Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 8 (2003) No. 1

Volume 8 (2003) pp 5 – 18
Title	CAPACITANCE AND RESISTANCE OF THE BILAYER LIPID MEMBRANE FORMED OF PHOSPHATIDYLCHOLINE AND CHOLESTEROL)
Authors	Monika Naumowicz¹, Aneta D. Petelska¹ and Zbigniew A. Figaszewski^1,2 *
Abstract	Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 mF/cm2, and resistance to 1.44x10⁴and 2.12x10⁶ W cm², respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (G3), capacitance (C3), and conductance ( 1 ) R3- , as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265´10^-6 mol/m², 0.54 mF/cm², 1.381´10^-6 W^-1 cm^-2 and 3.748´10⁷, respectively.
Adress and Contact Informations	¹Institute of Chemistry, University of Białystok, al. J. Piłsudskiego 11/4, 15-443 Białystok, Poland, ²Laboratory of Interfacial Electrochemistry, Faculty of Chemistry, University of Warsaw, ul. Pasteura 1, 02-093 Warszawa, Poland * Corresponding author, Fax: +4885 745 75 81, E-mail: elchem@uwb.edu.pl

Volume 8 (2003) pp 19 – 24
Title	LYSOSOMAL HIGH MOLECULAR WEIGHT MULTIENZYME COMPLEX
Authors	Halina Ostrowska*, Katarzyna Krukowska, Joanna Kalinowska, Mirosława Orłowska and Ilona Lengiewicz
Abstract	Three acidic glycosidases: b-galactosidase (b-GAL, EC 3.2.1.23), a- neuraminidase (NEUR, sialidase, EC 3.2.1.18), N-acetylaminogalacto-6-sulfate sulfatase (GALNS, EC 3.1.6.4) and serine carboxypepidase cathepsin A (EC 3.4.16.1) form a functional high molecular weight complex in the lysosomes. The major constituent of this complex is cathepsin A, the so-called “lysosomal protective protein” (PPCA). By forming a multienzyme complex, it protects the glycosidases from rapid intralysosomal proteolysis, and it is also required for the intracellular sorting and proteolytic processing of their precursors. In man, a deficiency of cathepsin A leads to a combined deficiency of b-GAL and NEUR activities, called “galactosialidosis”. Multiple mutations identified in the cathepsin A gene are the molecular basis of this lysosomal storage disease. This review describes the structural organization of the lysosomal high molecular weight multienzyme complex and the importance of the protective protein/cathepsin A in physiology and pathology.
Adress and Contact Informations	Department of Biology, Medical Academy of Białystok, Poland * Corresponding author, E-mail: halost@amb.edu.pl

Volume 8 (2003) pp 25 – 30
Title	DAMAGE TO THE ERYTHROCYTE MEMBRANE CAUSED BY CHLOROPHENOXYACETIC HERBICIDES
Authors	Piotr Duchnowicz and Maria Koter
Abstract	We studied the damage caused to erythrocyte membranes by chlorophenoxyacetic herbicides. An increase in haemolysis was observed. The compounds investigated caused lipid bilayer damage by lipid peroxidation, as well as an increase in membrane fluidity at the 16th carbon atom of fatty acids was observed. Metabolites caused damage to membrane proteins – the free SH group content was increased. Higher toxicity of metabolites compared to basic compounds was observed.
Adress and Contact Informations	Department of Biophysics of Environmental Protection, University of Łódź, Banacha 12/16, 90-247 Łódź, Poland

Volume 8 (2003) pp 31 – 40
Title	*DISTURBANCES OF STEM CIRCUMNUTATIONS EVOKED BY WOUND-INDUCED VARIATION POTENTIALS IN Helianthus annuus* L**
Authors	Maria Stolarz*, Halina Dziubińska, Maciej Krupa, Agnieszka Buda, Kazimierz Trębacz and Tadeusz Zawadzki
Abstract	The relationship between evoked electrical activity and stem movements in three-week old sunflowers was demonstrated. Electrical potential changes (recorded by Ag/AgCl extracellular electrodes) and time-lapse images (from a top view camera) were recorded and analyzed. A heat stimulus applied to the tip of one of the second pair of leaves evoked a variation potential, transmitted basipetally along one side of the stem. After stimulation, disturbances of circumnutations occurred. They included: changes in the period, disorders in the elliptical shape, and, in some cases, reversion of direction (of movement). We suggest that asymmetrically propagated variation potential induces asymmetric stem shrinking and bending, which strongly disturbs circumnutations. Our results confirm the involvement of electrical potential changes in the mechanism of stem nutations.
Adress and Contact Informations	Department of Biophysics, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, PL-20-033 Lublin, Poland * Corresponding author, E-mail: stolarzm@biotop.umcs.lublin.pl

