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At least 19 recordsLinked to original sources
PubMed2000

Predator-prey relations between age-1+ summer flounder (Paralichthys dentatus, Linnaeus) and age-0 winter flounder (Pseudopleuronectes americanus, Walbaum): predator diets, prey selection, and effects of sediments and macrophytes.

Laboratory experiments and weekly trammel net surveys in the Navesink River, New Jersey (USA) were used to examine the predator-prey interaction between age-1+ summer flounder (Paralichthys dentatus) and age-0 winter flounder (Pseudopleuronectes americanus). Winter flounder (24-67 mm TL) were the dominant piscine prey of summer flounder (n=95, 252-648 mm TL) collected in trammel nets. We observed a temporal shift in summer flounder diets from sand shrimp (Crangon septemspinosa) and winter flounder, dominant during June and early July, to blue crabs (Callinectes sapidus) and other fishes (primarily Atlantic silversides, Menidia menidia and Atlantic menhaden, Brevortia tyrannus) later in the summer. Variations in prey selection appeared to be related to changes in the spatial distribution of predators and spatio-temporal variation in prey availability. In laboratory experiments, summer flounder (271-345 mm total length, TL) preferred demersal winter flounder to a pelagic fish (Atlantic silversides) and a benthic invertebrate (sand shrimp) prey, and the vulnerability of winter flounder increased with increasing prey body size from 20 to 90 mm TL. Experiments testing habitat effects showed that mortality of winter flounder in three different size classes (20-29, 40-49, 60-69 mm TL) was not influenced by sediment grain sizes permitting differential burial of the prey. However, vegetation enhanced survival, with fish suffering lower mortality in eelgrass (Zostera marina, 15+/-0.04%) than in sea lettuce (Ulva lactuca, 38+/-0.04%) or bare sand (70+/-0.07%) when the macrophytes were planted to produce similar leaf surface areas (5000 cm(2) m(-2)). Prey vulnerability appeared to be related to the role of vision in the predator's attack strategy and prey activity levels.

Journal Article↗
PubMed2006

Detection of DNAs homologous to betanodavirus genome RNAs in barfin flounder Verasper moseri and Japanese flounder Paralichthys olivaceus.

I investigated the presence of DNA homologous to genome RNA1 and RNA2 (RNA1 DNA and RNA2 DNA) of betanodaviruses - the causative agent of viral nervous necrosis (VNN) --in eggs, sperm, ovarian cavity fluid, larvae, and juveniles of barfin flounder Verasper moseri and larvae and juveniles of Japanese flounder Paralichthys olivaceus collected at 6 sites in Hokkaido, Japan, from 1994 to 2001. RNA1 DNA and RNA2 DNA were detected by PCR in 13 and 33 % of barfin flounder samples and 0 and 69% of Japanese flounder samples, respectively. No infectious virus was detected by cell culture or by successive immunoblot against coat protein (genome RNA2 product) using an E-11 cell line, except for a virus present in 1 dead fish collected during an outbreak of VNN in 1995. Nucleotide sequence analysis showed that RNA1 DNA had a 82 to 96 % similarity to betanodavirus genome RNA1, and that RNA2 DNA had a 69 to 98 % similarity to RNA2. The detection rate of RNA2 DNA after intraperitoneal injection of betanodavirus strain HCF-1 into larvae and juveniles of the 2 flounder species was higher in samples from surviving fish than in the uninfected controls, whereas the detection rate of RNA1 DNA did not show a clear trend. Infectious virus was only detected in samples from fish that died subsequent to injection. Transfection assays of the viral genome RNAs into the barfin flounder cell line MK-1 and Japanese flounder cell lines H-1 and H-2 resulted in production of RNA2 DNA in all 3 cell lines. Quantitative measurement by ELISA revealed reverse transcriptase (RTase) activity. These results suggest that the DNA forms are produced and persist in the 2 flounder species as both clinical and subclinical infections, and do not lead to virion production.

Amino Acid Sequence↗
PubMed1986

Immunoglobulin levels of the winter flounder (Pseudopleuronectes americanus) and the summer flounder (Paralichthys dentatus) injected with the microsporidan parasite Glugea stephani.

