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      Molecular Characterization of Severin from Clonorchis sinensis Excretory/Secretory Products and Its Potential Anti-apoptotic Role in Hepatocarcinoma PLC Cells

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          Abstract

          Background

          Clonorchiasis, caused by the infection of Clonorchis sinensis ( C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma and hepatocellular carcinoma (HCC). It has been well known that the excretory/secretory products of C. sinensis ( CsESPs) play key roles in clonorchiasis associated carcinoma. From genome and transcriptome of C. sinensis, we identified one component of CsESPs, severin ( Csseverin), which had three putative gelsolin domains. Its homologues are supposed to play a vital role in apoptosis resistance of tumour cell.

          Methodology/Principal Findings

          There was significant similarity in tertiary structures between human gelsolin and Csseverin by bioinformatics analysis. We identified that Csseverin expressed at life stage of adult worm, metacercaria and egg by the method of quantitative real-time PCR and western blotting. Csseverin distributed in vitellarium and intrauterine eggs of adult worm and tegument of metacercaria by immunofluorence assay. We obtained recombinant Csseverin (r Csseverin) and confirmed that r Csseverin could bind with calciumion in circular dichroism spectrum analysis. It was demonstrated that r Csseverin was of the capability of actin binding by gel overlay assay and immunocytochemistry. Both Annexin V/PI assay and mitochondrial membrane potential assay of human hepatocarcinoma cell line PLC showed apoptosis resistance after incubation with different concentrations of r Csseverin. Morphological analysis, apoptosis-associated changes of mitochondrial membrane potential and Annexin V/PI apoptosis assay showed that co-incubation of PLC cells with r Csseverin in vitro led to an inhibition of apoptosis induced by serum-starved for 24 h.

          Conclusions/Significance

          Collectively, the molecular properties of Csseverin, a molecule of CsESPs, were characterized in our study. r Csseverin could cause obvious apoptotic inhibition in human HCC cell line. Csseverin might exacerbate the process of HCC patients combined with C. sinensis infection.

          Author Summary

          Clonorchis sinensis ( C. sinensis) has afflicted more than 35 million people in world and approximately 15 million in China, creating a socio-economic burden in epidemic regions. The infection of C. sinensis is highly related to cholangiocarcinoma and hepatocellular carcinoma (HCC). It has been documented that excretory/secretory products of C. sinensis ( CsESPs) involved in the pathogenesis of HCC. Csseverin, expressed at life stage of egg, metacercaria and adult worm, was a component of CsESPs. In the current study, we characterized the properties of Csseverin such as sequence signature, actin and calciumion binding activity. In addition, we demonstrated that Csseverin could cause apoptotic inhibition in spontaneously apoptotic human HCC cell line PLC cells by using morphological analysis, detection of the apoptosis-associated change of mitochondrial membrane potential (MMP) as well as Annexin V/PI apoptosis assay. Our study provided an exploratory sight view of mechanism involved in progress of carcinoma associated with the infection of C. sinensis and Csseverin might exacerbate the process of C. sinensis infected HCC patients.

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          Most cited references40

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          The Schistosoma japonicum genome reveals features of host-parasite interplay.

          (2009)
          Schistosoma japonicum is a parasitic flatworm that causes human schistosomiasis, which is a significant cause of morbidity in China and the Philippines. Here we present a draft genomic sequence for the worm. The genome provides a global insight into the molecular architecture and host interaction of this complex metazoan pathogen, revealing that it can exploit host nutrients, neuroendocrine hormones and signalling pathways for growth, development and maturation. Having a complex nervous system and a well-developed sensory system, S. japonicum can accept stimulation of the corresponding ligands as a physiological response to different environments, such as fresh water or the tissues of its intermediate and mammalian hosts. Numerous proteases, including cercarial elastase, are implicated in mammalian skin penetration and haemoglobin degradation. The genomic information will serve as a valuable platform to facilitate development of new interventions for schistosomiasis control.
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            JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis.

            The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (delta psi) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing deltapsi. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC6(3) - but not of R123 - only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing delta psi changes with flow cytometry, while the others show a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to delta psi, hypothesized that the fall in delta psi is one of the early events, if not one of the main causes, of apoptosis.
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              NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones.

              Excitatory amino acids act via receptor subtypes in the mammalian central nervous system (CNS). The receptor selectively activated by N-methyl-D-aspartic acid (NMDA) has been best characterized using voltage-clamp and single-channel recording; the results suggest that NMDA receptors gate channels that are permeable to Na+, K+ and other monovalent cations. Various experiments suggest that Ca2+ flux is also associated with the activation of excitatory amino-acid receptors on vertebrate neurones. Whether Ca2+ enters through voltage-dependent Ca2+ channels or through excitatory amino-acid-activated channels of one or more subtype is unclear. Mg2+ can be used to distinguish NMDA-receptor-activated channels from voltage-dependent Ca2+ channels, because at micromolar concentrations Mg2+ has little effect on voltage-dependent Ca2+ channels while it enters and blocks NMDA receptor channels. Marked differences in the potency of other divalent cations acting as Ca2+ channel blockers compared with their action as NMDA antagonists also distinguish the NMDA channel from voltage-sensitive Ca2+ channels. However, we now directly demonstrate that excitatory amino acids acting at NMDA receptors on spinal cord neurones increase the intracellular Ca2+ activity, measured using the indicator dye arsenazo III, and that this is the result of Ca2+ influx through NMDA receptor channels. Kainic acid (KA), which acts at another subtype of excitatory amino-acid receptor, was much less effective in triggering increases in intracellular free Ca2+.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                December 2013
                19 December 2013
                23 December 2013
                : 7
                : 12
                : e2606
                Affiliations
                [1 ]Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, People′s Republic of China
                [2 ]Key Laboratory of Tropical Diseases Control at Sun Yat-sen University, Ministry of Education, Guangzhou, People′s Republic of China
                McGill University, Canada
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: YH XY. Performed the experiments: XC SL. Analyzed the data: XC YH CL ZW JX MR. Contributed reagents/materials/analysis tools: XW PL WC LH SL XL JL MB. Wrote the paper: XC YH.

                Article
                PNTD-D-13-01393
                10.1371/journal.pntd.0002606
                3868641
                24367717
                1435aa33-4796-4fa3-9f12-33a848ab072b
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 September 2013
                : 28 October 2013
                Page count
                Pages: 10
                Funding
                This work was supported by the National Key Basic Research and Development Project (973 project; No. 2010CB530000), the National Natural Science Foundation of China (No. 81101270 and No. 81171602), the National S & T Major Program (2012ZX10004-220), the Fundamental Research Funds for the Central Universities (NO. 3164015). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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