Vol. 11 No. 3 September 2006

DOI: 10.2478/s11658-006-0025-3 Volume 11 (2006) pp 299 - 311
Title CAMPTOTHECIN INDUCES THE TRANSIT OF FasL TRIMERS TO THE CELL SURFACE IN APOPTOTIC HEp-2 CELLS
Authors Esteban Meza-Lamas1, Juan-José Bollain-Y-Goytia1, Roxana Ramirez-Sandoval, Sergio H Sánchez-Rodríguez, Erendira López-Robles, Esperanza Avalos-Díaz and Rafael Herrera-Esparza*
Abstract Fas ligand (L) is a membrane protein from the tumor necrosis factor (TNF) family. It induces apoptosis upon contact with its Fas/CD95/APO1 receptor. Trimerization of FasL on the surface of effector cells is essential in the binding of the Fas trimer of the target cells. The receptor then recruits an adaptor and caspase-like proteins which lead apoptosis. This paper reports on the fate of FasL in HEp-2 cells committed to apoptosis by induction with campthotecin. Our main results demonstrated that in non-apoptotic cells, FasL aggregates in the cytoplasm forming trimers of 120 kDa. Apoptosis increases the trimeric FasL species, but also induces its dissociation into monomers of 35 kDa. In conclusion, camptothecin appears to perturb the Fas and FasL segregation in the cytoplasm by promoting the transit of FasL to the cell surface, thus fostering a process of autocrine or paracrine apoptosis. FasL is trimerized prior to Fas/FasL complex formation, and after apoptosis, FasL undergoes an intense turnover.
Keywords FasL, Apoptosis, Oligomerization, Camptothecin, TUNEL
Address and Contact Information Department of Molecular Biology, CBE, Universidad Autónoma de Zacatecas, Guadalupe, Zacatecas, Mexico
1 Contributed equally
* Corresponding author: e-mail: herrerar@intranet.uaz.edu.mx
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DOI: 10.2478/s11658-006-0027-1 Volume 11 (2006) pp 312 - 325
Title The heterogeneity of ion channels in chromaffin granule membranes
Authors Renata Hordejuk1, Adam Szewczyk2 and Krzysztof Dołowy1*
Abstract Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic 86Rb+ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 + 10 pS, 220 + 8 pS, 152 + 8 pS and 13 + 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 + 8 pS and 31 + 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.
Keywords Chromaffin granule, Intracellular channel, Potassium channel, Chloride channel, Black lipid membrane
Address and Contact Information 1Department of Biophysics, Agriculture University SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland,
2Laboratory of Intracellular Ion Channels, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Pasteur 3, 02-093 Warsaw, Poland
* Corresponding author: tel: +48 22 593 8610; fax: +48 22 593 8619, e-mail: krzysztof_dolowy@sggw.pl
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0026-2 Volume 11 (2006) pp 326 - 337
Title PROTEIN PROFILING OF SICKLE CELL VERSUS CONTROL RBC CORE MEMBRANE SKELETONS BY ICAT TECHNOLOGY AND TANDEM MASS SPECTROMETRY #
Authors Jose Chou1,3,4, Pankaj K Choudhary2,3,4 and Steven R. Goodman1,3,4,5*
Abstract A proteomic approach using a cleavable ICAT reagent and nano-LC ESI tandem mass spectrometry was used to perform protein profiling of core RBC membrane skeleton proteins between sickle cell patients (SS) and controls (AA), and determine the efficacy of this technology. The data was validated through Peptide/Protein Prophet and protein ratios were calculated through ASAPratio. Through an ANOVA test, it was determined that there is no significant difference in the mean ratios from control populations (AA1/AA2) and sickle cell versus control populations (AA/SS). The mean ratios were not significantly different from 1.0 in either comparison for the core skeleton proteins (α spectrin, ß spectrin, band 4.1 and actin). On the natural-log scale, the variation (standard deviation) of the method was determined to be 14.1% and the variation contributed by the samples was 13.8% which together give a total variation of 19.7% in the ratios.
