Vol. 4 No. 4 December 1999

Volume 4 (1999) pp 525-536
Title ON IONIC TRANSPORT THROUGH A HIGH CONDUCTANCE LOCUST K+ CHANNEL IN VARIOUS VOLTAGES
Authors Zuzanna Siwy* and Zbigniew J. Grzywna
Abstract The influence of voltage on ionic transport through a high conductance locust K+ channel (BK channel) has been investigated. The nature of ionic flow has been examined by power spectrum, Hurst analysis, generalised entropy and surrogate data sets. The ordering influence of voltage on ionic current behaviour has been found.
Address and Contact Information Silesian University of Technology, Department of Physical Chemistry and Technology of Polymers, Strzody 9, 44-100 Gliwice, Poland
* Corresponding author, e-mail: siwy@zeus.polsl.gliwice.pl
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 537-551
Title ANNEXIN VI, A NOVEL ATP-DEPENDENT, PHOSPHOLIPID-BINDING PROTEIN
Authors Malgorzata Danieluk1, Aleksander F. Sikorski2, Slawomir Pikula1 and Joanna Bandorowicz-Pikula1,*
Abstract The determination of surface pressure (p) of a phosphatidylserine (PS) monolayer is used to study the interactions between specific phospholipid classes and various proteins. In the present study we show that ATP, but not ADP, in milimolar concentration ranges stimulate the increase of Dp in a PS monolayer evoked by annexin VI (AnxVI)/Ca2+ at a moderate initial p (p approx. 11 mN/m). The obtained results are consistent with ATP being a functional ligand for AnxVI. To further study the ATP binding site of AnxVI, we have used fluorescein 5’-isothiocyanate (FITC). This is useful in the characterization of nucleotide-binding sites of many membrane integral and cytosolic proteins. Under our experimental conditions FITC did not affect the binding of AnxVI to membranes but abolished the interaction of the protein with ATP insolubilized on agarose. This observation can be interpreted in terms of AnxVI possessing an ATP-binding site functionally similar to nucleotide-binding domains characterized in other ATP-dependent proteins. We also provide evidence that two AnxVI isoforms expressed constitutively in porcine liver differ from each other in respect to their ATP binding properties.
Address and Contact Information 1Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland,
2Institute of Biochemistry, University of Wroclaw, 63/67 Przybyszewskiego Street, 51-148 Wroclaw, Poland
* Corresponding author: fax: (+4822) 822 5342; e-mail: bandor@nencki.gov.pl
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 553-565
Title ION CURRENT FLUCTUATIONS IN ARTIFICIAL ION TRACK PORES — POWER SPECTRUM AND GENERALIZED ENTROPY
Authors Alexander Wolf1, Zuzanna Siwy2* , Yuri. E. Korchev3, Nicole Reber1 and Reimar Spohr1
Abstract The ion current through individual etched ion tracks (diameter ?50 nm, length ?12 ?m) in a poly (ethylene terephthalate) membrane is recorded at pH 7 as function of applied voltage (-5V to +5V) across the membrane. With increasing voltage, the ion current changes abruptly from random oscillations to structured fluctuations. The power spectrum and the generalized entropy of the recorded current reminds of the potassium channel of a locust muscle cell.
Address and Contact Information 1GSI Darmstadt, Planckstr. 1, D-64291 Darmstadt, Germany,
2Silesian University of Technology, Strzody 9, 44-100 Gliwice, Poland,
3Imperial College London, London W12 0NN, England
* Corresponding author
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 567-582
Title CHOLESTEROL-INDUCED VARIATIONS IN FLUCTUATIONS OF THE PORES IN BILAYER LIPID MEMBRANE
Authors Stanislawa Koronkiewicz and Krzysztof Bryl*
Abstract The constant-current chronopotentiometric measurements of egg yolk phosphatidylcholine bilayer membrane without and with cholesterol are presented. It was demonstrated that the constant-intensity current flow through the bilayer membranes without and with cholesterol generated the oscillating pores in their structures. The presence of cholesterol in the bilayer membrane increased the value of critical potential at which pores could be formed. The shift in the distribution of calculated pore radii towards smaller values was observed in the bilayer containing cholesterol. It was postulated that greater stability of bilayer with cholesterol resulted from increased critical pore radius (at which the bilayer would rupture). The implications of the membrane cholesterol for the application of constant-current method as a biotechnological tool for incorporating molecules of different size are discussed.
