Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase.

PubMed

Garscha, Ulrike; Romp, Erik; Pace, Simona; Rossi, Antonietta; Temml, Veronika; Schuster, Daniela; König, Stefanie; Gerstmeier, Jana; Liening, Stefanie; Werner, Markus; Atze, Heiner; Wittmann, Sandra; Weinigel, Christina; Rummler, Silke; Scriba, Gerhard K; Sautebin, Lidia; Werz, Oliver

2017-08-24

Arachidonic acid (AA) is metabolized to diverse bioactive lipid mediators. Whereas the 5-lipoxygenase-activating protein (FLAP) facilitates AA conversion by 5-lipoxygenase (5-LOX) to pro-inflammatory leukotrienes (LTs), the soluble epoxide hydrolase (sEH) degrades anti-inflammatory epoxyeicosatrienoic acids (EETs). Accordingly, dual FLAP/sEH inhibition might be advantageous drugs for intervention of inflammation. We present the in vivo pharmacological profile and efficiency of N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N'-(3,4-dichlorophenyl)urea (diflapolin) that dually targets FLAP and sEH. Diflapolin inhibited 5-LOX product formation in intact human monocytes and neutrophils with IC 50  = 30 and 170 nM, respectively, and suppressed the activity of isolated sEH (IC 50  = 20 nM). Characteristic for FLAP inhibitors, diflapolin (I) failed to inhibit isolated 5-LOX, (II) blocked 5-LOX product formation in HEK cells only when 5-LOX/FLAP was co-expressed, (III) lost potency in intact cells when exogenous AA was supplied, and (IV) prevented 5-LOX/FLAP complex assembly in leukocytes. Diflapolin showed target specificity, as other enzymes related to AA metabolism (i.e., COX1/2, 12/15-LOX, LTA 4 H, LTC 4 S, mPGES 1 , and cPLA 2 ) were not inhibited. In the zymosan-induced mouse peritonitis model, diflapolin impaired vascular permeability, inhibited cysteinyl-LTs and LTB 4 formation, and suppressed neutrophil infiltration. Diflapolin is a highly active dual FLAP/sEH inhibitor in vitro and in vivo with target specificity to treat inflammation-related diseases.

Lipoxygenase-mediated pro-radical effect of melatonin via stimulation of arachidonic acid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Radogna, F.; Sestili, P.; Martinelli, C.

We have shown that melatonin immediately and transiently stimulates intracellular free radical production on a set of leukocytes, possibly as a consequence of calmodulin binding. We show here that melatonin-induced ROS are produced by lipoxygenase (LOX), since they are prevented by a set of LOX inhibitors, and are accompanied by increase of the 5-LOX product 5-HETE. LOX activation is accompanied by strong liberation of AA; inhibition of Ca{sup 2+}-independent, but not Ca{sup 2+}-dependent, phospholipase A2 (PLA2), prevents both melatonin-induced arachidonic acid and ROS production, whereas LOX inhibition only prevents ROS, indicating that PLA2 is upstream with respect to LOX, asmore » occurs in many signaling pathways. Chlorpromazine, an inhibitor of melatonin-calmodulin interaction, inhibits both ROS and arachidonic acid production, thus possibly placing calmodulin at the origin of a melatonin-induced pro-radical pathway. Interestingly, it is known that Ca{sup 2+}-independent PLA2 binds to calmodulin: our results are compatible with PLA2 being liberated by melatonin from a steady-state calmodulin sequestration, thus initiating an arachidonate signal transduction. These results delineate a novel molecular pathway through which melatonin may participate to the inflammatory response.« less

Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism.

PubMed

Harizi, Hedi; Juzan, Monique; Moreau, Jean-François; Gualde, Norbert

2003-01-01

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.

BRP-187: A potent inhibitor of leukotriene biosynthesis that acts through impeding the dynamic 5-lipoxygenase/5-lipoxygenase-activating protein (FLAP) complex assembly.

PubMed

Garscha, Ulrike; Voelker, Susanna; Pace, Simona; Gerstmeier, Jana; Emini, Besa; Liening, Stefanie; Rossi, Antonietta; Weinigel, Christina; Rummler, Silke; Schubert, Ulrich S; Scriba, Gerhard K E; Çelikoğlu, Erşan; Çalışkan, Burcu; Banoglu, Erden; Sautebin, Lidia; Werz, Oliver

2016-11-01

The pro-inflammatory leukotrienes (LTs) are formed from arachidonic acid (AA) in activated leukocytes, where 5-lipoxygenase (5-LO) translocates to the nuclear envelope to assemble a functional complex with the integral nuclear membrane protein 5-LO-activating protein (FLAP). FLAP, a MAPEG family member, facilitates AA transfer to 5-LO for efficient conversion, and LT biosynthesis critically depends on FLAP. Here we show that the novel LT biosynthesis inhibitor BRP-187 prevents the 5-LO/FLAP interaction at the nuclear envelope of human leukocytes without blocking 5-LO nuclear redistribution. BRP-187 inhibited 5-LO product formation in human monocytes and polymorphonuclear leukocytes stimulated by lipopolysaccharide plus N-formyl-methionyl-leucyl-phenylalanine (IC 50 =7-10nM), and upon activation by ionophore A23187 (IC 50 =10-60nM). Excess of exogenous AA markedly impaired the potency of BRP-187. Direct 5-LO inhibition in cell-free assays was evident only at >35-fold higher concentrations, which was reversible and not improved under reducing conditions. BRP-187 prevented A23187-induced 5-LO/FLAP complex assembly in leukocytes but failed to block 5-LO nuclear translocation, features that were shared with the FLAP inhibitor MK886. Whereas AA release, cyclooxygenases and related LOs were unaffected, BRP-187 also potently inhibited microsomal prostaglandin E 2 synthase-1 (IC 50 =0.2μM), another MAPEG member. In vivo, BRP-187 (10mg/kg) exhibited significant effectiveness in zymosan-induced murine peritonitis, suppressing LT levels in peritoneal exudates as well as vascular permeability and neutrophil infiltration. Together, BRP-187 potently inhibits LT biosynthesis in vitro and in vivo, which seemingly is caused by preventing the 5-LO/FLAP complex assembly and warrants further preclinical evaluation. Copyright © 2016 Elsevier Inc. All rights reserved.

ALOX5AP Overexpression in Adipose Tissue Leads to LXA4 Production and Protection Against Diet-Induced Obesity and Insulin Resistance.

PubMed

Elias, Ivet; Ferré, Tura; Vilà, Laia; Muñoz, Sergio; Casellas, Alba; Garcia, Miquel; Molas, Maria; Agudo, Judith; Roca, Carles; Ruberte, Jesús; Bosch, Fatima; Franckhauser, Sylvie

2016-08-01

Eicosanoids, such as leukotriene B4 (LTB4) and lipoxin A4 (LXA4), may play a key role during obesity. While LTB4 is involved in adipose tissue inflammation and insulin resistance, LXA4 may exert anti-inflammatory effects and alleviate hepatic steatosis. Both lipid mediators derive from the same pathway, in which arachidonate 5-lipoxygenase (ALOX5) and its partner, arachidonate 5-lipoxygenase-activating protein (ALOX5AP), are involved. ALOX5 and ALOX5AP expression is increased in humans and rodents with obesity and insulin resistance. We found that transgenic mice overexpressing ALOX5AP in adipose tissue had higher LXA4 rather than higher LTB4 levels, were leaner, and showed increased energy expenditure, partly due to browning of white adipose tissue (WAT). Upregulation of hepatic LXR and Cyp7a1 led to higher bile acid synthesis, which may have contributed to increased thermogenesis. In addition, transgenic mice were protected against diet-induced obesity, insulin resistance, and inflammation. Finally, treatment of C57BL/6J mice with LXA4, which showed browning of WAT, strongly suggests that LXA4 is responsible for the transgenic mice phenotype. Thus, our data support that LXA4 may hold great potential for the future development of therapeutic strategies for obesity and related diseases. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

A medicinal extract of Scutellaria baicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5-lipoxygenase to reduce inflammation.

