A fatty acyl desaturase (fads2) with dual Δ6 and Δ5 activities from the freshwater carnivorous striped snakehead Channa striata.

There is a lack of understanding on how the environment and trophic niche affect the capability of long-chain polyunsaturated fatty acids (LC-PUFA) in freshwater carnivorous teleost. In this present study, we isolated and functionally characterised a fatty acyl desaturase (Fads) from the striped snakehead Channa striata. Sequence comparison and phylogenetic analysis suggested a Fads2 protein that is closely related to previously characterised Fads2 proteins from freshwater carnivorous and marine herbivorous fish species. We further demonstrated the capacity of Δ6 and Δ5 desaturation activities for this particular desaturase, with highest activities towards the conversion of omega-3 (n-3) polyunsaturated fatty acids (PUFA). Low Δ4 desaturation activity was also detected, although the significance of this at a physiological level remains to be studied. The expression of this striped snakehead Δ6/Δ5 fads2 gene was highest in brain, followed by liver and intestine. In liver, diet fortified with high LC-PUFA concentration impeded the expression of Δ6/Δ5 fads2 gene compared to vegetable oil (VO) based diets. The discovery of Δ6/Δ5 Fads2 desaturase here complements the previous discovery of a Δ4 Fads2 desaturase and an Elovl5 elongase, lending proof to the existence of all the required enzymatic machinery to biosynthesise LC-PUFA from C18 PUFA in a freshwater carnivorous species.

Echium oil increased the expression of a Δ4 Fads2 fatty acyl desaturase and the deposition of n-3 long-chain polyunsaturated fatty acid in comparison with linseed oil in striped snakehead (Channa striata) muscle.

Despite the potential of vegetable oils as aquafeed ingredients, a major drawback associated with their utilization is the inferior level of beneficial n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Echium oil (EO), which is rich in stearidonic acid (SDA, 18:4n-3), could potentially improve the deposition of n-3 LC-PUFA as the biosynthesis of LC-PUFA is enhanced through bypassing the rate-limiting ∆6 desaturation step. We report for the first time an attempt to investigate whether the presence of a desaturase (Fads2) capable of ∆4 desaturation activities and an elongase (Elovl5) will leverage the provision of dietary SDA to produce a higher rate of LC-PUFA bioconversion. Experimental diets were designed containing fish oil (FO), EO or linseed oil (LO) (100FO, 100EO, 100LO), and diets which comprised equal mixtures of the designated oils (50EOFO and 50EOLO) were evaluated in a 12-week feeding trial involving striped snakeheads (Channa striata). There was no significant difference in growth and feed conversion efficiency. The hepatic fatty acid composition and higher expression of fads2 and elovl5 genes in fish fed EO-based diets indicate the utilization of dietary SDA for LC-PUFA biosynthesis. Collectively, this resulted in a higher deposition of muscle eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) compared to LO-based diets. Dietary EO improved the ratio of n-3 LC-PUFA to n-6 LC-PUFA in fish muscle, which is desirable for human populations with excessive consumption of n-6 PUFA. This study validates the contribution of SDA in improving the content of n-3 LC-PUFA and the ratio of EPA to arachidonic acid (ARA, 20:4n-6) in a freshwater carnivorous species.

Potent PPARγ Ligands from Swietenia macrophylla Are Capable of Stimulating Glucose Uptake in Muscle Cells.

Numerous documented ethnopharmacological properties have been associated with Swietenia macrophylla (Meliaceae), with its seed extract reported to display anti-hypoglycemic activities in diabetic rats. In the present study, three compounds isolated from the seeds of S. macrophylla were tested on a modified ELISA binding assay and showed to possess PPARγ ligand activity. They were corresponded to PPARγ-mediated cellular response, stimulated adipocyte differentiation but produced lower amount of fat droplets compared to a conventional anti-diabetic agent, rosiglitazone. The up-regulation of adipocytes was followed by increased adipocyte-related gene expressions such as adiponectin, adipsin, and PPARγ. The S. macrophylla compounds also promoted cellular glucose uptake via the translocation of GLUT4 glucose transporter.

The capacity for long-chain polyunsaturated fatty acid synthesis in a carnivorous vertebrate: Functional characterisation and nutritional regulation of a Fads2 fatty acyl desaturase with Δ4 activity and an Elovl5 elongase in striped snakehead (Channa striata).

The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.

Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition.

Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

The ribosomal protein L19 mRNA is induced by copper exposure in the swordtail fish, Xiphophorus helleri.

Teleosts are useful vertebrate model species for understanding copper toxicity due to the dual entry route for copper intake via the gills and intestine. In this present study, we utilized the differential display reverse transcription-polymerase chain reaction to isolate potential novel hepatic genes induced by sublethal copper exposure in the freshwater swordtail fish, Xiphophorus helleri. Full length cloning of a cDNA fragment induced by copper exposure to 1 µg/ml during 24 h resulted in the positive identification of a hepatic ribosomal protein L19 (RPL19) gene. Further characterization of this gene revealed that its transcriptional expression was dependent on dosage and time of copper exposure. This study describes for the first time the involvement of RPL19 in copper toxicity, probably as a result of increase in ribosome synthesis rate to support activities such as cellular protein translation, transcriptional activation and mRNA stabilization during sublethal copper exposure.

Swietenia macrophylla extract promotes the ability of Caenorhabditis elegans to survive Pseudomonas aeruginosa infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Swietenia macrophylla or commonly known as big leaf mahogany, has been traditionally used as an antibacterial and antifungal agent. AIM OF THE STUDY: The unwanted problem of antibiotic resistance in many bacterial species advocates the need for the discovery of the new anti-infective drugs. Here, we investigated the anti-infective properties of Swietenia macrophylla with an assay involving lethal infection of Caenorhabditis elegans with the opportunistic human pathogen Pseudomonas aeruginosa. MATERIALS AND METHODS: Using a slow killing assay, Caenorhabditis elegans was challenged with an infective strain of Pseudomonas aeruginosa (PA14). The ability of Swietenia macrophylla seed ethyl acetate extract to promote the survival of infected worms was assessed by comparing the percentage of survival between extract treated and non-treated worm populations. The effect of Swietenia macrophylla towards PA14 growth, Caenorhabditis elegans feeding rate and degree of PA14 colonization in the worm gut was also evaluated. Lastly, using a fluorescent transgenic Caenorhabditis elegans strain and real time PCR, the effect of Swietenia macrophylla on the expression of lys-7, an immune response gene was also investigated. RESULTS: Our results demonstrate the ability of Swietenia macrophylla seed ethyl acetate extract in rescuing Caenorhabditis elegans from fatal PA14 infection. Consequently, we showed that the extract promotes the survival without exhibiting any bactericidal effect or perturbation of Caenorhabditis elegans feeding rate. We also showed that Swietenia macrophylla was able to restore the initially repressed lys-7 level in PA14 infected Caenorhabditis elegans. CONCLUSION: Swietenia macrophylla extract is able to enhance the ability of Caenorhabditis elegans to survive PA14 infection without directly killing the pathogen. We further showed that the extract boosted the expression of a gene pivotal for innate immunity in Caenorhabditis elegans. Collectively, these findings strongly suggest the presence of compounds within Swietenia macrophylla seed that either reduces Pseudomonas aeruginosa virulence and/or enhance host resistance.