Vol. 19 No. 4 December 2014

DOI: 10.2478/s11658-014-0208-2 Volume 19 (2014) pp 517-526
Title DYSREGULATION OF GENE EXPRESSION IN ABCC6 KNOCKDOWN HepG2 CELLS
Authors Rocchina Miglionico, Maria Francesca Armentano, Monica Carmosino, Antonella Maria Salvia, Flavia Cuviello, Faustino Bisaccia* and Angela Ostuni
Abstract ABCC6 protein is an ATP-dependent transporter that is mainly found in the basolateral plasma membrane of hepatocytes. ABCC6 deficiency is the primary cause of several forms of ectopic mineralization syndrome. Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of the elastic fibers in dermal, ocular and vascular tissues. Mutations in the mouse ABCC6 gene were also associated with dystrophic cardiac calcification. Reduced levels of ABCC6 protein were found in a β-thalassemic mouse model. Moreover, some cases of generalized arterial calcification in infancy are due to ABCC6 mutations. In order to study the role of ABCC6 in the pathogenesis of ectopic mineralization, the expressions of genes involved in this process were evaluated in HepG2 cells upon stable knockdown of ABCC6 by small hairpin RNA (shRNA) technology. ABCC6 knockdown in HepG2 cells causes a significant upregulation of the genes promoting mineralization, such as TNAP, and a parallel downregulation of genes with anti-mineralization activity, such as NT5E, Fetuin A and Osteopontin. Although the absence of ABCC6 has been already associated with ectopic mineralization syndromes, this study is the first to show a direct relationship between reduced ABCC6 levels and the expression of pro-mineralization genes in hepatocytes.
Keywords ABCC6, Knockdown, HepG2 cells, Gene expression, Mineralization, TNAP, NT5E, OPN, Fetuin A
Address and Contact Information Department of Sciences, University of Basilicata, Viale dell’Ateneo Lucano 10, 85100 Potenza, Italy
* Author for correspondence. Email: faustino.bisaccia@unibas.it; phone: +39 0971 205462; fax: +39 0971 205678

DOI: 10.2478/s11658-014-0210-8 Volume 19 (2014) pp 527-541
Title DOWNREGULATION OF KDR EXPRESSION INDUCES APOPTOSIS IN BREAST CANCER CELLS
Authors Xiao Zhang1,*, Yin-Lin Ge1,*, Shu-Ping Zhang2, Ping Yan3 and Run-Hua Tian
Abstract Angiogenesis plays a crucial role in the growth, invasion and metastasis of breast cancer. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are the key regulators of tumor angiogenesis. VEGFR-2, known as the kinase insert domain receptor (KDR), is a key receptor involved in malignant angiogenesis. We previously showed that knocking down KDR with short interference RNA (KDR-siRNA) markedly decreased KDR expression and suppressed tumor growth in a xenograft model. However, the mechanisms underlying the anti-cancer effects of KDR-siRNA are not clearly understood. This study aimed to elucidate the molecular mechanisms that induce apoptosis in human breast cancer MCF-7 cells after transfection with KDR-siRNA. We studied the effects of KDR-siRNA on proliferation, apoptosis, anti-apoptotic and pro-apoptotic proteins, mitochondrial membrane permeability, cytochrome c release and caspase-3 activity. The results indicated that KDR-siRNA treatment significantly inhibited the proliferation and induced the apoptosis of MCF-7 cells, reduced the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xl, and increased the level of the pro-apoptotic protein Bax, resulting in a decreased Bcl-2/Bax ratio. KDR-siRNA also enhanced the mitochondrial membrane permeability, induced cytochrome c release from the mitochondria, upregulated apoptotic protease-activating factor-1 (Apaf-1), cleaved caspase-3, and increased caspase-3 activity in MCF-7 cells. Furthermore, KDR-siRNA-induced apoptosis in MCF-7 cells was blocked by the caspase inhibitor Z-VAD-FMK, suggesting a role of caspase activation in the induction of apoptosis. These results indicate that the Bcl-2 family proteins and caspase-related mitochondrial pathways are primarily involved in KDR-siRNA-induced apoptosis in MCF-7 cells and that KDR might be a potential therapeutic target for human breast cancer treatments.
Keywords Apoptosis, Breast cancer, MCF-7 cells, Cytochrome c, Caspase-3, Mitochondrial pathway, KDR, VEGF, Short interfering RNA, siRNA
Address and Contact Information 1Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, 38 Dengzhou Road, Qingdao, China, 266021,
2Department of obstetrics, Qingdao Municipal Hospital, 5 Donghai middle Road, Qingdao, China, 266011,
3Department of Oral, Zichuan Hospital, 591 Zicheng Road, Zibo, China, 255100
* Authors for correspondence. Emails: zhx7605@126.com; geyinlin@126.com

