Vol. 15 No. 2 June 2010

DOI: 10.2478/s11658-010-0001-9 Volume 15 (2010) pp 177-195
Title A MICROARRAY GENE ANALYSIS OF PERIPHERAL WHOLE BLOOD IN NORMAL ADULT MALE RATS AFTER LONG-TERM GH GENE THERAPY
Authors Ying Qin* and Ya-Ping Tian*
Abstract The main aims of this study were to determine the effects of GH gene abuse/misuse in normal animals and to discover genes that could be used as candidate biomarkers for the detection of GH gene therapy abuse/misuse in humans. We determined the global gene expression profile of peripheral whole blood from normal adult male rats after long-term GH gene therapy using CapitalBio 27 K Rat Genome Oligo Arrays. Sixty one genes were found to be differentially expressed in GH gene-treated rats 24 weeks after receiving GH gene therapy, at a two-fold higher or lower level compared to the empty vector group (p < 0.05). These genes were mainly associated with angiogenesis, oncogenesis, apoptosis, immune networks, signaling pathways, general metabolism, type I diabetes mellitus, carbon fixation, cell adhesion molecules, and cytokine-cytokine receptor interaction. The results imply that exogenous GH gene expression in normal subjects is likely to induce cellular changes in the metabolism, signal pathways and immunity. A real-time qRT-PCR analysis of a selection of the genes confirmed the microarray data. Eight differently expressed genes were selected as candidate biomarkers from among these 61 genes. These 8 showed five-fold higher or lower expression levels after the GH gene transduction (p < 0.05). They were then validated in real-time PCR experiments using 15 single-treated blood samples and 10 control blood samples. In summary, we detected the gene expression profiles of rat peripheral whole blood after long-term GH gene therapy and screened eight genes as candidate biomarkers based on the microarray data. This will contribute to an increased mechanistic understanding of the effects of chronic GH gene therapy abuse/misuse in normal subjects.
Keywords Microarray, Peripheral whole blood, Growth hormone gene therapy, Biomarker genes
Address and Contact Information Department of Clinical Biochemistry, Chinese PLA General Hospital, 28 Fu-Xing Road, Beijing 100853, P.R. China
* Author for correspondence. e-mail address: yqrr54@yahoo.com.cn; TianYP301@tom.com; tel.: (86-10)-6693, 9449; fax: (86-10)-6693, 9374
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0005-5 Volume 15 (2010) pp 196-214
Title CHARACTERIZATION OF PROTEINS ASSOCIATING WITH 5’ TERMINUS OF PGHS-1 mRNA
Authors Natalia Bunimov and Odette Laneuville*
Abstract Induction of Prostaglandin Endoperoxide H Synthase-1 (PGHS-1) gene has been previously documented in a few studies during events such as development and cellular differentiation. However, molecular mechanisms governing the regulation of PGHS-1 gene expression and contributing to changes in protein levels are poorly understood. Using the MEG-01 cell model of PGHS-1 gene induction, our laboratory has previously demonstrated that the 5’UTR and the first two exons of PGHS-1 mRNA had a significant impact on decreasing the translational efficiency of a reporter gene and suggested that the presence of a secondary structure is required for conservation of this activity. This 5’end of PGHS-1 mRNA sequence has also been shown to associate with nucleolin protein. In the current study, we set to investigate the protein composition of the mRNP (messenger ribonucleoprotein) associating with the 5’end of PGHS-1 mRNA and to identify its protein members. RNA/protein binding assays coupled with LC-MS analysis identified serpin B1 and NF45 (nuclear factor 45) proteins as potential members of PGHS-1 mRNP complex. Immunoprecipitation experiments using MEG-01 protein extracts validated mass spectrometry data and confirmed binding of nucleolin, serpin B1, NF45 and NF90. The RNA fraction was extracted from immunoprecipitated mRNP complexes and association of RNA binding proteins, serpin B1, NF45 and NF90, to PGHS-1 mRNA target sequence was confirmed by RT-PCR. Together these data suggest that serpin B1, NF45 and NF90 associate with PGHS-1 mRNA and can potentially participate in the formation a single or a number of PGHS-1 ribonucleoprotein complexes, through nucleolin that possibly serves as a docking base for other protein complex members.
