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 Łódź, Banacha 12/16, 90-237
Łódź, Poland, 2Department of Digestive Tract Diseases, Medical University of
Łódź, Kopciłskiego 22, 90-153 Łódź, Poland, 3Department of Clinical
Pharmacology, Medical University of Łódź, Rewolucji 1905 37/39, 90-214
Łódź, 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 Łódź, Banacha 12/16,
90-237 Łódź, 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 Łódź,
S. Banacha 12/16, 90-237 Łódź, 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-Łaszewicz*, 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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