Address and Contact Information University of Agriculture and Technology, Department of Physics and Biophysics, 10-957 Olsztyn, Poland
* Corresponding author
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 583-597
Title PREPARATION OF PEG-GRAFTED IMMUNOMAGNETOLIPOSOMES AND THEIR APPLICATION IN CELL SORTING
Authors Joan Carles Domingo1*, Margarita Mercadal1, Jordi Petriz2, Joan Garcia2 and Maria Africa De Madariaga1
Abstract Immunomagnetic systems have been used for positive selection of the cell fraction from a mixture using appropiate surface markers with satisfactory results, as hematopoietic CD34+ cells. In this work, we report the development of poly(ethylene glycol) (PEG)-grafted immunoliposomes loaded with dextran-magne-tite particles as the separation vehicles for immunomagnetic separation techniques. The magnetic ferrofluid was encapsulated into PEG-liposomes by the FTS methodology. The magnetoliposomes had a liposomal size around 800 nm and a Fe/lipid molar ratio of 0.87?0.30, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes were prepared by coupling the My10 mAb and bound specifically to CD34+ KG-1a cells in culture and in mixtures with CD34- CHO cells. The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO with a 10% initial of CD34+ KG-1a cells. The purity of the MiniMACS-positive fraction and the capture efficiency depended on the liposome concentration and antibody density used, related to the nonspecific cell binding of immunomagnetoliposomes due to the ferrofluid adsorbed and to the presence of whole antibody molecules in the liposome surface. The CD34+ cells isolated retained viability with an estimated recovery of 45-50%.
Address and Contact Information 1Department of Biochemistry and Molecular Biology, Faculty of Chemistry, University of Barcelona, Martí i Franqués, 1, 08028 Barcelona, Spain,
2Department of Cryobiology and Celullar Therapy, Oncologic Research Institute,Autovía de Castelldefells, Km. 2,7, 08907-L'Hospitalet, Barcelona, Spain
*Corresponding author. Fax: 34-93-402.12.19; e-mail:domingo@sun.bq.ub.es
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 599-601
Title RAT BRAIN Ca2+- ATPase IS A SUBSTRATE FOR PROTEIN PHOSPHATASES PP1 AND PP2A
Authors Ludmila Zylinska*, Ewa Gromadzinska and Lilla Lachowicz
Abstract A possible role of serine/threonine protein phosphatases PP1 and PP2A in the regulation in vitro of the plasma membrane Ca2+-ATPase purified from rat cortical and cerebellar synaptosomal membranes was investigated. Calcium pump, the enzyme responsible for the maintenance of intracellular calcium homeostasis, is regulated by several mechanisms, including phosphorylation by protein kinases. Here we demonstrate that the protein phosphatases action decreased the activity of native Ca2+-ATPase, and increased the stimulatory effect of calmodulin. Moreover, the calcium pump dephosphorylated by PP1 and PP2A revealed the presence of the additional sites, accessible for a PKA-mediated phosphorylation. The subsequent PKA or PKC phosphorylation of the dephosphorylated cortical and cerebellar Ca2+-ATPase differentially regulated its hydrolytic activity. This study confirms that Ca2+-ATPase in nervous cell is phosphorylated in vivo, and shows for the first time that its activity may be directly regulated by PP1 and PP2A.