PubMed

Burnett, B P; Jia, Q; Zhao, Y; Levy, R M

2007-09-01

A mixed extract containing two naturally occurring flavonoids, baicalin from Scutellaria baicalensis and catechin from Acacia catechu, was tested for cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibition via enzyme, cellular, and in vivo models. The 50% inhibitory concentration for inhibition of both ovine COX-1 and COX-2 peroxidase enzyme activities was 15 microg/mL, while the mixed extract showed a value for potato 5-LOX enzyme activity of 25 microg/mL. Prostaglandin E2 generation was inhibited by the mixed extract in human osteosarcoma cells expressing COX-2, while leukotriene production was inhibited in both human cell lines, immortalized THP-1 monocyte and HT-29 colorectal adenocarcinoma. In an arachidonic acid-induced mouse ear swelling model, the extract decreased edema in a dose-dependent manner. When arachidonic acid was injected directly into the intra-articular space of mouse ankle joints, the mixed extract abated the swelling and restored function in a rotary drum walking model. These results suggest that this natural, flavonoid mixture acts via "dual inhibition" of COX and LOX enzymes to reduce production of pro-inflammatory eicosanoids and attenuate edema in an in vivo model of inflammation.

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1.

PubMed

Wecksler, Aaron T; Garcia, Natalie K; Holman, Theodore R

2009-09-15

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.

A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes

PubMed Central

Teder, Tarvi; Lõhelaid, Helike; Boeglin, William E.; Calcutt, Wade M.; Brash, Alan R.; Samel, Nigulas

2015-01-01

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using 18O-labeled substrate and incubations in H218O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom. PMID:26100625

Ketoconazole attenuates radiation-induction of tumor necrosis factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hallahan, D.E.; Virudachalam, S.; Kufe, D.W.

1994-07-01

Previous work has demonstrated that inhibitors of phospholipase A2 attenuate ionizing radiation-induced arachidonic acid production, protein kinase C activation, and prevent subsequent induction of the tumor necrosis factor gene. Because arachidonic acid contributes to radiation-induced tumor necrosis factor expression, the authors analyzed the effects of agents which alter arachidonate metabolism on the regulation of this gene. Phospholipase A2 inhibitors quinicrine, bromphenyl bromide, and pentoxyfylline or the inhibitor of lipoxygenase (ketoconazole) or the inhibitor of cycloxygenase (indomethacine) were added to cell culture 1 h prior to irradiation. Radiation-induced tumor necrosis factor gene expression was attenuated by each of the phospholipase A2more » inhibitors (quinicrine, bromphenylbromide, and pentoxyfylline). Furthermore, ketoconazole attenuated X ray induced tumor necrosis factor gene expression. Conversely, indomethacin enhanced tumor necrosis factor expression following irradiation. The finding that radiation-induced tumor necrosis factor gene expression was attenuated by ketoconazole suggests that the lipoxygenase pathway participates in signal transduction preceding tumor necrosis factor induction. Enhancement of tumor necrosis factor expression by indomethacin following irradiation suggests that prostaglandins produced by cyclooxygenase act as negative regulators of tumor necrosis factor expression. Inhibitors of tumor necrosis factor induction ameliorate acute and subacute sequelae of radiotherapy. The authors propose therefore, that ketoconazole may reduce acute radiation sequelae such as mucositis and esophagitis through a reduction in tumor necrosis factor induction or inhibition of phospholipase A2 in addition to its antifungal activity. 25 refs., 2 figs.« less

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

PubMed Central

Krug, H F; Hamm, U; Berndt, J

1988-01-01

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation. Images Fig. 4. PMID:3128272

Recent advances in the search for novel 5-lipoxygenase inhibitors for the treatment of asthma.

PubMed

Bruno, Ferdinando; Spaziano, Giuseppe; Liparulo, Angela; Roviezzo, Fiorentina; Nabavi, Seyed Mohammed; Sureda, Antoni; Filosa, Rosanna; D'Agostino, Bruno

2018-06-10

The products of 5-lipoxygenase are synthesized and released in the airway when an asthmatic reaction occurs. 5-lipoxygenase via arachidonic acid metabolism produces leukotrienes that mediate bronchoconstriction and inflammatory modifications essential in the pathophysiology of asthma. Until to now, only one approved 5-LO inhibitor, zileuton, can be found as a potential therapy for asthma. With the increasing number of indications for anti-leukotriene (anti-LT) drugs, the development of 5-LO inhibitor agents becomes increasingly important. The present MiniReview reports an update on 5-LO inhibitors currently under clinical investigation. In addition, the latest advances focused on the development of new 5-lipoxygenase inhibitors as asthma anti-inflammatory agents are also discussed. Copyright © 2017. Published by Elsevier Masson SAS.

[Inhibition of the lymphocyte response by metabolites released by the lipoxygenase of mouse macrophages].

PubMed

Gualde, N; Rigaud, M; Rabinovitch, H; Durand, J; Beneytout, J L; Breton, J C

1981-10-26

Arachidonic acid can be transformed into a series of metabolites by the lipoxygenase enzyme activity of Mouse peritoneal macrophages. The resulting metabolites inhibit tritiated thymidine uptake by Mouse splenocytes stimulated by ConA or PHA. They suppress the development of killer cells. When mice are injected with 15-hydroperoxide, their splenocytes show a decreased H3-thymidine uptake after lectin stimulation.

Stereocontrol of Arachidonic Acid Oxygenation by Vertebrate Lipoxygenases

PubMed Central

Jansen, Christian; Hofheinz, Katharina; Vogel, Robert; Roffeis, Jana; Anton, Monika; Reddanna, Pallu; Kuhn, Hartmut; Walther, Matthias

2011-01-01

Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10–30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms. PMID:21880725

Effect of extraction method on the concentrations of selected bioactive compounds in mandarin juice.

PubMed

Nogata, Yoichi; Ohta, Hideaki; Sumida, Takashi; Sekiya, Keizo

2003-12-03

A mandarin-type citrus fruit, ponkan (Citrus reticulata), was processed by in-line, chopper pulper, and hand-press extractions to investigate the effect of extraction method on the concentrations of bioactive compounds in processed juice. Concentrations of polymethoxylated flavones (tangeretin, nobiletin, and sinensetin) and beta-cryptoxanthin in juice, and inhibitory activities against arachidonate cyclooxygenase and lipoxygenases of the juice extract were analyzed. The juice processed by hand-press extraction contained the largest amounts of nobiletin (3.56 mg/100 mL), tangeretin (4.10 mg/100 mL), and sinensetin (0.13 mg/100 mL). Concentrations of beta-cryptoxanthin were 0.66, 0.59, 0.55, and 0.50 mg/100 mL in chopper pulper, in-line (5/64 in.), in-line (8/64 in.) and hand-press juices, respectively. Both extracts of in-line juices showed greater inhibitory activity toward platelet 12-lipoxygenase than the others. The inhibitory effect of hand-press juice extract on platelet cyclooxygenase activity was remarkable among juice extracts. All juice extracts effectively inhibited polymorphonuclear 5-lipoxygenase activity at nearly the same rate.

A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes.

PubMed

Shak, S; Perez, H D; Goldstein, I M

1983-12-25

We have found that a novel dioxygenation product of arachidonic acid, 8(S),15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid (8,15-diHETE), possesses chemotactic activity for human polymorphonuclear leukocytes comparable to that of leukotriene B4. Authentic 8,15-diHETE, identified by gas chromatography-mass spectrometry, was prepared by treating arachidonic acid with soybean lipoxygenase and was purified by reverse-phase high performance liquid chromatography. Using a "leading front" assay, 8,15-diHETE exhibited significant chemotactic activity at a concentration of 5.0 ng/ml. Maximum chemotactic activity was observed at a concentration of 30 ng/ml. The 8,15-diHETE generated by mixed human leukocytes after stimulation with arachidonic acid and the calcium ionophore, A23187, exhibited quantitatively similar chemotactic activity. Two synthetic all-trans conjugated isomers of 8,15-diHETE, however, were not chemotactic at concentrations up to 500 ng/ml. In contrast to its potent chemotactic activity, 8,15-diHETE (at concentrations up to 10 micrograms/ml) was relatively inactive with respect to its ability to provoke either degranulation or generation of superoxide anion radicals by cytochalasin B-treated leukocytes. Both leukotriene B4 and 8,15-diHETE may be important mediators of inflammation.

