincided well with the gene expressions in both rats. These results suggest that the low level of DAX-1 may enhance the transcriptional activity of SF-1 and expression of genes related to glucocorticoid synthesis, which are targeted by SF-1, in adrenals from SHR.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 39-45.

  Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential
Denisa Margina 1), Mihaela Ilie, Gina Manda, Ionela Neagoe, Magdalena Mocanu, Diana Ionescu, Daniela Gradinaru, Constanţa Ganea

1)Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, 6 Traian Vuia, sect 2, Bucharest, Romania.

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 47-55.

  Free-radical degradation of high-molar-mass hyaluronan induced by ascorbate plus cupric ions: Testing of stobadine and its two derivatives in function as antioxidants
Lubica Surovcikova-Machova 1), Katarina Valachova, Maria Banasova, Vladimir Snirc, Elena Priesolova, Milan Nagy, Ivo Juranek, Ladislav Soltes

1)Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, 84104 Bratislava, Slovak Republic.

Stobadine·2HCl and its two hydrophilic derivatives SM1dM9dM10·2HCl and SME1i-ProC2·HCl were tested in the function of antioxidants on hyaluronan (HA) degradation induced by the Weissberger oxidative system [ascorbate plus Cu(II)]. As a primary method, rotational viscometry was applied, where the substance tested was added before or 1 h after the initiation of HA degradation. The most effective scavengers of •OH and peroxy-type radicals were recorded to be stobadine·2HCl and SME1i-ProC2·HCl, respectively. The most effective scavenger, determined by applying the ABTS assay, was stobadine·2HCl.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 57-64.

  Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways
Letícia Roma 1), Kleber Souza, Everardo Carneiro, Antonio Boschero, José Bosqueiro

1)Laboratory of Endocrine Pancreas and Metabolism, Department of Physiology and Biophysics, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, Brazil.

Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondrial apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax α protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells, and mitochondria dysfunction might be involved in the deleterious effects after long-term GC treatment.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 65-76.

  Cytotoxicity and genotoxicity evaluation of antidote oxime HI-6 tested on eight cell lines of human and rodent origin
Hana Svobodova 1), Petr Jost, Rudolf Stetina

1)Faculty of Military Health Sciences, University of Defence, Trebesska 1575, 50001 Hradec Kralove, Czech Republic.

Oxime HI-6 is an efficient reactivator of the acetylcholinesterase inhibited by organophosphorous nerve agents. In this study we have estimated cytotoxicity of HI-6 by the colony forming assay and genotoxicity by the comet assay on human and rodent cell lines. IC50 of HI-6 assessed by the colony forming capacity was 3.59 mM for HeLa cells and 5.18 mM for a mouse cell line L929. Small difference in cytotoxicity was found among other cell lines tested: IC50 was 1.61 mM for human A549 cells, 1.14 mM for UROtse line, 1.96 mM and 1.71 mM for Chinese hamster cells AA8 and UV-20, respectively. The A549 cell viability measured with the MTT test was 5 times decreased comparing 2 and 24 hours of HI-6 oxime treatment. The 5 mM HI-6 concentration reduced the viability within 2 hours to 95% only, however, it induced a significant number of DNA breaks in mouse cells L929, and also in human UROtse and HepG2 cells. 1-β-D-arabinofuranosylcytosine (10−4 M) and hydroxyurea (10−2 M), supplemented to the cultivation medium, did not cause any significant accumulation of DNA breaks during treatment, which indicated that the nucleotide excision repair was not acting on the induced DNA damage.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 77-84.

  Alleviation of genotoxic effects of cyclophosphamide using encapsulation into liposomes in the absence or presence of vitamin C
Amany Tohamy 1), Amira Abdel Azeem, Medhat Shafaa, Wafaa Mahmoud

1)Zoology and Entomology Department, Faculty of Science, Helwan University, Cairo, Egypt.