Volume 8 (2003) pp 41 – 48
Title	CHARACTERIZATION OF THE HUMAN LACTOFERRIN (HLF) CELL LINE HLFK1, GENERATED IN CBA MICE
Authors	Maja Kocięba, Michał Zimecki and Grzegorz Chodaczek
Abstract	A human lactoferrin-specific cell line was generated in CBA mice, sensitized with 200 mg HLF in Freund’s complete adjuvant. HLFK1 cells derived from the lymph nodes of these mice were maintained using HLF as the antigen. HLF was added at the beginning of each 14-day restimulation cycle, at a concentration of 100 mg/ml. The presentation of the antigen to HLFK1 was demonstrated using glass-adherent lymphocytes from spleens (GAL) as the antigen-presenting cells (APC). The presentation of HLF by GAL was highly efficient; a very low concentration of the antigen (1 mg/ml) was enough to stimulate proliferation of the HLFK1 cell line. HLFK1 did not proliferate in the presence of ovalbumin or bovine lactoferrin (BLF), which is structurally related to HLF. However, we found that BLF caused a reduction in the proliferation of the HLFK1 cell line when BLF was added to the cultures together with the antigen – (HLF). On the other hand, proliferation of the HLFK1 cell line was not inhibited by pretreatment of the antigen-presenting cells or T cells with BLF. Therefore, we suggest that bovine lactoferrin may interfere with the binding or uptake of the antigen (HLF). Alternatively, BLF may nonspecifically inhibit the activation of the HLFK1 cell line.
Adress and Contact Informations	Department of Experimental Therapy, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland

Volume 8 (2003) pp 49 – 53
Title	PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR a IS DOWNREGULATED IN THE FAILING HUMAN HEART
Authors	Joanna Karbowska^1,2*, Zdzisław Kochan¹ and Ryszard T. Smolenski^1,2
Abstract	Cardiac hypertrophy in humans is associated with a decrease in myocardial fatty acid b-oxidation (FAO) and accompanying alterations in metabolic gene expression. Flux through the cardiac FAO pathway, which is the principal source of energy production in the adult mammalian heart, is tightly controlled in accordance with energy demands. In rodents, the FAO pathway is under control of a nuclear peroxisome proliferator-activated receptor a (PPARa). We sought to delineate the molecular regulatory events involved in the energy substrate preference switch from fatty acids to glucose during cardiac hypertrophic growth in humans. We analysed the amount of PPARa protein in human cardiac tissue. PPARa protein level was measured in homogenates prepared from left ventricular biopsies taken from five control donor hearts and compared to the amount of this transcription factor in biopsies from five patients with compensated end-stage heart failure (HF) at the time of transplantation. Using Western blot analysis with a monoclonal antibody against human PPARa, we observed a significant decrease (54%) in the mean amount of PPARa in the group of HF patients compared to that in the donor tissue. This study indicates that the decrease in cardiac PPARa transcription factor gene expression observed in the failing human heart could play an important role in a reduction in fatty acid utilisation by the adult heart during cardiac hypertrophy.
Adress and Contact Informations	¹Department of Biochemistry, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland, ²Heart Science Centre, National Heart & Lung Institute, Imperial College School of Medicine, Harefield Hospital, Middx, UK * Corresponding author, E-mail: jonka@amg.gda.pl

Volume 8 (2003) pp 55 – 61
Title	THE HEMOLYTIC AND PHYSIOLOGICAL ACTIVITIES OF MIXTURES OF SOME PHENOXY AND ORGANOPHOSPHOROUS HERBICIDES
Authors	Halina Kleszczyńska¹, Dorota Bonarska¹, Krzysztof Bielecki² and Janusz Sarapuk¹
Abstract	Experiments were performed investigating the potential to improve the biological activity of some phenoxy and organophosphorous compounds by using them in binary mixtures. The compounds were: 2,4-dichlorophenoxyacetic acid (1) and its sodium salt (2), dibutyl 1-butylamino-1-cyclohexanephosphonate (3) and diethyl 9-butylamino-9-fluorenephosphonate (4), all widely used as herbicides. There were two test methods: the inhibition of cucumber (Cucumis sativus) growth induced by one single herbicide or by equimolar binary mixtures of herbicides; and, in parallel, the hemolytic efficiency of separate compounds or their mixtures. The hemolytic properties of the compounds were studied as hemolysis is generally a good measure of their toxicity, especially in the case of lipophilic compounds. Pig erythrocytes were used as good models for the determination of toxicity and the kinetics of red blood cell hemolysis. In the plant-based experiments, binary mixtures were found to display additive type toxicity. The compounds’ hemolytic activities were of additive or antagonistic types. In some combinations, the addition of a second component did not change the hemolytic efficiency of the first component, and vice versa.
Adress and Contact Informations	¹Department of Physics and Biophysics, Agricultural University, Norwida 25, 50-375 Wrocław, Poland, ²Department of Botany and Plant Physiology, Agricultural University, Cybulskiego 32, 50-205 Wrocław, Poland

Volume 8 (2003) pp 63 – 67
Title	A MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL INVESTIGATION OF GUINEA PIG SKIN AFTER THE INTRODUCTION OF SUBSTANCE P AND VIP
Authors	Hanna Gendek-Kubiak*, Jacek Danowski and Bogumił L. Kmieć
Abstract	The aim of this study was to examine a morphological picture of guinea pig skin that had been injected with neuropeptides (NPS)2 – substance P (SP) and guinea pig vasoactive intestinal peptide (VIP) – to elucidate their local influence. Routine histological stainings were performed, together with immunohistochemical reactions for T cells and for macrophages. In the deeper layers of the skin, T cell and macrophagic infiltrations were observed. The intensity of these changes was greater 24 hours after injections than that observed at the third hour of the experiment.
Adress and Contact Informations	Department of Histology and Embryology, Medical University of Łódź, Narutowicza 60, 90-136 Łódź, Poland * Corresponding author, E-mail: h_kubiak@hotmail.com