A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE.

PubMed

Teder, Tarvi; Lõhelaid, Helike; Boeglin, William E; Calcutt, Wade M; Brash, Alan R; Samel, Nigulas

2015-08-07

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using (18)O-labeled substrate and incubations in H2(18)O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Lipocalin-2 Deficiency Attenuates Insulin Resistance Associated With Aging and Obesity

PubMed Central

Law, Ivy K.M.; Xu, Aimin; Lam, Karen S.L.; Berger, Thorsten; Mak, Tak W.; Vanhoutte, Paul M.; Liu, Jacky T.C.; Sweeney, Gary; Zhou, Mingyan; Yang, Bo; Wang, Yu

2010-01-01

OBJECTIVE The proinflammatory cytokines/adipokines produced from adipose tissue act in an autocrine and/or endocrine manner to perpetuate local inflammation and to induce peripheral insulin resistance. The present study investigates whether lipocalin-2 deficiency or replenishment with this adipokine has any impact on systemic insulin sensitivity and the underlying mechanisms. METHODS AND RESULTS Under conditions of aging or dietary-/genetic-induced obesity, lipocalin-2 knockout (Lcn2-KO) mice show significantly decreased fasting glucose and insulin levels and improved insulin sensitivity compared with their wild-type littermates. Despite enlarged fat mass, inflammation and the accumulation of lipid peroxidation products are significantly attenuated in the adipose tissues of Lcn2-KO mice. Adipose fatty acid composition of these mice varies significantly from that in wild-type animals. The amounts of arachidonic acid (C20:4 n6) are elevated by aging and obesity and are paradoxically further increased in adipose tissue, but not skeletal muscle and liver of Lcn2-KO mice. On the other hand, the expression and activity of 12-lipoxygenase, an enzyme responsible for metabolizing arachidonic acid, and the production of tumor necrosis factor-α (TNF-α), a critical insulin resistance–inducing factor, are largely inhibited by lipocalin-2 deficiency. Lipocalin-2 stimulates the expression and activity of 12-lipoxygenase and TNF-α production in fat tissues. Cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC), an arachidonate lipoxygenase inhibitor, prevents TNF-α expression induced by lipocalin-2. Moreover, treatment with TNF-α neutralization antibody or CDC significantly attenuated the differences of insulin sensitivity between wild-type and Lcn2-KO mice. CONCLUSIONS Lipocalin-2 deficiency protects mice from developing aging- and obesity-induced insulin resistance largely by modulating 12-lipoxygenase and TNF-α levels in adipose tissue. PMID:20068130

Protection of dopaminergic neurons by 5-lipoxygenase inhibitor.

PubMed

Kang, Kai-Hsiang; Liou, Horng-Hui; Hour, Mann-Jen; Liou, Houng-Chi; Fu, Wen-Mei

2013-10-01

Neuroinflammation and oxidative stress are important factors that induce neurodegeneration in age-related neurological disorders. 5-Lipoxygenase (5-LOX) is the enzyme responsible for catalysing the synthesis of leukotriene or 5-HETE from arachidonic acid. 5-LOX is expressed in the central nervous system and may cause neurodegenerative disease. In this study, we investigated the effect of the pharmacological inhibition of 5-lipoxygenase on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/MPP(+)-induced dopaminergic neuronal death in midbrain neuron-glia co-cultures and in mice. It was found that 5-LOX was over-expressed in astrocytes after the injection of MPTP into C57BL6 mice. MK-886, a specific inhibitor of 5-LOX activating protein (FLAP), significantly increased [(3)H]-dopamine uptake, a functional indicator of the integrity of dopaminergic neurons, in midbrain cultures or the SH-SY5Y human dopaminergic cell line following MPP(+) treatment. In addition, LTB₄, one of 5-LOX's downstream products, was increased in the striatum and substantia nigra following MPTP injection in mice. LTB₄ but not LTD₄ and 5-HETE enhanced MPP(+)-induced neurotoxicity in primary midbrain cultures. MK-886 administration increased the number of tyrosine hydroxylase-positive neurons in the substantia nigra and the dopamine content in the striatum in MPTP-induced parkinsonian mice. Furthermore, the MPTP-induced upregulation of LTB₄ in the striatum and substantia nigra was antagonised by MK-886. These results suggest that 5-LOX inhibitors may be developed as novel neuroprotective agents and LTB₄ may play an important pathological role in Parkinson's disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

Regulation of CRIg Expression and Phagocytosis in Human Macrophages by Arachidonate, Dexamethasone, and Cytokines

PubMed Central

Gorgani, Nick N.; Thathaisong, Umaporn; Mukaro, Violet R.S.; Poungpair, Ornnuthchar; Tirimacco, Amanda; Hii, Charles S.T.; Ferrante, Antonio

2011-01-01

Although the importance of the macrophage complement receptor immunoglobulin (CRIg) in the phagocytosis of complement opsonized bacteria and in inflammation has been established, the regulation of CRIg expression remains undefined. Because cellular activation during inflammation leads to the release of arachidonate, a stimulator of leukocyte function, we sought to determine whether arachidonate regulates CRIg expression. Adding arachidonate to maturing human macrophages and to prematured CRIg+ macrophages caused a significant decrease in the expression of cell-surface CRIg and CRIg mRNA. This effect was independent of the metabolism of arachidonate via the cyclooxygenase and lipoxygenase pathways, because it was not inhibited by the nonsteroidal anti-inflammatory drugs indomethacin and nordihydroguaiaretic acid. Studies with specific pharmacological inhibitors of arachidonate-mediated signaling pathways showed that protein kinase C was involved. Administration of dexamethasone to macrophages caused an increase in CRIg expression. Studies with proinflammatory and immunosuppressive cytokines showed that IL-10 increased, but interferon-γ, IL-4, and transforming growth factor-β1 decreased CRIg expression on macrophages. This down- and up-regulation of CRIg expression was reflected in a decrease and increase, respectively, in the phagocytosis of complement opsonized Candida albicans. These data suggest that a unique inflammatory mediator network regulates CRIg expression and point to a mechanism by which arachidonate and dexamethasone have reciprocal effects on inflammation. PMID:21741936

Isoliquiritigenin induces growth inhibition and apoptosis through downregulating arachidonic acid metabolic network and the deactivation of PI3K/Akt in human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ying; Zhao, Haixia; Wang, Yuzhong

