Vol. 9 No. 1 March 2004

Volume 9 (2004) pp 3-13
Title PLASMID CONDENSATION INDUCED BY CATIONIC COMPOUNDS: HYDROPHILIC POLYLYSINE AND AMPHIPHILIC CATIONIC LIPID
Authors Monika Chaszczewska-Markowska1, Maciej Ugorski1 and Marek Langner2,3
Abstract The construction of an efficient carrier for genetic material is a major research objective that needs to be achieved before gene therapy can become a viable pharmacological approach. Artificial aggregates containing nucleic acids are one of the options for the systemic delivery of genetic information. The diversity of functions the aggregate is expected to fulfill necessitates its complex architecture. In order to obtain a complex supramolecular aggregate, formed from elements that are themselves complex molecules, appropriate procedures based on the detailed understanding of processes at the molecular level are required. In this study, we investigated how the various properties of cationic compounds affect nucleic acid condensation. The combination of two condensing agents, differing in their affinity towards water, when mixed with plasmids, resulted in aggregates which are resistant to enzymatic digestion and which form particles with well-defined size distributions. Such uniform and well-defined complexes may subsequently be further modified in order to obtain a fully functional genetic material carrier.
Address and Contact Information 1Laboratory of Glycobiology, Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland,
2Institute of Physics, Wrocław University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland,
3Academic Centre for the Biotechnology of Lipid Aggregates, Przybyszewskiego 63/77, Wrocław, Poland
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Volume 9 (2004) pp 15-30
Title THE EFFECT OF TRIETHYLLEAD ON THE MOTILE ACTIVITY OF WALKER 256 CARCINOSARCOMA CELLS
Authors Jolanta Sroka1*, Rafał Kamiński1, Marta Michalik1, Zbigniew Madeja1, Stanisław Przestalski2 and Włodzimierz Korohoda1
Abstract The effect of triethyllead (TriEL) on the morphology and motile activity of Walker 256 carcinosarcoma cells was investigated. It was found that both 2 and 5 mM TriEL affected the cellular motility in a dose- and timedependent manner. Initially, 2 mM TriEL caused the formation of blebs instead of lamellipodia at the front of some cells and stimulated the migration of Walker cells, but after 2 hours of 2 mM TriEL treatment, a reduction of cellular motility was observed. In the presence of 5 mM TriEL, Walker 256 carcinosarcoma cells rounded up, and their rate of movement was reduced. Moreover, the treatment of Walker carcinosarcoma cells with TriEL caused the disruption of microtubules and affected the F-actin distribution at both concentrations. At a concentration of 2 mM TriEL, the actin staining intensity was greatest in the tail of front-tail polarised blebbing cells and the actin layer was very thin at the leading edge. The control cells showed linear cortical F-actin distribution and somewhat less intense cytoplasmic staining at the same TriEL concentration. Cells treated with 5 mM TriEL showed an under-membrane pattern of actin distribution.
Address and Contact Information 1Department of Cell Biology, Faculty of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland,
2Department of Physics and Biophysics, Agricultural University, Norwida 25, 50-375 Wrocław, Poland
* Corresponding author: e-mail: jola@mol.uj.edu.pl
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Volume 9 (2004) pp 31-45
Title DNA DAMAGE IN HUMAN COLONIC MUCOSA CELLS INDUCED BY BLEOMYCIN AND THE PROTECTIVE ACTION OF VITAMIN E
Authors Katarzyna Woźniak1, Michał Arabski1, Ewa Małecka-Panas2, Józef Drzewoski3 and Janusz Błasiak1*
Abstract Using the alkaline comet assay, we showed that bleomycin at 0.1-5 mg/ml induced DNA strand breaks and/or alkali-labile sites, measurable as the comet tail moment, in human colonic mucosa cells. This DNA damage was completely repaired during a 120-minute post-treatment incubation of the cells. Post-treatment of the bleomycin-damaged DNA with 3-methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage, indicating that the drug could induce alkylative bases in DNA. We did not observe any change in the comet tail moment in the presence of catalase. Vitamin E ((+)-a-tocopherol) decreased DNA damage induced by bleomycin. The results obtained suggest that hydrogen peroxide might not be involved in the formation of DNA lesions induced by bleomycin in the colonic mucosa cells, and that vitamin E may exert protective effects on these cells.
