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Publication Metadata only Functional characterization of recombinant gonad-inhibiting hormone (GIH) and implication of antibody neutralization on induction of ovarian maturation in marine shrimp(2014-05-20) Supattra Treerattrakool; Chanikarn Boonchoy; Sittichai Urtgam; Sakol Panyim; Apinunt Udomkit; Mahidol UniversityOvarian maturation in crustacean is controlled by an eyestalk neuropeptide called gonad-inhibiting hormone (GIH) that is presumed to inhibit vitellogenin synthesis. In this study, a recombinant protein of GIH of Penaeus monodon (rPem-GIH) was expressed in Pichia pastoris expression system and purified to homogeneity by reversed-phase SPE (C18). The purified rPem-GIH significantly reduced vitellogenin mRNA level in primary tissue culture derived from previtellogenic ovary of P. monodon broodstock by 45.7% compared with the untreated group. This effect was similar to that of partially purified optic lobe extract, and demonstrated that the rPem-GIH possessed gonad-inhibiting activity. A monoclonal antibody specific to Pem-GIH (anti-GIH mAb) was able to neutralize the activity of GIH from the partially purified optic lobe extract to about 58% by in vitro assay in primary tissue culture of the ovary. Peptide mapping revealed that amino acids 16 to 20 (MYNKV) of the mature Pem-GIH may serve as an epitope of anti-GIH mAb. A single injection of anti-GIH mAb into wild previtellogenic P. monodon was able to induce ovarian maturation and spawning at a comparable rate to the eyestalk ablation. Our results demonstrate neutralization effect of anti-GIH mAb on vitellogenesis inhibitory action of GIH, and thus provide a potential alternative to induce ovarian maturation in P. monodon broodstock. © 2014 Elsevier B.V.Publication Metadata only Characterization and organization of the U6 snRNA gene in zebrafish and usage of their promoters to express short hairpin RNA(2008-09-01) Surintorn Boonanuntanasarn; Sakol Panyim; Goro Yoshizaki; Suranaree University of Technology; Mahidol University; National University Corporation Tokyo University of Marine Science and TechnologyWe have characterized three U6 snRNA genes in zebrafish and randomly designated them as U6-1, U6-2, and U6-3. The U6-1 gene is closely related to the mammal U6 snRNA genes and that the U6-2 and U6-3 genes are more closely related to the Drosophila and Xenopus U6 snRNA genes. The upstream regulatory sequences were located based on their conserved position relative to the transcription start site. Furthermore, we speculate that the "CCAAT box" functions as the distal sequence element in the zebrafish U6 snRNA genes. Genomic BLASTn analysis revealed that at least 555 copies of the U6-1 gene are dispersed throughout the zebrafish genome, whereas the U6-2 and U6-3 genes are each present as a single copy. Three U6 snRNA genes are functionally expressed in various tissues. All three putative promoters were able to transcribe short hairpin RNA (shRNA) in zebrafish cell extracts. Our findings demonstrate that these putative promoters have the potential to be used for vector-based RNA interference (RNAi) in zebrafish. Another U6 snRNA was found from the genomic BLASTn search and designated as U6-4, demonstrating that there are four different types of zebrafish U6 snRNA genes. © 2008 Elsevier B.V. All rights reserved.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 Usage of putative zebrafish U6 promoters to express shRNA in Nile tilapia and shrimp cell extracts(2009-06-01) Surintorn Boonanuntanasarn; Sakol Panyim; Goro Yoshizaki; Suranaree University of Technology; Mahidol University; National University Corporation Tokyo University of Marine Science and TechnologyWe conducted in vitro transcription activities of the three zebrafish U6 putative promoters across species in cell extracts prepared from Nile tilapia (Oreochromis niloticus) and shrimps. The transcription efficiency of these putative U6 promoters in Nile tilapia cell extracts was similar to that of zebrafish cell extracts. In addition, all three zebrafish U6 snRNA promoters were able to express the shRNA in cell extracts prepared from two shrimp species, Penaeus monodon and Litopenaeus vannamei. However, the shRNA transcription products in shrimp cell extracts showed weaker signals. These U6 promoters could promote shRNA expression, suggesting that they have the potential for use for vector-based RNAi in Nile tilapia and shrimps. A putative U6 promoter would provide a powerful tool for long-term GKD in these aquaculture-related species. © 2009 Springer Science+Business Media B.V.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 Functional characterization of a cDNA encoding Piwi protein in Penaeus monodon and its potential roles in controlling transposon expression and spermatogenesis(2019-03-01) Suchitraporn Sukthaworn; Sakol Panyim; Apinunt Udomkit; Mahidol Universitytransposon mariner in shrimp testis. Investigation of the function of PmPiwi1 in spermatogenesis by sperm count showed significantly lower number of sperms in the spermatophore sac of PmPiwi1-knockdown shrimp compared with that in the control shrimp. OurPublication Metadata only Homologues of Piwi control transposable elements and development of male germline in Penaeus monodon(2020-12-01) Suchitraporn Sukthaworn; Sakol Panyim; Apinunt Udomkit; Mahidol Universityof transposons as PmPiwi2-knockdown shrimp showed a significant increase in the expression of gypsy2 retrotransposon and mariner element in the testis. In addition, a reduction of sperm numbers in the spermatophore of PmPiwi2-knockdown shrimp suggests that PmPublication Metadata only Induction of Ovarian Maturation and Spawning in Penaeus monodon Broodstock by Double-Stranded RNA(2011-04-01) Supattra Treerattrakool; Sakol Panyim; Apinunt Udomkit; Mahidol UniversityOvarian maturation in crustacean is under the control of gonad-inhibiting hormone (GIH); a neuropeptide secreted from X-organ sinus gland complex in eyestalks. Unilateral eyestalk ablation that partially destroys GIH source is therefore a general practice in Penaeus monodon hatchery to induce ovarian maturation and spawning. Our previous report showed that silencing of GIH expression by GIH-specific double-stranded RNA (GIH-dsRNA) resulted in an increased expression level of vitellogenin in P. monodon, thus suggesting that GIH-dsRNA could be an alternative method to induce ovarian maturation in female P. monodon broodstock. In this study, we further demonstrated that a single injection of GIH-dsRNA into previtellogenic female P. monodon at the concentration of 3 μg GIH-dsRNA per gram body weight of shrimp was able to inhibit GIH expression for a minimum of 30 days. This dsRNA-mediated GIH silencing led to ovarian maturation and eventual spawning in both domesticated and wild female broodstock, particularly with a comparable effect to eyestalk ablation in wild shrimp. This is the first report that demonstrates a potential strategy to induce ovarian maturation in female P. monodon broodstock by GIH-dsRNA and thus provides a possible substitute for the cruel and detrimental eyestalk ablation practice. © 2010 Springer Science+Business Media, LLC.Publication Metadata only Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp(2008-07-01) Chalermporn Ongvarrasopone; Mayuree Chanasakulniyom; Kallaya Sritunyalucksana; Sakol Panyim; Mahidol University; Thailand National Center for Genetic Engineering and BiotechnologyViral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp. © 2007 Springer Science+Business Media, LLC.Publication Metadata only Expression of biologically active crustacean hyperglycemic hormone (CHH) of Penaeus monodon in Pichia pastoris(2003-07-01) Supattra Treerattrakool; Apinunt Udomkit; Lily Eurwilaichitr; Burachai Sonthayanon; Sakol Panyim; Mahidol UniversityCrustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the α-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 μg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.