Arachidonic acid (AA)-derived eicosanoids and its downstream pathways have been demonstrated to play crucial roles in growth control of breast cancer. Here, we demonstrate that isoliquiritigenin, a flavonoid phytoestrogen from licorice, induces growth inhibition and apoptosis through downregulating multiple key enzymes in AA metabolic network and the deactivation of PI3K/Akt in human breast cancer. Isoliquiritigenin diminished cell viability, 5-bromo-2′-deoxyuridine (BrdU) incorporation, and clonogenic ability in both MCF-7 and MDA-MB-231cells, and induced apoptosis as evidenced by an analysis of cytoplasmic histone-associated DNA fragmentation, flow cytometry and hoechst staining. Furthermore, isoliquiritigenin inhibited mRNA expression of multiple forms of AA-metabolizing enzymes, including phospholipasemore » A2 (PLA2), cyclooxygenases (COX)-2 and cytochrome P450 (CYP) 4A, and decreased secretion of their products, including prostaglandin E{sub 2} (PGE{sub 2}) and 20-hydroxyeicosatetraenoic acid (20-HETE), without affecting COX-1, 5-lipoxygenase (5-LOX), 5-lipoxygenase activating protein (FLAP), and leukotriene B{sub 4} (LTB{sub 4}). In addition, it downregulated the levels of phospho-PI3K, phospho-PDK (Ser{sup 241}), phospho-Akt (Thr{sup 308}), phospho-Bad (Ser{sup 136}), and Bcl-x{sub L} expression, thereby activating caspase cascades and eventually cleaving poly(ADP-ribose) polymerase (PARP). Conversely, the addition of exogenous eicosanoids, including PGE{sub 2}, LTB{sub 4} and a 20-HETE analog (WIT003), and caspase inhibitors, or overexpression of constitutively active Akt reversed isoliquiritigenin-induced apoptosis. Notably, isoliquiritigenin induced growth inhibition and apoptosis of MDA-MB-231 human breast cancer xenografts in nude mice, together with decreased intratumoral levels of eicosanoids and phospho-Akt (Thr{sup 308}). Collectively, these data suggest that isoliquiritigenin induces growth inhibition and apoptosis through downregulating AA metabolic network and the deactivation of PI3K/Akt in human breast cancer. - Highlights: • Isoliquiritigenin induces growth inhibition and apoptosis in human breast cancer. • The proapoptotic action of isoliquiritigenin has been studied in vitro and in vivo. • Arachidonic acid metabolic network mediates isoliquiritigenin-induced apoptosis. • PI3K/Akt deactivation is asssociated with isoliquiritigenin-induced apoptosis. • Isoliquiritigenin may be a multi-target drug in the treatment of breast cancer.« less

Pharmacological modulation of lipopolysaccharide-induced pleural eosinophilia in the rat; a role for a newly generated protein.

PubMed

Bozza, P T; Castro-Faria-Neto, H C; Martins, M A; Larangeira, A P; Perales, J E; e Silva, P M; Cordeiro, R S

1993-06-01

Intrathoracic injection of endotoxin lipopolysaccharide, LPS into rats induced a dose-dependent increase in the number of eosinophils recovered from the pleural cavity. The pleural eosinophil accumulation peaked within 24-48 h, and returned to basal levels within 120 h. This phenomenon was accompanied by mononuclear cell infiltration, and preceded by massive neutrophil accumulation. Pretreatment with indomethacin, BW 755C (a dual cyclo/lipoxygenase inhibitor), BW A4C (a specific lipoxygenase inhibitor) or the platelet activating factor (PAF) antagonists WEB 2086 and PCA 4248 failed to inhibit the endotoxin-induced pleural eosinophilia, whilst dexamethasone (5-10 micrograms/cavity) or cycloheximide (14-28 micrograms/cavity) abolished this phenomenon. Transfer of the cell-free pleural washing from LPS-treated donor rats to normal recipient rats led to a two-fold increase in the eosinophil counts. Treatment of donors, but not recipients, with cycloheximide or dexamethasone inhibited the eosinophil accumulation induced by the pleural washings, indicating that the generation of the eosinophilotactic activity, but not its effects, depends on protein synthesis. This eosinophilotactic activity was maintained after lyophilization and heating (100 degrees C for 30 min), but was destroyed by trypsin. This substance has a molecular weight ranging between 10 and 50 kDa. The available data suggest that the late eosinophil accumulation induced by LPS is independent of arachidonic acid metabolites and PAF, and probably depends on a newly generated heat-stable soluble protein.

Omega-3 fatty acids are oxygenated at the n-7 carbon by the lipoxygenase domain of a fusion protein in the cyanobacterium Acaryochloris marina

PubMed Central

Gao, Benlian; Boeglin, William E.; Brash, Alan R.

2009-01-01

Lipoxygenases (LOX) are found in most organisms that contain polyunsaturated fatty acids, usually existing as individual genes although occasionally encoded as a fusion protein with a catalase-related hemoprotein. Such a fusion protein occurs in the cyanobacterium Acaryochloris marina and herein we report the novel catalytic activity of its LOX domain. The full-length protein and the C-terminal LOX domain were expressed in Escherichia coli, and the catalytic activities characterized by UV, HPLC, GC-MS, and CD. All omega-3 polyunsaturates were oxygenated by the LOX domain at the n-7 position and with R stereospecificity: α-linolenic and the most abundant fatty acid in A. marina, stearidonic acid (C18.4ω3), are converted to the corresponding 12R-hydroperoxides, eicosapentaenoic acid to its 14R-hydroperoxide, and docosahexaenoic acid to its 16R-hydroperoxide. Omega-6 polyunsaturates were oxygenated at the n-10 position, forming 9R-hydroperoxy-octadecadienoic acid from linoleic acid and 11R-hydroperoxy-eicosatetraenoic acid from arachidonic acid. The metabolic transformation of stearidonic acid by the full-length fusion protein entails its 12R oxygenation with subsequent conversion by the catalase-related domain to a novel allene epoxide, a likely precursor of cyclopentenone fatty acids or other signaling molecules (Gao et al, J. Biol. Chem. 284:22087-98, 2009). Although omega-3 fatty acids and lipoxygenases are of widespread occurrence, this appears to be the first description of a LOX-catalyzed oxygenation that specifically utilizes the terminal pentadiene of omega-3 fatty acids. PMID:19786119

5-Lipoxygenase Pathway, Dendritic Cells, and Adaptive Immunity

PubMed Central

Hedi, Harizi

2004-01-01

5-lipoxygenase (5-LO) pathway is the major source of potent proinflammatory leukotrienes (LTs) issued from the metabolism of arachidonic acid (AA), and best known for their roles in the pathogenesis of asthma. These lipid mediators are mainly released from myeloid cells and may act as physiological autocrine and paracrine signalling molecules, and play a central role in regulating the interaction between innate and adaptive immunity. The biological actions of LTs including their immunoregulatory and proinflammatory effects are mediated through extracellular specific G-protein-coupled receptors. Despite their role in inflammatory cells, such as neutrophils and macrophages, LTs may have important effects on dendritic cells (DC)-mediated adaptive immunity. Several lines of evidence show that DC not only are important source of LTs, but also become targets of their actions by producing other lipid mediators and proinflammatory molecules. This review focuses on advances in 5-LO pathway biology, the production of LTs from DC and their role on various cells of immune system and in adaptive immunity. PMID:15240920

The 5-lipoxygenase inhibitor RF-22c potently suppresses leukotriene biosynthesis in cellulo and blocks bronchoconstriction and inflammation in vivo.

PubMed

Schaible, Anja M; Filosa, Rosanna; Krauth, Verena; Temml, Veronika; Pace, Simona; Garscha, Ulrike; Liening, Stefanie; Weinigel, Christina; Rummler, Silke; Schieferdecker, Sebastian; Nett, Markus; Peduto, Antonella; Collarile, Selene; Scuotto, Maria; Roviezzo, Fioretina; Spaziano, Giuseppe; de Rosa, Mario; Stuppner, Hermann; Schuster, Daniela; D'Agostino, Bruno; Werz, Oliver

2016-07-15

5-Lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. Because LTs play pivotal roles in allergy and inflammation, 5-LO represents a valuable target for anti-inflammatory drugs. Here, we investigated the molecular mechanism, the pharmacological profile, and the in vivo effectiveness of the novel 1,2-benzoquinone-featured 5-LO inhibitor RF-22c. Compound RF-22c potently inhibited 5-LO product synthesis in neutrophils and monocytes (IC50⩾22nM) and in cell-free assays (IC50⩾140nM) without affecting 12/15-LOs, cyclooxygenase (COX)-1/2, or arachidonic acid release, in a specific and reversible manner, supported by molecular docking data. Antioxidant or iron-chelating properties were not evident for RF-22c and 5-LO-regulatory cofactors like Ca(2+) mobilization, ERK-1/2 activation, and 5-LO nuclear membrane translocation and interaction with 5-LO-activating protein (FLAP) were unaffected. RF-22c (0.1mg/kg; i.p.) impaired (I) bronchoconstriction in ovalbumin-sensitized mice challenged with acetylcholine, (II) exudate formation in carrageenan-induced paw edema, and (III) zymosan-induced leukocyte infiltration in air pouches. Taken together, RF-22c is a highly selective and potent 5-LO inhibitor in intact human leukocytes with pronounced effectiveness in different models of inflammation that warrants further preclinical analysis of this agent as anti-inflammatory drug. Copyright © 2016 Elsevier Inc. All rights reserved.

