Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: evolution of neurohypophysial hormone precursors.

We have cloned and determined the nucleotide sequences of cDNAs encoding precursors of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), from the hypothalamus of masu salmon, Oncorhynchus masou. The deduced amino acid sequences of masu salmon VT and IT precursors (proVT-I and proIT-I) are highly homologous to those of chum salmon proVT-I and proIT-I, respectively. The VT and IT precursors are composed of a signal peptide, hormone and neurophysin (NP), the middle portion of which is highly conserved among vertebrates. Both the NPs extend about 30 amino acids at the C-terminal. The extended C-terminals have a leucin-rich segment in the carboxyl-terminal, as copeptin of vasopressin precursor. Southern bot analysis showed the presence of two types of proVT genes (proVT-I and proVT-II) and proIT genes (proIT-I and proIT-II) in an individual masu salmon, as in a chum salmon. Southern blot analysis with proVT probes further suggested that at least two different types of proVT-I genes exist in a single masu salmon. Northern blot analysis indicated that proVT-I and proIT-I genes are expressed in the hypothalamus, whereas proVT-II and proIT-II genes are not expressed. Evolutionary distance between proVT-I and proIT-I genes was statistically estimated based on synonymous nucleotide substitution in the coding region of the cDNAs. The magnitude of distance between masu salmon proVT-I and proIT-I genes suggested that the highly conserved central portion of NPs resulted from a gene conversion event. Between masu salmon and chum salmon, evolutionary distance for proVT-I genes is about 6-fold larger than that for proIT-I genes.     

Cloning and sequence analyses of cDNAs encoding vasotocin and isotocin precursors of chum salmon,Oncorhynchus keta: evolutionary relationships of neurohypophysial hormone precursors

Summary The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.Abbreviations AVP vasopressin - C carboxyl - h human - IT isotocin - MT mesotocin - N amino - OXT oxytocin - S chum salmon - SDS sodium dodecyl sulfate - t toad - VT vasotocin     

Isolation and structures of coho salmon (Oncorhynchus kisutch) glucagon and glucagon-like peptide

Glucagon and glucagon-like peptide (GLP) containing 31 amino acids have been isolated from the principal islet of coho salmon (Oncorhynchus kisutch) by gel filtration of acid alcohol extracts followed by HPLC, and the complete amino acid sequence of both peptides has been determined. Salmon glucagon is a simple 29 residue peptide differing at 3 positions when compared to catfish glucagon and at 8 positions when compared to porcine glucagon. Salmon GLP differs at 6 positions when compared with the N-terminal 31 amino acids of the 34 amino acid catfish GLP. Both coho salmon glucagon and GLP cross-react weakly in our mammalian glucagon radioimmunoassay and therefore this technique could not be used to determine tissue content. Glucagon and GLP isolated amounted to 156 micrograms/g and 350 micrograms/g wet tissue, respectively.     

Expression of olfactory receptors in different life stages and life histories of wild Atlantic salmon (Salmo salar)

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. However, the key components of the molecular pathway involved in imprinting and homing are still unknown. If odorants are involved in salmon homing migration, then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, we examined the expression profiles of a suite of genes encoding olfactory receptors and other olfactory-related genes in the olfactory rosettes of different life stages in two anadromous and one non-anadromous wild Atlantic salmon populations from Newfoundland, Canada. We identified seven differentially expressed OlfC genes in juvenile anadromous salmon compared to returning adults in both populations of anadromous Atlantic salmon. The salmon from the Campbellton River had an additional 10 genes that were differentially expressed in juveniles compared to returning adults. There was no statistically significant difference in gene expression of any of the genes in the non-anadromous population (P < 0.01). The function of the OlfC gene products is not clear, but they are predicted to be amino acid receptors. Other studies have suggested that salmon use amino acids for imprinting and homing. This study, the first to examine the expression of olfactory-related genes in wild North American Atlantic salmon, has identified seven OlfC genes that may be involved in the imprinting and homeward migration of anadromous Atlantic salmon.     

Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factor I mRNA

Protein and cDNA sequence analysis have revealed that the insulin-like growth factor (IGF-I) has been highly conserved among several mammalian species. Using the combined techniques of polymerase chain reaction and molecular cloning, we have now obtained the cDNA sequence encoding preproIGF-I from a teleost species, Oncorhynchus kisutch (coho salmon). The 2020 nucleotide (nt) cloned cDNA sequence contains a 528 nt open reading frame encoding 176 amino acids in preproIGF-I and 175 nt and 1317 nt of flanking 5'- and 3'-untranslated regions, respectively. The deduced amino acid sequence of salmon IGF-I is highly conserved relative to its mammalian homologues and there are only 14 amino acid differences out of 70 between salmon and human IGF-I. Interestingly, the C-terminal E domain of salmon proIGF-I, which is presumed to be proteolytically cleaved during biosynthesis, also shows striking amino acid sequence homology with its mammalian counterpart, except for an internal 27 residue segment that is unique to salmon proIGF-I. Northern analysis revealed that salmon preproIGF-I mRNA consists predominantly of a single 3900 nt sized band although minor bands were also observed after prolonged autoradiographic exposure. The RNA analysis also revealed that the level of preproIGF-I mRNA is increased 6-fold in liver RNA isolated from salmon injected with bovine GH, as compared to untreated controls. These results demonstrate that the primary structure and regulated expression of IGF-I by GH have been conserved in teleosts.     

Species-dependent migration of fish hatching gland cells that commonly express astacin-like proteases in common

Two constituent proteases of the hatching enzyme of the medaka ( Oryzias latipes ), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above-mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish ( Brachydanio rerio ) and masu salmon ( Oncorynchus masou ) embryos. Using the amplified fragments as probes, two full-length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.     

Differences in digestion and absorption of dietary protein in Atlantic salmon (Salmo salar) with genetically different trypsin isozymes

Digestion and absorption of dietary protein were studied through facilitation of amino acid in the plasma and white muscle after a single feeding. The comparison was made between Atlantic salmon with and without trypsin isozyme TRP-2*92. Higher absorption of dietary protein was associated with the presence of the isozyme, as the post-prandial total levels of free amino acids (FAA) in both plasma and white muscle were significantly higher in salmon with the isozyme than those in salmon without it. Higher digestion rate of the dietary protein in salmon carrying the isozyme was indicated by faster elevation of essential FAA in the plasma and of overall FAA in their white muscle. Other indications which suggest differences in nitrogen metabolism between salmon with and without the isozyme were the observations of significant differences in (a) the levels of lysine, hydroxyproline, alanine, aspartic acid, β-alanine, threonine, valine and a nitrogen-containing compound taurine in plasma, and (b) the levels of alanine, glutamic acid, glycine and anserine in white muscle.
Trypsin activity in the pyloric caeca showed less response to feeding than that in the intestine, but it may have consequence for growth as its activity was significantly higher in growing fish than in non-growing fish.     

Changes in yeast amino acid pool with respiratory versus fermentative metabolism

The precursors of the amino acid yeast pool are intermediates of either the glycolytic or the tricarboxilic acid pathway (TCA). Accordingly, the influence of the metabolism (fermentative or respiratory) on the internal amino acid pool of the yeast Saccharmyces cerevisiae was established by measuring the intracellular amino acid concentration of the "grande" strain IF1256 and its "petite" mutant either in steady-state or when shifting from fermentative to respiratory conditions. Under steady-state conditions, when the cells only respire, there is a decrease in nearly all the amino acids whose precursors are intermediates of the glycolytic pathway. When the metabolism is exclusively fermentative, the opposite change takes place. This effect is not observed in those amino acids whose precursors come from the TCA cycle. However, in continuous culture and at dilution rates lower than 0.06 h(-1), there is an enormous increase in the concentration of all the amino acids in both strains, whatever their precursor, whereas, in batch cultures, both strains undergo variations in the concentration of most amino acids, when entering stationary growth phase. Results therefore indicate that, the fact that the cells ferment or respire effectively affect their amino acid pool according to their precursors coming from the glycolytic or the TCA pathway, but other parameters, such as growth rate or exponential versus stationary phase, have a much stronger effect on yeast amino acid concentration.     

