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General Physiology and Biophysics

Volume 27, 2008, No. 2


  Oxidative stress and thioredoxin system
M. Koháryová 1), M. Kolárová

1)Department of Biochemistry, Faculty of Natural Sciences, Commenius University, Bratislava, Slovakia.

Oxidative stress plays an important role in the modulation of several important physiological functions. On the other side, oxidative stress is accountable for development of many unphysiological changes, which can be deleterious for cells. Consequently, at the present time there is increased interest about study mechanisms and changes evocated by oxidative stress. Despite the highly oxidizing environment (21% oxygen, at sea level), at normal conditions, the cell cytoplasm of all aerobic organisms is reduced and proteins contain free sulfhydryl groups. In the cytoplasm, two major systems were identificated responsible for maintaining a reduced state: thioredoxin and glutathione/glutaredoxin system. Thioredoxin in bacteria, thanks to the low redox potential is the major dithiol reductant in the cytosol, or an advanced equivalent to dithiothreitol of cells (Holmgren 1985). Thioredoxin system acts the dominant role in many physiological processes (see below) and it is also a cell antioxidant.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 71-84.

  Relation of L-arginine to airway hyperreactivity
A. Strapková 1), M. Antošová, G. Nosáľová

1)Department of Pharmacology, Jessenius Faculty of Medicine, Comenius University, Sklabinská 26, 037 53 Martin, Slovakia.

The deficiency or the decrease in the bioavailability in basic substrate for nitric oxide synthesis – L-arginine can be one of factors contributing to the airway hyperreactivity. We studied the influence of L-arginine supplementation on the experimental airway hyperreactivity induced in guinea pigs by exposure to toluene vapours. L-arginine was administered before exposure in a dose of 300 mg/kg b.w. intraperitoneally during 3 or 17 days. After that the airway reactivity changes to histamine or acetylcholine were studied in in vitro conditions. In addition to that the tissue strips from exposed animals were incubated with L-arginine in concentration 10–4 mol/l. The administration of L-arginine during 3 days decreased the airway reactivity increased by irritant exposure. We recorded the decrease in the airway reactivity in animals with bronchial hyperreactivity after incubation of tissue strips with L-arginine, too. The pre-treatment of animals with L-arginine during 17 days did not affect the airway smooth muscle reactivity in larger extent. The exogenous administration of L-arginine resulted in a protective effect under the conditions of experimental airway hyperreactivity. The effect of supplementation was different depending on airway level and pre-treatment duration. The results refer to the importance of optimal L-arginine level for the control of bronchomotoric tone.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 85-91.

  Redistribution of cell death-inducing DNA fragmentation factor-like effector-a (CIDEa) from mitochondria to nucleus is associated with apoptosis in HeLa cells
E. Valoušková 1), K. Smolková, J. Šantorová, P. Ježek, M. Modrianský

1)Institute of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech Republic.

Cell death-inducing DFF[DNA fragmentation factor]-like effector-a (CIDEa), may initiate apoptosis by disrupting a complex consisting of 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). CIDEa, however, was found to be localized in mitochondria. We have performed immunodetection of CIDEa in whole cells and subcellular fractions of HeLa cells adapted for a tetracycline-inducible CIDEa expression. Using immunocytochemistry we observed redistribution, enhanced upon treatment with camptothecin or valinomycin, of CIDEa to nucleus. Similarly, CIDEa content increased in the nuclear fraction but decreased in cytosolic fraction in cells treated to initiate apoptosis. We hypothesize that CIDEa is sequestered in mitochondria while transfer of this potentially dangerous protein from mitochondria into nucleus intensifies or even initiates apoptosis.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 92-100.

  Production of reactive oxygen species after photodynamic therapy by porphyrin sensitizers
H. Kolarova 1), P. Nevrelova, K. Tomankova, P. Kolar, R. Bajgar, J. Mosinger

1)Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech Republic.

The objectives of this study was to investigate the production of reactive oxygen species (ROS) after photodynamic therapy (PDT) in vitro. We examined second generation sensitizers, porphyrines (TPPS4, ZnTPPS4 and PdTPPS4) and compared their effectivity on ROS generation in G361 cell line. Used porphyrines are very efficient water-soluble aromatic dyes with potential to use in photomedicine and have a high propensity to accumulate in the membranes of intracellular organelles like lysosomes and mitochondria. Interaction between the triplet excited state of the sensitizer and molecular oxygen leads to produce singlet oxygen and other ROS to induce cell death. Production of ROS was verificated by molecular probe CM-H2DCFDA and viability of cells was determined by MTT assay. Our results demonstrated that ZnTPPS4 induces the highest ROS production in cell line compared to TPPS4 and PdTPPS4 at each used concentration and light dose. These results consist with a fact that photodynamic effect depends on sensitizer type, its concentration and light dose.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 101-105.

  Protective effect of stobadine on NCV in streptozotocin-diabetic rats: augmentation by vitamin E
S. Skalska 1), Z. Kyselova, A. Gajdosikova, C. Karasu, M. Stefek, S. Stolc

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

Hyperglycaemia-induced oxidative stress makes an important contribution to the aetiology of diabetic neuropathy. Elevated reactive oxygen species (ROS) cause cumulative damage to neurons and Schwan cells, however, they also have a deleterious effect on nerve blood flow causing endoneurial hypoxia, which is responsible for early nerve conduction velocity (NCV) deficits and contributes to an increase in resistance to ischaemic conduction failure (RICF). We tested whether antioxidants – stobadine, vitamin E or the combination of these drugs, could prevent the early signs of neural dysfunction in animal model of diabetes in 8–9 weeks old male Wistar rats, made diabetic by streptozotocin (55 mg/kg i.v.) 4 months prior to testing. Neuropathy was evaluated electrophysiologically by measuring motor NCV and RICF of sciatic nerve in vitro. We observed that treatment with the combination of stobadine and vitamin E significantly (p < 0.001) reduced the NCV slowing in diabetic rats, although it did not fully prevent the NCV impairment. Significant effect (p < 0.05) was observed also in stobadine monotherapy. The RICF elevated in diabetic animals was not affected by any drug applied. This study confirmed that treatment with appropriate antioxidants, especially their combination could partially prevented the decrease in NCV in diabetic rats.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 106-114.

