Vol. 20 No. 4 December 2015

DOI: 10.1515/cmble-2015-0030 Volume 20 (2015) pp 535-548
Authors Eunyoung Hong1,2, Eunil Lee1,2,*, Joonhee Kim1, Daeho Kwon3 and Yongchul Lim4
Abstract The high frequency of intrinsic resistance to TNF-related apoptosis-inducing ligand (TRAIL) in tumor cell lines has necessitated the development of strategies to sensitize tumors to TRAIL-induced apoptosis. We previously showed that elevated pressure applied as a mechanical stressor enhanced TRAIL-mediated apoptosis in human lung carcinoma cells in vitro and in vivo. This study focused on the effect of elevated pressure on the sensitization of TRAIL-resistant cells and the underlying mechanism. We observed elevated pressure-induced sensitization to TRAIL-mediated apoptosis in Hep3B cells, accompanied by the activation of several caspases and the mitochondrial signaling pathway. Interestingly, the enhanced apoptosis induced by elevated pressure was correlated with suppression of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation and CREB without any change to other MAPKs. Phosphorylation of Bcl-2-associated death promoter (BAD) also decreased, leading to inhibition of the mitochondrial pathway. To confirm whether the activation of pERK1/2 plays a key role in the TRAIL-sensitizing effect of elevated pressure, Hep3B cells were pre-treated with the ERK1/2-specific inhibitor PD98059 instead of elevated pressure. Co-treatment with PD98059 and TRAIL augmented TRAIL-induced apoptosis and decreased BAD phosphorylation. The inhibition of ERK1/2 activation by elevated pressure and PD98059 also reduced BH3 interacting-domain death agonist (BID), thereby amplifying apoptotic stress at the mitochondrial level. Our results suggest that elevated pressure enhances TRAIL-induced apoptosis of Hep3B cells via specific suppression of ERK1/2 activation among MAPKs.
Keywords TRAIL, Elevated pressure, Mechanical stress, ERK1/2, Hepatocellular carcinoma, Apoptosis, BAD, CREB
Address and Contact Information 1Department of Preventive Medicine, School of Medicine, Korea University, Seoul, Korea,
2Department of Public Health, Graduate School of Medicine, Korea University, Seoul, Korea,
3Department of Microbiology, College of Medicine, Catholic Kwandong University, Gangneung, Korea,
4Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
* Author for correspondence. Email: eunil@korea.ac.kr; phone: +82 2 920 6170; fax: +82 2 927 7220

DOI: 10.1515/cmble-2015-0032 Volume 20 (2015) pp 549-561
Authors Branko Tomic* and Jelena Kusic-Tisma
Abstract Replication of DNA in multicellular organisms initiates from origin of replication (ori) sequences, which significantly differ in length and complexity. One of the best characterized is hamster dihydrofolate reductase (DHFR), which contains the ori-β sequence with several functionally relevant domains, such as an AT-rich region, dinucleotide repeat element (DNR), sequence-induced bend DNA (BEND) and a RIP60 protein-binding site (RIP60). Prior to initiation, ori sequences are recognized by origin recognition complex (ORC), which is a hetero hexamer complex that serves as the landing pad for proteins of the pre-replication complex. The function of each ORC subunit is still unclear. In this study, we analyze the function of subunit 4 of the human ORC complex (HsOrc4) in interaction with a plasmid bearing the ori-β DHFR sequence. We show that the topologically closed DHFR ori-β replicator contains a bubble-like structure within its AT-rich region and that it is reversibly modified in the interaction with HsOrc4. The non-canonical structure of the AT-rich region in the topologically closed ori sequence is recognized and changed by HsOrc4 using the energy of supercoiled DNA. These findings could help to further elucidate DNA replication and its possible association with human genetic diseases.
Keywords HsOrc4, ori-β DHFR, Replicator, DNA topology, Topoisomerase I, Mung bean nuclease, ORC complex, Supercoiled plasmid
Address and Contact Information Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Laboratory for Molecular Biology, P.O. Box 23, Vojvode Stepe 444a, 11010 Belgrade, Serbia
* Author for correspondence. Email: kobran@imgge.bg.ac.rs; phone: +381113976658; fax: +381113975808