Address and Contact Information 1Department of Molecular Genetics, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland,
2Department of Digestive Tract Diseases, Medical University of Łódź, Kopciłskiego 22, 90-153 Łódź, Poland,
3Department of Clinical Pharmacology, Medical University of Łódź, Rewolucji 1905 37/39, 90-214 Łódź, Poland
* Corresponding author; phone +48-42 635 4334, fax +48-42 635 4484, e-mail: januszb@biol.uni.lodz.pl
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Volume 9 (2004) pp 47-60
Title CAVEOLAE AS AN ADDITIONAL ROUTE FOR INFLUENZA VIRUS ENDOCYTOSIS IN MDCK CELLS
Authors Isabel Nunes-Correia1,2, Ana Eulalio1,2, Shlomo Nir3 and Maria C. Pedroso De Lima1,2,*
Abstract Clathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is internalized via clathrin-coated pits and/or caveolae in Madin Darby canine kidney (MDCK) cells. By applying pharmacological manipulations to selectively disrupt the cell internalization pathways, we found that, in MDCK cells, the influenza virus may be internalized via caveolae in addition to entry by clathrin-mediated endocytosis. However, a small contribution by another mode of entry, as recently proposed [Sieczkarski, S.B. and Whittaker, G.R., J. Virol. 76 (2002) 10455-10464], cannot be excluded.
Address and Contact Information 1Department of Biochemistry, University of Coimbra, Apartado 3126, 3000 Coimbra, Portugal,
2Center for Neuroscience and Cell Biology, University of Coimbra, 3000 Coimbra, Portugal,
3Seagram Center for Soil and Water Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel
* Corresponding author: Maria C. Pedroso de Lima; E-mail: mdelima@ci.uc.pt
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Volume 9 (2004) pp 61-67
Title CHANGES OF GABAA RECEPTOR ACTIVATION KINETICS IN HIPPOCAMPAL NEURONS CULTURED FOR DIFFERENT PERIODS OF TIME
Authors Jerzy W. Mozrzymas1* and Andrea Barberis1,2
Abstract Cell culture is a convenient model for pharmacokinetic studies, but during the culture period, GABAA receptors are likely to undergo different modulatory processes. In this study, the current responses to ultrafast GABA applications were recorded from patches excised from neurons cultured for either up to two days (short-term culture) or for more than two weeks (long-term culture). The dose-dependencies of the currentrising phases revealed significant differences between the two groups. In the short-term cultures, the responses to both saturating and non-saturating GABA concentrations were slower than in the case of the long-term cultures. We conclude that the GABAA receptors in cultured neurons undergo profound kinetic changes involving the modulation of the binding reaction and transitions between bound states.
Address and Contact Information 1Department of Biophysics, Wrocław Medical University, Chałubiłskiego 10, 50-368 Wrocław, Poland,
2Neuroscience Program and Istituto Nazionale Fisica della Materia (INFM) Unit, International School for Advanced Studies (SISSA), via Beirut 2-4, 34-014 Trieste, Italy
*Corresponding author, tel: +48 71 7841413, fax: +48 71 7840088, e-mail: mozrzy@biofiz.am.wroc.pl
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Volume 9 (2004) pp 69-81
Title SERA OF LUNG CANCER PATIENTS AFFECT THE RELEASE OF TH1, TH2 AND MONOCYTE-DERIVED CYTOKINES, AND THE EXPRESSION OF IL-2Ra BY NORMAL, STIMULATED MONONUCLEAR CELLS
Authors Magdalena Chechliłska1, Anna Duma1, Krystyna Świerkowska1, Janina Kamiłska2 and Jan Steffen1
Abstract We have shown that the sera of lung cancer patients affect the response of ConA-stimulated normal peripheral blood mononuclear cells by decreasing the expression of IL-2Ra and inhibiting the release of IL-1b and IL-2. A tendency to enhance the release of IL-6 was also observed. We conclude that an imbalance in the Th1/Th2 cytokine response, typical for cancer patients, may at least partly be related to soluble factors circulating in the patients’ blood. We discuss a putative role of serum IL-10, IL-1ra, and soluble IL-2Ra in the effects observed.