The microsporidan parasite G. stephani, has been found in the intestine of the American winter flounder Pseudopleuronectes americanus. A quantitative study of total serum immunoglobulin (Ig) levels has shown that an injection of this parasitic spore causes a decrease in serum IgM levels. The level of suppression seems to be related to the number of spores injected into the host, as well as the frequency of injections administered. As the dosage of spores injected into the winter flounder increased from 3 X 10(3)/ml to 3 X 10(8)/ml, the level of serum IgM's decreased proportionally. When a single IM injection of spores (3 X 10(6)/ml) was administered, the initial decrease in serum IgM was followed by a gradual recovery of total Ig levels by day 60. Repeated injections of G. stephani on days 21 and 42 respectively, causes an even greater decrease in total IgM levels. Indomethacin (a drug that inhibits prostaglandin activity), has been shown to negate the decrease in IgM levels caused by the G. stephani spores. When injected twice weekly, the indomethacin inhibited the decrease in serum IgM caused by the spore homogenate, with IgM levels comparable to those observed in control WF. This supports previous observations, that prostaglandins are implicated in the decrease in IgM levels induced by injection of the parasite. The effects of G. stephani on serum Ig levels does not seem to be species-specific. When the spores were injected into summer flounder (Paralichthys dentatus) together with an erythrocyte antigen, horse red blood cells (HRBC), a decrease in total IgM levels occurred, as well as a decrease in hemagglutination titers to HRBC similar to the decrease described in winter flounder.

Animals↗
PubMed1998

Recombinant flounder growth hormone from Escherichia coli: overexpression, efficient recovery, and growth-promoting effect on juvenile flounder by oral administration.

An efficient production method for recombinant flounder growth hormone (r-fGH) from Escherichia coli was developed and the biological activity of purified r-fGH was examined using juvenile flounder. The use of bicistronic construction in the expression plasmid resulted in the production of over 40% of the E. coli cellular protein as r-fGH. The r-fGH was recovered from cell lysates following inclusion body washing, solubilization and refolding in sodium dodecylsulfate (SDS) solution, and removal of contaminated proteins with secondary butanol treatment. The SDS content in purified r-fGH solution was adjusted to appropriate levels by diafiltration. More than 47% of the r-fGH was recovered from the E. coli cell lysates and the purity of recovered r-fGH was 98%. The oral administration of purified r-fGH to juvenile flounder, once a week for 4 weeks at a dosage of 40 micrograms r-fGH g-1 fish body weight, resulted in significant increases both in weight and length. These results of overexpression, simple purification with high recovery yield and purity, and good growth-promoting activity of the r-fGH suggest that the production scheme described in this study is useful for the potential application of r-fGH in fish farming.

Administration, Oral↗
PubMed2000

Selection of brood stock candidates of barfin flounder using an ELISA system with recombinant protein of barfin flounder nervous necrosis virus.

Barfin flounder nervous necrosis virus (BFNNV), the causative agent of viral nervous necrosis (VNN) of barfin flounder, is vertically transmitted from spawners to larvae. In the present study, an ELISA with a recombinant protein of BFNNV was performed for the detection of antibodies against BFNNV and applied for the selection of brood fish in order to prevent viral vertical transmissions. Brood stocks were divided into 4 groups based on ELISA antibody titers (< or = 10, 20, 40 and >40), and the BFNNV status of the brood stocks was determined by PCR. BFNNV was detected from the brood fish in the group with an antibody titer of >40 but not from those with titers < or = 10, 20 and 40. The offspring obtained from PCR-negative brood fish pairs in each group of ELISA antibody titers were subsequently reared for observation of VNN occurrence. VNN occurred in juveniles from 2 of 9 pairs of spawners with an antibody titer > or = 40, but did not occur in spawners with an antibody titer of < or = 10. Therefore, it was concluded that selection of brood fish using both the PCR test and ELISA antibody titers could help prevent vertical transmission of BFNNV in larval production of barfin flounder.

Animals↗
PubMed1998

Low inducibility of CYP1A activity by polychlorinated biphenyls (PCBs) in flounder (Platichthys flesus): characterization of the Ah receptor and the role of CYP1A inhibition.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.

Amino Acid Sequence↗
PubMed2002

Development of a time-resolved fluoroimmunoassay for insulins and its application to monitoring of insulin secretion induced by feeding in the barfin flounder, Verasper moseri.