Volume 8 (2003) pp 69 – 75
Title	THE EFFECT OF A SELECTIVE INHIBITION OF POTASSIUM CHANNELS ON THE RELAXATION INDUCED BY NITRIC OXIDE IN THE HUMAN PREGNANT MYOMETRIUM
Authors	Beata Modzelewska*, Tomasz Kleszczewski and Anna Kostrzewska
Abstract	The aim of this study was to investigate whether apamin-sensitive K+ channels play a role in the NO induced relaxation of the human pregnant myometrium. Concentration-response curves for sodium nitroprusside (SNP) (10^-9-10^-4 M) were constructed in the absence and presence of 10^-8M apamin and 10^-7 M charybdotoxin (CTX). Preincubation with apamin resulted in a significant attenuation of the relaxation caused by SNP, while pre-treatment with CTX insignificantly decreased the SNP induced relaxation. Our findings suggest that apamin-sensitive K⁺ channels exist in the human pregnant myometrium and play a role in modulation of the myometrium response to NO donors.
Adress and Contact Informations	Department of Biophysics, Medical University of Białystok, Mickiewicza 2A, 15-230 Białystok, Poland * Corresponding author; E-mail: beamo@amb.edu.pl

Volume 8 (2003) pp 77 – 84
Title	ASSOCIATING OLIGONUCLEOTIDES WITH POSITIVELY CHARGED LIPOSOMES
Authors	Piotr Jurkiewicz¹,*, Andrzej Okruszek², Martin Hof³ and Marek Langner^{1, 4}
Abstract	Oligonucleotides (ODNs) are short (up to 30 bases) fragments of single-stranded nucleic acids that are used as sequence specific regulators of gene expression and anti-sense based therapeutics. ODNs are frequently aggregated with particulates in order to improve their pharmacological characteristics. Complexes of ODN and lipid aggregates are among the most commonly mentioned in the literature. In order to control the formation and final properties of such aggregates, a detailed description of how ODN interacts with the lipid surface is needed. In this paper, we present the results of fluorescence measurements regarded an association of 20 base ODN, labelled with fluorescein, and a lipid surface containing various amount of positive charge. Unilamellar lipid vesicles were formed from egg phosphatidylcholine (PC) and various amounts of the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). It was found that about 20 mol% of DOTAP in the lipid bilayer suffices to obtain complete ODN association. This result was further confirmed via measurements performed by fluorescence correlation spectroscopy (FCS). These in turn showed that the diffusion time of labelled ODN in the presence of cationic liposomes decreases. Also, the particle number and count rate were reduced, concurring with conclusions derived from steadystate fluorescence spectroscopy results.
Adress and Contact Informations	¹Institute of Physics, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland, ²Centre for Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland, ³J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic and Center for Complex Molecular Systems and Biomolecules, Dolejškova 3, CZ-18223 Prague, Czech Republic, ⁴Academic Centre for the Biotechnology of Lipids Aggregates, Przybyszewskiego 63/67, 51-148, Wrocław, Poland * Corresponding author

Volume 8 (2003) pp 85 – 95
Title	THE NONALLOSTERIC MECHANISM OF ENZYME ACTIVITY REGULATION. IS IT THE ONLY TRUE MECHANISM ?
Authors	Marian Kuczek*
Abstract	The nonallosteric regulation mechanism of enzyme reaction velocity assumes that the substrate and enzyme interact via a metal cation and form simple and mixed, mono- and multi-nuclear complexes. A solution of equations for individual cases gives a function of initial reaction velocity at any given substrate or modifier concentration. This function can describe kinetic effects that are considered allosteric, as well as phenomena omitted by commonly-accepted models.
Adress and Contact Informations	Department of Experimental and Applied Biology, University of Opole, Kominka 4, 45-035 Opole, Poland * Fax: (+48 77) 4545467; E-mail: kuczek@uni.opole.pl

Volume 8 (2003) pp 97 – 103
Title	*THE EFFECT OF SELENIUM ON THE ACCUMULATION OF SOME METALS IN Zea mays* L. PLANTS TREATED WITH INDOLE-3-ACETIC ACID**
Authors	Krystyna Pazurkiewicz-Kocot¹, Witold Galas² and Andrzej Kita²
Abstract	In this study, we examined the relationship between the accumulation of NaHSeO3, the plant hormone (IAA), and some nutrient elements (K⁺, Na⁺, Ca²⁺) in the tissues of the roots, mesocotyls and leaves of Zea mays L. plants. Our experiments were carried out with eight- to nine-day old maize plants (Zea mays L. var K33xF2) grown on Hoagland's medium containing the standard macro- and microelements, IAA and NaHSeO₃. The accumulation of selenium, potassium, sodium and calcium in the seedlings was measured by emission spectroscopy using a spectrometer with excitation by the argon inductively coupled plasma technique (ICP-AES). We observed that when selenite and phytohormone (IAA) are present in the external medium of growing plants, they change the uptake and accumulation of some cations (K⁺, Na⁺, Ca²⁺) in the leaf, mesocotyl and root tissues. The change of transport of some nutrient elements is probably one of the first observed symptoms of selenium’s effects on plants.
Adress and Contact Informations	¹Department of Plant Physiology, Faculty of Biology and Environmental Protection, Silesian University, Jagiellońska 28, 40-032 Katowice, Poland, ²Department of Analytical Chemistry, Institute of Chemistry, Silesian University, Szkolna 7, 40-032 Katowice, Poland

