Vol. 19 No. 2 June 2014

DOI: 10.2478/s11658-014-0188-2 Volume 19 (2014) pp 181–200
Title STUDY ON THE ACTIVITY OF THE SIGNALING PATHWAYS REGULATING HEPATOCYTES FROM G0 PHASE INTO G1 PHASE DURING RAT LIVER REGENERATION
Authors Menghua Li1,2, Xiaochun Zhou1,2, Jinxin Mei1,2, Xiaofang Geng1,2, Yun Zhou1,2, Weimin Zhang2 and Cunshuan Xu1,2,*
Abstract Under normal physiological conditions, the majority of hepatocytes are in the functional state (G0 phase). After injury or liver partial hepatectomy (PH), hepatocytes are rapidly activated to divide. To understand the mechanism underlying hepatocyte G0/G1 transition during rat liver regeneration, we used the Rat Genome 230 2.0 Array to determine the expression changes of genes, then searched the GO and NCBI databases for genes associated with the G0/G1 transition, and QIAGEN and KEGG databases for the G0/G1 transition signaling pathways. We used expression profile function (Et) to calculate the activity level of the known G0/G1 transition signal pathways, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the interactions among these signaling pathways. The results of our study show that the activity of the signaling pathways of HGF, IL-10 mediated by p38MAPK, IL-6mediated by STAT3, and JAK/STAT mediated by Ras/ERK and STAT3 are significantly increased during the priming phase (2–6 h after PH) of rat liver regeneration. This leads us to conclude that during rat liver regeneration, the HGF, IL-10, IL-6 and JAK/STAT signaling pathways play a major role in promoting hepatocyte G0/G1 transition in the regenerating liver.
Keywords Rat liver regeneration, Signal transduction, Hepatocyte G0/G1 transition, HGF, IL-10, IL-6, JAK/STAT, p38MAPK, Ras/ERK, STAT3, Ingenuity pathway analysis 9.0 (IPA)
Address and Contact Information 1College of Life Science, Henan Normal University, Xinxiang 453007, P. R. China,
2Key Laboratory for Cell Differentiation Regulation, Xinxiang 453007, P. R. China
* Author for correspondence. Email: cellkeylab@126.com, phone: +086 373 3326001, fax: +086 373 3326524

DOI: 10.2478/s11658-014-0189-1 Volume 19 (2014) pp 201-214
Title TRANSPORT OF 3-BROMOPYRUVATE ACROSS THE HUMAN ERYTHROCYTE MEMBRANE
Authors Izabela Sadowska-Bartosz1,*, Mirosław Soszyński2, Stanisław Ułaszewski3, Young Ko4 and Grzegorz Bartosz1,2
Abstract 3-Bromopyruvic acid (3-BP) is a promising anticancer compound because it is a strong inhibitor of glycolytic enzymes, especially glyceraldehyde 3-phosphate dehydrogenase. The Warburg effect means that malignant cells are much more dependent on glycolysis thannormal cells. Potential complications of anticancer therapy with 3-BP are side effects due to its interaction with normal cells, especially erythrocytes. Transport into cells is critical for 3-BP to have intracellular effects. The aim of ourstudy was the kinetic characterization of 3-BP transport into human erythrocytes. 3-BP uptake by erythrocytes was linear within the first 3 min and pH-dependent. The transport rate decreased with increasing pH in the range of 6.0–8.0. The Km and Vm values for 3-BP transport were 0.89 mM and 0.94 mmol/(l cells x min), respectively. The transport was inhibited competitively by pyruvate and significantly inhibited by DIDS, SITS, and 1-cyano-4-hydroxycinnamic acid. Flavonoids also inhibited 3-BP transport: the most potent inhibition was found for luteolin and quercetin.
Keywords 3-Bromopyruvic acid, Erythrocytes, Polyphenols, Flavonoids, Transport, Monocarboxylate transporter, 1-Cyano-4-hydroxycinnamic acid, Luteolin, Quercetin
Address and Contact Information 1 Department of Biochemistry and Cell Biology, University of Rzeszów, Poland
2 Department of Molecular Biophysics, University of Łódź, Poland,
3 Institute of Genetics and Microbiology, University of Wrocław, Poland,
4 KoDiscovery LLC, UM BioPark, Innovation Center, Suites 502 E&F, 801 West Baltimore Street, Baltimore, MD 21201, USA
* Author for correspondence. Izabela Sadowska-Bartosz, Ph.D., Department of Biochemistry and Cell Biology, University of Rzeszów, Zelwerowicza 4, 35-601 Rzeszów, Poland. Email: sadowska@univ.rzeszow.pl; isadowska@poczta.fm; phone: +48 17 8755408, fax: +48 17 8721425