Keywords Prostaglandin endoperoxide H synthase-1, Cyclooxygenase-1, MEG-01, Megakaryoblastic cells, Untranslated region, Open reading frame, Messenger ribonucleoprotein, Serpin B1, Nuclear factor 45, Nuclear factor 90
Address and Contact Information Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, K1H 8M5 Canada
* Author for correspondence. e-mail: olaneuvi@uottawa.ca, tel.: 613-562-5800 x8402, fax: 613-562-5452
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0002-8 Volume 15 (2010) pp 215-233
Title THE PHYSIOLOGICAL AND MORPHOLOGICAL PHENOTYPE OF A YEAST MUTANT RESISTANT TO THE QUATERNARY AMMONIUM SALT N-(DODECYLOXYCARBOXYMETHYL)-N,N,N-TRIMETHYL AMMONIUM CHLORIDE
Authors Ewa Obłąk1*, Andrzej Gamian2,3, Ryszard Adamski1 and Stanisław Ułaszewski1
Abstract We investigated the action of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride) on Saccharomyces cerevisiae yeast cells. Changes in the yeast cell ultrastructure were confirmed by electron microscopy. We treated resistant mutant cells with QAS, and confirmed destruction of the mutant cytoplasm, an increase in the thickness of the cell wall, separation of the cell wall from the cytoplasm, and the accumulation of numerous lipid droplets. We also observed a relatively high production of lipids in the cells of the parental wild-type strain Σ1278b and in its IM-resistant (IMR) mutant in the presence of the QAS. The IMR mutant showed increased sensitivity to CaCl2 and SDS, and resistance to ethidium bromide, chloramphenicol, erythromycin and osmotic shock. It also tolerated growth at low pH. We suggest that the resistance to IM could be connected with the level of permeability of the cell membrane because the IMR mutant was sensitive to this compound in vivo in the presence of SDS and guanidine hydrochloride, which cause increased permeability of the cell plasma membrane.
Keywords Quaternary ammonium salts, Drug resistance, Saccharomyces cerevisiae
Address and Contact Information 1Institute of Genetics and Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
2Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland,
3Department of Medical Biochemistry, Wrocław Medical University, Chałubińskiego 10, 50-368 Wrocław, Poland
* Author for correspondence. e-mail: ewa.oblak@microb.uni.wroc.pl
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0006-4 Volume 15 (2010) pp 234-241
Title FLAVONOID TRANSPORT ACROSS RBE4 CELLS: A BLOOD-BRAIN BARRIER MODEL
Authors Ana Faria1,2*, Diogo Pestana1, Diana Teixeira1, Joana Azevedo2, Victor De Freitas2, Nuno Mateus2 and Conceicao Calhau1
Abstract There is a growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the Central Nervous System (CNS), which is associated with a number of pathophysiological processes, including Alzheimer’s and Parkinson’s diseases and cerebrovascular diseases. Identifying the mechanisms associated with phenolic neuroprotection has been delayed by the lack of information concerning the ability of these compounds to enter the CNS. The aim of this study was to evaluate the transmembrane transport of flavonoids across RBE-4 cells (an immortalized cell line of rat cerebral capillary endothelial cells) and the effect of ethanol on this transport. The detection and quantification of all of the phenolic compounds in the studied samples (basolateral media) was performed using a HPLC-DAD (Diode Array Detector). All of the tested flavonoids (catechin, quercetin and cyanidin-3-glucoside) passed across the RBE-4 cells in a time-dependent manner. This transport was not influenced by the presence of 0.1% ethanol. In conclusion, the tested flavonoids were capable of crossing this blood-brain barrier model.
Keywords Anthocyanin, Blood-brain barrier, Flavonol, 3-flavanol, RBE4, Transport
Address and Contact Information 1Department of Biochemistry (U38-FCT), Faculty of Medicine of the University of Porto, Al. Prof. Hernâni Monteiro, 4200-319 Porto, Portugal,
2Chemistry Investigation Centre (CIQ), Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
* Author for correspondence. e-mail: anafaria@med.up.pt, tel./fax: +351 22 551 36 24
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-004-6 Volume 15 (2010) pp 242-259
Title CIRCULATING NUCLEIC ACIDS AS A NEW DIAGNOSTIC TOOL
Authors Marketa Urbanova1,2, Jan Plzak1,3,4*, Hynek Strnad2 and Jan Betka1
Abstract The discovery of circulating nucleic acids in the 1940s opened up new possibilities for the non-invasive detection, monitoring and screening of various human disorders. Several tumour markers that enable early cancer detection or tumour behaviour prediction have been detected in the plasma of cancer patients. Maternal plasma analysis can be used to detect certain fetal abnormalities, with the quantification of cell-free nucleic acids used to screen for several pregnancy-associated disorders. Some other applications are in transplant monitoring and graft rejection assessment, and in certain medical emergencies such as trauma and burn severity stratification. Many studies have yielded promising results in this field, but the techniques have yet to be applied in routine clinical practice. Large-scale studies using similar technologies and a broad spectrum of patients are still needed to verify the results of the various studies.
