PubMed | Institute of Gyeongbuk Marine Bioindustry, National Institute of Fisheries Science NIFS, Hoseo University and Hankyong National University
Type: | Journal: General and comparative endocrinology | Year: 2016
We prepared monoclonal antibodies (mAbs) against a recombinant tethered follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica that was produced in Escherichia coli. Positive hybridomas (clones eFA-C5, eFA-C10, eFA-C11, eFA-C12, eFA-C13, and eFB-C14) were selected by using the eel FSH antigen in ELISA, and anti-eel FSH mAbs were purified from culture supernatants by performing affinity chromatography. Three of the 6mAbs were characterized and their isotypes were identified as IgG2b (eFA-C5 and eFA-C11) and IgG1 (eFB-C14). In western blotting assays, the mAbs recognized the antigen as a 24.3-kDa band, and further detected bands of 34 and 32kDa in the supernatants of CHO cells transfected with cDNA encoding tethered eel FSH/ and LH/, respectively. PNase F-mediated deglycosylation of the recombinant proteins resulted in a drastic reduction in their molecular weight, to 7-9kDa. The mAbs eFA-C5 and eFA-C11 recognized the eel FSH-subunit that is commonly encoded among glycoprotein hormones, whereas eFB-C14 recognized the eel FSH-subunit, and immunohistochemical analysis revealed that the staining by these mAbs was specifically localized in the eel pituitary. We also established an ELISA system for detecting rec-tethered FSH/ and LH/ produced from CHO cell lines. Measurement of biological activities in vitro revealed that only weak activity of rec-FSH/ was detected. The activity of rec-LH/ was found to be increased in a dose-dependent manner for eel oocyte maturation.
PubMed | National Institute of Fisheries Science NIFS, Chung - Ang University and Green Cross
Type: | Journal: Journal of biotechnology | Year: 2016
The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5 and 3-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to 30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring 30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.
PubMed | Gyeongsang National University, University of Miyazaki, National Institute of Fisheries Science NIFS, Copenhagen University and Waseda University
Type: | Journal: Fish & shellfish immunology | Year: 2016
Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced . Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the chains (Ig-a) in olive flounder. Interestingly, Ig-a
PubMed | National Institute of Fisheries Science NIFS and Sungkyunkwan University
Type: Journal Article | Journal: International journal of systematic and evolutionary microbiology | Year: 2016
A Gram-stain-negative, aerobic, non-motile and coccoid, ovoid or rod-shaped bacterial strain, designated RA4-7T, was isolated from the gut of an abalone (Haliotis discus hannai) collected from the sea around Jeju island, South Korea, and subjected to a polyphasic taxonomic study. RA4-7T grew optimally at 25C, at pH 7.0-7.5 and in the presence of 2.0-3.0% (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that RA4-7T represented a member of the genus Polaribacter. RA4-7T exhibited 16S rRNA gene sequence similarity values of 97.64 and 97.23% to Polaribacter atrinae WP25T and Polaribacter dokdonensis DSW-5T, respectively, and of 93.83-96.99% to the type strains of the other species of the genus Polaribacter. RA4-7T contained MK-6 as the predominant menaquinone and summed feature 3 (C16:17c and/or C16:16c), iso-C15:0 3-OH and iso-C15:0as the major fatty acids. The major polar lipids detected in RA4-7T were phosphatidylethanolamine and one unidentified lipid. The DNA G+C content of RA4-7T was 30.5 mol% and its DNA-DNA relatedness values with the type strains of P. atrinae and P. dokdonensis were 16 and 11%, respectively. Differential phenotypic properties, in combination with its phylogenetic and genetic distinctiveness, revealed that RA4-7T is separated from species of the genus Polaribacter with validly published names. On the basis of the data presented, RA4-7T represented a novel species of the genus Polaribacter, for which the name Polaribacterhaliotis sp. nov. is proposed. The type strain is RA4-7T (=KCTC 52418T=NBRC 112383T).
PubMed | Pukyong National University, National Institute of Fisheries Science NIFS and Dong Chang Co.
