Roberto de Antueno

Activity of human Δ5 and Δ6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids

Yeast co‐expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n‐3 and n‐6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Δ5 desaturase is active on 20:3n‐6 and 20:4n‐3. Endogenous Δ6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n‐6 or 20:5n‐3 which, through two sequential elongations, produced 24:4n‐6 and 24:5n‐3, respectively. Overall, our data suggest that a single human Δ6 desaturase is active on 18:2n‐6, 18:3n‐3, 24:4n‐6 and 24:5n‐3.

Liposome reconstitution of a minimal protein-mediated membrane fusion machine

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein‐mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion‐associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell–cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome–cell and liposome–liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14‐mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines.

Intracellular delivery of bovine lactoferricin's antimicrobial core (RRWQWR) kills T-leukemia cells.

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide with potent cytotoxic activity against cancer cells. The antimicrobial activity of LfcinB resides in its RRWQWR amino acid sequence (referred to here as LfcinB6); however, the anticancer activity of LfcinB6 is not known. Here, we show that free LfcinB6 did not kill T-leukemia or breast cancer cells but LfcinB6 was strongly cytotoxic when delivered to the cytosolic compartment by fusogenic liposomes. LfcinB6 bound weakly to isolated mitochondria but, unlike LfcinB, did not permeabilize mitochondria or cause cytochrome c to be released. Cathepsin B and caspase activity were important for cytotoxicity caused by intracellular LfcinB6 whereas reactive oxygen species were not involved. The mechanism of LfcinB6-induced cytotoxicity is therefore different from that of LfcinB. We suggest that LfcinB6, in combination with a fusogenic liposome delivery system that selectively targets malignant cells, has potential as a novel anticancer agent.

Relationship between mouse liver Δ9 desaturase activity and plasma lipids

This study was undertaken to investigate the total plasma fatty acid composition and the relationship between plasma triacylglycerol (TG) levels and liver Δ9 desaturase activity in mice fed n−3 and/or n−6 fatty acid or hydrogenated coconut oil (HCO) (maximum 25 mg/g) supplemented diets. Generally, plasma TG levels and Δ9 desaturase activity were inversely correlated with the ratio of the sum of long chain n−3 fatty acids to 18∶2n−6 and to the ratio of the sum of long chain n−3 fatty acids to 18∶n−3, but they were positively correlated with the ratio of products and substrates (18∶1/18∶0) of the enzyme in plasma total lipids. The n−3 fatty acid (mainly 20∶5n−3) enriched diet, when compared to the HCO diet at 21 d, caused a significant reduction in plasma TG levels but not in Δ9 desaturase activity. However, a marked reduction in plasma TG content (50–60%) and Δ9 desaturase activity (55–70%) was observed when both 20∶5n−3 and 18∶3n−6 were supplemented in the diet. The plasma TG levels and Δ9 desaturase activity rose again when the animals were fed the HCO diet or chow. The results suggest that low dose supplementation of a mixture of n−3 (mainly 20∶5n−3) and n−6 (18∶3n−6) fatty acids modified both plasma TG content and liver Δ9 desaturase activity, in parallel.

Nonclassical Resident Macrophages Are Important Determinants in the Development of Myocardial Fibrosis

Macrophages are increasingly recognized as a potential therapeutic target in myocardial fibrosis via interactions with fibroblasts. We have characterized macrophage depletion and inhibition of nonclassical macrophage migration, in addition to direct interactions between nonclassical macrophages and fibroblasts in angiotensin II (AngII)-mediated, hypertensive myocardial fibrosis. Macrophage depletion was achieved by daily i.v. clodronate liposomes (-1 day to +3 days) during AngII infusion. Cx3cr1(-/-) mice were used to inhibit nonclassical macrophage migration. Macrophage phenotype (F4/80, CD11b, Ly6C) was characterized by immunofluorescence and flow cytometry. Collagen was assessed by Sirius Red/Fast Green. Quantitative real-time RT-PCR was performed for transcript levels. AngII/wild-type (WT) mice displayed significant infiltrate and fibrosis compared with saline/WT, which was virtually ablated by clodronate liposomes independent of hypertension. In vitro data supported M2 macrophages promoting fibroblast differentiation and collagen production. AngII/Cx3cr1(-/-) mice, however, significantly increased macrophage infiltrate and fibrosis relative to AngII/WT. AngII/Cx3cr1(-/-) mice also showed an M1 phenotypic shift relative to WT mice in, which the predominant phenotype was Ly6C(low), CD206(+) (M2). Myocardial IL-1β was significantly up-regulated, whereas transforming growth factor β down-regulated with this M1 shift. We demonstrated that infiltrating macrophages are critical to AngII-mediated myocardial fibrosis by preventing the development of fibrosis after liposomal depletion of circulating monocytes. Our findings also suggest that some macrophages, namely M2, may confer a protective myocardial environment that may prevent excessive tissue injury.

