Activation of EP4 receptor limits transition of acute to chronic heart failure in lipoxygenase deficient mice.

Aim: Immune responsive 12/15 lipoxygenase (12/15LOX)-orchestrate biosynthesis of essential inflammation-resolution mediators during acute inflammatory response in post-myocardial infarction (MI). Lack of 12/15LOX dampens proinflammatory mediator 12-(S)-hydroxyeicosatetraenoic acid (12-(S)-HETE), improves post-MI survival, through the biosynthesis of endogenous mediators epoxyeicosatrienoic acids (EETs; cypoxins) to resolve post-MI inflammation. However, the mechanism that amplifies cypoxins-directed cardiac repair in acute heart failure (AHF) and chronic HF (CHF) remains of interest in MI-directed renal inflammation. Therefore, we determined the role of EETs in macrophage-specific receptor activation in facilitating cardiac repair in 12/15LOX deficient mice experiencing HF. Methods and Results: Risk-free young adult (8 -12 week-old) male C57BL/6J wild-type mice (WT; n = 43) and 12/15LOX-/- mice (n = 31) were subjected to permanent coronary artery ligation and monitored at day (d)1, d5 (as acute HF), and d28 to d56 (8 weeks; chronic HF) post-surgery maintaining no-MI mice that served as d0 naïve controls. Left ventricle (LV) infarcted area of 12/15LOX-/- mice displayed an increase in expression of prostanoid receptor EP4 along with monocyte chemoattractant protein-1 CCL2 in AHF and CHF. The transcriptome analysis of isolated leukocytes (macrophages/neutrophils) from infarcted LV revealed a higher expression of EP4 on reparative macrophages expressing MRC-1 in 12/15LOX-/- mice. Deletion of 12/15LOX differentially modulated the miRNA levels, downregulating miR-23a-3p (~20 fold; p < 0.05) and upregulating miR-125a-5p (~160 fold; p < 0.05) in AHF which promoted polarization of the macrophages towards reparative phenotype. Furthermore, 12/15LOX deletion markedly attenuated renal inflammation with reduced levels of NGAL and KIM-1 and apoptotic markers in the kidney during CHF. Conclusion: In risk-free mice during physiological cardiac repair, absence of 12/15LOX promoted reparative macrophages with marked activation of EP4 signaling thereby improving post-MI survival and limiting renal inflammation in acute and advanced HF. The future studies are warranted to advance the role of EETs in macrophage receptor biology.

Loss of 5-lipoxygenase activity protects mice against paracetamol-induced liver toxicity.

BACKGROUND AND PURPOSE: Paracetamol (acetaminophen) is the most widely used over-the-counter analgesic and overdosing with paracetamol is the leading cause of hospital admission for acute liver failure. 5-Lipoxygenase (5-LO) catalyses arachidonic acid to form LTs, which lead to inflammation and oxidative stress. In this study, we examined whether deletion or pharmacological inhibition of 5-LO could protect mice against paracetamol-induced hepatic toxicity. EXPERIMENTAL APPROACH: Both genetic deletion and pharmacological inhibition of 5-LO in C57BL/6J mice were used to study the role of this enzyme in paracetamol induced liver toxicity. Serum and tissue biochemistry, H&E staining, and real-time PCR were used to assess liver toxicity. KEY RESULTS: Deletion or pharmacological inhibition of 5-LO in mice markedly ameliorated paracetamol-induced hepatic injury, as shown by decreased serum alanine transaminase and aspartate aminotransferase levels and hepatic centrilobular necrosis. The hepatoprotective effect of 5-LO inhibition was associated with induction of the antitoxic phase II conjugating enzyme, sulfotransferase2a1, suppression of the pro-toxic phase I CYP3A11 and reduction of the hepatic transporter MRP3. In 5-LO(-/-) mice, levels of GSH were increased, and oxidative stress decreased. In addition, PPAR α, a nuclear receptor that confers resistance to paracetamol toxicity, was activated in 5-LO(-/-) mice. CONCLUSIONS AND IMPLICATIONS: The activity of 5-LO may play a critical role in paracetamol-induced hepatic toxicity by regulating paracetamol metabolism and oxidative stress.

