Cloning and characterisation of tandem-repeat type galectin in rainbow trout (Oncorhynchus mykiss)

Fish beta-galactoside binding lectin (galectin) cDNA was cloned from the cDNA library of rainbow trout (Oncorhynchus mykiss) head kidney. The clone contained a single open reading frame encoding 341 amino acids (aa) (38 kDa protein), including the initiator methionine. Significant sequence homology to mammalian galectin-9 (40-55% identity) was observed. Its amino acid sequence showed two distinct N- and C-terminal domains (148 and 130 aa, respectively) connected by a peptide linker (63 aa). The galectin contains two consensus WG-E-R/K motifs thought to play an essential role in sugar-binding, indicating that this lectin is a member of the tandem-repeat type galectins which have not been identified in fish. The 1.6 kDa mRNA of the lectin was found by Northern blot analyses to be widely expressed in the spleen, head kidney, thymus, peritoneal exudate cells, ovary, gills and heart. Southern blot analyses with the probe for C-terminal of the lectin showed the existence of two hybridising genes. These results suggest that rainbow trout has at least one tandem-repeat type galectin as well as proto-type galectin.     

Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss

Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing. Synthesis and processing of native piscine IL-1beta is poorly understood. In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx. 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli. Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus. Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted. These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.     

Cloning and characterization of the rainbow trout (Oncorhynchus mykiss) type II interleukin-1 receptor cDNA.

A homologue of mammalian type II interleukin-1 receptor (IL-1RII) was isolated from a rainbow trout cDNA library by differential hybridization using a suppression subtractive hybridization generated probe enriched for sequences upregulated after immune stimulation. The trout cDNA has an ORF encoding 441 amino acids, and represents the first piscine IL-1 receptor described. The predicted amino-acid sequence has 29 and 26% identity with human and mouse IL-1RII, respectively. The trout IL-1 receptor has a domain organization similar to that of mammalian type II receptor, with a short cytoplasmic tail of 24 amino acids. These results suggest that type II receptor is also present in lower vertebrates, and therefore the duplication of an ancestral gene that generated type I and type II IL-1 receptors occurred prior to the time mammals emerged.     

Cloning, expression analysis and bioactivity studies of rainbow trout (Oncorhynchus mykiss) interleukin-22

This report describes the cloning and characterisation of rainbow trout (Oncorhynchus mykiss) interleukin (IL)-22, and presents studies of the functional activity of its recombinant protein for the first time in a non-mammalian species. The predicted IL-22 coding region consists of 522 nucleotides which translates into a 173 amino acid protein, that contains an IL-10 family signature which is reasonably well conserved with other vertebrate IL-22 molecules. Expression analysis in tissues from healthy fish revealed a higher constitutive expression of IL-22 in mucosal tissues, suggesting a potentially important role in mucosal immunity. In vitro studies demonstrated that IL-22 expression was induced significantly by PHA and PMA in splenocyte primary cultures 4h post-stimulation. Expression was also induced in the spleen upon infection of fish with the Gram-negative bacterium Yersinia ruckeri, suggesting a potential role of IL-22 in vivo in defence against bacterial diseases. The Escherichia coli produced recombinant IL-22 enhanced the expression of a number of antimicrobial peptides, promoting host innate immunity against microbes and revealing a biological similarity with its mammalian counterpart.     

The functional characterisation of CK-1, a putative CC chemokine from rainbow trout (Oncorhynchus mykiss)

Recently a number of cytokine homologs have been cloned in teleost fish, including several that resemble chemokines, but to date few have been confirmed using functional assays. Chemokines are a family of cytokines that are able to induce chemotaxis in leucocytes. In this study CK-1, a rainbow trout chemokine, was functionally characterised. Recombinant CK-1 is able to attract rainbow trout peripheral blood leucocytes (PBL) in a micro-chemotaxis chamber. A greater number of PBLs migrated in response to CK-1 than to negative controls, either media alone or equivalent concentrations of beta2M, while comparable numbers migrated to the positive control, recombinant human C5a. The tissue distribution of CK-1 mRNA was also assessed by Northern blotting of RT-PCR and showed that expression is constitutive in the liver and gut, and is inducible by intraperitoneal injection of phytohemagglutinin in PBL and the head-kidney. Continuous cell lines generated from the gut and pituitary gland of the rainbow trout also express CK-1 message, whilst Southern analysis shows that CK-1 is a single copy gene. Finally, CK-1 shows the greatest amino acid similarity CCL20/LARC/Mip-3alpha as well as similar gene structure and expression pattern.     