Keywords Circulating nucleic acids, RNA, DNA, Diagnostics, Cancer
Address and Contact Information 1Charles University, 1st Faculty of Medicine, Department of Otorhinolaryngology and Head and Neck Surgery, Faculty Hospital Motol, Institute of Postgraduate Medicine, V Uvalu 84, 15006 Prague 5, Czech Republic,
2Academy of Sciences of the Czech Republic, Institute of Molecular Genetics, Department of Genomics and Bioinformatics, Videnska 1083, 14220 Prague 4, Czech Republic,
3Charles University, 2nd Faculty of Medicine, Centre of Cell Therapy and Tissue Repair, V Uvalu 84, 15006 Prague 5, Czech Republic,
4 Charles University, 1st Faculty of Medicine, Institute of Anatomy, U Nemocnice 3, 12800 Prague 2, Czech Republic
* Author for correspondence. e-mail: jan.plzak@lf1.cuni.cz
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0003-7 Volume 15 (2010) pp 260-271
Title GLOBAL MAPPING OF ZBTB7A TRANSCRIPTION FACTOR BINDING SITES IN HepG2 CELLS
Authors Xuyu Zu1, Lingling Yu1, Yiming Sun3, Jing Tian1, Feng Liu1, Qinsheng Sun1, Shengnan He1, Guang Sun1, Weishi Luo1 and Yuyang Jiang1,2*
Abstract ZBTB7A is a known proto-oncogene that is implicated in carcinogenesis and cell differentiation and development. Fully understanding the function of ZBTB7A in cellular processes could provide useful strategies for cancer treatment and development-associated disease therapy. Here, global mapping of ZBTB7A transcription factor binding sites was developed by utilizing microarray technology in HepG2 cells. The data obtained from the microarrays was further validated via chromatin immunoprecipitation-PCR (ChIP-PCR) and real time-PCR, and it was revealed that ZBTB7A may be one of the regulators of neural development. ZBTB7A target signal pathways were identified in signal pathway and GO (Gene Ontology) analyses. This is the first report on the global mapping of ZBTB7A downstream direct targets, and these findings will be useful in understanding the roles of ZBTB7A in cellular processes.
Keywords ZBTB7A, Proto-oncogene, Transcription factor, ChIP-on-chip analysis, Signal pathway
Address and Contact Information 1The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong 518055, People’s Republic of China,
2School of Medicine, Tsinghua University, Beijing 100084, People’s Republic of China,
3Medical Systems Biology Research Center, Tsinghua University, Beijing 100084, People’s Republic of China, National Engineering Research Center for Beijing Biochip Technology (NERCBBT), 18 Life Science Parkway, Beijing 102206, People's Republic of China
* Author for correspondence. e-mail: jiangyy@sz.tsinghua.edu.cn, tel: +86 755 26036017, fax: +86 755 26032094
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0007-3 Volume 15 (2010) pp 272-295
Title INHIBITOR OF DIFFERENTIATION 1 (ID1) PROMOTES CELL SURVIVAL AND PROLIFERATION OF PROSTATE EPITHELIAL CELLS
Authors Michelle Schmidt1, Ananthi J. Asirvatham2 and Jaideep Chaudhary2*
Abstract Id1 (inhibitor of differentiation 1) is a member of the bHLH protein family. Consistent with its role in promoting proliferation and inhibiting differentiation, Id1 expression is low or negligible in normal prostate epithelial cells but is high in prostate cancer. Ectopic expression of Id1 in normal prostate epithelial cells could therefore provide a model for understanding early events involved in initiation of prostate cancer. Over-expression of Id1 immortalized but did not transform ventral prostate epithelial cells (Id1-RPE). Immortalization was associated with decreased Cdkn2a, Cdkn1a, androgen receptor and increased Tert expression. Gene expression profiling over successive doublings was used to identify transcriptomic changes involved during immortalization (Tieg, Jun, alpha actin, Klf10, Id2) and in maintaining the immortalized phenotype (Igfbp3, Igfbp5, Mmp2, Tgfb3). Network analysis indicated that Id1 promotes cancer/tumor morphology, cell cycle and epithelial to mesenchymal transition by influencing AP1, tnf, tgfß, PdgfBB and estradiol pathways. During immortalization, the expression of majority of differentially expressed genes reduced over progressive doublings suggesting a decline in transcriptional regulatory mechanisms. The associated molecular/gene expression profile of Id1-RPE cells provides an opportunity to understand the molecular pathways associated with prostate epithelial cell survival and proliferation.
