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General Physiology and Biophysics


Volume 28, 2009, No. 4

Content:


  Carboxymethylated tetrahydropyridoindoles as aldose reductase inhibitors: in vitro selectivity study in intact rat erythrocytes in relation to glycolytic pathway
Maria Juskova 1), Vladimir Snirc, Alena Gajdošíková, Andrej Gajdosik, Ludmila Krizanova, Milan Stefek

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


Oxidative stress and polyol pathway hypotheses are generally accepted in the etiology of diabetic complications. Recently, novel carboxymethylated pyridoindoles, structural analogues of the efficient chain-breaking antioxidant stobadine, were designed, synthesised and characterised as prospective aldose reductase inhibitors endowed with antioxidant activity. Of them (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 1) and (2-phenethyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 2) were found to be the most efficient inhibitors of aldose reductase with the corresponding IC50 values in a micromolar region. The aim of this work was to study cellular uptake of the novel pyridoindole derivatives and their effect on the complex metabolism of glucose in isolated rat erythrocytes under euglycaemic conditions. Glycolysis was shown to be the sole process responsible for the observed clearance of glucose. The compounds studied were avidly taken up by the cells, yet they did not significantly affect glucose consumption and lactate production nor did they affect osmotic fragility of the erythrocytes. On balance, the present experimental findings indicate that compounds 1 and 2, efficient inhibitors of aldose reductase, are selective in relation to the glycolytic pathway of glucose elimination. This conclusion supports current preclinical development of novel carboxymethylated tetrahydropyridoindoles as promising aldose reductase inhibitors for pharmacological prevention and treatment of diabetic complications.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 325-330.

 
  Expression and purification of recombinant NFI proteins for functional analysis
Gabriel Kollárovič 1), Dušana Majera, Katarína Luciaková, Peter Baráth

1)Laboratory of Molecular Biology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia. gabriel.kollarovic@savba.sk.


Nuclear factor I (NFI) is a transcription factor playing wide role in signal transduction pathways and developmental processes in higher eukaryotes. In order to produce recombinant NFI proteins for functional and structural studies, full length cDNAs of individual isoforms were subcloned into pETM30 vector and expressed in Escherichia coli. Although the fusion proteins containing both glutathione S-transferase (GST) and His6 tags at the N-terminus could be overexpressed in detectable amounts, they were found mainly, if not exclusively, in insoluble form. Purification yield was improved by modification of cell disruption procedure and by the use of detergent Tween 20. The final purification strategy represents a triple-helix affinity chromatography consisting of prepurification of bacterial lysate on Heparin-Sepharose with subsequent immobilized metal affinity and glutathione affinity chromatography. Heparin chromatography was crucial for obtaining active NFI proteins, whereas the other steps significantly improved the purity of isolated proteins. As demonstrated by EMSA and DNase I protection assay, the recombinant proteins were able to recognize their cognate DNA sequences.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 331-339.

 
  Self-consistent equation of plant cell growth
Mariusz Pietruszka 1)

1)Department of Plant Physiology, Faculty of Biology and Environmental Protection, University of Silesia, ul. Jagiellonska 28, PL-40032 Katowice, Poland. mariusz.pietruszka@us.edu.pl.


We introduce a transcendental equation, describing the subsequent stages of plant cell/organ growth. Starting from empirically verified conclusions originating from the central limit theorem we also insert the influence of temperature on elongation growth to receive a time-dependent equation of growth parameterized by temperature. This self-consistent equation evolves with time using three cardinal parameters: t0, T0 and V0. They represent the time of maximum expansion rate, the growth optimum temperature and the corresponding volume, respectively. Experimental determination of these cardinal values enables evaluation of the dynamic extensibility coefficient Φ = ΦT(t) in time and temperature domain.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 340-346.

 
  Influence of pyridoxylidene aminoguanidine on biomarkers of the oxidative stress and selected metabolic parameters of rats with diabetes mellitus
Zuzana Országhová 1), Anna Liptáková, Jana Muchová, Oľga Uličná, Oľga Vančová, Monika Sivoňová, Peter Božek, Jozef Čársky, Zdeňka Ďuračková

1)Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University, Sasinkova 2, 813 72 Bratislava, Slovakia. zdenka.durackova@fmed.uniba.sk.


