Vol.8 No.3 September 2003

Volume 8 (2003) pp 639-648
Title THE KINETICS OF HAEMOLYSIS OF SPHEROCYTIC ERYTHROCYTES
Authors Grzegorz Paździor1, Marek Langner1,2, Anna Chmura1, Dżamila Bogusławska3, Elżbieta Heger3, Anna Chorzalska4 and Aleksander F. Sikorski2,4
Abstract Spherocytosis is a hereditary disease. It results from mutations in genes that encode proteins participating in the attachment of the membrane skeleton to the plasma membrane bilayer of the erythrocyte. In affected cells, interaction between the spectrin-actin meshwork and integral membrane proteins is altered. This results in the weakening of plasma membrane mechanical resistance and diminishing its elasticity. Since defective cells are prone to mechanical destruction and phagocytosis in the spleen, the fraction of morphologically-altered erythrocytes is rather small; this in turn means such an examination is prone to errors. In this paper, we describe a simple method which could be useful in the identification of red blood cells with altered osmotic properties. The method is based on the measurement of the amount of light scattered by a suspension of the red blood cells, during which cells are exposed to osmotic stress in the stopped-flow regime. The obtained plots are fitted to a mathematical formula, the parameters of which can be used as quantitative indicators of the changes in red blood cells' osmotic features. Two types of spherocytotic samples were examined: those with a proven deficiency in ankyrin and those with a decrease in the band 3 anion transporting protein. The presented data show that this method gives a reliable indication of altered osmotic properties of the spherocytic cells.
Address and Contact Information 1Institute of Physics, Wrocław University of Technology, Wrocław, Poland,
2Academic Centre for Biotechnology of Lipid Aggregates, Wrocław, Poland,
3Institute of Biotechnology and Environmental Sciences, University of Zielona Góra, Poland,
4Institute of Biochemistry and Molecular Biology, University of Wrocław, Poland
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Volume 8 (2003) pp 649-653
Title A POSSIBLE ORIGIN OF THE TOROCYTE-LIKE SHAPE OF VESICLES DERIVED FROM THE TRANSVERSE TUBULE IN THE TRIAD JUNCTION OF SKELETAL MUSCLE
Authors Aleš Iglič1, Miha Fošnarič1 and Veronika Kralj-Iglič2
Abstract The origin of characteristic torocyte-like shape of vesicles derived from transverse tubule in triad junction of skeletal muscles is studied theoretically. Two possible mechanisms are suggested. The first is the minimization of membrane bending energy where the special intermediate molecular structures in the central region of the vesicle is assumed to protect the opposing bilayers to come in the direct contact. The second mechanism is based on the assumption that the characteristic shape of the vesicles may be explained by non-homogenous lateral distribution of anisotropic membrane components.
Address and Contact Information 1Faculty of Electrical Engineering, University of Ljubljana, SI-1000, Ljubljana, Slovenia,
2Faculty of Medicine, University of Ljubljana and University Clinical Centre, SI-1000, Ljubljana, Slovenia
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Volume 8 (2003) pp 655-665
Title APOPTOSIS AND CLONOGENIC SURVIVAL IN THREE TUMOUR CELL LINES EXPOSED TO GAMMA RAYS OR CHEMICAL GENOTOXIC AGENTS
Authors Sławomir Kumala1, Paweł Niemiec2, Maria Wideł1, Ronald Hancock3 and Joanna Rzeszowska-Wolny1*
Abstract We compared the extent to which apoptosis is induced and clonogenicity reduced in three tumour cell lines-the human melanoma Me45 and promyelocytic leukaemia HL-60, and the rat rhabdomyosarcoma R1-after exposure to the anticancer drugs etoposide and cis-platinum or to gamma radiation; each induces different types of DNA damage. Cells which readily underwent apoptosis did not necessarily show a correlated loss of clonogenicity; for example, Me45 cells showed the highest sensitivity to all three agents in clonogenic assays but much lower levels of apoptotic cells than R1 or HL-60 cells. These results show that the efficiency of the eradication of clonogenic cells by genotoxic agents does not solely depend on the induction of apoptotic processes, and suggest that the induction of apoptosis and suppression of clonogenicity are independent processes.