  Oxidative alternations in rat heart homogenate and mitochondria during ageing
E. Babusikova 1), M. Jesenak, P. Racay, D. Dobrota, P. Kaplan

1)Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Malá Hora 4, 036 01 Martin, Slovakia.

Our understanding of the role played by reactive oxygen and nitrogen species in disease pathology and ageing is still insufficient. Reactive oxygen species and reactive nitrogen species can initiate protein and lipid oxidative damage that may be the most important contribution to ageing and age-related heart diseases. In the present study, we investigated the effect of ageing on oxidative damage of protein amino acid residues and lipids in heart homogenate and mitochondria of 4- and 26-month-old Wistar rats. Levels of dityrosine and levels of lysine conjugates increased in heart homogenate during ageing, although levels of conjugated dienes did not change. We observed significantly oxidative modification of tryptophan in heart mitochondria and increased levels of dityrosine with advancing age. However, levels of lysine conjugates, conjugated dienes as well as relative level of cytochrome c oxidase were unchanged in heart mitochondria during ageing. The results of this study suggest a different mechanism of oxidative modification in heart compartments during ageing and moreover, mitochondria and other cellular compartments are targets for oxidative modifications.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 115-120.

  Study of high deprenyl dose on the preimplantation embryo development and lymphocyte DNA in rat
J. Mihalik 1), P. Kravčuková, T. Špakovská, M. Mareková, K. Schmidtová

1)Department of Anatomy, Medical Faculty, P. J. Šafárik University, Šrobárova 2, 040 01 Košice, Slovakia.

To investigate the role of potent MAO-B inhibitor deprenyl in fertilized females, we have evaluated the effect of chronic treatment with deprenyl at a high dosage on preimplantation embryo development and DNA damage in blood lymphocytes in Wistar rats. We have found that the number of embryos isolated from both uterus and oviduct per rat was significantly lower in the experimental group. Almost 14% of embryos in experimental animals were flushed from oviducts compared to 1.95% of those in the control rats. Morphological analysis of embryos isolated from deprenyl-treated animals had revealed impaired rates in the distribution pattern compared with controls. But deprenyl administration had no significant effect on the mean number of cells in morulae or even blastocysts. On the other hand, analysis of cell number distribution in blastocysts using the chi-square test indicated a significantly decreased cell proliferation in the experimental group. Despite the harmful impact of deprenyl on rat preimplantation embryo development, deprenyl administration significantly decreased the DNA damage in blood lymphocytes as was scored employing Comet Assay. Our description of the adverse effects of deprenyl administration on rat preimplantation embryo development compared to the protective effects on the lymphocyte DNA is very important because deprenyl is still widely used in human medicine as a treatment. Potential mechanisms mediating deprenyl-induced impaired preimplantation embryo development are proposed.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 121-126.

  S100A1 deficiency results in prolonged ventricular repolarization in response to sympathetic activation
G. Ackermann 1), A. Domenighetti, A. Deten, I. Bonath, I. Marenholz, T. Pedrazzini, P. Erne, C. Heizmann

1)Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, Steinwiesstrasse 75, 8032 Zürich, Switzerland.

S100A1 is a Ca2+-binding protein and predominantly expressed in the heart. We have generated a mouse line of S100A1 deficiency by gene trap mutagenesis to investigate the impact of S100A1 ablation on heart function. Electrocardiogram recordings revealed that after β-adrenergic stimulation S100A1-deficient mice had prolonged QT, QTc and ST intervals and intraventricular conduction disturbances reminiscent of 2 : 1 bundle branch block. In order to identify genes affected by the loss of S100A1, we profiled the mutant and wild type cardiac transcriptomes by gene array analysis. The expression of several genes functioning to the electrical activity of the heart were found to be significantly altered. Although the default prediction would be that mRNA and protein levels are highly correlated, comprehensive immunoblot analyses of salient up- or down-regulated candidate genes of any cellular network revealed no significant changes on protein level. Taken together, we found that S100A1 deficiency results in cardiac repolarization delay and alternating ventricular conduction defects in response to sympathetic activation accompanied by a significantly different transcriptional regulation.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 127-142.

  Pharmacological inhibitors of JNK and ERK kinases SP600125 and U0126 are not appropriate tools for studies of drug metabolism because they activate aryl hydrocarbon receptor
P. Bachleda 1), Z. Dvořák

1)2nd Department of Surgery, University Hospital Olomouc, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic.

Mitogen-activated protein kinases (MAPKs) are important regulators of aryl hydrocarbon receptor (AhR). An immense progress in MAPKs' biochemistry was attained with the discovery of their specific pharmacological inhibitors. Unfortunately, the inhibitors of JNK and ERK MAPKs, i.e. SP600125 and U0126, respectively, affect AhR-CYP1A signaling pathway because they are partial agonists of AhR and induce CYP1A genes. This implies that SP600125 and U0126 are inappropriate tools for studies of the role of MAPKs in AhR regulation. The results from studies using SP600125 or U126, past or future, should be interpreted with prudence regarding their stimulatory effects on AhR-CYP1A pathway.

General Physiology and Biophysics. Volume 27, 2008, No. 2: 143-145.