Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 7 (2002) No. 1

Volume 7 (2002) pp 7-18
Title	*A USEFUL PROTOCOL FOR IN SITU* RT-PCR ON PLANT TISSUE**
Authors	Ewa Urbanczyk-Wochniak, Marcin Filipecki and Zbigniew Przybecki
Abstract	This study describes an effective method of in situ RT-PCR (RT- ISPCR) that was developed to localize gene expression in plant tissues. This RT- PCR technique was performed on sectioned tissues of female buds of the cucumber GY3 inbred line. The CUS1 gene, encoding the MADS-box type (agamus-like) protein, the expression pattern of which was described earlier [10], was used as a marker gene for optimisation of steps in the in situ RT-PCR inside the cells. For the identification of RT-PCR products inside the cells of the female buds, they were fixed in FAA solution, embedded in Paraplast Plus and cut into 7mm thick sections which were dewaxed by immersion in HistoClear and dehydrated with ethanol. They were washed in water, then in 0.02M HCl, 2xSSC and PBS buffer. In the next step of tissue pretreatment, the sections were digested with 1% pectinase. As shown, the pectinase treatment proved to be a crucial step in the tissue preparation procedure to get successful RT-PCR products. After washing in PBS buffer, the sections were digested with protease K followed by incubation with RNase-free DNase I, and subsequently washed in 2xSSC, 1xSSC and 0.5xSSC and finally in DEPC-treated water. Then the sections were covered with 50ml of the RT-PCR reaction mixture supplemented with 0.5mM digoxigenin dUTP and sealed with a coverslip. After amplification in situ the PCR products were identified with anti-digoxigenin antibody (Roche Molecular Biochemicals), conjugated with alkaline phosphatase. The data obtained showed that specific signals reflecting CUS1 gene expression were detected in the female flower buds of cucumber. The specificity of the in situ RT-PCR protocol was confirmed by dot blot hybridization of RT-PCR products with CUS1 cDNA probe.
Adress and Contact Informations	Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horticulture, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warsaw, Poland

Volume 7 (2002)pp 19-29/td>
Title	THE PRODUCTION OF TRANSGENIC POTATO PLANTS EXPRESSING HUMAN a-INTERFERON USING LIPOFECTIN- MEDIATED TRANSFORMATION
Authors	Wagdy A. Sawahel*
Abstract	The lipofectin system was used to transform potato protoplasts with plasmid DNA (pIG3031), which contains human a-interferon cDNA and codes for the selectable neomycin phosphotransferase II gene (NPT II). Both genes are under the control of the bi-directional plant active transcriptional promoter from Agrobacterium tumefaciens T-DNA. The criteria of phenotype stability after selective pressure removal, in vitro activity assay of NPT II, the biological analysis of a-interferon activity, Southern blot analysis and RNA slot blot were used to confirm the mitotic stability of the foreign gene and its expression and stable integration into the host genome. These studies demonstrate that human a-interferon cDNA can be correctly expressed in potato cells.
Adress and Contact Informations	Microbial Genetics Department, Genetic Engineering & Biotechnology Division, National Research Center, Dokki, Cairo, Egypt * Present address: Institute of Microbiology and Genetics, Vienna Biocenter, Developmental Genetics & Plant Biotechnology Section, University of Vienna, Bohrgasse 9, A-1030 Vienna, Austria

Volume 7 (2002)pp 31-38
Title	THE MECHANISM OF THE HEMOLYTIC ACTIVITY OF POLYENE ANTIBIOTICS
Authors	Agnieszka Knopik-Skrocka and Jozef Bielawski
Abstract	The kinetics of the filipin-, amphotericin B- and nystatin-induced hemolysis of human erythrocytes were investigated. Filipin-induced hemolysis is of the damage type. It is an all-or-none process, partly inhibited by Ca²⁺ or Ba²⁺ but not by Mg²⁺, Na⁺ or SO₄ 2-. The hemolytic activity of filipin is explained by the formation of large aggregates within the erythrocyte membrane in the form of large perforations, permeable to substances of low molecular weight as well as to macromolecules, including hemoglobin. In isotonic KCl solution, both amphotericin B and nystatin, at low concentrations, form smaller aggregates within the membranes. As a result, the permeability of the membranes to KCl increases and hemolysis occurs. However, the kinetics of the hemolysis induced by the two polyenes is complex. The process shows some features of the permeability type and some of the damage type. It is suggested that amphotericin B and nystatin may simultaneously form a number of transport systems, differing in their molecular organisation and hemolytic activity. Their participation in erythrocyte membrane permeability can be modified by small changes in membrane organisation and the chemical composition of the incubation medium. In isotonic solutions of divalent cation chlorides, and at higher antibiotic concentration, additional aggregates, allowing divalent cations to permeate, appear. These structures do not permit SO₄ 2- to permeate.
Adress and Contact Informations	Department of Cytology and Histology, A. Mickiewicz University, 61-701 Poznan, Fredry 10, Poland

