Vol. 20 No. 2 June 2015

DOI: 10.1515/cmble-2015-0017 Volume 20 (2015) pp 177-195
Authors Vincent Gerard Francis and Sathyanarayana N. Gummadi*
Abstract Human phospholipid scramblase 1 (hPLSCR1) is a Ca2+-dependent protein known to scramble phospholipids in the plasma membrane resulting in loss of membrane asymmetry. It has been reported that hPLSCR1 exhibits Ca2+-independent activity at low pH. However, the conformational changes induced at low pH leading to functional activation are not known. Our results showed that recombinant hPLSCR1 was functionally activated at low pH, which is similar to the behavior of natively extracted hPLSCR1. Tryptophan fluorescence measurements showed a decrease in Ca2+-binding affinity at low pH, although not at pH 5.5. Far and near UV-CD revealed that low pH induced structural changes, with a significant increase in the β-sheet content of the protein. At the physiological level, decreased hPLSCR1 expression was observed after a period of exposure to low pH. The effect occurred at the promoter level. The expression levels of hPLSCR1 directly correlated with the sensitivity of HEK293 to apoptosis. Based on these results, we conclude that the mechanisms of Ca2+- and pH-induced functional activation of hPLSCR1 are different and that hPLSCR1 expression regulated by low pH could provide insights into the role of hPLSCR1 in cancer progression.
Keywords Human phospholipid scramblase, Low pH, Tryptophan fluorescence, Promoter, Gene expression, CD spectroscopy, Apoptosis assay, c-FOS, Phosphatidylcholine, Phosphatidylserine, Calcium binding
Address and Contact Information Applied and Industrial Microbiology Laboratory, Bhupat and Jyoti Mehta School of Biosciences, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
* Author for correspondence. Email: gummadi@iitm.ac.in, phone: 91-44-2257-4114, fax: 91-44-2257-4102

DOI: 10.1515/cmble-2015-0011 Volume 20 (2015) pp 196-212
Authors Shuhei Yamada*
Abstract Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix of animal tissues. CS chains are covalently bound to a core protein to form a proteoglycan, which is involved in various biological events including cell proliferation, migration, and invasion. Their functions are executed by regulating the activity of bioactive proteins, such as growth factors, morphogens, and cytokines. This review article focuses on the catabolism of CS. This catabolism predominantly occurs in lysosomes to control the activity of CS-proteoglycans. CS chains are fragmented by endo-type glycosidase(s), and the resulting oligosaccharides are then cleaved into monosaccharide moieties from the nonreducing end by exoglycosidases and sulfatases. However, the endo-type glycosidase responsible for the systemic catabolism of CS has not yet been identified. Based on recent advances in studies on hyaluronidases, which were previously considered to be hyaluronan-degrading enzymes, it appears that they recognize CS as their original substrate rather than hyaluronan and acquired hyaluronan-hydrolyzing activity at a relatively late stage of evolution.
Keywords Chondroitin sulfate, Hyaluronan, Glycosaminoglycan, Catabolism, Hydrolase, Hyaluronidase, Proteoglycan, Mucopolysaccharidosis
Address and Contact Information Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, 468-8503, Japan
¶ Invited paper
* E-mail: shuheiy@meijo-u.ac.jp, phone: 81-(52)-839-2650, fax: 81-(52)-834-8090

DOI: 10.1515/cmble-2015-0013 Volume 20 (2015) pp 213-221
Authors Anna Wiktorowska-Owczarek1,* and Jacek Owczarek2
Abstract Prostaglandin E2 (PGE2) is generated in various cells, including endothelial cells, and is responsible for various functions, such as vascular relaxation and angiogenesis. Effects of PGE2 are mediated via receptors EP1-EP4, among which EP2 and EP4 are coupled to Gs protein which activates adenylate cyclase (AC) and cAMP synthesis. The aim of this work was to study the ability of human microvascular endothelial cells (HMEC-1) to synthesize cAMP in the presence of PGE2, and to determine the effect of hypoxia on the PGE2- stimulated cAMP level. It was decided to evaluate the effect of PGE2 on the secretion of VEGF, an inducer of angiogenesis. In summary, our findings show that PGE2 induces cAMP production, but hypoxia may impair PGE2-stimulated activity of the AC-cAMP signaling pathway. These results suggest that the cardioprotective effect of PGE2/EP4/cAMP may be attenuated during ischemia. Furthermore, this study indicates that the pro-angiogenic effect of PGE2 is not associated with VEGF secretion in HMEC-1 cells.
Keywords Hypoxia, CoCl2, Prostaglandin E2, Receptor EP4, Adenylate cyclase, cAMP, VEGF, Angiogenesis, Endothelial cells, HMEC-1 cells
Address and Contact Information 1Department of Pharmacology and Toxicology, Medical University of Lodz, Poland,
2Department of Pharmacogenetics, Chair of Biopharmacy, Medical University of Lodz, Poland
* Author for correspondence. Department of Pharmacology and Toxicology, Medical University of Lodz, 90-752 Lodz, Zeligowskiego Street 7/9, Poland, email: anna.wiktorowska-owczarek@umed.lodz.pl, phone/fax +48 42 639 32 90

