18 results
Search Results
Now showing 1 - 10 of 18
Publication Metadata only Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon)(2002-01-01) Premruethai Supungul; Sirawut Klinbunga; Rath Pichyangkura; Sarawut Jitrapakdee; Ikuo Hirono; Takashi Aoki; Anchalee Tassanakajon; Chulalongkorn University; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University; National University Corporation Tokyo University of Marine Science and TechnologyAn expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 × 105. Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported. © Springer-Verlag New York Inc.Publication Metadata only Successful yellow head virus infection of Penaeus monodon requires clathrin heavy chain(2015-01-01) Pratsaneeyaporn Posiri; Hidehiro Kondo; Ikuo Hirono; Sakol Panyim; Chalermporn Ongvarrasopone; Mahidol University; National University Corporation Tokyo University of Marine Science and Technology© 2014 Elsevier B.V. Viral disease caused by the Yellow head virus (YHV) had great impact on economic loss in the aquaculture industry. Prevention or curing YHV disease is still not possible due to the lack of understanding of the basic mechanisms of YHV infection. In this report, the endocytosis inhibitors (chlorpromazine (CPZ), amiloride and methyl-β-cyclodextrin (MβCD)) were used to identify the cellular entry pathway of YHV. Pretreating shrimp with CPZ but not amiloride or MβCD followed by YHV challenge resulted in a significant reduction of YHV levels, suggesting that YHV entered the shrimp cells via clathrin-mediated endocytosis. Next, the major component of the clathrin-coated vesicle, Penaeus monodon clathrin heavy chain (PmCHC) was cloned and characterized. The complete coding sequence of PmCHC is 5055. bp encoding a putative protein of 1684 amino acids. Specific silencing of PmCHC mRNA by dsRNA-PmCHC showed an inhibition of YHV replication for 48. h post YHV injection as well as exhibiting a delay in shrimp mortality. These results indicated that PmCHC was an essential component for YHV infection of shrimp cells.Publication Metadata only Molecular cloning and characterization of Mj-mov-10, a putative RNA helicase involved in RNAi of kuruma shrimp(2015-05-01) Amnat Phetrungnapha; Hidehiro Kondo; Ikuo Hirono; Sakol Panyim; Chalermporn Ongvarrasopone; Naresuan University; National University Corporation Tokyo University of Marine Science and Technology; Mahidol University© 2015 Elsevier Ltd. Identification and characterization of the RNAi-related genes is the key to understanding RNAi mechanism in shrimp. In this study, we have identified and characterized a novel putative RNA helicase gene, Mj-mov-10 from the kuruma shrimp, Marsupenaeus japonicus and its implication in shrimp RNAi was demonstrated. The full-length Mj-mov-10 gene contained 3536. bp, including 239bp of 5'UTR, 2895bp of the open reading frame (ORF) and 402. bp of 3'UTR, respectively. An ORF of Mj-mov-10 could be translated to a 109-kDa protein which consists of a single helicase core domain containing seven signature motifs of the RNA helicase superfamily-1. Mj-MOV-10 protein shared 47% and 40% identity with mammalian MOV-10 and plant SDE3, respectively. Expression of Mj-mov-10 gene was significantly up-regulated upon dsRNA and white spot syndrome virus (WSSV) challenge. Invivo gene knockdown of Mj-mov-10 resulted in an increase of a susceptibility of shrimp to WSSV infection. Our results implied the functional significance of Mj-MOV-10 in dsRNA-mediated gene silencing and antiviral defense mechanism in shrimp.Publication Metadata only Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells(2020-01-01) Iyapa Palang; Ikuo Hirono; Saengchan Senapin; Wanna Sirimanapong; Boonsirm Withyachumnarnkul; Rapeepun Vanichviriyakit; National University Corporation Tokyo University of Marine Science and Technology; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Prince of Songkla University; AquaAcademy Farm© 2020 John Wiley & Sons Ltd Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.Publication Metadata only Enhancement of shrimp immunity against white spot syndrome virus by Macrobrachium rosenbergii nodavirus-like particle encapsulated VP28 double-stranded RNA(2015-09-01) Pitchanee Jariyapong; Wattana Weerachatyanukul; Sataporn Direkbusarakom; Ikuo Hirono; Suwit Wuthisuthimethavee; Charoonroj Chotwiwatthanakun; Walailak University; Mahidol University; National University Corporation Tokyo University of Marine Science and Technology© 2015 Elsevier B.V. We investigated the efficiency and downstream effects of Macrobrachium rosenbergii nodavirus-like particle (MrNv-VLP) encapsulated VP28 double-stranded RNA against white spot syndrome virus (WSSV) in shrimp. Our results showed that the VP28 gene of WSSV was significantly silenced at 72. h post-viral challenge in group pre-treated with the VLP encapsulated VP28 dsRNA. At 24. h post-treatment, the amount of VP28 dsRNA was significantly higher (two-fold) in encapsulated VP28 dsRNA-VLP pre-treated shrimp compared with