Volume 7 (2002)pp 49-59
Title	STABLE GENETIC TRANSFORMATION OF GARLIC PLANTS USING PARTICLE BOMBARDMENT
Authors	Wagdy A. Sawahel*
Abstract	The improvement of garlic plants (Allium sativum L.) via biotechnological approaches is currently limited by the lack of an applicable direct gene transfer system. In this paper, we present the development of a genetic transformation system using particle bombardment for gene del ivery and immature clove-derived callus as the gene target. Plasmid DNA (pBI221.23), containing the selectable ” hpt„ gene for hygromycin resistance and the reporter ” gus! gene, was delivered into callus tissue that had been previously treated with aurintricarboxylic acid as an endogenous nuclease inhibitor. The transformed calli were selected using hygromycin B, regenerated, and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical ’-glucuronidase assay. The results indicated that biolistic transformation can lead to the transfer, expression and stable integration of a DNA fragment into garlic chromosomal DNA. The relative simplicity of this system is a good recommendation for its future use in the production of genetically modified garlic plants.
Adress and Contact Informations	Microbial Genetics Department, Genetic Engineering & Biotechnology Division, National Research Center, Dokki, Cairo, Egypt * Present address: Institute of Microbiology and Genetics, Vienna Biocenter, Developmental Genetics & Plant Biotechnology Section, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria

Volume 7 (2002)pp 61-67
Title	FATTY ACID PROFILING: ITS USEFULNESS IN THE EVALUATION OF MICROBIAL ASSOCIATIONS WITH THE GREEN MICROALGA Apatococcus constipatus
Authors	Robert Zarnowski*
Abstract	To determine differences in microbial community structures, fatty acids from two strains of the green microalga Apatococcus constipatus were isolated and identified by instrumental means. The main fatty acid s found were 16:0 and 14:0. These predominant acids represented more than 53% of the total fatty acid (content in both algal isolates. In addition, saturated fatty acids were present in much greater quantity than unsaturated ones. Differences between the strains in the composition of other, modified fatty acids were also evident. The occurrence of fatty acid biomarkers characteristic of certain taxonomic groups confirmed the presence of Gram-positive and Gram-negative bacteria, and fungi. Those observed variations were undoubtedly due to distinct community structures of symbiotic microorganisms living in close associations with the alga. The results presented here indicate that different isolates of the same alga might exhibit different microbial community structures.
Adress and Contact Informations	Department of Agricultural Microbiology, Agricultural University, Grunwaldzka 53, 50-375 Wroc“aw, Poland * E-mail: robert@ozi.ar.wroc.pl

The International Conference on

LIFE SCIENCES

September 22-26 2001, Gozd Martuljek, SLOVENIA

The international conference on Life Sciences, 2001 was organized by the Slovenian Biophysical Society

The following societies also participated in the organization of conference:
Slovene Association of Human Genetics of the Slovene Medical Society
Slovenian Pharmacological Society
Slovenian Genetic Society
Slovenian Society for Medical and Biological Engineering
Slovenian Society of Toxicology.