DOI: 10.2478/s11658-014-0190-8 Volume 19 (2014) pp 215-232
Title WDR5, ASH2L, AND RBBP5 CONTROL THE EFFICIENCY OF FOS TRANSCRIPT PROCESSING
Authors Peik Lin Teoh1,2 and Andrew D. Sharrocks2
Abstract H3K4 trimethylation is strongly associated with active transcription. The deposition of this mark is catalyzed by SET-domain methyltransferases, which consist of a subcomplex containing WDR5, ASH2L, and RBBP5 (the WAR subcomplex); a catalytic SET-domain protein; and additional complexspecific subunits. The ERK MAPK pathway also plays an important role in gene regulation via phosphorylation of transcription factors, co-regulators, or histone modifier complexes. However, the potential interactions between these two pathways remain largely unexplored. We investigated their potential interplay in terms of the regulation of the immediate early gene (IEG) regulatory network. We found that depletion of components of the WAR subcomplex led to increased levels of unspliced transcripts of IEGs that did not necessarily reflect changes in their mature transcripts. This occurs in a manner independent from changes in the H3K4me3 levels at the promoter region. We focused on FOS and found that the depletion of WAR subcomplex components affected the efficiency of FOS transcript processing. Our findings show a new aspect of WAR subcomplex function in coordinating active transcription with efficient pre-mRNA processing.
Keywords RBBP5, ASH2L, WDR5, Histone methyltransferase, Immediate early gene, Splicing
Address and Contact Information 1 Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia,
2 Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, U.K.
* Author for correspondence. Email: peiklin@ums.edu.my, phone: +6088-320000 ext. 8471, fax: +6088-320993

DOI: 10.2478/s11658-014-0191-7 Volume 19 (2014) pp 233-242
Title MOLECULARLY TARGETING THE PI3K-Akt-mTOR PATHWAY CAN SENSITIZE CANCER CELLS TO RADIOTHERAPY AND CHEMOTHERAPY
Authors Ziwen Wang1,*, Yujung Huang2 and Jiqiang Zhang3,*
Abstract Radiotherapy and chemotherapeutic agents that damage DNA are the current major non-surgical means of treating cancer. However, many patients develop resistances to chemotherapy drugs in their later lives. The PI3K and Ras signaling pathways are deregulated in most cancers, so molecularly targeting PI3K-Akt or Ras-MAPK signaling sensitizes many cancer types to radiotherapy and chemotherapy, but the underlying molecular mechanisms have yet to be determined. During the multi-step processes of tumorigenesis, cancer cells gain the capability to disrupt the cell cycle checkpoint and increase the activity of CDK4/6 by disrupting the PI3K, Ras, p53, and Rb signaling circuits. Recent advances have demonstrated that PI3K-Akt-mTOR signaling controls FANCD2 and ribonucleotide reductase (RNR). FANCD2 plays an important role in the resistance of cells to DNA damage agents and the activation of DNA damage checkpoints, while RNR is critical for the completion of DNA replication and repair in response to DNA damage and replication stress. Regulation of FANCD2 and RNR suggests that cancer cells depend on PI3K-Akt-mTOR signaling for survival in response to DNA damage, indicating that the PI3K-AktmTOR pathway promotes resistance to chemotherapy and radiotherapy by enhancing DNA damage repair.
Keywords PI3K, Akt, Target of rapamycin, Ribonucleotide reductase, p53, FANCD2, Drug resistance, DNA damage response, Chemotherapy, Radiotherapy, ATM
Address and Contact Information 1 Department of Preventive Medicine, College of Military Preventive Medicine, Third Military Medical University, Chongqing 400038, China,
2 Department of Environmental Hygiene, College ofMilitary Preventive Medicine, Third Military Medical University, Chongqing 400038, China,
3 Department of Neurobiology, Third Military Medical University, Chongqing 400038, China
* Authors for correspondence. Zhang, J.: email: zhangjqtmmu@yahoo.com, phone: (86)-23-68752223, Wang, Z.: email: wangziwen8080@163.com, phone: (86) 9679-9893