Volume 8 (2003) pp 105 – 110
Title	pH-DEPENDENT INFLUENCE OF A QUATERNARY AMMONIUM SALT AND AN AMINOESTER ON THE YEAST Saccharomyces cerevisiae ULTRASTRUCTURE
Authors	Ewa Obłąk, Ryszard Adamski And Tadeusz M. Lachowicz
Abstract	Quaternary ammonium salts inhibited the growth of yeast especially at pH higher (pH 8) than optimal. It was postulated that compounds integrate with the cell membrane and interfere with its functions. The yeast cell ultrastructure investigated under an electron microscope confirms this hypothesis. A relatively high percentage of cells treated at pH 6 with the quaternary ammonium salt of alanine derivative (DMALM-12) at the minimal inhibitory concentration showed an irregularity in the cell shape. No such irregularity was observed in the control. Besides, in the cells treated with the drug, practically no lipid droplets were seen at all. Inside the control cells, electron-dense round bodies were clearly seen and interpreted as vacuoles. These bodies were absent in the cells treated with DMALM-12. Although the yeast cells growing at pH 8 showed a more or less normal shape, they seemed to have difficulty in budding – no fully developed buds were found in the preparations. Only some convexities of the cell wall were seen that could be the beginning of budding which stopped early after the start. Some changes in the round bodies interpreted as vacuoles were visible: they were less dense and full of granules.
Adress and Contact Informations	Institute of Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland

Volume 8 (2003) pp 111 – 120
Title	*THE DUAL MECHANISM OF THE ANTIFUNGAL EFFECT OF NEW LYSOSOMOTROPIC AGENTS ON THE SACCHAROMYCES CEREVISIAE* RXII STRAIN**
Authors	Anna Krasowska¹*, Lucyna Chmielewska¹, Jacek Łuczyński², Stanisław Witek² and Karel Sigler³
Abstract	Quinacrine was used to visualize the intracellular pH changes in the yeast strain Saccharomyces cerevisiae RXII occurring after exposure to four recently-synthesized lysosomotropic drugs: DM-11, PY-11, PYG-12s and DMAL-12s. The cells took up quinacrine, mostly accumulating it in their vacuoles. DM-11 and PY-11 gave rise to diffuse quinacrine fluorescence throughout the cells, with the vacuoles staining to a somewhat greater extent than the cytosol. This quinacrine-detected overall acidification of the cell interior is very probably caused by blocking of plasma membrane H+-ATPase. PYG-12s gave rise to a strong vacuolar accumulation of the dye. Like the vacuolar ATPase inhibitor bafilomycin A1, DMAL-12s strongly lowered the intensity of quinacrine fluorescence. Owing to its low pKa, it can penetrate rapidly into the cells and may inhibit vacuolar H+-ATPase and prevent quinacrine-detectable vacuolar acidification without causing strong cell acidification. Since these drugs were found to penetrate into the cells, their lack of effect may reflect a higher resistance of both plasma membrane H+-ATPase and vacuolar ATPase to the drugs. Our data indicate that the lysosomotropic drugs under study have a dual action. On entering the cell, they cause intracellular acidification, very probably by inhibiting plasma membrane H+-ATPase and curtailing active proton pumping from the cells. Furthermore, they interfere with the function of V-type ATPase, causing vacuolar alkalinization and eventually cell death.
Adress and Contact Informations	¹Institute of Microbiology, Wrocław University, Przybyszewskiego 63-77, 51- 148 Wrocław, Poland, ²Chemistry Dept., Technical University of Wrocław, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland, ³Institute of Microbiology, Acad. Sci. Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic * Corresponding author, E-mail: aniak@microb.uni.wroc.pl

Volume 8 (2003) pp 121 – 126
Title	MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN HUMAN FIBROBLAST LINES INDUCED BY ANTHRACYCLINES DURING APOPTOSIS
Authors	Katarzyna Kania, Sylwia Dragojew and Zofia Jóźwiak*
Abstract	We show that treating human trisomic fibroblasts with anthracyclines – aclarubicin, daunorubicin and idarubicin – leads to certain changes in these cells; namely the activation of caspase 3, morphological changes and an increase in the level of intracellular calcium. These results suggest that anthracycline drugs are also able to induce apoptosis in pathological, trisomic cells.
Adress and Contact Informations	Department of Thermobiology, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland *Corresponding author; E-mail: zjozwiak@biol.uni.lodz.pl, Fax: (42) 6354473

