Biochemical and molecular studies of the polyunsaturated fatty acid desaturation pathway in fish

Fish have an absolute dietary requirement for certain polyunsaturated fatty acids (PUFA) termed “essential fatty acids” (EFA) that include members of both the n-6 and n-3 series typified by linoleic acid, 18:2n-6, and α-linolenic acid, 18:3n-3. However, the biologically active forms of EFA are generally the C20 and C22 metabolites of 18:2n-6 and 18:3n-3, viz. 20:4n-6, 20:5n-3 and 22:6n-3. Some fish species can convert C18 PUFA to the C20 and C22 PUFA through a series of alternating desaturation and chain elongation reactions mediated by microsomal systems containing elongases and Δ6 and Δ5 fatty acid desaturases. In species that cannot perform these conversions, the C20 and C22 PUFA themselves are dietary EFA and their C18 homologues do not satisfy EFA requirements. The extent to which the foregoing statements apply quantitatively to a given fish species varies widely. Therefore, a vital area in lipid nutrition in fish is the provision of sufficient amounts of the correct EFA to satisfy the requirements for normal growth and development, requirements that can vary quantitatively during the life of the fish and are particularly important factors in larval marine fish. This paper reviews the work on defining and characterising the fatty acid desaturation and elongation pathway in fish. Biochemical studies have been advanced by the use of cell cultures which have elucidated key parts of the pathway. Thus, the presence of the so-called Sprecher shunt, where 22:6n-3 is produced from 20:5n-3 through two successive elongations and a Δ6 desaturase followed by peroxisomal chain shortening, was demonstrated in trout. Similarly, the block in the pathway in marine and/or piscivorous fish could be due to either a deficiency of C18-20 elongase or Δ5 desaturase and this varies between different marine species. Recent work has focussed on the molecular biology of the pathway with the cloning of fatty acid desaturases and elongases from a variety of fish species. Zebrafish have been used as a model species and a unique desaturase possessing both Δ6 and Δ5 activity along with an elongase with very high C18-20 activity have been cloned and characterised. Understanding this pathway is of increased importance due to the current dependence of salmonid and marine fish aquaculture on fish oil, the supply of which is becoming increasingly limited and unsustainable, necessitating the use in fish feeds of sustainable plant oils, rich in C18 PUFA, but devoid of C20 and C22 PUFA

Electron localisation function in current-density-functional theory

We present a generalisation of the electron localisation function (ELF) to current-density-functional theory as a descriptor for the properties of molecules in the presence of magnetic fields. The resulting current ELF (cELF) is examined for a range of small molecular systems in field strengths up to B0 = 235 kT (one atomic unit). The cELF clearly depicts the compression of the molecular electronic structure in the directions perpendicular to the applied field and exhibits a structure similar to that of the physical current densities. A topological analysis is performed to examine the changes in chemical bonding upon application of a magnetic field

Zebrafish cDNA encoding multifunctional fatty acid elongase involved in production of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids

Enzymes that increase the chain length of fatty acids are essential for biosynthesis of highly unsaturated fatty acids. The gLELO gene encodes a protein involved in the elongation of polyunsaturated fatty acids in the fungus Mortierella alpina. A search of the Genbank database identified several EST sequences, including one obtained from zebrafish (Danio rerio), with high similarity to gLELO. The full-length transcript, ZfELO, encoding a polypeptide of 291 amino acid residues was isolated from zebrafish liver cDNA. The predicted amino acid sequence of the open reading frame (ORF) shared high similarity with the elongases of C. elegans and human. When expressed in Saccharomyces cerevisiae, the zebrafish ORF conferred the ability to lengthen the chain of a range of C18, C20 and C22 polyunsaturated fatty acids, indicating that biosynthesis of 22:6n-3 from 18:3n-3 via a 24-carbon intermediate is not only feasible, but that one elongase enzyme can perform all three elongation steps required. The zebrafish enzyme was also able to elongate monounsaturated and saturated fatty acids, and thus demonstrates a greater level of promiscuity in terms of substrate use than any elongase enzyme described previously

Modulation of type IV collagenase and plasminogen activator in a hamster fibrosarcoma by basement membrane components and lung fibroblasts.