Volume 7 (2002) pp 71
Title	PREFACE
Authors	Dr. Marjeta Sentjurc and Dr. Janez Strancar
	The International conferences on Life Sciences are traditional annual meetings of Slovenian scientists and scientists from around the globe working in different fields of life sciences (from physics, chemistry, biology to pharmacy, medicine, biotechnology etc). Each year, one of the Societies from these fields takes on the organization of the conference. This year, the organization was performed by the Slovenian Biophysical Society. The idea for the Life sciences meetings originated in 1992. The basic aim was and remains to gather the scientists from Slovenia and neighbouring countries, which work in similar fields of research but use different approaches and techniques, once per year in order to give them the chance to beco me acquainted with the researches and techniques used by others, and to discuss issues of common scientific interest. Although each year0s conference is organised by a different Society, the scientific program should be of common interest. As the result of these meetings several interdisciplinary research projects have been initiated between different scientific institutes and clinics in Slovenia and neighbouring countries. The connective theme of Life Sciences, 2001 was The biophysics of membranes, and it covered the following topics: membrane structure and function, membranes and signal transduction, electroporation of membranes, receptors and ionic channels, genetic diseases related to membrane functions, and some other related themes: theory modelling and computer simulations, cell biophysics, toxicology, magnetic resonance imaging, radiation physics, biomechanics, and the biomechanical properties of the failing left ventricle. The plasma membrane is highly selective filter device for active transport and signal transduction. It controls the entry of nutrients to cells and the exit of waste products from cells, generates differences in ion concentration between the interior and exterior of cells, and acts as a sensor for exterior signals, allowing the cell to change in response to environmental conditions. The alteration and damage of cell membranes influence many intracellular metabolic processes. Since the biological membrane can be connected to many pathologic changes in cells and organisms, an understanding of its structure and function and its interaction with biologically active substances will lead to better protection from and therapy of some deleterious diseases. Interdisciplinary collaboration between scientists with different educational backgrounds is necessary to more rapidly solve problems in the field of membrane research, and we hope that this meeting and the publications which follow will contribute to a leap forward in membrane research and result in fruitful collaborations.

Volume 7 (2002) pp 73-175
Invited Lectures
Lectures List	THE BIOMECHANICS OF STANDING AND BALANCING IN PARALYZED PEOPLE Tadej Bajd, Matjaž Mihelj and Zlatko Matjačić - p74 SOME MULTIPLE-TIME-SCALE PROBLEMS IN MOLECULAR DYNAMICS Dušanka Janežič - p78 MAGNETIC RESONANCE IMAGING OF ARTERIAL THROMBI AND ITS POSSIBLE CORRELATION TO FIBRINOLYTIC TREAMENT Matija Kozak, Aleš Blinc, Igor Serša, Urša Mikac and Miloš Šurlan - p82 STRUCTURAL STUDIES ON MEMBRANE PROTEINS USING NON-LINEAR SPIN LABEL EPR SPECTROSCOPY Tibor Páli and Derek Marsh - p87 FUNCTIONAL MDR1 POLYMORPHISMS (G2677T AND C3435T) AND TCF4 MUTATIONS IN COLORECTAL TUMORS WITH HIGH MICROSATELLITE INSTABILITY Uroš Potočnik, Metka Ravnik-Glavač and Damjan Glavač - p92 MEMBRANE DESTABILIZATIONS SUPPORTING ELECTRO-PERMEABILIZATION Justin Teissié - p96 A GENERALIZED MATHEMATICAL MODEL OF BIOLOGICAL OSCILLATORS Tuong Ton-That - p101

