Vol. 15 No. 4 September 2010

DOI: 10.2478/s11658-010-0026-0 Volume 15 (2010) pp 517-529
Title THE PROLIFERATION AND DIFFERENTIATION OF OSTEOBLASTS IN CO-CULTURE WITH HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS: AN IMPROVED ANALYSIS USING FLUORESCENCE-ACTIVATED CELL SORTING
Authors Yu Zhang1,2,3, Andreas Schedle3, Michael Matejka1, Xiaohui Rausch-Fan1 and Oleh Andrukhov1*
Abstract The interaction of osteoblasts and endothelial cells plays a pivotal role in osteogenesis. This interaction has been extensively studied using their direct co-culture in vitro. However, co-culture experiments require clear discrimination between the two different cell types in the mixture, but this was rarely achieved. This study is the first to use fluorescence-activated cell sorting (FACS) for the separation and quantitative analysis of the proliferation and differentiation of MG-63 cells grown in direct co-culture with human umbilical vein endothelial cells (HUVECs). The cells of the MG-63 cell line have properties consistent with the characteristics of normal osteoblasts. We labeled HUVECs with fluorescent antibody against CD31 and used FACS to measure the proportions of each cell type and to separate them based on their different fluorescence intensities. The rate of proliferation of the MG-63 cells was estimated based on a count of the total viable cells and the proportion of MG-63 cells in the mixture. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), collagen type 1 (Coll-1) and osteocalcin (OC) in the MG-63 cells were measured via real-time PCR after the separation via FACS. We found that HUVECs stimulated the proliferation of the MG-63 cells after 72 h of co-culture, and inhibited it after 120 h of co-culture. The mRNA expression levels of ALP and Coll-1 significantly increased, whereas that of OC significantly decreased in MG-63 after co-culture with HUVECs. Using FACS for the quantitative analysis of the proliferation and differentiation of osteoblasts directly interacting with endothelial cells could have merit for further co-culture research.
Keywords Osteoblasts, Endothelial cells, Co-culture, Proliferation, Differentiation
Address and Contact Information 1Department of Periodontology, Bernhard Gottlieb University Clinic of Dentistry, Medical University of Vienna, Waehringer Strasse 25a, Vienna 1090, Austria,
2Peking University School and Hospital of Stomatology, Beijing 100081, China,
3Central Research Unit, Bernhard Gottlieb University Clinic of Dentistry, Medical University of Vienna, Vienna 1090, Austria
* Author for correspondence. e-mail: oleh.andrukhov@meduniwien.ac.at, tel.: +43 1 427767144, fax: +43 1 427767294
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DOI: 10.2478/s11658-010-0025-1 Volume 15 (2010) pp 530-540
Title ZNF300, A RECENTLY IDENTIFIED HUMAN TRANSCRIPTION FACTOR, ACTIVATES THE HUMAN IL-2Rß PROMOTER THROUGH THE OVERLAPPING ZNF300/EGR1 BINDING SITE
Authors Lu Xue§, Hongling Qiu§, Jian Ma, Mingxiong Guo and Wenxin Li*
Abstract ZNF300 was recently identified as a member of the human KRAB/C2H2 zinc finger protein family. Little is known about the role of ZNF300 in human gene regulation networks. In this study, the DNA-binding property of ZNF300 was further analyzed. We found that the recombinant ZNF300 could bind to the binding site 5’-GCGGGGGCG-3’ of Egr1, another member of the KRAB/C2H2 zinc finger protein family. Similarly, recombinant Egr1 also showed a similar binding affinity to the ZNF300 binding site 5’-CTGGGGGCG-3’. Bioinformatics analysis revealed that there is an overlapping ZNF300/Egr1 binding site in the human IL-2Rß promoter region, which was previously known to be recognized by endogenous Egr1. Electrophoretic mobility shift assays showed that endogenous ZNF300 could also bind to this site. A transient transfection assay revealed that both ZNF300 and Egr1 could transactivate the IL-2Rß promoter, and that the activation was abrogated by a mutation of residues in the overlapping ZNF300/Egr1 binding site. Co-expression of ZNF300 and Egr1 led to enhanced IL-2Rß promoter activity. Thus, ZNF300 is likely to be another regulator of the human IL-2Rß promoter
Keywords ZNF300, Egr-1, IL-2Rß promoter, Activation
Address and Contact Information State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, P.R. China
§These authors contributed equally to this work
* Author for correspondence. e-mail: liwxlab@whu.edu.cn, tel.: +86 (27) 6875-2831, fax: +86 (27) 6875-2146
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DOI: 10.2478/s11658-010-0028-y Volume 15 (2010) pp 541-550
Title ZFAT IS ESSENTIAL FOR ENDOTHELIAL CELL ASSEMBLY AND THE BRANCH POINT FORMATION OF CAPILLARY-LIKE STRUCTURES IN AN ANGIOGENESIS MODEL
Authors Yasuhiro Yoshida1,2,§, Toshiyuki Tsunoda1,3,§, Yasuo Takashima1,3, Takahiro Fujimoto1,3, Keiko Doi1,3, Takehiko Sasazuki4, Masahide Kuroki3, Akinori Iwasaki2 and Senji Shirasawa1,3 *
Abstract ZFAT, originally identified as a susceptibility gene for autoimmune thyroid disease, encodes a transcriptional regulator with one AT-hook and 18 C2H2- type zinc-finger domains. It is highly conserved among species. Here, we demonstrate that ZFAT is clearly expressed in human umbilical vein endothelial cells (HUVECs). Furthermore, we show that endothelial cell assembly and the branch point formation of capillary-like structures in HUVECs is impaired by the reduction of ZFAT expression through the use of ZFAT-miRNAs, whereas differences in cell proliferation or apoptotic features were not observed after the reduction in ZFAT expression. These results suggest that ZFAT may have critical roles in the capillary-like network formation that is involved in vascular remodeling. Elucidating the ZFAT-mediated transcriptional network will lead to a better understanding of the molecular mechanisms of angiogenesis.
