Vol. 16 No. 4 December 2011

DOI: 10.2478/s11658-011-0021-0 Volume 16 (2011) pp 515-538
Title QUANTITATIVE AND KINETIC PROFILE OF Wnt/ß-CATENIN SIGNALING COMPONENTS DURING HUMAN NEURAL PROGENITOR CELL DIFFERENTIATION
Authors Orianne Mazemondet1,4,§, Rayk Hubner1,§, Jana Frahm1, Dirk Koczan2, Benjamin M. Bader3, Dieter G. Weiss3, Adelinde M. Uhrmacher4, Moritz J. Frech1, Arndt Rolfs1,* and Jiankai Luo1,*
Abstract ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/ß-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/ß-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized ß-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and ß-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/ß-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/ß-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.
Keywords Wnt/ß-catenin pathway, Spatio-temporal dynamics, Quantitative kinetics
Address and Contact Information 1Albrecht-Kossel-Institute for Neuroregeneration, Centre for Mental Health Disease, University of Rostock, Gehlsheimer Strasse 20, 18147 Rostock,Germany,
2Proteome Center Rostock, University of Rostock, Schillingallee 69, 18055 Rostock, Germany,
3Institute of Biological Sciences, Cell Biology and Biosystems Technology, University of Rostock, 18059 Rostock, Germany,
4Modelling and Simulation Group, Institute of Computer Science, University of Rostock, Albert-Einstein-Str. 21, 18059 Rostock, Germany
§Both authors contributed equally to this work and should be considered co-first authors
* Authors for correspondence. Arndt Rolfs: e-mail: arndt.rolfs@med.uni-rostock.de, phone: 0049-3814949540, fax: 0049-3814949542, Jiankai Luo: e-mail: jiankai.luo@uni- rostock.de, phone: 0049-3814949629, fax: 0049-3814944899
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DOI: 10.2478/s11658-011-0022-z Volume 16 (2011) pp 539-563
Title ERp57/GRP58: A PROTEIN WITH MULTIPLE FUNCTIONS
Authors Carlo Turano*, Elisa Gaucci, Caterina Grillo and Silvia Chichiarelli
Abstract The protein ERp57/GRP58 is a stress-responsive protein and a component of the protein disulfide isomerase family. Its functions in the endoplasmic reticulum are well known, concerning mainly the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class 1. However, ERp57 is present in many other subcellular locations, where it is involved in a variety of functions, primarily suggested by its participation in complexes with other proteins and even with DNA. While in some instances these roles need to be confirmed by further studies, a great number of observations support the participation of ERp57 in signal transduction from the cell surface, in regulatory processes taking place in the nucleus, and in multimeric protein complexes involved in DNA repair.
Keywords Protein disulfide isomerases, Calcitriol, STAT3, Cellular stress, Signal transduction, DNA repair
Address and Contact Information Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Sapienza – Universita di Roma, Italy
* Author for correspondence. e-mail: carlo.turano@uniroma1.it, phone: +39 06 49910576, fax: +39 06 4440062
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DOI: 10.2478/s11658-011-0023-y Volume 16 (2011) pp 564-575
Title 17ß-ESTRADIOL PROMOTES CELL PROLIFERATION IN RAT OSTEOARTHRITIS MODEL CHONDROCYTES VIA PI3K/AKT PATHWAY
Authors Jia Gu Huang1,3, Chun Xia1*, Xin Peng Zheng1, Ting Ting Yi2, Xiao Yong Wang3, Gang Song2 and Bing Zhang2*
Abstract Osteoarthritis (OA) is the most common cause of musculoskeletal pain and disability. The importance of chondrocytes in the pathogenesis of OA is unequivocal. 17ß-estradiol (E2) has a potential protective effect against OA. However, the mechanism of E2 in OA chondrocytes remains unclear. In this study, we investigated the regulative effect of E2 on cell growth and the relationship between E2 and the PI3K/Akt pathway in rat OA model chondrocytes (pretreated with interleukin-1ß). We found that E2 induced chondrocyte proliferation, and increased the expression level of Akt simultaneously, especially the expression level of P-Akt. Furthermore, the inhibition of P-Akt could block chondrocyte proliferation induced by E2. These results suggest that PI3K/Akt activation induced by E2 may be an important factor in the mechanism of E2 in cell proliferation in rat OA model chondrocytes, and help further understanding the role of E2 in OA progression.