Volume 8 (2003) pp 127 – 131
Title	CARBAMYLATION OF PROTEINS LEADS TO ALTERATIONS IN THE MEMBRANE STRUCTURE OF ERYTHROCYTES
Authors	Anna Pieniążek and Krzysztof Gwoździński
Abstract	The effect of the sodium cyanate-induced carbamylation (carbamoylation) of proteins in erythrocytes was studied using spin labelling and spectrophotometric methods. The experiments were conducted in whole blood and in erythrocytes in phosphate buffer using 25 mmol/L of sodium cyanate. Lipid membrane fluidity was determined using three spin-labelled fatty acids: 5-, 12- and 16-doxylstearic acids (5-DS, 12-DS, 16-DS). Internal viscosity was measured with Tempamine, using also EPR spectroscopy. Osmotic fragility was determined spectrophotometrically. Incubation of whole blood with sodium cyanate led to an increase in lipid membrane fluidity in the deeper region of the lipid layer, indicated by 12- and 16-doxylstearic acid, and a decrease near the surface (5-DS). Statistically significant results were obtained for the internal viscosity and osmotic fragility of erythrocytes. An increase in internal viscosity and increase in osmotic fragility were found in erythrocytes after incubation of whole blood, as well as in erythrocytes incubated with sodium cyanate in buffer. Alterations in internal viscosity were stronger in erythrocytes incubated with sodium cyanate in blood than in erythrocytes in the buffer. On the other hand, higher osmotic fragility was observed for erythrocytes in the buffer.
Adress and Contact Informations	Department of Molecular Biophysics University of Łódź, 90-273 Łódź, Poland

Volume 8 (2003) pp 133 – 140
Title	52 kD Ro/SS-A LOCALIZES TO PUNCTATE STRUCTURES IN THE CYTOPLASM OF EPITHELIAL CELLS
Authors	Adaniel P. Mccauliffe¹ and Anna Woźniacka²*
Abstract	Autoantibodies directed against 52 kD and 60 kD Ro/SS-A are frequently found in the sera of patients with lupus erythematosus and Sjögren’s syndrome-related disorders. Their location in the cell is subject to continuous debate in literature. It has been postulated that 52 kD Ro (52 Ro) co-localizes with the 60 kD Ro autoantigen in the nucleus, while others demonstrated that 52 Ro is primarily cytoplasmic. In order to resolve this controversy, 52 Ro protein was tagged with green fluorescence protein, overexpressed in A431 keratynocytes, and its location determined using fluorescence confocal microscopy. The intracellular location of the fusion protein was revealed via GFP autofluorescene and indirect immunofluorescence microscopy, using purified anti-52 Ro antibodies. The cellular locations of native 52 Ro in normal human keratinocytes, and in human A431 keratinocyte and HepG2 hepatocyte cell lines were similarly determined by utilizing 2 human anti-52 Ro antibodies purified from two different non-overlapping fragments of recombinant 52 Ro. In addition, colocalization of 52 Ro with mitochondria, lysosomes and endosomes was evaluated. It was found that both the 52 Ro-GFP fusion protein and the native 52 Ro localize in discrete cytoplasmic punctate structures separately from the mitochondria, lysosomes and endosomes. Furthermore, human autoantibodies that are reactive with denaturation-sensitive epitopes on 52 Ro recognize these cytoplasmic punctate structures, whereas antibodies directed against denaturation-resistant 52 Ro epitopes do not. This explains why the previously used antibody against denaturation-resistant 52 Ro epitopes failed to detect the protein in such punctate structures.
Adress and Contact Informations	¹Department of Dermatology, University of North Carolina at Chapel Hill, USA ²Department of Dermatology, Medical University of Łódź, Krzemieniecka 5, 60-017 Łodź, Poland * Corresponding author, Tel/Fax: (+48 42) 688 45 65, E-mail: wozniacka@bmp.net.pl

Volume 8 (2003) pp 141 – 146
Title	THE INFLUENCE OF X-RAYS ON HUMAN ERYTHROCYTES. PRIMARY RADICALS
Authors	Marta Gonciarz-Wach and Zofia Szweda-Lewandowska
Abstract	The effects on human erythrocytes of water-derived radicals generated by X-rays were studied under anaerobic conditions and in the presence of oxygen. Erythrocyte damage was estimated on the basis of the reduced GSH and MetHb content in the erythrocytes, the -SH group content in the membrane proteins and the amount of K+ released from the erythrocytes. The results obtained show that the level of reduced GSH was the most sensitive indicator of erythrocyte damage by X-rays followed by the efflux of K+. The processes of GSH oxidation took place most rapidly under air. At a dose of 100 Gy, the level of GSH fell to about 50%, whereas under argon and N2O to about 75% and 65%, respectively. A slight increase in the efflux of K+ was observed in preparations irradiated under air. However, when erythrocytes were irradiated under argon and N2O, the loss of K+ occurred at a dose 8-times higher. Changes in the remaining parameters occurred at considerably higher doses. On the basis of the results obtained one can say that oxygen is a factor increasing the toxicity of ˙OH radicals towards erythrocytes; however, e– aq present in the system can cause a decrease in damage to certain cellular components.
Adress and Contact Informations	Department of Molecular Biology, University of Łódź, Banacha 12/16, Poland