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

Liver Δ5 and Δ6 desaturase activity differs among laboratory rat strains

This study was designed to examine the variations among rat strains in hepatic fatty acid desaturase activities and to determine the correlations between the activities of these enzymes and the levels of each microsomal fatty acid. Wistar rats from two different sources as well as Long-Evans and Sprague-Dawley rats were selected to assess, under standard and identical experimental conditions, the liver Δ5 and Δ6 desaturase activities. Both desaturase activities were significantly reduced by 56% in Sprague-Dawley rats when compared to BB-Wistar control rats, whereas intermediate reduced values were detected in Wistar (CR) and Long-Evans strains. The activities of Δ5 and Δ6 desaturases were significantly and positively correlated with each other. However, no significant correlations were detected between either Δ5 or Δ6 desaturase activities and levels of any of their fatty acid substrates or any other of the major microsomal fatty acids. Fatty acid composition of microsomal total lipids showed strain dependency. A positive correlation was detected between the microsomal levels of the two major final products of both desaturases, namely 20∶4n−6 and 22∶6n−3. In general, the sum of n−3 or n−6 fatty acids but not the ratio of one to the other, varied among rat strains. The study demonstrated that Δ6 and Δ5 desaturase activities are strain-related. The data also suggested that (i) the desaturation activity should be measured and not predicted from the fatty acid composition and (ii) different rat strains should be used for lipid metabolic studies before conclusions are drawn for rats in general.

Activity and mRNA abundance of Δ-5 and Δ-6 fatty acid desaturases in two human cell lines

We analyzed fatty acid biosynthesis in Chang and ZR‐75‐1 cells. Both cell lines could desaturate and further elongate substrates for Δ‐5 desaturase. ZR‐75‐1 but not Chang cells showed Δ‐6 desaturation of 18:2n‐6, 18:3n‐3, 24:4n‐6 and 24:5n‐3. In both cell lines, the mRNA abundance can be related to Δ‐5 or Δ‐6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Δ‐6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.

Citral reduces breast tumor growth by inhibiting the cancer stem cell marker ALDH1A3.

Breast cancer stem cells (CSCs) can be identified by increased Aldefluor fluorescence caused by increased expression of aldehyde dehydrogenase 1A3 (ALDH1A3), as well as ALDH1A1 and ALDH2. In addition to being a CSC marker, ALDH1A3 regulates gene expression via retinoic acid (RA) signaling and plays a key role in the progression and chemotherapy resistance of cancer. Therefore, ALDH1A3 represents a druggable anti‐cancer target of interest. Since to date, there are no characterized ALDH1A3 isoform inhibitors, drugs that were previously described as inhibiting the activity of other ALDH isoforms were tested for anti‐ALDH1A3 activity. Twelve drugs (3‐hydroxy‐dl‐kynurenine, benomyl, citral, chloral hydrate, cyanamide, daidzin, DEAB, disulfiram, gossypol, kynurenic acid, molinate, and pargyline) were compared for their efficacy in inducing apoptosis and reducing ALDH1A3, ALDH1A1 and ALDH2‐associated Aldefluor fluorescence in breast cancer cells. Citral was identified as the best inhibitor of ALDH1A3, reducing the Aldefluor fluorescence in breast cancer cell lines and in a patient‐derived tumor xenograft. Nanoparticle encapsulated citral specifically reduced the enhanced tumor growth of MDA‐MB‐231 cells overexpressing ALDH1A3. To determine the potential mechanisms of citral‐mediated tumor growth inhibition, we performed cell proliferation, clonogenic, and gene expression assays. Citral reduced ALDH1A3‐mediated colony formation and expression of ALDH1A3‐inducible genes. In conclusion, citral is an effective ALDH1A3 inhibitor and is able to block ALDH1A3‐mediated breast tumor growth, potentially via blocking its colony forming and gene expression regulation activity. The promise of ALDH1A3 inhibitors as adjuvant therapies for patients with tumors that have a large population of high‐ALDH1A3 CSCs is discussed.

In vitro conversion of saturated to monounsaturated fatty acid by Ehrlich ascites cells

In this paper, evidence is presented on the capacity of Ehrlich ascites cells to synthesize in vitro monounsaturated fatty acids from radioactive palmitate. Localization of the double bond was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas liquid chromatography. These results proved that Ehrlich ascites cells have a Δ9 desaturase that catalyzes the biosynthesis of palmitoleic acid from palmitic acid and oleic and vaccenic acid via elongation-desaturation and desaturation-elongation, respectively, using palmitic acid as substrate. Furthermore, it is shown that, as in the hepatic cells, Δ9 desaturase enzyme activity of the tumoral cells is associated with the endoplasmic reticulum. The electron transport components involved in the desaturase system, i.e., NADH-cytochrome bs reductase and NADH-cytochrome c reductase, were also measured. The activities of these enzymes do not appear to be rate-limiting in the desaturase activity of these tumoral cells.