Address and Contact Information 1Department of Experimental and Clinical Radiobiology, Center of Oncology, Wybrzeże AK 15, 44-100 Gliwice, Poland,
2Department of Chemistry and General Biochemistry, Silesian Medical Academy, 40-752 Katowice, Poland,
3Laval University Cancer Research Centre, 9 rue MacMahon, Quebec, Canada G1R 2J6
* Corresponding author: tel: (+4832) 278-96-77, fax: (+4832) 231-35-12, e-mail: jwolny@io.gliwice.pl
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Volume 8 (2003) pp 667-680
Title THE ASSOCIATION OF GLYCOLYTIC ENZYMES WITH CELLULAR AND MODEL MEMBRANES
Authors Jan Gutowicz1 and Grzegorz Terlecki2
Abstract This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity.
Address and Contact Information 1Department of Physico-chemistry of Microorganisms, Institute of Genetics and Microbiology, Przybyszewskiego 63/77, Wrocław University, Poland,
2Department of Medical Biochemistry, Chałubinskiego 10, Wrocław Medical University, Poland
* Corresponding author, E-mail: gutowicz@microb.uni.wroc.pl
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Volume 8 (2003) pp 681-687
Title THE EFFECTS OF HISTAMINE INJECTION ON THE HISTOCHEMISTRY OF THE GUINEA PIG PARATHYROID GLAND
Authors Jacek Danowski, Hanna Gendek-Kubiak and Bogumił L. Kmieć
Abstract Histochemical studies on the activity of some neuropeptides (PGP, CT, NPY, SP and βE) and enzymes (AP, AlP, SDH and MAO) were performed on guinea pig parathyroid glands after injections of histamine, histamine receptor blockers and muscarinic receptor blocker. Under conditions of histamine shock, the immunoreactivity of CT and the reactivity of SDH and MAO were found to decrease together with an increase in the activity of βE and AP. The reactions for SP, NPY, PGP and AlPdid not change in any of the groups.
Address and Contact Information Department of Cytophysiology, Embryology and Histology, Chair of Morphology, Medical University, Narutowicza 60, 90-136 Łódź, Poland
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Volume 8 (2003) pp 689-697
Title UNIQUE SPECTROSCOPIC PROPERTIES OF SYNTHETIC 15-cis b-CAROTENE, AN IMPORTANT COMPOUND IN PHOTOSYNTHESIS, AND A MEDICINE FOR PHOTOPROTECTIVE FUNCTION
Authors Grażyna Ewa Białek-Bylka1*, Beata Jazurek1, Roman Dedic2, Jan Hala2 and Andrzej Skrzypczak3
Abstract The (11Bu +) energy of synthetic 15-cis b-carotene exhibits a linear dependence on (n2-1)/(n2+2) in non-polar and polar solvents; in this it is similar to (that of) all-trans b-carotene. The point of intersection is at (n2-1)/(n2+2) = 0.3 for both isomers. The microenvironment of 15-cis b-carotene in the Photosystem II reaction center was established as having a mean refractive index 1.473. Persistent spectral hole burning with a very broad (30 nm) hole observed around 500 nm (corresponding to an extremely short excited lifetime 9 fs) indicates that 15-cis b-carotene has/displays very efficient photoprotective quenching.
