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General Physiology and Biophysics


Volume 25, 2006, No. 3

Content:


  Atomic force microscopy and FT-IR spectroscopy investigations of human heart valves
M Jastrzebska 1), J Zalewska-Rejdak, I Mróz, B Barwinski, R Wrzalik, A Kocot, J Nozynski

1)Department of Biophysics, Faculty of Pharmacy, Medical University of Silesia, Ostrogórska 30, 41–200 Sosnowiec, Poland. maja@slam.katowice.pl.


Human aortic, mitral, tricuspid and pulmonary heart valves were investigated by the contact mode atomic force microscopy (AFM) in air, and using FT-IR spectroscopy in the frequency range 950–4000 cm-1. Heart valves were collected post mortem from 65–78 years old patients who died from non-cardiac diseases. All of the examined valves showed considerable heterogeneity in the surface topography of collagen fibrils as well as in their organization on the tissue surface. The AFM images revealed areas with significantly different spatial organization of the collagen fibril bundles. We observed zones with multidirectional, stacked collagen fibrils as well as areas of thin fibrils packed regularly, densely and “in phase”. The majority of the collagen fibrils reproduced the typical transverse D-banding pattern, with the band interval varying in rather wide range of 70–90 nm. Using AFM imaging, objects that correspond to some pathological states of heart valves at their early stages, i.e. some forms of mineral deposits, were observed. The FT-IR spectra allowed us to recognize main components, i.e. collagen and elastin, in di.erent layers (ventricularis, fibrosa) of the valve leaflets as well as they gave also support for the presence of mineral deposits on the valve surface. The presented results showed, that the AFM imaging and FT-IR spectroscopy can be applied as a complementary methods for structural characterization of heart valves at the molecular and supramolecular levels.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 231-244.

 
  Oxidation pattern of the anticancer drug ellipticine by hepatic microsomes – similarity between human and rat systems
M Stiborová 1), L Bořek-Dohalská, D Aimová, V Kotrbová, K Kukačková, K Janouchová, M Rupertová, H Ryšlavá, J Hudeček, E Frei

1)Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2, Czech Republic. stiborov@natur.cuni.cz.


Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N2-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N2-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N2-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 245-261.

 
  Oxaliplatin, an anticancer agent that affects both Na+ and K+ channels in frog peripheral myelinated axons
E Benoit 1), S Brienza, J Dubois

1)Laboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040, CNRS, bât. 32–33, 91198 Gif-sur-Yvette cedex, France. benoit@nbcm.cnrs-gif.fr.


The use of oxaliplatin, a relatively new chemotherapeutic agent, is somewhat limited since it produces a specific peripheral neuropathy regarding other neurotoxic anticancer platinum analogues. In order to investigate the mechanism of such a peripheral neuropathy, the effects of 1–100 µmol/l oxaliplatin were assessed on the nodal ionic currents of single frog myelinated axons as a model of peripheral excitable membranes. Oxaliplatin decreased both Na+ and K+ currents in a dose-dependent manner and within 5–10 min, without producing any marked changes in the current kinetics. It was about three to eight times more effective in reducing the Na+ than the K+ current. In addition, it shifted the voltage-dependence of both Na+ and K+ conductances towards negative membrane potentials. A negative shift in the steady-state inactivation-voltage curve of the peak Na+ current was also observed in the presence of oxaliplatin. These effects were not reversed by washing the myelinated axons with an oxaliplatin-free solution for at least 30 min. It is concluded that oxaliplatin modifies the voltage-dependent ionic channels mainly by altering the external surface membrane potential. The knowledge of such a mechanism may help to counteract the neurotoxic action of this anticancer agent.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 263-276.

 
  Effects of N-acyl-2-hydroxymethyl aziridines on in vitro proliferative responses of human lymphocytes stimulated by mitogens
A Baba 1), W Medjahed, H Merzouk, J Kajima Mulengi, J Belleville, J Prost

1)Laboratoire de Physiologie Animale et Biochimie, Département de Biologie, Faculté des Sciences, Université de Tlemcen, Algérie. fz_babaahmed@mail.univ-tlemcen.dz.