Keywords Proteomics, Cleavable ICAT, Ion trap mass spectrometry, RBC membrane skeleton, Sickle cell
Address and Contact Information 1Department of Molecular and Cell Biology,
2Department of Mathematical Sciences,
3The Institute of Biomedical Sciences and Technology,
4University of Texas at Dallas, Richardson, Texas, USA,
5Department of Cell Biology, UT Southwestern Medical Center, Dallas, Texas, USA
# Invited paper
* Corresponding author; email: sggoodm@utdallas.edu
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DOI: 10.2478/s11658-006-0028-0 Volume 11 (2006) pp 338 - 347
Title STUDIES ON GENETIC CHANGES IN RYE SAMPLES (Secale cereale L.) MAINTAINED IN A SEED BANK
Authors Katarzyna J. Chwedorzewska1*, Piotr T. Bednarek1*, Renata Lewandowska1, Paweł Krajewski2 And Jerzy Puchalski1
Abstract The aim of this study was to identify genetic changes in rye seeds induced by natural ageing during long-term storage and consecutive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples varying in their initial viability after one and three cycles of reproduction was analyzed by AFLP (amplified fragment length polymorphism) fingerprinting. Seven EcoRI/MseI primer combinations defined 663 fragments, and seven PstI/MseI primer combinations defined 551 fragments. The variation in the frequency of the seventy-four EcoRI/MseI bands was statistically significant between samples. These changes could be attributed to genetic changes occurring during storage and regeneration. However, the PstI/MseI fragments appeared to be uninfluenced by seed ageing, regeneration and propagation. A combined Principle Coordinate Analysis revealed differences between samples with different initial viability. We showed that materials with low initial viability differ in their response from highly viable ones, and that the changes exhibited in the former case are preserved through regeneration cycles.
Keywords Rye, Secale cereale L., Seed storage and propagation, AFLP
Address and Contact Information 1Botanical Garden – Centre for Biological Diversity Conservation of the Polish Academy of Sciences, Prawdziwka 2, 02-973 Warsaw, Poland,
2Institute of Plant Genetics, Polish Academy of Science, Strzeszyńska 34, Poznań, Poland
* Addresses for correspondence: Katarzyna J. Chwedorzewska, Departament of Antarctic Biology PAS, Ustrzycka 10/12, 02-141 Warsaw, Poland, kchwedorzewska@go2.pl; Piotr T. Bednarek Plant Breeding and Acclimatization Institute, 05-870 Warsaw, Radzików, Poland
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0029-z Volume 11 (2006) pp 348 - 359
Title GENOTYPING OF RECOMBINANT Pichia pastoris STRAINS
Authors Jose M. Viader-Salvadó*, Eddy L. Cab-Barrera, Luis J. Galán-Wong and Martha Guerrero-Olazaran
Abstract A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.
Keywords Pichia pastoris, Amplified-fragment length polymorphism (AFLP), Clonal variation
Address and Contact Information Instituto de Biotecnología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Ave. Pedro de Alba s/n, Col. Ciudad Universitaria, 66450 San Nicolás de los Garza, N.L., Mexico
* Corresponding author: e-mail: jviader@fcb.uanl.mx
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0030-6 Volume 11 (2006) pp 360 - 375
Title A COMPARISON OF THE in vitro ANTIMICROBIAL ACTIVITY OF LIPOSOMES CONTAINING MEROPENEM AND GENTAMICIN
Authors Zuzanna Drulis-Kawa1,3*, Jerzy Gubernator2,4, Agata Dorotkiewicz-Jach1, Włodzimierz Doroszkiewicz1 and Arkadiusz Kozubek2
Abstract The antimicrobial activity of eight cationic, two neutral and three anionic liposome compositions containing meropenem and gentamicin was tested in vitro in broth and serum medium. The cationic formulations showed better antibacterial efficacy against both Gram-positive and Gram-negative bacteria than the anionic and neutral ones, regardless of the encapsulated drug. The most effective formulations were the cationic PC/DOPE/DOTAP 3:4:3 and PC/Chol/DOTAP 3:4:3, as the MICs with meropenem were 2 to 4 times lower than those of the free drug.
Keywords Liposomes, Meropenem, Gentamicin, Bacteria
Address and Contact Information 1Institute of Genetics and Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
2Institute of Biochemistry and Molecular Biology, University of Wrocław, 51-148 Wrocław, Poland,
3Lower Silesian Centre of Paediatrics in Wrocław, 51-147 Wrocław, Poland,
4Academic Centre for Biotechnology of Supramolecular Lipid Aggregates, 51-148 Wrocław, Poland
* Corresponding author: tel./fax: +48 71 325-2151; e-mail: kawa@microb.uni.wroc.pl
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0031-5 Volume 11 (2006) pp 376 - 383
Title THE EFFECT OF AUXINS (IAA AND 4-CL-IAA) ON THE REDOX ACTIVITY AND MEDIUM pH OF Zea mays L. ROOT SEGMENTS
Authors Halina Lekacz* and Waldemar Karcz
Abstract Indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl- IAA) were tested at different concentrations and times for their capacity to change the redox activity and medium pH of maize root segments. The doseresponse surfaces (dose-response curves as a function of time) plotted for redox activity and changes in medium pH (expressed as ΔpH) had a similar shape for both auxins, but differed significantly at the optimal concentrations. With 4-Cl- IAA, the maximal values of redox activity and medium pH changes were observed at 10-10 M, which was a 100-fold lower concentration than with IAA. Correlations were observed between redox activity and medium pH changes at the optimal concentrations of both IAA and 4-Cl-IAA. The results are discussed herein, taking into account both the concentration of the auxins and the effects produced by them.