Address and Contact Information 1Department of Immunology and 2Department of Tumour Markers, The Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland
* Corresponding author: tel./fax +48 22 6449085, e-mail: chech@coi.waw.pl
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Volume 9 (2004) pp 83-94
Title NICKEL IMPAIRS THE REPAIR OF UV- AND MNNG-DAMAGED DNA
Authors Katarzyna Woźniak and Janusz Błasiak*
Abstract Nickel(II) is reported to be genotoxic, but the mechanisms underlying its genotoxicity are largely unknown. It can interfere with DNA repair and this may contribute to its genotoxicity. We studied the effect of nickel chloride on the repair of DNA damaged by UV radiation or N-methyl-N-nitro-Nnitrosoguanidine (MNNG) in human lymphocytes using the alkaline comet assay. Nickel(II) at 1 mM caused an accumulation of DNA breaks during repair incubation, which could follow from the inhibition of the polymerization/ligation step of UV-damaged DNA repair. On the other hand, nickel(II) inhibited the formation of transient DNA breaks brought by the repair process after incubation with MNNG at 5 mM, which might follow from interference with the recognition/incision step of excision repair. Additionally, nickel at 1 mM inhibited the activity of formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (Alk A), enzymes involved in DNA excision repair. A decrease in endonuclease III (Endo III) activity was observed at 2 and 5 mM of nickel chloride. Our results suggest that nickel(II) at non-cytotoxic concentrations can inhibit various steps of DNA excision repair, and this may contribute to its genotoxicity.
Address and Contact Information Department of Molecular Genetics, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland
*Corresponding author: tel: +48-42 635 43 34, fax: +48-42 635 44 84, e-mail januszb@biol.uni.lodz.pl
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Volume 9 (2004) pp 95 - 105
Title TWO SEQUENCES ENCODING CHALCONE SYNTHASE IN YELLOW LUPIN (Lupinus luteus L.) MAY HAVE EVOLVED BY GENE DUPLICATION
Authors Dorota Narożna1, Jakub Paś2, Jolanta Schneider1 and Cezary J. Mądrzak1*
Abstract Two full copy cDNA sequences encoding chalcone synthase (CHS) were selected from a yellow lupin (Lupinus luteus L.) root and nodule cDNA library, and sequenced. Analysis of their open reading frames gave evidence that both encode the functional enzyme. Sequence alignment and phylogenetic studies on the DNA and protein level of these clones compared to the sequences of chalcone synthases from 54 other plant species reveal the possibility that lupin chalcone synthase is encoded by a multigene family consisting of at least two distinct genes that probably diverged by gene duplication. The duplication event is estimated to have taken place about 16 million years ago.
Address and Contact Information 1Department of Biochemistry and Biotechnology, August Cieszkowski Agricultural University of Poznań, Wołyńska 35, 60-637 Poznań, Poland,
2BioInfoBank Institute, Limanowskiego 24A, 60-744 Poznań, Poland
*Corresponding author
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Volume 9 (2004) pp 107-122
Title THE EVALUATION OF THE GENOTOXICITY OF TWO COMMONLY USED FOOD COLORS: QUINOLINE YELLOW (E 104) AND BRILLIANT BLACK BN (E 151)
Authors Violetta K. Macioszek* and Andrzej K. Kononowicz**
Abstract Additives, especially colors, are in widespread use in the food industry. With the exception of the quinolines, food colors are relatively weak mutagens and are certified as safe additives despite reports that some people have allergic reactions to them. The number of food additives is still on the increase, and research on their potential mutagenic/carcinogenic activity in vivo is very expensive. Using two different cellular model systems, human lymphocytes in vitro and Vicia faba root tip meristems of in vivo, we evaluated the potential cytological and genotoxic effects of two dyes: Quinoline Yellow (E 104) and Brilliant Black BN (E 151). Two relatively new, very sensitive and rapid tests – the micronucleus and Comet assays – were used in this study. The data provided in this paper showed the genotoxic effects of the two analyzed food colors, and confirmed the diagnostic value of the MN and Comet assays for screening potentially genotoxic substances.