Type: | Journal: Fish & shellfish immunology | Year: 2016
An 8-week feeding trial was conducted to evaluate the effects of dietary probiotics on growth performance and non-specific immune responses in starry flounder, Platichthys stellatus. Fish averaging 46.50.65g (meanSD) were fed one of the six experimental diets; one control (Cont), and five other diets were prepared by supplementing single-probiotics 1 (Bacillus subtilis; SP
PubMed | National Institute of Fisheries Science NIFS and Hanyang University
Type: | Journal: Chemosphere | Year: 2016
Legacy and new persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs) were measured in sediments near a wastewater treatment plant (WWTP) outfall in a semi-enclosed bay, to investigate the current contamination and temporal changes in these contaminants associated with regulation activities in Korea. The concentrations of most of the POPs showed clear decreasing trends with an increase in the distance from the WWTP outfall, indicating that the WWTP discharges greatly contributed to the sediment contamination by POPs. Highly significant correlations were found for most of the POPs, indicating a common source for sediment contamination. Significant declines were found in the concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), dioxin-like polychlorinated biphenyls (DL-PCBs), polybrominated diphenyl ethers (PBDEs), and PAHs in the sediments collected between 2005 and 2013. This result suggested that legislative actions (regulation of the PCDD/Fs in flue gas, total pollution load management, and whole effluent toxicity for WWTP discharges) and change of fuels, were likely to be effective at reducing the POP and PAH levels in sediments during the past several years. The different compositional profiles of the PCDD/Fs and PAHs between 2005 and 2013 implied changes in and/or additional sources of these contaminants. Despite a decline in the PCDD/Fs over time, the present levels of PCDD/Fs in the sediment exceeded some of the sediment quality guidelines suggested by the National Oceanic and Atmospheric Administration.
Choi M.,National Institute of Fisheries Science NIFS |
Lee I.-S.,National Institute of Fisheries Science NIFS |
Jung R.-H.,National Institute of Fisheries Science NIFS
Food Chemistry | Year: 2016
A rapid automated extraction and cleanup method using selective pressurized liquid extraction was developed and validated for 14 organochlorine pesticides in fish. The lipid-removal efficiencies achieved by adding alumina, Florisil, acid-treated silica gel, and silica gel to the extraction cell were determined and optimized. In the optimized method, fish (2-3 g) was placed above alumina (30 g) in the extraction cell, then the sample was extracted using a 7:3 mixture of hexane and dichloromethane. The method was validated using certified reference materials (NIST SRM 1946 and 1974c), spiked fish, and four lipid-rich fish samples. The mean low- and high-concentration spike recoveries were 91% and 93% with RSD < 20%, respectively. Measured concentrations of target OCPs showed good agreement with the certified concentrations in certified reference materials. It suggests the good accuracy and precision of the SPLE method. The proposed method met the most important requirements of an extraction and cleanup procedure, including having a short preparation (cleanup and concentration) time and minimal sample contamination and being able to be automated. © 2016 Elsevier Ltd. All rights reserved.
PubMed | National Institute of Fisheries Science NIFS and Pusan National University
Type: | Journal: The Science of the total environment | Year: 2016
The occurrence trends and effects of 30 human and veterinary pharmaceuticals, including antibiotics, anthelmintics, anti-inflammatory drugs, and -blockers, in the marine environment, with a focus on seawater, sediment, cultured fish, and their feed collected from coastal and fish farm areas in the southern sea of Korea, were investigated. The concentrations of total pharmaceuticals in coastal area seawater (mean: 533ng/L) were higher than those in fish farm seawater (mean: 300ng/L), while the opposite trend (coastal area: 136ng/gdrywt
PubMed | National Institute of Fisheries Science NIFS
Type: | Journal: Veterinary microbiology | Year: 2016
A strain of viral haemorrhagic septicaemia virus (VHSV) was isolated from cultured olive flounder (Paralichthys olivaceus) during epizootics in South Korean. This strain showed high mortality to olive flounder in in vivo challenge experiment. The complete genomic RNA sequences were determined and phylogenetic analysis of the amino acid sequences of glycoprotein revealed that this isolate was grouped into genotype IVa of genus Novirhabdovirus. Expression profile of genes in olive flounder was analyzed at day 1 and day3 after infection with this VHSV isolate by using cDNA microarray containing olive flounder 13K cDNA clones. Microarray analysis revealed 785 up-regulated genes and 641 down-regulated genes by at least two-fold in virus-infected fish compared to healthy control groups. Among 785 up-regulated genes, we identified seven immune response-associated genes, including the interferon (IFN)-induced 56-kDa protein (IFI56), suppressor of cytokine signaling 1 (SOCS1), interleukin 8 (IL-8), cluster of differentiation 83 (CD83), -globin (HBA), VHSV-induced protein-6 (VHSV6), and cluster of differentiation antigen 9 (CD9). Our results confirm previous reports that even virulent strain of VHSV induces expression of genes involved in protective immunity against VHSV.