DOI: 10.2478/s11658-014-0211-7 Volume 19 (2014) pp 542-560
Title IN VITRO EFFECTS OF PROLONGED EXPOSURE TO QUERCETIN AND EPIGALLOCATECHIN GALLATE ON THE PERIPHERAL BLOOD MONONUCLEAR CELL MEMBRANE
Authors Denisa Margina1, Mihaela Ilie1,*, Gina Manda2, Ionela Neagoe2, Rucsandra Danciulescu-Miulescu3, Carmen Nicoleta Purdel1 and Daniela Gradinaru1,
Abstract The study aimed to assess biophysical changes that take place in the peripheral blood mononuclear cell (PBMC) membranes when exposed in vitro to 10 µM quercetin or epigallocatechin gallate (EGCG) for 24 and 48 h. PBMCs isolated from hypercholesterolemia patients were compared to those from normocholesterolemia subjects. The membrane fluidity and transmembrane potential were evaluated and the results were correlated with biochemical parameters relevant to oxidative stress, assessed in the patients’ plasma. The baseline value of PBMC membrane anisotropy for the hypercholesterolemia patients was lower than that of the control group. These results correlated with the plasma levels of advanced glycation end products, which were significantly higher in the hypercholesterolemia group, and the total plasma antioxidant status, which was significantly higher in normocholesterolemia subjects. In the case of normocholesterolemia cells in vitro, polyphenols induced a decrease in membrane anisotropy (7.25–11.88% at 24 h, 1.82–2.26% at 48 h) and a hyperpolarizing effect (8.30–8.90% at 24 h and 4.58–13.00% at 48 h). The same effect was induced in hypercholesterolemia cells, but only after 48 h exposure to the polyphenols: the decrease in membrane anisotropy was 5.70% for quercetin and 2.33% for EGCG. After 48 h of in vitro incubation with the polyphenols, PBMCs isolated from hypercholesterolemia patients exhibited the effects that had been registered in cells from normocholesterolemia subjects after 24 h exposure. These results outlined the beneficial action of the studied polyphenols, quercetin and EGCG, as dietary supplements in normocholesterolemia and hypercholesterolemia patients.
Keywords Membrane fluidity, Transmembrane potential, Total antioxidant status, Advanced glycation end products, Hypercholesterolemia, Quercetin, Epigallocatechin gallate
Address and Contact Information 1Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, 6 Traian Vuia St., 020956 Bucharest, Romania,
2Victor Babeş National Institute of Pathology, Bucharest, 99-101 Splaiul Independentei, 050096 Bucharest, Romania,
3N. Paulescu National Institute for Diabetes, Nutrition and Metabolic Diseases, 5-7 Ion Movila St., 020475 Bucharest, Romania
* Author for correspondence. Email: mihaela.ilie@umf.ro; phone/fax: +40213111152