Oxidative damage is considered to play an important role in the pathogenesis of several diseases, such as diabetes mellitus (DM), atherosclerosis, cardiovascular complications and chronic renal failure. DM is associated with the oxidative stress and formation of advanced glycation end products (AGEs). Different drugs inhibit oxidative stress and formation of advanced glycation end products. Aminoguanidine (AG) has been proposed as a drug of potential benefit in prophylaxis of the complications of DM. Recent reports show a pro-oxidant activity of AG. Therefore we examined the effect of structural analogue of AG, its Schiff base with pyridoxal – pyridoxylidene aminoguanidine (PAG) on the level of selected markers of oxidative stress. We found that PAG decreased total damage to DNA in controls as well as in diabetic group of rats. However, we also found that PAG supplementation increases susceptibility of lipoproteins to oxidation and formation of conjugated dienes in both, diabetic as well as control animals. Its administration to diabetic rats decreases antioxidant capacity of plasma. Therefore, it is necessary to search for other structural modifications of AG that would combine its higher anti-diabetic activity with less toxicity.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 347-355.

 
  EPR signal reduction kinetic of several nitroxyl derivatives in blood in vitro and in vivo
Zhivko Zhelev 1), Ken-Ichiro Matsumoto, Veselina Gadjeva, Rumiana Bakalova, Ichio Aoki, Antoaneta Zheleva, Kazunori Anzai

1)Department of Biophysics, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan. zh_zhelev@yahoo.com.


The present study is focused on the mechanism(s) of electron-paramagnetic resonance (EPR) signal reduction kinetic of several nitroxyl radicals and nitroxyl-labeled anticancer drugs in physiological solutions in the context of their application for evaluation of oxidation/reduction status of blood and tissues – an important step in biomedical diagnostics and planning of therapy of many diseases. The nitroxyl derivatives were characterized with different size and water-solubility. Some of them are originally synthesized. In buffer, in the absence of reducing and oxidizing equivalents, the EPR signal intensity of all nitroxyls was constant with the time. In serum and cell cultured medium, in an absence of cells and in a negligible amount of reducing and oxidizing equivalents, there was no significant EPR signal reduction, too. In vitro (in freshly isolated blood samples), the EPR signal intensity was characterized with slow decrease within 30 min, presumably as a result of interaction between the nitroxyl derivative and blood cells. The EPR spectrum of hydrophobic nitroxyls showed a slight anisotropy in cell-containing solutions and it did not changed in non-cell physiological solutions. This suggests for a limited motion of more hydrophobic nitroxyls through their preferable location in cell membranes. In vivo (in the bloodstream of mice under anesthesia), the EPR signal reduction kinetic was characterized by two phases: i) a rapid enhancement within 30 s as a result of increasing of nitroxyl concentration in the bloodstream after its intravenous injection, followed by ii) a rapid decrease (∼80–100%) within 2–5 min, presumably as a result of transportation of nitroxyl in the tissues. The hydrophobic nitroxyls were characterized with stronger and faster decrease in EPR signal intensity in the blood in vivo, as a result of their higher cell permeability, rapid clearance from the bloodstream and/or transportation in the surrounding tissues. The hydrophilic nitroxyls persist in the bloodstream (in their radical form) for a comparatively long time. The data suggest that the hydrophobic cell-permeable nitroxyl derivatives are most appropriate for evaluation of cell and tissue oxidation/reduction status, while the hydrophilic nitroxyls (impermeable for cell membranes or with very slow cell permeability) are most appropriate for evaluation of oxidation/reduction status of blood using EPR imaging.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 356-362.

 
  Vitamin D3 affects expression of thyroid hormone receptor alpha and deiodinase activity in liver of MNU-treated Sprague-Dawley rats
Dana Macejová 1), Slavomíra Ondková, Július Brtko

1)Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlárska 3, 833 06 Bratislava, Slovakia. ueenmace@savba.sk.