Address and Contact Information 1Faculty of Technical Physics, Poznań University of Technology, 60-965 Poznań, Nieszawska 13 a, Poland,
2Faculty of Mathematics and Physics, Charles University, CZ 12116, Prague 2, Ke Karlovu 3, Czech Republic,
3Faculty of Chemical Technology, Poznań University of Technology, 60-965 Poznań, Marii Skłodowskiej-Curie 2, Poland
*Corresponding author, E-mail: bialek@phys.put.poznan.pl
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Volume 8 (2003) pp 699-711
Title BACTERIOPHAGES PROVIDE REGULATORY SIGNALS IN MITOGEN-INDUCED MURINE SPLENOCYTE PROLIFERATION
Authors Michał Zimecki1, Beata Weber-Dąbrowska1, Marzanna Łusiak-Szelachowska1, Marian Mulczyk1, Janusz Boratyński1, Gryzelda Poźniak2, Danuta Syper1 and Andrzej Górski1,3
Abstract The aim of this investigation was to reveal the regulatory properties of bacteriophage preparations in a model of mitogen-induced splenocyte proliferation in mice. We showed that sepharose 4B-purified preparations of the Staphylococcus aureus phage A20/R exhibited costimulatory activity in splenocyte proliferation induced by suboptimal (0.25 mg/ml) concentrations of ConA. On the other hand, the purified phage fraction was regulatory with regard to splenocyte proliferation induced by the optimal (2.5 mg/ml) ConA concentration. We also showed that the phage preparation can elicit IL-6 production in splenocyte cultures and enhance ConA-induced production of that cytokine. Furthermore, the phages preferentially induced IL-6 production in adherent splenocytes and increased levels of that cytokine in cultures of peritoneal cells from mice and rats. This phenomenon may explain the costimulatory activity of phages in the model described.
Address and Contact Information 1Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland,
2Institute of Organic and Polymer Technology, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland,
3Transplantation Institute, Medical University of Warsaw, Nowogrodzka 59,02-006 Warszawa, Poland
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Volume 8 (2003) pp 713-714
Title THE INFLUENCE OF PHYTOHORMONES ON THE PROPERTIES OF WHEAT PHOSPHOLIPID MONOLAYERS AT THE WATER-AIR INTERFACE.
Authors Maria Filek1*, Barbara Gzyl2 and Anna Dudek1
Abstract The surface behaviour of monolayers of wheat phospholipids in the presence of phytohormones introduced into the water phase was studied using Langmuir's method. The phospholipids were extracted from the plasmalemma of non-embryogenic (NE) and embryogenic (E) calli initiated from two types of explant: immature inflorescences (inf) and embryos (emb). The surface properties were investigated in model systems of monolayers of mixed phospholipids with: 1) natural amphiphile composition (PL); 2) a determined hydrophobic part (16:0) and the natural percentage composition of the hydrophilic part (PPL); and 3) a determined hydrophilic part (PC) and the natural percentage composition of the hydrophobic part (HPL). The lower limit values of the molecular area (Alim) were observed for NE rather than for E monolayers in all the investigated systems (PL, PPL and HPL). The collapse pressure (πcoll) of the monolayer decreased in the order PPL>PL>HPL, indicating the high stability of monolayers containing saturated hydrocarbon chains. The injection of non-surface-active phytohormones into the water subphase and the subsequent formation of natural and also artificial phospholipid monolayers of E and NE causes a decrease in monolayer stability against collapse and molecular close packing. As a result of their amphipathic (hydrophilic-hydrophobic) structure, the surface properties of E phospholipids are probably optimal for these systems.
Address and Contact Information 1Department of Plant Physiology Polish Academy of Sciences, Podłużna 3, 30-239 Kraków, Poland,
2 Department of Physical Chemistry, Jagiellonian University, Ingardena 3, Kraków, Poland
*Corresponding author: Fax: 4812-4253202; E-mail: mariafilek@excite.com
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Volume 8 (2003) pp 727-734
Title MODULATION OF THE MITOTIC ACTIVITY AND POPULATION OF THE MAST CELLS IN THE ORAL MUCOSA BY SUBSTANCE P
Authors Marcin Kozakiewicz1 and Andrzej Godlewski2
Abstract To assess the effect of substances inducing mast cell degranulation (substance P and granuliberin R) on the mitotic indices of the gingiva stratified epithelium, basal cells from rats were studied in vivo. Seventy Lewis male rats were used in the study. The rats received injections of either 0.1 ml 0.9% NaCl (l0 rats), or substance P (10-4, l0-6, 10-8 g/ml) (30 rats), or granuliberin R (10-4, l0- 6, 10-8 g/ml) (30 rats) into their mandibular gingiva in the vicinity of the right mental foramen. The mitotic index of keratinocytes was established after the kolchicine arrest (2 hours prior to material collection i.p. injection) . The number of cells in metaphase was counted on 1000 consecutive basal layer cells after hematoxilin and eosin section staining. Mast cells were revealed using pinocyanol erythrosinate according to Bensley. Numerical density and morphometric features were analyzed. Substance P and granuliberin R injected into the gingiva affect the mast cells and the basal cell proliferation of the gingival epithelium. The diminished mitotic activity of basal layer cells was accompanied by degranulation and/or migration of mast cells under the basal membrane of the epithelium. After administration of high doses of granuloliberin R, mast cells were found in the deep connective tissue alligned towards the epithelium. A neuromediator from the trigeminal nerve (substance P) and substances from mast cells actively interfere in the proliferation of oral keratinocytes and the activity of connective tissue cells.
