Vol. 20 No. 5 December 2015

DOI: 10.1515/cmble-2015-0042 Volume 20 (2015) pp 699-716
Title HOW TASTE WORKS: CELLS, RECEPTORS AND GUSTATORY PERCEPTION
Authors Dariusz Kikut-Ligaj1,* and Joanna Trzcielińska-Lorych2
Abstract The sensitivity of taste in mammals varies due to quantitative and qualitative differences in the structure of the taste perception organs. Gustatory perception is made possible by the peripheral chemosensory organs, i.e., the taste buds, which are distributed in the epithelium of the taste papillae of the palate, tongue, epiglottis, throat and larynx. Each taste bud consists of a community of ~100 cells that process and integrate taste information with metabolic needs. Mammalian taste buds are contained in circumvallate, fungiform and foliate papillae and react to sweet, salty, sour, bitter and umami stimuli. The sensitivity of the taste buds for individual taste stimuli varies extensively and depends on the type of papillae and the part of the oral cavity in which they are located. There are at least three different cell types found in mammalian taste buds: type I cells, receptor (type II) cells and presynaptic (type III) cells. This review focuses on the biophysiological mechanisms of action of the various taste stimuli in humans. Currently, the best-characterized proteins are the receptors (GPCR). In addition, the activation of bitter, sweet and umami tastes are relatively well known, but the activation of salty and sour tastes has yet to be clearly explained.
Keywords Taste papillae, Taste buds, Taste perception, Basic taste qualities, Types of cells located in the taste buds, Activation of taste receptor cells, Epithelial sodium channels, ENaC, Bitter, Sweet and umami taste receptors, TASRs, Extra-oral tissue TAS2Rs, Calcium homeostasis modulator 1 channel, CALHM1
Address and Contact Information 1Department of Department of Natural Science and Quality Assurance, Faculty of Commodity Science, University of Economics, Al. Niepodległości 10, 61-875 Poznań, Poland,
2Department of Histology and Embryology, Poznań University of Medical Sciences, ul.Święcickiego 6, 60-781 Poznań, Poland
* Author for correspondence. Email: dariusz.kikut@ue.poznan.pl; phone: +48 618569043; fax: +48 618543993

DOI: 10.1515/cmble-2015-0040 Volume 20 (2015) pp 717-735
Title NON-COOPERATIVE IMMOBILIZATION OF RESIDUAL WATER BOUND IN LYOPHILIZED PHOTOSYNTHETIC LAMELLAE
Authors Hubert Harańczyk1,*, Ewelina Baran1, Piotr Nowak1, Małgorzata Lorek-Wojciechowska1, Anna Leja1, Dorota Zalitacz1 and Kazimierz Strzałka2
Abstract This study applied 1H-NMR in time and in frequency domain measurements to monitor the changes that occur in bound water dynamics atdecreased temperature and with increased hydration level in lyophilizates of native wheat photosynthetic lamellae and in photosynthetic lamellae reconstituted from lyophilizate. Proton relaxometry (measured as free induction decay = FID) distinguishes a Gaussian component S within the NMR signal (o). This comes from protons of the solid matrix of the lamellae and consists of (i) an exponentially decaying contribution L1 from mobile membrane protons, presumably from lipids, and from water that is tightly bound to the membrane surface and thus restricted in mobility; and (ii) an exponentially decaying component L2 from more mobile, loosely bound water pool. Both proton relaxometry data and proton spectroscopy show that dry lyophilizate incubated in dry air, i.e., at a relative humidity (p/p0) of 0% reveals a relatively high hydration level. The observed liquid signal most likely originates from mobile membrane protons and a tightly bound water fraction that is sealed in pores of dry lyophilizate and thus restricted in mobility. The estimations suggest that the amount of sealed water does not exceed the value characteristic for the main hydration shell of a phospholipid. Proton spectra collected for dry lyophilizate of photosynthetic lamellae show a continuous decrease in the liquid signal component without a distinct freezing transition when it is cooled down to -60ºC, which is significantly lower than the homogeneous ice nucleation temperature [Bronshteyn, V.L. et al. Biophys. J. 65 (1993) 1853]
Keywords Wheat photosynthetic lamellae, Membrane lyophilizate, Bound water fractions, Bound water freezing, Bound water overcooling, 1H-NMR relaxometry, 1H-NMR spectroscopy, FID moment expansion, Thylakoid lyophilizate molecular mobility, CPMG
Address and Contact Information 1Institute of Physics, Jagiellonian University, ul. Prof. Stanisława Łojasiewicza 11, 30-348 Kraków, Poland,
2Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland
* Author for correspondence. Email: hubert.haranczyk@uj.edu.pl

