Lipoxygenase Synthesis Research Articles

Patatin-like lipolytic acyl hydrolases and galactolipid metabolism in marine diatoms of the genus Pseudo-nitzschia

Diatoms are eukaryotic microalgae that play a pivotal role in biological and geochemical marine cycles. These microorganisms are at the basis of the trophic chain and their lipids are essential components (e.g. eicosapentaenoic acid, EPA) of aquatic food webs. Galactolipids are the primary lipid components of plastid membranes and form the largest lipid family of diatoms. As source of polyunsaturated fatty acids (PUFAs), these compounds are also involved in the synthesis of lipoxygenase (LOX) products such as non-volatile oxylipins and polyunsaturated aldehydes. Here, we report the first identification of two genes, namely PmLAH1 and PaLAH1, coding for lipolytic enzymes in two diatoms of the genus Pseudo-nitzschia. Functional and modeling studies evidence a patatin-like domain endowed with galactolipase and phospholipase activity at the C-terminus of both proteins. Homologues of Pseudo-nitzschia LAH1 genes were retrieved in other diatom species so far sequenced in agreement with conservation of the functional role of these proteins within the lineage.

Leptin Elicits LTC4 Synthesis by Eosinophils Mediated by Sequential Two-Step Autocrine Activation of CCR3 and PGD2 Receptors.

Leptin is a cytokine, produced mainly by mature adipocytes, that regulates the central nervous system, mainly to suppress appetite and stimulate energy expenditure. Leptin also regulates the immune response by controlling activation of immunomodulatory cells, including eosinophils. While emerging as immune regulatory cells with roles in adipose tissue homeostasis, eosinophils have a well-established ability to synthesize pro-inflammatory molecules such as lipid mediators, a key event in several inflammatory pathologies. Here, we investigated the impact and mechanisms involved in leptin-driven activation of eicosanoid-synthesizing machinery within eosinophils. Direct in vitro activation of human or mouse eosinophils with leptin elicited synthesis of lipoxygenase as well as cyclooxygenase products. Displaying selectivity, leptin triggered synthesis of LTC4 and PGD2, but not PGE2, in parallel to dose-dependent induction of lipid body/lipid droplets biogenesis. While dependent on PI3K activation, leptin-driven eosinophil activation was also sensitive to pertussis toxin, indicating the involvement of G-protein coupled receptors on leptin effects. Leptin-induced lipid body-driven LTC4 synthesis appeared to be mediated through autocrine activation of G-coupled CCR3 receptors by eosinophil-derived CCL5, inasmuch as leptin was able to trigger rapid CCL5 secretion, and neutralizing anti-RANTES or anti-CCR3 antibodies blocked lipid body assembly and LTC4 synthesis induced by leptin. Remarkably, autocrine activation of PGD2 G-coupled receptors DP1 and DP2 also contributes to leptin-elicited lipid body-driven LTC4 synthesis by eosinophils in a PGD2-dependent fashion. Blockade of leptin-induced PGD2 autocrine/paracrine activity by a specific synthesis inhibitor or DP1 and DP2 receptor antagonists, inhibited both lipid body biogenesis and LTC4 synthesis induced by leptin stimulation within eosinophils. In addition, CCL5-driven CCR3 activation appears to precede PGD2 receptor activation within eosinophils, since neutralizing anti-CCL5 or anti-CCR3 antibodies inhibited leptin-induced PGD2 secretion, while it failed to alter PGD2-induced LTC4 synthesis. Altogether, sequential activation of CCR3 and then PGD2 receptors by autocrine ligands in response to leptin stimulation of eosinophils culminates with eosinophil activation, characterized here by assembly of lipidic cytoplasmic platforms synthesis and secretion of the pleiotropic lipid mediators, PGD2, and LTC4.

