Subcellular localization of Na+/K+-ATPase isoforms resolved by in situ hybridization chain reaction in the gill of chum salmon at freshwater and seawater.

The Na+/K+-ATPase (NKA) α1-isoforms were examined by in situ hybridization chain reaction (ISHCR) using short hairpin DNAs, and we showed triple staining of NKA α1a, α1b, and α1c transcripts in the gill of chum salmon acclimated to freshwater (FW) and seawater (SW). The NKA α1-isoforms have closely resembled nucleotide sequences, which could not be differentiated by conventional in situ hybridization. The ISHCR uses a split probe strategy to allow specific hybridization using regular oligo DNA, resulting in high specificity at low cost. The results showed that NKA α1c was expressed ubiquitously in gill tissue and no salinity effects were observed. FW lamellar ionocytes (type-I ionocytes) expressed cytoplasmic NKA α1a and nuclear NKA α1b transcripts. However, both transcripts of NKA α1a and α1b were present in the cytoplasm of immature type-I ionocytes. The developing type-I ionocytes increased the cytoplasmic volume and migrated to the distal region of the lamellae. SW filament ionocytes (type-II ionocytes) expressed cytoplasmic NKA α1b transcripts as the major isoform. Results from morphometric analysis and nonmetric multidimensional scaling indicated that a large portion of FW ionocytes was NKA α1b-rich, suggesting that isoform identity alone cannot mark the ionocyte types. Both immature or residual type-II ionocytes and type-I ionocytes were found on the FW and SW gills, suggesting that the chum salmon retains the potential to switch the ionocyte population to fit the ion-transporting demands, which contributes to their salinity tolerance and osmoregulatory plasticity.

Transduction of Brain Neurons in Juvenile Chum Salmon (Oncorhynchus keta) with Recombinant Adeno-Associated Hippocampal Virus Injected into the Cerebellum during Long-Term Monitoring.

Corpus cerebelli in juvenile chum salmon is a multiprojective region of the brain connected via afferent and efferent projections with the higher regions of the brainstem and synencephalon, as well as with multiprojection regions of the medulla oblongata and spinal cord. During the postembryonic development of the cerebellum in chum salmon, Oncorhynchus keta, the lateral part of the juvenile cerebellum gives rise to the caudomedial part of the definitive cerebellum, which is consistent with the data reported for zebrafish and mouse cerebellum. Thus, the topographic organization of the cerebellum and its efferents are similar between fish (chum salmon and zebrafish) and mammals, including mice and humans. The distributions of recombinant adeno-associated viral vectors (rAAVs) after an injection of the base vector into the cerebellum have shown highly specific patterns of transgene expression in bipolar neurons in the latero-caudal lobe of the juvenile chum tectum opticum. The distribution of rAAVs in the dorsal thalamus, epithalamus, nucleus rotundus, and pretectal complex indicates the targeted distribution of the transgene via the thalamo-cerebellar projections. The detection of GFP expression in the cells of the epiphysis and posterior tubercle of juvenile chum salmon is associated with the transgene's distribution and with the cerebrospinal fluid flow, the brain ventricles and its outer surface. The direct delivery of the rAAV into the central nervous system by intracerebroventricular administration allows it to spread widely in the brain. Thus, the presence of special projection areas in the juvenile chum salmon cerebellum, as well as outside it, and the identification of the transgene's expression in them confirm the potential ability of rAAVs to distribute in both intracerebellar and afferent and efferent extracerebellar projections of the cerebellum.

A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, Oncorhynchus keta, Using Mouse Adeno-Associated Viral Vectors.