DOI: 10.1515/cmble-2015-0043 Volume 20 (2015) pp 736-742
Title COEXISTENCE OF RARE VARIANT HbD PUNJAB [α2β2121(Glu→Gln)] AND ALPHA 3.7 kb DELETION IN A YOUNG BOY OF HINDU FAMILY IN WEST BENGAL, INDIA
Authors Arijit Ghosh1, Jayasri Basak1,* and Ashis Mukhopadhyay2
Abstract HbD Punjab is a variant of hemoglobin which occurs as a result of mutation in codon 121 (GAA>CAA) of the β-globin gene, which replaces glutamic acid with glutamine (Glu→Gln). The heterozygous state of HbD does not produce any clinical or hematological symptoms, although its association with HbS and thalassemia produces clinically significant but less severe conditions. The homozygous state produces mild hemolytic anemia and mild to moderate splenomegaly. Alpha-thalassemia is haracterized by reduction or absence of the α-globin chains due to deletional or non-deletional mutations of α-globin genes located on chromosome 16. The present study describes a Hindu family where both HbD Punjab and alpha 3.7 kb deletion are present among the members in the heterozygous and double heterozygous state. Comparison of clinical and hematological parameters between the heterozygous and double heterozygous state of HbD and the alpha 3.7 kb deletion is also discussed here. According to our study, the prevalence rate of HbD Punjab is very low, i.e. 0.06%
Keywords HbD Punjab, Alpha 3.7 kb deletion, Hemoglobinopathy, Double heterozygous, DNA sequencing, Hindu family
Address and Contact Information 1Department of Molecular Biology, Netaji Subhash Chandra Bose Cancer Research Institute (NCRI),16 A Park Lane, Kolkata – 700 016, India,
2Department of Medical Oncology, NCRI, Kolkata, India
* Author for correspondence. Email: ncri.molecularbiology@gmail.com, bjayasri_2000@yahoo.com, phone: +91 33 2227 6161, mobile: +91 98 3006 8536, fax: +91 33 2226 4704.

DOI: 10.1515/cmble-2015-0045 Volume 20 (2015) pp 743-756
Title SOMATIC STEM CELL AGING AND MALIGNANT TRANSFORMATION – IMPACT ON THERAPEUTIC APPLICATION
Authors Marcela Kuniakova, Lenka Oravcova, Zuzana Varchulova- Novakova, Diana Viglaska and Lubos Danisovic*
Abstract Somatic stem cells possess unique properties of self-renewal and plasticity which make them promising candidates for use in tissue engineering and regenerative medicine, in addition to serving as efficient delivery vehicles in site-specific therapy. In the case of therapeutic application, it is essential to isolate and culture stem cells in vitro, to obtain them in sufficient quantities. Although long-term cultivation provides an adequate number of cells, it has been shown that this approach is associated with increased risk of transformation of cultured cells, which presents a significant biological hazard. This article reviews information about biological features and cellular events which occur during long-term cultivation of somatic stem cells, with respect to their safe utilization in potential clinical practice.
Keywords Stem cells, Aging, Malignant transformation, Therapeutic application, Biological safety
Address and Contact Information Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University in Bratislava, Sasinkova 4, 811 08 Bratislava, Slovak Republic
* Author for correspondence. Email: lubos.danisovic@fmed.uniba.sk; phone: + 42 1259357215