Keywords Redox activity, Medium pH changes, Root segments, Zea mays L., Auxins, Dose-response surface
Address and Contact Information University of Silesia, Faculty of Biology, Department of Plant Physiology, ul. Jagiellońska 28, 40-032 Katowice, Poland
* Corresponding author; e-mail: hlekacz@us.edu.pl
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0032-4 Volume 11 (2006) pp 384 - 395
Title THE ATYPICAL PATTERN OF CELL DEATH IN B16F10 MELANOMA CELLS TREATED WITH TNP-470.
Authors Marcin Okrój1*, Dorota Stawikowska1, Ewa M. Słomińska2 Andrzej Myśliwski3 and Jacek Bigda1
Abstract TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.
Keywords Reactive oxygen species, Angiogenesis, Melanoma, Apoptosis
Address and Contact Information 1Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology,
2Department of Biochemistry, Faculty of Medicine,
3Department of Histology, Faculty of Medicine, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland
* Corresponding author; tel: +46 40 337830, fax: +46 40 337043, e-mail: m arcin.okroj@med.lu.se
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0033-3 Volume 11 (2006) pp 396 - 407
Title THE BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARY OF THE NARROW-LEAFED LUPIN (Lupinus angustifolius L.)
Authors Andrzej Kasprzak1, Jan Šafář2, Jaroslav Janda2, Jaroslav Doležel2, Bogdan Wolko1* and Barbara Naganowska1
Abstract The narrow-leafed lupin possesses valuable traits for environmentfriendly agriculture and for the production of unconventional agricultural products. Despite various genetic and environmental studies, the breeding of improved cultivars has been slow due to the limited knowledge of its genomic structure. Further advances in genomics require, among other things, the availability of a genomic DNA library with large inserts. We report here on the construction of the first DNA library cloned in a BAC (bacterial artificial chromosome) vector from diploid Lupinus angustifolius L. cv. Sonet. The high molecular weight DNA used for its preparation was isolated from interphase nuclei that were purified by flow cytometry. The library comprises 55,296 clones and is ordered in 144×384-well microtitre plates. With an average insert size of 100 kb, the library represents six haploid genome equivalents. Thanks to the purification of the nuclei by flow cytometry, contamination with chloroplast DNA and mitochondrial DNA was negligible. The availability of a BAC library opens avenues for the development of a physical contig map and positional gene cloning, as well as for the analysis of the plant’s genome structure and evolution.
Keywords BAC, Genomic DNA library, Lupinus angustifolius, Narrow-leafed lupin
Address and Contact Information 1Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland,
2Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Sokolovská 6, CZ-772 00 Olomouc, Czech Republic
* Corresponding author: e-mail: bwol@igr.poznan.pl
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0034-2 Volume 11 (2006) pp 408 - 423
Title ERK IS INVOLVED IN THE DIFFERENTIATION INDUCED BY DIALLYL DISULFIDE IN THE HUMAN GASTRIC CANCER CELL LINE MGC803
Authors Hui Ling, Liang-Yun Zhang, Qi Su*, Ying Song, Zhao-Yang Luo, Xiu Tian Zhou, Xi Zeng, Jie He, Hui Tan and Jing-Ping Yuan
Abstract Diallyl disulfide (DADS) is a major constituent of garlic. Previously, we found that DADS both inhibited proliferation in human gastric cancer cells in vitro and in vivo, and induced G2/M arrest. In this study, we investigated whether this differentiation effect was induced by DADS in human gastric cancer MGC803 cells, and whether it was related to an alteration in ERK activity. The results showed that the growth of MGC803 cells was inhibited by DADS. Cells treated with DADS displayed a lower nucleocytoplasmic ratio and tended to form gland and intercellular conjunction structures. The ConAmediated cell agglutination ratio and cells’ ALP specific activity decreased. In MGC803 cells, dye transfer was limited to a few cells neighbouring the dyeinjected cell and to a depth of 1-2 layers beneath the scrape site. However, after treatment with DADS, the LY (Lucifer Yellow) was transferred to several cells immediately neighbouring the microinjected cell and to a depth of 2-4 cell layers from the scrape site. This indicated that DADS induced differentiation in MGC803 cells. Western blot analysis revealed that although DADS did not influence the quantity of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls. At 30 mg·L-1, DADS inhibited the activation of ERK1/2 in 15-30 min. These results suggested that the DADS-induced differentiation of MGC803 cells involved an alteration of the ERK1/2 signaling pathway.
