Molecular cloning and brain distribution of three types of gonadotropin-releasing hormone from mummichog Fundulus heteroclitus.

Complementary DNAs encoding gonadotropin-releasing hormone (GnRH) precursors were cloned from the mummichog Fundulus heteroclitus brain, showing that this species has three GnRH forms, i.e. medaka Oryzias latipes GnRH (mdGnRH), chicken GnRH-II (cGnRH-II) and Atlantic salmon Salmo salar GnRH (sGnRH). The F. heteroclitus prepro GnRHs have common structural architectures of vertebrate GnRHs, consisting of the signal peptide, 10 amino acids of mature peptide, GKR sequence and GnRH-associated peptide (GAP). Phylogenetic analysis of fish prepro GnRHs showed that F. heteroclitus mdGnRH is a homologue of sbGnRHs and mdGnRHs of other acanthopterygian. Quantitative real-time PCR revealed that mdGnRH was abundantly expressed in the olfactory bulb and in olfactory lobe areas and is expressed in the pituitary. The cGnRH-II was mainly expressed in the midbrain and interbrain areas, and the sGnRH was expressed not only in the olfactory bulb but also in other regions of the brain. These results suggest that the mdGnRH is involved in the stimulation of gonadotrophs in the pituitary, whereas cGnRH-II and sGnRH are involved in neurotransmission and neuromodulation.

Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region.

Pseudomonas plecoglossicida is the agent of bacterial haemorrhagic ascites (BHA) in freshwater fish farming in Japan. To develop a rapid identification and detection method for P. plecoglossicida, a PCR amplification technique targeting the chromosomal DNA region coding the B subunit of the DNA gyrase (gyrB) was used. The nucleotide sequences of gyrB were determined in nine isolates of P. plecoglossicida and two other Pseudomonas species. On the basis of these determined sequences and the gyrB sequences of other Pseudomonas species or fish pathogenic bacteria deposited in international nucleotide sequence databases (GenBank/EMBL/DDBJ), PCR primers PL-G1F, PL-G1R, PL-G2F and PL-G2R were designed for specific amplification of the partial gyrB of P. plecoglossicida. The specificity of these primers in amplifying the gyrB of P. plecoglossicida was verified using selected strains of related bacterial species. The nested PCR technique was used to detect P. plecoglossicida from kidney and intestine of ayu. Primer pair PL-G1F and PL-G1R was used for the external PCR, and primer pair PL-G2F and PL-G2R for the internal PCR. Of 10 ayu juveniles, expected size PCR products were observed from intestine and kidney samples in one and two specimens, respectively. The PCR technique with primers based on the gyrB sequence is thus useful for the diagnosis of BHA.

PCR-RFLP genotypes associated with quinolone resistance in isolates of Flavobacterium psychrophilum.

A novel genotyping method for epizootiological studies of bacterial cold-water disease caused by Flavobacterium psychrophilum and associated with quinolone resistance was developed. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was performed on 244 F. psychrophilum isolates from various fish species. PCR was performed with primer pair GYRA-FP1F and GYRA-FP1R amplifying the A subunit of the DNA gyrase (GyrA) gene, which contained the quinolone resistance determining region. Digestion of PCR products with the restriction enzyme Mph1103I showed two genotypes, QR and QS. The difference between these genotypes was amino acid substitutions at position 83 of GyrA (Escherichia coli numbering). The genotype QR indicated an alanine residue at this position associated with quinolone resistance in F. psychrophilum isolates. Of the 244 isolates tested in this study, the number of QR genotype isolates was 153 (62.7%). In isolates from ayu (n=177), 146 (82.5%) were genotype QR. With combination of this technique and previously reported PCR-RFLP genotyping, eight genotypes were observed in F. psychrophilum isolates. Using this genotyping system, the relationships between genotype and host fish species, or locality of isolation, were analysed and are discussed.

Detection and identification of Flavobacterium psychrophilum from gill washings and benthic diatoms by PCR-based sequencing analysis.

