Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 11 (2006) No. 4

DOI: 10.2478/s11658-006-0036-0 Volume 11 (2006) pp 449 - 460
Title	Schistosoma japonicum: THE DESIGN AND EXPERIMENTAL EVALUATION OF A MULTIVALENT DNA VACCINE
Authors	Chunyan Li^1,3, Longjiang Yu¹*, Zhi Liu¹, Lu Zhu¹, Yuan Hu², Min Zhu¹, Xiaohua Zhu², Youen Shi² and Sijin Meng^1,3
Abstract	The aim of this study was to construct and evaluate the immunity efficacy of the DNA multivalent vaccine pVIVO₂S_jFABP-23. The vaccine was constructed and produced as follows. Forty BALB/c mice were divided into four groups designated pVIVO₂, pVIVO₂S_j23, pVIVO²S_jFABP and pVIVO₂S_jFABP-23. Each mouse was immunized with 100 μg of the corresponding plasmid DNA by intramuscular injection. 28 days postvaccination, the mice were challenged with S. japonicum cercariae, and the worm and egg burdens were determined 42 days post-challenge. Serum samples were collected from all the mice before and after vaccination and at the end of the experiment, and used for antibody detection. The IFN-γ and IL-4 levels were quantified in the supernatants of specifically stimulated spleen cells. The number of worms was reduced by 52%, 40% and 42% in mice respectively immunized with pVIVO₂S_jFABP-23, pVIVO₂S_j23 or pVIVO₂S_jFABP. A respective 61%, 38% and 39% egg reduction was determined relative to those mice that only received the empty pVIVO₂ plasmid. pVIVO₂S_jFABP-23 immunization increased IgG levels against SWAP and SEA. Increased IFN-γ levels were detected in the supernatant of specific stimulated spleen cells from mice immunized with the 3 different constructs. The multivalent DNA vaccine developed induced higher levels of protection than the two monovalent tested vaccines.
Keywords	DNA vaccine, Schistosoma japonicum, Sj23, SjFABP, Protective efficacy
Adress and Contact Informations	¹Life school, Huazhong University of Science and Technology, Luoyu Road 1037, Wuhan 430074, PR China, ²Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 13,Wuhan 430030,PR China, ³Department of Sports Kinetics, Wuhan Institute of Physical Education, Luoyu Road 461, Wuhan 430079, PR China * Corresponding author; tel: 86-27-87792264, fax: 86-27-87792265, e-mail: y ulj@mail.hust.edu.cn

DOI: 10.2478/s11658-006-0038-y Volume 11 (2006) pp 461 - 474
Title	OSTEOBLAST DIFFERENTIATION OF NIH3T3 FIBROBLASTS IS ASSOCIATED WITH CHANGES IN THE IGF-I/IGFBP EXPRESSION PATTERN
Authors	Basem M. Abdallah*
Abstract	Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are essential regulators for osteoblast proliferation and differentiation. It has been reported that Dexamethasone (Dex), an active glucocorticoid (GC) analogue, synergizes the stimulatory effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast differentiation in the mouse fibroblastic cell line NIH3T3. I investigated whether this stimulatory effect is associated with changes in the expression pattern of the IGF/IGFBP system. Quantitative realtime PCR technology was used to quantify the gene expression levels of the IGF-system during osteoblast differentiation and in response to 1,25(OH)2D3 or Dex alone under serum-containing and serum-free culture conditions. Interestingly, NIH3T3 was shown to express high mRNA levels of IGF-I, IGF-II and IGFBP-5, and low levels of both IGFBP-2 and -6. During osteoblast differentiation (days 6-12), IGF-I mRNA was repressed by more than 60%, while the transcript of IGFBP-5 was markedly up-regulated, by more than 50- fold. Similarly, treatment with Dex alone resulted in a dose- and time-dependent increase in the expression of IGFBP-5 and a decrease in IGF-I mRNA. Treatment with 1,25(OH)2D3 alone increased the mRNA levels of IGF-I and IGFBP-6 by around 4- and 7-fold, respectively, in a dose- and time-dependent manner. In conclusion, my data demonstrated that osteoblast differentiation of NIH3T3 is associated with changes in the expression pattern of IGFs/IGFBPs, which are regulated by glucocorticoid in the presence of 1,25(OH)2D3. Modulation of the IGF/IGFBP levels by glucocorticoid might suggest important roles for the IGF-system in mediating the osteoblast differentiation of the NIH3T3 cell line.
Keywords	IGFs/IGFBPs, NIH3T3, Gene expression, Osteoblast differentiation
Adress and Contact Informations	Department of Biochemistry, Faculty of Science, Helwan University, Cairo, Egypt *Present address: Department of Endocrinology and Metabolism, University Hospital of Odense, Medical Biotechnology Center, SDU, DK-5000 Odense C, Denmark, tel: +45 65503057, fax: +45 6 5503950, e-mail: babdallah@health.sdu.dk

