A hyperosmotic stress-induced mRNA of carp cell encodes Na(+)- and Cl(-)-dependent high affinity taurine transporter.

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.

Structure of a full-length cDNA clone for the pro-alpha1(V/XI) collagen chain of red seabream.

The cDNA of type V/XI collagen alpha1 (rsCOL) chain has been isolated from cells established from eyed-period eggs of red seabream, Pagrus major, and sequenced. The amino acid sequence deduced from red seabream alpha1(V/XI) chain resembles that of type XI collagen alpha1 chain. On the other hand, tissue distribution of rsCOL resembles that of type V collagen based on RT-PCR analysis. This is the first report of the cloning of the full-length cDNA of type V/XI collagen alpha1 chain from fish.

Sequence and expression of a cDNA encoding the red seabream androgen receptor.

The cDNA of the androgen receptor (AR) has been isolated from the ovary of red seabream, Pagrus major, and sequenced. The amino acid sequence of red seabream AR (rsAR) shows about 45% identity with those of Xenopus, rat, mouse, and human ARS. It is shown that rsAR has the ability to trans-activate the responsive gene depending on the presence of androgen.

Sequence and expression of a cDNA encoding the red sea bream androgen receptor.

The cDNA of the androgen receptor (AR) has been isolated from the ovary of red sea bream, Pagrus major, and sequenced. The amino acid sequence of red sea bream AR (rsAR) shows about 45% identity with that of Xenopus, rat, mouse, and human AR. It is shown that rsAR has the ability to trans-activate the responsive gene depending on the presence of androgen.

Fish glucocorticoid receptor with splicing variants in the DNA binding domain.

Here we describe the isolation of a rainbow trout cDNA containing an entire GR coding region. Although the encoded protein is highly homologous to other GRs, especially in its DNA binding domain, it contains a nine amino acid insertion between the two zinc fingers. This novel form is found in all rainbow trout tissues examined; however, the testis also contains a splice variant lacking this insert, making it completely continuous to other GRs. In transient transfection assays of cultured cells, the two rainbow trout GR variants activated transcription from the glucocorticoid-responsive mouse mammary tumor virus promoter to comparable levels.

Purification and properties of a novel latent proteinase showing myosin heavy chain-degrading activity from threadfin-bream muscle.

A novel latent proteinase of which activity was induced by heating in the presence of NaCl was purified to homogeneity from threadfin-bream muscle by a combination of DEAE-cellulose, Con A-Sepharose, Arg-Sepharose, and Shim-pack HAC chromatographies. This proteinase was a glycoprotein having a monomeric subunit structure; Mr was estimated to be 77,000 on SDS-PAGE analysis. The proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA as well as myosin heavy chain in the presence of 2-4% NaCl at pH 7.0 and at 60 degrees C, optimally. The proteinase was classified as serine proteinase based on the effects of soybean trypsin inhibitor, leupeptin, and antipain.

Structure and expression of carp mitogen-activated protein kinases homologous to mammalian JNK/SAPK.

Two distinct stress-activated protein kinase (JNKa and b) cDNAs were isolated from a carp ovary cDNA library. These cDNAs contained a full-length open reading frame encoding 427 amino acid residues with a predicted mass of 48.6 kDa. The deduced amino acid sequences of JNKa and b were 95.8% identical, with 18 residues replaced, and showed a high degree of sequence similarity to mammalian JNK/SAPK subgroup including the common dual phosphorylation motif of TPY. By Northern blot analysis, the carp JNKs were found to be abundant in the brain and ovary. Detailed study by RT-PCR assay revealed ubiquitous expression of JNKb, although expression of JNKa was specific to the brain and ovary. The high level expression of both JNKa and b in the ovary implies that JNKs play an important role in egg maturation or ectogenetic early development.

Significant amount of multicatalytic proteinase identified on membrane from human erythrocyte.