DOI: 10.1515/cmble-2015-0044 Volume 20 (2015) pp 757-772
Title 1H NMR-BASED METABOLIC PROFILING FOR EVALUATING POPPY SEED RANCIDITY AND BREWING
Authors Ewa Jawień1, Adam Ząbek1, Stanisław Deja2, Marcin Łukaszewicz3 and Piotr Młynarz1*
Abstract Poppy seeds are widely used in household and commercial confectionery. The aim of this study was to demonstrate the application of metabolic profiling for industrial monitoring of the molecular changes which occur during minced poppy seed rancidity and brewing processes performed on raw seeds. Both forms of poppy seeds were obtained from a confectionery company. Proton nuclear magnetic resonance (1H NMR) was applied as the analytical method of choice together with multivariate statistical data analysis. Metabolic fingerprinting was applied as a bioprocess control tool to monitor rancidity with the trajectory of change and brewing progressions. Low molecular weight compounds were found to be statistically significant biomarkers of these bioprocesses. Changes in concentrations of chemical compounds were explained relative to the biochemical processes and external conditions.The obtained results provide valuable and comprehensive information to gain a better understanding of the biology of rancidity and brewing processes, while demonstrating the potential for applying NMR spectroscopy combined with multivariate data analysis tools for quality control in food industries involved in the processing of oilseeds. This precious and versatile information gives a better understanding of the biology of these processes.
Keywords Metabolomics, NMR spectroscopy, Poppy seeds, Rancidity, Brewing, Bioprocess quality control, System biology, Chemometrics
Address and Contact Information 1Bioorganic Chemistry Group, Department of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland,
2Department of Analytical and Ecological Chemistry, Opole University, pl. Kopernika 11a, 45-040 Opole, Poland,
3Faculty of Biotechnology, University of Wrocław F. Joliot-Curie 14A, 50-383 Wrocław, Poland
* Author for correspondence. E-mail: piotr.mlynarz@pwr.edu.pl, phone/fax: +48-71-3204597

DOI: 10.1515/cmble-2015-0047 Volume 20 (2015) pp 773-787
Title HOMOLOGY ARMS OF TARGETING VECTORS FOR GENE INSERTIONS AND CRISPR/Cas9 TECHNOLOGY: SIZE DOES NOT MATTER; QUALITY CONTROL OF TARGETED CLONES DOESS
Authors Silvia Petrezselyova1, Slavomir Kinsky1, Dominika Truban1, Radislav Sedlacek1, Ingo Burtscher2 and Heiko Lickert2,*
Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.
Keywords CRISPR/Cas9, Genome editing, Reporter, Targeting vector, Homology arms, Embryonic stem cell
Address and Contact Information 1Institute of Molecular Genetics of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic,
2Institute of Stem Cell Research, Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
* Author for correspondence. Email: heiko.lickert@helmholtz-muenchen.de, phone: +49-89-3187-3867, fax: +49-89-3187-2060

DOI: 10.1515/cmble-2015-0048 Volume 20 (2015) pp 788-797
Title DEATH DOMAIN ASSOCIATED PROTEIN (Daxx), A MULTI-FUNCTIONAL PROTEIN
Authors Shuang-Yang Tang1,2,3, Yan-Ping Wan1,2,3,* and Yi-Mou Wu1,2,3,*
Abstract Death domain associated protein (Daxx), a multi-functional protein, plays an important role in transcriptional regulation, cell apoptosis, carcinogenesis, anti-virus infection and so on. However, its regulatory mechanisms for both cell survival and apoptosis remain largely obscure. Our review of recent studies shows that Daxx has many interesting functional dualities and can provide a reference for further research on Daxx.
Keywords Death domain associated protein, Function, Mutation, Expression, Localization, Interaction, Modification, Regulation, Transcription, Apoptosis
Address and Contact Information 1Institute of Pathogenic Biology, University of South China,
2Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control,
3Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Hengyang 421001, P. R. China
* Author for correspondence. E-mail: wanyy1991@aliyun.com.cn; phone: +86 0734- 8282907

DOI: 10.1515/cmble-2015-0046 Volume 20 (2015) pp 798-815
Title In silico SCREENING OF ALLEGED miRNAs ASSOCIATED WITH CELL COMPETITION: AN EMERGING CELLULAR EVENT IN CANCER
Authors Manish Patel1, Bhavesh Antala1 and Neeta Shrivastava1,2*
Abstract Cell competition is identified as a crucial phenomenon for cancer and organ development. There is a possibility that microRNAs (miRNAs) may play an important role in the regulation of expression of genes involved in cell competition. In silico screening of miRNAs is an effort to abridge, economize and expedite the experimental approaches to identification of potential miRNAs involved in cell competition, as no study has reported involvement of miRNAs in cell competition to date. In this study, we used multiple screening steps as follows: (i) selection of cell competition related genes of Drosophila through a literature survey; (ii) homology study of selected cell competition related genes; (iii) identification of miRNAs that target conserved cell competition-related genes through prediction tools; (iv) sequence conservation analysis of identified miRNAs with human genome; (v) identification of conserved cell competition miRNAs using their expression profiles and exploration of roles of their homologous human miRNAs. This study led to the identification of nine potential cell competition miRNAs in the Drosophila genome. Importantly, eighteen human homologs of these nine potential Drosophila miRNAs are well reported for their involvement in different types of cancers. This confirms their probable involvement in cell competition as well, because cell competition is well justified for its involvement in cancer initiation and maintenance.
Keywords Cancer, MicroRNA, Drosophila melanogaster, Cell competition, Organ development, PITA Top, PicTar, TargetScan, FlyAtlas, ClustalW
Address and Contact Information 1Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, C/o B.V. Patel PERD Centre, Thaltej, Ahmedabad-380054, Gujarat, India,
2Department of Pharmacognosy and Phytochemistry, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad-380054, Gujarat, India
* Author for correspondence. Email: manpatel2@gmail.com; neetas@perdcentre.com, phone: +917927439375, fax: +917927450449