DOI: 10.1515/cmble-2015-0016 Volume 20 (2015) pp 222-236
Authors Vasileios Kordinas1,*, George Tsirpanlis2, Chryssoula Nicolaou1, Margarita Zoga1, Anastasios Ioannidis3, Vasiliki Ioannidou1, Sotiris Bersimis4, Christina Petrihou2, Lefkothea Savva2, Nicholaos J. Legakis1 and Stylianos Chatzipanagiotou1
Abstract Telomerase is involved in the elongation of telomeres. It remains active in very few types of cell in mature organisms. One such cell type is the lymphocytes. In this study, we investigated the activity and expression of telomerase in lymphocytes from renal failure patients and compared it to that for normal controls. Inflammation status was determined at the same time. The enzyme activity was measured using PCR-ELISA with peripheral blood mononuclear cells (PBMCs) from three groups: 53 healthy individuals, 50 patients with chronic kidney disease (CKD) and 50 dialysis patients. In the same cell populations, the expression of the reverse transcriptase of the human telomerase gene (hTERT) was measured via real-time PCR. The inflammation status of these individuals was determined by calculating the interleukin 6 (IL-6), IL-10, C-reactive protein (CRP) and tumor necrosis factor alpha (TNF-a) serum concentrations via ELISA. The lowest levels of telomerase activity were detected in CKD, and this group had the highest IL-6 and CRP values and the lowest hTERT expression. The dialysis group showed significant differences in comparison to the normal subjects and to the CKD patients. Further studies are warranted in order to explore the way inflammation influences telomerase activity and hTERT expression.
Keywords CKD, Cytokines, Dialysis, hTERT, Inflammation, PBMCs, Real-time PCR, Telomerase
Address and Contact Information 1Department of Clinical Microbiology and Medical Biopathology, Aeginition Hospital, Medical School, University of Athens, Athens, Greece,
2Department of Nephrology, General Hospital of Athens, “G. Gennimatas”, Athens, Greece
3Department of Nursing, Sparta General Hospital Building Complex, University of Peloponnese, Faculty of Human Movement and Quality of Life Sciences, Sparta, Greece,
4Department of Statistics and Insurance Science, University of Piraeus, Piraeus, Greece
* Author for correspondence. Email: kordinasv@med.uoa.gr

DOI: 10.1515/cmble-2015-0010 Volume 20 (2015) pp 237-247
Authors Hirak Kumar Barman*, Ramya Mohanta, Swagat Kumar Patra, Vemulawada Chakrapani, Rudra Prasanna Panda, Swapnarani Nayak, Sasmita Jena, Pallipuram Jayasankar and Priyanka Nandanpawar
Abstract We previously characterized the β-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The β-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of β-actin gene expression across eukaryotes. Furthermore, the isolated β-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.
Keywords β-actin promoter, Labeo rohita, Rohu, Transgenic, GFP, Spermatogonial cells, HEK293, K562, SF21, Transfection protocol
Address and Contact Information Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India
* Author for correspondence. Email: hk.barman@cifamail.in or hirak.barman@icar.gov.in, phone: +916742465446, +916742465414, fax: +916742465407

DOI: 10.1515/cmble-2015-0014 Volume 20 (2015) pp 248-264
Authors Daria Głogocka1,2, Magdalena Przybyło1,2 and Marek Langner1,2
Abstract Biological systems are characterized by directional and precisely controlled flow of matter and information along with the maintenance of their structural patterns. This is possible thanks to sequential transformations of information, energy and structure carried out by molecular machines. The new perception of biological systems, including their mechanical aspects, requires the implementation of tools and approaches previously developed for engineering sciences. In this review paper, a biological system is presented in a new perspective as an ensemble of coordinated molecular devices functioning in the limited space confined by the biological membrane. The working of a molecular machine is presented using the example of F0F1 ATPase, and the general conditions necessary for the coordination of a large number of functional units are described.
Keywords Molecular machines, Molecular mechanics, Central dogma of biology, F0F1 ATPase, Interdependence of energy-matter-information fluxes
Address and Contact Information 1Institute of Biomedical Engineering and Measurements, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland,
2Lipid Systems Sp. z o.o., ul. Duńska 9, 54-427 Wrocław, Poland
* Author for correspondence. Email: daria.glogocka@pwr.edu.pl, tel. +48 71 320 4155