DOI: 10.1515/cmble-2015-0033 Volume 20 (2015) pp 562-570
Authors Sabina Galiniak1, Grzegorz Bartosz1,2 and Izabela Sadowska-Bartosz1*
Abstract The role of metal (especially) iron ions has been postulated to play a prominent role in protein glycation, suggesting antiglycating effectiveness of metal chelators. However, this rule may not apply to all model glycation systems. We found that metal chelators are not effective in prevention of glycation of bovine serum albumin (BSA) in vitro, and there is no correlation between the antiglycating effects of 32 compounds and their iron chelation activity as measured with the ferrozine test. These data indicate that the glycation of BSA in vitro is iron-independent and is not a proper system to study the role of metals in protein glycation.
Keywords Bovine serum albumin, Chelation, Ferrozine, Fructose, Glucose, Glycation, Glyoxal, Iron, Methylglyoxal, Ribose
Address and Contact Information 1Department of Biochemistry and Cell Biology, University of Rzeszów, Poland,
2Department of Molecular Biophysics, University of Łódź, Poland
* 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: 10.1515/cmble-2015-0031 Volume 20 (2015) pp 571-585
Authors Vincent Digiacomo1, Ivan A. Gando1, Lisa Venticinque1, Alicia Hurtado1 and Daniel Meruelo1,2,*
Abstract The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.
Keywords 37-kDa laminin receptor, 67-kDa laminin receptor, RPSA, 67LR, 37LRP, Non-integrin laminin receptor, LAMR1, p40, SUMO
Address and Contact Information 1New York University School of Medicine, Department of Pathology, 550 First Avenue, New York, NY 10016, USA.
2NYU Cancer Institute, 550 First Avenue, New York, NY 10016, USA
* Author for correspondence. Email: daniel.meruelo@nyumc.org; phone: +1 212 263 5599

DOI: 10.1515/cmble-2015-0034 Volume 20 (2015) pp 586-596
Authors Yang Wang1, Xianqiong Zou1, Yong Guo1,2,3,*, Lu Wang1, Yongming Liu1, Qiangcheng Zeng2 and Xizheng Zhang3
Abstract MicroRNAs (miRNAs) are important regulators of cell proliferation, differentiation and function. Mechanical strain is an essential factor for osteoblast proliferation and differentiation. A previous study revealed that a physiological mechanical tensile strain of 2500 microstrain (με) at 0.5 Hz applied once a day for 1 h over 3 consecutive days promoted osteoblast differentiation. However, the mechanoresponsive miRNAs of these osteoblasts were not identified. In this study, we applied the same mechanical tensile strain to in vitro cultivated mouse MC3T3-E1 pre-osteoblasts and identified the mechanoresponsive miRNAs. Using miRNA microarray and qRT-PCR assays, the expression patterns of miRNAs were evaluated and 5 of them were found to be significantly different between the mechanical loading group and the control group: miR-3077-5p, 3090-5p and 3103-5p were significantly upregulated and miR-466i-3p and 466h-3p were downregulated. Bioinformatics analysis revealed possible target genes for these differentially expressed miRNAs. Some target genes correlated with osteoblast differentiation. These findings indicated that the mechanical strain changed the expression levels of these miRNAs. This might be a potential regulator of osteoblast differentiation and responses to mechanical strain.
Keywords Mechanical strain, 2500 microstrain (με), Mouse MC3T3-E1 pre-osteoblasts, MicroRNA, Osteoblast differentiation, Microarray, qRT-PCR, Bioinformatics, Regulator, Mechanoresponsive
Address and Contact Information 1College of Biotechnology, Guilin Medical University, Guilin, 541004, Guangxi, China,
2Shandong Provincial Key Laboratory of Functional Macromolecular Biophysics, Institute of Biophysics, Dezhou University, Dezhou, China,
3Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, China
* Author for correspondence. Email: guoyong74@163.com; phone: +86-013788568212; fax: +86-07733680230