Address and Contact Information Department of Cytogenetics and Plant Molecular Biology, University of Łódź, S. Banacha 12/16, 90-237 Łódź, Poland
*Current address: Institute of Plant Biochemistry, Department of Stress and Developmental Biology, Weinberg 3, 06 120 Halle, Germany;
e-mail: vmaciosz@ipb-halle.de
**Corresponding author: e-mail: akononow@biol.uni.lodz.pl.
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Volume 9 (2004) pp 123-133
Title MAR/SAR ELEMENTS FLANK THE RAT HST70 GENE TRANSCRIPTION UNIT
Authors Wiesława Widłak1* and Piotr Widłak2
Abstract The rat hst70 gene is specifically expressed in spermatocytes and spermatids. This tissue-specific expression of the gene is primarily mediated through cis-acting elements located within the 0.4 kb segment upstream of the coding region, including two transcription initiation sites. Here, we study the 5’ and 3’ distal elements flanking the hst70 gene and find that they possess structural motifs characteristic of MAR/SAR elements, and exhibit enhanced affinities for nuclear matrix binding in vitro. Such elements bind efficiently to matrices from either the testis or the liver, i.e. tissues where the gene is either fully active or repressed, although one subfragment in the 5’ region was identified as exhibiting testis-specific interactions. Surprisingly, the activity of the CAT reporter gene was repressed in testis-transient transfection assays when the hst70 promoter sequences were extended into the 5’ MAR/SAR.
Address and Contact Information 1Department of Tumor Biology and 2Department of Experimental and Clinical Radiobiology, Center of Oncology, Maria Skłodowska-Curie Memorial Institute, Wybrzeże AK 15, 44-100 Gliwice, Poland
*Corresponding author: wwidlak@io.gliwice.pl
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Volume 9 (2004) pp 135-143
Title A HIGH FREQUENCY OF APOPTOSIS WAS FOUND IN CULTURES OF LYMPHOCYTES ISOLATED FROM THE VENOUS BLOOD OF CHILDREN BORN WITH A LOW BIRTH WEIGHT
Authors Ewa Barg1*, Kazimierz Gąsiorowski2, Barbara Brokos2, Anna Świędrych3 and Katarzyna Skórkowska3
Abstract Children born with a low birth weight (below 2500g) exhibit a slower rate of development, and a greater tendency towards morbidity and mortality, together with a deficit of weight and height. One reason could be an increase in the level of cell elimination by apoptosis. The aim of this study was to evaluate and compare the incidence of apoptotic and necrotic (dead) cells in cultures of peripheral blood lymphocytes obtained from children born with a low birth weight and from children with a normal birth weight. Peripheral blood lymphocytes were obtained by venipuncture (10 ml) and isolated using the density gradient centrifugation method. The lymphocytes were cultured for 48 h in a culture medium containing low concentrations of fetal calf serum. A comparison study was performed between low birth weight children and normal birth weight children and the susceptibility of their lymphocytes to apoptosis and to necrosis in serum-deficient feeding culture conditions. The amount of apoptotic cells and the percentage of dead cells were significantly higher in cultures of lymphocytes obtained from low birth weight children than in cultures from normal birth weight children. The two estimated parameters inversely correlated with the concentration of fetal calf serum in the culture medium. Pulsed field gel electrophoresis showed increased DNA degradation patterns in the cultures of lymphocytes obtained from low birth weight children. Our results should be perceived as an indication that, under worse feeding conditions, the elimination of cells by apoptosis and by necrosis is significantly higher for lymphocytes of low birth weight children than for those of normal birth weight children. The enhanced elimination of lymphocytes is related to a greater susceptibility to infections, especially of the respiratory tract, as established in the retrospective analysis of the anamneses of the examined group of low birth weight children.
Address and Contact Information 1Department of Endocrinology for Children and Adolescents, Wrocław Medical University, Wrocław, Poland;
2Department of Basic Medical Sciences, Wrocław Medical University, Wrocław, Poland;
3Departament of Angiology, Arterial Hypertension and Diabetology, Wrocław Medical University, Wrocław, Poland
*Corresponding author, fax: (48 71) 3280682; e-mail: ebarg@dilnet.wroc.pl
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Volume 9 (2004) pp 145-152
Title THE ACTIVITY OF a1,6-FUCOSYLTRANSFERASE DURING HUMAN MEGAKARYOCYTIC DIFFERENTIATION
Authors Urszula Bany-Łaszewicz*, Joanna Kamiłska, Edyta Klimczak-Jajor and Jerzy KoÂścielak
Abstract α1,6-Fucosyltransferase (6FucT, E.C. 2.4.1.68) is one of the enzymes involved in the synthesis of N-linked glycans of the GpIIb/IIIa complex (CD41a) which is present on megakaryocytes (MKs) and platelets. In this study, we examined 6FucT activity in ex vivo cultures of immunoselected cord blood CD34+ cells grown in a medium promoting megakaryocytopoiesis. Our results show that the activity of 6FucT increased ahead of, and thereafter concomitantly with, cells expressing the CD41a antigen. When the CD41a+ subpopulation of cells was immunoselected (using anti-CD61 i.e. anti-GpIIIa antibodies), its 6FucT activity increased proportionally to the yield of CD61+ cells. Taking into account the heavy load of 6FucT in platelets and megakaryocytes, we regard this enzyme as a candidate for the earliest marker of MK-commitment in cultured hematopoietic stem cells. Such a marker should allow an earlier detection and earlier transplantation of patients’ own, ex vivo expanded, Mk progenitors.