A soybean cultivar lacking lipoxygenase 2 and 3 has similar calcium bioavailability to a commercial variety despite higher calcium absorption inhibitors.

The aim of this study was to evaluate calcium bioavailability of a new soybean variety without 2 lipoxygenases with better taste and flavor than a commercial variety containing all 3 isozymes. Using the femur (45)Ca uptake method, calcium absorption from a new Brazilian variety, UFV-116, was compared to a common Brazilian variety, OCEPAR 19. Male Sprague-Dawley growing rats weighing 150 to 170 g (10/group) received test meals of whole fat soy flour prepared from UFV-116 or OCEPAR-19 seeds labeled with 10 microCi of (45)Ca. Femurs were removed after 48 h for determination of (45)Ca uptake. Calcium fractional absorption was equivalent between the 2 varieties. The higher oxalate:calcium molar ratio and the higher content of oxalate and phytate (P < 0.05) found in the UFV-116 variety did not affect calcium absorption. Therefore, the new variety is a comparable source of high bioavailable calcium.

IL-4-induced peroxisome proliferator-activated receptor gamma activation inhibits NF-kappaB trans activation in central nervous system (CNS) glial cells and protects oligodendrocyte progenitors under neuroinflammatory disease conditions: implication for CNS-demyelinating diseases.

Th2 phenotype cytokine, IL-4, plays an important role in the regulation of Th1 cell responses and spontaneous remission of inflammatory CNS demyelinating diseases such as multiple sclerosis (MS). In this study we demonstrate IL-4-induced down-regulation of inducible NO synthase (iNOS) expression and survival of differentiating oligodendrocyte progenitors (OPs) in proinflammatory cytokine (Cyt-Mix)-treated CNS glial cells, which is a condition similar to that observed in the brain of a patient with MS. IL-4 treatment of Cyt-Mix-treated CNS glial cells significantly decreased iNOS expression/NO release with a parallel increase in survival of differentiating OPs. IL-4 effects were concentration-dependent and could be reversed by anti-IL-4R Abs. The use of inhibitors for Akt, p38 MAPK, and peroxisome proliferator-activated receptor gamma (PPAR-gamma) antagonist revealed that inhibition of Cyt-Mix-induced iNOS expression and survival of differentiating OPs by IL-4 is via PPAR-gamma activation. There was a coordinate increase in the expression of both PPAR-gamma and its natural ligand-producing enzyme 12/15-lipoxygenase (12/15-LOX) in IL-4-treated cells. Next, EMSA, immunoblots, and transient cotransfection studies with reporter plasmids (pNF-kappaB-Luc and pTK-PPREx3-Luc) and 12/15-LOX small interfering RNA revealed that IL-4-induced PPAR-gamma activation antagonizes NF-kappaB transactivation in Cyt-Mix-treated astrocytes. In support of this finding, similarly treated 12/15-LOX(-/-) CNS glial cells further corroborated the result. Furthermore, there was reversal of IL-4 inductive effects in the brain of LPS-challenged 12/15-LOX(-/-) mice when compared with LPS-challenged wild-type mice. Together, these data for the first time demonstrate the inhibition of Cyt-Mix-induced NF-kappaB transactivation in CNS glial cells by IL-4 via PPAR-gamma activation, hence its implication for the protection of differentiating OPs during MS and other CNS demyelinating diseases.

[Development of a new type soybean germplasm with null lipoxygenase isozymes].

Soybean protein is a kind of high-quality protein composed of balanced amino acids. It contains all kinds of amino acids, especially eight amino acids necessary for human, but also contains some components that are not good for human and affect food quality, such as Lipoxygenase (Lox) and Trypsin inhibitor (Ti). Nutritional value and processing quality of soybean can be improved by means of development of new variety with null Lox and Ti. In this paper, a new type soybean germplasm with null lipoxygenase isozymes was developed by Institute of Crop Sciences, Chinese Academy of Agricultural Sciences through years of biochemical marker assistant selection for null lipoxygenase by means of isoelectric focusing-polyacrylamid gel electrophoresis (IEF-PAGE) in the hybrid progenies of "96P17" (Female parent, a null lipoxygenase 2.3 line) and "93704" (Male parent, a null lipoxygenase 1.3 line). It is the first new soybean germplasm with null Lox1.2.3 genes in our country, which will contribute to soybean breeding for high quality, soybean production and utilization. In this paper, the development process of new type soybean germplasm is described.