Address and Contact Information 1Department of Maxillofacial Surgery, Institute of Surgery, Medical University of Łódź, Kopcińskiego 22, 90-153 Łódź, Poland,
2Department of Histology and Embryology, Medical University of Łódź, Narutowicza 60, 90-136 Łódź, Poland
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Volume 8 (2003) pp 735-741
Title FIBROBLAST CELL SHAPE AND ADHESION IN VITRO IS ALTERED BY OVEREXPRESSION OF THE 7A AND 7B ISOFORMS OF PROTOCADHERIN 7, BUT NOT THE 7C ISOFORM
Authors Kenichi Yoshida*
Abstract Protocadherins (Pcdhs) are a family of cadherins considered to play an important role in the cell-cell adhesion of specific neurons in the central nervous system. Of the reported Pcdhs, relatively little is known about the functional role of protocadherin 7 (Pcdh7), and there is no evidence of Pcdh7 mediated cell-cell adhesion. To date, three splicing variants are known; they may have different effects on cell phenotype. We report here that mouse fibroblast L cells stably overexpressing the Pcdh7 isoforms 7a and 7b, but not 7c, showed a morphological change and Ca2+ dependent cell adhesion.
Address and Contact Information Genetic Diagnosis, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
* E-mail: yoshidak@ims.u-tokyo.ac.jp
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Volume 8 (2003) pp 743-748
Title INSERTION OF A REAMPLIFICATION ROUND INTO THE ISSR-PCR PROTOCOL GIVES NEW FLAX FINGERPRINTING PATTERNS.
Authors Ivo Wiesner* and Dana Wiesnerová
Abstract We expanded the basic ISSR-PCR protocol by an additional PCR reamplification round in order to detect whether increased PCR productivity would give new bands in ISSR patterns. We found that the reamplification step had a prominent impact on the quality of the inter-simple-sequence repeat (ISSR) PCR patterns of flax, depending on the particular primer used for PCR amplification. We could clearly distinguish between two types of reamplification effect. Most ISSR primers (16 out of 21) gave no reamplification effect as usual, but five primers (23.8%) provided a new ISSR fingerprinting pattern after the 2nd reamplification round, leaving the previous 1st round pattern completely blank. Therefore, we recommend the expansion of a basic ISSR-PCR protocol for another reamplification round in order to mine out full the fingerprinting potential from ISSR-PCR method.
Address and Contact Information The Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Branišovská 31, České Budějovice, CZ 370 05, Czech Republic
* Corresponding author, E-mail: nfix@jcu.cz
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Volume 8 (2003) pp 749-775
Title A NEW DOUBLE-CHAMBER MODEL OF ION CHANNELS. BEYOND THE HODGKIN AND HUXLEY MODEL
Authors Krzysztof Dołowy
Abstract This paper proposes a new double-chamber model (DCM) of ion channels. The model ion channel consists of a series of three pores alternating with two chambers. The chambers are net negatively charged. The chamber's electric charge originates from dissociated amino acid side chains and is pH dependent. The chamber's net negative charge is compensated by cations present inside the chamber and in a diffuse electric layer outside the chamber. The pore's permeability is constant independent of time. One pore of the sodium channel and one of the potassium channel is a voltage-sensing pore. Due to the channel's structure, ions flow through the pores and chambers in a timedependent manner. The model reproduces experimental voltage clamp and action potential data. The current flowing through a single sodium channel is less then one femtoampere. The DCM is considerably simpler then the Hodgkin and Huxley model (HHM) used to describe the electrophysiological properties of an axon. Unlike the HHM, the DCM can explain refractoriness, anode break excitation, accommodation and the effect of pH and temperature on the channels without additional parameters. In the DCM, the axon membrane shows repetitive activity depending on the channel density, sodium to potassium channel ratio and external potassium concentration. In the DCM, the action potential starts from 'hot spot areas' of higher channel densities and a higher sodium to potassium channel ratio, and then propagates through the whole axon.