A nested polymerase chain reaction (PCR) amplification technique was used to detect Flavobacterium psychrophilum from washings of fish gill surfaces and benthic diatoms as environmental samples. Gill washing samples were prepared from kawamutsu, Zacco temminckii (Temminck & Schlegel) and oikawa, Z. platypus (Temminck & Schlegel). Benthic diatom samples were collected from stone surfaces. All samples were collected from rivers in Wakayama Prefecture, Japan from November 2003 to January 2004. Following simple DNA extraction using a chelating resin, nested PCR techniques targeting 16S-rDNA and gyrB regions were performed, and PCR products were cloned and sequenced. With nested PCR amplification for the 16S-rDNA gene, ambiguous PCR products were detected from two of six samples, and by cloning and sequencing analysis were found not to be DNA fragments amplified from F. psychrophilum. Using nested PCR for the gyrB gene, however, five of six samples were clearly positive for F. psychrophilum in agarose gel electrophoresis, and were found to be identical with nucleotide sequences of F. psychrophilumgyrB deposited in DNA databases by sequencing analysis. Results indicate that nested PCR for the gyrB region is a useful technique to detect low levels of F. psychrophilum from environmental samples contaminated with many other organisms.

Characterization of matrix metalloproteinase in flatfish embryo undergoing formation of lenses.

Members of the matrix metalloproteinase (MMP) family are responsible for breakdown of extracellular matrix components involved in morphogenetic remodeling of animal embryogenesis. The highly sensitive assay of MMP using synthetic fluorescence-quenching substrate was employed to detect and to characterize a veiled MMP activity expressed in Japanese flounder embryos undergoing formation of lenses. The MMP activity was enhanced in proportion to increasing protein amounts of the embryonic lysate over 5 microg, and this reaction was proceeded in a time-dependent manner and with increasing substrate concentrations. Almost 2-fold increase in the embryonic MMP activity occurred by treatment with 4-aminophenylmercuric acetate, but the activity was markedly suppressed by metal chelating reagents. These enzymatic characteristics are apparently consistent with those of mammalian embryonic MMPs, particularly MMP-9. The characterized MMP activity was highly expressed at the specificstage during embryogenesis, indicating that this MMP may be involved in formation of lenses.

Characterization of Japanese eel immunoglobulin M and its level in serum.

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790,000; it contained an equimolar heavy chain and light chain with molecular weights of 72,000 and 25,000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by D-mannose and could be abolished by alpha-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml(-1); it amounted to 10.3% of the total serum protein.

Purification and Characterization of alpha1-Proteinase Inhibitor from Carp (Cyprinus carpio) Serum.

: alpha1-Proteinase inhibitor was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 17-fold and a 3% recovery rate. The inhibitor was estimated to have molecular weight of 55,000 under reducing and nonreducing conditions, indicating its composition of a single polypeptide. The inhibitor immunologically crossreacted faintly with carp muscular serine proteinase inhibitor but had no crossreactivity with serine proteinase inhibitors from other species. Carp serum inhibitor exhibited marked stability over broad pH ranges of 4.0 to 10.0 and temperatures below 55 degrees C. The inhibitor potently inhibited the activities of carp intestinal and fish myofibril-binding proteinases, and its respective inhibitions of trypsin-type and carp muscular proteinases were more severe than those of chymotrypsin-type and white croaker muscular proteinases. Its inhibitions were similar to those of bovine pancreatic trypsin and alpha-chymotrypsin, and the amount required to completely inactivate 0.2 µg of each of these two proteinases was evaluated as 0.43 to 0.45 µg. This indicates a molar ratio close to 1:1 during combination of the inhibitor with each proteinase. In addition, its ability to form irreversible complexes with the proteinases was observed electrophoretically and immunologically under denaturing and reducing conditions.

Purification and Characterization of Serum Serpin from Carp (Cyprinus carpio).

: A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 6.2-fold and a 3% recovery rate after separation from alpha1-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific antitryptic activity of alpha1-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently larger than that of alpha1-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically crossreacted with serpin62 and had no crossreactivity with fish serum alpha1-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic activity physicochemically and immunologically.

Purification and characterization of a novel dipeptidase from carp ordinary muscle.