non-encapsulated VP28 dsRNA pre-treated shrimp. Using quantitative real time polymerase chain reaction (PCR), we found higher and longer expression of RNA-induced silencing complex (RISC) and immune-related genes in shrimp pre-treated with encapsulated VP28 dsRNA compared to the control groups. Increasing expression of apoptotic-related genes was also associated with the encapsulated VP28 dsRNA-VLP pretreatment group at 24. h post-WSSV challenge. These results indicate that MrNv-VLP was able to efficiently deliver VP28 dsRNA into shrimp tissues, which in turn, triggered a better anti-viral response. This may represent a novel strategy for aquaculture disease management. Statement of relevance: This study developed methods for WSSV protection.Publication Metadata only Delivery of double stranded RNA by Macrobrachium rosenbergii nodavirus-like particles to protect shrimp from white spot syndrome virus(2015-01-01) Pitchanee Jariyapong; Charoonroj Chotwiwatthanakun; Sataporn Direkbusarakom; Ikuo Hirono; Suwit Wuthisuthimethavee; Wattana Weerachatyanukul; Walailak University; Mahidol University; National University Corporation Tokyo University of Marine Science and Technology© 2014 Elsevier B.V. We tested the use of Macrobrachium rosenbergii nodavirus-like particles (MrNv-VLPs) as a delivery mechanism to carry therapeutic agents against white spot syndrome in shrimp. We used constructed double-stranded RNA called VP28 (VP28 dsRNA) against WSSV envelope genes to confer protection against the pathogen. Results showed that MrNv-VLP was able to encapsulate VP28 dsRNA. Using enhanced green fluorescent protein (EGFP) as a reporter, we found that VLP penetrated various shrimp tissues including the muscle, hepatopancreas, and gill. A statistically significant relative survival rate of 44.5% was obtained in the group of shrimp receiving encapsulated VP28 dsRNA-VLP after WSSV challenge as compared to 100% mortality in the control shrimp at 7. days post-infection. Shrimp treated with EGFP dsRNA loaded into MrNv-VLPs showed relatively similar motility rates as those of controls. Moreover, MrNv-VLP encapsulation improved VP28 dsRNA efficiency against WSSV. A higher survival rate of 16.7% was observed in the group of shrimp receiving encapsulated VP28 dsRNA-VLP when compared to those receiving naked VP28 dsRNA. These results indicate that MrNv-VLP is a good candidate for use as a therapeutic delivery system against shrimp diseases due to its self-reassembly property, broad target of various shrimp tissues and immune enhancement.Publication Metadata only Gene silencing of a prophenoloxidase activating enzyme in the shrimp, Penaeus monodon, increases susceptibility to Vibrio harveyi infection(2009-07-01) Walaiporn Charoensapsri; Piti Amparyup; Ikuo Hirono; Takashi Aoki; Anchalee Tassanakajon; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; National University Corporation Tokyo University of Marine Science and TechnologyThe prophenoloxidase (proPO) activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase (PO), is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases (clip-SPs). In this study, a cDNA encoding a proPO activating enzyme (PPAE) from the black tiger shrimp, Penaeus monodon, designated as PmPPAE1, was cloned and characterized. The full-length cDNA contains an open reading frame (ORF) of 1392 bp encoding a predicted protein of 463 amino acids including an 18 amino acid signal peptide. The PmPPAE1 protein exhibits a characteristic sequence structure of clip-SPs consisting of the clip domain at the N-terminus and a SP domain at the C-terminus. Sequence analysis showed that PmPPAE1 exhibited the highest amino acid sequence similarity (70%) to a PPAE of the crayfish, Pacifastacus leniusculus. PmPPAE1 mRNA is abundantly expressed in hemocytes, and this is regulated after systemic Vibrio harveyi infection supporting that it is an immune-responsive gene. RNA interference-mediated suppression of PmPPAE1, performed by injection of double-stranded RNA (dsRNA) corresponding to the PmPPAE1 gene into shrimp, resulted in a significant reduction of PmPPAE1 but not other clip-SP and related gene transcript levels of P. monodon, suggesting gene-specific knockdown. RNAi-mediated silencing of PmPPAE1 gene significantly decreased the total PO activity (36.7%) in shrimp and additionally increased the mortality of V. harveyi infected shrimp, the latter of which correlated with an increase in the number of viable bacteria in the hemolymph. These results indicate that PmPPAE1 functions in the proPO system and is an important component in the shrimp immune system. © 2009 Elsevier Ltd. All rights reserved.Publication Metadata only WSV399, a viral tegument protein, interacts with the shrimp protein PmVRP15 to facilitate viral trafficking and assembly(2016-06-01) Phattarunda Jaree; Saengchan Senapin; Ikuo Hirono; Chu Fang Lo; Anchalee Tassanakajon; Kunlaya Somboonwiwat; Chulalongkorn University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; National University