DOI: 10.2478/s11658-014-0212-6 Volume 19 (2014) pp 561-575
Title REGULATION OF lncRNA EXPRESSION
Authors Zhuomin Wu, Xiaoxia Liu, Li Liu, Houliang Deng, Jingjing Zhang, Qian Xu, Bohong Cen and Aimin Ji*
Abstract Long non-coding RNAs (lncRNAs) are series of transcripts with important biological functions. Various diseases have been associated with aberrant expression of lncRNAs and the related dysregulation of mRNAs. In this review, we highlight the mechanisms of dynamic lncRNA expression. The chromatin state contributes to the low and specific expression of lncRNAs. The transcription of non-coding RNA genes is regulated by many core transcription factors applied to protein-coding genes. However, specific DNA sequences may allow their unsynchronized transcription with their location-associated mRNAs. Additionally, there are multiple mechanisms involved in the post-transcriptional regulation of lncRNAs. Among these, microRNAs might have indispensible regulatory effects on lncRNAs, based on recent discoveries.
Keywords lncRNAs, Mechanisms, Dynamic expression, Chromatin state, Transcription factor, Specific DNA sequence, microRNA, Post-transcriptional regulation
Address and Contact Information Center for Drug Research and Development, Zhujiang Hospital, Southern Medical University
* Author for correspondence. Email: wzm71@fimmu.com; phone: +86 02061643500; fax: +86 02084300639

DOI: 10.2478/s11658-014-0213-5 Volume 19 (2014) pp 576-589
Title ATTENUATION OF ENOYL COENZYME A HYDRATASE SHORT CHAIN 1 EXPRESSION IN GASTRIC CANCER CELLS INHIBITS CELL PROLIFERATION AND MIGRATION IN VITRO
Authors Xiao-San Zhu*, Peng Gao, Yi-Chen Dai*, Jun-Pei Xie, Wei Zeng and Qing-Na Lian
Abstract Enoyl coenzyme A hydratase short chain 1 (ECHS1) is an important part of the mitochondrial fatty acid β-oxidation pathway. Altered ECHS1 expression has been implicated in cancer cell proliferation. This study assessed ECHS1 expression in human gastric cancer cell lines and investigated the effects of ECHS1 knockdown on gastric cancer cell proliferation and migration. The human gastric cancer cell lines SGC-7901, BGC-823 and MKN-28, and the immortalized human gastric epithelial mucosa GES-1 cell line were analyzed for ECHS1 protein levels using western blot. The effectiveness of ECHS1-RNA interference was also determined using western blot. Proliferation and migration of the siECHS1 cells were respectively measured with the CCK-8 and transwell assays. Phosphorylation of PKB and GSK3β was assessed using western blot. ECHS1 protein levels were significantly higher in poorly differentiated cells than in well-differentiated cells and immortalized gastric epithelial mucosa cells. Stable expression of ECHS1 shRNA was associated with an over 41% reduction in the ECHS1 protein levels of siECHS1 cells. Constitutive knockdown of the ECHS1 gene in siECHS1 cells was associated with significantly inhibited cell proliferation and migration. We also observed decreased levels of PKB and GSK3β phosphorylation in siECHS1 cells. ECHS1 expression is increased in human gastric cancer cells. Increased ECHS1 expression activates PKB and GSK3β by inducing the phosphorylation of the two kinases. ECHS1 may play important roles in gastric cancer cell proliferation and migration through PKB- and GSK3β-related signaling pathways.
Keywords ECHS1, PKB, GSK3β, Gastric cancer, Cell proliferation, Cell migration, Interference, β-Oxidation pathway, Signaling pathways, Cells
Address and Contact Information Department of Gastroenterology, Chenggong Hospital Affiliated to Xiamen University, Xiamen, Fujian, 361003, China
* Authors for correspondence. Email: dyichen@sina.com, phone: +86-592-6335542, fax: +86-592-6335542; zhuxs223@126.com, phone: +86-592-2020390, fax: +86-592-6335702