8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-γ-linolenic acid: a distinctive transformation compared with S-lipoxygenases

Conversion of fatty acid hydroperoxides to epoxyalcohols is a well known secondary reaction of lipoxygenases, described for S-specific lipoxygenases forming epoxyalcohols with a trans-epoxide configuration. Here we report on R-specific lipoxygenase synthesis of a cis-epoxyalcohol. Although arachidonic and dihomo-γ-linolenic acids are metabolized by extracts of the Caribbean coral Plexaura homomalla via 8R-lipoxygenase and allene oxide synthase activities, 20:3ω6 forms an additional prominent product, identified using UV, GC-MS, and NMR in comparison to synthetic standards as 8R,9S-cis-epoxy-10S-erythro-hydroxy-eicosa-11Z,14Z-dienoic acid. Both oxygens of (18)O-labeled 8R-hydroperoxide are retained in the product, indicating a hydroperoxide isomerase activity. Recombinant allene oxide synthase formed only allene epoxide from 8R-hydroperoxy-20:3ω6, whereas two different 8R-lipoxygenases selectively produced the epoxyalcohol.A biosynthetic scheme is proposed in which a partial rotation of the reacting intermediate is required to give the observed erythro epoxyalcohol product. This characteristic and the synthesis of cis-epoxy epoxyalcohol may be a feature of R-specific lipoxygenases.

A gain‐of‐function allele of TPC1 activates oxylipin biogenesis after leaf wounding in Arabidopsis

Jasmonates, potent lipid mediators of defense gene expression in plants, are rapidly synthesized in response to wounding. These lipid mediators also stimulate their own production via a positive feedback circuit, which depends on both JA synthesis and JA signaling. To date, molecular components regulating the activation of jasmonate biogenesis and its feedback loop have been poorly characterized. We employed a genetic screen capable of detecting the misregulated activity of 13-lipoxygenase, which operates at the entry point of the jasmonate biosynthesis pathway. Leaf extracts from the Arabidopsis fou2 (fatty acid oxygenation upregulated 2) mutant displayed an increased capacity to catalyze the synthesis of lipoxygenase (LOX) metabolites. Quantitative oxylipin analysis identified less than twofold increased jasmonate levels in healthy fou2 leaves compared to wild-type; however, wounded fou2 leaves strongly increased jasmonate biogenesis compared to wounded wild-type. Furthermore, the plants displayed enhanced resistance to the fungus Botrytis cinerea. Higher than wild-type LOX activity and enhanced resistance in the fou2 mutant depend fully on a functional jasmonate response pathway. The fou2 mutant carries a missense mutation in the putative voltage sensor of the Two Pore Channel 1 gene (TPC1), which encodes a Ca(2+)-permeant non-selective cation channel. Patch-clamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. The results indicate that cation fluxes exert strong control over the positive feedback loop whereby JA stimulates its own synthesis.

Towards prevention

Towards prevention

Metabolism of arachidonic acid by canine polymorphonuclear leukocytes synthesis of lipoxygenase and omega-oxidized metabolites

Both polymorphonuclear (PMN) leukocytes and metabolites of arachidonic acid, especially lipoxygenase products, have been reported to contribute to myocardial damage after coronary artery occlusion and reperfusion. While canine models of myocardial ischemia were used in many of these studies, very little is known about arachidonic acid metabolism by canine PMNs. Moreover, it is unclear whether arachidonic acid metabolites released by canine PMNs affect vascular tone. Therefore, we characterized arachidonic acid metabolism by canine PMNs and determined the effect of these metabolites on vascular tone of isolated canine coronary arteries. Suspensions of canine PMNs were incubated with [ 14C]arachidonic acid and the calcium ionophore A23187. The incubation media was extracted, and the metabolites resolved by HPLC. 20-Hydroxy-leukotriene B 4 (LTB 4), 12,20-dihydroxyeicosatetraenoic acid (diHETE), LTB 4, 12-hydroxyheptadeclatrienoic acid (HHT), and 12-( S)-hydroxyeicosatetraenoic acid (HETE) were isolated, and their structures confirmed by gas chromatography/mass spectrometry. There was also evidence for the formation of 20-HETE, thromboxane B 2 (TXB 2), 5-HETE, and several isomers of LTB 4. None of the arachidonic acid metabolites that were isolated from incubates of canine PMNs augmented vascular tone, but material migrating with 12,20-diHETE relaxed canine coronary arteries. Authentic 12( S),20-diHETE also produced a concentration-related relaxation of canine coronary artery. 12( R), 20-diHETE was inactive. 20-HETE inhibited A23187-induced PMN aggregation. Thus, arachidonic acid is metabolized in canine PMNs through the cyclooxygenase, lipoxygenases and cytochrome P-450 pathways. Whether these metabolites contribute to myocardial injury remains to be determined.

Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries.

Coronary vascular injury promotes blood cell-vessel wall interactions that influence arachidonic acid metabolism and coronary blood flow patterns. Since lipoxygenase and cytochrome P-450 epoxygenase metabolites of arachidonic acid are synthesized by vascular and inflammatory cells and have a variety of important biological actions, we investigated the metabolism of arachidonic acid by these pathways in normal and stenosed, endothelially injured canine coronary arteries. We found and confirmed by gas chromatography/mass spectrometry that primarily 12- and 15-hydroxyeicosatetraenoic acids (HETEs) are synthesized by both coronary artery segments. Lesser amounts of 11-, 9-, 8-, and 5-HETEs are also produced. 15-Ketoeicosatetraenoic acid is also synthesized. The synthesis of 14C-HETEs is fivefold to 10-fold greater by the stenosed than the normal coronary artery. Specific radioimmunoassays indicated that the stenosed coronary artery synthesized 93 +/- 14 and 1,102 +/- 154 ng/g of tissue of 15- and 12-HETE, respectively, while the normal coronary artery produced 17 +/- 3 and 162 +/- 68 ng/g of tissue of 15- and 12-HETE, respectively. Products comigrating with 14,15-; 11,12-; 8,9-; and 5,6-epoxyeicosatrienoic acids (EETs) and the corresponding dihydroxyeicosatrienoic acids (DHETs) were detected predominantly in stenosed coronary arteries by high-pressure liquid chromatography. The structures of the EETs were confirmed by GC/MS. The EETs and prostaglandin I2 produced endothelium-independent, concentration-related relaxations of dog coronary artery rings. These data indicate that normal and stenotic coronary arteries metabolize arachidonic acid to HETEs, DHETs, and EETs along with prostaglandins; however, the synthesis of these metabolites is greater in the stenosed, endothelially injured vessel. The EETs may be synthesized during the development of cyclic flow variations and counteract the vasoconstrictor effects of thromboxane A2.

Alteration of the synthesis of lipoxygenase in the early stages of soybean cotyledon culture

A detailed study of lipoxygenase (EC 1.13.11.12) synthesis in cotyledons of soybean [Glycine max (L.) Merr. cv. Century] cultured in vitro for up to 40 h showed that synthesis of this protein, measured by in vivo [35S]‐methionine labelling in connection with immunological methods and cell‐free translation of mRNA, underwent a large transient reduction in the first 4 h of culturing and gradually increased in the following 36 h. Northern blot hybridizations with lipoxygenase cDNA clones showed that the decrease in translational activity was the consequence of a considerable reduction in lipoxygenase mRNA in the cotyledons. From these results we conclude that the transient decline in lipoxygenase synthesis in excised soybean cotyledons is regulated at the RNA level. Similarly judged from the analysis of patterns of uni‐dimensional gel electrophoresis, the synthesis of a few other polypeptides decreased during the first 4 h of culture as well, while several others increased; in cotyledons cultured for 20 to 40 h the protein‐synthesis pattern had returned to that in freshly excised cotyledons. An acclimation period of ca 1 day seems to be needed for isolated soybean cotyledons to stabilize and to resume regular RNA and protein synthesis.

Changes in lipoxygenase activity during seedling development of Lupinus albus

A lipoxygenase preparation was obtained from Lupinus albus seeds and was shown to differ from previously characterized lipoxygenase. This study describes changes in lipoxygenase activity during seedling development of Lupinus albus. The enzyme activity shows a decrease from 0–6 h postgermination (about 15%), is roughly constant or even rises slightly from 6–30 h and then shows a large increase between 30 and 48 h (about 50%). Enzymatically active proteins from 48 h-old seedlings were isolated and the increase of enzyme activity was mainly due to the presence of two components with maximum activity at pH 6 and pH 8.5, respectively. When arachidonic acid was used as substrate, the two enzymatic activities produce 15 HPETE. The increase in lipoxygenase activity during seedling development was inhibited by cycloheximide. Cordycepin appears to have no direct effect on lipoxygenase synthesis in vivo at the studied doses.