Ubiquitin genes in rainbow trout (Oncorhynchus mykiss)

Ubiquitin is a small protein involved in intracellular proteolysis. It is highly conserved throughout eukaryotic phyla and has been detected in such diverse species as yeast, barley, Drosophila and man. A previous study showed that chromatin of rainbow trout testis contains free ubiquitin with a sequence similar to that of other phyla. In the present study, which focused on rainbow trout but included eleven other species, it is shown that fish ubiquitin genetic organisation and expression are similar to those of other phylogenetic groups through the following set of observations: (a) Multiple loci were detected, (b) These loci encode repeats of ubiquitin, (c) Although the DNA sequences are not conserved, the encoded amino acid sequences are fully conserved, (d) The expression of ubiquitin was influenced by cell culture conditions and viral infection.     

Cloning and characterization of glucose transporter in teleost fish rainbow trout (Oncorhynchus mykiss)

The facilitated diffusion of monosaccharides across the plasma membrane is mediated by glucose transporters (GLUTs). In contrast to mammals, the glucose transport system of lower vertebrates remains unexplored. We detected glucose transport activity in rainbow trout embryos. Two GLUTs sharing 83% amino acid identity were cloned from juvenile fish, these have been denoted OnmyGLUT1A and OnmyGLUT1B. In adult trout OnmyGLUT1A is predominantly expressed in the heart with low expression in other tissues. An inverse terminal repeat of a Tc1-like transposable element was found in the 3'-untranslated region of OnmyGLUT1B. Phylogenetic analysis suggested that rainbow trout genes share a common ancestor with higher vertebrate GLUT1. We also found GLUT genes in several salmonid species.     

Peripheral serotonin dynamics in the rainbow trout (Oncorhynchus mykiss)

Serotonin (5-hydroxytryptamine, 5-HT) occurs in a wide range of tissues throughout the body of the rainbow trout. Results reported here indicate that the main peripheral sources of serotonin are the intestinal tract and the gill epithelium (levels above 1500 ng/g). The high intestinal serotonin concentration is mostly due to serotoninergic nerve fibres, which are present at high density in the intestinal wall. Only about 2% of serotonin is associated with mucosal enterochromaffin cells. In the remaining tissues studied serotonin concentration was below 160 ng/g: the highest concentrations were seen in the anterior and posterior kidneys, followed by the liver, heart, and spleen. 5-Hydroxyindolacetic acid (5-HIAA) levels, except in plasma, were generally lower than serotonin levels, and were below our detection limits in heart, spleen and posterior kidney. Acute d-fenfluramine treatment (5 or 15 mg/kg i.p.) significantly increased 5-HIAA/5-HT ratio in the anterior intestine, pyloric caeca and plasma. Serotonin released from intestinal serotoninergic fibres in response to d-fenfluramine treatment is metabolized locally, and only a small part reaches the blood, from where it can be taken up and metabolized by other peripheral tissues, such as the liver and gill epithelium. The non-metabolized serotonin pool in the blood appears to be located extracellularly, not intracellularly as in mammals. In view of these findings, we present an overview of peripheral serotonin dynamics in rainbow trout.     