Address and Contact Information Department of Biophysics, Warsaw Agricultural University - SGGW, Rakowiecka 26/30, 02-528 Warszawa, Poland
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Volume 8 (2003) pp 777-781
Title THE ACTIVITY OF ANTIOXIDANT ENZYMES IN Arabidopsis thaliana EXPOSED TO COLCHICINE AND H2O2
Authors Maria Drążkiewicz1, Ewa Skórzyńska-Polit1, Małgorzata Wanke2 And Ewa Świeżewska2
Abstract Studies on the possible interference of colchicine and H2O2 with the activity of some antioxidant enzymes were carried out on Arabidopsis thaliana v. Columbia grown in Murashige and Skooge nutrient medium. Measurements of superoxide dismutase (SOD), guaiacol peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT) activities were conducted spectrophotometrically. In the presence of colchicine, SOD activity increased, while CAT, APX and POX activities decreased. Inhibitory H2O2 effects on the activity of the enzymes were found. Colchicine pre-treatment resulted in an increase in CAT activity and a further increase in SOD activity in plants treated with H2O2
Address and Contact Information 1Department of Plant Physiology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland,
2Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
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Volume 8 (2003) pp 783-792
Title CONCANAVALIN A-AGAROSE REMOVES MANNAN IMPURITIES FROM AN EXTRACELLULARLY EXPRESSED PICHIA PASTORIS RECOMBINANT PROTEIN
Authors Małgorzata Palczewska1, *, Gyula Batta2 And Patrick Groves1, **
Abstract Pichia pastoris secretes few native proteins. However, the more than 1 g l-1 of extracellularly expressed mannan interfered with the purification of our extracellularly expressed, non-glycosylated recombinant protein. Concanavalin A-agarose removed more than 95% of the unwanted mannan as monitored by phenol reaction. A 13C-based NMR assay confirmed this improvement. Concanavalin A-agarose can assist the purification of extracellular expressed, non-glycosylated proteins from yeasts.
Address and Contact Information 1Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland,
2Research Group for Antibiotics of the Hungarian Academy of Sciences, University of Debrecen, Egyetem tér. 1, H-1040, Debrecen, Hungary
*Current address: Centro Nacional de Biotechnologia (C.S.I.C), Campus Cantoblanco, 28049 Madrid, Spain.
**Corresponding author at current address: Centro de Investigaciones Biológicas (C.S.I.C.), Ramiro de Maeztu 9, 28040 Madrid, Spain; email: pdgroves@cib.csic.es
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Volume 8 (2003) pp 793-798
Title REP-PCR FINGERPTINTING AS A TOOL FOR THE ANALYSIS OF GENOMIC DIVERSITY IN Escherichia coli STRAINS ISOLATED FROM AN AQUEOUS/FRESHWATER ENVIRONMENT
Authors Katarzyna Baldy-Chudzik, Joanna Niedbach and Michał Stosik
Abstract The rep-PCR fingerprinting method, with the support of ERIC and REP primers, was used to analyse the genomic diversity of 93 E. coli strains isolated from lake water samples drawn at two different depths. The applied UPGMA for DNA analysis did not reveale any genomic similarities between the 48 E. coli strains derived from the subsurface-zone water and the 43 of the bottom-zone water. The considerable genomic diversity of the E. coli of the surface zone was expressed as a dendrogram in the form of 8 similarity groups comprising strains isolated from samples drawn over one month. The bottomzone strains, which display a lesser degree of genomic diversity (5 similarity groups), showed distinct common features in their DNA fingerprints. In the similarity dendrogram for the bottom-zone, strains derived in different months of sampling were segregated into the same similarity groups. Applying REP primers in rep-PCR generates more complex fingerprints increasing the discriminatory power of the analysis, whereas the ERIC primer generates less complex fingerprint patterns, and is thus clearer to interpret.