Omega-oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site.

PubMed Central

Ivanov, I; Schwarz, K; Holzhütter, H G; Myagkova, G; Kühn, H

1998-01-01

During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the omega-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of omega-modified fatty acids by 15-lipoxygenases and found that omega-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, omega-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403-->Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of omega-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the omega-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases. PMID:9820810

Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

PubMed

Kulkarni, Shilpa; Das, Sudipto; Funk, Colin D; Murray, Diana; Cho, Wonhwa

2002-04-12

The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.

A Single Amino Acid Difference between Mouse and Human 5-Lipoxygenase Activating Protein (FLAP) Explains the Speciation and Differential Pharmacology of Novel FLAP Inhibitors.

PubMed

Blevitt, Jonathan M; Hack, Michael D; Herman, Krystal; Chang, Leon; Keith, John M; Mirzadegan, Tara; Rao, Navin L; Lebsack, Alec D; Milla, Marcos E

2016-06-10

5-Lipoxygenase activating protein (FLAP) plays a critical role in the metabolism of arachidonic acid to leukotriene A4, the precursor to the potent pro-inflammatory mediators leukotriene B4 and leukotriene C4 Studies with small molecule inhibitors of FLAP have led to the discovery of a drug binding pocket on the protein surface, and several pharmaceutical companies have developed compounds and performed clinical trials. Crystallographic studies and mutational analyses have contributed to a general understanding of compound binding modes. During our own efforts, we identified two unique chemical series. One series demonstrated strong inhibition of human FLAP but differential pharmacology across species and was completely inactive in assays with mouse or rat FLAP. The other series was active across rodent FLAP, as well as human and dog FLAP. Comparison of rodent and human FLAP amino acid sequences together with an analysis of a published crystal structure led to the identification of amino acid residue 24 in the floor of the putative binding pocket as a likely candidate for the observed speciation. On that basis, we tested compounds for binding to human G24A and mouse A24G FLAP mutant variants and compared the data to that generated for wild type human and mouse FLAP. These studies confirmed that a single amino acid mutation was sufficient to reverse the speciation observed in wild type FLAP. In addition, a PK/PD method was established in canines to enable preclinical profiling of mouse-inactive compounds. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemoprevention of Prostate Cancer Initiation in a Novel Transgenic Mouse Model by Targeting 15-Lipoxygenase-1

DTIC Science & Technology

2009-02-01

2005) 15-lipoxygenase metabolites of γ-linolenic acid / eicosapentaenoic acid suppress growth and arachidonic acid metabolism in human prostatic...found primarily in fish oils, comprise the -3 family: Alpha linolenic acid (ALA or -96 LNA; 18:3-3), eicosapentaenoic acid (EPA; 20:5-3), and... eicosapentaenoic acid (EPA) also 122 serves as a substrate for 15-LO-1 and COX-2, but metabolism of EPA by these enzymes results in 123 the formation of

Chemical composition and lipoxygenase activity in soybeans (Glycine max L. Merr.) submitted to gamma irradiation

NASA Astrophysics Data System (ADS)

Barros, Érica Amanda de; Broetto, Fernando; Bressan, Dayanne F.; Sartori, Maria M. P.; Costa, Vladimir E.

2014-05-01

Soybeans are an important food due to their functional and nutritional characteristics. However, consumption by western populations is limited by the astringent taste of soybeans and their derivatives which results from the action of lipoxygenase, an enzyme activated during product processing. The aim of this study was to evaluate the effect of gamma irradiation on the chemical composition and specific activity of lipoxygenase in different soybean cultivars. Soybeans were stored in plastic bags and irradiated with doses of 2.5, 5 and 10 kGy. The chemical composition (moisture, protein, lipids, ashes, crude fiber, and carbohydrates) and lipoxygenase specific activity were determined for each sample. Gamma irradiation induced a small increase of protein and lipid content in some soybean cultivars, which did not exceed the highest content of 5% and 26%, respectively, when compared to control. Lipoxygenase specific activity decreased in the three cultivars with increasing gamma irradiation dose. In conclusion, the gamma irradiation doses used are suitable to inactivate part of lipoxygenase while not causing expressive changes in the chemical composition of the cultivars studied.

Dioxygenase Activity of Epidermal Lipoxygenase-3 Unveiled

PubMed Central

Zheng, Yuxiang; Brash, Alan R.

2010-01-01

Epidermal lipoxygenase-3 (eLOX3) exhibits hydroperoxide isomerase activity implicated in epidermal barrier formation, but its potential dioxygenase activity has remained elusive. We identified herein a synthetic fatty acid, 9E,11Z,14Z-20:3ω6, that was oxygenated by eLOX3 specifically to the 9S-hydroperoxide. Reaction showed a pronounced lag phase, which suggested that eLOX3 is deficient in its activation step. Indeed, we found that high concentrations of hydroperoxide activator (e.g. 65 μm) overcame a prolonged lag phase (>1 h) and unveiled a dioxygenase activity with arachidonic acid; the main products were the 5-, 9-, and 7-hydroperoxyeicosatetraenoic acids (HPETEs). These were R/S mixtures (ranging from ∼50:50 to 73:27), and as the bis-allylic 7-HPETE can be formed only inside the enzyme active site, the results indicate there is oxygen availability along either face of the reacting fatty acid radical. That the active site oxygen supply is limited is implied from the need for continuous re-activation, as carbon radical leakage leaves the enzyme in the unactivated ferrous state. An Ala-to-Gly mutation, known to affect the positioning of O2 in the active site of other lipoxygenase enzymes, led to more readily activated reaction and a significant increase in the 9R- over the 5-HPETE. Activation and cycling of the ferric enzyme are thus promoted using the 9E,11Z,14Z-20:3ω6 substrate, by continuous hydroperoxide activation, or by the Ala-to-Gly mutation. We suggest that eLOX3 represents one end of a spectrum among lipoxygenases where activation is inefficient, favoring hydroperoxide isomerase cycling, with the opposite end represented by readily activated enzymes in which dioxygenase activity is prominent. PMID:20921226

Aryl-acetic and cinnamic acids as lipoxygenase inhibitors with antioxidant, anti-inflammatory, and anticancer activity.

PubMed

Hadjipavlou-Litina, Dimitra; Pontiki, Eleni

2015-01-01

Cinnamic acids have been identified as interesting compounds with cytotoxic, anti-inflammatory, and antioxidant properties. Lipoxygenase pathway, catalyzing the first two steps of the transformation of arachidonic acid into leukotrienes is implicated in several processes such as cell differentiation, inflammation and carcinogenesis. Development of drugs that interfere with the formation or effects of these metabolites would be important for the treatment of various diseases like asthma, psoriasis, ulcerative colitis, rheumatoid arthritis, atherosclerosis, cancer, and blood vessel disorders. Till now, asthma consists of the only pathological case in which improvement has been shown by lipoxygenase LO inhibitors. Thus, the research has been directed towards the development of drugs that interfere with the formation of leukotrienes. In order to explore the anti-inflammatory and cytotoxic effects of antioxidant acrylic/cinnamic acids a series of derivatives bearing the appropriate moieties have been synthesized via the Knoevenagel condensation and evaluated for their biological activities. The compounds have shown important antioxidant activity, anti-inflammatory activity and very good inhibition of soybean lipoxygenase while some of them were tested for their anticancer activity.

Hydroperoxyeicosatetraenoic acids. Potent inhibitors of lymphocyte responses.