DOI: 10.2478/s11658-014-0193-5 Volume 19 (2014) pp 243-248
Title THE INFLUENCE OF HETEROCYCLIC COMPOUND–PAMAM DENDRIMER COMPLEXES ON EVOKED ELECTRICAL RESPONSES IN SLICES OF HYPOXIC BRAIN TISSUE
Authors Vladimir I. Potkin1, Dzmitry Shcharbin2,*, Andrey A. Denisov3, Svetlana G. Paschkevich3, Maria Bryszewska4, Sergey K. Petkevich1, Alexey V. Kletskov1, Dmitri O. Lapotko5, Vladimir V. Kazbanov3, Tatiana A. Gurinovich3 and Vladimir A. Kulchitsky3
Abstract We used complexes between a fourth generation polyamidoamine (PAMAM) dendrimer and one of two heterocyclic compounds – 1-(6-hydroxyhexyl)-3-(5-phenyl-isoxazole-3-yl)-urea or 5-phenyl-isoxazole-3-carboxylic acid – to reduce oxygen consumption in transverse slices of the hippocampus taken from 4-week old male rats. In vitro electrophysiological experiments revealed that the inhibitory effect of the hypoxic state on the evoked responses was enhanced in the presence of the complexes.The data were analyzed in terms of the potential antitumor effects of these complexes.
Keywords Hypoxia, Dendrimer, Brain slices, Postsynaptic potential, Antitumor effects, Heterocycles, Isoxazoles
Address and Contact Information 1 Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus,
2 Institute of Biophysics and Cell Engineering of National Academy of Sciences of Belarus, Minsk, Belarus,
3 Institute of Physiology of National Academy of Sciences of Belarus, Minsk, Belarus,
4 Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland,
5 Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, USA
* Author for correspondence. Dr. Dzmitry Shcharbin, Institute of Biophysics and Cell Engineering of NASB, Akademicheskaja str. 27, Minsk, 220072, Belarus. Email: d.shcharbin@mail.ru, phone: (37517) 2842358, fax: (37517) 2842359

DOI: 10.2478/s11658-014-0194-4 Volume 19 (2014) pp 249-261
Title CA-125 OF FETAL ORIGIN CAN ACT AS A LIGAND FOR DENDRITIC CELL-SPECIFIC ICAM-3-GRABBING NON-INTEGRIN
Authors Ninoslav Mitić*, Bojana Milutinović and Miroslava Janković
Abstract CA-125 (coelomic epithelium-related antigen) forms the extracellular portion of transmembrane mucin 16 (MUC16). It is shed after proteolytic degradation. Due to structural heterogeneity, CA-125 ligand capacity and biological roles are not yet understood. In this study, we assessed CA-125 as a ligand for dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which is a C-type lectin showing specificity for mannosylated and fucosylated structures. It plays a role as a pattern recognition molecule for viral and bacterial glycans or as an adhesion receptor. We probed a human DC-SIGN-Fc chimera with CA-125 of fetal or cancer originusing solid- or fluid-phase binding and inhibition assays. The results showed that DC-SIGN binds to CA-125 of fetal origin and that this interaction is carbohydrate-dependent. By contrast, cancerderived CA-125 displayed negligiblebinding. Inhibition assays indicated differences in the potency of CA-125 to interfere with DC-SIGN binding to pathogen-related glycoconjugates, such as mannan and Helicobacter pylori antigens. The differences in ligand properties between CA-125 of fetal and cancer origin may be due to specificities of glycosylation. This might influence various functions of dendritic cells based on their subset diversity and maturation-related functional capacity.
Keywords CA-125, Mucin 16, DC-SIGN, C-type lectin, Carbohydrate binding, Pathogen-related glycoconjugates, Mannan, Helicobacter pylori
Address and Contact Information University of Belgrade, Institute for the Application of Nuclear Energy, INEP, Department for Immunochemistry and Glycobiology, Banatska 31b, 11080 Zemun, Serbia
* Author for correspondence. Email: ninoslavm@inep.co.rs, phone: +381 11 2199 949, fax: +381 11 261 8724