DOI: 10.2478/s11658-014-0215-3 Volume 19 (2014) pp 590-600
Title PROMOTED MEGAKARYOCYTIC DIFFERENTIATION OF K562 CELLS THROUGH OXIDATIVE STRESS CAUSED BY NEAR ULTRAVIOLET IRRADIATION
Authors Retno Wahyu Nurhayati, Yoshihiro Ojima, Naoki Nomura and Masahito Taya*
Abstract Reactive oxygen species (ROS) have been proven to be important activators for various cellular activities, including cell differentiation. Several reports showed the necessity of ROS during cell differentiation of the megakaryocytic (MK) lineage. In this study, we employed near ultraviolet (near-UV) irradiation to generate endogenous oxidative stress in an MK differentiation process of K562 cells with phorbol 12-myristate 13-acetate (PMA) induction. A significant increase in the intracellular ROS level was detected on day 1 after near-UV irradiation. In the initial stage of differentiation, a shifted fraction of G1 and G2 phase cells was obtained using near-UV irradiation, giving an increased percentage of G2 phase cells (up from 31.1 to 68.7%). The near-UV irradiation-induced upregulation of the p21 gene, which is a cell cycle inhibitor, suggested that the G2 phase cells were prevented from undergoing cell division. It was found that the percentage of high ploidy (8N and 16N) cells was enhanced significantly at the later stage of the K562 cell culture with near-UV irradiation. Moreover, time-lapse analysis showed that near-UV irradiation encouraged the expression of CD41, a specific surface marker of megakaryocytes. This is the first report that the elevated oxidative stress through the near-UV irradiation promoted the MK differentiation of PMA-induced K562 cells.
Keywords Near ultraviolet, Irradiation, K562 cells, Megakaryocytic differentiation, Polyploidization, Reactive oxygen species, Cell cycle, Phorbol 12-myristate 13-acetate
Address and Contact Information Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1–3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan
* Author for correspondence. Email: taya@cheng.es.osaka-u.ac.jp; phone: +81-6-6850- 6251; fax: +81-6-6850-6254

DOI: 10.2478/s11658-014-0214-4 Volume 19 (2014) pp 601-610
Title Ano1 IS A BETTER MARKER THAN c-Kit FOR TRANSCRIPT ANALYSIS OF SINGLE INTERSTITIAL CELLS OF CAJAL IN CULTURE
Authors Raúl Loera-Valencia1,2,*, Xuan-Yu Wang2, George W.J. Wright2, Carlos Barajas-López1 and Jan D. Huizinga2
Abstract The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.
Keywords Interstitial cells of Cajal, c-Kit, Ano1, Multiplexed RT-PCR, Single-cell PCR, Transcriptional regulation, ICC marker, Small intestine, Primary cultures, Pacemaker cells
Address and Contact Information 1División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, Col. Lomas 4a Sección, C.P.78216 San Luis Potosí, SLP, México,
2Farncombe Family Digestive Health Research Institute, Department of Medicine, McMaster University, HSC-3N8, 1200 Main Street West, Hamilton, ON, L8N 3Z5, Canada
* Author for correspondence. Email: raul.loera@ipicyt.edu.mx; phone: +52 444 834 2000 x2033; fax: +52 444 834 2010