DOI: 10.2478/s11658-006-0039-x Volume 11 (2006) pp 475 - 487
Title	A COMPARATIVE GENE-EXPRESSION ANALYSIS OF CD34+ HEMATOPOIETIC STEM AND PROGENITOR CELLS GROWN IN STATIC AND STIRRED CULTURE SYSTEMS
Authors	Qunliang Li^1,2, Qiwei Liu¹, Haibo Cai¹ and Wen-Song Tan¹*
Abstract	Static and stirred culture systems are widely used to expand hematopoietic cells, but differential culture performances are observed between these systems. We hypothesize that these differential culture outcomes are caused by the physiological responses of CD34⁺ hematopoietic stem and progenitor cells (HSPCs) to the different physical microenvironments created in these culture devices. To understand the genetic changes provoked by culture microenvironments, the gene expression profiling of CD34⁺ HSPCs grown in static and stirred culture systems was compared using SMART-PCR and cDNA arrays. The results revealed that 103 and 99 genes were significantly expressed in CD34+ cells from static and stirred systems, respectively. Of those, 91 have similar levels of expression, while 12 show differential transcription levels. These differentially expressed genes are mainly involved in anti-oxidation, DNA repair, apoptosis, and chemotactic activity. A quantitative molecular understanding of the influences of growth microenvironments on transcriptional events in CD34+ HSPCs should give new insights into optimizing culture strategies to produce hematopoietic cells.
Keywords	CD34⁺ hematopoietic stem and progenitor cells, Ex vivo expansion, Culture microenvironment, SMART-PCR, cDNA array
Adress and Contact Informations	¹The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, P.R. China, ²School of Chemistry & Chemical Engineering, Guangxi University, 100 Daxue Road, Nanning 530004, P.R. China * Author for correspondence; e-mail: wstan@ecust.edu.cn, phone: +86 21 64250948; fax: +86 21 64252250

DOI: 10.2478/s11658-006-0040-4 Volume 11 (2006) pp 488 - 505
Title	ENDOPLASMIC RETICULUM STRESS AND APOPTOSIS
Authors	Jitka Faitova, Daniel Krekac, Roman Hrstka and Borivoj Vojtesek*
Abstract	Cell death is an essential event in normal life and development, as well as in the pathophysiological processes that lead to disease. It has become clear that each of the main cellular organelles can participate in cell death signalling pathways, and recent advances have highlighted the importance of the endoplasmic reticulum (ER) in cell death processes. In cells, the ER functions as the organelle where proteins mature, and as such, is very responsive to extracellular-intracellular changes of environment. This short overview focuses on the known pathways of programmed cell death triggering from or involving the ER.
Keywords	Endoplasmic reticulum, Apoptosis, p53, Scotin
Adress and Contact Informations	Department of Pathology and Experimental Oncology, Masaryk Memorial Cancer Institute, Zluty Kopec 7, 656 53 Brno, Czech Republic * Author for correspondence; e-mail: vojtesek@mou.cz