A novel dipeptidase was purified to homogeneity from the crude extract of carp ordinary muscle with an increase in specific activity of 4041-fold and a 4% recovery rate. The enzyme was determined to have molecular weights of 54,000 after reduction and 106,000 without reduction, indicating that it is composed of two sulfide-linking molecules of subunit peptide chains of identical molar size. The optimum hydrolysis pH and temperature of the enzyme were evaluated by l-leucine-glycine to be pH 8.5 and 40 degreesC, respectively, and it was markedly stable in the weak alkaline region and at temperatures below 30 degreesC. Dipeptide hydrolysis of the enzyme was inhibited by metalloprotease inhibitors, sulfide-specific reagents, metal chelating reagents and reductants. In addition, sulfide-affinity bivalent metals potently inactivated the enzyme, while Mg2+ and Mn2+ activated it to different extents, and Mn2+ was also effective on the restoration of the nearly completely inactivated enzyme. The enzyme had a broad range of action on dipeptides that are composed of only l-amino acids, such as l-leucine, l-methionine, l-phenylalanine, and l-valine at the C-terminal and l-alanine, l-leucine, l-methionine, and l-valine at the N-terminal, but it had no catalytic action on dipeptides containing l-proline and d-amino acids, tripeptides, and peptide derivatives.

Distinctive catalytic actions of carp dipeptidases from ordinary muscle and intestine.

Although carp muscular and intestinal dipeptidases are metalloenzymes acting only on dipeptides, some structural and enzymatic differences occur between them. The present study verifies distinctive actions during dipeptide hydrolysis by these enzymes in terms of their kinetic characterization. The intestinal enzyme was differentially inhibited by EDTA and 1,10-phenanthroline, whereas these compounds induced a similar level inhibition of the muscular enzyme. The Km and Vmax values of both enzymes for l-leucine-glycine hydrolysis varied during incubations with 1,10-phenanthroline and EDTA, and the Km or Vmax values of the intestinal enzyme increased or remained the same with increasing concentrations of 1,10-phenanthroline, respectively. Analysis of the kinetic parameters indicated that Co2+ and Mn2+ had noncompetitive effects on the muscular enzyme and that a noncompetitive activation on the intestinal enzyme was stimulated by 1.5 mM of Mg2+ and with increasing concentrations of Mn2+. The muscular enzyme acted on a wide range of l-configuration dipeptides, whereas the intestinal enzyme acted only on a select range of dipeptides with a hydrophobic amino acid at the N-terminal position. The Kcat/Km values of both enzymes for dipeptide hydrolysis showed that highly hydrophobic dipeptides served as their preferential substrates. Other kinetic parameters demonstrated distinctive hydrolytic action of the two enzymes on these dipeptides: a strong affinity of the low catalytic rate muscular enzyme, and a weak affinity of the high catalytic rate intestinal enzyme.

Susceptibility of Opioid Peptides and Myofibrillar Proteins to Carp Cathepsin L.

The ability of carp cathepsin L to degrade opioid peptides and separate myofibrillar proteins was examined. The enzyme differentially released fragments from leucine-enkephalin, alpha-neoendorphin, and dynorphin, in which the common cleavage site of the enzyme was determined to be a Gly2-Gly3 bond. A Leu5-Arg6 bond in alpha-neoendorphin and dynorphin was also commonly cleaved by the enzyme. At a protein ratio of the enzyme to myofibrillar protein of 1:500, the preferences of the enzyme appeared to be myosin > tropomyosin > troponins T, I, and C > actin > alpha-actinin. Myosin heavy chain was completely degraded into some fragments by the enzyme within 30 min, and all of them underwent further degradation during subsequent incubation. Although troponins and tropomyosin were also completely degraded by the enzyme within 24 h, a small amount of actin and a large amount of alpha-actinin remained undegraded.

Epidermal proteinases in the European eel.

A fluorescent-sensitive assay demonstrated the exhaustive detection of proteinase activities in the dorsal skin of the European eel. Two distinct skin extracts were prepared from skin mucus and epidermal cell layers with no mutual contamination, so that the latter extract contained significant susceptibility of all tested substrates. Optimum hydrolysis pH's for susceptible substrates were found in acidic and neutral ranges, and optimum hydrolysis temperatures for the same substrates fell mainly in the 40 degrees-50 degrees C range. In addition, diverse inhibitory influences on these hydrolyses were prompted by several proteinase inhibitors and metal chlorides, and some other reagents specifically affected the individual hydrolysis; among them, the inhibitions of all activities by p-tosyl-L-phenylalanyl-chloromethylketone CdCl2, CuCl2, HgCl2, and ZnCl2 were remarkable. Antipain, iodoacetamide, and CoCl2 induced a severe inhibition of all except three activities, whereas N-ethylmaleimide markedly inhibited only these three activities. These findings suggest that epidermal cell layers of the European eel retain a party of proteolytic enzymes, and this party is judged from their exhibiting specificities to be composed of four distinct proteinases, presumably cathepsins L and B, a serine proteinase, and an aminopeptidase.