Isolation and characterization of chum salmon growth hormone

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by iso-electric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 microgram/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.     

Two differing salmon GnRH precursor mRNAs are co-expressed in the brain of sockeye salmon (Oncorhynchus nerka)

The localization of two salmon-type gonadotropin-releasing hormone (sGnRH) precursors, pro-sGnRH-I (short type) and pro-sGnRH-II (long type), was investigated by using in situ hybridization techniques in the brain of the landlocked sockeye salmon, Oncorhynchus nerka. We used 30-mer oligonucleotide probes complementary to pro-sGnRH-I and pro-sGnRH-II cDNA. No significant differences were observed in the localization of sGnRH neurons expressing pro-sGnRH-I and pro-sGnRH-II mRNAs; both were expressed in the olfactory nerve, the olfactory bulbs, the regions between the olfactory bulb and telencephalon, the ventral telencephalon, the preoptic area, and the hypothalamus. Almost all sGnRH neurons examined co-expressed both precursors. The expression of two sGnRH precursors in the same neuron and the wide distribution of such neurons in the brain suggest that there are no functional differences between the two precursors.     

Partial amino acid sequences of several globin chains from the sockeye salmon, Oncorhynchus nerka

1. Partial amino acid sequences for several sockeye salmon hemoglobin beta-chains have been determined and compared to several other fish beta-chain sequences. 2. A 90% homology exists between the sockeye cathodal (C1) beta-chain and the trout Hb I beta-chain for residues 1-19. 3. The sockeye salmon anodal (A1-3) beta-chain is virtually identical to the trout HB IV beta-chain for the first 55 amino acid residues. 4. The alpha-chains of the sockeye salmon appear to be acetylated at the N-terminal position and about 0.6% of the sockeye hemoglobin is glycosylated.     

鱼类斯钙素的研究进展

本文概述了近年来有关硬骨鱼类斯坦尼氏小体（ｃｏｒｐｕｓｃｌｅｓｏｆＳｔａｎｎｉｕｓ,ＣＳ）分泌激素———斯钙素（ｓｔａｎｎｉｏｃａｌｃｉｎ,ＳＴＣ）的研究进展。ＳＴＣ是糖蛋白类激素,为同型二聚体,其表观分子量在天然状态下从４６（大麻哈鱼）～５６（虹鳟）ｋＤａ,还原状态下则为２３～２８ｋＤａ。ＳＴＣ单体的氨基酸序列分析表明,大麻哈鱼、银大麻哈鱼和澳大利亚鳗鲡的氨基酸残基数分别为１７９、２２３和２３１个。研究还表明,ＳＴＣ的分泌受血钙浓度的调节,并且胆碱能神经参与ＳＴＣ的释放。     

