Molecular cloning and characterization of PLCB1 (phospholipase C, beta 1) gene from the olive flounder, Paralichthys olivaceus

PLCB1 (phospholipase C, beta 1) cDNA was cloned from olive flounder (Paralichthys olivaceus) cDNA via rapid amplification of cDNA ends (RACE). The cDNA for olive flounder PLCB1 (PoPLCB1) encodes for a polypeptide of 1,244 amino acids in length containing a well-conserved PH domain, catalytic X and Y domains, a C2 domain. From the sequence information of the BAC library, we assembled a contig containing the whole flounder PLCB1 cDNA sequences, and determined the exon/intron structure of the gene spanning > 110,743 bp DNA. PoPLCB1 gDNA sequences demonstrated the new sequence (exon 15), which has only been observed in the fish, is located between the X and Y domain of the PLCB1, and that PoPLCB1 exists as two splice variants-PoPLCB1a (1,244 amino acids) and PoPLCB1b (1,210 amino acids). Phylogenic analysis and sequence comparison of PoPLCB1 with other PLC isozymes showed a close relationship with the PLCB1 isozyme. Tissue-specific mRNA of PoPLCB1 was expressed predominantly in the brain and heart tissues. Between the two splicing variants of PoPLB1 in RT-PCR by tissue, PoPLCB1a showed a major expression pattern in more diverse types of tissues than the PoPLCB1b. PoPLCB1 gene expression was compared with that of the inflammatory cytokines IL-1β and TNF-α in infected spleen and kidney tissues via real-time RT-PCR assays following stimulation with LPS. After the stimulation, the expression of PoPLCB1 increased significantly prior to IL-1B and TNF-α expression. This provided direct evidence suggesting that PoPLCB1 may perform a crucial role in immune responses against pathogens and in inflammation.     

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus)

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.     

Effect of a probiotic strain, Enterococcus faecium, on the immune responses of olive flounder (Paralichthys olivaceus)

The present study was aimed to investigate the effect of a probiotic, Enterococcus faecium, on the immune responses against infection with the marine fish pathogen Lactococcus garvieae in olive flounder (Paralichthys olivaceus). The immune responses were assessed by lysozyme activity, complement activity, protease activity, and expression of proinflammatory cytokines by RT-PCR. The lysozyme and complement activities were increased between 9 to 15 and 9 to 13 days, respectively, and antiprotease activity was slightly elevated after 5 days of probiotic treatment. The TNF-alpha and IL-1beta expressions were observed from kidney and spleen. The results of this study reveal that E. faecium induces immune-responsible materials and protects olive flounder from lactococcosis.     

Molecular cloning and characterization of cathepsin F gene from olive flounder Paralichthys Olivaceus

We isolated a homologue of cathepsin F from cDNA library of olive flounder liver. A 2,077 kb full-length cDNA encoding a predicted polypeptide of 474 amino acids was sequenced. The flounder cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 17 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 244 amino acids and the domain of the mature protease comprising 213 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the ‘catalytic triad’. The cathepsin F protein showed 49–99% amino acid sequence identity with other known cathepsin F sequences. An in vivo expression study showed that cathepsin F mRNA was expressed predominantly in brain, liver, eye and heart, and moderately in other tissues. The accumulation of cathepsin F mRNA in early stage of development increased with development. This expression pattern suggests that flounder cathepsin F has been implicated in the growth and reproduction regulation.     

An immune responsive complement factor D/adipsin and kallikrein-like serine protease (PoDAK) from the olive flounder Paralichthys olivaceus

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6–36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro.     

Immunization of olive flounder (Paralichthys olivaceus) with an auxotrophic Edwardsiella tarda mutant harboring the VHSV DNA vaccine

The aims of the present study were to find more powerful promoter for DNA vaccines in olive flounder (Paralichthys olivaceus) and to evaluate the availability of the auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine against VHSV in olive flounder. The marine medaka (Oryzias dancena) β-actin promoter was clearly stronger than cytomegalovirus (CMV) promoter when the vectors were transfected to Epithelioma papulosum cyprini (EPC) cells or injected into the muscle of olive flounder, suggesting that marine medaka β-actin promoter would be more appropriate promoter for DNA vaccines in olive flounder than CMV promoter. Olive flounder immunized with the Δalr Δasd E. tarda harboring viral hemorrhagic septicemia virus (VHSV) DNA vaccine vector driven by the marine medaka β-actin promoter showed significantly higher serum neutralization titer and higher survival rates against challenge with VHSV than fish immunized with the bacteria carrying VHSV DNA vaccine vector driven by CMV promoter. These results indicate that auxotrophic E.?tarda mutant harboring marine medaka β-actin promoter-driven DNA vaccine vectors would be a potential system for prophylactics of infectious diseases in olive flounder.     

