Barramundi (Lates calcarifer) desaturase with Δ6/Δ8 dual activities.

Barramundi is a commercially farmed fish in Australia. To examine the potential for barramundi to metabolise dietary α-linolenic acid (ALA, 18:3 n-3), the existence of barramundi desaturase enzymes was examined. A putative fatty acid Δ6 desaturase was cloned from barramundi liver and expressed in yeast. Functional expression revealed Δ6 desaturase activity with both the 18 carbon (C(18)) and C(24) n-3 fatty acids, ALA and 24:5 n-3 as well as the C(18) n-6 fatty, linoleic acid (LA, 18:2 n-6). Metabolism of ALA was favoured over LA. The enzyme also had Δ8 desaturase activity which raises the potential for synthesis in barramundi of omega-3 (n-3) long chain polyunsaturated fatty acids from ALA via a pathway that bypasses the initial Δ6 desaturase step. Our findings not only provide molecular evidence for the fatty acid desaturation pathway in the barramundi but also highlight the importance of taking extracellular fatty acid levels into account when assessing enzyme activity expressed in Saccharomyces cerevisiae.

Fatty acid desaturase 2 promoter mutation is not responsible for Δ6-desaturase deficiency.

Dietary essential polyunsaturated fatty acids (PUFAs) require fatty acid desaturases (FADS) for conversion to long-chain PUFAs (LCPUFAs), which are critical for many aspects of human health. A Δ6-desaturase deficiency in a single patient was attributed to an insertion mutation in the FADS2 promoter. Later population studies have shown this thymidine nucleotide (T) insertion to be a common polymorphism (rs3834458). We examined correlations between rs3834458 variants and fatty acid evidence of FADS2 activity in a cohort of rheumatoid arthritis patients selected for low or nil consumption of n-3 LCPUFA as fish or fish oil. The presence of the T allele was associated with higher FADS2 activity, as indicated by higher conversion of plasma n-3 PUFA to LCPUFA. However, the T-insertion/deletion polymorphism did not affect FADS2 promoter activity in luciferase reporter assays in HepG2 or NIH/3T3 cells. Our results indicate that the polymorphism rs3834458 does not appear to directly affect FADS2 promoter activity and is not responsible for a previously reported Δ6-desaturase deficiency.

Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis.

BACKGROUND: Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA. METHODOLOGY/PRINCIPAL FINDINGS: The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C(20) and C(22) polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3. CONCLUSIONS: The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.

Endothelial cell COX-2 expression and activity in hypoxia.

The clinical experience with selective cyclooxygenase (COX)-2 inhibitors reveals there are important protective roles for COX-2 in the cardiovascular system. This study examined the response to hypoxia of endothelial cell eicosanoid synthesis with respect to the role of COX-2 and its molecular regulation in hypoxia. Human umbilical vein endothelial cells (HUVEC) were exposed to hypoxia and the effects on COX-2, prostacyclin (PGI(2)) and thromboxane (TXA(2)) synthesis were examined. COX-2 promoter constructs were used to examine the role of Hypoxia Inducible Factors (HIFs) in COX-2 responses to hypoxia. Hypoxia caused an increase in PGI(2) synthesis, but not TXA(2) synthesis. PGI(2), but not TXA(2) synthesis, was absolutely dependent on upregulation of COX-2 by hypoxia. Mutations of transcription factor binding sites in the promoter showed a lack of involvement of NFkappaB in the response to hypoxia, but suggested involvement of HIFs. Transfection of HUVEC with HIF expression vectors increased activity of the promoter construct and increased native COX-2 expression in normoxia. EMSA showed HIF binding in nuclear extracts of hypoxic HUVEC to a region of the COX-2 promoter. The endothelial cell response to hypoxia involves increased production of the anti-thrombotic eicosanoid, PGI(2), which is dependent on COX-2 upregulation by a HIF-mediated process.

Effects of hypoxia on the expression and activity of cyclooxygenase 2 in fibroblast-like synoviocytes: interactions with monocyte-derived soluble mediators.

