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General Physiology and Biophysics

Volume 32, 2013, No. 2


  Potential uses of G-quadruplex-forming aptamers
Viktor Viglasky 1), Tibor Hianik

1)Department of Biochemistry, Institute of Chemistry, Faculty of Sciences, P. J. Šafárik University, Košice, Slovak Republic.

Guanine quadruplex (G-quadruplex) structures are one of a number of structures which are capable of adopting aptamers. G-rich DNA or RNA has an increased propensity to form quadruplex structures which have unusual biophysical and biological properties. G-rich aptamers which form G-quadruplexes have several advantages over unstructured sequences: G-quadruplexes are non-immunogenic, thermodynamically and chemically stable and they have both higher resistance to various serum nucleases and an enhanced cellular uptake. These advantages have led to a number of synthetic oligonucleotides being studied for their potential use as therapeutic agents for cancer therapy and in the treatment of various other diseases. In addition to their suitability in the fields of medicine and biotechnology, these, highly specified, aptameric G-quadruplexes also have great potential in the further development of nano-devices; e.g. basic components in microarrays, microfluidics, sandwich assays and electrochemical biosensors. This review summarizes the biophysical properties of G-quadruplexes and highlights the importance of the stability and recognition properties of aptamers. Examples of the application of aptamers in medical therapy and in biosensors are also discussed.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 149-172.

  Influence of naturally occurring antioxidants on magnetic nanoparticles: Risks, benefits, and possible therapeutic applications
Štefan Durdík 1), Hanka Vrbovská, Adam Olas, Melánia Babincová

1)St. Elizabeth Cancer Institute, Bratislava, Slovak Republic.

We have studied interaction of well known antioxidant L-ascorbic acid with magnetic nanoparticles containing insoluble Fe(III) in their core. In analogy with ferritin, mobilization of iron in the form of water soluble Fe(II) was observed, especially pronounced at higher temperatures. In the presence of hydrogen peroxide cytotoxic hydroxyl radicals are produced. These results suggest possible harmful effects of widely used magnetic nanoparticles as a MRI contrast agents in combination with overload of organism with ascorbic acid in some specific conditions, like fever of patient. On the other hand combination of magnetic nanoparticles and ascorbic acid may be used for a cancer therapy using alternating magnetic field for the release of Fe(II) via Néel relaxation of magnetic moment of used nanoparticles. We have further found that lipoic acid is an efficient antioxidant scavenging hydroxyl radicals produced by Fenton reaction from Fe(II).

General Physiology and Biophysics. Volume 32, 2013, No. 2: 173-177.

  Bcl-2 proapoptotic proteins distribution in U-87 MG glioma cells before and after hypericin photodynamic action
Lucia Balogová 1), Mária Maslaňáková, Lenka Dzurová, Pavol Miškovský, Katarína Štroffeková

1)Department of Biophysic, Institute of Physical Sciences, P. J. Safarik University, Kosice, Slovak Republic.

Apoptosis is a key process in the development and maintenance of tissue homeostasis. This process of controlled cell death is tightly regulated by a balance between cell survival and damage signals. We focused our attention towards one apoptotic pathway, the intrinsic mitochondrial one where Bcl-2 family of proteins plays the major role. We are particularly interested in two pro-apoptotic players Bak and Bax from this family. Here we investigated their role in apoptosis triggered by photodynamic action. Targeted photodynamic therapy (PDT) is a promising approach to diagnose and treat different types of cancer. We show the localization of Bax and Bak in U-87 MG human glioma cells incubated with photosensitizer hypericin (Hyp) before and after photodynamic action. Apoptotic stimulus by Hyp photodynamic action causes Bax translocation into mitochondria. However our results suggest that under these conditions there are two populations of mitochondria: one which contains Bax and Bak simultaneously, and is almost exclusively localized near the plasma membrane; the other which contains Bax only and is distributed throughout the cell. The different protein content and spatial distribution of these two populations suggest that they can play different roles in response to apoptotic stimuli.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 179-187.

  The physical properties of lipid monolayers and bilayers containing calixarenes sensitive to cytochrome c
Pavol Vitovič 1), Veronika Šubjaková, Tibor Hianik

1)Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic.