Keywords Id1, Prostate, Epithelial cells, Immortalization, Cancer
Address and Contact Information 1Sleep Research Laboratory, Washington State University, Spokane, WA, USA,
2Department of Biological Sciences, Center for Cancer Research and Therapeutics Development, Clark Atlanta University, 223 James P. Brawley Dr. SW, Atlanta, GA 30314, USA
* Author for correspondence. e-mail: jchaudhary@cau.edu, tel.: 404 880 6821, fax: 404 880 8065
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0009-1 Volume 15 (2010) pp 296-310
Title THE ANTI-APOPTOTIC PROTEIN LIFEGUARD IS EXPRESSED IN BREAST CANCER CELLS AND TISSUES
Authors Vesna Bucan*, Kerstin Reimers, Claudia Yu Choi, Mau-Thek Eddy and Peter M. Vogt
Abstract Lifeguard (LFG) is an anti-apoptotic protein that inhibits Fas- mediated death in tumour cells. However, the molecular function of human LFG in the carcinogenesis of human breast cells is uncertain. We studied the expression and function of endogenous LFG in four breast cancer cell lines (MCF-7, MDA-MB-231, T-47D and HS 578T), a human breast epithelial cell line (HS 578Bst), and in healthy and cancerous breast tissues. Molecular (Western blot and RT-PCR) and immunohistochemical techniques were used to investigate the LFG expression. To investigate the breast cancer cell proliferation in the presence of Fas, we performed fluorescent cell viability assays. The possible association of Fas with LFG was analyzed by immunofluorescence microscopy. In this paper, we provide convincing evidence that LFG is overexpressed in several human breast cancer cell lines. More importantly, we found that the LFG expression correlates with high tumour grades in primary breast tumours. Finally, we demonstrated that Fas sensitivity is reduced in breast cancer cell lines expressing LFG. Our results indicated that LFG is strongly expressed in breast cancer epithelial cells. Moreover, the overexpression of LFG correlated with tumour grade and reduced Fas sensitivity. Our findings support the idea that LFG may have a role in the downregulation of apoptosis in breast cancer cells.
Keywords Apoptosis, Lifeguard, Fas, Breast cancer
Address and Contact Information Department of Plastic, Hand and Reconstructive Surgery, Medical School of Hannover, Podbielskistraße 380, D-30659 Hannover, Germany
* Author for correspondence: Dr. Vesna Bucan, Abteilung für Plastische, Hand- und Wiederherstellungschirurgie, Medizinische Hochschule Hannover, Carl-Neubergstr. 1, 30625 Hannover, Germany, e-mail: Bucan.vesna@mh-hannover.de, tel.: 0511-532-8856, fax: 0511-532-8890
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0010-8 Volume 15 (2010) pp 311-341
Title MOLECULAR SYSTEMATICS: A SYNTHESIS OF THE COMMON METHODS AND THE STATE OF KNOWLEDGE
Authors Diego San Mauro1 * and Ainhoa Agorreta1,2
Abstract The comparative and evolutionary analysis of molecular data has allowed researchers to tackle biological questions that have long remained unresolved. The evolution of DNA and amino acid sequences can now be modeled accurately enough that the information conveyed can be used to reconstruct the past. The methods to infer phylogeny (the pattern of historical relationships among lineages of organisms and/or sequences) range from the simplest, based on parsimony, to more sophisticated and highly parametric ones based on likelihood and Bayesian approaches. In general, molecular systematics provides a powerful statistical framework for hypothesis testing and the estimation of evolutionary processes, including the estimation of divergence times among taxa. The field of molecular systematics has experienced a revolution in recent years, and, although there are still methodological problems and pitfalls, it has become an essential tool for the study of evolutionary patterns and processes at different levels of biological organization. This review aims to present a brief synthesis of the approaches and methodologies that are most widely used in the field of molecular systematics today, as well as indications of future trends and state-of-the-art approaches.