Address and Contact Information Neurochemical Laboratory, Department of Biochemistry, Medical University, 6 Lindley Street, 90-131 Łódz, Poland
* Corresponding author: e-mail: luska@psk2.am.lodz.pl
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 611-623
Title BIOPHYSICAL CHARACTERISATION OF LIPOSOMAL DELIVERY SYSTEMS FOR LIPOPHILIC DRUGS: CYCLOSPORIN A AS AN EXAMPLE
Authors Alfred Fahr1 and Gerald Reiter2
Abstract Liposomal formulations of cyclosporin A have been investigated in a series of studies. One of the most used arguments for the development of this pharmaceutical preparation was the high lipophilicity of cyclosporin A, which ensures an encapsulation of cyclosporin A in the liposomal bilayer. From this study on the interaction of cyclosporin with lipids spread as a monolayer on the air/water interface we demonstrate, that one molecule of cyclosporin A occupies an area of 260 ?2 in the lipid monolayer. These data correlate well with molecular modelling data, calculating a cross-sectional area of 230 ?2 for a cyclosporin A molecule. Calculation of a partition coefficient using these data at different surface pressures results in a value of about 4000 at a surface pressure of about 31 mN/m. This partition coefficient is also found in bilayer membranes supporting the notion, that phospholipid bilayer systems have a surface pressure of about 32 mN/m. If the lipid:cyclosporin A ratio exceeds a value of 20, the lipid monolayer becomes nonlinear. Cyclosporin A starts to detoriate liposomal membranes at similar lipid:cyclosporin ratios. It could also be concluded from our measurements at the air/water interface, that cyclosporin A is able to exchange binding places between membranes rapidly with a time constant of about 2.5 min. Lipid monolayer experiments are not only an easy and fast method to obtain information about molecule interactions with lipid membranes, but is also a suitable method to develop liposomal formulations of drugs.
Address and Contact Information 1Institute of Pharmaceutical Technology and Biopharmaceutics, Philipps-University Marburg; Ketzerbach 63, D-35032 Marburg, Germany,
2Institute of Physical Chemistry, University of Vienna, Althanstrasse 14, POB 217 A-1091 Vienna, Austria
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 625-630
Title PICOSECOND LASER PULSES MEDIATED DRUG RELEASE FROM MAGNETOLIPOSOMES
Authors Melánia Babincová1, Danuta Leszczynska2, Phoumyphoune Sourivong3 And Peter Babinec1
Abstract Liposomes made from dipalmitoyl-phosphatidylcholine and containing 6-carboxyfluorescein and dextran-magnetite entrapped in their aqueous interior compartments have been irradiated with a picosecond laser pulses. Substantial amounts of carboxyfluorescein were released in response to a single picosecond laser pulse and almost complete release was achieved using four laser pulses, which may be useful for laser induced delivery of therapeutic agents and other applications of lasers in biological systems.
Address and Contact Information 1Department of Biophysics and Chemical Physics, Comenius University, MFF UK, Mlynská dolina F1, 842 15 Bratislava, Slovakia,
2Department of Civil Engineering, FAMU-FSU College of Engineering, Tallahassee, FL 32310, USA, 3Physics Department, University of North Dakota, Grand Fork, ND 58202, USA
[Rozmiar: 1332 bajtów]

Volume 4 (1999) pp 631-645
Title ORGANIZATION OF THE Drosophila GENOME
Authors Nicolas Jullien1 , Alain Henaut2 and Raymond Miassod1*
Abstract The distribution of (A,T)-rich regions has been investigated on a 835kb DNA fragment (D835), from the Drosophila X sex chromosome, unsequenced but provided with detailed physical maps for restriction enzymes recognizing (A,T)- or (C,G)- or (mixte)-motifs, and on several sequenced DNA fragments, 100-600 kb long, from the autosomal chromosomes. Numerous (A,T)-rich regions are present in all DNA fragments. Their size varies from 0.2 kb to several kb in all cases, except for D835 where some of them extend to 20-30 kb. The relationship between these (A,T)-rich regions and several chromosome landmarks has been examined in the particular case of D835. Topo II in-vitro sites are randomly distributed with regard to (A,T)-richness. However, transcription units and repeated regions are significantly localized outside (A,T)-rich regions. On the opposite, SARs and ARSs are mostly localized within (A,T)-rich regions. Lastly, topo II in-vivo sites are almost exclusively localized in (A,T)-rich regions. Speculations are proposed on why and how (A,T)-rich regions may have appeared during the emergence of Drosophila genome from a primitive genome.
Address and Contact Information 1Laboratoire de Génétique et Physiologie du Développement, IBDM, Luminy, case 907, 13288 Marseille, Cedex 9, France
2Centre de Génétique Moléculaire du CNRS, Laboratoire associé a l'Université Pierre-et-Marie Curie et a l'Université Versailles-Saint-Quentin, F-91118 Gif-sur-Yvette Cedex, France
[Rozmiar: 1332 bajtów]