Long-chain polyunsaturated fatty acids (LC-PUFA) are essential nutrients for membrane function, development, and immunity in animals. Polychaetes are promising sources of LC-PUFA due to their endogenous biosynthetic capacity, but optimal feeding strategies to enhance their nutritional value remain poorly understood. We investigated the nutritional regulation of LC-PUFA biosynthesis in the marine polychaete Platynereis dumerilii fed three diets differing in fatty acid (FA) profiles: spinach, yeast, and commercial fish feed. Juveniles were cultured for 37 days at 35‰ salinity, and survival, growth, total lipids, FA profiles, and expression of elongases, desaturases, and putative transcription factors were analysed. Survival rates were high across all diets (93.3-95.0%), while growth was significantly reduced in spinach-fed worms, which showed a lower specific growth rate (1.5 ± 0.1% day-1) than worms fed yeast (2.2 ± 0.1% day-1) or fish feed (2.0 ± 0.1% day-1). Diet strongly affected FA composition: fish feed increased n-3 LC-PUFA, spinach elevated α-linolenic acid, and yeast enhanced n-6 LC-PUFA. Despite low dietary LC-PUFA, eicosapentaenoic acid levels remained stable, indicating active endogenous synthesis. Gene expression responses were diet-dependent, with elongase Elovl2/5 expression higher in fish-feed-fed worms (0.8 ± 0.3) than in yeast-fed worms (0.4 ± 0.1), while the front-end desaturase Fed1 (Δ5 activity) and methyl-end desaturase ω des1 (Δ12 activity) showed their highest expression in spinach-fed worms (3.9 ± 1.8 and 2.4 ± 1.0, respectively). Overall, P. dumerilii adjusted its lipid metabolism in response to dietary inputs, demonstrating transcriptional plasticity and reinforcing its potential as a sustainable source of LC-PUFA.
Serine racemase (SR) catalyzes the interconversion of l-serine and d-serine and can also catalyze β-elimination of serine to pyruvate and ammonia. In mammals, however, the presence and enzymatic function of SR have been reported mainly in mouse, rat, and human. It is therefore unclear whether functional SR is broadly conserved across mammals or whether its enzymatic properties differ among lineages. To address this question, we produced and purified eleven SRs from representative eutherians, marsupials, and a monotreme and compared their catalytic properties. All enzymes examined showed both racemization and β-elimination activities. Together with the phylogenetic relationships, these activities suggest that functional SR was present before the monotreme-therian divergence and retained in the lineages examined. Specific activities and kinetic parameters nevertheless differed among species. Residues involved in cofactor and substrate binding were broadly conserved, whereas variation at the substrate-recognition region around positions 150-152 may contribute to the observed kinetic differences. Analysis of public transcriptome data further suggested species differences in tissue expression patterns. Together, these findings show conservation of SR function across the mammalian lineages examined, with species-dependent differences in catalytic properties and expression.
The permanently sub-zero temperatures and high oxygen solubility of the Southern Ocean promote the formation of reactive oxygen species (ROS) in the tissues of resident organisms, imposing persistent oxidative stress. Antarctic notothenioid fish have evolved efficient antioxidant systems to counter this pressure. This study reports the first molecular characterisation of the catalase (cat) transcript and deduced protein (CAT) sequence, together with tissue-specific CAT activity, in two Antarctic species with contrasting physiological traits: the red-blooded notothenioid Trematomus bernacchii and the haemoglobin-less icefish Chionodraco hamatus. Integrating cDNA sequencing with transcriptomic data, we reconstructed full-length cat coding sequences, performed Bayesian and maximum-likelihood phylogenetic analyses, and compared electrostatic surface properties using structural modelling. The cat transcript levels and CAT activity were quantified in liver, gills, heart, and skeletal muscle. Catalytic, NADPH-binding, and substrate-channel residues were fully conserved across Antarctic and non-Antarctic teleosts, consistent with strong purifying selection. T. bernacchii displayed a markedly more negative electrostatic surface than the icefish and non-Antarctic species, a pattern consistent with the increased surface acidity typical of cold-adapted proteins and with the higher oxidative burden of the red-blooded condition. The icefish showed a near-neutral electrostatic profile, consistent with its haemoglobin-less physiology. The liver was the principal site of both cat transcript accumulation and CAT activity in both species. Non-hepatic tissues showed a translation index below unity, consistent with post-transcriptional and/or post-translational regulation, with maximum translational efficiency shifting from the liver in T. bernacchii to the heart in C. hamatus. These findings suggest that enzyme surface properties and tissue-specific translational regulation may jointly contribute to antioxidant homeostasis under extreme marine conditions.
Fish skin mucus covers the body surface, serving as a physical and chemical first line of defense at the interface with the aquatic environment. Epidermal mucus provides a protective cushioning layer and contains numerous bioactive molecules involved in immune defense, antimicrobial activity, and tissue repair. During body-surface injury, direct contact between the plasma components and the mucus layer may influence plasma clotting. However, the role of epidermal mucus in this process remains poorly understood. This study investigated, under in vitro conditions, the effects of epidermal mucus from Japanese flounder (Paralichthys olivaceus) on plasma coagulation and examined coagulation-modulating components through biochemical fractionation. The addition of crude mucus to the flounder plasma shortened the fibrin clotting time and accelerated thrombin substrate cleavage under both prothrombin time-like and activated partial thromboplastin time-like conditions. Affinity fractionation of crude mucus using benzamidine-Sepharose showed that both the bound and unbound fractions retained procoagulant activity, suggesting that the observed activity was not solely attributable to serine protease-like components. Furthermore, size-based fractionation of the crude mucus using an ultrafiltration membranes and size-exclusion chromatography showed that procoagulant activity was distributed across multiple fractions in the approximate 29 to >2000 kDa range, whereas lower-molecular-weight fractions showed anticoagulant activity. These findings suggest that modulation of coagulation by mucus may contribute to the balance between local clot formation and the prevention of excessive coagulation at the epithelial surface.
Telomerase reverse transcriptase (tert), which encodes the major catalytic subunit of telomerase, is thought to play a central role in the regulation of telomerase activity. However, its transcriptional regulatory mechanisms remain unclear. In this study, we investigated the transcriptional regulatory mechanisms of tert in EPC (Epithelioma papulosum cyprini) cells, a teleost fish cell line derived from fathead minnow (Pimephales promelas) epithelium and the mechanisms underlying the maintenance of telomerase activity. A series of deletion mutants spanning the 2 kb upstream region of the fathead minnow tert transcription start site was inserted into the pGL3-Basic vector. Following transfection into EPC cells, the mutants were analyzed using a luciferase assay. A cellular myelocytomatosis (c-Myc) transcription factor binding site was identified in the region extending from -227 to -620 upstream of the tert transcription start site. Luciferase activity decreased significantly following introduction of site-specific mutations into the c-Myc binding site or treatment with the c-Myc inhibitor 10058-F4. In contrast, qRT-PCR analysis revealed no significant decrease in endogenous tert expression following the addition of 10058-F4 to EPC cells, suggesting that either DNA methylation in the region -1160 to -1420 upstream of tert or c-Myc binding, or both, may contribute to tert expression, and that this redundancy maintains tert expression. Furthermore, cycloheximide chase assays and real-time quantitative telomeric repeat amplification protocol suggest that high telomerase activity in EPC cells may also be maintained by the high stability of the telomerase protein.