A time-resolved fluoroimmunoassay (TR-FIA) system was developed to quantify insulin levels in the barfin flounder. This TR-FIA system is a solid-phase assay based on competition of unlabeled insulins and biotinylated barfin flounder insulin-II against an anti-barfin flounder insulin-II antibody. The minimum detectable level of barfin flounder insulin-I and -II in this TR-FIA was 10 pg/well which corresponded to 1.0 ng/ml, and insulin-II showed slightly higher crossreactivity than insulin-I. The accuracy of this TR-FIA was assured by specificity test, validation test, and recovery test using plasma added insulin-II. The results indicated the high specificity and sufficient accuracy of this assay system for insulin level measurement. This system was applied to the measurement of plasma insulin levels of fed and fasted barfin flounders. Plasma insulin levels (average +/- SEM) in fed flounders reached a maximum 2 h (9.3 +/- 1.7 ng/ml) and decreased gradually thereafter, while those in fasted flounders remained at low levels (1.1 +/- 0.1-2.0 +/- 0.2 ng/ml) during the experiment. After removing proteins by acidification and subsequent gel filtration, plasma samples taken from fed and fasted flounders at 2 h after feeding were fractionated separately by reversed-phase HPLC. In fed flounders, insulin immunoreactivity was detected in fractions corresponding to those of insulin-I or -II. The ratio of integrated insulin immunoreactivities of each peak was 0.378 +/- 0.044 (average +/- SD). This value was in good agreement with those (0.355 +/- 0.019) of absorbance areas of each insulin from Brockmann body extracts of the barfin flounder on reversed-phase HPLC. In fasted flounders, very weak insulin immunoreactivities were observed at retention times corresponding to those of insulin-I and -II. These results indicated that both insulin-I and -II were secreted into the blood being induced by feeding stimulation with approximately the same ratio as that of the quantities harbored in the Brockmann body.

Animals↗
PubMed1998

Isolation of cDNAs for trypsinogen from the winter flounder, Pleuronectes americanus.

Knowledge of the timing of digestive enzyme expression in developing larvae is essential for evaluating the appropriateness of formulated larval diets. Since little is known at the molecular biological level about the ontogeny of digestive enzyme function in flatfish, we have isolated cDNA clones for key digestive enzymes such as trypsinogen. Portions of trypsinogen genes have been amplified from winter flounder cDNA libraries by PCR using primers based on sequence motifs conserved among trypsinogen genes from other organisms. The PCR products were sequenced, cloned, and used as radioactively labeled probes to screen the libraries. Three distinct trypsinogen cDNAs have been isolated, representing the first cDNAs for winter flounder digestive enzymes. The first type (Flounder 1) is very similar to a trypsinogen sequence reported from a related flounder, Pleuronectes platessa. The second type (Flounder 2) is related to sequences reported from Atlantic salmon, cod, and the Antarctic fish, Paranotothenia magellanica. The third type (Flounder 3) shows limited similarity to the Antarctic fish trypsinogen sequence. All three cDNAs encode a cleavable signal sequence at the amino-terminal end, and Flounder 1 and 2 contain the characteristic acidic sequence for activation of the trypsinogen proenzyme to trypsin. Southern hybridization experiments indicate that Flounder 1 and 3 are single-copy genes, whereas Flounder 2 may be present in more than one copy. In addition, Flounder 2 detects homologs in a wide variety of other fish species. The sequence information will be used to establish RT-PCR assays for larval winter flounder (Pleuronectes americanus) digestive enzyme expression.

Journal Article↗
PubMed2004

Organochlorine contaminants in flounder (Platichthys flesus) and mullet (Mugil cephalus) from Douro estuary, and their use as sentinel species for environmental monitoring.