Corporation Tokyo University of Marine Science and Technology; National Cheng Kung University© 2016 Elsevier Ltd. Viral responsive protein 15 (PmVRP15) has been identified as a highly up-regulated gene in the hemocyte of white spot syndrome virus (WSSV)-infected shrimp Penaeus monodon. However, the function of PmVRP15 in host-viral interaction was still unclear. To elucidate PmVRP15 function, the interacting partner of PmVRP15 from WSSV was screened by yeast two-hybrid assay and then confirmed by co-immunoprecipitation (Co-IP). Only WSV399 protein was identified as a PmVRP15 binding protein; however, the function of WSV399 has not been characterized. Localization of WSV399 on the WSSV virion was revealed by immunoblotting analysis (in vitro) and immunoelectron microscopy (in vivo). The results showed that WSV399 is a structural protein of the WSSV virion and is particularly located on the tegument. Gene silencing of wsv399 in WSSV-infected shrimp reduced the percentage of cumulative mortality by 74%, although the expression level of a viral replication marker gene, vp28, was not changed suggesting that WSV399 might not involved in viral replication but viral assembly. Because it has already been known that tegument proteins function in capsid transport during viral trafficking and assembly, interaction between PmVRP15 on hemocyte nuclear membrane and the WSV399 viral tegument protein suggests that PmVRP15 might be required for trafficking and assembly of WSSV during infection.Publication Metadata only Homology modeling and virtual screening for antagonists of protease from yellow head virus(2014-01-01) Sasimanas Unajak; Orathai Sawatdichaikul; Napat Songtawee; Siriluk Rattanabunyong; Anchalee Tassnakajon; Nontawith Areechon; Ikuo Hirono; Hidehiro Kondo; Pongsak Khunrae; Triwit Rattanarojpong; Kiattawee Choowongkomon; Kasetsart University; Mahidol University; Chulalongkorn University; National University Corporation Tokyo University of Marine Science and Technology; King Mongkuts University of Technology ThonburiYellow head virus (YHV) is one of the causative agents of shrimp viral disease. The prevention of YHV infection in shrimp has been developed by various methods, but it is still insufficient to protect the mass mortality in shrimp. New approaches for the antiviral drug development for viral infection have been focused on the inhibition of several potent viral enzymes, and thus the YHV protease is one of the interesting targets for developing antiviral drugs according to the pivotal roles of the enzyme in an early stage of viral propagation. In this study, a theoretical modeling of the YHV protease was constructed based on the folds of several known crystal structures of other viral proteases, and was subsequently used as a target for virtual screening - molecular docking against approximately 1364 NCI structurally diversity compounds. A complex between the protease and the hit compounds was investigated for intermolecular interactions by molecular dynamics simulations. Five best predicted compounds (NSC122819, NSC345647, NSC319990, NSC50650, and NSC5069) were tested against bacterial expressed YHV. The NSC122819 showed the best inhibitory characteristic among the candidates, while others showed more than 50 % of inhibition in the assay condition. These compounds could potentially be inhibitors for curing YHV infection. © 2014 Springer-Verlag.Publication Metadata only Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon(2008-02-01) Piti Amparyup; Hidehiro Kondo; Ikuo Hirono; Takashi Aoki; Anchalee Tassanakajon; Thailand National Center for Genetic Engineering and Biotechnology; Mahidol University; National University Corporation Tokyo University of Marine Science and TechnologyA novel crustin-like antimicrobial peptide (Crus-likePm) was identified from haemocytes of Penaeus monodon. The deduced amino acid sequence of a Crus-likePm consists of 124 amino acid residues of the mature peptide and a signal peptide of 17 amino acid residues. The mature peptide contains a glycine-rich domain at the N-terminus and 12 conserved cysteine residues containing a single WAP domain at the C-terminus. Phylogenetic tree and sequence comparison clearly confirmed a distinct between a Crus-likePm and other shrimp crustins. Genomic organization and upstream region of a Crus-likePm gene was investigated. The gene consisted of two exons and one intron. The 5′-flanking regions of a Crus-likePm gene contain multiple putative transcription factor binding sites. mRNA transcript of a Crus-likePm was found to be abundantly expressed in haemocyte and highly up-regulated after Vibrio harveyi injection. The mature Crus-likePm was cloned into the pET28b with an N-terminal hexa-histidine tag fused in-frame, and expressed in E. coli. The purified recombinant Crus-likePm showed strong antimicrobial activity against both Gram-positive and Gram-negative bacteria including V. harveyi, a major pathogenic bacteria in shrimp aquaculture. © 2007 Elsevier Ltd. All rights reserved.