DOI: 10.2478/s11658-014-0195-3 Volume 19 (2014) pp 262-276
Title MONITORING OF MEMBRANE PHOSPHOLIPID SCRAMBLING IN HUMAN ERYTHROCYTES AND K562 CELLS WITH FM1-43 – A COMPARISON WITH ANNEXIN V-FITC
Authors Anna Wróbel1, Małgorzata Bobrowska-Hägerstrand2, Christer Lindqvist2 and Henry Hägerstrand2,*
Abstract The styryl dye FM1-43 becomes highly fluorescent upon binding to cell membranes. The breakdown of membrane phospholipid asymmetry in ionophorestimulated T-lymphocytes further increases this fluorescence [Zweifach, A. Biochem. J. 349 (2000) 255]. In this study, the capacity of FM1-43 to monitor membrane phospholipid scrambling was explored using flow cytometry in human erythrocytes and human erythrocyte progenitor K562 cells. The Ca2+-dependent phosphatidylserine-specific probe annexin V-FITC was used for comparison. The presented data show that the loss ofphospholipid asymmetry that could be induced in human erythrocytes by elevated intracellular Ca2+ or by structurally different membrane intercalated amphiphilic compounds increases the FM1-43 fluorescence two- to fivefold. The profile of FM1-43 fluorescence for various treatments resembles that of phosphatidylserine exposure reported by annexin V-FITC. FM1-43 detected the onset of scrambling more efficiently than annexin V-FITC. The amphiphile-induced scrambling was shown to be a Ca2+-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca2+-release from intracellular stores and by Ca2+ and ionophore A23187 treatment showed that the increase inFM1-43 fluorescence correlated well with the number of annexin V-FITC-detected phosphatidylserine-positive cells. The results presented here show the usefulness of FM1-43 as a Ca2+-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.
Keywords FM1-43, Phospholipid scrambling, Annexin V-FITC, Phosphatidylserine exposure, Human erythrocytes, K562 cells, Ionophores, Amphiphiles
Address and Contact Information 1 Institute of Physics, Wrocław University of Technology, 50–370 Wrocław, Poland,
2 Department of Biosciences, Åbo Akademi University, 20520 Åbo/Turku, Finland
Invited paper
* Author for correspondence. Department of Biosciences, Cell Biology, Åbo Akademi University, Artillerigatan 6, FIN-20520 Åbo/Turku, Finland. Email: hhagerst@abo.fi, phone: +358-2-2154089, fax: +358-2-2154748