DOI: 10.1515/cmble-2015-0035 Volume 20 (2015) pp 597-611
Authors Hang Wang1, Ke-Yin Cai1, Wei Li2 and Hao Huang3,*
Abstract Endothelial progenitor cells (EPCs) play a fundamental role in neoangiogenesis and tumor angiogenesis. Through the sphingosine-1-phosphate receptor 3 (S1PR3), sphingosine-1-phosphate (S1P) can stimulate the functional capacity of EPCs. Platelet-derived growth factor receptor-beta (PDGFR-β) contributes to the migration and angiogenesis of EPCs. This study aimed to investigate whether S1P induces the migration and angiogenesis of EPCs through the S1PR3/PDGFR-β/Akt signaling pathway. We used the Transwell system and the Chemicon In Vitro Angiogenesis Assay Kit with CAY10444 (an S1PR3 antagonist), AG1295 (a PDGFR kinase inhibitor) and sc-221226 (an Akt inhibitor) to examine the role of the S1PR3/PDGFR-β/Akt pathway in the S1P-induced migration and angiogenesis of EPCs.
Keywords Akt signaling pathway, Angiogenesis, Crosstalk, EPCs, Migration, PDGFR-β, S1P, S1PR3
Address and Contact Information 1Cadre Ward Two, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China,
2Coronary Heart Disease Diagnosis and Treatment Center, the 305 Hospital of the Chinese People’s Liberation Army, Beijing, China,
3Clinic Center, Shenzhen Hornetcorn Biotechnology Company, Ltd, Shenzhen, China
* Author for correspondence. Email: huanghao@hornetcorn.com; phone: +86-0755- 21672023-825; fax: +86-0755-21672020

DOI: 10.1515/cmble-2015-0036 Volume 20 (2015) pp 612-625
Authors Marius Mihăşan*
Abstract The omnipresence of Arthrobacter species in polluted and toxic soils indicates their great potential in environmental biotechnologies, but practical applications of these bacteria are scarce mainly due to the availability of useful genetic engineering tools. Although many fully sequenced Arthrobacter genomes have been deposited in GenBank, little is known about the biology of their plasmids, especially the core functions: replication and partition. In this study the available Arthrobacter plasmid sequences were analyzed in order to identify their putative replication origin. At least the oris from the cryptic plasmids pXZ10142, pCG1, and pBL1 appear to work in this genus. Based on ParA homolog sequences, the Arthrobacter specific plasmids were classified into 4 clades. Iteron-like sequences were identified on most of the plasmids, indicating the position of the putative Arthrobacter specific oris. Although attempts were made to identify the core gene set required for plasmid replication in this genus, it was not possible. The plasmid proteomes showed a rather low similarity.
Keywords Arthrobacter, Plasmids, Replication, Partition, Classification
Address and Contact Information Biochemistry and Molecular Biology Laboratory, Faculty of Biology Alexandru Ioan Cuza University of Iaşi, Copou Bvd, No. 22B, 700506 Romania
* Author for correspondence. email: marius.mihasan@uaic.ro, phone:+40(0)232201102 – 2434, fax: +40(0)232201472