DOI: 10.1515/cmble-2015-0051 Volume 20 (2015) pp 816-824
Title MGL INDUCES NUCLEAR TRANSLOCATION OF ENDOG AND AIF IN CASPASE-INDEPENDENT T CELL DEATH
Authors Qingpan Bu, Jianhui Wang, Yi Zheng, Yingying Zou and Min Wei*
Abstract Macrophage galactose-type lectin (MGL) participates in the regulation of T cell apoptosis, but the exact death pathway remains unclear. Here, we demonstrated that MGL-induced T cell death occurs in a caspase-independent manner. Furthermore, MGL treatment triggers the translocation of endonuclease G (EndoG) and apoptosis-inducing factor (AIF) from the mitochondria to the nucleus. Because galectin-1 (Gal-1) can also initiate similar mitochondrial events, we speculate that this death pathway may be widely used by the lectin family.
Keywords MGL, Gal-1, Lectin, T cell apoptosis, Caspase, EndoG, AIF
Address and Contact Information School of Life Sciences, Northeast Normal University, #5268, Renmin Street, Changchun, Jilin 130024, P.R. China
* Author for correspondence. e-mail: weim750@nenu.edu.cn

DOI: 10.1515/cmble-2015-0049 Volume 20 (2015) pp 825-839
Title CERIVASTATIN REPRESSES ATHEROGENIC GENE EXPRESSION THROUGH THE INDUCTION OF KLF2 VIA ISOPRENOID METABOLIC PATHWAYS
Authors Jiyuan Zhao1,2,*, Selvamuthu K. Natarajan2, Nicolas Chronos2 and Jai Palsingh2,*
Abstract Earlier clinical studies have reported that cerivastatin has an anti-atherosclerotic effect that is unique among the statins. In our study, human THP-1 macrophage cells were used to study the effects of various statins on the expressions of the atherosclerotic genes and Kruppel-like factor 2 (KLF2). Cerivastatin significantly inhibited the two atherosclerotic genes, monocyte chemoattractant protein-1 (MCP-1) and C-C chemokine receptor type 2 (CCR2) at both the mRNA and protein levels, while the other statins did not. Accordingly, cerivastatin was also the most potent inducer of KLF2 transcription in the macrophages. An siRNA-induced reduction in KLF2 expression blocked the inhibition of MCP-1 and CCR2 by cerivastatin. When the cells were further treated with mevalonate, farnesylpyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP), the effects of cerivastatin on KLF2, MCP-1 and CCR2 were obviously reversed. Thus, the results showed that cerivastatin was a potent inhibitor of the inflammation genes MCP-1 and CCR2 through the induction of KLF2. The regulation of MCP-1, CCR2 and KLF2 by cerivastatin was isoprenoid pathway dependent. Our studies suggest that the effect of cerivastatin on atherosclerotic genes and KLF2 expression may contribute to the cardioprotection observed in reported clinical studies.
Keywords Cerivastatin, Macrophage, Inflammation, KLF2, MCP-1, CCR2, Gene expression, Isoprenoid pathway
Address and Contact Information 1School of Medicine, Ningbo University, Ningbo, Zhejiang, People's Republic of China,
2Saint Joseph’s Translational Research Institute, Atlanta, GA, USA
* Authors for correspondence. Email: zhaojiyuan@nbu.edu.cn (J. Zhao); jsingh@sjha.org (J. Singh); phone: +86-574-8760-9596 (J. Zhao)