Keywords ZFAT, HUVECs, Endothelial cell assembly, Branch point formation, Vascular remodeling, Angiogenesis
Address and Contact Information 1Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan,
2Department of Thoracic, Endocrine and Pediatric Surgery, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan,
3Center for Advanced Molecular Medicine, Fukuoka University, Fukuoka 814-0180, Japan,
4Kyushu University, Fukuoka 812-8582, Japan
§ These authors contributed equally to this work.
* Author for correspondence. e-mail: sshirasa@fukuoka-u.ac.jp
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DOI: 10.2478/s11658-010-0027-z Volume 15 (2010) pp 551-563
Title LEPTIN REGULATES MMP-2, TIMP-1 AND COLLAGEN SYNTHESIS VIA p38 MAPK IN HL-1 MURINE CARDIOMYOCYTES
Authors Kristin Schram, Sabrina De Girolamo, Siham Madani, Diana Munoz, Farah Thong and Gary Sweeney*
Abstract A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observed that leptin enhanced collagen synthesis determined by both proline incorporation and picrosirius red staining of conditioned media. Pro-collagen type-I and pro-collagen type-III expression, measured by real-time PCR and Western blotting were also increased by leptin, effects which were again attenuated by SB203580. In summary, these results demonstrate the potential for leptin to play a role in mediating myocardial ECM remodeling and that the p38 MAPK pathway plays an important role in mediating these effects.
Keywords Leptin, Obesity, Heart failure, Extracellular matrix
Address and Contact Information Department of Biology, York University, Toronto, Canada, M3J 1P3
* Author for correspondence. e-mail: gsweeney@yorku.ca, tel.: 416 736 2100 ext 66635, fax: 416 736 5698
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DOI: 10.2478/s11658-010-0030-4 Volume 15 (2010) pp 564-581
Title THE in vitro EFFECTS OF 2-METHOXYESTRADIOL-BIS-SULPHAMATE ON CELL NUMBERS, MEMBRANE INTEGRITY AND CELL MORPHOLOGY, AND THE POSSIBLE INDUCTION OF APOPTOSIS AND AUTOPHAGY IN A NON-TUMORIGENIC BREAST EPITHELIAL CELL LINE
Authors Michelle H. Visagie and Annie M. Joubert*
Abstract 2-methoxyestradiol (2ME2) exerts estrogen receptor-independent anti-proliferative, anti-angiogenic and anti-tumor activity in vitro and in vivo. Due to its low bioavailability and rapid metabolic degradation, several analogues have been developed in recent years. 2-methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE) is a bis-sulphamoylated derivative of 2ME2 with anti- proliferative activity. The aim of this study was to investigate cell signaling events induced by 2-MeOE2bisMATE in a non-tumorigenic cell line (MCF-12A) by analysing its influence on cell number, morphology and membrane integrity, and the possible induction of apoptosis and autophagy. Dose- and time-dependent studies revealed that 48 h exposure to 2-MeOE2bisMATE (0.4 µM) resulted in a decrease in cell numbers to 79%. A slight increase in the level of lactate dehydrogenase production was observed in the 2-MeOE2bisMATE-treated cells. Morphological studies revealed an increase in the number of cells in metaphase. Hallmarks of apoptosis were also found, namely nuclear fragmentation and apoptotic bodies. In addition, increased lysosomal staining was observed via fluorescent microscopy, suggesting the induction of another type of cell death, namely autophagy. Since 2-MeOE2bisMATE is regarded as a potential anti-cancer agent, it is also imperative to investigate the susceptibility of non-tumorigenic cells to its influence. The data generated from this study contributes to the understanding of the action that 2-MeOE2bisMATE exerts on the non-tumorigenic MCF-12A breast epithelial cell line.