DOI: 10.2478/s11658-006-0041-3 Volume 11 (2006) pp 506 - 525
Title	CURRENT CONCEPTS IN APOPTOSIS: THE PHYSIOLOGICAL SUICIDE PROGRAM REVISITED
Authors	Indrajit Chowdhury¹, Binu Tharakan² and Ganapathy K. Bhat¹*
Abstract	Apoptosis, or programmed cell death (PCD), involves a complex network of biochemical pathways that normally ensure a homeostatic balance between cellular proliferation and turnover in nearly all tissues. Apoptosis is essential for the body, as its deregulation can lead to several diseases. It plays a major role in a variety of physiological events, including embryonic development, tissue renewal, hormone-induced tissue atrophy, removal of inflammatory cells, and the evolution of granulation tissue into scar tissue. It also has an essential role in wound repair. The various cellular and biochemical mechanisms involved in apoptosis are not fully understood. However, there are two major pathways, the extrinsic pathway (receptor-mediated apoptotic pathway) and the intrinsic pathway (mitochondria-mediated apoptotic pathway), which are both well established. The key component in both is the activation of the caspase cascade. Caspases belong to the family of proteases that ultimately, by cleaving a set of proteins, cause disassembly of the cell. Although the caspase-mediated proteolytic cascade represents a central point in the apoptotic response, its initiation is tightly regulated by a variety of other factors. Among them, Bcl-2 family proteins, TNF and p53 play pivotal roles in the regulation of caspase activation and in the regulation of apoptosis. This review summarizes the established concepts in apoptosis as a physiological cell suicide program, highlighting the recent and significant advances in its study.
Keywords	Apoptosis, Programmed cell death, Pathways, Caspases, Bcl-2, p53, TNF, Apaf
Adress and Contact Informations	¹Department of Obstetrics and Gynecology, Morehouse School of Medicine, Atlanta, GA, USA, ²Department of Neurology, Scott and White Clinic, The Texas A & M University Health Science Center, College of Medicine, Temple, Texas, USA * Author for correspondence; e-mail: gbhat@msm.edu

DOI: 10.2478/s11658-006-0042-2 Volume 11 (2006) pp 526 - 535
Title	AtGRP7 IS INVOLVED IN THE REGULATION OF ABSCISIC ACID AND STRESS RESPONSES IN ARABIDOPSIS
Authors	Shuqing Cao*, Li Jiang, Shiyong Song, Ran Jing and Guosheng Xu
Abstract	The Arabidopsis AtGRP7 gene, encoding a glycine-rich RNA-binding protein, has been shown to be involved in the regulation of a circadian-regulated negative feedback loop. However, little is known about the role of AtGRP7 in mediating abscisic acid (ABA) and stress responses. Here, we show that AtGRP7 plays a role in both. AtGRP7 was repressed by ABA, high salt and mannitol. Disruption of AtGRP7 by T-DNA insertion led to hypersensitive responses to ABA in both seed germination and root growth assays. The atgrp7-1 mutant was also hypersensitive to osmotic stress conditions, such as high salt and high concentrations of mannitol. In addition, the atgrp7-1 mutant plants accumulated significantly higher transcript levels of two ABA- and stress-inducible genes, RD29A and RAB18, compared with the wild-type plants. Taken together, these results suggest that AtGRP7 is involved in the regulation of ABA and stress responses.
Keywords	AtGRP7 gene, Abscisic acid, Osmotic stress
Adress and Contact Informations	School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, Anhui 230009, China * Author for correspondence; e-mail: shuqing.cao@163.com, phone: +86-551-2901505- 8 416

DOI: 10.2478/s11658-006-0044-0 Volume 11 (2006) pp 536 - 556
Title	PLANT DEHYDRINS – TISSUE LOCATION, STRUCTURE AND FUNCTION
Authors	Tadeusz Rorat*
Abstract	Dehydrins (DHNs) are part of a large group of highly hydrophilic proteins known as LEA (Late Embryogenesis Abundant). They were originally identified as group II of the LEA proteins. The distinctive feature of all DHNs is a conserved, lysine-rich 15-amino acid domain, EKKGIMDKIKEKLPG, named the K-segment. It is usually present near the C-terminus. Other typical dehydrin features are: a track of Ser residues (the S-segment); a consensus motif, T/VDEYGNP (the Y-segment), located near the N-terminus; and less conserved regions, usually rich in polar amino acids (the Φ-segments). They do not display a well-defined secondary structure. The number and order of the Y-, S- and K-segments define different DHN sub-classes: Y_nSK_n, Y_nK_n, SK_n, K_n and K_nS. Dehydrins are distributed in a wide range of organisms including the higher plants, algae, yeast and cyanobacteria. They accumulate late in embryogenesis, and in nearly all the vegetative tissues during normal growth conditions and in response to stress leading to cellular dehydration (e.g. drought, low temperature and salinity). DHNs are localized in different cell compartments, such as the cytosol, nucleus, mitochondria, vacuole, and the vicinity of the plasma membrane; however, they are primarily localized to the cytoplasm and nucleus. The precise function of dehydrins has not been established yet, but in vitro experiments revealed that some DHNs (YSK_n-type) bind to lipid vesicles that contain acidic phospholipids, and others (K_nS) were shown to bind metals and have the ability to scavenge hydroxyl radicals [Asghar, R. et al. Protoplasma 177 (1994) 87-94], protect lipid membranes against peroxidation or display cryoprotective activity towards freezing-sensitive enzymes. The SKn- and K-type seem to be directly involved in cold acclimation processes. The main question arising from the in vitro findings is whether each DHN structural type could possess a specific function and tissue distribution. Much recent in vitro data clearly indicates that dehydrins belonging to different subclasses exhibit distinct functions.
Keywords	Dehydration stress, Drought, Cold acclimation, Freezing tolerance, LEA proteins, Dehydrin
Adress and Contact Informations	Institute of Plant Genetics, PAS, Strzeszyńska 34, 60-479 Poznań, Poland * E-mail: tror@igr.poznan.pl