Impact of the suppression of lipoxygenase and hydroperoxide lyase on the quality of the green odor in green leaves.

Most of the volatile compounds responsible for the "green" notes to the aroma of fruits and vegetables are produced by the degradation of polyunsaturated fatty acids through the lipoxygenase pathway. The most determinant steps of this pathway are the peroxidation of free linoleic or linolenic acid by the action of lipoxygenase and then the lysis of the resulting hydroperoxides through a reaction catalyzed by the hydroperoxide lyase. This work analyzes the impact of the depletion of these enzymes on the volatile composition of leaves from potato plants. A characterization of the volatile profiles of the different potato mutants, a study of the metabolism of radiolabeled linoleic acid, and a determination of lipoxygenase activity have been carried out. The depletion of hydroperoxide lyase induced an increase in the lipoxygenase activity and the content of C5 volatiles, whereas the lipoxygenase silencing caused a severe decrease in the amount of volatiles produced by the leaves and always in the intensity of their aroma. The changes in the sensory evaluation of leaf aroma, as correlated to depletion of the two enzymes, have been investigated. The perspectives of producing vegetable products with a modified aroma by genetic engineering are discussed in light of the statistical results.

A lipase-inhibiting protein from lipoxygenase-deficient soybean seeds.

A lipase-inhibiting protein was isolated from lipoxygenase (LOX)-deficient soybean seeds. The molecular mass of the protein was 56.0-kDa and the N-terminal amino acid was blocked. The protein was identified by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The masses of the lysyl endopeptidase-digested peptides of the 56.0-kDa inhibiting protein were almost identical to the calculated masses of the theoretically predicted lysyl endopeptidase-treated peptides of beta-amylase from soybean seed. In a previous paper (Biosci. Biotechnol. Biochem., 62, 1498-1503, 1998), we reported that LOX-1, an isozyme of soybean seed LOX, inhibited hydrolysis of soybean oil by pancreatic lipase. Purified beta-amylase also inhibited lipase activity, although the magnitude of inhibition was weaker than that by LOX-1. Thus, there are at least two lipase-inhibiting proteins, one is a LOX and the other is a beta-amylase, in soybean seed.

Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells.

Nordihydroguaiaretic acid (NDGA) induces apoptosis in a variety of cell lines. The mechanism(s) of this effect is not known, although the focus has been on the ability of NDGA to inhibit lipoxygenase (LOX) activities. In the present study, NDGA-induced apoptosis was studied in a murine hematopoietic cell line, FL5.12. Although this cell line lacks detectable LOX protein or activities, NDGA (10 microM) was able to induce apoptosis. There was a massive loss of mitochondrial membrane potential by 4 h after the addition of NDGA, suggesting that this organelle might be targeted by NDGA. A pro-oxidant NDGA effect has been suggested as playing a role in apoptosis. This was supported by the findings that glutathione disulfide levels were increased by 4 h following treatment with 10 microM NDGA, that pretreatment with N-acetylcysteine completely blocked the NDGA-induced loss of membrane potential and apoptosis, and that lipid peroxidation was enhanced in cells treated with NDGA. However, no evidence of increased levels of reactive oxygen could be seen in NDGA-treated cells loaded with dichlorofluorescin diacetate or dihydrorhodamine and analyzed by flow cytometry. Bcl-X(L) protein levels were unaffected by NDGA treatment. Caspase-3 was rapidly activated with a peak at 8 h after FL5.12 cells were treated with NDGA. Ac-DEVD-CHO (25 microM) and boc-asp-FMK (20 microM) both inhibited caspase-3 enzyme activity by 97% 8 h after NDGA treatment. Boc-asp-FMK, a more general caspase inhibitor, delayed NDGA-induced apoptosis while Ac-DEVD-CHO, a more specific inhibitor of caspase-3, had no effect. These results suggest that NDGA-induced apoptosis happens through reactions that depolarize mitochondria, oxidize glutathione and lipids, but do not generate significant amounts of free reactive oxygen species.