Morphological and functional development of the thyroid tissue in rainbow trout (Oncorhynchus mykiss) embryos

The development of the thyroid tissue in rainbow trout (Oncorhynchus mykiss) embryos between 20 and 88 days postfertilization (dpf) was studied using traditional histological methods in combination with immunostaining using antibodies raised against L-thyroxine (T4) and triiodo-L-thyronine (T3). In 20 dpf embryos, the thyroid primordium appeared as a simple tube aligned with the dorsal surface of the ventral aorta. The tubular primordium becomes progressively bifurcated at each end and follicles are formed by pinching off balls of cells from the tips of the bifurcations. The secretory activity of the thyroid tissue appears first as a synthesis phase, evident at 30 dpf, characterized by T4 and T3 immunostaining present only in the follicle lumen. A later hormone release or secretory phase was first evident for T4 immunostaining at 43 dpf and for T3 immunostaining at 46 dpf and was characterized by the presence of immunostaining both within the follicle lumen and also in the cytoplasm of some (but not all) thyrocytes. This stage was also coincident with a marked increase in the number of pituitary thyrotrophs. The proportion of embryos exhibiting immunostained thyrocytes increased progressively from 43 to 52 dpf, at which stage all embryos had T4- and T3-immunostained thyrocytes.     

Regulation of the rainbow trout (Oncorhynchus mykiss) hepatic beta2-adrenoceptor by adrenergic agonists

The characteristics of hepatic beta(2)-adrenoceptors (AR) were examined in rainbow trout (Oncorhynchus mykiss) chased once per day to exhaustion for up to 7 days or fed the repartitioning agents clenbuterol (CLEN) or ractopamine (RACT) that function in mammals as beta-agonists. A one-day chase and feeding the CLEN for 37 days resulted in a significant 27% and 33% decrease, respectively, in the number of CGP-binding sites (B(max)) with no significant change in affinity (Kd) of hepatic beta(2)-ARs. Despite the significant decrease in beta(2)-AR numbers with CLEN feeding, no significant differences were found for either beta(2)-AR mRNA levels or adenylyl cyclase (ACase) activities. In addition, CLEN displayed only partial agonist activities as it was found to be more effective at blocking isoproterenol-stimulated cAMP production in isolated hepatocytes than stimulating cAMP production. The small affects of RACT may be related to its low active stereoisomer content and low affinity for the trout beta(2)-AR. Agonist regulation of the trout hepatic beta(2)-ARs may involve down-regulation of the receptors without affecting responsiveness.     

Testis transplantation in male rainbow trout (Oncorhynchus mykiss)

The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n = 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P: < 0.05) smaller than the intact testes (transplants = 1.2 g; intact = 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%).     

Monoclonal antibodies to lymphocytes of rainbow trout (Oncorhynchus mykiss)

Monoclonal antibodies (Mabs) to lymphocytes of rainbow trout have been developed by immunisation with synthetic peptides, prepared from selected parts of the alpha- and beta-gene sequences of the T-cell receptor (TCR). Mab 1C2 (TCR beta immunisation) identified lymphocytes in blood (11%), spleen (18%) and in thymus (9%) in flow cytometry analysis (FCM). Immune complexes of lymphocytes coupled to Mab 1C2 was used for further immunisations resulting in numerous supernatants reactive with lymphocytes in FCM, of which Mabs 7A5 and 8H4 were selected for further characterisation. Mab 7A5 identified 31% of lymphocytes in blood and 9% in the spleen. Mab 8H4 labelled 61% and 85% of lymphocytes in the same organs. Mab 8H4 reacted with the majority of the lymphocytes in the thymus (98%). Mabs 1C2, 7A5 and 8H4 recognised surface markers on both Ig(-) and Ig(+) lymphocytes in peripheral blood and in spleen in double staining experiments. An increased proportion of Ig(-) lymphocytes were identified when Ig(+) lymphocytes were eliminated by immunomagnetic separation. No cross-reactivity of Mabs 1C2, 7A5 or 8H4 to anti-thrombocyte Mabs was detected. Mab 1C2 captured molecules of about 40 and also of 55-60kDa, in an immunoprecipitation assay. Mab 7A5 recognised an antigen of approximately 75-80kDa and Mab 8H4 identified proteins of about 70, 100 and 150kDa. Immunohistochemical staining by Mab 8H4 of fixed thymus, revealed a strong labelling of lymphoid cells in the outer zones of thymus. The 8H4 positive lymphoid cells surrounds circular structures, which were not labelled by Mab 8H4. These distinctly appearing structures have a similar shape as nurse cells described in mammals.