1α,25-dihydroxyvitamin D3 and its analogue, Seocalcitol (EB1089), are able to reverse or slow the process of carcinogenesis in experimental models and cell cultures. The aim of this study was to investigate the effect of administration vitamin D or Seocalcitol to female Sprague-Dawley rats with 1-methyl-1-nitrosourea (MNU)-induced carcinogenesis of mammary glands on binding characteristics and mRNA levels of thyroid hormone receptors (TRs). Chemopreventive administration of vitamin D caused significant reduction of animal body weight. The expression of TRα mRNA was significantly higher in liver of animals treated with vitamin D after detection of first tumour. In our experiment, administration of vitamin D or Seocalcitol significantly reduced KA (group MNU+Seo; MNU+D) and increased Bmax (group MNU+Seo) of thyroid receptors in liver when compared to healthy animals. We show that the activity type I 5’-deiodinase was significantly decreased in livers of animals treated with vitamin D. The data from our in vivo experiment has clearly shown, for the first time, that vitamin D but not Seocalcitol i) may affect the body weight of animals, ii) can cause an increase in the expression of TRα in rat liver, remaining the functionality of the TRs unaffected, and iii) is responsible for type I iodothyronine 5’-deiodinase activity decrease in rat liver, remaining the expression of the enzyme unaffected.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 363-370.

 
  Ca2+ signaling in mouse cardiomyocytes with ablated S100A1 protein
Konstantin Gusev 1), Gabriele Ackermann, Claus Heizmann, Ernst Niggli

1)Department of Physiology, University of Bern, Bühlplatz 5, CH-3012 Bern, Switzerland. niggli@pyl.unibe.ch.


S100A1 is a Ca2+-binding protein expressed at high levels in the myocardium. It is thought to modulate the Ca2+ sensitivity of the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors or RyRs) and its expression has been shown to be down regulated in various heart diseases. In this study we used S100A1 knock-out (KO) mice to investigate the consequences of chronic S100A1 deficiency on Ca2+ cycling in ventricular cardiomyocytes. Confocal Ca2+ imaging showed that field-stimulated KO myocytes had near normal Ca2+ signaling under control conditions but a blunted response to β-adrenergic stimulation with 1 µmol/l isoproterenol (ISO). Voltage-clamp experiments revealed that S100A1-deficient cardiomyocytes have elevated ICa under basal conditions. This larger Ca2+ influx was accompanied by augmented Ca2+ transients and elevated SR Ca2+ content, without changes in macroscopic excitation-contraction coupling gain, which suggests impaired fractional Ca2+ release. Exposure of KO and WT cells to ISO led to similar maximal ICa. Thus, the stimulation of the ICa was less pronounced in KO cardiomyocytes, suggesting that changes in basal ICa could underlie the reduced β-adrenergic response. Taken together, our findings indicate that chronic absence of S100A1 results in enhanced L-type Ca2+ channel activity combined with a blunted SR Ca2+ release amplification. These findings may have implications in a variety of cardiac pathologies where abnormal RyR Ca2+ sensitivity or reduced S100A1 levels have been described.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 371-383.

 
  Lipoamide dehydrogenase and diaphorase catalyzed conversion of some NO donors to NO and reduction of NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)
Alena Stibingerová 1), Hana Velvarská, Klára Kynčlová, Běla Marounková, Marcela Špundová, Gustav Entlicher

1)Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 40 Prague 2, Czech Republic. entg@seznam.cz.


One of the key functions of nitric oxide (NO) in human is to dilate blood vessels. We tested glycerol trinitrate (GTN) and other well-known NO donors together with those bearing a >C=N-OH group for possible conversion to NO (or nitrites, respectively) by diaphorase (DP) and lipoamide dehydrogenase (LAD). Both, DP and LAD were unable to convert formamidoxime (FAM), acetone oxime (AC), acetohydroxamic acid (AHA) and Nω-hydroxy-L-arginine (L-NOHA). On the other hand, we observed good conversion of GTN without the requirement of superoxide anion. However, superoxide anion participated to a varying extent in the conversion of other donors (formaldoxime (FAL), acetaldoxime (AO), nitroprusside (NP), S-nitrosoglutathione (SNOG), S-nitroso-N-acetylpenicillamine (SNAP) and hydroxylamine (HA)). All DP- and LAD-mediated reactions were inhibited by diphenyleneiodonium chloride (DPI), (an inhibitor of flavine enzymes), in a concentration-dependent manner. For these inhibition reactions we determined Ki and IC50 values. In addition, we found that conversion of SNOG was significantly accelerated by glutathione reductase (GTR). Like with DP, 2-phenyl- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) was reduced also by LAD and thioredoxin reductase (TRR). In summary, we found that LAD significantly accelerates the conversion of a defined subset of NO donors to NO, especially GTN, and eliminates the NO scavenging effect of PTIO.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 384-390.

