Michel Rigaud

Transformation of arachidonic acid into monohydroxy-eicosatetraenoic acids by mouse peritoneal macrophages

Mouse peritoneal macrophages synthesize 6 monohydroxylated eicosatetraenoic acids when incubated with exogenous arachidonic acid. These compounds were identified by chromatographic techniques (high pressure liquid chromatography and high efficiency glass capillary column gas chromatography and mass spectrometry. The chromatographic and spectrometric data are presented. These results show that peritoneal macrophages constitute one of the best systems to study in evaluating the metabolism of oxygenated products of arachidonic acid.

Inhibition of lipoxygenase activity and HL60 leukemic cell proliferation by ursolic acid isolated from heather flowers (Calluna vulgaris)

A compound was isolated and purified from heather flowers (Calluna vulgaris) based on its ability to inhibit lipoxygenase activity. This molecule was characterized as ursolic acid by GC-MS. Ursolic acid was found to be an inhibitor of both potato tuber 5-lipoxygenase and soybean 15-lipoxygenase with IC50 values of 0.3 mM. Ursolic acid also inhibits lipoxygenase activity in mouse peritoneal macrophages at 1 microM and HL60 leukemic cells growth (IC50 = 0.85 microM) as well as their DNA synthesis (IC50 = 1 microM). The possible role of lipoxygenase inhibition in the proliferation of leukemic cells is discussed.

Transformation of arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid by mouse peritoneal macrophages

Abstract Mouse peritoneal macrophages were incubated at 37°C for 30 min with arachidonic acid (all- cis -5,8,11,14-eicosatetraenoic acid). Oxygenation of arachidonic acid in mouse peritoneal macrophages occurs by two major pathways: fatty acid cyclooxygenase and lipoxygenase. The major metabolite of the latter is 12-hydroxy-5,8,10,14-eicosatetraenoic acid which was identified by gas liquid chromatography on high resolution glass capillary column and mass spectrometry.

Basal level of human platelet prostaglandins: PGE1 is more elevated than PGE2

Radioimmunoassays of platelet prostaglandins E1 and F1 alpha in platelet rich plasma or platelet suspension, demonstrate that both PGE1 and PGF1 alpha are present at higher concentrations than prostaglandins E2 and F2 alpha. Gas chromatography--mass spectrometry determinations of prostaglandins E1 and E2 in resting washed platelets confirm this difference. Lastly, there is a greater incorporation of [1--14C] acetate into prostaglandins E1 and F1 alpha compared to that into prostaglandins E2 and F2 alpha.

Molecular characterization of L2 lipoxygenase from maize embryos.

We investigated the expression and accumulation pattern of lipoxygenaseisoforms throughout the maize plant life. Two forms of lipoxygenase L1and L2 have been identified as acidic proteins of 100 kDa (pI 6.4) and90 kDa (pI 5.5-5.7) which accumulate in dry embryos and in variousorgans of maize seedlings. In young embryos, only the L2 form wasdetected and accumulation of L2 mRNA decreased during embryodevelopment. Identification of lipoxygenases from in vivo and in vitro synthesized proteins indicates that similar levels of both L1and L2 forms accumulated during treatment with abscisic acid, (ABA)gibberellic acid (GA3) and jasmonic acid (JA). However,differences in the activity of both enzymes were detected. By using anantiserum directed against purified L2 we isolated and characterized apartial cDNA clone of maize embryos encoding a lipoxygenase. The deducedamino acid sequence of L2 cDNA shares 78% identity with the rice L2protein, and 51-56% identity with lipoxygenases from thedicotyledonous plants soybean and Arabidopsis/. DNA blotanalysis indicated that maize contains a family of lipoxygenase geneswhich are presently being characterized.

LIPOXYGENASES FROM ZEA MAYS L. PURIFICATION AND PHYSICOCHEMICAL CHARACTERISTICS

Abstract Maize ( Zea mays L.) seeds after 5 d germination contain at least two lipoxygenase isoenzymes, L1 and L2. Enzymes were extracted from acetone powder with 0.1 M sodium acetate (pH 4.5) buffer, supplemented with a non-ionic detergent (0.1% Brij 99) and DETAPAC (0.1 mM). The pI of L1 is 6.40 and isoelectric focusing of L2 results in two peaks with pI values in close proximity: 5.55 and 5.70. L1 and L2 are monomeric proteins of M r 100000 and 90000, respectively. Linoleic acid, 18:2( n - 6), is a good substrate for L2, but L1 has more affinity for α-linolenic acid, 18:3( n − 3). These kinetic studies could indicate a different functional role of the two isoenzymes.

Dual metabolic pathways of 12-HETE in rat aortic smooth muscle cells

12(S)-HETE, a major lipoxygenase-derived compound from arachidonic acid is incorporated and metabolized by vascular smooth muscle cells via beta-oxidation. We have now identified for the first time in this cell type 12(S)-HETE metabolites formed by a combination of reductase and oxidation pathways. HPLC and GC-MS analysis of time-course experiments allow us to characterize two different metabolic pathways: a direct peroxisomal beta-oxidation of 12(S)-HETE leading to the formation of 16:3 (8-OH) which accumulates first and a reduction of one of the conjugated double bonds of 12(S)-HETE giving the dihydro-intermediate 20:3(12-OH) that transiently accumulates before being converted itself by peroxisomal beta-oxidation to 16:2(8-OH). Taken together these results may suggest that the transient accumulation of 20:3(12-OH) through transcellular metabolism of 12(S)-HETE may represent a part of the modulatory effect of 12(S)-HETE on vascular function.

Enzymatic synthesis of structural analogs of PAF-acether by phospholipase D-catalysed transphosphatidylation

PAF-acether can be transformed into analogs by the phospholipase D enzyme activity of Streptomyces sp. In this reaction choline is replaced by primary cyclic alcohols (acceptors). The reaction products, cyclic phospholipid and phosphatidic acid, were separated by silicic acid chromatography. This procedure enabled us to synthetize five analogs of PAF-acether, with a cyclic ring structure. The primary cyclic alcohols used in this work were: 3-(2-hydroxyethyl)-indol, OH-Et-I; 2-(hydroxymethyl)-1,4-benzodioxan, OH-Met-BZD; N-(2-hydroxyethyl)-phthalimide, OH-Et-PHT; 2-(2-thienyl)-ethanol, Th-EtOH; (1-R)-(-)-Nopol, R-NOP.