To date, data on the presence of adenoviral receptors in fish are very limited. In the present work, we used mouse recombinant adeno-associated viral vectors (rAAV) with a calcium indicator of the latest generation GCaMP6m that are usually applied for the dorsal hippocampus of mice but were not previously used for gene delivery into fish brain. The aim of our work was to study the feasibility of transduction of rAAV in the mouse hippocampus into brain cells of juvenile chum salmon and subsequent determination of the phenotype of rAAV-labeled cells by confocal laser scanning microscopy (CLSM). Delivery of the gene in vivo was carried out by intracranial injection of a GCaMP6m-GFP-containing vector directly into the mesencephalic tegmentum region of juvenile (one-year-old) chum salmon, Oncorhynchus keta. AAV incorporation into brain cells of the juvenile chum salmon was assessed at 1 week after a single injection of the vector. AAV expression in various areas of the thalamus, pretectum, posterior-tuberal region, postcommissural region, medial and lateral regions of the tegmentum, and mesencephalic reticular formation of juvenile O. keta was evaluated using CLSM followed by immunohistochemical analysis of the localization of the neuron-specific calcium binding protein HuCD in combination with nuclear staining with DAPI. The results of the analysis showed partial colocalization of cells expressing GCaMP6m-GFP with red fluorescent HuCD protein. Thus, cells of the thalamus, posterior tuberal region, mesencephalic tegmentum, cells of the accessory visual system, mesencephalic reticular formation, hypothalamus, and postcommissural region of the mesencephalon of juvenile chum salmon expressing GCaMP6m-GFP were attributed to the neuron-specific line of chum salmon brain cells, which indicates the ability of hippocampal mammal rAAV to integrate into neurons of the central nervous system of fish with subsequent expression of viral proteins, which obviously indicates the neuronal expression of a mammalian adenoviral receptor homolog by juvenile chum salmon neurons.

Cardiovascular Active Peptides of Marine Origin with ACE Inhibitory Activities: Potential Role as Anti-Hypertensive Drugs and in Prevention of SARS-CoV-2 Infection.

Growing interest in hypertension-one of the main factors characterizing the cardiometabolic syndrome (CMS)-and anti-hypertensive drugs raised from the emergence of a new coronavirus, SARS-CoV-2, responsible for the COVID19 pandemic. The virus SARS-CoV-2 employs the Angiotensin-converting enzyme 2 (ACE2), a component of the RAAS (Renin-Angiotensin-Aldosterone System) system, as a receptor for entry into the cells. Several classes of synthetic drugs are available for hypertension, rarely associated with severe or mild adverse effects. New natural compounds, such as peptides, might be useful to treat some hypertensive patients. The main feature of ACE inhibitory peptides is the location of the hydrophobic residue, usually Proline, at the C-terminus. Some already known bioactive peptides derived from marine resources have potential ACE inhibitory activity and can be considered therapeutic agents to treat hypertension. Peptides isolated from marine vertebrates, invertebrates, seaweeds, or sea microorganisms displayed important biological activities to treat hypertensive patients. Here, we reviewed the anti-hypertensive activities of bioactive molecules isolated/extracted from marine organisms and discussed the associated molecular mechanisms involved. We also examined ACE2 modulation in sight of SARS2-Cov infection prevention.

The Olfactory Transcriptome and Progression of Sexual Maturation in Homing Chum Salmon Oncorhynchus keta.

Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone ß subunit (lhß) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17ß-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation.

Effect of Marine Collagen Peptides on Physiological and Neurobehavioral Development of Male Rats with Perinatal Asphyxia.

Asphyxia during delivery produces long-term deficits in brain development. We investigated the neuroprotective effects of marine collagen peptides (MCPs), isolated from Chum Salmon skin by enzymatic hydrolysis, on male rats with perinatal asphyxia (PA). PA was performed by immersing rat fetuses with uterine horns removed from ready-to-deliver rats into a water bath for 15 min. Caesarean-delivered pups were used as controls. PA rats were intragastrically administered with 0.33 g/kg, 1.0 g/kg and 3.0 g/kg body weight MCPs from postnatal day 0 (PND 0) till the age of 90-days. Behavioral tests were carried out at PND21, PND 28 and PND 90. The results indicated that MCPs facilitated early body weight gain of the PA pups, however had little effects on early physiological development. Behavioral tests revealed that MCPs facilitated long-term learning and memory of the pups with PA through reducing oxidative damage and acetylcholinesterase (AChE) activity in the brain, and increasing hippocampus phosphorylated cAMP-response element binding protein (p-CREB) and brain derived neurotrophic factor (BDNF) expression.

Comparative studies of oral administration of marine collagen peptides from Chum Salmon (Oncorhynchus keta) pre- and post-acute ethanol intoxication in female Sprague-Dawley rats.