Substrate specificity of carp Cyprinus carpio cathepsin H with methylcoumarylamide substrates.

Cathepsin H was purified from the crude extract of carp (Cyprinus carpio) hepatopancreas by a reformed method involving six stages, and the specific activity increased about 11,500-fold with a 23% recovery. Of varying fluorescent synthetic substrates tested, carp cathepsin H possessed an ability to hydrolyze four N-terminal unblocked substrates those are composed of a single amino acid bound to 4-methylcoumaryl-7-amide (MCA), namely Leu-MCA, Arg-MCA, Lys-MCA and Ala-MCA. In contrast the enzyme was only marginally able to hydrolyze an unblocked substrate such as Pro-Phe-Arg-MCA and totally unable to degrade all blocked derivative employed. From the kinetic constants with four unblocked substrates, car cathepsin H had the highest affinity toward Leu-MCA with a Km value of 35.4 microM. Besides both the hydrolytic rates and molecular activities of the enzyme decreased from Lys-MCA > Arg-MCA > Ala-MCA > Leu MCA as judged by their Vmax and Kcat values, respectively. Otherwise, optimal pHs for hydrolysis of cathepsin H were different for four substrates. The enzyme exhibited maximum level of the activity at pH 6.5 for Arg-MCA and Lys-MCA and at pH 7.0 for Leu-MCA and Ala-MCA.

Purification and characterization of cathepsin L from hepatopancreas of carp Cyprinus carpio.

Cathepsin L was purified from carp hepatopancreas by a method involving ammonium sulfate precipitation and a series of column chromatographies, in which the enzyme had an affinity toward Concanavalin A and Cibacron Blue F3GA. Its homogeneity was established by Native-PAGE, but two protein bands corresponding to molecular masses of 30,000 (single chain) and 24,000 (heavy chain) migrated on SDS-PAGE. The enzyme exhibited a maximum activity for carbobenzoxy-L-phenylalanyl-L-arginyl-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA) at pH 5.5-6.0 and 50 degrees C and the remarkable stability at pH 5.0-6.5 and below 40 degrees C. All tested cysteine protease inhibitors and TLCK and chymostatin markedly inhibited its activity, whereas the other serine protease inhibitors and a metalloprotease inhibitor negligibly affected it. In addition, several metal compounds reduced either its activity or stability to differing extents. Although EDTA alone caused an only marginal activation of the enzyme, its maximum activation required both 2 mM cysteine and 1 mM EDTA. The enzyme had an ability to hydrolyze three peptidyl-MCA substrates including Z-Phe-Arg-MCA, but all kinetic constants indicate that Z-Phe-Arg-MCA is the optical substrate to the enzyme.

Purification and characterization of cathepsin H from hepatopancreas of carp Cyprinus carpio.

1. Cathepsin H was purified about 5400-fold from hepatopancreas of carp (Cyprinus carpio) by the method involving ammonium sulfate fractionation, and chromatography on S-Sepharose, DEAE-Sephacel, Ultrogel AcA54, Concanavalin A-Sepharose 4B and GPC on Protein-Pak 125. 2. The purified cathepsin H gave a single protein band on analytical-PAGE, but migrated as two bands of 27,000 and 23,000 mol. wt on SDS-PAGE. 3. Cathepsin H had a pH and temperature optimum of 6.5 and 45 degrees C using Arg-MCA as a substrate, respectively, and was activated by sulfhydryl compounds and inhibited by cysteine protease inhibitors and metal compounds having high reactivities at cysteine residue. 4. The carp hepatopancreas cathepsin H immunoreacted with the monospecific antibody against rat liver cathepsin H, and did not react with the antibodies against carp hepatopancreas cathepsins B and L by the method of immunoelectrophoretic blotting.