Keywords Osteoarthritis, Rat osteoarthritis model chondrocyte, 17ß-estradiol, Estrogen receptor, Akt, Cell proliferation, PI3K/Akt pathway
Address and Contact Information 1Zhongshan Hospital, Xiamen University, Fujian, China,
2Medical School, Xiamen University, Fujian, China,
3Ningde Municipal Hospital, Ningde, Fujian, China
* Authors for correspondence. e-mails - C. Xia: chunxia@xmu.edu.cn; B. Zhang: cristal66@xmu.edu.cn, phone.: +86 5925921461; fax: +86 5922188680
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DOI: 10.2478/s11658-011-0024-x Volume 16 (2011) pp 576-594
Title GENERALIZED STERN MODELS OF THE ELECTRIC DOUBLE LAYER CONSIDERING THE SPATIAL VARIATION OF PERMITTVITY AND FINITE SIZE OF IONS IN SATURATION REGIME
Authors Ekaterina Gongadze1, Ursula Van Rienen1 and Ales Iglic2*
Abstract The interaction between a charged metal implant surface and a surrounding body fluid (electrolyte solution) leads to ion redistribution and thus to formation of an electrical double layer (EDL). The physical properties of the EDL contribute essentially to the formation of the complex implant-biosystem interface. Study of the EDL began in 1879 by Hermann von Helmholtz and still today remains a scientific challenge. The present mini review is focused on introducing the generalized Stern theory of an EDL, which takes into account the orientational ordering of water molecules. To ascertain the plausibility of the generalized Stern models described, we follow the classical model of Stern and introduce two Langevin models for spatial variation of the relative permittivity for point-like and finite sized ions. We attempt to uncover the subtle interplay between water ordering and finite sized ions and their impact on the electric potential near the charged implant surface. Two complementary effects appear to account for the spatial dependency of the relative permittivity near the charged implant surface – the dipole moment vectors of water molecules are predominantly oriented towards the surface and water molecules are depleted due to the accumulation of counterions. At the end the expressions for relative permittivity in both Langevin models were generalized by also taking into account the cavity and reaction field.
Keywords Spatial variation of permittivity, Generalized Stern models, Water dipoles, Charged implant surface, Osteoblasts, Cell-implant interactions, Langevin model, Langevin-Bikerman model, Booth model, Gongadze-Iglic model
Address and Contact Information 1Institute of General Electrical Engineering, University of Rostock, Albert-Einstein-Straße 2, 18051 Rostock, Germany,
2Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, SI-1000 Ljubljana, Slovenia
* Author for correspondence. e-mail: ales-iglic@fe.uni-lj.si , phone: +386 1 4768 825
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DOI: 10.2478/s11658-011-0025-9 Volume 16 (2011) pp 595-609
Title NON-ERYTHROID BETA SPECTRIN INTERACTING PROTEINS AND THEIR EFFECTS ON SPECTRIN TETRAMERIZATION
Authors Akin Sevinc and Leslie W.-M. Fung*
Abstract With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697-2145) of non-erythroid beta spectrin (ßII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with ßII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with ßII-C (IPßII-C s). The proteins include a fragment (residues 38-284) of "THAP domain containing, apoptosis associated protein 3, isoform CRA g", "glioma tumor suppressor candidate region gene 2" (residues 1-478), a fragment (residues 74-442) of septin 8 isoform c, a fragment (residues 704-953) of "coatomer protein complex, subunit beta 1, a fragment (residues 146-614) of zinc-finger protein 251, and a fragment (residues 284-435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these ßII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPßII-C s were able to bind ßII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of ßII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and ßII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.