Volume 8 (2003) pp 147 – 159
Title	IS A FLUID-MOSAIC MODEL OF BIOLOGICAL MEMBRANES FULLY RELEVANT? STUDIES ON LIPID ORGANIZATION IN MODEL AND BIOLOGICAL MEMBRANES
Authors	Anna Wiśniewska¹, Jolanta Draus¹ and Witold K. Subczynski²
Abstract	The basic concept of the fluid-mosaic model of Singer and Nicolson, an essential point of which is that the membrane proteins are floating in a sea of excess lipid molecules organized in the lipid bilayer, may be misleading in understanding the movement of membrane components in biological membranes that show distinct domain structure. It seems that the lipid bilayer is an active factor in forming the membrane structure, and the lipid composition is responsible for the presence of domains in the membrane. The main role in the process of domain formation is played by cholesterol and sphingolipids. The results presented here show that in a binary mixture of cholesterol and unsaturated phospholipids, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase. This forms cholesterol-enriched domains or clustered cholesterol domains due to the lateral nonconformability between the rigid planar ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at double bond position. These cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by specific proteins which selectively locate in these domains and stabilize them as a result of protein-protein interaction. Such lipid domains are called “rafts” and have been shown to be responsible both for signal transduction to and from the cell and for protein sorting. We also looked at whether polar carotenoids, compounds showing some similarities to cholesterol and affecting membrane properties in a similar way, would also promote domain formation and locate preferentially in one of the lipid phases. Our preliminary data show that in the presence of cholesterol, lutein (a polar carotenoid) may segregate out from saturated lipid regions (liquid-ordered phase) and accumulate in the regions rich in unsaturated phospholipids forming carotenoid-rich domains there. Conventional and pulse EPR (electron paramagnetic resonance) spin labeling techniques were employed to assess the molecular organization and dynamics of the raft-constituent molecules and of the raft itself in the membrane.
Adress and Contact Informations	¹Biophysics Department, Institute of Molecular Biology and Biotechnology, Jagiellonian University, Krakow, Poland, ²Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

Volume 8 (2003) pp 161 – 170
Title	ORGANIZATION OF ANTIBIOTIC AMPHOTERICIN B IN MODEL LIPID MEMBRANES. A MINI REVIEW
Authors	Wiesław I. Gruszecki¹*, Mariusz Gagoś², Monika Hereć¹ and Peter Kernen³
Abstract	Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of fungal infections. According to the general understanding, the mode of action of AmB is directly related to the molecular organization of the drug in the lipid environment, in particular to the formation of pore-like molecular aggregates. Electronic absorption and fluorescence techniques were applied to investigate formation of molecular aggregates of AmB in the lipid environment of liposomes and monomolecular layers formed at the argon-water interface. It appears that AmB dimers, stabilized by van der Waals interactions, are present in the membrane environment along with the aggregates formed by a greater number of molecules. Linear dichroism measurements reveal that AmB is distributed between two fractions of molecules, differently oriented with respect to the bilayer. Molecules in one fraction remain parallel to the plane of the membrane and molecules in the other one are perpendicular. Scanning Force Microscopy imaging of the surface topography of the monolayers formed with AmB in the presence of lipids reveals formation of pore-like structures characterized by the external diameter close to 17 Å and the internal diameter close to 6 Å. All the findings are discussed in terms of importance of the molecular organization of AmB in the pharmacological action, as well as of the toxic side effects of the drug.
Adress and Contact Informations	¹Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, 20-031 Lublin, Poland, ²Department of Physics, Agricultural University, Lublin, Poland, 3Zyomyx, Inc., 26101 Research Road, Hayward, CA 94545, USA *Corresponding author, Fax + (4881) 537 61 91, E-mail: wieslaw@tytan.umcs.lublin.pl

Volume 8 (2003) pp 171 – 177
Title	THE NITROXIDES PIROLIN AND PIROLID PROTECT THE PLASMA MEMBRANES OF RAT CARDIOMYOCYTES AGAINST DAMAGE INDUCED BY ANTHRACYCLINES
Authors	Aneta Koceva-Chyła¹, Adam Sokal², Katarzyna Kania¹, Krzysztof Gwoździński³ and Zofia Jóźwiak¹
Abstract	This study was performed to evaluate the protective effects of pyrroline and pyrrolidine nitroxides Pirolin, PL, and Pirolid, PD, on the plasma membranes of rat cardiomyocytes treated in vitro with anthracycline drugs aclarubicin (ACL) and doxorubicin (DOX). The influence of two concentrations of drugs (10 and 20 μM) and nitroxides (0.1 and 1 mM) as well as their combinations (a drug and a nitroxide) on membrane fluidity was investigated. The plasma membranes of cardiomyocytes were labelled with a hydrophobic fluorescence probe 12-AS and membrane fluidity was estimated on the basis of the fluorescence anisotropy of the probe. We found that aclarubicin and doxorubicin induced a significant dose-dependent decrease in membrane fluidity, whereas the nitroxides (PL and PD) caused its increase. Preincubation of cardiomyocytes with Pirolin entirely protected plasma membranes of these cells against damage caused by DOX. In the same conditions no protective effect of Pirolid was observed. What is more, Pirolid in combination with DOX caused fluidisation of the plasma membranes of cardiomyocytes. Both nitroxides at low concentration (0.1 mM) protected plasma membranes against rigidification induced by aclarubicin, while high concentration (1 mM) was ineffective and caused fluidisation of the plasma membranes of cardiomyocytes.
Adress and Contact Informations	¹Department of Thermobiology and ³Department of Molecular Biophysics, University of Łódź, Łódź, Poland, ²Silesian Centre of Heart Disease, Zabrze, Poland