Keywords Molecular systematics, Phylogenetic inference, Molecular evolution, Phylogeny, Evolutionary analysis, Evolutionary hypothesis, Divergence time.
Address and Contact Information 1Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom,
2Department of Zoology and Ecology, University of Navarra, Irunlarrea s/n, 31008 Pamplona, Spain
* Author for correspondence: e-mail: d.san-mauro@nhm.ac.uk, tel.: +44 (0)20 7942 5315, fax: +44 (0)20 7942 5054
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0008-2 Volume 15 (2010) pp 342-355
Title STAT3 KNOCKDOWN BY siRNA INDUCES APOPTOSIS IN HUMAN CUTANEOUS T-CELL LYMPHOMA LINE Hut78 VIA DOWNREGULATION OF Bcl-xL
Authors Navin K. Verma*, Anthony M. Davies, Aideen Long, Dermot Kelleher and Yuri Volkov
Abstract Cutaneous T-cell lymphomas (CTCLs) are non-Hodgkin's lymphomas resulting from clonal expansion and localization of malignant T-lymphocytes to the skin. CTCL cells have defective apoptosis. Signal transducers and activators of transcription (STAT) are a family of transcription factors known to play important roles in the development and progression of several human cancers by promoting cell proliferation and protecting against apoptosis. In this study, we investigated the specific role of STAT3, a major component of the STAT family, in growth and survival of human CTCL cell line Hut78. Western immunoblot analysis showed elevated expression of STAT3 and phospho-STAT3(Y705) in human CTCL cells as compared to freshly isolated peripheral blood lymphocytes (PBLs). Specific knockdown of STAT3 expression in Hut78 cells by RNA interference induced morphological and biochemical changes indicating apoptotic cell death. Moreover, STAT3 inhibition downregulated the expression of Bcl2 family of anti-apoptotic gene Bcl-xL. These observations suggest that STAT3 is required for the survival of CTCL cells and strongly indicate that targeting STAT3 using siRNA techniques may serve a novel therapeutic strategy for the treatment of CTCL.
Keywords Apoptosis, Cutaneous T-cell lymphoma (CTCL), siRNA, STAT3
Address and Contact Information Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin 8, Ireland
* Author for correspondence. e-mail: verman@tcd.ie, tel: +353-1-8963350
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0011-7 Volume 15 (2010) pp 356-364
Title HOMOLOGOUS LIVER PARENCHYMAL CELL-CELL ADHESION MEDIATED BY AN ENDOGENOUS LECTIN AND ITS RECEPTOR
Authors Saswati Banerjee1 and Gopal Chandra Majumder1,2*
Abstract Many studies have implicated cell-surface lectins in heterologous cell-cell adhesion, but little is known about the participation of lectins in cellular adhesion in homologous cells. Here, we show the development of a cell model for investigating the direct role of a cell-surface lectin in homologous cell-cell adhesion. Parenchymal cells were isolated from caprine liver using a perfusion buffer, and dispersed in a chemically defined modified Ringer’s solution. These cells undergo autoagglutination in the presence of Ca2+. The autoagglutinated cells can be dissociated specifically with D-galactose (50 mM), which also inhibits the liver cell autoagglutination event. The blood serum protein fetuin has no effect on liver cell autoagglutination, whereas desialylated fetuin (100 µM), with its terminal D-galactose residue, showed a high affinity for blocking the autoagglutination event. The data demonstrates the occurrence of a Ca2+-dependent D-galactose-specific lectin and a lectin receptor on the parenchymal cells. Furthermore, it shows that the observed autoagglutination event is caused by the interaction of the cell-surface lectin with its receptor on the neighbouring homologous cells. The data supports the view that homologous cell-cell contact in mammalian tissues is triggered by such lectin-receptor interaction and that the previously reported cell-surface adhesive proteins serve as a secondary force to strengthen cell adhesion. This cell model could be extremely useful for investigating the direct role of cell-surface lectin and its receptor in homologous cell adhesion in a variety of tissues under normal and pathological conditions.
Keywords Autoagglutination, Homologous cell-cell adhesion, Lectin, Lectin receptor, Liver, Parenchymal cell
Address and Contact Information 1Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata - 700 032, India,
2Centre for Rural and Cryogenic Technologies, Jadavpur University, Kolkata - 700 032, India
* Author for correspondence. e-mail: majumdergc42@yahoo.co.in
[Rozmiar: 1332 bajtów]