Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus.

Interferon γ (IFN-γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN-γ is best described in humans, but less of IFN-γ in fish species has been described at protein level. In the present study, IFN-γ from Gadus macrocephalus (GmIFN-γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN-γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN-γ signature sequence at the C-terminal. GmIFN-γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN-γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN-γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET-32a-GmIFN-γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA-GmIFN-γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN-γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1-GmIFN-γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN-γ protein could also induce MICA expression, indicating that GmIFN-γ could stimulate immune response. The yeast GS115 with GmIFN-γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN-γ was upregulated significantly; however, genes of the interferon type-I signal pathway were not well stimulated.

Purification, structural characterization, and anticoagulant activity evaluation of chondroitin sulfate from codfish (Gadus macrocephalus) bones.

Chondroitin sulfate (CCS) was purified from discarded codfish (Gadus macrocephalus) bones, and its chemical structure and anticoagulant activity were assessed. CCS was obtained via enzymatic lysis and ion-exchange column chromatography, with a yield of approximately 0.15%. High-performance gel performance chromatography revealed CCS to be a largely homogeneous polysaccharide with a relatively low molecular weight of 12.3 kDa. FT-IR spectroscopy, NMR spectroscopy, and SAX-HPLC indicated that CCS was composed of monosulfated disaccharides (ΔDi4S 73.85% and ΔDi6S 19.06%) and nonsulfated disaccharides (ΔDi0S 7.09%). In vitro anticoagulation analyses revealed that CCS was able to significantly prolong activated partial thromboplastin time (APTT) and thrombin time (TT) (p < 0.05). At a CCS concentration of 5 µg/mL and 25 µg/mL, APTT and TT were approximately 1.08 and 1.12 times higher, respectively, compared to the negative control group. The results indicated that CCS might offer value as a dietary fiber supplement with the potential to prevent the incidence of coagulation-related thrombosis.

Isolation and Characterization of a Novel Sialoglycopeptide Promoting Osteogenesis from Gadus morhua Eggs.

Gadus morhua eggs contain several nutrients, including polyunsaturated fatty acids, lecithin and glycoproteins. A novel sialoglycopeptide from the eggs of G. morhua (Gm-SGPP) was extracted with 90% phenol and purified by Q Sepharose Fast Flow (QFF) ion exchange chromatography, followed by S-300 gel filtration chromatography. Gm-SGPP contained 63.7% carbohydrate, 16.2% protein and 18.6% N-acetylneuraminic acid. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that Gm-SGPP is a 7000-Da pure sialoglycopeptide. ß-elimination reaction suggested that Gm-SGPP contained N-glycan units. Amino acid N-terminal sequence analysis indicated the presence of Ala-Ser-Asn-Gly-Thr-Gln-Ala-Pro amino acid sequence. Moreover, N-glycan was connected at the third Asn location of the peptide chain through GlcNAc. Gm-SGPP was composed of D-mannose, D-glucuronic acid and D-galactose. Fourier transform-infrared spectroscopy (FT-IR), 1H-nuclear magnetic resonance spectroscopy (1H-NMR) and methylation analysis were performed to reveal the structure profile of Gm-SGPP. In vitro results showed that the proliferation activity of MC3T3-E1 cells was significantly promoted by Gm-SGPP. In vivo data revealed that Gm-SGPP increased the calcium and phosphorus content of tibias and promoted longitudinal bone growth in adolescent rats.

Effects of xylitol and stevioside on the physical and rheological properties of gelatin from cod skin.

Jelly and confectionery products are high in sugar and calories. Xylitol and stevioside are natural low-calorie sweeteners and they can be used as an alternative; however, their effects on fish gelatin are unknown. The gelatin was extracted from cod skins and added to xylitol or stevioside (0, 2, 6, 10, 14, and 20% (w/v)) to form gel products. This paper investigated how xylitol and stevioside affected the physical and rheological behaviors of fish gelatin, such as color, gel strength, texture profile analysis, storage modulus (G'), loss modulus (G″), and viscosity. Results showed that the change of color and viscosity in gel products were similar when various concentrations of xylitol or stevioside were added to the fish gelatin. But the effects of xylitol/stevioside on texture profile analysis and G', G″ were different, which might due to the structure variation in xylitol and stevioside. The linear structure of xylitol resulted in ionic interaction, hydrogen bonding, van der Waals forces, and hydrophobic association between xylitol and fish gelatin. Therefore, xylitol is a promising sweetener substitute, which was probably related to its greater solubility and number of -OH groups.

Striatal mitochondria response to 3-nitropropionic acid and fish oil treatment.