Keywords Brain beta spectrin, Spectrin tetramerization, Brain proteins, Yeast three-hybrid, Library screening, Spectrin interacting proteins
Address and Contact Information Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, MC 111, Chicago, IL 60607, USA
* Author for correspondence. e-mail: lfung@uic.edu, phone: 312-355-5516, fax: 312-996-0431
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DOI: 10.2478/s11658-011-0026-8 Volume 16 (2011) pp 610-624
Title THE DETERMINATION OF CHANGES IN THE EXPRESSION OF GENES FOR SELECTED SPECIFIC TRANSCRIPTIONAL FACTORS IN in vitro DUCTAL BREAST CANCER CELLS UNDER THE INFLUENCE OF PACLITAXEL
Authors Marta Ziaja-Sołtys* and Jolanta Rzymowska
Abstract This study aimed to determine the changes in the expression of genes for selected specific transcriptional factors that have both activating and repressing functions in in vitro ductal breast cancer cells, under the influence of paclitaxel, applying the microarray technique. The cells are treated with 60 ng/ml and 300 ng/ml doses of paclitaxel that correspond to those applied in breast cancer therapy. About 60 ng/ml doses of paclitaxel cause a statistically significant increase in expression of all the 16 analysed genes coding transcriptional factors, ranging from 1.84-fold (for PO4F2) to 4.65-fold (for LMO4) (p < 0.05) in comparison with the control cells, and enhanced the taxane mechanism of action. The 300 ng/ml doses of paclitaxel cause a cytotoxic effect in the cells. In this article, we argue that these changes in gene expression values may constitute prognostic and predictive factors in ductal breast cancer therapy.
Keywords Breast cancer, Transcriptional factors, Gene expression, Microarrays, Anti-tumour, Treatment, Paclitaxel, Microtubules, Cytotoxic effect, Apoptosis
Address and Contact Information Medical University of Lublin, Department of Biology and Genetics, 20-093 Lublin, ChodĄki 4A, Poland
* Author for correspondence. marta.ziaja-soltys@umlub.pl
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DOI: 10.2478/s11658-011-0027-7 Volume 16 (2011) pp 625-637
Title DU-145 PROSTATE CARCINOMA CELLS THAT SELECTIVELY TRANSMIGRATE NARROW OBSTACLES EXPRESS ELEVATED LEVELS OF Cx43
Authors Katarzyna Szpak§, Ewa Wybieralska§, Ewa Niedziałkowska§, Monika Rak, Iga Bechyne, Marta Michalik, Zbigniew Madeja and Jarosław Czyż*
Abstract The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the “leading front” formation during cancer invasion
Keywords Cancer invasion, Cell heterogeneity, Cell motility, Cx43, Gap junctions, Metastasis, Prostate cancer, Transmigration
Address and Contact Information Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387, Cracow, Poland
§ Contributed equally
* Author for correspondence. e-mail: jarek.czyz@uj.edu.pl, phone: +48 126646146, fax: +48 126646902
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DOI: 10.2478/s11658-011-0028-6 Volume 16 (2011) pp 638-651
Title FUNCTIONAL CHARACTERIZATION OF HUMAN KINDLIN-2 CORE PROMOTER IDENTIFIES A KEY ROLE OF SP1 IN KINDLIN-2 TRANSCRIPTIONAL REGULATION
Authors Ammad Aslam Khan1, Takashi Shimokawa1, Staffan Stromblad1 and Hongquan Zhang1,2*
Abstract Kindlin-2 is a recently identified FERM and PH domain containing integrin interacting protein. Kindlin-2 is ubiquitously expressed in normal tissues. So far, much effort has been spent exploring the functional aspects of Kindlin-2. However, the transcriptional regulation of Kindlin-2 has not yet been investigated. In this study we identified and functionally characterized the promoter of the human Kindlin-2 gene. We show that the core promoter of Kindlin-2 is a 39 base pair long GC rich fragment located -122/-83 upstream of the Kindlin-2 transcription start site. Functional characterization of this core promoter region by both in silico as well as in vitro/in vivo analysis shows that the transcription factor SP1 plays an important role in regulation of Kindlin-2 expression.