Comunications
List of Comm.	INTRAVASCULAR PLUG FORMATION INDUCED BY POLY-APS IS THE PRINCIPAL MECHANISM OF THE TOXIN'S LETHALITY IN RATS/RAT TISSUES Matjaž Bunc, Lucija Šarc, Janez Rozman, Tom Turk, Kristina Sepčič and Dušan Šuput. - p106 THE INFLUENCE OF THE BIOMECHANICAL PARAMETERS OF THE HIP ON THE OUTCOME OF TREATMENT OF HIPS SUBJECT TO AVASCULAR NECROSIS OF THE FEMORAL HEAD Drago Dolinar, Veronika Kralj-Iglič, Aleš Iglič, Vane Antolič, Srečko Herman and Vinko Pavlovčič - p109 EXAMINATION OF FRAGMENT DOSE CONTRIBUTION IN HEAVY ION RADIOTHERAPY Marko Giacomelli, Radomir Ilić and Jure Skvarč - p112 FREQUENCY ENCODING OF INTRACELLULAR Ca²⁺ SIGNALS Vladimir Grubelnik and Marko Marhl - p115 CROW FOR LARGE SCALE MACROMOLECULAR SIMULATIONS Milan Hodošček, Urban Borštnik and Dušanka Janežič - p118 STUDY OF WATER MIGRATION BY MICRO MR IMAGING Tadej Kokalj, Peter Jevnikar and Igor Serša - p120 CYCLOPHOSPHAMIDE METABOLITES PERFORATE THE APICAL PLASMA MEMBRANES OF UROTHELIAL CELLS IN RATS Daša Koprivec and Kristijan Jezernik - p123 THE FORMATION OF AN INVERTED HEXAGONAL PHASE FROM THYLAKOID MEMBRANES UPON HEATING Zoltán Kóta, Balázs Szalontai, Magdolna Droppa, Gábor Horváth and Tibor Páli - p126 THE SMALL WORLD IN BIOPHYSICAL SYSTEMS STRUCTURAL PROPERTIES OF GLYCOLYSIS AND THE TCA CYCLE IN ESCHERICHIA COLI Borut Krajnc and Marko Marhl - p129 SPHERICITY OF THE FEMORAL HEAD ON ANTERIOR-POSTERIOR RADIOGRAPHS OF DYSPLASTIC HIPS Blaž Mavčič, Vane Antolič, Aleš Iglič and Veronika Kralj-Iglič - p132 CALCULATION OF SMALL ANGLE SCATTERING INTENSITIES FROM MOLECULAR DYNAMICS SIMULATION Franci Merzel and Jeremy C. Smith - p134 ELECTRIC CURRENT DENSITY IMAGING OF TABLET DISSOLUTION Urša Mikac, Alojz Demšar, Igor Serša and Franci Demšar - p136 RENAL INJURIES INDUCED BY CHRONIC INTOXICATION WITH MICROCYSTINS Aleksandra Milutinović, Bojan Sedmak, Irena Horvat-Žnidaršič and Dušan Šuput - p139 EPR SPECTROSCOPY OF COMMON NITRIC OXIDE - SPIN TRAP COMPLEXES Saviana Nedeianu and Tibor Páli - p142 HOW SHOULD DYSPLASTIC HUMAN HIPS BE EVALUATED? Borut Pompe, Vane Antolič, Aleš Iglič, Anton Jaklič, Veronika Kralj-Iglič and Blaž Mavčič - p144 THE SPLIT INTEGRATION SYMPLECTIC METHOD Matej Praprotnik and Dušanka Janežič - p147 THE RELATIONSHIP BETWEEN INNERVATION DENSITY AND DYNAMIC BALANCE FUNCTION Darja Rugelj - p149 THE INFLUENCE OF AMINO ACID SIDE CHAINS ON WATER BINDING TO THE COPPER(II) IN BIS(N,N-DIMETHYL-L-?-ISOLEUCINATO)-COPPER(II): AN EPR AND MOLECULAR MECHANICS STUDY Jasmina Sabolović and Vesna Noethig-Laslo - p151 QUANTIFICATION OF THE MR IMAGES OF HYDROGELS BY NMR RELAXATION MEASUREMENTS Ana Sepe, Saša Baumgartner, Julijana Kristl, Gojmir Lahajnar and Orest Jarh - p154 SHAPE OF VESICLES IN FLOW-THEORETICAL ANALYSIS France Sevšek - p157 HEART RATE VARIABILITY - A SHAPE ANALYSIS OF LORENZ PLOTS France Sevšek and Miroljub Jakovljević - p159 SELECTIVE RADIOGRAPHY OF 10B DISTRIBUTION IN ORGANS USING COLD AND THERMAL NEUTRON BEAMS Jure Skvarč, Marko Giacomelli, Hironobu Yanagië and Guido Kühne - p162 THE INFLUENCE OF Na⁺ IONS ON HISTAMINE SECRETION FROM MAST CELLS INDUCED BY NGF OR COMPOUND 48/80 Mateja Štempelj, Marija Čarman-Kržan and Ilonka Ferjan - p165 ELECTRON PARAMAGNETIC RESONANCE CHARACTERIZATION OF THE EXOPOLYSACCHARIDE LAYER PRODUCED BY BACTERIA David Stopar, Tatjana Trček and Janez Štrancar - p167 THE EFFECT OF PROPRIOCEPTIVE TRAINING ON NEURO-MUSCULAR FUNCTION IN PATIENTS WITH PATELLAR PAIN Vojko Strojnik, Rok Vengust And Vinko Pavlovčič - p170 ALTERNATING ELECTRIC FIELDS STIMULATE ATP SYNTHESIS IN ESCHERICHIA COLI Alexis Zrimec, Igor Jerman and Gojmir Lahajnar - p172

1ST POLISH MOLECULAR BIOTECHNOLOGY CONFERENCE

and

3RD SEMINAR OF POLISH BIOTECHNOLOGY STUDENTS' SCIENTIFIC CIRCLES

18-20th october 2001, GDAŃSK

Volume 7 (2002) pp 176
Late abstract
Abstracts List	*MICROSATELLITE POLYMORPHISM SELECTING LOCI IN A JAPANESE QUAIL (COTURNIX JAPONICA) POPULATION* Joanna Gruszczyńska, Elżbieta Michalska and Katarzyna Sobierajska - p176

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