DOI: 10.2478/s11658-014-0192-6 Volume 19 (2014) pp 277-283
Title FANNIN-LUBBOCK-I [α2β2119(GLY>ASP)], A RARE MUTATION IN THE β-GLOBIN GENE, HAS BEEN DETECTED FOR THE FIRST TIME IN A HINDU BRAHMIN FAMILY IN WEST BENGAL, INDIA
Authors Jayasri Basak1,*, Deboshree M. Bhattacharyya1 and Ashis Mukhopadhyay2
Abstract This study aims to describe the hemoglobin Fannin-Lubbock-I, which has a rare mutation substituting the amino acid glycine with aspartic acid at codon 119 of the β-globin chain. A Bengalee Hindu Brahmin family from Kolkata in West Bengal was the focus of this study. Molecular analysis using ARMS-PCR and direct DNA sequencing revealed the presence of a GGC > GAC mutation in codon 119 of the β-globin gene in a heterozygote state in three women of the same family. This is the first report of the hemoglobin FanninLubbock-I from India. Our results will help to identify this mutation, which is relatively infrequent in our population.
Keywords Hb Fannin-Lubbock-I, Bengalee, Brahmin family, β-globin gene, Rare mutation, Thalassemia, West Bengal, India, ARMS-PCR, Sequencing
Address and Contact Information 1 Department of Molecular Biology, Netaji Subhas Chandra Bose Cancer Research Institute (NCRI), 16A Park Lane, Kolkata 700016, India
2 Department of Medical Oncology, NCRI, Kolkata, India
* Author for correspondence. Email: hmcwt@dataone.in, ncri.molecularbiology@gmail.com, bhattacharyya.deboshree@gmail.com, phone: +91-33- 22276161, fax: +91-33- 22264704

DOI: 10.2478/s11658-014-0196-2 Volume 19 (2014) pp 284-296
Title THE EFFECT OF CARBOHYDRATE MOIETY STRUCTURE ON THE IMMUNOREGULATORY ACTIVITY OF LACTOFERRIN IN VITRO
Authors Michał Zimecki1,*, Jolanta Artym1, Maja Kocięba1, Maria Duk2 and Marian L. Kruzel3
Abstract The aim of this study was to evaluate the immunoregulatory effects of recombinant human lactoferrin (rhLF) in two in vitro models: (1) the secondary humoral immune response to sheep erythrocytes (SRBC); and (2) the mixed lymphocyte reaction (MLR). We compared the non-sialylated glycoform of rhLF as expressed by glycoengineered Pichia pastoris with one that was further chemically sialylated. In an earlier study, we showed that sialylated rhLF could reverse methotrexate-induced suppression of the secondary immune response of mouse splenocytes to SRBC, and thatthe phenomenon is dependent on the interaction of lactoferrin (LF) with sialoadhesin (CD169). We found that the immunorestorative activity of sialylated rhLF is also dependent on its interaction with the CD22 antigen, a member of the immunoglobulin superfamily that is expressed by B lymphocytes. We also demonstrated that only sialylated rhLF was able to inhibit the MLR reaction. MLR was inhibited by bovine lactoferrin (bLF), a glycoform that has a more complex glycan structure. Desialylated bLF and lactoferricin, a bLF-derived peptide devoid of carbohydrates, did not express such inhibitory activity. We showed that the interaction of LF with sialic acid receptors is essential for at least some of the immunoregulatory activity of this glycoprotein.
Keywords Human recombinant lactoferrin, CD22, Sialic acid, Humoral immune response, Mixed lymphocyte reaction, CD169, Glycoproteins, Immune suppression
Address and Contact Information 1 Department of Experimental Therapy, Institute of Immunology and Experimental Therapy, Weigla 12, 53-114 Wrocław, Poland,
2 Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Weigla 12, 53-114 Wrocław,
3 University of Texas, Health Science Center, Houston, Texas, USA
* Author for correspondence. Email: zimecki@iitd.pan.wroc.pl