Identification of proteoglycan from salmon nasal cartilage

There has been no structural information about the core protein of salmon nasal cartilage proteoglycan although its physiological activities have been investigated. Internal amino acid sequencing using nano-LC/MS/MS revealed that the salmon proteoglycan was aggrecan. Primer walk sequencing based on the amino acid information determined that the salmon aggrecan cDNA is comprised of 4207 bp nucleotides predicted to encode 1324 amino acids with a molecular mass of 143,276. It exhibited significant similarities to predicted pufferfish aggrecan, zebrafish similar to aggrecan, zebrafish aggrecan, bovine aggrecan and human aggrecan isoform 2 precursor; whose amino acid identities were 56%, 55%, 49%, 31% and 30%, respectively. Salmon cartilage aggrecan had globular domains G1, G2 and G3 as in mammalian aggrecans. Neither the putative keratan sulfate attachment domain enriched with serine, glutamic acid and proline, nor the putative chondroitin sulfate attachment domain with repeating amino acid sequence containing serine–glycine, found in mammalian aggrecans were observed in salmon, however, random serine–glycine (or glycine–serine) sequences predicted to the sugar chain attachment sites were observed. Based on cDNA analysis and amino acid analysis after β-elimination, the ratio of serine attached to sugar chains was calculated to be approximately 37.7% of total serine, that is, 46 of 123 serine residues.     

High levels of MHC class II allelic diversity in lake trout from Lake Superior

Sequence variation in a 216 bp portion of the major histocompatibility complex (MHC) II B1 domain was examined in 74 individual lake trout (Salvelinus namaycush) from different locations in Lake Superior. Forty-three alleles were obtained which encoded 71-72 amino acids of the mature protein. These sequences were compared with previous data obtained from five Pacific salmon species and Atlantic salmon using the same primers. Although all of the lake trout alleles clustered together in the neighbor-joining analysis of amino acid sequences, one amino acid allelic lineage was shared with Atlantic salmon (Salmo salar), a species in another genus which probably diverged from Salvelinus more than 10-20 million years ago. As shown previously in other salmonids, the level of nonsynonymous nucleotide substitution (dN) exceeded the level of synonymous substitution (dS). The level of nucleotide diversity at the MHC class II B1 locus was considerably higher in lake trout than in the Pacific salmon (genus Oncorhynchus). These results are consistent with the hypothesis that lake trout colonized Lake Superior from more than one refuge following the Wisconsin glaciation. Recent population bottlenecks may have reduced nucleotide diversity in Pacific salmon populations.     

Synthesis of biologically active tritiated amylin and salmon calcitonin analogues

Amylin is a hormone belonging to the calcitonin protein family of peptides. To facilitate receptor screening studies, alternatively radiolabeled and biologically active amylin and salmon calcitonin analogues were synthesized by reductive methylation. Free amino groups of amylin and salmon calcitonin were methylated by reaction of peptides with formaldehyde and sodium [(3)H]borohydride. Radioactively labeled peptides were purified by size exclusion chromatography followed by HPLC. Analysis by MALDI-TOF mass spectrometry of purified amylin and salmon calcitonin peptides revealed incorporation of both two and four tritiated methyl groups per peptide molecule. Specific activities of 22.6 and 23.2 GBq/mmol were measured for amylin and salmon calcitonin, respectively. Methylation of rat amylin and salmon calcitonin did not affect their biological activities as both retained their potency to inhibit insulin-stimulated glycogen synthesis in isolated rat soleus muscle. The synthesis of these tritiated analogues provides an alternative chemically stable radiolabeled ligand which may be useful in exploring receptor interactions within the calcitonin peptide family.     

Formation of heterocyclic amines using model systems

Initially, modeling was used to identify the mutagenic heterocyclic amines and their precursors. Major precursors have been shown to be single amino acids or amino acids together with creatine or creatinine. There is also evidence that Maillard reactions are involved since heating sugar and amino acids together with creatine or creatinine has been shown to produce several of the mutagenic heterocyclic amines, especially the aminoimidazoazaarenes (AIA compounds), e.g., IQ, MeIQ, MeIQx, DiMeIQx and PhIP. Due to a low yield in the model systems, the mechanisms behind the formation of the mutagenic heterocyclic amines are still unclear and need further substantiation. The fact that some AIA compounds are also produced in the absence of sugar casts some doubts on an obligatory participation of the Maillard reaction; alternative routes might exist. Further work using isotopically labeled precursors needs to be done and so far such work has only been performed for PhiP. The formation of mutagenic heterocyclic amines is dependent on time, temperature, pH, concentration of the precursors, type of amino acid, and the presence of certain divalent ions. Water may have an impact both as a temperature regulator and as a solvent medium for the reactants.     