Arachidonic acid metabolism in isolated pancreatic islets: II. The effects of glucose and of inhibitors of arachidonate metabolism on insulin secretion and metabolite synthesis

Isolated pancreatic islets from the rat incubated with 28 mM glucose have been found to secrete more insulin and to synthesize greater amounts of arachidonate lipoxygenase and cyclooxygenase products than islets incubated with 3 mM glucose. This effect was not apparent in studies examining metabolism of radiolabeled arachidonate and was revealed only when the metabolites were quantitated with mass spectrometric measurements. That the glucose-induced synthesis of arachidonate metabolites may participate in insulin secretion was suggested by studies with inhibitors of arachidonate metabolism. Eicosa 5,8,11,44 tetrynoic acid (ETYA) suppressed glucose-induced insulin secretion by 63–74% at a concentration (20 μM) which inhibited the synthesis of arachidonate lipoxygenase and cyclooxygenase products by 90%. Indomethacin (10 μM) completely prevented islet synthesis of cyclooxygenase products but did not influence glucose-induced insulin secretion. Although indomethacin did not inhibit the conversion of exogenous, 3H-labeled arachidonate to [ 3H]12-HETE, it did significantly inhibit (41–72%) the synthesis of 12-HETE from endogenous precursor. This is presumed to reflect indirect effects of indomethacin on hydrolysis of arachidonate from phospholipids, as recently reported in platelets. These studies constitute the first demonstration that glucose stimulates the synthesis of a lipoxygenase product (12-HETE) from endogenous arachidonate by isolated islets, and that suppression of 12-HETE synthesis with ETYA reduces glucose-induced insulin secretion from isolated islets.

Lipoxygenase mRNA in rabbit reticulocytes. Its isolation, characterization and translational repression.

The synthesis of the erythroid lipoxygenase, an enzyme which is of importance for the degradation of mitochondria during the maturation of reticulocytes to erythrocytes, was studied in reticulocytes from bone marrow and in density-separated fractions from peripheral blood of anemic rabbits. Lipoxygenase mRNA was enriched to about 75% by digestion of polysomes with protease K, poly(U)-Sepharose chromatography and repeated sucrose gradient centrifugation. From sucrose gradient centrifugation, electrophoresis and electron microscopy a molecular weight of about 10(6) was calculated. Synthesis of lipoxygenase is absent in erythroblasts, in very young reticulocytes obtained from bone marrow, or in the lightest fractions of reticulocytes from the peripheral blood. More mature blood reticulocytes show a considerable synthesis of the enzyme. The induction of the synthesis of the lipoxygenase seems to be initiated when reticulocytes have reached the peripheral blood. It is shown that lipoxygenase mRNA is present in reticulocytes as a translationally inactive free cytoplasmic messenger ribonucleoprotein (mRNP) particle. After deproteinization isolated mRNA obtained from masked mRNP codes for authentic lipoxygenase in a cell-free protein-synthesizing system of reticulocytes.

On the physiological significance of dark reversion of phytochrome in the mustard seedling.

Abstract—We present physiological evidence using the threshold control of lipoxygenase synthesis† by Pfr in the mustard seedling (LOG response) that there is no dark reversion of phytochromewhich would be relevant for this response. Such Ptr which can be detected with the lipoxygenase response disappears exclusively through degradation with a half‐life of 45 min at 25°C. De novo synthesis of P*r in the hypocotylar hook takes place at a constant rate (zero order rate constant) irrespective of the level of Pr or P*tot, i.e. there is no detectable feedback control of Pr synthesis during the period of experimentation. The data of the present paper are consistent with a quantitative phytochrome model (Scheme 1) which has been advanced and treated quantitatively in the companion paper (Oelze‐Karow and Mohr, 1976).

A new approach to explain the ?high irradiance responses? of photomorphogenesis on the basis of phytochrome

A new approach to explain the “high irradiance responses” of photomorphogenesis on the basis of phytochrome is described. Irradiance and wave-length dependency of different phytochrome reaction models are analyzed. 1) An open phytochrome model which is based on spectophotometric data (Schafer, 1972; Schafer et al., 1972; Schafer and Mohr, 1974) and describes only the induction phenomena is presented; 2) A closed phytochrome-receptor model which is based on the analysis of phytochrome-receptor interactions (Quail et al., 1973a. Quail and Schafer, 1974) describes the principal behavior of high irradiance responses, namely, optimum quantum responsivity in the far-red and blue part of the visible spectrum and the irradiance dependency. This model cannot account for the induction phenomena; 3) An open phytochrome-receptor model which describes both the properties of the induction responses, as well as those of the high irradiance responses, is tested for its physiological relevance by comparing the predictions based on this model with the measured data for phytochrome controlled anthocyanin synthesis (Wagner and Mohr, 1966) and suppression of lipoxygenase synthesis in the mustard seedling (Oelze and Mohr, 1973).