DOI: 10.1515/cmble-2015-0037 Volume 20 (2015) pp 626-646
Authors Jason D. Coombes1,§, Galina Schevzov1,§, Chin-Yi Kan2, Carlotta Petti2, Michelle F. Maritz2, Shane Whittaker1, Karen L. Mackenzie2 and Peter W. Gunning1,*
Abstract Extensive re-organisation of the actin cytoskeleton and changes in the expression of its binding proteins is a characteristic feature of cancer cells. Previously we have shown that the tropomyosin isoform Tpm3.1, an integral component of the actin cytoskeleton in tumor cells, is required for tumor cell survival. Our objective was to determine whether cancer cells devoid of Tpm3.1 would evade the tumorgenic effects induced by H-Ras transformation. The tropomyosin isoform (Tpm) expression profile of a range of cancer cell lines (21) demonstrates that Tpm3.1 is one of the most broadly expressed Tpm isoform. Consequently, the contribution of Tpm3.1 to the transformation process was functionally evaluated. Primary embryonic fibroblasts isolated from wild type (WT) and Tpm3.1 knockout (KO) mice were transduced with retroviral vectors expressing SV40 large T antigen and an oncogenic allele of the H-Ras gene, H-RasV12, to generate immortalized and transformed WT and KO MEFs respectively. We show that Tpm3.1 is required for growth factor-independent proliferation in the SV40 large T antigen immortalized MEFs, but this requirement is overcome by H-Ras transformation. Consistent with those findings, we found that Tpm3.1 was not required for anchorage independent growth or growth of H-Ras-driven tumors in a mouse model. Finally, we show that pERK and Importin 7 protein interactions are significantly decreased in the SV40 large T antigen immortalized KO MEFs but not in the H-Ras transformed KO cells, relative to control MEFs. The data demonstrate that H-Ras transformation overrides a requirement for Tpm3.1 in growth factor-independent proliferation of immortalized MEFs. We propose that in the SV40 large T antigen immortalized MEFs, Tpm3.1 is partly responsible for the efficient interaction between pERK and Imp7 resulting in cell proliferation, but this is overidden by Ras transformation.
Keywords Actin, Cytoskeleton, Tropomyosin, Cancer, Tumor, ERK, Ras, Transformation
Address and Contact Information 1Oncology Research Unit, School of Medical Sciences, UNSW Australia, Sydney, NSW 2052, Australia,
2Cancer Cell Development Group, Children's Cancer Institute Australia, Lowy Cancer Research Centre, UNSW Australia, Sydney, NSW 2052, Australia
§ Authors contributed equally to this paper
* Author for correspondence. phone: +61-2-9385 2471, fax: +61-2-9385 0022, Email: p.gunning@unsw.edu.au

DOI: 10.1515/cmble-2015-0038 Volume 20 (2015) pp 647-662
Authors Kartiga Natarajan1,2, Gokila Devi Mathialagan1, Somasundaram Raghavan1,3 and Narkunaraja Shanmugam1,*
Abstract Malondialdehyde (MDA) is a highly reactive endogenous product of thromboxane synthesis in the prostagland and lipid peroxidation by reactive oxygen species. Elevated MDA levels occur in diabetes and atherosclerotic plaques. The aim of this study was to examine the molecular mechanisms of MDA-induced IL-17E cytokine expression and its effect on T-cell differentiation. Real-time PCR, RT-PCR and ELISA were used to assess the expression of IL-17 family cytokines in Jurkat T-cells and human peripheral blood lymphocytes (PBLCs) from diabetic subjects. Luciferase reporter assays were used for the promoter activation study. Pharmacological inhibitors were used for signaling pathway experiments. FACS analyses were used to measure the Th1, Th2 and Th17 subset levels. MDA induced significant (2- to 3-fold; p < 0.01) generationof IL-17E mRNA in a dose- and time-dependent manner in Jurkat T-cells and PBLCs. Elevated IL-17E mRNA levels were found in the lymphocytes from diabetic subjects. The increased IL-17E protein and mRNA levels correlate well with serum MDA levels from diabetic patients. Transient transfection of plasmid containing the minimum IL-17E promoter region (pIL-17E-Luc) showed a significant (2-fold; p < 0.01) increase in luciferase activity. Pretreatment of lymphocytes with pharmacological inhibitors showed the involvement of antioxidant, NF-κB, p38MAPK, PKC and ERK signaling pathways. Quantification of the Th1, Th2 and Th17 cell population in PBLCs via FACS analyses revealed an increase in the Th17 subset. These results show that MDA transcriptionally upregulates the expression of IL-17E in lymphocytes and alters lymphocyte differentiation towards the pathogenic Th17 subset.
Keywords Malondialdehyde, IL-17E, Lymphocyte, Th cells, Skewing, mRNA expression, Promoter activation, Diabetes, Inflammation, Advanced glycation/lipoxidation
Address and Contact Information 1Diabetes and Cardiovascular Research Lab, Department of Biomedical Science, Bharathidasan University, Tiruchirappalli 620 024, India,
2Current address: Department of Cellular and Molecular Biology, University of Texas Health Science Center, USA,
3Current address: Department of Physiology, University of Tennessee Health Science Center, USA
* Author for correspondence. Email: nshanmugam@bdu.ac.in; phone: 91-431-2407072 ext. 431; fax: 91-431-2407045