 
  Multidrug resistant P-glycoprotein positive L1210/VCR cells are also cross-resistant to cisplatin via a mechanism distinct from P-glycoprotein-mediated drug efflux activity
Lenka Gibalová 1), Jan Sedlak, Martina Labudová, Miroslav Barancik, Alena Reháková, Albert Breier, Zdena Sulová

1)Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia. zdena.sulova@savba.sk.


P-glycoprotein (P-gp, a drug transporter found in the plasma membrane)-mediated multidrug resistance of leukemia cells represents a real obstacle in the effective chemotherapeutic treatment of leukemia. While cisplatin (CisPt) is known to be a substance that is untransportable by P-gp, P-gp positive cells were often found to be resistant to CisPt. The aim of the current paper is to study this phenomenon using P-gp positive mouse leukemia cells L1210/VCR in which the overexpression of P-gp was induced by its ability to adapt to growth on vincristine (VCR). L1210/VCR cells are also resistant to CisPt. However, resistance to this substance could not be reversed by addition of the known P-gp inhibitor verapamil. CisPt induced more pronounced entry into apoptosis, as measured using the annexin V/propidium iodide kit, in sensitive L1210 cells than in resistant L1210/VCR cells. In addition, CisPt induced an increase in the proportion of L1210 cells that were in the g2 phase of the cell cycle when compared to L1210/VCR cells, as measured by staining with propidium iodide. Similarly, a higher release of cytochrome c from the mitochondria to the cytosol was induced by CisPt treatment in L1210 than in L1210/VCR cells. While similar levels of Bax and Bcl-2 proteins were observed in sensitive and resistant cells, CisPt induced a more pronounced decrease of the Bcl-2 levels in L1210 cells than in L1210/VCR cells. Consistent with this observation, CisPt induced a larger decrease of the Bcl-2 content in the Bcl-2:Bax heterooligomer in L1210 cells than in L1210/VCR cells. Moreover, CisPt induced a similar apoptotic DNA fragmentation pattern in both resistant and sensitive cells. All of the above observations indicated that L1210/VCR cells are also resistant to CisPt and that this resistance is related to the differences in the regulatory mechanisms responsible for CisPt-induced apoptosis in L1210/VCR cells without any contribution from the drug efflux activity of P-gp.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 391-403.

 
  Cyclic AMP increases cytoplasmic free calcium in renin-secreting cells from rat kidney
Julia Laske-Ernst 1), Marc Chmielnicki, Ulrich Quast, Ulrich Russ

1)Department of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty, University of Tübingen, Wilhelmstr. 56, D-72074 Tübingen, Germany. ulrich.russ@uni-tuebingen.de.


The renin-secreting juxtaglomerular cells (JGC) in the media of the afferent arteriole at the vessel pole are the major source of circulating renin. The control of renin secretion is complex with increases in cAMP being the major stimulus and increases in intracellular free Ca2+ concentration ([Ca2+]i) being inhibitory. We measured [Ca2+]i in the afferent arteriole from mostly JGC. Manoeuvres that increase cAMP (e.g. isoproterenol) or dibutyryl-cAMP elicited an increase in [Ca2+]i which was ∼40% of that induced by angiotensin II (3 nmol/l). The Ca2+ response occurred in 50–90% of the cases, and increasing the stimulus increased responder frequency but not response size. The response was (almost) abolished by removal of extracellular Ca2+, prevented by inhibitors of store-operated Ca2+ channels (Gd3+ and 2-aminoethoxydiphenyl-borate), but was unaffected by isradipine or protein kinase A inhibitors. It was not produced by an activator of EPACs (exchange protein activated by cAMP) and was not accompanied by changes in membrane potential. The data suggest that in rat JGC, cAMP, perhaps directly, activates store-operated Ca2+ channels to increase [Ca2+]i. One could speculate that this increase in [Ca2+]i serves to finely adjust the stimulating effect cAMP-increasing signals on the renin-angiotensin system.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 404-413.