BACKGROUND: Mitochondrial dysfunction is involved in neurodegenerative diseases, such as Huntington's disease (HD). 3-Nitropropionic acid (3-NP) is a mitochondrial toxin that specifically inhibits complex II of the electron transport chain (ETC) and is used to generate an experimental model of HD. OBJECTIVE: To study the effect of fish liver oil (FO) over the mitochondrial dysfunction induced via partial ETC inhibition by 3-NP. METHODS: This study was performed in rats and consisted of two phases: (i) administration of increasing doses of 3-NP and (ii) administration of FO for 14 days before to 3-NP. The rats' exploratory activity; complex I, II, III, and IV activities; and rearing behavior were observed. Additionally, the number of TUNEL-positive cells and various mitochondrial parameters, including oxygen consumption, transmembrane potential, adenosine triphosphate synthesis, and ETC activity, were measured. RESULTS: We observed that FO exerted a protective effect against the 3-NP-induced toxicity, although complex II inhibition still occurred. Instead, this effect was related to strengthened mitochondrial complex III and IV activities. DISCUSSION: Our results show that FO exerts a beneficial prophylactic effect against mitochondrial damage. Elucidating the mechanisms linking the effects of FO with its prevention of neurodegeneration could be the key to developing recommendations for FO consumption in neurological pathologies.

Estimating the impact of petroleum substances on survival in early life stages of cod (Gadus morhua) using the dynamic energy budget theory.

To estimate the impact of accidental oil-spills on cod fisheries a model framework is developed in which a Dynamic Energy Budget (DEB) model is applied to assess mortality caused by petroleum substances in early life stages. In this paper we report on a literature search and DEB analyses, aiming for cod specific DEB-parameters. Furthermore, we explored the relevance of Fathead minnow DEB-parameters as surrogate by comparing LC50 values calculated from DEB-parameters with literature. Cod specific DEB-parameters could not be estimated based on available literature. LC50 values calculated from Fathead minnow DEB-parameters were higher than literature LC50 for early life stages of fish. Applying an extrapolation factor of 50 to the DEB-parameters resulted in LC50 values that were below literature irrespective of life stage. Therefore, we propose to use the last as an estimate for early life stages in cod and recommend relevant experiments with individual petroleum substances on cod.

Acetylcholinesterase activity in the host-parasite system of the cod Gadus morhua and acanthocephalan Echinorhynchus gadi from the southern Baltic Sea.

Acetylcholinesterase (AChE) activity measurement is widely used as a specific biomarker of neurotoxic effects. The aim of this study was to evaluate AChE activity in a host fish (the cod) and its acanthocephalan parasite Echinorhynchus gadi from the southern Baltic. AChE activity in hosts and parasites was inversely related: the highest cod AChE activity corresponded to the lowest E. gadi enzymatic activity and vice versa ("mirror effect"). This is the first report on the simultaneous application of this biomarker in cod and its acanthocephalan parasites. Results obtained for the host-parasite system are complementary and provide comprehensive information about the response of this biomarker. Analysis of the system allows for detection of a greater number of factors influencing AChE activity in the marine environment than separate analysis of the host and parasites. Thus, AChE activity measurement in a host-parasite system may be considered to be a promising tool for biomonitoring.

Melanin-concentrating hormone (MCH) and gonadotropin-releasing hormones (GnRH) in Atlantic cod, Gadus morhua: tissue distributions, early ontogeny and effects of fasting.

Melanin-concentrating hormone (MCH) is classically known for its role in regulating teleost fish skin color change for environmental adaptation. Recent evidence suggests that MCH also has appetite-stimulating properties. The gonadotropin-releasing hormone (GnRH) peptide family has dual roles in endocrine control of reproduction and energy status in fish. Atlantic cod (Gadus morhua) are a commercially important aquaculture species inhabiting the shores of Atlantic Canada. In this study, we examine MCH and GnRH transcript expression profiles during early development as well as in central and peripheral tissues and quantify juvenile Atlantic cod MCH and GnRH hypothalamic mRNA expressions following food deprivation. MCH and GnRH3 cDNAs are maternally deposited into cod eggs, while MCH has variable expression throughout early development. GnRH2 and GnRH3 mRNAs "turn-on" during mid-segmentation once the brain is fully developed. For both MCH and GnRH, highest expression appears during the exogenous feeding stages, perhaps supporting their functions as appetite regulators during early development. MCH and GnRH transcripts are found in brain regions related to appetite regulation (telencephalon/preoptic area, optic tectum/thalamus, hypothalamus), as well as the pituitary gland and the stomach, suggesting a peripheral function in food intake regulation. Atlantic cod MCH mRNA is upregulated during fasting, while GnRH2 and GnRH3 transcripts do not appear to be influenced by food deprivation. In conclusion, MCH might be involved in stimulating food intake in juvenile Atlantic cod, while GnRHs may play a more significant role in appetite regulation during early development.