Keywords Kindlin-2, FERMT2, PLEKHC1, Mig-2, Transcription factor SP1, Promoter, Transcription start site, Gene regulation, Cis-acting elements, CpG island, Cell migration, Integrin, Gene expression
Address and Contact Information 1Karolinska Institutet, Center for Biosciences, Department of Biosciences and Nutrition, Novum, 141 83 Huddinge, Sweden,
2Peking University School of Basic Medical Sciences, Laboratory of Molecular Cell Biology and Tumor Biology and Key Laboratory of Carcinogenesis and Translational Research, The Ministry of Education, Beijing 100191, P.R. China
* Author for correspondence: e-mail: HUHongquan.Zhang@bjmu.edu.cnU, phone: 0086-10-82802424
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DOI: 10.2478/s11658-011-0029-5 Volume 16 (2011) pp 652-668
Title REAL-TIME PCR FOR THE DETECTION OF PRECISE TRANSGENE COPY NUMBER IN DURUM WHEAT
Authors Agata Gadaleta*, Angelica Giancaspro, Maria Francesca Cardone and Antonio Blanco
Abstract Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs. This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.
Keywords Durum wheat, Transgenic plants, Biolistic method, Glutenin subunits, Real-time PCR, Southern analysis, Transgene copy number, Quantitative PCR assay, Transgene stable expression, HMW-GS genes
Address and Contact Information Department of Environmental and Agro-Forestry Biology and Chemistry, Section of Genetics and Plant Breeding, University of Bari Aldo Moro, Via Amendola 165/A-70126 Bari, Italy
* Author for correspondence. e-mail: agata.gadaleta@agr.uniba.it, phone: +39 080 544 2995, fax: +39 080 544 2200
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DOI: 10.2478/s11658-011-0030-z Volume 16 (2011) pp 669-688
Title INHIBITOR-2 INDUCED M-PHASE ARREST IN XENOPUS CYCLING EGG EXTRACTS IS DEPENDENT ON MAPK ACTIVATION
Authors Arian Khandani1*, Mahmood Mohtashami2 and Anne Camirand3
Abstract The evolutionarily-conserved protein phosphatase 1 (PP1) plays a central role in dephosphorylation of phosphoproteins during the M phase of the cell cycle. We demonstrate here that the PP1 inhibitor inhibitor-2 protein (Inh-2) induces an M-phase arrest in Xenopus cycling egg extracts. Interestingly, the characteristics of this M-phase arrest are similar to those of mitogen-activated protein kinase (p42MAPK)-induced M-phase arrest. This prompted us to investigate whether Inh-2-induced M-phase arrest was dependent on activation of the p42MAPK pathway. We demonstrate here that MAPK activity is required for Inh-2-induced M-phase arrest, as inhibition of MAPK by PD98059 allowed cycling extracts to exit M phase, despite the presence of Inh-2. We next investigated whether Inh-2 phosphorylation by the MAPK pathway was required to induce an M-phase arrest. We discovered that while p90Rsk (a MAPK protein required for M-phase arrest) is able to phosphorylate Inh-2, this phosphorylation is not required for Inh-2 function. Overall, our results suggest a novel mechanism linking p42MAPK and PP1 pathways during M phase of the cell cycle
Keywords Inhibitor-2, PP-1 phosphatase, MAPK kinase, p90Rsk, M-phase arrest, Xenopus cycling egg extract
Address and Contact Information 1Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7,
2Department of Immunology, University of Toronto and Sunnybrook Research Institute, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5
3Lady Davis Institute for Medical Research, McGill University, Montréal, Québec, Canada H3T 1E2
* Author for correspondence. e-mail:arian.khandani@sickkids.ca
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