Address and Contact Information Institute of Biotechnology and Environmental Sciences, University of Zielona Góra, Monte Cassino 21b, 65-561 Zielona Góra, Poland
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Volume 8 (2003) pp 799-808
Title A COMPETITOR DNA TEMPLATE FOR THE MOLECULAR QUANTIFICATION OF THE HEPATITIS B VIRUS
Authors Małgorzata Sidorkiewicz1, Aneta Bzorska1, Barbara Józwiak1, Maciej Jabłkowski2, Janusz Szemraj1 and Urszula Lewandowska1
Abstract The molecular determination of viral load in the serum represents the most valuable prognostic marker of HBV infection. In this paper, a new molecular assay for the quantitative measurement of HBV presence is described. It is based on PCR performing with a HBV-specific competitor DNA template. For the construction of the DNA template, a HBV DNA-originated 436 bp DNA fragment was modified by introducing a 110 bp deletion and cloned into pUC19. The resulting vector serves as the competitor DNA template in the competitive PCR. Post-PCR, the competitor DNA generates an amplified fragment of 306 bp; it could be easily distinguished from the product generated from the viraloriginated DNA product (416 bp) when the same primers are used. The quantitative ratio between the two products enables the quantitative determination of viral load. The range of the HB-PCR assay is from 3 x 104 to 6 x 1010 particles/ml. A serum HBV load determination performed by HB-PCR assay indicated a close correlation with the results of the Quantiplex HBV DNA assay (bDNA). The HB-PCR assay is cheap, reliable and easy to use in any laboratory working with PCR methods.
Address and Contact Information 1Department of Medical Biochemistry, 2Department of Infectious Diseases, Medical University of Łódź, 92-215 Łódź, Poland
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Volume 8 (2003) pp 809-824
Title PLANT CHITINASES-REGULATION AND FUNCTION
Authors Anna Kasprzewska
Abstract The aim of this review is to present the current state of knowledge on plant chitinases and their regulation and function. Chitinases are up-regulated by a variety of stress conditions, both biotic and abiotic, and by such phytohormones as ethylene, jasmonic acid, and salicylic acid. Like other PR proteins, chitinases play a role in plant resistance against distinct pathogens. Moreover, by reducing the defence reaction of the plant, chitinases allow symbiotic interaction with nitrogen-fixing bacteria or mycorrhizal fungi. However, recent investigations have shown that these enzymes are also involved in numerous physiological events. The involvement of chitinases in development and growth processes is also described.
Address and Contact Information Department of Plant Resistance Physiology, Institute of Experimental Plant Biology, Warsaw University, Miecznikowa 1, 02-096 Warsaw, Poland
* Corresponding author, E-mail: jaga@biol.uw.edu.pl
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Volume 8 (2003) pp 825-830
Title THE APPLICATION OF FPLC CHROMATOGRAPHY (MONO Q) FOR THE PURIFICATION OF WHEAT RNA POLYMERASES II AND III
Authors Paweł Glanc1, Mariola Galbas1, Piotr Dullin1, Marlena Szalata2 and Ryszard Słomski1,2
Abstract Transcription is the main step in the regulation of gene expression. To study this process in vitro, it is necessary to obtain highly purified RNA polymerases. Here, we describe a method of RNA polymerase purification using a Mono Q FPLC column. Using Mono Q column chromatography accelerates the purification process and separates RNA polymerase II from RNA polymerase III with good yield.