OBJECTIVE: Rheumatoid synovium is characterized by hyperplasia of fibroblast-like (type B) synoviocytes (FLS), infiltration with mononuclear leukocytes, and tissue hypoxia. Although the latter is well documented, it has received little attention in dissection of the biochemical events that mediate the inflammatory lesion in rheumatoid arthritis (RA). Therefore, this study was designed to assess the effect of hypoxia on FLS responses to the monokine interleukin-1beta (IL-1beta) and to monocyte conditioned medium. METHODS: FLS obtained from serial cultures of synovial fluid aspirates were treated with IL-1beta or monocyte conditioned medium, under normoxia and hypoxia. RESULTS: In hypoxia, transcription of cyclooxygenase 2 (COX-2), expression of COX-2 protein, and production of COX-2-derived eicosanoids and matrix metalloproteinase (MMP) activity by FLS were all increased in response to IL-1beta. In contrast to our recent observations concerning monocytes, there was no change in COX-2 message stability and cytosolic phospholipase A2 activity in the FLS under hypoxia. Treatment of monocyte conditioned medium with an IL-1beta blocking antibody showed that most of the effect of the conditioned medium was attributable to IL-1beta. CONCLUSION: The findings suggest that hypoxia is an important factor in aggravating the inflammatory lesion in RA, through increased production of COX-2-derived nociceptive eicosanoids and increased release of tissue-damaging MMPs.

Identification of AtNDI1, an internal non-phosphorylating NAD(P)H dehydrogenase in Arabidopsis mitochondria.

Plant mitochondria contain non-phosphorylating NAD(P)H dehydrogenases (DHs) that are not found in animal mitochondria. The physiological function, substrate specificity, and location of enzymes within this family have yet to be conclusively determined. We have linked genome sequence information to protein and biochemical data to identify that At1g07180 (SwissProt Q8GWA1) from the Arabidopsis Genome Initiative database encodes AtNDI1, an internal NAD(P)H DH in Arabidopsis mitochondria. Three lines of evidence are presented: (a). The predicted protein sequence of AtNDI1 has high homology with other designated NAD(P)H DHs from microorganisms, (b). the capacity for matrix NAD(P)H oxidation via the rotenone-insensitive pathway is significantly reduced in the Atndi1 mutant plant line, and (c). the in vitro translation product of AtNDI1 is imported into isolated mitochondria and located on the inside of the inner membrane.

Effects of hypoxia on monocyte inflammatory mediator production: Dissociation between changes in cyclooxygenase-2 expression and eicosanoid synthesis.

Blood-derived monocytes are found at sites of inflammation as well as in solid tumors and atherosclerotic arteries. They are an abundant source of inflammatory eicosanoids such as prostaglandin E2 (PGE2) and thromboxane A2, which are formed via arachidonic acid (AA) metabolism by cyclooxygenase-1/2 (COX-1/2). In vitro studies of inflammatory mediator production are conducted invariably in room air, which does not reflect the oxygen tensions found in monocyte-containing lesions, which are frequently hypoxic. In this work we examined the effects of hypoxia at levels reported in these lesions, on monocyte COX-2 expression, the related events that lead to eicosanoid synthesis, and relationships with tumor necrosis factor (TNF)-alpha synthesis. In fresh human monocytes exposed to hypoxia (1% O2), there was an increase in COX-2 protein compared with cells in normoxia, and this was attributable to increased transcription and mRNA stability. However, the synthesis of PGE2 and thromboxane A2 was reduced in hypoxia and did not reflect the increased level of COX-2. Monocytes prelabeled with [3H]AA followed by lipopolysaccharide stimulation in the presence of hypoxia showed a reduced release of AA compared with cells in normoxia. In addition, hypoxia resulted in decreased phosphorylation of the p44/42 mitogen-activated protein kinase and of cytosolic phospholipase A2. Hypoxia also increased TNF-alpha synthesis, which appeared to play a role in COX-2 expression, and the observed increase TNF-alpha synthesis appeared to result from reduced PGE2 synthesis. Overall, the results suggest the existence of an autocrine loop of regulation between monocyte eicosanoid and TNF-alpha production, which is dysregulated in hypoxia and establishes hypoxia as being an important environmental determinant of inflammatory mediator production.