DOI: 10.1515/cmble-2015-0008 Volume 20 (2015) pp 265-278
Authors Rongqiang Yang1, Xin Jiang2, Rui Ji1, Lingbin Meng3, Fuli Liu4, Xiaolei Chen5,* and Ying Xin6,*
Abstract Pituitary adenylate cyclase activating polypeptide (PACAP) is widely expressed in the central and peripheral nervous system. PACAP can initiate multiple signaling pathways through binding with three class B G-protein coupled receptors, PAC1, VPAC1 and VPAC2. Previous studies have revealed numerous biological activities of PACAP in the nervous system. PACAP acts as a neurotransmitter, neuromodulator and neurotrophic factor. Recently, its neuroprotective potential has been demonstrated in numerous in vitro and in vivo studies. Furthermore, evidence suggests that PACAP might move across the blood-brain barrier in amounts sufficient to affect the brain functions. Therefore, PACAP has been examined as a potential therapeutic method for neurodegenerative diseases. The present review summarizes the recent findings with special focus on the models of Alzheimer’s disease (AD) and Parkinson’s disease (PD). Based on these observations, the administered PACAP inhibits pathological processes in models of AD and PD, and alleviates clinical symptoms. It thus offers a novel therapeutic approach for the treatment of AD and PD.
Keywords Pituitary adenylate cyclase activating polypeptide, Neurodegenerative diseases, Neuroprotective, Neurotrophic, Alzheimer’s disease, Parkinson’s disease
Address and Contact Information 1Department of Biochemistry and Molecular Biology, University of Louisville, School of Medicine, Louisville, KY 40202, USA,
2Department of Radiation Oncology, The First Hospital of Jilin University, Changchun, 130021, China,
3Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40202, USA,
4Department of Physiology and Neurobiology, Geisel Medical School at Dartmouth, Lebanon, NH 03756, USA,
5Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA,
6Ying Xin, Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Changchun 130021, China
* Authors for correspondence. Email: xiaolei.chen@dartmouth.edu; r0yang02@louisville.edu

DOI: 10.1515/cmble-2015-0015 Volume 20 (2015) pp 279-293
Authors Siti Nurbaya Oslan1,2, Abu Bakar Salleh1,2,6*, Raja Noor Zaliha Raja Abd Rahman1,3,6, Thean Chor Leow1,4,6, Hafizah Sukamat2 and Mahiran Basri1,5,6
Abstract Pichia guilliermondii strain SO isolated from spoiled orange was developed for use as an alternative expression host by using Pichia pastoris as the model of the experiment. This is the first study to report on the capability of P. guilliermondii SO as a host to express thermostable T1 lipase from Geobacillus zalihae. Alcohol oxidase and formaldehyde dehydrogenase promoters were present in the yeast genome. Interestingly, the recombinant yeast [SO/pPICZαB/T1-2 (SO2)] took only 30 h to reach optimal production with minimal methanol induction [1.5% (v/v)] in YPTM medium, as compared to P. pastoris, which took longer to reach its optimal condition. The purification yield of the His-tagged fusion lipase was 68.58%, with specific activity of 194.58 U/mg. The optimum temperature was 65°C at pH 9 in glycine-NaOH buffer, and it was stable up to 70°C in a wide pH range from pH 5 to 12. In conclusion, a newly isolated yeast from spoiled orange has been proven suitable for use as an expression host.
Keywords Pichia guilliermondii, Expression, Thermostable lipase, Alcohol oxidase, Geobacillus zalihae, Yeast, Pichia pastoris, His-tagged fusion protein, Methylotrophic yeast, Local host
Address and Contact Information 1Enzyme and Microbial Technology Research Center,
2Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,
3Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences,
4Department of Cell & Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,
5Department of Chemistry, Faculty of Science,
6Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
* Author for correspondence. Email: abubakar@upm.edu.my, phone: 603-8947 1053, fax: 603-8946 7590