Purification and characterization of an antimicrobial histone H1-like protein and its gene from the testes of olive flounder, Paralichthys olivaceus

An approximately 21?kDa antimicrobial protein was purified from an acidified testis extract of olive flounder, Paralichthys olivaceus, by ion-exchange and C(18) reversed-phase HPLC. A comparison of the N-terminal amino acid sequence with those of other known antimicrobial polypeptides revealed high homology between this antimicrobial protein and other histone H1 molecules; thus, it was designated flounder histone H1-like protein (fH1LP). fH1LP showed potent antimicrobial activity against Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 2.8-30.0?μg/ml), Gram-negative bacteria, including Aeromonas hydrophila, Escherichia coli D31, Vibrio parahaemolyticus (MECs, 1.4-12.0?μg/ml), and Candida albicans (MEC, 2.0?μg/ml). cDNA cloning and tissue distribution studies of fH1LP indicated that it is constitutively expressed in testis and ovary. The fH1LP expression level was significantly dependent on developmental stage, and decreased dramatically after hatching. However, lipopolysaccharide stimulation did not induce fH1LP mRNA in other immune organs, including the kidney and spleen. These results suggest that fH1LP plays an important role in innate immunity in fish during reproduction, including mating, fertilization, and hatching.     

Molecular Cloning of Growth Hormone Complementary DNA in Barfin Flounder (Verasper moseri)

The olive flounder (family Paralichthidae; Paralichthys olivaceus) growth hormone (ofGH) appears to be the most derived among known growth hormones, with the deletion of 14 consecutive amino acids in the carboxy-terminal region. To ascertain if this deletion is common to all flounders, growth hormone complementary DNA of the barfin flounder (bfGH) (family Pleuronectidae; Verasper moseri) has been cloned. It was amplified by polymerase chain reaction using single-strand cDNA from the pituitary gland. Excluding the poly(A) tail, the bfGH cDNA is 919 nucleotides long and contains a 609-bp open reading frame encoding a putative signal peptide of 17 amino acids and a mature protein of 186 amino acids. Northern blot analysis detected 1.0 kb of bfGH messenger RNA in the pituitary gland, which is a reasonable value considering the poly(A) tail. The deduced amino acid sequence of bfGH has 78% identity with the sequence of ofGH. A major difference is the presence of a 14 amino acid segment (140–153) in bfGH, as in other growth hormones, suggesting that this deletion in the olive flounder occurred after the divergence of the Pleuronectoidae. Received May 7, 1999; accepted July 13, 1999.     

Identification of two differentially expressed forms of progranulin mRNA in the teleost fish Paralichthys olivaceus

Two forms of progranulin mRNA were isolated from kidney and spleen cDNA libraries of the olive flounder, Paralichthys olivaceus. The 3'-untranslated regions (3'-UTRs) of these flounder progranulin (f-pgrn) mRNAs differed in a 20-nucleotide sequence element (5'-AACTGATTACGTTCAACAAC-3') that was present in one mRNA (designated f-pgrn type II) and not in the other (designated f-pgrn type I). Both mRNA sequences contained an open reading frame encoding a 289-amino-acid polypeptide of approximately 33 kDa. Southern blot analysis of the P. olivaceus flounder genome using an f-pgrn cDNA probe and a PCR-based approach identified a single copy of f-pgrn corresponding to the type II mRNA. The expression profiles of the two types of f-pgrn mRNA differed from each other and were tissue- and condition-dependent. The type II mRNA was detected abundantly in studied tissues (gill, kidney, spleen, and intestine) of non-stimulated healthy flounders. The type I mRNA was rarely expressed in any tissues of healthy flounders, but it was continuously increased in the examined tissues of flounders after the intraperitoneal injection of lipopolysaccharide. On the other hand, the expression of type II mRNA was decreased in inverse proportion to the type I mRNA in the LSP-stimulated flounders. These results suggest that type I and type II f-pgrn mRNA are translated into different proteins with different activities in the immune system of flounder.     

Isolation, modulatory functions on murine B cell development and antigen-specific immune responses of BP11, a novel peptide from the chicken bursa of Fabricius

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds which plays important roles in B lymphocyte differentiation. Here, a new bursal peptide (BP11) with the amino acid sequence DVAGKLPDNRT was identified and characterized from BF. It was proved that BP11 promoted CFU pre-B formation, and regulated B cell differentiation, including increase the percentage of immature and mature B cells in BM cells co-cultured with IL-7. BP11 also exerted immunomodulatory function on antigen-specific immune responses in BALB/c mice immunized with inactivated influence virus (AIV, H9N2 subtype) vaccine, including enhancing AIV-specific antibody and cytokine production. Furthermore, it was noteworthy that BP11 stimulated antibody productions and potentiates the Th1 and Th2-type immune responses in dose-dependent manner in chicken. These results suggested that BP11 might be highly relevant for the development of avian immune system.