DOI: 10.2478/s11658-014-0197-1 Volume 19 (2014) pp 297-314
Title POLY(L-LACTIDE-CO-GLYCOLIDE) THIN FILMS CAN ACT AS AUTOLOGOUS CELL CARRIERS FOR SKIN TISSUE ENGINEERING
Authors Aleksandra Zuber1, Julia Borowczyk1, Eliza Zimolag1, Malgorzata Krok2, Zbigniew Madeja1, Elzbieta Pamula2 and Justyna Drukala1,*
Abstract Degradable aliphatic polyesters such as polylactides, polyglycolides and their copolymers are used in several biomedical and pharmaceutical applications. We analyzed the influence of poly(L-lactide-co-glycolide) (PLGA) thin films on the adhesion, proliferation, motility and differentiation of primary human skin keratinocytes and fibroblasts in the context of their potential use as cell carriers for skin tissue engineering. We did not observe visible differences in the morphology, focal contact appearance, or actin cytoskeleton organization of skin cells cultured on PLGA films compared to those cultured under control conditions. Moreover, we did not detect biologically significant differences in proliferative activity, migration parameters, level of differentiation, or expression of vinculin when the cells were cultured on PLGA films and tissue culture polystyrene. Our results indicate that PLGA films do not affect the basic functions of primary human skin keratinocytes and fibroblasts and thus show acceptable biocompatibility in vitro, paving the way for their use as biomaterials for skin tissue engineering.
Keywords Burns, Skin regeneration, Wound healing, Keratinocytes, Fibroblasts, Biomaterials, PLGA, Tissue engineering
Address and Contact Information 1 Laboratory of Cell & Tissue Engineering, Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology and Malopolska Centre of Biotechnology, Jagiellonian University, Krakow 30-387, Poland,
2 Department of Biomaterials, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, Krakow 30-059, Poland
* Author for correspondence. Email: justyna.drukala@uj.edu.pl, phone: +48126646145, fax: +48126646902

DOI: 10.2478/s11658-014-0199-z Volume 19 (2014) pp 315-329
Title MiR-30b IS INVOLVED IN METHYLGLYOXAL-INDUCED EPITHELIAL–MESENCHYMAL TRANSITION OF PERITONEAL MESOTHELIAL CELLS IN RATS
Authors Hong Liu1, Ning Zhang2 and Da Tian3,*
Abstract Epithelial–mesenchymal transition (EMT) of peritoneal mesothelial cells (PMC) is a major contributor to the pathogenesis of peritoneal fibrosis. EMT is at least in part caused by repeated exposure to glucose degradation products (GDPs), such as methylglyoxal (MGO). MiRNA contributes greatly to the EMT of PMCs. In this study, we tried to profile whether differences exist between the peritoneal membrane (PM) miRNA expression seen in control rats and that seen in rats injected intraperitoneally with MGO. We assessed whether miR-30b has a possible role in MGO-induced EMT of PMCs in rats. Comparative miRNA expression array and real-time PCR analyses were conducted for the control group at the start of the experiment and for the MGO group after 1 and 2 weeks. During the second week, the MGO rats were treated with: a chemically modified antisense RNA oligonucleotide (ASO) complementary to the mature miR-30b (ASO group); an miR-30b mismatch control sequence (MIS group); or a citrate buffer (EMT group). Bioinformatic analyses indicated that the 3’ untranslated region (3’-UTR) of bone morphogenetic protein 7 (BMP7) mRNA did contain a putative binding site for miR-30b. We also tried to investigate whether miR-30b targeted BMP7 in vitro by transfection. Of the upregulated miRNAs, miR-30b expression demonstrated the greatest increase. The administration of miR-30b ASO for two weeks significantly reduced α-SMA excretion and upregulated E-cadherin and BMP-7 expression. Our in vitro study showed that miR-30b directly targeted and inhibited BMP7 by binding to its 3’-UTR. Our results revealed that miR-30b is involved in MGO-induced EMT of PMCs in rats
Keywords Methylglyoxal, miR-30b, Epithelial-mesenchymal transition, Peritoneal dialysis, Rats, Peritoneal fibrosis, MicroRNA, Bone morphogenetic protein 7, E-cadherin, Peritoneal membrane
Address and Contact Information 1 Department of Emergency Medicine, People’s Hospital of Lishui, Zhejiang Province, 15 Dazhong Street, Liandu District, Lishui, 323000, Zhejiang Province, P.R. China,
2 Department of Critical Care Medicine, People’s Hospital of Lishui, Zhejiang Province, 15 Dazhong Street, Liandu District, Lishui, 323000, Zhejiang Province, P.R. China,
3 Department of Neurosurgery, People’s Hospital of Lishui, Zhejiang Province, 15 Dazhong Street, Liandu District, Lishui, 323000, Zhejiang Province, P.R. China
* Author for correspondence. Email: datiandeemail@163.com, phone: 86 578-2780150