Volume 8 (2003) pp 179 – 183
Title	EFFECTS OF PYRROLINE AND PYRROLIDINE NITROXIDES ON LIPID PEROXIDATION IN HEART TISSUE OF RATS TREATED WITH DOXORUBICIN
Authors	Aneta Koceva-Chyła¹, Krzysztof Gwoździński², Agata Kochman³, Aneta Stolarska² and Zofia Jóźwiak¹
Abstract	Protection from doxorubicin-induced lipid peroxidation in vivo by two pyrroline and pyrrolidine nitroxides, Pirolin, PL, and Pirolid, PD, was examined in the heart tissue of rats treated with this drug. The level of lipid peroxidation was estimated on the basis of MDA content. A considerable (threefold) increase in the MDA amount was found in heart homogenates from rats injected with doxorubicin, whereas no significant changes in MDA content compared to control were observed in cardiomyocytes treated with the nitroxides (Pirolin or Pirolid) only. Pirolin injected simultaneously with doxorubicin showed antioxidative effect and markedly attenuated lipid peroxidation in the heart tissue caused by this drug. In contrast to Pirolin, structurally related Pirolid was ineffective in the protection of heart myocytes from DOX-induced lipid peroxidation.
Adress and Contact Informations	¹Department of Thermobiology and ²Department of Molecular Biophysics, University of Łódź, Łódź, Poland, ³Department of Pathological Anatomy, Medical University of Wrocław, Wrocław, Poland

Volume 8 (2003) pp 185 – 193
Title	TESTING CMS-P-LINKED AFLPs FOR SELECTION OF RYE HYBRID COMPONENTS
Authors	Piotr Tomasz Bednarek¹*, Anna Dąbkowska1, Irena Kolasińska² And Paweł Krajewski³
Abstract	Application of AFLPs linked to pollen fertility restoration and nonperforming genes evaluated in the C394-F2 hybrid was studied using a set of male sterile lines in the sterilising Pampa cytoplasm, several restorers and maintainer lines and, finally, two inbred lines backcrossed into cms-P, cms-R, cms-S and cms-C cytoplasms each. The set of male sterile lines based on the Pampa cytoplasm exhibited gradual variation in their ability to restore pollen fertility (starting from low and closing with high) in crosses with three unrelated restorers. Variations in the AFLPs between the analysed materials were observed, however, no clustering of the lines according to their sterile and fertile phenotypes was observed. The same markers, when applied to the population restorer (cv. Walet) that formed the C394-F2 cross permitted identification of plants with genotypes that could be recognized as restorers.
Adress and Contact Informations	¹Botanical Garden – Center for Biological Diversity Conservation PAS, Prawdziwka 2, 02-973 Warszawa, Poland, ²Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland, ³Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, Poznań, Poland * Corresponding author, E-mail: tmol.ob@ihar.edu.pl

Volume 8 (2003) pp 195 – 214
Title	COMPREHENSIVE ANALYSIS OF ALL TRIPLE HELICAL REPEATS IN b-SPECTRINS REVEALS PATTERNS OF SELECTIVE EVOLUTIONARY CONSERVATION
Authors	Anthony J. Baines*
Abstract	The spectrin superfamily (spectrin, a-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, a and b chains contain multiple copies of this repeat. b- spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical b-spectrins have 17 repeats; b-heavy spectrins have 30. Here, the repeats of five human b-spectrins, plus b-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical b are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind a-spectrin. Repeats 1 of b- spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of b-spectrin repeat 1 detects a-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.
Adress and Contact Informations	Randall Centre for Molecular Mechanisms of Cell Function, King’s College London, Guy’s Campus, London SE1 1UL, UK and Department of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK. * E-mail: A.J.Baines@ukc.ac.uk