In order to monitor the presence of organic pollutants in Douro estuary (NW Portugal), two sentinel fish species, grey mullet (Mugil cephalus) and flounder (Platichthys flesus), were periodically sampled from May 2001 to April 2002. At all sampling periods, several specimens of both species were allowed to depurate in clean sea water. Levels of PCBs and DDTs were quantified in liver and muscle of both species. The accumulation of PCBs was higher in muscle of mullet, with a maximum of 345 ng/g dw, than in flounder, with a maximum 52ng/g dw. In the liver, flounder showed the highest levels (811 ng/g dw). Of the 18 congeners analysed, CBs 180 (hepta), 153 and 138 (hexachlorobiphenyls) were predominant in the tissue of both species. The maximum concentration of tDDT was measured in flounder liver (301ng/g dw). In contrast, a 10-fold higher tDDT was recorded in mullet muscle (63-69 ng/g dw and 8-16 ng/g dw for mullet and flounder, respectively). The hepatic ethoxyresorufin O-deethylase (EROD) activity was periodically determined. Consistently, a 10-fold higher enzymatic activity was present in mullet in comparison with flounder (1536 pmol/min/mg protein in mullet and 156 pmol/min/mg protein in flounder). In mullet, no correlation could be found between EROD activity and gonado-somatic index (GSI) or hepato-somatic index (HSI). On the contrary, during the reproductive season, female flounder showed a negative correlation between EROD activity and GSI. Despite being in clean sea water for 1 month period, no significant decrease in the tissue content of PCBs and tDDT was found. However, mullet's EROD activity followed a clear pattern, with a decrease enzymatic activity after being in captivity. Female flounder displayed a similar trend during the resting season. Yet, during the reproductive season, an increased EROD activity was recorded after being in captivity for 1 month, which may be associated with a modulation effect of steroids on CYP 1A1. Histological analyses of gonad revealed that 21% of male mullet displayed testis-ova, while no male flounder was found to show gonadal abnormalities. Overall, the study reports the accumulation of significant levels of PCBs and DDTs in the tissues of the studied species. It also provides important evidences supporting the use of grey mullet as a sentinel species for monitoring the presence of organic contaminants and xeno-estrogenic pollution in southern European estuaries.

Analysis of Variance↗
PubMed2001

Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein.

In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene. The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns. A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme. Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes. In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene. A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene. Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda. Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25 degrees C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria. This is the first report of a g-type lysozyme gene other than for reported avian species.

Animals↗
PubMed1991

Erythrocyte micronuclei in winter flounder (Pseudopleuronectes americanus): results of field surveys during 1980-1988 from Virginia to Nova Scotia and in Long Island Sound.

Erythrocyte micronuclei (MN) frequencies of 280 winter flounder (Pseudopleuronectes americanus) from Chesapeake Bay to the southern Scotian Shelf including Long Island Sound were measured. The MN data were combined with data previously reported for 224 flounder and the combined data sets were analyzed. Incidences of MN were elevated sixfold in flounder from the New York Bight Apex as compared to frequencies for fish from the inshore Gulf of Maine and Block Island Sound, and twice those found in Georges Bank and Long Island Sound flounder. Inshore New Jersey fish had higher MN frequencies than those from inshore Gulf Of Maine and Block Island Sound. The occurrence of MN in flounder from inshore Virginia was higher than in flounder from inshore Gulf of Maine and Block Island Sound. The large subset of stations from Long Island Sound indicated higher frequencies of MN in flounder from Hempstead and Shoreham as compared to most other sites in the Sound. There was no significant difference in the frequency of MN between males and females, but males had significantly more MN than sexually immature flounder. Flounder over the entire sampling area had increased levels of MN during the fall months of the year. This is probably influenced by the maternal cycle of gonadal maturation.

Animals↗
PubMed2001

Cloning of Japanese flounder Paralichthys olivaceus CD3 cDNA and gene, and analysis of its expression.

Two distinct CD3 homologue cDNAs, CD3-1 and CD3-2, were isolated from a Japanese flounder leukocyte cDNA library. CD3-1 consisted of 961 bp encoding 178 amino acid residues, and CD3-2 consisted of 927 bp encoding 182 amino acid residues. The two deduced amino acid sequences had an identity of 95.1%, and neither had N-linked glycosylation sites. The identities between the Japanese flounder CD3s and previously reported CD3s (CD3 epsilon, CD3 gamma, or CD3 delta) of Xenopus laevis, chicken, and various mammals were approximately 25%. The Japanese flounder CD3s had an extracellular domain, a CXXCXE motif, and an immunoreceptor tyrosine-based activation motif (ITAM), each of which are important characteristics of CD3 chains. Furthermore, the positions of four cysteine residues in the extracellular domain were preserved in both of the Japanese flounder CD3s. A phylogenetic tree based on the amino acid sequences confirmed that the Japanese flounder CD3s are closer to CD3 epsilon than to CD3 gamma and CD3 delta. However, the gene structure of Japanese flounder CD3 is identical to the chicken and Xenopus CD3 gamma/delta genes and the mammalian CD3 delta gene. Southern blot hybridization and the DNA sequence of the CD3 gene of homocloned Japanese flounder indicated that the CD3 gene exists as a single copy. Southern blot hybridization also showed the presence of a polymorphic variant of Japanese flounder CD3. An RT-PCR analysis detected Japanese flounder CD3 mRNA in several organs that contained lymphocytes. The proportion of CD3-positive cells in the peripheral blood leukocytes was 34.9%.