DOI: 10.1515/cmble-2015-0039 Volume 20 (2015) pp 663-684
Authors Agata Wawrzkiewicz-Jałowiecka*, Przemysław Borys and Zbigniew J. Grzywna
Abstract We model the activity of an ion channel gate by Langevin dynamics in a logarithmic potential. This approach enables one to describe the power-law dwell-time distributions of the considered system, and the long-term correlations between the durations of the subsequent channel states, or fractal scaling of statistical characteristics of the gate’s movement with time. Activity of an ion channel gate is described as an overdamped motion of the reaction coordinate in a confining logarithmic potential, which ensures great flexibility of the model. Depending on the chosen parameters, it allows one to reproduce many types of gate dynamics within the family of non-Markovian, anomalous conformational diffusion processes. In this study we apply the constructed model to large-conductance voltage and Ca2+-activated potassium channels (BKCa). The interpretation of model assumptions and parameters is provided in terms of this biological system. Our results show good agreement with the experimental data.
Keywords Langevin dynamics, Logarithmic potential, BK channels, Channel gate, Cell membrane fluctuations, Subdiffusion, Hurst analysis, Memory effect
Address and Contact Information Section of Physics and Applied Mathematics, Silesian University of Technology, 44-100 Gliwice, Ks. M. Strzody 9, Poland
* Author for correspondence. email: agata.wawrzkiewicz@polsl.pl; phone: +48 32 237 12 85; fax: +48 32 237 17 22

DOI: 10.1515/cmble-2015-0041 Volume 20 (2015) pp 685-697
Authors Chaitali Botre1, Arjun Shahu1, Neeraj Adkar2, Yogesh Shouche3, Saroj Ghaskadbi1 and Richa Ashma1,*
Abstract Oxidative stress plays an important role in the development of osteoporosis. The present cross-sectional study focuses on mapping single nucleotide polymorphisms (SNPs) in the mitochondrial manganese superoxide dismutase (SOD2) gene in Asian Indians. The bone mineral density (BMD) of study subjects was assessed by dual x-ray absorptiometry. Individuals were classified as normal (n = 82) or osteoporotic (n = 98). Biochemical parameters such as vitamin D, total oxidant status (TOS) and SOD2 enzyme activity were estimated from plasma samples. Semi-quantitative PCR was carried out using GAPDH as an endogenous control. Genomic DNA was isolated from whole blood and SNPs were evaluated by PCR sequencing. Thirteen SNPs are reported in the examined region of the SOD2 gene, out of which in our samples SNPs rs5746094 and rs4880 were found to be polymorphic. Allele G of rs5746094 (intronic) and allele C of rs4880 (exonic) are significantly higher in the osteoporotic individuals. Presence of allele C of rs4880 and increased level of TOS among osteoporotic individuals were found to be associated with disease risk.
Keywords Osteoporosis, Superoxide dismutase 2, MnSOD, rs4880, Oxidative stress, SNPs, Total oxidant status, rs5746094
Address and Contact Information 1Department of Zoology, Savitribai Phule Pune University, Pune – 411 007, Maharashtra, India,
2SaiShree Hospital and Joint Replacement Center, Aundh Pune – 411 007, India,
3Microbial Culture Collection, Indian Institute of Science Education and Research, Pune – 411008, India
* Author for correspondence. Email: richaashma@unipune.ac.in; phone 91-02025601436 ext. 41; fax 91-20-25690617