[Defects in the prostaglandin system. IX. Lipoxygenase defect of thrombocytes in a patient with polycythemia vera]. / Defekte im Prostaglandinsystem. IX. Lipoxygenasedefekt der Thrombozyten bei einem Patienten mit Polycythaemia vera.

Patients with the myeloproliferative syndrome (MPS) often show morphological and functional platelet abnormalities and an increased incidence of lowered cyclooxygenase- and/or lipoxygenase activity. We present the case history of a 68-year-old male patient with polycythaemia vera in whom an absolute absence of platelet lipoxygenase activity has been detected for the first time in the literature.

Lipoxygenase activities of human platelets and their subcellular fractions: comparison between lipoxygenase-deficient platelets and normal platelets.

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.

Deficient induction of leukotriene synthesis in human neutrophils by lipoxygenase-deficient platelets.

The effect of human platelets with deficient lipoxygenase activities on leukotriene B4 (LTB4) synthesis by neutrophils was studied. When arachidonic acid (AA) metabolites obtained from the incubation of washed normal neutrophils and platelets with N-formylmethionylleucylphenylalanine (FMLP), cytochalasin B, and AA were analyzed by reversed-phase high-performance liquid chromatography, the synthesis of 5-lipoxygenase products, including LTB4, was remarkably stimulated by platelets, with their maximal effect at a ratio of platelets to neutrophils of 15:1. However, the use of lipoxygenase-deficient platelets obtained from four patients with myeloproliferative disorders instead of normal platelets showed the deficient production of 5-lipoxygenase-derived products, whereas platelets with normal lipoxygenase activities obtained from MPD patients stimulated the 5-lipoxygenase pathway similarly to the way in which normal platelets did. The addition of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a labile AA metabolite via the platelet lipoxygenase pathway, could activate the 5-lipoxygenase pathway in neutrophils incubated with FMLP, cytochalasin B and AA, but its stable end product, 12-hydroxyeicosatetraenoic acid, could not. Thus, it is suggested that lipoxygenase-deficient platelets did not sufficiently stimulate LTB4 synthesis during platelet-neutrophil interactions because of defective formation of 12-HPETE. This altered interaction between platelets and neutrophils through the lipoxygenase pathway might result in deficient responses at sites of thrombosis or inflammation in patients with deficient platelet lipoxygenase activities.

Deficiency of platelet lipoxygenase activity in myeloproliferative disorders.

The myeloproliferative disorders are characterized by frequent bleeding and thrombotic complications, which have been attributed to abnormal platelet function. In 24 of 60 patients studied, reduced activity of the platelet lipoxygenase pathway for oxygenation of arachidonic acid was revealed by a new direct assay. This assay measured arachidonic acid-induced oxygen consumption in platelets preincubated with aspirin to block cyclooxygenase activity. Patients with secondary polycythemia or thrombocytosis had normal lipoxygenase activity . In the cells of seven of eight patients with lipoxygenase deficiency arachidonic acid induced increased synthesis of thromboxane, the major cyclooxygenase product. Nevertheless, patients with deficient lipoxygenase activity tended to have episodes of hemorrhage rather than thrombosis. Bleeding complications occurred in 67 per cent of patients with lipoxygenase deficiency, but in only 19 per cent of those with normal lipoxygenase activity (P less than 0.001). In contrast, 13 per cent of lipoxygenase-deficient patients, but 31 per cent of patients with other myeloproliferative disorders, had thromboembolic complications. Measurement of platelet lipoxygenase activity may be of diagnostic value in distinguishing myeloproliferative disorders from secondary thrombocytosis or polycythemia. Lipoxygenase deficiency may prove to be a useful natural model for investigating the role of lipoxygenase products in hemostasis.