DOI: 10.1515/cmble-2015-0052 Volume 20 (2015) pp 840-866
Title PROTEASOMES RAISE THE MICROTUBULE DYNAMICS IN INFLUENZA A (H1N1) VIRUS-INFECTED LLC-MK2 CELLS
Authors Flora De Conto1,*, Carlo Chezzi1, Alessandra Fazzi1, Sergey V. Razin2, Maria Cristina Arcangeletti1, Maria Cristina Medici1, Rita Gatti3 and Adriana Calderaro1
Abstract The dynamics of microtubule networks are known to have an impact on replication of influenza A virus in some cellular models. Here we present evidence suggesting that at late stages of LLC-MK2 cell infection by influenza A (H1N1) virus the ubiquitin-proteasome protein degradation system participates in destabilization of microtubules, and favours virus replication. Chemical inhibition of proteasome activity partially suppresses influenza A virus replication, while stimulation of proteasome activity favours influenza A virus replication. Conversely, in another cellular model, A549 cells, inhibitors and activators of proteasomes have a small effect on influenza A virus replication. These data suggest that influenza A virus might take selective advantage of proteasome functions in order to set up a favourable cytoskeletal “environment” for its replication and spread. Furthermore, the relationship between influenza virus and the host cell is likely to depend on both the cellular model and the virus strain.
Keywords Influenza A virus, Microtubule cytoskeleton, Viral nucleoprotein, Virus-host interaction, Proteasomes, Acetylated alpha-tubulin, Microtubule-associated protein 4, Gene expression, MG132, IU1
Address and Contact Information 1Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy,
2Institute of Gene Biology, Russian Academy of Sciences and Lomonosow Moscow State University, Moscow, Russia,
3Department of Biomedical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy
* Author for correspondence. Unit of Microbiology and Virology, Department of Clinical and Experimental Medicine, University of Parma, Viale Antonio Gramsci 14, 43126 Parma, Italy. E-mail: flora.deconto@unipr.it, phone: +39 0521 033499, fax: +39 0521 993620

DOI: 10.1515/cmble-2015-0050 Volume 20 (2015) pp 867-918
Title PURINERGIC SIGNALING AND THE FUNCTIONING OF THE NERVOUS SYSTEM CELLS
Authors Kamila Puchałowicz, Irena Baranowska-Bosiacka*, Violetta Dziedziejko and Dariusz Chlubek
Abstract Purinergic signaling in the nervous system has been the focus of a considerable number of studies since the 1970s. The P2X and P2Y receptors are involved in the initiation of purinergic signaling. They are very abundant in the central and peripheral nervous systems, where they are expressed on the surface of neurons and glial cells – microglia, astrocytes, oligodendrocytes and Schwann cells and the precursors of the latter two. Their ligands – extracellular nucleotides – are released in the physiological state by astrocytes and neurons forming synaptic connections, and are essential for the proper functioning of nervous system cells. Purinergic signaling plays a crucial role in neuromodulation, neurotransmission, myelination in the CNS and PNS, intercellular communication, the regulation of ramified microglia activity, the induction of the response to damaging agents, the modulation of synaptic activity and other glial cells by astrocytes, and the induction of astrogliosis. Understanding these mechanisms and the fact that P2 receptors and their ligands are involved in the pathogenesis of diseases of the nervous system may help in the design of drugs with different and more effective mechanisms of action.
Keywords Astrocytes, ATP, Calcium waves, Extracellular nucleotides, Microglia, Myelination, Neurons, Neurotransmission, P2X receptors, P2Y receptors
Address and Contact Information Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, 72 Powstańców Wlkp. St., 70-111 Szczecin, Poland
* Author for correspondence. Email: irena.bosiacka@pum.edu.pl; phone: +48-91-466-15-15; fax: +48-91-466-15-15