Multicatalytic proteinase (MCP) was solubilized from human erythrocyte membrane with 0.1% Triton X-100 and purified to homogeneity using a combination of DEAE-cellulose, hydroxylapatite, and Ultrogel AcA34 chromatographies. This membranous MCP had similar properties to MCP purified in parallel from the cytosol. Both MCPs had a molecular mass of 570 kDa, were composed of apparently nine subunits of 22-36 kDa and had trypsin- and chymotrypsin-like activities. These activities were latent and required heating for the induction. However, slight differences were observed in the effects of reagents (DFP, monoiodoacetic acid, Mg2+, and Ca2+) between membranous and cytosolic MCP. The amount of MCP identified on membranes was estimated to be three-quarters or one-half of that found in the cytosol based on its trypsin- or chymotrypsin-like activity, respectively.

Existence of two isoforms of extracellular signal-regulated kinase in fish.

Full-length cDNAs for extracellular signal-regulated kinases (ERK1 and ERK2) were isolated from a carp ovary cDNA library. The deduced amino acid sequences of carp ERK1 (cERK1) and ERK2 (cERK2) exhibited high degrees of homology to the known sequences of the ERK group. Northern blot analysis showed that cERK1 mRNA was not expressed in a tissue-specific manner, though the level of expression of cERK2 mRNA varied among tissues. Western blot analysis of the brain, kidney, and ovary confirmed the expression of cERK1 and cERK2 in carp. Our findings indicate that two isoforms of ERK, ERK1 and ERK2, exist in fish.

Transgenic medaka overexpressing a melanin-concentrating hormone exhibit lightened body color but no remarkable abnormality.

Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide that concentrates melanin granules in the melanophores and lightens the body color of a fish. To investigate the utility of MCH as a reporter gene, a transgenic medaka strain overexpressing the MCH gene was established and its phenotypic features were examined. The salmon MCH gene driven by cytomegalovirus promoter was injected into 100 fertilized eggs of the HNI-1 medaka strain, which exhibits black body color. One F(0) female transmitted the transgene and a lightened body color phenotype to the F(1) generation. A homozygous transgenic strain was established by crossing F(2) fish homozygous for the transgene. Expression of the transgene was detected in several organs by Northern blotting. The melanin granules of transgenics were highly shrunk. Bioassay using scales confirmed the secretion of MCH into blood, and the MCH concentration was estimated between 0.5 and 5 microM. Development, growth, feeding behavior, and reproduction of transgenics did not differ significantly among transgenic and nontransgenic siblings. The result whereby enhanced MCH expression induced a change in body color, but no remarkable abnormality, suggests the usefulness of MCH as a novel reporter gene with unique features.

Comparison of calpain I and calpain II from carp muscle.

1. The content of calpain II is 3.4 times more than that of calpain I when estimated by the elution profiles from a column of DEAE-cellulose. 2. Calpain I required 1 mM Ca2+ and calpain II required 5 mM Ca2+ to show the full activities. These data demonstrated that Ca2+-sensitivities of both calpains were lower than those of mammalian calpains, respectively. 3. The optimum caseinolytic activity was pH 7.2 for calpain I and pH 7.5 for calpain II. 4. The molecular weight of calpain I was estimated to be 110 k and that of calpain II to be 120 k by gel filtration. 5. Calpain I was much more heat-stable than calpain II around 50-60 degrees C. 6. Both calpains were sensitive to calpastatin, an endogenous inhibitor for calpain.

Inhibitory effect of carp muscle trypsin inhibitor on mammalian pancreatic proteinases.

Carp muscle trypsin inhibitor showed an inhibitory effect on bovine trypsin, bovine alpha-chymotrypsin and porcine elastase in a non-competitive, competitive and competitive manner, respectively. The inhibitor formed a stable complex with the above proteinases which was not dissociated in the presence of 2-mercaptoethanol and SDS. The true target proteinase for carp muscle trypsin inhibitor, as yet unknown, seems to be an alpha-chymotrypsin- or elastase-like enzyme rather than trypsin, judging from the manner of inhibition.