The present study aimed to evaluate the effect of an oral administration of marine collagen peptides (MCPs) pre- and post-acute ethanol intoxication in female Sprague-Dawley (SD) rats. MCPs were orally administered to rats at doses of 0 g per kg bw, 2.25 g per kg bw, 4.5 g per kg bw and 9.0 g per kg bw, prior to or after the oral administration of ethanol. Thirty minutes after ethanol treatment, the effect of MCPs on motor incoordination and hypnosis induced by ethanol were investigated using a screen test, fixed speed rotarod test (5 g per kg bw ethanol) and loss of righting reflex (7 g per kg bw ethanol). In addition, the blood ethanol concentrations at 30, 60, 90, and 120 minutes after ethanol administration (5 g per kg bw ethanol) were measured. The results of the screen test and fixed speed rotarod test suggested that treatment with MCPs at 4.5 g per kg bw and 9.0 g per kg bw prior to ethanol could attenuate ethanol-induced loss of motor coordination. Moreover, MCP administered both pre- and post-ethanol treatment had significant potency to alleviate the acute ethanol induced hypnotic states in the loss of righting reflex test. At 30, 60, 90 and 120 minutes after ethanol ingestion at 5 g per kg bw, the blood ethanol concentration (BEC) of control rats significantly increased compared with that in the 4.5 g per kg bw and 9.0 g per kg bw MCP pre-treated groups. However, post-treatment with MCPs did not exert a significant inhibitory effect on the BEC of the post-treated groups until 120 minutes after ethanol administration. Therefore, the anti-inebriation effect of MCPs was verified in SD rats with the possible mechanisms related to inhibiting ethanol absorption and facilitating ethanol metabolism. Moreover, the efficiency was better when MCPs were administered prior to ethanol.

A chronic oral toxicity study of marine collagen peptides preparation from chum salmon (Oncorhynchus keta) skin using Sprague-Dawley rat.

Due to the increased consumption of marine collagen peptides preparation (MCP) as ingredients in functional foods and pharmaceuticals, it was necessary to carry out safety requirements in the form of an oral chronic toxicity assessment. In order to define the oral chronic toxicity of MCP, a 24-month feeding study of MCP was carried out. Sprague-Dawley (S-D) rats at the age of four-week of both sexes were treated with MCP at the diet concentrations of 0%, 2.25%, 4.5%, 9% and 18% (wt/wt). The actual food intake and bodyweight of the individual animals were recorded periodically until sacrifice. Blood and urine samples were collected for serum chemistry evaluations and urinalysis. Throughout the experimental period, there was no toxicologically significant difference between the vehicle and MCP-treated animals with respect to the survival rate, body weight, food consumption, urinalysis, clinical biochemistry parameter and relative organ weight in either sex. Moreover, incidences of non-neoplastic lesions in MCP-treated groups did not significantly increase compared with the control group. Under the present experimental conditions, no higher risk of chronic toxic effects was observed in MCP-treated rats at the diet concentrations of 2.25%, 4.5%, 9% and 18% (wt/wt) than in the rats fed with basal rodent diet.

Oral administration of marine collagen peptides from Chum Salmon skin enhances cutaneous wound healing and angiogenesis in rats.

BACKGROUND: A wound is a clinical entity which often poses problems in clinical practice. The present study was aimed to investigate the wound healing potential of administering marine collagen peptides (MCP) from Chum Salmon skin by using two wound models (incision and excision) in rats. RESULTS: Ninety-six animals were equally divided into the two wound models and then within each model animals were randomly divided into two groups: vehicle-treated group and 2 g kg(-1) MCP-treated group. Wound closure and tensile strength were calculated. Collagen deposition was assessed by Masson staining and hydroxyproline measurement. Angiogenesis was assessed by immunohistological methods. MCP-treated rats showed faster wound closure and improved tissue regeneration at the wound site, which was supported by histopathological parameters pertaining to wound healing. MCP treatment improved angiogenesis and helped form thicker and better organised collagen fibre deposition compared to vehicle-treated group. CONCLUSION: The results show the efficacy of oral MCP treatment on wound healing in animals.

Marine collagen peptides prepared from chum salmon (Oncorhynchus keta) skin extend the life span and inhibit spontaneous tumor incidence in Sprague-Dawley Rats.

To observe the effects of marine collagen peptides (MCPs) prepared from chum salmon (Oncorhynchus keta) skin on life span and spontaneous tumor incidence, Sprague-Dawley rats were fed diets supplemented with MCP at concentrations of 0%, 2.25%, 4.5%, and 9% (wt/wt) from the age of 4 weeks until natural death. There were 40 rats in each group (male:female ratio = 1:1). The results showed that the MCP did not significantly influence body weight or food consumption of rats of either sex throughout the life span; it did dose-dependently inhibit the age-related decrease in the activities of antioxidant enzymes and the age-related increase in the levels of lipid peroxidation product in both sexes. MCP notably increased the mean life span, the life span of the last 30% of the survivors, and the maximal life span; it decreased overall spontaneous tumor incidence of both sexes with significance in the 4.5% and 9% MCP-treated male groups and 9% MCP-treated female group. Compared to the control group, the incidence of death from tumors was decreased in MCP groups in comparison with the control group of both sexes. Therefore, we concluded that MCPs dose-dependently increase life span and decrease spontaneous tumor incidence in Sprague-Dawley rats. Moreover, the antioxidative property of MCPs may be responsible for the increased life span and protection against tumor development.