Amino Acid Sequence↗
PubMed2004

Characterization of structure and expression of the growth hormone receptor gene of the Japanese flounder (Paralichtys olivaceus).

Growth hormone receptor (GHR) cDNA and gene of the Japanese flounder (Paralicthys olivaceus) were cloned and their molecular structures were characterized. The 641 amino acid sequence predicted from the cDNA sequence showed more than 75% overall sequence similarity with GHRs of other teleosts such as turbot and goldfish, and contained common structural features of vertebrate GHRs. The extracellular domain of flounder GHR had three pairs of cysteines and an FGEFS motif with a replacement E to D. The cytoplasmic domain contained two conserved motifs referred to as box 1 and box 2. The flounder GHR gene was cloned by PCR using primers designed from the sequence of the GHR cDNA. The GHR gene was composed of 10 exons. The sequence of exon 1 corresponded to the 5'-untranslated region of the cDNA, and exons 2-6 encoded most parts of the extracellular domain. The transmembrane domain was found in exon 7, and the intracellular domain was encoded in exons 8-10. Exon 10 also encoded the 3'-untranslated region. Comparison of the flounder GHR gene with the human GHR gene shows that the flounder gene contains no exons corresponding to exon 3 of the human GHR gene, and that the region corresponding to exon 10 in the human GHR gene is encoded by exons 9 and 10 in the flounder GHR gene. These findings indicate that the flounder GHR gene diverged from those of mammalian and avian GHR genes, especially in the organization of the exons encoding the cytoplasmic domain. In addition to the regular form of GHR mRNA, a 3'-truncated form lacking the region derived from exons 9 and 10 was detected as a minor species in the liver by RT-PCR and by RNase protection assay. RT-PCR analysis showed that both the regular and the 3'-truncated GHR mRNAs are expressed in a wide range of flounder tissues with the highest levels being found in the liver. The 5'-flanking region of the flounder GHR gene was cloned by inverse PCR, and three transcription start points were identified with similar frequency by RNase protection assay.

5' Flanking Region↗
PubMed2000

Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus.

We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were approximately 20-30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-alpha. The Japanese flounder TNF gene has a length of approximately 2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-alpha. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-alpha gene. There are simple CA or AT dinucleotide repeats in the 5'-upstream and 3'-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-alpha than mammalian lymphotoxin-alpha, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.

Amino Acid Sequence↗
PubMed2004

Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin.

Flounder (Paralichthys olivaceus) Immunoglobulins (Igs) were purified from the serum of mouse IgG-immunized flounder by using affinity chromatography. Under denaturing conditions in SDS-PAGE, the flounder Igs appeared to be composed of 2 heavy (H) chains (72 and 77 kDa) and two light (L) chains (26 and 28 kDa). Monoclonal antibodies (MAbs) were produced by the fusion of myeloma cells (SP2/0) with Balb/c mouse spleen cells that were previously sensitized against affinity-purified flounder Igs. In a Western blot analysis, the produced MAbs, FIM511, FIM519, and FIM562 recognized both the 72 and 77 kDa H chains, 26 kDa, and 28 kDa L chain, respectively. Mouse antiserum against flounder Igs reacted more strongly with the L chain of 28 kDa than with 26 kDa, suggesting that the 28 kDa molecule is more immunogenic than the 26 kDa L chain molecule. In a FACS analysis, the ratios of the Ig+ cell population in the flounder head kidney and spleen cells were 49% and 24%, respectively. Unexpectedly, however, the ratios of the Ig+ B-like cell population in the flounder were not significantly augmented, even after the immunization of an immunogenic antigen. This suggests that the humoral immune response in fish could be considerably different from that in mammals. The produced MAbs in this study would be useful in characterizing flounder Ig+ B-like cells and in developing flounder Ig detecting an immunoassay system.