Comparison of muscle proteinase activity among fish species.

The effects of temperature during storage, portion of muscle and growth stage of fish on the activity level of carp muscle acid (cathepsin D, EC 3.4.23.5), neutral and alkaline proteinases were examined. Icing storage, freezer storage and portion of muscle did not affect each proteinase activity (P less than 0.05), but acid proteinase activity was affected by growth stage (P less than 0.05) and the level decreased from small to large fish. The activity levels of three kinds of proteinases were compared among species (P less than 0.05) and the following order was obtained. Acid proteinase, white croaker = rainbow trout = carp greater than sardine = common mackerel, sardine greater than lizardfish, common mackerel = lizardfish. Neutral proteinase, rainbow trout greater than carp = white croaker greater than lizardfish = sardine = common mackerel. Alkaline proteinase, rainbow trout = sardine greater than white croaker = carp = common mackerel greater than lizardfish.

Detection and some properties of calpain II (high-Ca2+-requiring form of calpain) in carp (Cyprinus carpio) erythrocytes.

In order to examine the existence of calpain I, a low (micromolar)-Ca2+-requiring form of calpain, in fish tissues, carp erythrocytes were chosen as the experimental material, since only calpain I is known to exist in mammalian erythrocytes. By DEAE-cellulose chromatography, calpain and calpastatin (specific inhibitor for calpain) were separated from carp erythrocyte hemolysate. Carp erythrocyte calpain is classified as calpain II, a high (millimolar)-Ca2+-requiring form of calpain, from the result of Ca2+-requirement for the activity.

Purification and some properties of a trypsin inhibitor from carp (Cyprinus carpio) muscle.

A trypsin inhibitor was purified from carp muscle to apparent homogeneity by the successive chromatographies of DEAE-cellulose, DEAE-Sepharose CL-6B, Con A-Sepharose, Ultrogel AcA 44 and hydroxylapatite. The mol. wt of the inhibitor was estimated to be 58,000 by SDS-polyacrylamide gel electrophoresis or 50,000 by gel filtration. The inhibitor seemed to form a 1:1 stoichiometric complex with trypsin, alpha-chymotrypsin and elastase, respectively. Carp muscle trypsin inhibitor was likely to be identical with serum alpha 1-proteinase inhibitor judging from its glycoprotein nature, mol. wt and the inhibition stoichiometry.

High molecular weight heat-stable alkaline proteinase from white croaker and chum salmon muscle: comparison of the activating effects by heating and urea.

Effects of heating and urea on the heat-stable alkaline proteinase from white croaker and chum salmon muscle were compared in order to know the regulating mechanism of the proteinase. Chum salmon proteinase required a higher temperature for activity and was more heat-stable than white croaker proteinase. In the presence of 5M urea, the activity was observed to some degree at 37 degrees C only in white croaker proteinase, while both proteinases lost their activities at usual assay temperature around 60 degrees C. These results suggest that the stability of the regulatory and catalytic subunits of the proteinases is somewhat different among fish species.

The possible existence of a heat-stable alkaline proteinase (HAP) in rat skeletal muscle.

1. A unique caseinolytic activity was found in the crude extract from chicken and rat skeletal muscle. Hardly any activity was detected at physiological assay temperatures at pH 8.0 but did well at around 60 degrees C. 2. The activity partially purified from rat skeletal muscle showed optimum pH at around 8.0 at 60 degrees C. It hardly hydrolyzed casein below 50 degrees C, but in the presence of 5 M urea it showed relatively high activity at 30 degrees C. The activity was completely stable at 50 degrees C for 1 hr. 3. The activity seems to be contained in a high mol. wt (450,000) protein from the elution volume and is due to cysteine proteinase from the effect of inhibitors. 4. The above properties agreed with those of the heat-stable alkaline proteinase (HAP) of fish purified homogeneously by electrophoresis. This seems to suggest that HAP may also exist in rat skeletal muscle.