PubMed

Gualde, N; Chable-Rabinovitch, H; Motta, C; Durand, J; Beneytout, J L; Rigaud, M

1983-03-01

Arachidonic acid can be transformed into a series of HPETEs by the lipoxygenase enzyme activity of mouse peritoneal macrophages. These resulting HPETEs inhibit some mouse lymphocyte responses. When mice are injected with 15-L-HPETE, their splenocytes show a decreased [3H]thymidine uptake after lectin stimulation.

cPLA2α Gene Activation by IL-1β is Dependent on an Upstream Kinase pathway, Enzymatic Activation and Downstream 15-lipoxygenase Activity: A Positive Feedback Loop

PubMed Central

Walters, Jewell N.; Bickford, Justin S.; Beachy, Dawn E.; Newsom, Kimberly J.; Herlihy, John-David H.; Peck, Molly V.; Qiu, Xiaolei; Nick, Harry S.

2011-01-01

Cytosolic phospholipase A2α (cPLA2α) is the most widely studied member of the Group IV PLA2 family. The enzyme is Ca2+-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA2α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA2α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca2+ levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA2α is phosphorylated within 10 min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA2α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA2α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA2α→15-LOX2-dependent, positive feedback loop where a protein’s enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus. PMID:21771656

Impact of myeloperoxidase-derived oxidants on the product profile of human 5-lipoxygenase.

PubMed

Zschaler, Josefin; Dorow, Juliane; Schöpe, Louisa; Ceglarek, Uta; Arnhold, Jürgen

2015-08-01

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6 E,8 Z,11 Z,14 Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A4. In neutrophils, LTA4 is further converted to the potent chemoattractant LTB4. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography-electrospray ionization-tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB4 in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB4, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO-H2O2-Cl(-) system when glucose oxidase and glucose were applied as a source of H2O2. This was necessary because of a strong impairment of 5-LOX activity by H2O2. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils. Copyright © 2015 Elsevier Inc. All rights reserved.

Levels of prostaglandin E metabolite and leukotriene E(4) are increased in the urine of smokers: evidence that celecoxib shunts arachidonic acid into the 5-lipoxygenase pathway.

PubMed

Duffield-Lillico, Anna J; Boyle, Jay O; Zhou, Xi Kathy; Ghosh, Aradhana; Butala, Geera S; Subbaramaiah, Kotha; Newman, Robert A; Morrow, Jason D; Milne, Ginger L; Dannenberg, Andrew J

2009-04-01

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) play a role in inflammation and carcinogenesis. Biomarkers that reflect tobacco smoke-induced tissue injury are needed. In this study, levels of urinary prostaglandin E metabolite (PGE-M) and leukotriene E(4) (LTE(4)), biomarkers of the COX and 5-LO pathways, were compared in never smokers, former smokers, and current smokers. The effects of celecoxib, a selective COX-2 inhibitor, on levels of PGE-M and LTE(4) were determined. Baseline levels of PGE-M and LTE(4) were positively associated with smoking status; levels of PGE-M and LTE(4) were higher in current versus never smokers. Treatment with 200 mg celecoxib twice daily for 6 +/- 1 days led to a reduction in urinary PGE-M levels in all groups but exhibited the greatest effect among subjects with high baseline PGE-M levels. Thus, high baseline PGE-M levels in smokers reflected increased COX-2 activity. In individuals with high baseline PGE-M levels, treatment with celecoxib led to a significant increase in levels of urinary LTE(4), an effect that was not found in individuals with low baseline PGE-M levels. In conclusion, increased levels of urinary PGE-M and LTE(4) were found in human smokers, a result that may reflect subclinical lung inflammation. In individuals with high baseline levels of PGE-M (elevated COX-2 activity), celecoxib administration shunted arachidonic acid into the proinflammatory 5-LO pathway. Because 5-LO activity and LTE(4) have been suggested to play a role in cardiovascular disease, these results may help to explain the link between use of COX-2 inhibitors and cardiovascular complications.

Molecular basis for the catalytic inactivity of a naturally occurring near-null variant of human ALOX15.

PubMed

Horn, Thomas; Ivanov, Igor; Di Venere, Almerinda; Kakularam, Kumar Reddy; Reddanna, Pallu; Conrad, Melanie L; Richter, Constanze; Scheerer, Patrick; Kuhn, Hartmut

2013-12-01

Mammalian lipoxygenases belong to a family of lipid-peroxidizing enzymes, which have been implicated in cardiovascular, hyperproliferative and neurodegenerative diseases. Here we report that a naturally occurring mutation in the hALOX15 gene leads to expression of a catalytically near-null enzyme variant (hGly422Glu). The inactivity may be related to severe misfolding of the enzyme protein, which was concluded from CD-spectra as well as from thermal and chemical stability assays. In silico mutagenesis experiments suggest that most mutations at hGly422 have the potential to induce sterical clash, which might be considered a reason for protein misfolding. hGly422 is conserved among ALOX5, ALOX12 and ALOX15 isoforms and corresponding hALOX12 and hALOX5 mutants also exhibited a reduced catalytic activity. Interestingly, in the hALOX5 Gly429Glu mutants the reaction specificity of arachidonic acid oxygenation was shifted from 5S- to 8S- and 12R-H(p)ETE formation. Taken together, our data indicate that the conserved glycine is of functional importance for these enzyme variants and most mutants at this position lose catalytic activity. © 2013.

Arachidonate Metabolism in Breast Cancer Cultures: Identification of Antagonists/Agonist for Possible Intervention Strategies.

DTIC Science & Technology

1995-10-01

Curcumin ) and phospholipase A2 inhibition (dexamethasone) were both ineffective at usable concentrations. Lipoxygenase products can also be generated by...S. Weeks, C. L. Hill, R. F. Schinazi (1992). Synthesis, Charaterization, and Anti-human Immunodeficiency Virus Activity of Water- soluble Salts of

Virtual screening using the ligand ZINC database for novel lipoxygenase-3 inhibitors.

PubMed

Monika; Kour, Janmeet; Singh, Kulwinder

2013-01-01

The leukotrienes constitute a group of arachidonic acid-derived compounds with biologic activities suggesting important roles in inflammation and immediate hypersensitivity. Epidermis-type lipoxygenase-3 (ALOXE3), a distinct subclass within the multigene family of mammalian lipoxygenases, is a novel isoenzyme involved in the metabolism of leukotrienes and plays a very important role in skin barrier functions. Lipoxygenase selective inhibitors such as azelastine and zileuton are currently used to reduce inflammatory response. Nausea, pharyngolaryngeal pain, headache, nasal burning and somnolence are the most frequently reported adverse effects of these drugs. Therefore, there is still a need to develop more potent lipoxygenase inhibitors. In this paper, we report the screening of various compounds from the ZINC database (contains over 21 million compounds) using the Molegro Virtual Docker software against the ALOXE3 protein. Screening was performed using molecular constraints tool to filter compounds with physico-chemical properties similar to the 1N8Q bound ligand protocatechuic acid. The analysis resulted in 4319 Lipinski compliant hits which are docked and scored to identify structurally novel ligands that make similar interactions to those of known ligands or may have different interactions with other parts of the binding site. Our screening approach identified four molecules ZINC84299674; ZINC76643455; ZINC84299122 & ZINC75626957 with MolDock score of -128.901, -120.22, -116.873 & - 102.116 kcal/mol, respectively. Their energy scores were better than the 1N8Q bound co-crystallized ligand protocatechuic acid (with MolDock score of -77.225 kcal/mol). All the ligands were docked within the binding pocket forming interactions with amino acid residues.

Induced resistance in soybean toHelicoverpa zea: Role of plant protein quality.