Cyclophosphamide (CP) is a widely used anticancer and immunosuppressant that induces oxidative stress. To ameliorate the side effects resulted from CP treatment, liposomes were tested as an efficient drug delivery system with or without vitamin C as an antioxidant. CP resulted in clastogenic and cytotoxic effects that significantly increased for the total chromosomal aberrations as well as the numerical ones in the CP group (150.8 and 6, respectively) than the control group (6.6 and 0.0) as mean values at p < 0.05. Micronucleus assay showed a significant increased micronucleated polychromatic erythrocytes percentage (MNPCEs% = 11.7%) and a significant decrease of polychromatic to normochromatic erythrocytes ratio (0.551) when compared to the group treated with liposomised CP and vitamin C (3.44%; 0.795, respectively) at p < 0.05. Also, the total glutathione S-transferase activity as a body antioxidant enzyme was decreased from 52.2 in the control to 16.09 nmol/min/mg protein in CP group at p < 0.05, while the highly significant amelioration results were observed in the liposomised vitamin C and CP group (40.88 nmol/min/mg protein). Our findings support the potential use of CP in a liposomal formulation doped with vitamin C to diminish the potential side effects of the agent.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 85-91.

  Glutamate receptors and the airways hyperreactivity
Anna Strapkova 1), Martina Antošová

1)Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic.

It is proposed the link between the hyperactivity of NMDA receptors and airway hyperresponsiveness. We investigated the effect of agents modulating the activity of NMDA receptors in the ovalbumin-induced airway hyperreactivity in guinea pigs. The airways hyperreactivity was influenced by the agonist (NMDA) and selective antagonist – competitive (AP-5) and non-competitive (MK-801) of NMDA receptors. Airway responsiveness to histamine or acetylcholine was evaluated in in vitro conditions. NMDA administration caused the increase of tracheal smooth muscle response in ovalbumin-induced hyperreactivity to acetylcholine. MK 801 as well as AP-5 provoked the decrease of reactivity mainly to acetylcholine in tracheal smooth muscle, while the former, non-competitive antagonist was more effective. We recorded more pronounced response in tracheal than in lung tissue smooth muscle with more considerable response to acetylcholine than to histamine. The results of experiments show the modification of airway smooth muscles responses by agents modulating the activity of NMDA receptors. They confirm the possibility of NMDA receptors participation in experimental airway hyperreactivity. The results enlarge information regarding the link of the inflammatory diseases and glutamatergic system.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 93-100.

  Proton magnetic resonance spectroscopy and its diagnostically important metabolites in the brain
Michal Bittšanský 1), Desanka Výbohová, Dušan Dobrota

1)Department of Medical Biochemistry, Jessenius Faculty of Medicine, Comenius University, Martin, Slovak Republic.

This review provides a brief summary of the physical basis of magnetic resonance spectroscopy (1H MRS) and its application in the human brain. We discuss the chemical structure, signal properties, biological function, normal spatial distribution and diagnostic potential of the more significant metabolites detectable in brain tissue: N-acetylaspartate, N-acetylaspartylglutamate, choline-containing substances, creatine, phosphocreatine, myo-inositol, glutamine and glutamate. In conclusion, a few notes on the importance of proper spectral quantification and contemporary trends in 1H MRS are presented.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 101-112.

  Estimation of rate constants of drug binding to open channels of cardiac transient outward current
Jiří Šimurda 1), Milena Šimurdová

1)Department of Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

An improved approach to the evaluation of the rate constants of drugs binding to the open channel underlying the transient outward potassium current It0 is described. It is based on an analysis of a quantitative model formulated by a set of twelve differential equations. The rate constants are calculated from the time constants resulting from an approximation of the time course of apparent inactivation of the recorded It0 by two exponentials in the absence and by three exponentials in the presence of a blocking agent. The model study confirmed significantly higher accuracy in comparison with the existing electrophysiological method.

General Physiology and Biophysics. Volume 31, 2012, No. 1: 113-116.