We studied physical properties of the monolayers and bilayer lipid membranes (BLM) formed by calix[6]arene carboxylic acid derivative (CX) and its mixtures with diphytanoylphosphatidylcholine (DPhPC) by means of measurement surface pressure, surface dipole potential and electrostriction. CX forms stable monolayers at an air-water interface and complexes in mixed monolayers contained DPhPC. Calixarenes increase the elastic moduli of lipid monolayers as well as BLM. Cytochrome c (cyt c) specifically binds to CX by incorporation of the amino groups of lysine residues at the protein surface. This binding affected the physical properties of CX monolayers depending on their initial surface pressure. Addition of cyt c into the water subphase induced increase of surface pressure of CX monolayers at relatively low initial pressure (15 mN/m) when monolayer was in liquid expanded state (LE). This may be due interaction of positively charged cyt c with negatively charged carboxylic groups of CX and also by its penetration into the air-water interface. However, much subtle changes were observed for higher initial surface pressure (20 and 35 mN/m) when monolayer is in liquid condensed (LC) and solid (S) state, respectively. Lysine induced substantially lower changes in surface pressure in comparison with that of cyt c.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 189-200.

  Solubilization of poorly soluble photosensitizer hypericin by polymeric micelles and polyethylene glycol
Diana Búzová 1), Peter Kasák, Pavol Miškovský, Daniel Jancura

1)Department of Biophysics, P. J. Safarik University, Kosice, Slovak Republic.

Hypericin (Hyp) is a promising photosensitizer for photodiagnostic and photodynamic therapy of cancer. However, Hyp has a large conjugated system and in aqueous solutions forms insoluble aggregates which do not possess biological activity. This makes intravenous injection of Hyp problematic and restricts its medical applications. To overcome this problem, Hyp is incorporated into drug delivery systems which can increase its solubility and bioavailability. One of the possibilities is utilization of polymeric micelles. The most used hydrophilic block for preparation of polymeric micelles is polyethylen glycol (PEG). PEG is a polymer which for its lack of immunogenicity, antigenicity and toxicity obtained approval for use in human medicine. In this work we have studied the solubilization of Hyp aggregates in the presence of PEG-PE and PEG-cholesterol micelles. The concentration of polymeric micelles which allows total monomerization of Hyp corresponds to the critical micellar concentration of these micelles (~10−6 M). We have also investigated the effect of the molecular weight and concentration of PEG on the transition of aggregated Hyp to its monomeric form. PEGs with low molecular weight (< 1000 g/mol) do not significantly contribute to the solubilization of Hyp. However, PEGs with molecular weight > 2000 g/mol efficiently transform Hyp aggregates to the monomeric state of this photosensitizer.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 201-208.

  Attenuation of the insulin amyloid aggregation in presence of Fe3O4-based magnetic fluids
Katarina Siposova 1), Eva Bystrenova, Andrea Antosova, Martina Koneracka, Vlasta Zavisova, Peter Kopcansky, Zuzana Gazova

1)Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Košice, Slovak Republic.

Presence of protein amyloid deposits is associated with pathogenesis of amyloid-related diseases. Insulin amyloid aggregates have been reported in a patient with diabetes undergoing treatment by injection of insulin. We have investigated the interference of insulin amyloid aggregation with two Fe3O4-based magnetic fluids. The magnetic fluids are able to inhibit insulin amyloid fibrillization and promote disassembly of amyloid fibrils. The cytotoxic effect of amyloid fibrils is attenuated in presence of magnetic fluids probably due to reduction of the fibrils. We suggest that present findings propose the potential use of Fe3O4-based magnetic fluids as the therapeutic agents targeting insulin-associating amyloidosis.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 209-214.

  Polyanion induced circular dichroism of Thioflavin T
Diana Fedunova 1), Peter Huba, Jaroslava Bagelova, Marian Antalik

1)Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 04001 Kosice, Slovak Republic.

Thioflavin T (ThT) is amyloid specific fluorescence dye possessing the properties of molecular rotor. We have shown that Thioflavin T forms complexes with non-peptide polyanions heparin, polyadenylate and polystyrene sulphonate by means of absorption spectroscopy. In the presence of chiral polyanions - heparin and polyadenylate - induced optical activity of ThT occurs whereas interaction with achiral polystyrene sulphonate (PSS) does not lead to production of induced circular dichroism signal. The positively charged ThT forms centre for ordered binding of chiral polyanion. Similarly, complexation of structurally different chromophore 9-aminoacridine with polyanions has led to induction of optical activity only in the presence of chiral ones. We suggest that, primarily, the optical activity of environment plays important role in inducing optical activity of achiral compounds.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 215-219.