DOI: 10.1515/cmble-2015-0054 Volume 20 (2015) pp 919-936
Title THE EFFECT OF CULTUREWARE SURFACES ON FUNCTIONAL AND STRUCTURAL COMPONENTS OF DIFFERENTIATED 3T3-L1 PREADIPOCYTES
Authors Nela Pavlikova1,§, Martin Weiszenstein2,§, Jan Pala2, Petr Halada3, Ondrej Seda4, Moustafa Elkalaf5, Jan Trnka5, Jan Kovar1 and Jan Polak2,6,*
Abstract Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.
Keywords Cell culture, Proteomics, Cultureware surface, Preadipocytes, 2-D electrophoresis, Mass spectrometry, Adipocyte, Lipids, Adipose tissue, Lipolysis
Address and Contact Information 1Department of Biochemistry, Cell and Molecular Biology – Division of Cell and Molecular Biology, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic,
2 Department of Sport Medicine, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
3 Laboratory of Molecular Structure Characterization, Institute of Microbiology, The Czech Academy of Sciences, Prague, Czech Republic,
4 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic,
5 Laboratory for Metabolism and Bioenergetics, Third Faculty of Medicine, Charles University in Prague, Prague, Czech Republic,
6 Center of Toxicology and Health Safety, The National Institute of Public Health, Prague, Czech Republic
§ These authors contributed equally to this study
* Author for correspondence. Jan Polak, MD, PhD, Center for Research on Diabetes, Nutrition and Metabolism, Third Faculty of Me dicine, Charles University in Prague, Ruska 10, Prague 10, 100 00, Czech Republic. Ema il: jan.polak@lf3.cuni.cz, phone: +420 731 181 599, fax: +420 267 102 210

DOI: 10.1515/cmble-2015-0053 Volume 20 (2015) pp 937-947
Title RAMIPRIL INHIBITS HIGH GLUCOSE-STIMULATED UP-REGULATION OF ADHESION MOLECULES VIA THE ERK1/2 MAPK SIGNALING PATHWAY IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS
Authors Moo Hyun Kim1,§, Hae Min Kang2,§, Chae-Eun Kim1, Seongho Han3,* and Sung-Whan Kim4,*
Abstract Ramipril has recently been shown to have anti-atherogenic properties. However, the specific mechanisms underlying these effects remain unclear. The purpose of this study was to determine the effects of ramipril on induction of adhesion molecules in human umbilical vein endothelial cells (HUVECs) using high-glucose (HG) conditions and to investigate possible underlying molecular mechanisms. The effects of ramipril on expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 production, and ERK phosphorylation were examined in HG-induced HUVECs with inhibitors targeting the mitogen-activated protein kinase (MAPK) signaling pathway. HG induced the expression of the adhesion molecules ICAM-1 and VCAM-1. Pretreatment with ramipril drastically inhibited ICAM-1 and VCAM-1 production in a time- and dose-dependent manner. Moreover, upon investigating the effects of ramipril on the MAPK/extracellular signal-regulated kinase (ERK) signaling pathway, we found that ramipril completely inhibited HG-induced phosphorylation of ERK1/2. ERK inhibitors completely prevented the inhibitory effect of HG. This study demonstrated that ramipril reduces HG-stimulated induction of ICAM-1 and VCAM-1 expression via MAPK signaling, which may be useful for inhibition of atherosclerosis.
Keywords ACE inhibitor, Adhesion, Atherosclerosis, Cardiovascular risk, Endothelial cells, ERK pathway, Glucose, Inhibitor, MAPK, Ramipril
Address and Contact Information 1Department of Cardiology, College of Medicine, Dong-A University, Busan, Korea,
2 Department of Ophthalmology, International St. Mary’s Hospital, College of Medicine, Catholic Kwandong University, Incheon, Korea,
3 Department of Family Medicine, College of Medicine, Dong-A University, Busan, Korea,
4 Department of Medicine, College of Medicine, Catholic Kwandong University, Gangneung, Korea
§ These authors equally contributed to this work
* Authors for correspondence. Sung-Whan Kim Ph.D., 25, Simgok-ro 100beon-gil, Seo-gu, International St. Mary’s Hospital, Incheon, Korea 404-190, phone: + 82 (32) 290-2616, fax: +82 (32) 290-2620, email: swkim@cku.ac.kr or Seongho Han MD, Ph.D., Department of Family Medicine, College of Medicine, Dong-A University, Busan, Korea, phone: + 82 (51) 240-5293, fax: +82 (51) 240-8282, email: drhans@dau.ac.kr