Isolation, primary structure, and biological and immunological properties of pink and chum salmon insulins

1. Insulins have been isolated from islet tissue of pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon. The primary structure of chum and pink salmon insulins was found to be identical. Compared to the amino acid sequence of human insulin, the salmon insulins under study differed at 14 positions. 2. Biological activity of pink salmon insulin was 83% of that of standard porcine insulin. 3. The immunological properties of fish insulins were investigated in specific radioimmunoassay (RIA) systems, based on porcine and pink salmon insulins. 4. A significant difference in the antigenic determinants of these fish and mammalian hormones was revealed.     

cDNA cloning and sequence analysis of preproendothelin-1 (PPET-1) from salmon, Oncorhynchus keta

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).     

Characterization of parameters for in vitro culture of isolated ovarian follicles of greenback flounder Rhombosolea tapirina

Isolated ovarian follicles of greenback flounder Rhombosolea tapirina were incubated with a variety of gonadotropins (GtHs) and steroid precursors for periods of up to 42 h, and levels of free and glucuronated testosterone (T) and 17beta-estradiol (E(2)) in the medium, and free T and E(2) from inside follicles were measured by RIA. Short incubations (6 h) generated increases in T and E(2) in response to steroid precursors, but not human chorionic GtH (hCG), or salmon or carp GtH. At incubation times of 18 h, all GtHs stimulated T and, or E(2) production, whereas after 42-h incubation, GtH effects on E(2) production had disappeared. Steroid precursors remained effective at 18 and 42 h. T and E(2) glucuronides were formed in small quantities but did not account for loss of treatment effects at long incubation times. Instead, this could be explained by accumulation of E(2) in controls as a result of continued basal steroid production. Follicles absorbed substantial amounts of both endogenous and exogenous steroid from the medium, however, this did not appear to have any influence on changes in treatment effects with incubation time. Flounder follicles were most sensitive to hCG, followed by salmon and carp GtH at approximately 10-fold higher concentrations. Ovarian segments were not sensitive to any GtH but did convert exogenous steroid precursors indicating that tissue access by GtH may be a limiting factor under certain in vitro conditions. HCG augmented the conversion of 17-hydroxyprogesterone (17P) to T but not T to E(2), consistent with the relative GtH-insensitivity of aromatase in other species. Follicles converted a range of steroid precursors with equal competence, indicating that no step in the cleavage pathway is strongly rate-limited, and that choice of precursor is unlikely to affect the assessment of steroidogenic activity.     

Role of amino acid insertions on intermolecular forces between arginine peptide condensed DNA helices: implications for protamine-DNA packaging in sperm

In spermatogenesis, chromatin histones are replaced by arginine-rich protamines to densely compact DNA in sperm heads. Tight packaging is considered necessary to protect the DNA from damage. To better understand the nature of the forces condensing protamine-DNA assemblies and their dependence on amino acid content, the effect of neutral and negatively charged amino acids on DNA-DNA intermolecular forces was studied using model peptides containing six arginines. We have previously observed that the neutral amino acids in salmon protamine decrease the net attraction between protamine-DNA helices compared with the equivalent homo-arginine peptide. Using osmotic stress coupled with x-ray scattering, we have investigated the component attractive and repulsive forces that determine the net attraction and equilibrium interhelical distance as a function of the chemistry, position, and number of the amino acid inserted. Neutral amino acids inserted into hexa-arginine increase the short range repulsion while only slightly affecting longer range attraction. The amino acid content alone of salmon protamine is enough to rationalize the forces that package DNA in sperm heads. Inserting a negatively charged amino acid into hexa-arginine dramatically weakens the net attraction. Both of these observations have biological implications for protamine-DNA packaging in sperm heads.