Volume 8 (2003) pp 215 – 219
Title	THE EFFECT OF HYPOCHLORITE ON HUMAN ERYTHROCYTES PRETREATED WITH X-RADIATION
Authors	Anita Krokosz*
Abstract	Both hypochlorite and ionizing radiation induce oxidation processes of biomolecules. The effects are dependent to a large degree on the dose of the oxidizing agent. Previously we observed that split doses of gamma radiation caused lower hemolysis than the same but single doses. The critical factors influencing the occurrence of this effect were: the value of the first dose and the time between the doses. In this work we examined the effect of gamma radiation (40-400 Gy) on hemolysis of human erythrocytes induced by hypochlorite. Erythrocytes in PBS, hematocrit 2 %, were irradiated with doses of 40, 200 or 400 Gy. The dose-rate was 23.8 Gy/min. Cell suspensions were stirred during irradiation. After irradiation the erythrocytes were incubated for 1, 3 or 4 hours at room temperature and then hypochlorite was added to a 250 microM concentration. Control samples were erythrocytes treated only with NaOCl. The level of hemolysis was determined after NaOCl addition. Hemolysis of erythrocytes preirradiated with the dose of 400 Gy was lower than hemolysis of erythrocytes treated only with NaOCl. The effect was dependent on the time between the end of irradiation and the addition of NaOCl. In contrast, slightly higher hemolysis was observed for erythrocytes preirradiated with lower (40 or 200 Gy) doses of radiation. The observed effect is similar to that obtained for radiation-induced hemolysis. It suggests that ionizing radiation may induce structural and/or functional changes in erythrocytes, which make the cell more resistant to further oxidative damage.
Adress and Contact Informations	Department of Molecular Biophysics, University of Łódź, ul. Banacha 12/16, 90-237 Łódź, Poland * E-mail: krokosz@biol.uni.lodz.pl

Volume 8 (2003) pp 221 – 229
Title	CYSTOCYTE AND LYMPHOCYTE DERIVED FUSOMES/SPECTROSOMES: ANALOGIES AND DIFFERENCES: A MINI-REVIEW
Authors	Patrycja M. Dubielecka¹, Katarzyna Stebelska¹, Bożena Jaźwiec² and Aleksander F. Sikorski^1,3*
Abstract	Structures analogous to Drosophila spectrosomes were found in mammalian lymphocytes. Repasky and colleagues discovered an intracellular spectrin-rich structure in lymphoid cells, which had far-reaching parallels with the fusome/spectrosome of D. melanogaster germ cells. This fact implies that spectrosomes may be characteristic not only of insect germ cells, but also that an analogous structure may play an important role in other cell types. The term “spectrosome” was first used by Lin and Spradling in 1995 to describe a large sphere of fusomal material in D. melanogaster germline stem cells and their differentiated daughter cells – cytoblasts. In the D. melanogaster ovary, membrane skeletal proteins such as ankyrin, a/b spectrin as well as adducin-like Hts protein(s) were found in this specific organelle – spectrosome/fusome. These orgalelles are involved in the creation of mitotic spindles and D. melanogaster cyst formation and oocyte differentiation, but the role of analogous spectrinbased aggregates found in nucleated cells still remains unclear.
Adress and Contact Informations	¹University of Wrocław, Institute of Biochemistry and Molecular Biology, Laboratory of Cytobiochemistry, Przybyszewskiego 63/77, 51-148 Wrocław, Poland, ²Wrocław Medical University, Department of Haematology, Pasteura 4, 50-367 Wrocław, Poland, ³Academic Centre for Biotechnology of Lipid Aggregates, Przybyszewskiego 63/77, 51-148 Wrocław, Poland *Corresponding author: E-mail: afsbc@ibmb.uni.wroc.pl

Volume 8 (2003) pp 231 – 241
Title	RESOLVING THE IONOTROPIC RECEPTOR KINETICS AND MODULATION IN THE TIME SCALE OF SYNAPTIC TRANSMISSION
Authors	Maria Pytel¹*, Katarzyna Mercik^1,2 and Jerzy W. Mozrzymas¹
Abstract	Synaptic transmission plays a crucial role in signal transduction in the adult central nervous system. It is known that synaptic transmission can be modulated by physiological and pathological processes and a number of factors including metal ions, pH, drugs, etc. The patch-clamp technique allows to measure postsynaptic currents, but the mechanism of these currents modulation remains unclear. The estimated value of neurotransmitter transient indicates that this time course is very short and the activation of postsynaptic receptors is extremely non-equilibrient. The ultrafast perfusion system makes it possible to mimic synaptic conditions and, additionally, the agonist concentration can be controlled, which is very important for pharmacokinetic studies. In the present paper, examples of pharmacological modulation of mIPSC kinetics and currents evoked by ultrafast agonist application are presented.
Adress and Contact Informations	¹Department of Biophysics, Wrocław Medical University, Chałubińskiego 10, 50-368 Wrocław, Poland, ²Institute of Physics, Technical University of Wrocław, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland * Corresponding author: E-mail: maja@biofiz.am.wroc.pl

Volume 8 (2003) pp 243 – 247
Title	INDUCTION AND DECAY OF THERMOTOLERANCE IN HUMAN ERYTHROCYTES DETERMINED BY HEMOLYSIS
Authors	Małgorzata Rogozińska and Maria Koter
Abstract	Hemolysis was used as an endpoint for the measurement of damage to the plasma membrane in human erythrocytes after a single or a double heat shock. The thermotolerance of erythrocytes is a transitional phenomenon, reaching its maximum at a 3-hour incubation at 37°C between the heat shocks.
Adress and Contact Informations	Department of Biophysics of Environmental Protection, University of Łódź, ul. Banacha 12/16, 90–237 Łódź, Poland

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