Keywords 2-methoxyestradiol-bis-sulphamate, Apoptosis, Autophagy
Address and Contact Information Department of Physiology, University of Pretoria, Pretoria, P.O. Box 2034, Pretoria, 0001, South Africa
* Author for correspondence. e-mail address: annie.joubert@up.ac.za, tel.: +27 12 319 2246, fax: +2712 321 1679
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DOI: 10.2478/s11658-010-0029-x Volume 15 (2010) pp 582-599
Title THE MOLECULAR CLONING OF GLIAL FIBRILLARY ACIDIC PROTEIN IN Gekko japonicus AND ITS EXPRESSION CHANGES AFTER SPINAL CORD TRANSECTION
Authors Dehong Gao1, Yongjun Wang2, Yan Liu2, Fei Ding2, Xiaosong Gu2*, and Zhengli Li1*
Abstract The glial fibrillary acidic protein (GFAP) is an astrocyte-speci?c member of the class III intermediate filament proteins. It is generally used as a specific marker of astrocytes in the central nervous system (CNS). We isolated a GFAP cDNA from the brain and spinal cord cDNA library of Gekko japonicus, and prepared polyclonal antibodies against gecko GFAP to provide useful tools for further immunochemistry studies. Both the real-time quantitative PCR and western blot results revealed that the expression of GFAP in the spinal cord after transection increased, reaching its maximum level after 3 days, and then gradually decreased over the rest of the 2 weeks of the experiment. Immunohistochemical analyses demonstrated that the increase in GFAP-positive labeling was restricted to the white matter rather than the gray matter. In particular, a slight increase in the number of GFAP positive star-shaped astrocytes was detected in the ventral and lateral regions of the white matter. Our results indicate that reactive astrogliosis in the gecko spinal cord took place primarily in the white matter during a short time interval, suggesting that the specific astrogliosis evaluated by GFAP expression might be advantageous in spinal cord regeneration.
Keywords Glial fibrillary acidic protein, Gecko, Regeneration, Spinal cord, Real-time PCR
Address and Contact Information 1Department of Anatomy, Tongji Medical College of Huazhong University of Science & Technology, Wuhan, HB 430030, P.R. China,
2Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226007, P.R. China
*Authors for correspondence. e-mail. lizhengli123@hotmail.com (Zhengli Li) and neurongu@public.nt.js.cn (Xiaosong Gu)
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DOI: 10.2478/s11658-010-0031-3 Volume 15 (2010) pp 600-610
Title TRPC EXPRESSION IN MESENCHYMAL STEM CELLS
Authors Frederic Torossian1,2, Aurelie Bisson1, Jean-Pierre Vannier2, Olivier Boyer1 and Marek Lamacz1,*
Abstract Transient receptor potential canonical (TRPC) channels are key players in calcium homeostasis and various regulatory processes in cell biology. Little is currently known about the TRPC subfamily members in mesenchymal stem cells (MSC), where they could play a role in cell proliferation. We report on the presence of TRPC1, 2, 4 and 6 mRNAs in MSC. Western blot and immunofluorescence staining indicate a membrane and intracellular distribution of TRPC1. Furthermore, the decrease in the level of TRPC1 protein caused by RNA interference is accompanied by the downregulation of cell proliferation. These results indicate that MSC express TRPC1, 2, 4 and 6 mRNA and that TRPC1 may play a role in stem cell proliferation.
Keywords Mesenchymal stem cells, TRPC1, Cell proliferation
Address and Contact Information 1Inserm, U905, University of Rouen, IFRMP, Institute for Biomedical Research, Rouen, France,
2EA 3829, University of Rouen, Institute for Biomedical Research, Rouen, France
* Author for correspondence: Marek Lamacz, Inserm U905, Faculty of Medicine and Pharmacy, 22 bd Gambetta, F-76000 Rouen, France, e-mail: marek.lamacz@univ-rouen.fr, tel.: 33-2-35-14-85-27, fax 33-2-32-88-81-86
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DOI: 10.2478/s11658-010-0032-2 Volume 15 (2010) pp 611-629
Title A FUNCTIONAL ANALYSIS OF G23A POLYMORPHISM AND THE ALTERNATIVE SPLICING IN THE EXPRESSION OF THE XPA GENE
Authors Dorota Butkiewicz1, Małgorzata Krześniak1, Rasa Vaitiekunaite1, Bożena Sikora1, Elise D. Bowman2, Curtis C. Harris2 and Marek Rusin1*
Abstract The XPA gene has a commonly occurring polymorphism (G23A) associated with cancer risk. This study assessed the functional significance of this polymorphism, which is localised near the translation start codon. Lymphoblastoid cell lines with alternative homozygous genotypes showed no significant differences in their XPA levels. The luciferase reporter assay detected no functional difference between the two sequences. Unexpectedly, we found that the alternatively spliced form of XPA mRNA lacked a part of exon 1. Only the reading frame downstream of codon Met59 was preserved. The alternative mRNA is expressed in various human tissues. The analysis of the 5’cDNA ends showed similar transcription start sites for the two forms. The in vitro expression of the alternative XPA labelled with the red fluorescent protein (mRFP) showed a lack of preferential nuclear accumulation of the XPA isoform. The biological role of the alternative XPA mRNA form remains to be elucidated.