DOI: 10.2478/s11658-014-0216-2 Volume 19 (2014) pp 611-622
Title GENE EXPRESSION PATTERNS IN BONE FOLLOWING LIPOPOLYSACCHARIDE STIMULATION
Authors Jing Yang, Nan Su, Xiaolan Du and Lin Chen*
Abstract Bone displays suppressed osteogenesis in inflammatory diseases such as sepsis and rheumatoid arthritis. However, the underlying mechanisms have not yet been clearly explained. To identify the gene expression patterns in the bone, we performed Affymetrix Mouse Genome 430 2.0 Array with RNA isolated from mouse femurs 4 h after lipopolysaccharide (LPS) administration. The gene expressions were confirmed with real-time PCR. The serum concentration of the N-terminal propeptide of type I collagen (PINP), a bone- formation marker, was determined using ELISA. A total of 1003 transcripts were upregulated and 159 transcripts were downregulated (more than twofold upregulation or downregulation). Increased expression levels of the inflammation-related genes interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were confirmed from in the period 4 h to 72 h after LPS administration using real-time PCR. Gene ontogene analysis found four bone-related categories involved in four biological processes: system development, osteoclast differentiation, ossification and bone development. These processes involved 25 upregulated genes. In the KEGG database, we further analyzed the transforming growth factor β (TGF-β) pathway, which is strongly related to osteogenesis. The upregulated bone morphogenetic protein 2 (BMP2) and downregulated inhibitor of DNA binding 4 (Id4) expressions were further confirmed by real-time PCR after LPS stimulation. The osteoblast function was determined through examination of the expression levels of core binding factor 1 (Cbfa1) and osteocalcin (OC) in bone tissues and serum PINP from 4 h to 72 h after LPS administration. The expressions of OC and Cbfa1 decreased 6 h after administration (p < 0.05). Significantly suppressed PINP levels were observed in the later stage (from 8 h to 72 h, p < 0.05) but not in the early stage (4 h or 6 h, p > 0.05) of LPS stimulation. The results of this study suggest that LPS induces elevated expressions of skeletal system development- and osteoclast differentiation-related genes and inflammation genes at an early stage in the bone. The perturbed functions of these two groups of genes may lead to a faint change in osteogenesis at an early stage of LPS stimulation. Suppressed bone formation was found at later stages in response to LPS stimulation.
Keywords Lipopolysaccharide, Microarray, Bone, Mouse, Inflammation, Bone morphogenetic protein 2, N-terminal propeptide of type I collagen, Osteogenesis, Core binding factor 1, Osteocalcin
Address and Contact Information State Key Laboratory of Trauma, Burns and Combined Injury, Center of Bone Metabolism and Repair, Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China
* Author for correspondence. Email: linchen70@tmmu.edu.cn; phone: 86-23-68702991, fax: 86-23-68702991

DOI: 10.2478/s11658-014-0217-1 Volume 19 (2014) pp 623-637
Title THE EFFECT OF BUFFALO CD14 shRNA ON THE GENE EXPRESSION OF TLR4 SIGNAL PATHWAY IN BUFFALO MONOCYTE/MACROPHAGES
Authors Xiangping Li1,*, Meiqing Li1, Shihai Huang2, Shuye Qiao1, Zhaoxian Qin3, Chao Kang2 and Deshun Shi1,*
Abstract CD14 plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, resulting in inflammation. Upstream inhibition of the inflammation pathway mediated by bacterial LPS, toll-like receptor 4 (TLR4) and cluster of differentiation antigen 14 (CD14) was proven to be an effective therapeutic approach for attenuating harmful immune activation. To explore the effect of CD14 downregulation on the expression of TLR4 signaling pathway-related genes after LPS stimulation in buffalo (Bubalus bubalis) monocyte/macrophages, effective CD14 shRNA sequences were screened using qRT-PCR and FACS analysis with buffalo CD14 shRNA lentiviral recombinant plasmids (pSicoR-GFP-shRNA) and buffalo CD14 fusion expression plasmids (pDsRed-N1-buffalo CD14) co-transfected into HEK293T cells via liposomes. Of the tested shRNAs, shRNA-1041 revealed the highest knockdown efficiency (p < 0.01). When buffalo peripheral blood monocyte/macrophages were infected with shRNA-1041 lentivirus and stimulated with LPS, the expression of endogenous CD14 was significantly decreased by CD14 shRNA (p < 0.01), and the mRNA expression levels of TLR4, IL-6 and TNF-α were also significantly downregulated compared to the control groups (p < 0.01). These results demonstrated that the knockdown of endogenous CD14 had clear regulatory effects on the signal transduction of TLR4 after stimulation with LPS. These results may provide a better understanding of the molecular mechanisms of CD14 regulation in the development of several buffalo diseases.
Keywords Buffalo CD14 gene, shRNA, TLR4 signal pathway, Gene expression, Monocyte/macrophages
Address and Contact Information 1State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China,
2College of life science and technology, Guangxi University, Nanning, Guangxi, China,
3Guangxi Institute of Animal Science, Nanning, Guangxi, China
* Authors for correspondence. Email: xiangpingli@163.com, ardsshi@gxu.edu.cn