  Hydroxyl radicals’ production and ECG parameters during ischemia and reperfusion in rat, guinea pig and rabbit isolated heart
Hana Paulova 1), Tibor Stracina, Jiri Jarkovsky, Marie Novakova, Eva Taborska

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

Ischemic and reperfusion injury is a serious condition related to numerous biochemical and electrical abnormalities of the myocardium. It has been repeatedly studied in various animal models. In this study, the production of hydroxyl radicals and electrophysiological parameters were compared in three species. Rat, guinea pig and rabbit isolated hearts were perfused according to Langendorff under strictly identical conditions. The heart rate and arrhythmia were monitored during ischemia and reperfusion periods at defined time intervals; the production of hydroxyl radical was determined by HPLC as 2.5-dihydroxybenzoic acid (2.5-DHBA) formed by salicylic acid hydroxylation. Relationship between arrhythmias and production of 2.5-DHBA was studied. The inter-species differences were observed in timing of arrhythmias onset and their severity, and in the production of 2.5-DHBA in both ischemia and reperfusion. The most considerable changes were observed in rats, where arrhythmias appeared early and with highest severity during ischemia on one side and the regular rhythm was restored early and completely during reperfusion. The corresponding changes in the production of 2.5-DHBA were observed. It can be concluded that rat isolated heart is the most suitable model for evaluation of ischemia/reperfusion injury under given experimental conditions.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 221-228.

  Effect of TiO2 nanoparticles on emotional behavior and biochemical parameters in adult Wistar rats
Salem Amara 1), Wahid Khemissi, Imen Mrad, Naima Rihane, Imen Ben Slama, Lassaad Mir, Mustapha Jeljeli, Khemais Ben Rhouma, Hafedh Abdelmelek, Mohsen Sakly

1)Laboratoire de Physiologie Intégrée, Faculté des Sciences de Bizerte, 7021 Jarzouna, Tunisia.

The rapidly developing field of nanotechnology is becoming a potential source for human exposure to nanoparticles. Titanium dioxide (TiO2) nanoparticles have been widely produced in industrial processes for several years. The aim of this study was to investigate the effects of TiO2 nanoparticles on plasmatic biochemical parameters and the emotional behavior in adult Wistar rats. Rats were treated by intraperitoneal injection of TiO2 nanoparticles (20-30 nm) at a dose of 25 mg/kg. For toxicity evaluation of nanoparticles sample, body weight, organ coefficient, blood biochemistry panel assay (AST, ALT, LDH, uric acid, creatinine, and glucose content) and emotional behavior parameters were determined. Sub-acute TiO2 nanoparticles treatment decreased the body weight, but increased the relative brain weight. Biochemical assessment in plasma samples showed that TiO2 nanoparticles injection increased uric acid concentration and AST activity in rats. However, the same treatment decreased the creatinine level, but had no effect on glucose concentration, ALT and LDH activity. The emotional behavior of control and treated rats was tested in elevated plus-maze. Interestingly, our results showed that TiO2-treated rats spent more time in the secured closed arms and entered the anxiogenic open arms less frequently than control. Our results suggest that TiO2 nanoparticles intoxication could altered biochemical parameters related to changes in organ function and leads to emotional behavior impairment of rats.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 229-234.

  Long-term effect of prazosin and losartan administration on blood pressure, heart, carotid artery, and acetylcholine induced dilation of cardiovascular system of young Wistar rats and SHR
Frantisek Kristek 1), Magdalena Malekova, Sona Cacanyiova

1)Institute of Normal and Pathological Physiology, "Centre of excellence for examination of regulatory role of nitric oxide in civilisation diseases", Slovak Academy of Sciences, Sienkiewiczova 1, 813 71 Bratislava, Slovak Republic.