Keywords Diallyl disulfide, Stomach neoplasm, Differentiation, ERK
Address and Contact Information Cancer Research Institute, Nanhua University, Hengyang City, Hunan Province 421001, China
* Corresponding author; tel: +86-734-8281547, fax: +86-734-8281547, e-mail: suqi1@hotmail.com, nhdxlh@163.com
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0035-1 Volume 11 (2006) pp 424 - 437
Title ENHANCED EXPRESSION OF SELENOCYSTEINE LYASE IN ACUTE GLOMERULONEPHRITIS AND ITS REGULATION BY AP-1
Authors Claudia Jafari, Ulf Panzer, Oliver M. Steinmetz, Gunther Zahner, Rolf A.K. Stahl and Sigrid Harendza*
Abstract Acute glomerulonephritis can lead to chronic glomerulonephritis or resolve without permanent damage to the kidneys. Differential gene expression was studied in a model of acute and chronic glomerulonephritis to identify factors influencing the course of glomerulonephritis towards healing or chronification. One of the differentially expressed genes was identified as SCL, encoding selenocysteine lyase. Its expression was higher in acute glomerulonephritis and lower in chronic glomerulonephritis. The transcriptional regulation of SCL was studied in vitro in rat mesangial cells (MC). SCL RNA expression increased eight-fold compared to the baseline after stimulation with interleukin-1ß (IL-1ß) for three hours. Luciferase expression and gel shift experiments revealed an enhancer element between bp -152 and -298 of the SCL 5’-regulatory region, with protein binding to an AP-1 binding site that may be involved in the regulation of SCL-RNA in vivo in an endogenous feedback mechanism to the inflammatory reaction in acute glomerulonephritis, leading to resolution of this disease.
Keywords Selenocysteine lyase, Glomerulonephritis, Differential display, Inflammation, Mesangial cells, AP-1, Transcription
Address and Contact Information Universitätsklinikum Hamburg-Eppendorf, Zentrum für Innere Medizin, III. Medizinische Klinik, Martinistr. 52, 20246 Hamburg, Germany
* Corresponding author: tel: +49 40 42803 3908, fax: +49 40 42803 5186, e-mail: h arendza@uke.uni-hamburg.de
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-006-0037-z Volume 11 (2006) pp 438 - 448
Title EMULSIONS OF OIL FROM Adenanthera pavonina L. SEEDS AND THEIR PROTECTIVE EFFECT
Authors Anna Jaromin1*, Robert Żarnowski2 and Arkadiusz Kozubek1
Abstract In our previous study, we developed very stable formulations of submicron oil-in-water emulsions from Adenanthera pavonina L. (family Leguminosae, subfamily Mimosoideae) seed oil, stabilised with soybean lecithin (SPC). Continuing our research, we introduced an additional co-emulsifier, Tween 80, to those formulations in order to decrease the size of the emulsion particles and improve their stability. Formulations with a mean particle size ranging from 43.6 to 306.5 nm and a negative surface charge from -45.3 to -28.5 mV were obtained. Our stability experiments also revealed that most of the tested formulations had a very good degree of stability over a 3-month storage period, both at 4oC and at room temperature. Since many intravenous injectable drugs exhibit lytic activity against erythrocytes, we examined this activity for the emulsion form of cardol, a natural compound with already proven hemolytic properties. The incorporation of this agent into the emulsion caused an evident decrease in hemolytic activity (97-99%). This highly protective effect, observed against sheep erythrocytes, was independent of both the composition and the particle size of the emulsions used. Our studies suggest that nonionic surfactant/phospholipid-based emulsions containing this edible oil of A. pavonina L. may be useful as an alternative formulation matrix for pharmaceutical, nutritional or cosmetic applications of otherwise membrane-acting components.
Keywords Adenanthera pavonina L., Submicron oil-in-water emulsion, Hemolysis, Cardol
Address and Contact Information 1Department of Lipids and Liposomes, Institute of Biochemistry and Molecular Biology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
2Department of Medicinal Microbiology & Immunology, 424 Medical Science Centre, University of Wisconsin – Madison, 1300 University Ave, Madison, WI 53706, USA
* Corresponding author; e-m ail: ajaromin@ibmb.uni.wroc.pl
[Rozmiar: 1332 bajtów]