DOI: 10.2478/s11658-006-0043-1 Volume 11 (2006) pp 557 - 569
Title	IS A GENETIC DEFECT IN Fkbp6 A COMMON CAUSE OF AZOOSPERMIA IN HUMANS?
Authors	Toshinobu Miyamato¹,*^# Hisashi Sato^1#, Leah Yogev², Sandra Kleiman², Mikio Namiki³, Eitetsu Koh³, Naoko Sakugawa¹, Hiroaki Hayashi¹, Mutsuo Ishikawa¹, Dolores J. Lamb⁴ and Kazuo Sengoku¹
Abstract	FK506-binding protein 6 (Fkbp6) is a member of a gene family containing a prolyl isomerase/FK506-binding domain and tetratricopeptide protein-protein interaction domains. Recently, the targeted inactivation of Fkbp6 in mice has been observed to result in aspermic males and the absence of normal pachytene spermatocytes. The loss of Fkbp6 results in abnormal pairing and a misalignment of the homologous chromosomes, and in non-homologous partner switches and autosynapsis of the X chromosome cores in meiotic spermatocytes. In this study, we analyzed whether human FKBP6 gene defects might be associated with human azoospermia. We performed a mutation analysis in all the coding regions of the human FKBP6 gene in 19 patients with azoospermia resulting from meiotic arrest. The expression of the human FKBP6 gene was specific to the testis, and a novel polymorphism site, 245C��G (Y60X) could be found in exon 3. Our findings suggest that the human FKBP6 gene might be imprinted in the testis based on an analysis using two polymorphism sites.
Keywords	Azoospermia, FKBP6, Genomic imprinting, Meiosis, Polymorphism
Adress and Contact Informations	¹Department of Obstetrics and Gynecology, Asahikawa Medical College, Midorigaokahigashi 2-1-1-1, Asahikawa 078-8510, Japan, ²Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, Tel Aviv, 64239, Israel, ³Department of Intrgrated Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Science, Kanazawa, 920- 8641, Japan, ⁴Scott Department of Urology, and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, 77030, USA * Author for correspondence: e-mail: toshim@asahikawa-med.ac.jp, phone: +81-166-68- 2562, fax: +81-166-68-2569 # These authors equally contributed to this paper

DOI: 10.2478/s11658-006-0045-z Volume 11 (2006) pp 570 - 578
Title	Helicobacter pylori INFECTION CAN MODULATE THE SUSCEPTIBILITY OF GASTRIC MUCOSA CELLS TO MNNG
Authors	Michał Arabski¹, Paweł Kazmierczak², Maria Wiśniewska-Jarosińska³, Zbigniew Morawiec⁴, Alina Morawiec-Bajda⁵, Grażyna Klupińska³, Józef Drzewoski⁵, Jan Chojnacki³ and Janusz Błasiak¹*
Abstract	The pathogenesis of stomach cells can be associated with their susceptibility to exogenous dietary irritants, like nitrosamines such as dimethylnitrosamines (DMNA), and to the effects of non-dietary factors, including Helicobacter pylori infection. We used N-methyl-N’-nitro N-nitrosoguanidyne (MNNG) as a surrogate agent that induces a spectrum of DNA damage similar to DMNA. Using the alkaline comet assay, we showed that antioxidants – vitamins C and E, quercetin, and melatonin – reduced the genotoxic effect of MNNG in H. pylori-infected and non-infected human gastric mucosa cells (GMCs). To compare the sensitivity of the stomach and the blood, the experiment was also carried out in peripheral blood. We observed a higher level of DNA damage induced by MNNG in H. pylori-infected than in noninfected GMCs. We did not note any difference in the efficacy of the repair of the damage in either type of GMC. H. pylori infection may play an important role in the pathogenesis of GMCs, as it can modulate their susceptibility to dietary mutagens/carcinogens, thus contributing to gastric cancer.
Keywords	Helicobacter pylori, DNA damage, DNA repair, MNNG, Melatonin, Vitamin C, Vitamin E, Quercetin
Adress and Contact Informations	¹Department of Molecular Genetics, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland, ²Outpatient Gastroenterology Department, St John of God’s Hospital, Kosynierów Gdyńskich 61, 93-357 Łódź, Poland, ³Department of Gastroenterology and Internal Diseases, Medical University, 90-647 Łódź, Plac Hallera 1, Łódź, Poland, ⁴N. Copernicus Hospital, Pabianicka, Łódź, Poland, Department of Head and Neck Cancer, Medical University of Łódź, Łódź, Poland, ⁵Depatment of Diabeteology and Internal Diseases, Medical University of Łódź, Rewolucji 1905 37/39, 90-214 Łódź, Poland * Author for correspondence; phone: +48 42 635 4334, fax: +48-42 635 4484, e-mail: j anuszb@biol.uni.lodz.pl