DOI: 10.1515/cmble-2015-0056 Volume 20 (2015) pp 948-964
Title THE EFFECT OF NICOTINE ON THE EXPRESSIONS OF THE α7 NICOTINIC RECEPTOR GENE AND BAX AND BCL-2 PROTEINS IN THE MAMMARY GLAND EPITHELIAL-7 BREAST CANCER CELL LINE AND ITS RELATIONSHIP TO DRUG RESISTANCE
Authors Naghmeh Aali1,3 and Gholamreza Motalleb1,2,*
Abstract The binding of nicotine with nicotinic acetylcholine receptors (nAChRs) stimulates cell division and increases drug resistance in cancer. Experiments with specific inhibitors such as RNAi, hexamethonium, and α-bungarotoxin showed that α7 nicotinic receptor plays a key role in the pro-proliferation activity of nicotine. However, the mechanism of nicotine in the progress of breast cancer, the commonest malignancy in women, remains unknown. This study focuses on the effect of nicotine on the expressions of the α7 nicotinic receptor gene and Bax and Bcl-2 proteins in mammary gland epithelial-7 (MCF-7) breast cancer cells and its relationship to drug resistance. To evaluate the effect on drug resistance, human mammary gland epithelial adenocarcinomas from the MCF-7 line were exposed to 100 μl of nicotine at a concentration of 9.2 mg/ml for varying periods of time. Then, the cells were treated with 1, 2, 3 or 5 μl/ml of doxorubicin, either with or without the continued presence of nicotine. Cell viability was determined using the MTT assay. The biochemical parameters of apoptosis, including the expressions of Bax, Bcl-2 and α7 nicotinic receptor proteins were determined via western blotting, and the α7 nicotinic receptor gene expression level was assessed via real-time qPCR using the 2-ΔΔCt method. Differences in the target gene expression levels were evaluated with ANOVA with p ≤ 0.05 considered significant. We found a novel and effective signaling pathway of nicotine in the MCF-7 breast cancer cell line. The levels of α7 nicotinic receptor and Bcl-2 protein increased but the Bax protein levels decreased, while the α7 nicotinic receptor gene expression level was not significantly different compared with the control.
Keywords Breast cancer, α7 nicotinic receptor gene, Bcl-2, Bax, Doxorubicin, Apoptosis, Drug resistance, MTT assay, qPCR, Western blotting
Address and Contact Information 1Department of Biology, Faculty of Sciences, University of Zabol, Zabol, Iran,
2Center of Agricultural Biotechnology, University of Zabol, Zabol, Iran,
3Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Iran
* Author for correspondence. Email: rezamotalleb@gmail.com; phone: +98 54 31232180; fax: +98 54 31232180

DOI: 10.1515/cmble-2015-0057 Volume 20 (2015) pp 965-973
Title THE CLONING, EXPRESSION AND PURIFICATION OF RECOMBINANT HUMAN NEURITIN FROM Escherichia coli AND THE PARTIAL ANALYSIS OF ITS NEUROBIOLOGICAL ACTIVITY
Authors Yuanyuan Li1,§, Juan Tang2,§, Yunhua Zhang1,§, Haiyan Wang1, Wumei Yuan1, Na Yu1, Xing Luo1, Xiaoling Xu3, Jin Huang1,* and Lei Yang3,*
Abstract Neuritin (Nrn1) is a neurotrophic factor that plays various roles in neural development and synaptic plasticity. In this study, the NRN1 gene was cloned and expressed in Escherichia coli and then recombinant neuritin protein was purified so that its neurobiological activity could be evaluated. The protein, which was obtained at a concentration of 0.45 mg/ml and > 90% purity, had the predicted molecular weight of 30 kDa, as determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis confirmed that an anti-neuritin antibody could recognize the fusion protein. Subsequent functional analyses revealed that recombinant neuritin promoted neurite outgrowth in embryonic chicken dorsal root ganglia and PC12 cells. These results suggest that recombinant neuritin protein could be a valuable tool for inducing neurite regeneration, for instance in cases of spinal cord injury or neurological diseases.
Keywords Neuritin, Expression, Refolding, Purification, PC12 cells, Dorsal root ganglion (DRG), Neurite outgrowth, Escherichia coli
Address and Contact Information 1The Key Laboratory of Xinjiang Endemic & Ethnic Diseases and Department of Biochemistry, Shihezi University School of Medicine, Shihezi, Xinjiang, P. R. China,
2 The First Affiliated Hospital of Shihezi University School of Medicine, Shihezi, Xinjiang, P. R. China,
3 Hangzhou Normal University, Hangzhou, Zhejiang, P. R. China
§ These authors contributed equally to this work.
* Authors for correspondence. Jin Huang – email: huangjin623@163.com, Lei Yang – email: 20080009@hznu.edu.cn