DOI: 10.1515/cmble-2015-0018 Volume 20 (2015) pp 294-309
Authors Ramamoorthy M. Kalidas1, Subramanian Elaiya Raja1 and Sivasubramaniam Sudhakar1*
Abstract Lamin A is an intermediate filament protein which is cleaved by the enzyme, FACE 1 at VTRSY↓L. The cleavage is the final step in the production of the mature lamin A protein. The mature lamin A protein localizes in the inner membrane of the nucleus. The mutation in the lamin A gene causes many diseases, including accelerated aging. It is known that the protein is not expressed in neuronal cells of the brain. Many splicing variants of the lamin A gene have been reported. In this study, the amino acid sequence VTRSY (a penta-peptide repeat) was found in three different sites of the C-terminal end of the lamin A protein, the protein expressed in cells of ear cartilage tissues is shorter than the protein expressed in cells of the skin tissues. Using two lamin A antibodies, it was found that the amino acid sequence between penta-peptide 2 and 3 is missing in lamin A protein that was expressed in the cells of mouse ear cartilage tissue, besides the RT-PCR data confirmed that the corresponding coding sequence between the penta repeat 2 and 3 is intact. Cleavage may occur at the penta-peptide (VTRSY) at site 3 in the lamin A tail of mouse ear cartilage.
Keywords Lamin A, Skin, Ear cartilage, Endoproteolytic cleavage, Nucleoskeleton, Cleavage, Prelamin A, Post-translational modification
Address and Contact Information 1Department of Biotechnology, Manonmaniam Sundaranar University, Abisekapatti, Tirunelveli 627012, Tamilnadu, India
* Author for correspondence. E-mail: sudhakarmsu@yahoo.com, phone: +91 9940998936, +91 9487999622

DOI: 10.1515/cmble-2015-0020 Volume 20 (2015) pp 310-322
Authors Valter Lubrano1*, Morena Gabriele2, Maria Rita Puntoni3, Vincenzo Longo2 and Laura Pucci2
Abstract Previous studies evidenced a significant reduction in serum cholesterol levels during an episode of acute inflammation. The aim of the present study was to verify the hypothesis of a regulatory role of cytokines through an in vitro model that simulates a situation of vascular inflammation and high levels of LDL or lipoperoxides. Human microvascular endothelial cells-1 were used in all experiments. The cells were exposed for 24 h to increasing doses of LDL, oxidized lipoprotein, and 8-isoprostane (in the absence or presence of SQ29.548, a TXA 2 receptor antagonist). Moreover, LDL receptor and oxidized lipoprotein receptor expression analyzed after endothelial cells’ incubation with increasing doses of interleukin-6. The ELISA test and quantitative real-time PCR were performed. Endothelial cells showed a significant increase in interleukin-6 medium levels associated with LDL, oxidized LDL and with the degree of oxidation (absence or presence of SQ29.548), while 8-isoprostane did not. Treatment of human microvascular endothelial cells-1 for 24 h with increasing doses of interleukin-6 significantly enhanced LDL receptor and oxidized lipoprotein receptor-1 mRNA expression. Our data suggest the presence of a compensatory mechanism. The induction of a significant increase of IL-6 does not seem to be caused by the presence of the biological activity of 8-isoprostane.
Keywords Cytokines, Isoprostanes, Lipids, Oxygen radicals, Endothelial cells, LOX-1, LDLR
Address and Contact Information 1Fondazione G. Monasterio CNR-Regione Toscana, Pisa, Italy,
2Istituto di Biologia e Biotecnologia Agraria, CNR, Pisa, Italy,
3Istituto di Fisiologia Clinica, CNR, Pisa, Italy
* Author for correspondence. Fondazione G. Monasterio CNR-Regione Toscana, Via Moruzzi n° 1, 56100 - Pisa, Italy, e-mail: walterl@ifc.cnr.it, phone: +390503152199, fax: +39-050-3153454

DOI: 10.1515/cmble-2015-0021 Volume 20 (2015) pp 323-349
Authors Dan Qin1,2 and Cunshuan Xu1,2,*
Abstract Long non-coding RNAs (lncRNAs) have attracted considerable attention recently due to their involvement in numerous key cellular processes and in the development of various disorders. New high-throughput methods enable their study on a genome-wide scale. Numerous lncRNAs have been identified and characterized as important members of the biological regulatory network, with significant roles in regulating gene expression at the epigenetic, transcriptional and post-transcriptional levels. This paper summarizes the diverse mechanisms of action of these lncRNAs and looks at the study strategies in this field. A major challenge in future study is to establish more effective bioinformatics and experimental methods to explore the functions, detailed mechanisms of action and structures deciding the functional diversity of lncRNAs, since the vast majority remain unresolved.
Keywords LncRNAs, Characterizations, Functions, Mechanisms, Gene expression regulation, High-throughput techniques, Bioinformatics, Higher structure, Disease
Address and Contact Information 1State Key Laboratory Cultivation Base for Cell Differentiation Regulation, Henan Normal University, Xinxiang 453007, P.R. China,
2College of Life Science, Henan Normal University, Xinxiang 453007, P. R. China
* Author for correspondence. Email: cellkeylab@126.com; phone: +86 03733326001, fax: +86 03733326524