PubMed

Bi, J L; Felton, G W; Mueller, A J

1994-01-01

Resistance in soybean toHelicoverpa zea is comprised of both constitutive and inducible factors. In this study, we investigated the induction of resistance byH. zea in both greenhouse and field studies. In a greenhouse experiment, fourth-instarH. zea growth rates were reduced by 39% after 24 hr feeding and by 27% after 48 hr when larvae fed on previously wounded V3 foliage (cv. Forrest) compared with undamaged foliage. In a field study, the weight gain by larvae was more than 52% greater when larvae fed for 72 hr on undamaged R2/R3 soybean plants (cv. Braxton) compared to those that fed on previously wounded plants. A significant component of the induced resistance is due to a decline in the nutritional quality of foliar protein following foliar damage byH. zea. Foliar protein was extracted from damaged and undamaged foliage and incorporated into artificial diets. Larval growth was reduced 26% after four days and 49% after seven days on diets containing protein from damaged plants compared to larvae feeding on foliar protein from undamaged plants. Chemical analyses of protein quality also indicated a decline in quality in damaged plants compared to unwounded plants. Increases in lipoxygenase activity (53%), lipid peroxidation products (20%), and trypsin inhibitor content (34%) were observed in protein from wounded plants. Moreover, a 5.9% loss in free amines and 19% loss in total thiols occurred in protein from wounded plants. Larval feeding causes a significant increase in foliar lipoxygenase activity that varied among genotypes. Lipoxygenase isozymes were measured at pH 5.5, pH 7.0, and pH 8.5 in V3 stage plants of Forrest, Hark, D75-1069, and PI 417061 genotypes. Lipoxygenase activity in each genotype was significantly increased after 72 hr of larval feeding at each pH level tested, with the exception of lipoxygenase isozymes at pH 5.5 in genotype PI 417061. Larval feeding on R2/R3 stage plants (field-grown cv. Braxton) for six days also increased foliar lipoxygenase activity.

Identification of Protein Targets of 12/15-Lipoxygenase-Derived Lipid Electrophiles in Mouse Peritoneal Macrophages Using Omega-Alkynyl Fatty Acid.

PubMed

Isobe, Yosuke; Kawashima, Yusuke; Ishihara, Tomoaki; Watanabe, Kenji; Ohara, Osamu; Arita, Makoto

2018-04-20

The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of highest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Here, we describe a chemoproteomic method to identify the targets of enzymatically generated LDE. By treating mouse peritoneal macrophages with omega-alkynyl arachidonic acid (aAA), we identified a series of proteins adducted by LDE generated through a 12/15-LOX catalyzed reaction. Pathway analysis revealed a dramatic enrichment of proteins involved in energy metabolism and found that glycolytic flux and mitochondrial respiration were significantly affected by the expression of 12/15-LOX. Our findings thus highlight the utility of chemoproteomics using aAA for identifying intracellular targets of enzymatically generated LDE.

Pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase.

PubMed Central

McMillan, R. M.; Spruce, K. E.; Crawley, G. C.; Walker, E. R.; Foster, S. J.

1992-01-01

1. This paper describes the pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase which is undergoing clinical evaluation. 2. ICI D2138 potently inhibited leukotriene synthesis in murine peritoneal macrophages (IC50 = 3 nM) and human blood (IC50 = 20 nM). In human and dog blood, ICI D2138 did not inhibit thromboxane B2 synthesis at a concentration of 500 microM, thus the selectivity ratio (cyclo-oxygenase: 5-lipoxygenase) was greater than 20,000. In contrast, zileuton (a 5-lipoxygenase inhibitor also undergoing clinical evaluation) exhibited a selectivity ratio of 15-100. 3. ICI D2138 potently and dose-dependently inhibited ex vivo leukotriene B4 (LTB4) synthesis by rat blood with ED50 values of 0.9, 4.0 and 80.0 mg kg-1 p.o. at 3, 10 and 20 h respectively after dosing. Similar activity was observed for inhibition of LTB4 production in a zymosan-inflamed rat air pouch model. Zileuton produced ED50 values of 5 and 20 mg kg-1 at 3 and 10 h respectively. 4. Oral administration of 1, 3 or 10 mg kg-1 ICI D2138 to dogs produced maximal inhibition of ex vivo LTB4 synthesis by blood for 5, 9 and 31 h respectively. A dose of 5 mg kg-1 p.o. of zileuton caused maximal inhibition of LTB4 for 24 h. 5. Oral administration of 10 mg kg-1 ICI D2138 caused total inhibition of LTB4 production in zymosan-inflamed rabbit knee joint. 6. Topical administration of ICI D2138 to rabbit skin caused a dose-related inhibition of arachidonic acid-induced plasma extravasation with an ID30 of 1.08 nmol per site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1334748

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events.

PubMed

Timár, J; Tóth, S; Tóvári, J; Paku, S; Raz, A

1999-01-01

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1-5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCalpha to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilbert, Nathaniel C.; Rui, Zhe; Neau, David B.

The enzyme 5-lipoxygenase (5-LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15-LOX, is also required for synthesis of the anti-inflammatory lipoxins. The catalytic activity of 5-LOX is regulated through multiple mechanisms, including Ca{sup 2+}-targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15-LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5-LOX activity remaining; synthesis of the anti-inflammatory lipoxin A4more » from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable-5-LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5-LOX at S663 could not only down-regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15-LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.« less

Pharmacological studies on proglumetacin maleate, a new non-steroidal anti-inflammatory drug (4). Mode of action on anti-inflammatory activity.

PubMed

Ono, N; Yamasaki, Y; Yamamoto, N; Sunami, A; Miyake, H

1986-11-01

The possible mechanism of the anti-inflammatory activity of proglumetacin maleate (PGM), a new indomethacin (IND) derivative interacting with arachidonic acid (AA) metabolism, was investigated to elucidate the contributions of PGM itself and its two major metabolites, desproglumideproglumetacin maleate (DPP) and IND. PGM caused much less inhibition of PGE2 formation by sheep seminal vesicle microsomes (IC50 = 310 microM) and TXB2 formation by a washed rabbit platelet suspension (IC50 = 6.3 microM) than IND. DPP also caused less inhibition of cyclooxygenase than IND. Moreover, PGM had less effect on sodium arachidonate (SAA)-induced rat platelet aggregation ex vivo and AA-induced sudden death in rabbits than IND. These results show that PGM has anti-inflammatory activity after its conversion to the active metabolite IND. However, the inhibitory effects of PGM and DPP were as strong as that of IND on SAA- or collagen-induced rabbit platelet aggregation in vitro. These activities are considered to be associated with platelet membrane interaction. Moreover, unlike IND, PGM (IC50 = 1.5 microM) and DPP (IC50 = 16.3 microM) strongly inhibited 5-HETE formation by the cytosol of guinea pig polymorphonuclear leukocytes. This unique activity of PGM on 5-lipoxygenase may contribute to its anti-inflammatory activity.

mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

PubMed

De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

2010-07-01

Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Arachidonic acid can function as a signaling modulator by activating the TRPM5 cation channel in taste receptor cells.

PubMed

Oike, Hideaki; Wakamori, Minoru; Mori, Yasuo; Nakanishi, Hiroki; Taguchi, Ryo; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

2006-09-01

Vertebrate sensory cells such as vomeronasal neurons and Drosophila photoreceptor cells use TRP channels to respond to exogenous stimuli. In mammalian taste cells, bitter and sweet substances as well as some amino acids are received by G protein-coupled receptors (T2Rs or T1Rs). As a result of activation of G protein and phospholipase Cbeta2, the TRPM5 channel is activated. Intracellular Ca(2+) is known to be a TRPM5 activator, but the participation of lipid activators remains unreported. To clarify the effect of arachidonic acid on TRPM5 in taste cells, we investigated the expression profile of a series of enzymes involved in controlling the intracellular free arachidonic acid level, with the result that in a subset of taste bud cells, monoglyceride lipase (MGL) and cyclooxygenase-2 (COX-2) are expressed as well as the previously reported group IIA phospholipase A(2) (PLA(2)-IIA). Double-labeling analysis revealed that MGL, COX-2 and PLA(2)-IIA are co-expressed in some cells that express TRPM5. We then investigated whether arachidonic acid activates TRPM5 via a heterologous expression system in HEK293 cells, and found that its activation occurred at 10 microM arachidonic acid. These results strongly suggest the possibility that arachidonic acid acts as a modulator of TRPM5 in taste signaling pathways.