Aziridines have been shown to possess marked immunotropic activity. The aim of this work was to study the in vitro effects of different concentrations of three novel aziridines, 2-hydroxy-methyl-1-(N-phtaloylglycyl) aziridine (aziridine 1), 2-hydroxy-methyl-1-(N-phtaloylalanyl) aziridine (aziridine 2) and 2-hydroxy-methyl-1-(N-phtaloylphenylalanyl) aziridine (aziridine 3), on the proliferative responses of human lymphocytes stimulated by mitogens (concanavalin A (Con A) and lipopolysaccharide (LPS)), and interleukin-2 (IL-2), interleukin-6 (IL-6) secretion. The results showed that aziridines 1 and 3 significantly stimulated the resting and Con A or LPS lymphocyte proliferation at concentrations between 1 µmol/l and 1 mmol/l, in a dose-dependent manner, the action of aziridine 3 being the highest. They also increased IL-2 and IL-6 secretion. However, aziridine 2 had no effect on the resting lymphocyte proliferation in the absence of mitogens, at any concentration used, reduced Con A-stimulated T lymphocyte proliferation and LPS- stimulated B lymphocyte proliferation in a dose dependent manner and diminished IL-2 and IL-6 production. None of the three aziridines affected cell viability. In conclusion, the three aziridines used in this study displayed immunomodulatory properties. Aziridines 1 and 3 are potentially immunostimulant while aziridine 2 is immunosuppressive and could be used to provide nonspecific cell-mediated immune responses.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 277-287.

 
  Changes in the function and ultrastructure of vessels in the rat model of multiple low dose streptozotocin-induced diabetes
R Sotnikova 1), S Skalska, L Okruhlicova, J Navarova, Z Kyselova, J Zurova, M Stefek, R Hozova, V Nosalova

1)Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava 4, Slovakia. exfarosa@savba.sk.


In this study we investigated functional changes in the femoral artery and ultrastructural alterations in mesenteric vessels and capillaries in the rat model of multiple low dose streptozotocin (STZ)-induced diabetes. Participation of oxidative stress in this model of diabetes was established by studying the effect of the pyridoindole antioxidant stobadine (STB) on diabetes-induced impairment. Experimental diabetes was induced by i.v. bolus of STZ (20 mg/kg) given for three consecutive days to male rats. At the 12th week following STZ administration, the animals revealed typical signs of diabetes, such as polyphagia, polydypsia and polyuria. There was no weight gain in the diabetic groups throughout the experiment. No exitus occurred in any group. Diabetes was characterised with high levels of plasma glucose, no significant changes in lipid metabolism, decreased serum levels of glutathione, increased serum levels of the lysosomal enzyme N-acetyl-β-D-glucosaminidase (NAGA), injured endothelial relaxant capacity of the femoral artery and alterations in ultrastructure of mesenteric arteries and capillaries. Antioxidant STB in the dose of 25 mg/kg body weight i.p. (5 times per week) did not influence glucose levels, however, it mitigated biochemical, functional and ultrastructural changes induced by diabetes, suggesting a role of reactive oxygen species in diabetes-induced tissue damage.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 289-302.

 
  H/D isotope effects on protein hydration and interaction in solution
A Goryunov 1)

1)Molecular Biophysics Group, Institute of Biology, Karelian Research Centre, Russian Academy of Sciences, Pushkinskaya 11, 185910 Petrozavodsk, Russia. goryunov@krc.karelia.ru.


An approach has been suggested to study the H/D isotope effect on protein-water and protein-protein intermolecular interactions by determining the content of non-freezing water using low-temperature 1H NMR in mixed (H2O/D2O) water solutions. Direct data are obtained on the amount of H2O adsorbed (absolute hydration) in presence of the heavy isotope (deuterium D), and isothermals of H2O/D2O fractionation at protein surface groups are presented for temperatures between –10 °C and –35 °C and solutions of varying composition. The fractionation factor, &phis; = [x/(1 – x)]/[x0/(1 – x0)], where x and x0 are the fractions of deuterons in hydration and bulk water, respectively, appeared to be extremely high: &phis; ≫ 1 at 0.03 < x0 < 0.10. The high values of &phis; indicate a decrease in apparent hydration of protein molecules. A probable reason of the effect can be an inter-protein molecular solvent-mediated interaction induced by D2O. The excess of &phis; over 1 appears to provide a quantitative estimate of the fraction of hydration water affected by such interaction.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 303-311.