The long-term effects of prazosin and losartan administration on blood pressure, trophicity of the heart and carotid arteries, and responses of the cardiovascular system to acetylcholine, were studied in Wistar rats and spontaneously hypertensive rats (SHRs). Four-week-old rats were treated with prazosin (10 mg/kg b.w./day in tap water) or losartan (20 mg/kg b.w./day in tap water) for 5–6 weeks. BP was measured by plethysmographic method. Ten animals of each group were subjected to in vivo studies and subsequent to morphological investigations. The right jugular vein was cannulated for administration of acetylcholine (0.1, 1, and 10 µg). After perfusion with a glutaraldehyde fixative (120 mmHg), the carotid arteries were embedded in Durcupan ACM, and the inner diameter (ID), wall thickness (WT) (tunica intima and media), cross sectional area (CSA) (tunica intima and media), and WT/ID ratio were calculated. In Wistar rats and SHRs, prazosin and losartan administration produced a decrease in the blood pressure and trophicity of the heart. In Wistar rats, both drugs decreased the WT, CSA, and the WT/ID ratio. In addition, these drugs increased the circumferential stress of the artery without affecting the ID. In contrast, in the SHRs, only losartan administration produced these effects. Importantly, both the drugs improved the responses to acetylcholine in SHRs.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 235-243.

  Mesenchymal stem cells in prostate cancer have higher expressions of SDF-1, CXCR4 and VEGF
Guanxiong Ding 1), Lujia Wang, Hua Xu, Zheng Xu, Chenchen Feng, Qiang Ding, Zujun Fang, Zhong Wu, Haowen Jiang, Jianfeng Xu, Peng Gao

1)Department of Urology, Huashan Hospital, Fudan University, Shanghai, China.

Our previous study found that the activity of PCa-MSCs, which could stimulate the cell proliferation of RM-1, was significantly different compared to BMMSCs. Our results indicated that it could be mediated in part by growth factors/chemokines, which were involved in the different activity between two kinds of MSCs (PCa-MSCs and BMMSCs). Normal MSCs (BMMSCs) were isolated from the femur, tibia of the normal mice; prostate tumor MSCs (PCa-MSCs) were obtained from the mice implanted with prostate tumor. Analysis of the expression of SDF-1, CXCR4, VEGF、bFGF and vWF of two kinds of MSCs were examined by ELISA, Realtime-PCR and Western blotting. The expressions of SDF-1 and CXCR4 in PCa-MSCs were higher compared to BMMSCs. Expressions of bFGF and vWF were higher in PCa-MSCs yet the difference did not reach statistical significance. The expression of VEGF was significantly higher in PCa-MSCs. Our data showed that activity of PCa-MSCs was significantly improved compared with BMMSCs, which seemed to have an intrinsic, cell-specific capacity localized to PCa. It could be induced by some factors or chemokines such as SDF-1, CXCR4, and VEGF. The possible role of PCa-MSCs in the process of PCa development needed further clarification.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 245-250.

  The long-term administration of Orai 1 antagonist possesses antitussive, bronchodilatory and anti-inflammatory effects in experimental asthma model
Martina Šutovská 1), Michaela Kocmálová, Marián Adamkov, Desanka Výbohová, Pavol Mikolka, Daniela Mokrá, Jozef Hatok, Martina Antošová, Soňa Fraňová

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

The best-studied store-operated Ca2+ channels (SOCs), Ca2+ release activated Ca2+ (CRAC) channels, are activated by depleting endoplasmic reticulum Ca2+ pool and mediate Ca2+ influx vitally important for Ca2+ restoration and many cellular function. CRAC channels were identified on immune and airway smooth muscle (ASM) cells. Emerging evidence points to its involvement in allergic airways diseases. This article evaluated therapeutic potency of CRAC antagonist in experimental animal model of allergic asthma. Allergic asthma, induced by repetitive exposure of guinea pigs to ovalbumine, was followed by 14 days therapy by CRAC antagonist (3-fluoropyridine-4-carboxylic acid, FPCA). In vivo changes of specific airways resistance (sRaw) evaluated bronchodilatory effect of FPCA and salbutamol. The method of citric acid-induced cough reflex assessed antitussive activity of FPCA and codeine. The measurement of exhaled NO (ENO), expression of inducible NO-synthase (iNOS) by RT-PCR and immunohistochemical staining of airways tissue verified anti-inflammatory effect of FPCA. Long-term administration of FPCA resulted in significant cough suppression and bronchodilation, both comparable to the effect of control drugs. FPCA significantly decreased ENO and iNOS expression, which together with immunohistochemical analysis validated its anti-inflammatory effect. Presented data confirmed CRAC channels as a promising target for treatment of respiratory diseases associated with allergic inflammation.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 251-259.