Address and Contact Information Department of Biochemistry, Institute of Hematology and Blood Transfusion, Chocimska 5, 00-957 Warsaw, Poland
Corresponding author; tel: 048-22-8489515, fax: 048-22-8480637, e-mail: ubany@ihit.waw.pl
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Volume 9 (2004) pp 153-165
Title THE NUCLEAR PROTEIN P30 SPECIFICALLY INTERACTS WITH A NUCLEAR MATRIX ATTACHMENT REGION FROM THE RAT GENOME
Authors Anton Fedorov1*, Dmitri Lukyanov1, Jacek Rogoliłski2, Piotr Widłak2, Olga Podgornaya1 and Joanna Rzeszowska-Wolny2
Abstract In our previous study, a 454 bp DNA fragment was isolated from rat genomic DNA as an element which interacts with nuclear matrix proteins, i.e. a Matrix Associated Region (MAR). Computer analyses revealed that the right half of this fragment, named RME (Rat MAR Element), possesses a high matrix association potential and is likely to be responsible for the matrix association of the whole sequence. RME was used as a probe in an electrophoretic mobility shift assay (EMSA), and with the use of Southwestern blotting, a rat liver nuclear protein which binds specifically to it was identified. Its molecular mass was estimated by SDS-PAGE as 30 kDa (p30). Polyclonal antibodies raised against protein-RME complexes caused a super-shift of specific complexes in EMSA, and bound to p30 in nuclear extracts of rat liver in Western blotting. The immunofluorescence labelling of a rat embryonic fibroblast cell monolayer with anti-p30 antibody revealed a mainly intranuclear pattern of staining.
Address and Contact Information 1Institute of Cytology, Russian Academy of Science, Tikhoretsky pr. 4, 194064 St.Petersburg, Russia,
2Institute of Oncology, Department of Experimental and Clinical Radiobiology, 44-100 Gliwice, Poland
*Corresponding autor, tel: (812) 247-7450, fax: (812) 247-0341, e-mail: a_slon@pochtamt.ru
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Volume 9 (2004) pp 167-186
Title NMR STUDIES OF CALCIUM-BINDING TO MUTANT a-SPECTRIN EF-HANDS
Authors Alexei V. Buevich1#, Susanne Lundberg3, Ingmar Sethson1, Ulf Edlund1 and Lars Backman2*
Abstract The co-operative calcium binding mechanism of the two C-terminal EF-hands of human aII-spectrin has been investigated by site-specific mutagenesis and multi-dimensional NMR spectroscopy. To analyse the calcium binding of each EF-hand independently, two mutant structures (E33A and D69S) of wild type a-spectrin were prepared. According to NMR analysis both E33A and D69S were properly folded. The unmutated EF-hand in these mutants remained nearly intact and active in calcium binding, whereas the mutated EFhand lost its affinity for calcium completely. The apparent calcium binding affinity of the E33A mutant was much lower compared to the D39S mutant (~2470 mM and ~240 mM, respectively). When the chemical shift perturbations were followed upon calcium titration, a positive correlation between the D69S mutant and the binding of the first calcium ion to the wild type was revealed. These observations showed that the first EF-hand in spectrin binds the first calcium ion and thereby triggers a conformational change that allows the second calcium ion to bind to the other EF-hand
Address and Contact Information 1Organic Chemistry and 2Biochemistry, Department of Chemistry, Umea University, SE-901 87 Umea,
3Swedish Defence Research Agency, FOI NBC defence, SE-901 82 Umea, Sweden
* Corresponding author, e-mail: lars.backman@chem.umu.se
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