DOI: 10.2478/s11658-006-0046-y Volume 11 (2006) pp 579 - 593
Title	A NEW METHOD FOR THE PREPERATIVE AND ANALYTICAL ELECTROPHORESIS OF CELLS
Authors	Anna Wilk, Kinga Rośkowicz and Włodzimierz Korohoda
Abstract	In this paper, a new method is described for the horizontal electrophoresis of cells on a density cushion under near-isopycnic conditions. When cell sedimentation is minimized, the electrophoresis of red blood cells (RBC) used as model cells within an anti-convective porous matrix (with pores over 300 μm in diameter) was capable of separating a mixture of human and chicken RBC according to their electrophoretic mobilities. Samples taken from the separated RBC bands show over 90% purity for each species. The simultaneous electrophoresis of several RBC samples carried out under identical conditions permitted the use of comparative data based on the electrophoretic mobility of cells which differ in their surface properties. We believe that this relatively simple system, in which cell sedimentation and convection are minimized, has the potential to be modified and adapted for the separation of other cell types/organelles.
Keywords	Cell electrophoresis, Cell separation, Cell surface, Red blood cells
Adress and Contact Informations	Department of Cell Biology, Faculty of Biotechnology, Jagiellonian University ul. Gronostajowa 7, 30-378 Kraków, Poland * Author for correspondence: e-mail: korohoda@moj.uj.edu.pl

DOI: 10.2478/s11658-006-0047-x Volume 11 (2006) pp 594 - 615
Title	PHYLOGENETIC ANALYSES WITHIN THREE SECTIONS OF THE GENUS Vicia
Authors	Tomasz Sakowicz¹ and Tomasz Cieślikowski²
Abstract	The averaged genomic similarities based on multilocus randomly amplified polymorphic DNA (RAPD) were calculated for eight species representing three sections of the genus Vicia: faba, bithynica and narbonensis. The frequency of appearance of the sequences corresponding to 25 decamers selected at random from genomes of different Fabace species was checked, and a high correlation with the frequency observed for Vicia allowed us to assume their similar weight in typing Vicia species. The RAPD-based similarity coefficients compared with those related to whole genome hybridization with barley rDNA and those based on restriction fragment length polymorphism (RFLP) revealed similar interspecies relationships. The averaged RAPD-based similarity coefficient (Pearson’s) was 0.68 for all the species, and was sectionspecific: 0.43 (bithynica), 0.50 (faba) and 0.73 (narbonensis). The averaged similarity coefficient for V. serratifolia (0.63) placed it apart from the rest (0.75) of its section. The results correspond to the interspecies relationships built upon non-genetic data. The averaged similarity coefficient for particular RAPD was related to the presence and type of tandemly repeated motif in a primer: 0.7-0.8 for heterodimers (GC, AG, CA, GT, CT), 0.5-0.6 for homodimers (CC, GG) and 0.6 for no repeat, indicating the sensitivity of diversity range to the type of target sequences.
Keywords	Vicia, Average similarity, Multilocus diversity, RAPD
Adress and Contact Informations	¹University of Łódź, Department of Cytogenetics and Plant Molecular Biology, 90-237 Łodź, Banacha 12/16, Poland, ²Centre for Medical Biology Polish Academy of Sciences, 93-232 Łódź, Lodowa 106, Poland * Author for correspondence: e-mail: tomeksakowicz@wp.pl

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