  3T3-L1 adipocytes possess anandamide- and epinephrine-responsive machinery for MDM2 distribution to the plasma membrane
Yasuhito Ohsaka 1), Hoyoku Nishino

1)Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan.

The effects of biomolecules on peripheral tissues and their responsive machinery are not well understood. We examined MDM2 level in the plasma membrane (PM) and total MDM2 level of 3T3-L1 adipocytes treated with biomolecular anandamide, epinephrine, and other agents for 15 min. We also examined biomolecular responses in cells treated with mithramycin A, a binding inhibitor, or cells exposed to cooling and cell viability. Immunoblotting revealed that PM MDM2 level increased and total MDM2 level was not altered following treatment with anandamide, epinephrine, capsaicin, CL316243, and aluminum fluoride. PM MDM2 distribution caused by a biomolecular concentration was maintained by treatment with mithramycin A and exposure of cells to 28°C or 32°C but not to 18°C, and PM MDM2 levels after treatment with high concentrations of biomolecules were altered upon exposure to the inhibitor and mild hypothermia. These conditions did not decrease cell viability. Our findings indicate that 3T3-L1 adipocytes possess molecular machinery that responds differentially to anandamide and epinephrine under the inhibitor treatment and cool temperature conditions and that is sensitive to other agents (which mimic biomolecular responses); these machineries can induce subcellular alterations in molecular interactions. We provide information helpful for clarifying biomolecular responsive machinery present in 3T3-L1 adipocytes.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 261-275.

  Effects of caloric restriction on oxidative stress parameters
Marija Stankovic 1), Dusan Mladenovic, Milica Ninkovic, Danijela Vucevic, Tina Tomasevic, Tatjana Radosavljevic

1)Institute of Pathophysiology, Faculty of Medicine, University of Belgrade, Dr Subotica 9, Belgrade, Serbia.

Moderate caloric restriction prolongs lifespan. Changes in oxidative stress and hormesis may be involved in this process. The aim of this study is to examine the effects of different levels of chronic caloric restriction (CR) and acute fasting on stress response and oxidative stress parameters in rat liver and plasma. Forty-two rats were divided into groups: control group, calorie-restricted groups with intake of 80–90%, 60–70%, 40–50%, 20–30% of daily caloric needs and acute fasting group. To determine alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD) activity, concentration of corticosterone, nitrites and nitrates (NOx), malondialdehyde (MDA) and glutathione (GSH), liver samples and blood were collected. Increase in plasma corticosterone concentration and AST and ALT activity was found in severe CR. Ingestion 40–50% daily caloric needs or less increased liver MDA and NOx concentration and decreased SOD activity. Ingestion 60–70% daily caloric needs increased Mn-SOD activity, GSH and NOx. In acute fasting group and group taking 20–30% daily caloric needs, GSH was significantly lower than in control group. Severe CR and acute fasting increase oxidative damage and decrease antioxidative capacity of hepatocytes. Moderate CR increases antioxidative capacity of hepatocytes due to increase in Mn-SOD activity and GSH concentration, which might have a role in anti-aging and hormetic mechanism of CR.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 277-283.

  Ischemia-reperfusion injury of the isolated diabetic rat heart: effect of the antioxidant stobadine
Zuzana Broskova 1), Zuzana Kyselova, Vladimir Knezl

1)Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak Republic.

The etiology of diabetic complications is strongly associated with increased oxidative stress. The aim of the present study was to evaluate the effect of the potent antioxidant stobadine (STB) on global ischemia-reperfusion cardiac injury in the rat model of diabetes mellitus (DM). Diabetes was induced by multiple low doses of streptozotocin. The effect of STB was compared with that of a high dose of α-lipoic acid (ALA). All experiments were performed on isolated Langendorff-perfused hearts 10 weeks after streptozotocin administration. Diabetic hearts showed to be more resistant to ischemia-reperfusion than the control hearts, as shown by the reduced number of reperfusion dysrhythmias. The effect of the therapy with ALA (100 mg/kg i.p., 5 times a week during 8 weeks) was comparable to that of STB (25 mg/kg i.p., 5 times a week during 8 weeks) resulting in lowering the heart rate and coronary flow as well as the number of serious reperfusion dysrhythmias. Though the protective effect of STB on the reperfusion-induced dysrhythmias was comparable with that of ALA, both substances failed to enhance functional recovery of the diabetic rat heart.

General Physiology and Biophysics. Volume 32, 2013, No. 2: 285-292.