Address and Contact Information 1Department of Biochemistry and Biotechnology, Agricultural University, Wołyńska 35, 60-637 Poznań, Poland, 2Institute of Human Genetics, Polish Academy of Sciences, Strzeszyńska 32, 60-479 Poznań, Poland
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Volume 8 (2003) pp 831-840
Title THE DNA-BINDING CAPACITY OF GENETIC VARIANTS OF THE BOVINE STAT5A TRANSCRIPTION FACTOR
Authors Krzysztof Flisikowski, Małgorzata Szymanowska and Lech Zwierzchowski
Abstract The STATs are a family of transcription factors. STAT5A, previously known as MGF, transduces prolactin signals to the milk protein genes. Here, we describe the detection of nucleotide sequence polymorphism in exon 16 of the bovine STAT5A gene, coding for the SH2 domain. SSCP was found in a 281-bp PCR amplified gene fragment, lying between positions 12,525 and 12,806, and encompassing parts of intron 15 and exon 16 of the bovine STAT5A gene (GenBank AJ 237937). Three SSCP patterns (genotypes) were identified in a group of 108 animals of different cattle breeds. The DNA sequencing showed that they differed by a CCT deletion at position from 12,549 in intron 15, and a T C substitution at position 12,743 in exon 16. The latter mutation changes an amino acid sequence in the STAT5A protein-a Val/Ala substitution at position 686. Since T C substitution creates a new MslI site, genetic variants in the bovine STAT5A gene can be distinguished with RFLP analysis. The frequency of alleles T and C varied between the different cattle breeds studied; the CC genotype was the least frequent and the frequency of alleles T and C was 0.842 and 0.158, respectively. Proteins were extracted from the cell nuclei of liver tissues derived from bulls of different STAT5A genotypes and subjected to EMSA in order to study if the amino acid substitution might change the DNAbinding capacity of STAT5A transcription factor. Statistically significant (p<0.05) differences in nuclear protein binding to DNA were observed between genotypes TT and CC; nuclear proteins derived from CC animals always showed less DNA protein complexing than those of TT animals. EMSA competition experiments confirmed that STAT5 transcription factors take part in the formation of the DNA-protein complexes.
Address and Contact Information Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzębiec, 05-552 Wólka Kosowska, Poland
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Volume 8 (2003) pp 841-848
Title THE RATE CONSTANTS OF THE REACTION OF HYDROXYL RADICALS (°OH) WITH ALCOHOL DEHYDROGENASE AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
Authors Aleksandra Kowalczyk* And Mieczysław Puchała
Abstract The rate constants of the reactions of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase with hydroxyl radicals were determined using the method of steady-state competitive reactions. Ethanol was used as a scavenger of hydroxyl radicals. The rate constants of the reactions of hydroxyl radicals with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were found to be 2.8·1012dm3mol-1s-1, and 1.6·1012dm3mol-1s-1, respectively.
Address and Contact Information Department of Molecular Biophysics, University of Łódź, 90-237 Łódź, Banacha 12/16, Poland
*Corresponding author, E-mail: olakow@biol.uni.lodz.pl
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Volume 8 (2003) pp 849-856
Title THE HISTOLOGY OF PIGMENTED BOMIRSKI MELANOMA GROWING IN THE HAMSTER EYE-PRELIMINARY RESULTS
Authors Bożena Romanowska-Dixon1, Krystyna Urbańska2* , Martyna Elas2 and Tadeusz Cichocki3
Abstract We demonstrated that a quickly growing hamster skin melanoma developed a tumor after autologous implantation into the anterior chamber of the eye. Tumor cells were seen invading all the surrounding tissues, including the iris, ciliary body, choroid and cornea. Histological examination confirmed the presence of numerous blood vessels of large diameter. Their walls were very thin, thus only the endothelium could be identified using light microscopy. Macrophages, microemboli and extravasations were present within the tumor mass.
Address and Contact Information 1Department of Ophthalmology Collegium Medicum,
2Faculty of Biotechnology,
3Department of Histology Collegium Medicum, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland
* Corresponding author, E-mail: urbanska@awe.mol.uj.edu.pl
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