The effect of basement membrane components (laminin, fibronectin and type IV collagen) and lung fibroblasts on type IV collagenase and plasminogen activator activity was investigated in a primary HSV-2-induced hamster fibrosarcoma, and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. Fibronectin and type IV collagen were ineffective at influencing the expression of either type IV collagenase or plasminogen activator activity. Laminin, however, at concentrations of 1-10 micrograms ml-1 added to the serum-free culture supernatants, increased the release of type IV collagenase by up to 100% for the parental cell line. Three highly metastatic sublines (two from in vivo origin and one from in vitro cloning) showed increases of up to 300%. Non-metastatic sublines (two from in vivo origin and one from in vitro cloning), however, showed no increase in type IV collagenase activity. Plasminogen activator release from either the parental line cell or its metastatic sublines and clones, was unaffected by the addition of laminin. Addition of tumour cells to lung fibroblast monolayers resulted in an increased expression of PA activity in the supernatant, whilst type IV collagenase activity was reduced

Highly unsaturated fatty acid synthesis in vertebrates: new insights with the cloning and characterization of a delta6 desaturase of Atlantic salmon

Fish are an important source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids that are crucial to the health of higher vertebrates. The synthesis of HUFA involves enzyme-mediated desaturation, and a ∆5 fatty acyl desaturase cDNA has been cloned from Atlantic salmon (Salmo salar) and functionally characterized previously. Here we report cloning and functional characterisation of a ∆6 fatty acyl desaturase of Atlantic salmon, and describe its genomic structure, tissue expression and nutritional regulation. A salmon genomic library was screened with a salmon ∆5 desaturase cDNA and positive recombinant phage isolated and subcloned. The full-length cDNA for the putative fatty acyl desaturase was shown to comprise 2106bp containing an ORF of 1365 bp specifying a protein of 454 amino acids (GenBank accession no. AY458652). The protein sequence included three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing the haem-binding motif HPGG, all of which are characteristic of microsomal fatty acid desaturases. Functional expression showed that this gene possessed predominantly ∆6 desaturase activity. Screening and sequence analysis of the genomic DNA of a single fish revealed that the ∆6 desaturase gene comprised 13 exons in 7965 bp of genomic DNA. Quantitative real time PCR assay of gene expression in Atlantic salmon showed that both ∆6 and ∆5 fatty acyl desaturase genes, and a fatty acyl elongase gene, were highly expressed in intestine, liver and brain, and less so in kidney, heart, gill, adipose tissue, muscle and spleen. Furthermore, expression of both ∆6 and ∆5 fatty acyl desaturase genes in intestine, liver, red muscle and adipose tissue was higher in salmon fed a diet containing vegetable oil than in fish fed a diet containing fish oil

Effects of diets containing vegetable oil on expression of genes involved in highly unsaturated fatty acid biosynthesis in liver of Atlantic salmon (Salmo salar)

Fish are an important dietary source of the long-chain C20 and C22 highly unsaturated fatty acids (HUFA), arachidonate (20:4n-6), eicosapentaenoate (20:5n-3) and docosahexaenoate (22:6n-3), that are crucial to the health of higher vertebrates and that can be beneficial in human diets. Δ5 and Δ6 fatty acid desaturases, and fatty acid elongases are critical enzymes in the biosynthetic pathways of HUFA from shorter chain C18 polyunsaturated fatty acids (PUFA) such as linoleic (18:2n-6) and α-linolenic (18:3n-3) acids. Recently, full-length cDNAs for fatty acid desaturase and elongase enzymes have been cloned from Atlantic salmon. Functional characterisation of the desaturase revealed n-3 Δ5 desaturase activity, whereas the elongase had broad substrate specificity for PUFA with a range of chain lengths from C18 to C22. The study described here was primarily focused on the nutritional regulation of genes involved in the HUFA biosynthetic pathway in Atlantic salmon. A feeding trial was performed whereby salmon smolts in seawater pens were fed for 40 weeks on five different diets. The diets consisted of a control diet containing fish oil (FO) and four diets in which the FO was replaced in a graded manner by linseed oil (LO). Specifically, in terms of added oils, the five diets were 100% FO (FO), 100%LO (LO100) and FO/LO in ratios of 3:1 (LO25), 1:1 (LO50) and 1:3 (LO75). Fish were sampled at 20 and 40 weeks, and samples of liver were collected for lipid analyses and total RNA extraction. Hepatocytes were also prepared and the activity of the HUFA biosynthetic pathway determined. Expression of fatty acid desaturase and elongase genes was determined by quantitative real time PCR and the ratio of the copy number of the targeted gene against that of β-actin was calculated. The results showed that after 20 weeks of feeding, desaturase and elongase gene expression in liver was increased in a graded manner by increasing dietary LO. Expression of both genes was positively and negatively correlated with dietary 18:3n-3 and n-3 HUFA, respectively. By 40 weeks of feeding, expression of neither gene showed the same degree of correlation with dietary fatty acid composition. In contrast, activity of the HUFA biosynthetic pathway, which showed some association with diet at 20 weeks, was positively and significantly correlated with dietary LO after 40 weeks of feeding. Elongation activity reflected the overall activity of the HUFA biosynthetic pathway to a greater degree than Δ5 desaturation activity. The possible mechanisms underlying the observed results and the regulation of the HUFA biosynthetic pathway are discussed

Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis niloticus)

The desaturation and elongation of [1-14C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level

Site-selective spectroscopy and level ordering in C-phycocyanin

We present a combined fluorescence and hole-burning study of the biliprotein C-phycocyanin. Sharp zero-phonon holes compare with a broad structureless fluorescence. This finding is rationalized in terms of the special level structure in this pigment, the fast energy-transfer processes and a lack of correlation of the energies of the emissive states

Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexanoic acids from alpha-linolenic acid in Atlantic salmon (Salmo salar)

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFA) and, with declining fisheries, farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term, and will constrain continuing growth of aquaculture activities. A greater understanding of how fish metabolise and biosynthesise HUFA may lead to effective use of more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimising the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterisation of two genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase cDNA, SalDes, was isolated that included an open reading frame (ORF) of 1362 bp specifying a protein of 454 amino acids. The protein sequence included all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and an N-terminal cytochrome b5 domain containing a haem-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast, Saccharomyces cerevisiae, showed SalDes is predominantly an omega-3 Δ5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from α-linolenic acid (18:3n-3). The desaturase showed only low levels of Δ6 activity towards C18 polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that includes an ORF of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo includes characteristic features of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast, SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C18 > C20 > C22. Thus, all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3 are displayed by this one polypeptide product

Factors affecting dairy farmers' attitudes towards antimicrobial medicine usage in cattle in England and Wales

There has been growing concern about bacterial resistance to antimicrobials in the farmed livestock sector. Attention has turned to sub-optimal use of antimicrobials as a driver of resistance. Recent reviews have identified a lack of data on the pattern of antimicrobial use as an impediment to the design of measures to tackle this growing problem. This paper reports on a study that explored use of antibiotics by dairy farmers and factors influencing their decision-making around this usage. We found that respondents had either recently reduced their use of antibiotics, or planned to do so. Advice from their veterinarian was instrumental in this. Over 70% thought reducing antibiotic usage would be a good thing to do. The most influential source of information used was their own veterinarian. Some 50% were unaware of the available guidelines on use in cattle production. However, 97% thought it important to keep treatment records. The Theory of Planned Behaviour was used to identify dairy farmers’ drivers and barriers to reduce use of antibiotics. Intention to reduce usage was weakly correlated with current and past practice of antibiotic use, whilst the strongest driver was respondents’ belief that their social and advisory network would approve of them doing this. The higher the proportion of income from milk production and the greater the chance of remaining in milk production, the significantly higher the likelihood of farmers exhibiting positive intention to reduce antibiotic usage. Such farmers may be more commercially minded than others and thus more cost-conscious or, perhaps, more aware of possible future restrictions. Strong correlation was found between farmers’ perception of their social referents’ beliefs and farmers’ intent to reduce antibiotic use. Policy makers should target these social referents, especially veterinarians, with information on the benefits from, and the means to, achieving reductions in antibiotic usage. Information on sub-optimal use of antibiotics as a driver of resistance in dairy herds and in humans along with advice on best farm practice to minimise risk of disease and ensure animal welfare, complemented with data on potential cost savings from reduced antibiotic use would help improve poor practice