Keywords Single nucleotide polymorphism, XPA, Kozak sequence, Alternative splicing, Cancer risk, Reporter assay
Address and Contact Information 1Department of Tumour Biology, Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology, Gliwice Branch, Wybrzeże Armii Krajowej 15, 44-101 Gliwice, Poland, 2Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, 37 Convent Drive, Bethesda MD 20892-4258, USA
* Author for correspondence. e-mail: rusinm@rocketmail.com, tel.: + 48 32 2789806, fax: + 48 32 2789840
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DOI: 10.2478/s11658-010-0033-1 Volume 15 (2010) pp 630-650
Title In vitro AND in vivo MATRIX METALLOPROTEINASE EXPRESSION AFTER PHOTODYNAMIC THERAPY WITH A LIPOSOMAL FORMULATION OF AMINOLEVULINIC ACID AND ITS METHYL ESTER
Authors Beata Osiecka1, Kamil Jurczyszyn2, Krzysztof Symonowicz1, Andrzej Bronowicz1, Paweł Ostasiewicz1 , Elżbieta Czapińska3, Katarzyna Hotowy3, Małgorzata Krzystek-Korpacka3, Elżbieta Gębarowska4, Ilona Iżykowska4, Piotr Dzięgiel4, Grzegorz Terlecki3 and Piotr Ziółkowski1,*
Abstract Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including non- melanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.
Keywords Photodynamic therapy, Aminolevulinic acid, Methyl aminolevulinate, Liposomes, Metalloproteinases
Address and Contact Information 1Department of Pathology,
2Department of Dental Surgery,
3Department of Clinical Biochemistry,
4Department of Histology and Embryology, Wrocław
Medical University, Marcinkowskiego 1 Street, Wrocław, Poland
* Author for correspondence. e-mail: ziolkows@interia.pl, tel.: (+48) 71 784-12-15
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DOI: 10.2478/s11658-010-0034-0 Volume 15 (2010) pp 651-664
Title RIBOSOMAL DNA, TRI- AND BI-PARTITE PERICENTROMERES IN THE PERMANENT TRANSLOCATION HETEROZYGOTE Rhoeo spathacea
Authors Hieronim Golczyk1*, Robert Hasterok2 and Marek Szklarczyk3
Abstract High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A3-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.
Keywords Pericentromere, Permanent translocation heterozygosity, rDNA, Rhoeo
Address and Contact Information 1Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Grodzka 52, 31-044 Kraków, Poland,
2Department of Plant Anatomy and Cytology, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland,
3Department of Genetics, Plant Breeding and Seed Science, Agricultural University of Krakow, Al. 29 Listopada 54, 31-425 Kraków, Poland
* Author for correspondence. e-mail: h.golczyk@wp.pl, tel./fax +48-12-4228107
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DOI: 10.2478/s11658-010-0035-z Volume 15 (2010) pp 665-678
Title THE DOMAIN STRUCTURE OF Entamoeba a-ACTININ2
Authors Barbara Addario and Lars Backman*
Abstract Entamoeba histolytica, a major agent of human amoebiasis, expresses two distinct forms of a-actinin, a ubiquitous actin-binding protein that is present in most eukaryotic organisms. In contrast to all metazoan a-actinins, in both isoforms the intervening rod domain that connects the N-terminal actin-binding domain with the C-terminal EF-hands is much shorter. It is suggested that these a-actinins may be involved in amoeboid motility and phagocytosis, so we cloned and characterised each domain of one of these a-actinins to better understand their functional role. The results clearly showed that the domains have properties very similar to those of conventional a-actinins.
Keywords a-actinin, Spectrin repeat, Actin-binding protein
Address and Contact Information Department of Chemistry, Biochemistry, Umea University, SE-901 87 Umea, Sweden
* Author for correspondence. e-mail: lars.backman@chem.umu.se, tel.: (46) 90 786 5847, fax: (46) 90 786 7655
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