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration.

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River-Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na(+),K(+)-ATPase activity in the coastal SW fish and the plasma Na(+) levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.

The function of rhamnose-binding lectin in innate immunity by restricted binding to Gb3.

L-rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3, on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs.

Chum salmon trypsin-catalyzed preferential formation of peptides containing D-amino acid.

Chum salmon trypsin-catalyzed peptide synthesis has been studied by using nine series of "inverse substrates," i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- and m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters derived from Nalpha-(tertbutyloxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. All substrates tested in this study undergo less enantioselective coupling reaction, and the coupling product was the favorably obtained D-series rather than L-series (in the present case; Nalpha-Boc-D-Ala and Nalpha-Boc-L-Ala). The optimum condition for the coupling reaction was studied by changing the organic solvent, buffer solution, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.

Molecular cloning and expression of an otolith matrix protein cDNA from the rainbow trout, Oncorhynchus mykiss.

The fish otolith is a hard tissue consisting of calcium carbonate and organic matrices. The matrix proteins play important roles in otolith formation, but little is known about the nature of these proteins. In this study, matrix proteins were extracted from the otoliths of rainbow trout, Oncorhynchus mykiss, and chum salmon, Oncorhynchus keta. EDTA-soluble matrix proteins were separated by SDS-PAGE, revealing two major components in the otoliths of both species with apparent molecular masses of 55 and 43 kDa. N-terminal and some internal amino acid sequences of the 55-kDa otolith matrix protein were determined. A cDNA fragment encoding this protein of O. mykiss was amplified by reverse transcription PCR using two degenerate primers designed from the amino acid sequences. A cDNA encoding this protein was obtained by screening a saccular cDNA library using the amplified cDNA fragment as a probe. Nucleotide sequence analysis revealed that the cDNA clone has a sequence of 2.5 kb and the open reading frame encoding 344 amino acid residues. Northern blot analysis showed that mRNA of this protein is expressed specifically in the sacculus, and consistently during the day.

Miltpain, new cysteine proteinase from the milt of chum salmon, Oncorhynchus keta.

A new cysteine proteinase, salmon miltpain, was isolated and purified from the milt of chum salmon (Oncorhynchus keta). Native molecular mass was estimated as 67,000 by gel filtration column chromatography (Shodex WS2003) and 22,300 by SDS-polyacrylamide gel electrophoresis. Isoelectoric point was determined to be 3.9 by isoelectric focusing. The first 15 amino acid residues in the N-terminal region were LPSFLY-AEMVGYNIL. The cysteine proteinase, which had a pH optimum of 6.0 for Z-Arg-Arg-MCA hydrolysis, required a thiol-reducing reagent for activation and was inhibited by E-64, iodacetamide, CA-074 Me, TLCK, TPCK and ZPCK. The cysteine proteinase exhibited unique substrate specificity toward paired basic residues such as Lys-Arg, Arg-Arg at the subsites of P2-P1 and had a K(m) of 16.3 microM and kcat of 20.3 s-1 with Z-Arg-Arg-MCA as substrate and a K(m) of 52.9 microM and kcat of 1.79 s-1 with Z-Phe-Arg-MCA. This proteinase was found to considerably hydrolyze basic proteins such as histone, salmine and clupaine but not milk casein.

Gonadotropin-releasing hormone (GnRH): expression during salmon migration.

In the seaward migrating chum salmon, immunocytochemical and in situ hybridization techniques revealed isolated GnRH neurons at the base of the nasal epithelium, along the nervus terminalis and as ganglia at the rostroventral (gROB) and caudalmost (GT) olfactory bulb. A novel GnRH ganglion was seen at the cribriform bone (gCB). GnRH immunoreactivity but not the hybridization signal was detected in the midbrain neurons. During the migratory period, there were trends towards an increase in GnRH mRNA in the gCB and the gROB and a significant surge in plasma thyroid hormones was also evident. Therefore, we hypothesise thyroid hormones might be crucial for the increased tendency of GnRH expression and the migratory behavior of chum salmon.