Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a Sciaenops ocellatus ISG15 homologue that is involved in host immune defense against bacterial infection

ISG15 is an interferon-stimulated gene that encodes a ubiquitin-like protein. ISG15 homologues have been identified in a number of fish species, some of which are known to be regulated at expression level by virus infection and lipopolysacchande (LPS) treatment However, the relationship between ISG15 and live bacterial infection has not been investigated in piscine models. In this study, an ISG15 homologue, SoISG15, was identified from red drum Scraeriops ocellaws and analyzed at expression and functional levels The open reading frame ofSolSG15 is 477 base pairs (bp) and mtronless, with a 5'-untranslated region (UTR) of 91 bp and a 3'-UTR of 415 bp The deduced amino acid sequence of S0ISG15 shares 60-67% overall identities with the ISG15 of several fish species. S0ISG15 possesses two conserved ubiquinn-like domains and the canonical ubiquitin conjugation motif, LRGG, at the C-terminus. Expressional analysis showed that constitutive expression of SolSG15 was highest in blood and lowest in kidney Experimental challenges with LPS and bacterial pathogens induced significant S0ISG15 expression in the kidney but not in the liver Similar differential induction was also observed at cellular level with primary hepatocytes and head kidney (HK) lymphocytes. Poly(' C), however, effected drastic induction of S0ISG15 expression in kidney and liver at both tissue and cellular levels. Immunoblot analysis showed that S0ISG15 was secreted by cultured HK lymphocytes into the extracellular milieu. Recombinant S0ISG15 expressed in and purified from Eschenclua colt was able to enhance the respiratory burst activity, acid phosphatase activity, and bactericidal activity of HK macrophages. Taken together, the results of this study indicated that SoISG 15 possesses apparent immunological property and is likely to be involved in host immune defense against bacterial infection. (C)2010 Elsevier Ltd All rights reserved.

Construction and analysis of an experimental Streptococcus iniae DNA vaccine

Streptococcus iniae is a severe aquaculture pathogen that can also infect humans and animal. A putative secretory antigen, Slat 0, was identified from a pathogenic S. iniae strain by in vivo-induced antigen technology. Using turbot as an animal model, the immunoprotective effect of Sia10 was examined as a DNA vaccine in the form of plasmid pSia10, which expresses sia10 under the cytomegalovirus immediate-early promoter. In fish vaccinated with pSia10, transcription of sia10 was detected in muscle, liver, spleen, and kidney at 7, 14, 21, 28, 35, 42, and 49 days post-vaccination. In addition, production of Sia10 protein was also detected in the muscle tissues of pSia10-vaccinated fish. Fish vaccinated with pSia10 exhibited a relative percent survival (RPS) of 73.9% and 92.3%, respectively, when challenged with high and low doses (producing a cumulative mortality of 92% and 52%, respectively, in the control groups) of S. iniae. Immunological and transcriptional analyses showed that vaccination with pSia10(i) induced much stronger chemiluminescence response and significantly higher levels of nitric oxide production and acid phosphatase activity in head kidney macrophages; (ii) caused the production of specific serum antibodies, which afforded apparent immunoprotection when transferred passively into naive fish; and (iii) upregulated the expression of the genes encoding proteins that are possibly involved in both innate and adaptive immune responses. Taken together, these results indicated that pSia10 is an effective vaccine candidate and may be used in the control of S. iniae infection in aquaculture. (C) 2010 Elsevier Ltd. All rights reserved.

Characterization of muscle-regulatory gene, MyoD, from flounder (Paralichthys olivaceus) and analysis of its expression patterns during embryogenesis

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

Identification and analysis of a Scophthalmus maximus ferritin that is regulated at transcription level by oxidative stress and bacterial infection

Ferritins are conserved Iron storage proteins that exist in most living organisms and play an essential role in Iron homeostasis. In this study, we reported the identification and analysis a ferritin M subunit, SmFerM, from turbot Scophthalmus maximus. The full length cDNA of SmFerM contains a 5'-untranslated region (UTR) of 232 bp, an open reading frame (ORF) of 531 bp, and a 3'-UTR of 196 bp The ORF encodes a putative protein of 176 amino acids, which shares extensive sequence identities with the M terrains of several fish species. In silico analysis identified in SmFerM both the ferroxidase center of mammalian H ferritins and the iron nucleation site of mammalian L ferritins. Quantitative real time reverse transcriptase-PCR analysis indicated that SmFerM expression was highest in muscle and lowest in heart and responded positively to experimental challenges with bacterial pathogens and poly(I center dot C) Exposure of cultured turbot hepatocytes to treatment of stress inducers (iron, copper, and H2O2) significantly upregulated the expression of SmFerM in a dose dependent manner. Iron chelating analysis showed that recombinant SmFerM purified from Escherichia coli exhibited apparent iron binding activity. These results suggest that SmFerM is a functional M ferritin and is likely to play a role in iron sequestration and protection against oxidative stress and microbial infection (C) 2010 Elsevier Inc All rights reserved

Expression, purification of recombinant flounder MRF4 protein in Escherichia coli and analysis of its polyclonal antibodies

MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (130). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.

Fractionation and analysis of Artemisia capillaris Thunb. by affinity chromatography with human serum albumin as stationary phase

A method for the screening and analysis of biologically active compounds in traditional Chinese medicine is proposed. Affinity chromatography using a human serum albumin (HSA) stationary phase was applied to separate and analyze the bioactive compounds from Artemisia capillaris Thunb. Five major peaks and several minor peaks were resolved based on their affinity to HSA, two of them were identified as scoparone (SCO, 6,7-dimethoxycoumarin) and capillarisin (CAP). CAP shows a much higher affinity to HSA than SCO. The effects of acetonitrile concentration, eluent pH, phosphate concentration and temperature on the retention behaviors of several major active components were also investigated, and it was found that hydrophobicity and eluent pH play major roles in changing retention values. The results demonstrate that the affinity chromatography with a HSA stationary phase is an effective way for analyzing and screening biologically active compounds in traditional Chinese medicine. (C) 2000 Elsevier Science B.V. All rights reserved.

Screening and analysis of biologically active compounds in Angelica sinensis by molecular biochromatography

A novel strategy for the screening and analysis of biologically active compounds in traditional Chinese medicine by molecular biochromatography is proposed. Molecular biochromatography with human serum albumin (HSA) immobilized on silica as stationary phase was used to screen and analyse the bioactive compounds in the typical Chinese medicine of Angelica sinensis (Oliv.) Diels. Ten peaks showed retention on this column, which is based on their affinity for HSA. Ferulic acid and liguistilide were identified as the principal active components, which agrees very well with the results in the literature. A quality control method was also developed based on the simultaneous determination the concentrations of ferulic acid and liguistilide in solutions of Angelica sinensis (Oliv.) Diels extracted with water and methanol. It was observed that the concentrations of ferulic acid and liguistilide in solution extracted with methanol were 2 and 53 times higher, respectively, than those with water. It was shown that molecular biochromatography is an effective way of analysing and screening biologically active compounds in traditional Chinese medicine.

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC

Biofingerprinting chromatogram, analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein. cell. etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA. respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated. © 2005 Elsevier B.V. All rights reserved.