QUANTITATIVE CORRELATION BETWEEN SPECTROPHOTOMETRIC PHYTOCHROME ASSAY AND PHYSIOLOGICAL RESPONSE

Abstract— Apparent synthesis* of the enzyme lipoxygenase in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (Pfr ground state)† through a threshold (all‐or‐none) mechanism. This response was used to determine physiologically the photostationary states, Λ that is, the [Pfr]/[Ptot] ratios established by different wavelengths in the red and far‐red range of the spectrum, including the standard red and far‐red sources used in this laboratory (Mohr, 1966). Under the premises (for which justification has been given on previous occasions) that the [Pfr]/[Ptot] ratio for standard red light is 0.8, and that the decay of Pfr is a first‐order process with a half‐life of 45 min, the [Pfr]/[Ptot] ratios determined physiologically by means of the lipoxygenase response agree with the [Pfr]/[Ptot] ratios determined spectrophotometrically by Hartmann and Spruit (cf. Fig. 9 in Hanke et al., 1969) in hypocotyl hooks of mustard seedlings. In the hook the fr, that is, the [Pfr]/[Ptot] ratio for standard far‐red, is found to be 0.023. In the cotyledons, this ratio is several times higher (Schafer et al., 1972). The conclusion that apparent lipoxygenase synthesis in the cotyledons is controlled by phytochrome located in the hook has been substantiated by further spectrophotometric (Schäfer et al., 1973) and physiological experiments (H. Oelze‐Karow and H. Mohr, in preparation). The minimum steepness of the threshold was determined. An increase of the Pfr level from 118 (relative units) to 130 leads to an instantaneous and total suppression of apparent lipoxygenase synthesis; a corresponding decrease from 130 (relative units) to 118 leads to an immediate resumption of apparent LOG synthesis at full speed. It is concluded that an explanation of the experimental facts requires a cooperative effect on the level of Pfr, a high degree of synchrony on the cellular and organismic level and rapid communication between the hypocotyl hook and the cotyledons.*The term ‘apparent synthesis’ is used operationally in the present paper to denote any increase of enzyme activity, although de novo synthesis of lipoxygenase has not so far been rigorously demonstrated. The usual inhibitor experiments (cf. Oelze‐Karow et al., 1970) have led to the conclusion that intact RNA and protein synthesis is required for an increase of lipoxygenase activity.

Nachweis einer Schwellenwertsregulation durch Phytochrom bei der Photomodulation des Hypokotylstreckungswachstums von Senfkeimlingen (Sinapis alba L.)

The inhibition of hypocotyl lengthening in intact mustard seedlings is controlled by two photosensitive systems which can be experimentally separated. 1. Kinetics of the growth response in the dark after red and far-red irradiation (Figs. 1, 2) demonstrate the operation of Pfr in the ground state via a threshold mechanism similar to the regulation of lipoxygenase synthesis in the mustard cotyledons (c. f. ref. [27]). This threshold mechanism determines the duration of the growth inhibition (Δt) following irradiation (Fig. 1, 2). Δt is dependent on the relative Pfr concentration at the beginning of the dark period and on the half life of Pfr destruction, but it is independent of the quantum flux density of far-red light (Fig. 4). The effect of 5 min red light on Δt can be fully reversed by 5 min far-red light (Fig. 3). The data reveal a quantitative relationship between the relative Pfr concentration and the photomorphogenetic response, Δt (Fig. 6). This relationship may explain in principle the logarithmic correlation between the percentage of phytochrome converted to Pfr by an initial irradiation and the subsequent response which has also been reported in the literature. 2. In continuous far-red light the velocity constant of the steady state growth is controlled by a “high intensity reaction” which shows the usual logarithmic dependence on quantum flux density (Fig. 4, 5), but no phytochrome destruction is apparent (Fig. 2). According to Hartmann (ref.[11–13]) this “high intensity reaction” can also be attributed to Pfr, which in this case acts through some excited state, P fr * .