 
  Influence of membrane phospholipid composition and structural organization on spontaneous lipid transfer between membranes
R Pankov 1), T Markovska, P Antonov, L Ivanova, A Momchilova

1)Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University, Dragan Tzankov str. 8, 1164 Sofia, Bulgaria. rpankov@biofac.uni-sofia.bg.


Investigations were carried out on the influence of phospholipid composition of model membranes on the processes of spontaneous lipid transfer between membranes. Acceptor vesicles were prepared from phospholipids extracted from plasma membranes of control and ras-transformed fibroblasts. Acceptor model membranes with manipulated levels of phosphatidylethanolamine (PE), sphingomyelin and phosphatidic acid were also used in the studies. Donor vesicles were prepared of phosphatidylcholine (PC) and contained two fluorescent lipid analogues, NBD-PC and N-Rh-PE, at a self-quenching concentration. Lipid transfer rate was assessed by measuring the increase of fluorescence in acceptor membranes due to transfer of fluorescent lipid analogues from quenched donor to unquenched acceptor vesicles. The results showed that spontaneous NBD-PC transfer increased upon fluidization of acceptor vesicles. In addition, elevation of PE concentration in model membranes was also accompanied by an increase of lipid transfer to all series of acceptor vesicles. The results are discussed with respect to the role of lipid composition and structural order of cellular plasma membranes in the processes of spontaneous lipid exchange between membrane bilayers.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 313-324.

 
  Tetraphenylphosphonium-selective electrode as a tool for evaluating mitochondrial permeability transition pore function in isolated rat hepatocytes
A Lábajová 1), J Kofránek, P Křiváková, Z Červinková, Z Drahota

1)Institute of Pathophysiology, 1st Faculty of Medicine, Charles University, U Nemocnice 5, 120 00 Prague 2, Czech Republic. Anna.Labajova@lf1.cuni.cz.


The changes in mitochondrial membrane potential (Δψm) were used as an indicator for evaluating the mitochondrial permeability transition pore (MPTP) function. We found that in situ mitochondria in digitonin-permeabilized hepatocytes were coupled and responded to the addition of substrates, inhibitors and uncouplers. Ca2+-induced Δψm dissipation was caused by MPTP opening because this process was inhibited by cyclosporin A. MPTP opening was enhanced by the pro-oxidant tert-butyl hydroperoxide.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 325-331.

 
  Ghrelin suppression of potassium currents in smooth muscle cells of human mesenteric artery
M Mladenov 1), K Hristov, D Duridanova

1)Institute of Biology, Faculty of Natural Sciences and Mathematics, "Sv. Kiril i Metodij" University, Arhimedova 6, P.O.Box 162, Skopje, Republic of Macedonia. mitkoivan@yahoo.com.


Ghrelin is a 28-amino acid peptide hormone which modulates many physiological functions including cardiovascular homeostasis. Here we report some novel findings about the action of ghrelin on smooth muscle cells (SMC) freshly isolated from human mesenteric arteries. Ghrelin (10-7 mol/l) significantly suppressed the iberiotoxin-blockable component of potassium currents (IK) and depolarized the cell membrane, while having no effect on Ca2+ currents. Inhibition of inositol-trisphosphate (IP3)-activated Ca2+ release channels, depletion of sarcoplasmic reticulum (SR) Ca2+ stores, blockade of phospholipase D (PLD) or protein kinase C (PKC) each abolished the effect of ghrelin on IK, while the inhibition of phospholipase C (PLC) did not. These data imply that in human mesenteric artery SMC ghrelin suppresses IK via PLD, PKC and SR Ca2+-dependent signaling pathway.

General Physiology and Biophysics. Volume 25, 2006, No. 3: 333-338.