DOI: 10.2478/s11658-014-0219-z Volume 19 (2014) pp 638-648
Title RAPID PROLIFERATION OF ACTIVATED LYMPH NODE CD4+ T CELLS IS ACHIEVED BY GREATLY CURTAILING THE DURATION OF GAP PHASES IN CELL CYCLE PROGRESSION
Authors Takuya Mishima§, Shoko Toda§, Yoshiaki Ando, Tsukasa Matsunaga and Manabu Inobe*
Abstract Peripheral T cells are in G0 phase and do not proliferate. When they encounter an antigen, they enter the cell cycle and proliferate in order to initiate an active immune response. Here, we have determined the first two cell cycle times of a leading population of CD4+ T cells stimulated by PMA plus ionomycin in vitro. The first cell cycle began around 10 h after stimulation and took approximately 16 h. Surprisingly, the second cell cycle was extremely rapid and required only 6 h. T cells might have a unique regulatory mechanism to compensate for the shortage of the gap phases in cell cycle progression. This unique feature might be a basis for a quick immune response against pathogens, as it maximizes the rate of proliferation.
Keywords CD4+ T cells, Cell cycle, G0/G1 transition, Gap phase, In vitro, PMA plus ionomycin, Proliferation, T cell activation
Address and Contact Information Laboratory of Human Molecular Genetics, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 Japan
§These authors contributed equally to this work
* Author for correspondence. Email: inobem@p.kanazawa-u.ac.jp; phone: +81-76-264-6272; fax: +81-76-234-4427

DOI: 10.2478/s11658-014-0218-0 Volume 19 (2014) pp 649-658
Title THE PPARα PATHWAY IN Vγ9Vδ2 T CELL ANERGY
Authors Mary Poupot1,2,3,*, Frédéric Boissard1,2,3, Delphine Betous1,2,3, Laure Bardouillet4, Séverine Fruchon5, Fatima L’Faqihi- Olive5, Frédéric Pont1, Mourad Mekaouche4, Sophie Ingoure6, Hélène Sicard6, Guy Dubreuilh4 and Jean-Jacques Fournié1,2,3
Abstract Phosphoantigens (PAgs) activate Vγ9Vδ2 T lymphocytes, inducing their potent and rapid response in vitro and in vivo. However, humans and non- human primates that receive repeated injections of PAgs progressively lose their Vγ9Vδ2 T cell response to them. To elucidate the molecular mechanisms of this in vivo desensitization, we analyzed the transcriptome of circulating Vγ9Vδ2 T cells from macaques injected with PAg. We showed that three PAg injections induced the activation of the PPARα pathway in Vγ9Vδ2 T cells. Thus, we analyzed the in vitro response of Vγ9Vδ2 T cells stimulated with a PPARα agonist. We demonstrated that in vitro PPARα pathway activation led to the inhibition of the BrHPP-induced activation and proliferation of human Vγ9Vδ2 T cells. Since the PPARα pathway is involved in the antigen-selective desensitization of human Vγ9Vδ2 T cells, the use of PPARα inhibitors could enhance cancer immunotherapy based on Vγ9Vδ2 T cells.
Keywords Activation, Gamma-delta T-lymphocyte, Immunotherapy, Phosphoantigen, TCR, PPARα
Address and Contact Information 1INSERM UMR1037-Cancer Research Center of Toulouse,
2ERL 5294 CNRS, BP3028, CHU Purpan, F-31300 Toulouse, France,
3Université Toulouse III Paul-Sabatier, 31300 Toulouse, France,
4Station de Primatologie, UPS 846, Centre National de la Recherche Scientifique, F-13790 Rousset sur Arc, France,
5INSERM U1043-CPTP Toulouse, F-31300 France,
6Innate Pharma, F-13276 Marseille, France
* Author for correspondence. Email: mary.poupot@inserm.fr, phone: +335 8274 1662