Animals↗
PubMed2000

Molecular Cloning and Novel Repeated Sequences of a C-type Lysozyme Gene in Japanese Flounder (Paralichthys olivaceus).

A clone of a DNA fragment of approximately 10 kb that included a c-type lysozyme gene of Japanese flounder (Paralichthys olivaceus) was isolated from a Japanese flounder genomic DNA library. This clone was subcloned, and the nucleotide sequence of the c-type lysozyme gene was determined. The flounder c-type lysozyme gene, which consisted of 3,617 bp, was found to be composed of four exons and three introns. The transcription start site was determined to be 44 bp upstream of the ATG codon by primer extension analysis. The number of exons and introns and the positions of the catalytic residues of the flounder gene were the same as those of the human and chicken c-type lysozyme genes. However, introns 1 and 3 of the flounder gene were shorter, and intron 2 was longer, than those in the other reported vertebrates genes. There were no Alu repeated sequences in the flounder lysozyme gene, as determined for the human lysozyme gene, but 45 repeated sequences were found in the second intron of the flounder lysozyme gene. We suggest that the length of intron 2 in the Japanese flounder c-type lysozyme gene depends on the presence of this repeated sequence. Furthermore, Southern blot analysis revealed that the c-type lysozyme gene was probably a single copy in the genomic DNA of homo-cloned Japanese flounder.

Journal Article↗
PubMed2005

Molecular characterization of the Japanese flounder, Paralichthys olivaceus, CD3epsilon and evolution of the CD3 cluster.

A second CD3 gene, i.e. CD3epsilon, has been cloned and sequenced in Japanese flounder. The full length cDNA is 1006 bp and encodes 164 amino acids. When compared with other known CD3epsilon peptide sequences, the most conserved region of the Japanese flounder CD3epsilon chain peptide is the cytoplasmic domain and the least conserved is the extracellular domain. A phylogenetic analysis based on the deduced amino acid sequence grouped the two Japanese flounder CD3 sequences with CD3epsilon and CD3gamma(delta, respectively. The Japanese flounder CD3epsilon gene has Lyf-1, GATAs, Oct-1, CEBPs, AP-1, and NF-AT but lacks TATA and CCAAT elements in the 5' flanking region. The Japanese flounder CD3 cluster (consisting of CD3epsilon and CD3gamma(delta) spans only 10.4 kb. The two genes are oppositely transcribed only 3.8 kb apart. Both Japanese flounder CD3 genes have five exons. The two Japanese flounder CD3 genes were predominantly expressed in PBLs, kidney, spleen, and gills. A polyclonal rabbit antiserum that reacts with the CD3 marker on human T cells also reacted with Japanese flounder CD3epsilon. The epitope highly conserved between mammalian and non-mammalian CD3epsilons, this antibody bound to a single 15 kDa peptide.

Amino Acid Sequence↗
PubMed2006

Molecular cloning and characterization of Toll-like receptor 9 in Japanese flounder, Paralichthys olivaceus.

Toll-like receptor (TLR) 9 cDNA and gene were cloned from Japanese flounder, Paralichthys olivaceus. The Japanese flounder TLR9 cDNA encodes 1065 amino acids. The leucine-rich domain (LRD) and the Toll/interleukin-1 receptor (TIR) domain found in other vertebrate TLR9s were conserved in Japanese flounder TLR9. The gene is composed of three exons and two introns. The Japanese flounder tumor necrosis factor (TNF) gene promoter was activated in Japanese flounder TLR9-transformed hirame natural embryo (HINAE) cells upon stimulation with synthesized CpG oligodeoxynucleotide (ODN), but not by stimulation with GpC ODN. The Japanese flounder TLR9 gene was highly expressed in epithelial and lymphoid organs, such as the gills, intestines, kidney, spleen and stomach in an apparently healthy fish. The mRNA copy numbers of Japanese flounder TLR9 and its adapter protein, the myeloid differentiation factor 88 (MYD88) were increased in some organs including blood, gill, kidney and spleen after Edwardsiella tarda challenge. Immunohistochemical analysis revealed that TLR9 and MYD88 were expressed in the same cells of kidney. Few TLR9-expressing cells were found in gill, kidney and spleen in healthy Japanese flounder, but many were found in these organs after E. tarda challenge and were coincident with lesions that had been colonized by the bacteria.

Amino Acid Sequence↗