 
  Effect of static magnetic field and/or cadmium in the antioxidant enzymes activity in rat heart and skeletal muscle
Salem Amara 1), Catherine Garrel, Alain Favier, Khemais Ben Rhouma, Mohsen Sakly, Hafedh Abdelmelek

1)Salem Amara, Laboratoire de Physiologie Intégrée, Faculté des Sciences de Bizerte, 7021 Jarzouna, Tunisia. amara_salem_fsb@yahoo.fr.


Currently, environmental and industrial pollution along with increase and causes multiple stress conditions, the combined exposure to magnetic field and other toxic agents is recognised as an important research area, with a view to better protecting human health against their probable unfavourable effects. In the present study, we investigated the effect of co-exposure to static magnetic field (SMF) and cadmium (Cd) on the antioxidant enzymes activity and the malondialdehyde (MDA) concentration in rat skeletal and cardiac muscles. The exposure of rats to SMF (128 mT, 1 h/day during 30 consecutive days) decreased the activities of glutathione peroxidase (GPx) and the superoxide dismutase (CuZn-SOD) in heart muscle. Sub-chronic exposure to SMF increased the MDA concentration in rat cardiac muscle. Cd treatment (CdCl2, 40 mg/l, per os) during 4 weeks decreased the activities of catalase (CAT) in skeletal muscle and the CuZn-SOD in the heart. Moreover, Cd administration increased MDA concentration in the both structures. The combined effect of SMF (128 mT, 1 h/day during 30 consecutive days) and Cd (40 mg/l, per os) disrupt the antioxidant enzymes activity in rat skeletal and cardiac muscles. Moreover, we noted a huge increase in MDA concentration in the heart and skeletal muscle compared to control group. Thus it is possible that the SMF- and/or Cd-induced depletion of antioxidant enzymes activity in muscle tissues might, like the enhanced lipid peroxidation, importantly contribute to oxidative damage. The combined effect of SMF and Cd altered significantly the antioxidant enzymatic capacity and induced lipid peroxidation in both skeletal and cardiac muscle.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 414-419.

 
  Extremely low frequency magnetic field exposure affects DnaK and GroEL expression in E. coli cells with impaired heat shock response
Brunella Del Re 1), Pamela Marcantonio, Ferdinando Bersani, Gianfranco Giorgi

1)Department of Evolutionary Experimental Biology, University of Bologna, via Selmi 3, 40126 Bologna, Italy. gianfranco.giorgi@unibo.it.


In our earlier experiments, we found that extremely low frequency magnetic fields (ELF-MF) affect heat shock protein (HSP) expression in wild type Escherichia coli cells. In the present work we investigate the ability of ELF-MF exposure to trigger an increase of DnaK and GroEL protein levels also in E. coli cells not exhibiting the classic heat shock response (HSR) when subjected to a 42°C heat stress. We find that these cells, although lacking a HSR to heat shock treatment, show an enhancement of DnaK and GroEL protein levels after 30 or 90 min sinusoidal ELF-MF exposure (50 Hz, 1 mT). This result suggests that the HSP induction pathway triggered by ELF-MF exposure could be different from that elicited by heat shock treatment.

General Physiology and Biophysics. Volume 28, 2009, No. 4: 420-424.

 
  Malignant Triton tumour exhibits a complete expression pattern of nuclear retinoid and rexinoid receptor subtypes
Július Brtko 1), Daniela Sejnová, Slavomíra Ondková, Dana Macejova

1)Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia. Julius.Brtko@savba.sk.



General Physiology and Biophysics. Volume 28, 2009, No. 4: 425-427.