DOI: 10.2478/s11658-014-0220-6 Volume 19 (2014) pp 659-674
Title THE ROLE OF ALK-1 AND ALK-5 IN THE MECHANOSENSING OF CHONDROCYTES
Authors Patricia Sanz-Ramos1, Javier Dotor2 and Iñigo Izal-Azcárate1,*
Abstract We aim to demonstrate the role of Alk receptors in the response of hydrogel expansion. Chondrocytes from rat knees were cultured onto plastic and hydrogel surfaces. Alk-1 and Alk-5 were overexpressed or silenced and the effects on cells during expansion were tested and confirmed using peptide inhibitors for TGFβ. Overexpression of Alk-5 and silencing of Alk-1 led to a loss of the chondrocyte phenotype, proving that they are key regulators of chondrocyte mechanosensing. An analysis of the gene expression profile during the expansion of these modified cartilage cells in plastic showed a better maintenance of the chondrocyte phenotype, at least during the first passages. These passages were also assayed in a mouse model of intramuscular chondrogenesis. Our findings indicate that these two receptors are important mediators in the response of chondrocytes to changes in the mechanical environment, making them suitable targets for modulating chondrogenesis. Inhibition of TGFβ could also be effective in improving chondrocyte activity in aged or expanded cells that overexpress Alk-1.
Keywords Chondrocyte, Mechanosensing, Mechanobiology, Mechanotransduction, Collagen hydrogels, TGFβ, Differentiation, Signalling, Cartilage, Chondrogenesis
Address and Contact Information 1Laboratory for Orthopaedic Research, University of Navarra, School of Medicine, Pamplona, Spain,
2DIGNA Biotech, Pamplona, Spain
* Author for correspondence. inizal@me.com, phone: +34 48 425600 (6403), fax: +34 48 425649

DOI: 10.2478/s11658-014-0221-5 Volume 19 (2014) pp 675-691
Title FUNPRED-1: PROTEIN FUNCTION PREDICTION FROM A PROTEIN INTERACTION NETWORK USING NEIGHBORHOOD ANALYSIS
Authors Sovan Saha1, Piyali Chatterjee2, Subhadip Basu3,*, Mahantapas Kundu3 and Mita Nasipuri3
Abstract Proteins are responsible for all biological activities in living organisms. Thanks to genome sequencing projects, large amounts of DNA and protein sequence data are now available, but the biological functions of many proteins are still not annotated in most cases. The unknown function of such non-annotated proteins may be inferred or deduced from their neighbors in a protein interaction network. In this paper, we propose two new methods to predict protein functions based on network neighborhood properties. FunPred 1.1 uses a combination of three simple-yet-effective scoring techniques: the neighborhood ratio, the protein path connectivity and the relative functional similarity. FunPred 1.2 applies a heuristic approach using the edge clustering coefficient to reduce the search space by identifying densely connected neighborhood regions. The overall accuracy achieved in FunPred 1.2 over 8 functional groups involving hetero-interactions in 650 yeast proteins is around 87%, which is higher than the accuracy with FunPred 1.1. It is also higher than the accuracy of many of the state-of-the-art protein function prediction methods described in the literature. The test datasets and the complete source code of the developed software are now freely available at https://code.google.com/p/cmater-bioinfo/.
Keywords Protein interaction network, Protein function prediction, Functional groups, Neighborhood analysis, Relative functional similarity, Edge clustering coefficient
Address and Contact Information 1Department of Computer Science and Engineering, Dr. Sudhir Chandra Sur Degree Engineering College, Dumdum, Kolkata-700074, India,
2Department of Computer Science and Engineering, Netaji Subhash Engineering College, Garia, Kolkata-700152, India,
3Department of Computer Science and Engineering, Jadavpur University, Kolkata-700032, India
* Author for correspondence. Email: subhadip@cse.jdvu.ac.in