Kinetic investigation of human 5-lipoxygenase with arachidonic acid.

PubMed

Mittal, Monica; Kumar, Ramakrishnan B; Balagunaseelan, Navisraj; Hamberg, Mats; Jegerschöld, Caroline; Rådmark, Olof; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

2016-08-01

Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A4, a pivotal intermediate in the biosynthesis of the leukotrienes, a family of proinflammatory lipid mediators. 5-LOX has thus gained attention as a potential drug target. However, details of the kinetic mechanism of 5-LOX are still obscure. In this Letter, we investigated the kinetic isotope effect (KIE) of 5-LOX with its physiological substrate, arachidonic acid (AA). The observed KIE is 20±4 on kcat and 17±2 on kcat/KM at 25°C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35°C. Also, we observed low Arrhenius prefactor ratio (AH/AD=0.21) and a small change in activation energy (Ea(D)-Ea(H)=3.6J/mol) which suggests that 5-LOX catalysis involves tunneling as a mechanism of H-transfer. The measured KIE for 5-LOX involves a change in regioselectivity in response to deuteration at position C7, resulting in H-abstraction form C10 and formation of 8-HETE. The viscosity experiments influence the (H)kcat, but not (D)kcat. However the overall kcat/KM is not affected for labeled or unlabeled AA, suggesting that either the product release or conformational rearrangement might be involved in dictating kinetics of 5-LOX at saturating conditions. Investigation of available crystal structures suggests the role of active site residues (F421, Q363 and L368) in regulating the donor-acceptor distances, thus affecting H-transfer as well as regiospecificity. In summary, our study shows that that the H-abstraction is the rate limiting step for 5-LOX and that the observed KIE of 5-LOX is masked by a change in regioselectivity. Copyright © 2016 Elsevier Ltd. All rights reserved.

ω-Alkynyl lipid surrogates for polyunsaturated fatty acids: free radical and enzymatic oxidations.

PubMed

Beavers, William N; Serwa, Remigiusz; Shimozu, Yuki; Tallman, Keri A; Vaught, Melissa; Dalvie, Esha D; Marnett, Lawrence J; Porter, Ned A

2014-08-13

Lipid and lipid metabolite profiling are important parameters in understanding the pathogenesis of many diseases. Alkynylated polyunsaturated fatty acids are potentially useful probes for tracking the fate of fatty acid metabolites. The nonenzymatic and enzymatic oxidations of ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were compared to that of linoleic and arachidonic acid. There was no detectable difference in the primary products of nonenzymatic oxidation, which comprised cis,trans-hydroxy fatty acids. Similar hydroxy fatty acid products were formed when ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid were reacted with lipoxygenase enzymes that introduce oxygen at different positions in the carbon chains. The rates of oxidation of ω-alkynylated fatty acids were reduced compared to those of the natural fatty acids. Cyclooxygenase-1 and -2 did not oxidize alkynyl linoleic but efficiently oxidized alkynyl arachidonic acid. The products were identified as alkynyl 11-hydroxy-eicosatetraenoic acid, alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid, and alkynyl prostaglandins. This deviation from the metabolic profile of arachidonic acid may limit the utility of alkynyl arachidonic acid in the tracking of cyclooxygenase-based lipid oxidation. The formation of alkynyl 11-hydroxy-8,9-epoxy-eicosatrienoic acid compared to alkynyl prostaglandins suggests that the ω-alkyne group causes a conformational change in the fatty acid bound to the enzyme, which reduces the efficiency of cyclization of dioxalanyl intermediates to endoperoxide intermediates. Overall, ω-alkynyl linoleic acid and ω-alkynyl arachidonic acid appear to be metabolically competent surrogates for tracking the fate of polyunsaturated fatty acids when looking at models involving autoxidation and oxidation by lipoxygenases.

Arachidonic acid and lipoxin A4 attenuate alloxan-induced cytotoxicity to RIN5F cells in vitro and type 1 diabetes mellitus in vivo.

PubMed

Gundala, Naveen K V; Naidu, Vegi G M; Das, Undurti N

2017-03-01

We studied whether polyunsaturated fatty acids (PUFAs) can protect rat insulinoma (RIN5F) cells against alloxan-induced apoptosis in vitro and type 1 diabetes mellitus (type 1 DM) in vivo and if so, mechanism of this beneficial action. In vitro study was conducted using RIN5F cells while in vivo study was performed in Wistar rats. The effect of PUFAs, cyclo-oxygenase and lipoxygenase inhibitors, various eicosanoids and PUFAs metabolites: lipoxin A4 (LXA4), resolvin D2 and protectin against alloxan-induced cytotoxicity to RIN5F cells and type 1 DM was studied. Expression of PDX1, P65 NF-kB and IKB in RIN5F cells and Nrf2, GLUT2, COX2, iNOS protein levels in the pancreatic tissue and plasma glucose, insulin and tumor necrosis factor-α and antioxidants, lipid peroxides and nitric oxide were measured. Of all, arachidonic acid (AA) was found to be the most effective against alloxan-induced cytotoxicity to RIN5F cells and preventing type 1 DM. Both cyclo-oxygenase and lipoxygenase inhibitors did not block the beneficial actions of AA in vitro and in vivo. Alloxan inhibited LXA4 production by RIN5F cells and in alloxan-induced type 1 DM Wistar rats. AA-treatment restored LXA4 levels to normal both in vitro and in vivo. LXA4 protected RIN5F cells against alloxan-induced cytotoxicity and prevented type 1 DM and restored expression of Nrf2, Glut2, COX2, and iNOS genes and abnormal antioxidants to near normal. AA seems to bring about its beneficial actions against alloxan-induced cytotoxicity and type 1 DM by enhancing the production of LXA4. © 2016 BioFactors, 43(2):251-271, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

Arachidonate 15-Lipoxygenase 2 as an Endogenous Inhibitor of Prostate Cancer Development

DTIC Science & Technology

2006-03-01

dehydrogenase; NHP, normal human prostate epithelial cells; PCa, prostate cancer; NLS, nuclear localization signal; PPAR -, peroxisome proliferator...cloned, i.e., 15-LOX2sv-a/b/c, are mostly excluded from the nucleus. A potential bi-partite nuclear localization signal (NLS...only partially involved in the nuclear import of 15-LOX2. To elucidate the relationship between nuclear localization , enzymatic activity, and tumor

Up-Regulated Expression of AOS-LOXa and Increased Eicosanoid Synthesis in Response to Coral Wounding

PubMed Central

Lõhelaid, Helike; Teder, Tarvi; Tõldsepp, Kadri; Ekins, Merrick; Samel, Nigulas

2014-01-01

In octocorals, a catalase–like allene oxide synthase (AOS) and an 8R-lipoxygenase (LOX) gene are fused together encoding for a single AOS-LOX fusion protein. Although the AOS-LOX pathway is central to the arachidonate metabolism in corals, its biological function in coral homeostasis is unclear. Using an acute incision wound model in the soft coral Capnella imbricata, we here test whether LOX pathway, similar to its role in plants, can contribute to the coral damage response and regeneration. Analysis of metabolites formed from exogenous arachidonate before and after fixed time intervals following wounding indicated a significant increase in AOS-LOX activity in response to mechanical injury. Two AOS-LOX isoforms, AOS-LOXa and AOS-LOXb, were cloned and expressed in bacterial expression system as active fusion proteins. Transcription levels of corresponding genes were measured in normal and stressed coral by qPCR. After wounding, AOS-LOXa was markedly up-regulated in both, the tissue adjacent to the incision and distal parts of a coral colony (with the maximum reached at 1 h and 6 h post wounding, respectively), while AOS-LOXb was stable. According to mRNA expression analysis, combined with detection of eicosanoid product formation for the first time, the AOS-LOX was identified as an early stress response gene which is induced by mechanical injury in coral. PMID:24551239

Lens lipids.

PubMed

Zelenka, P S

1984-11-01

Lens cells can synthesize, degrade, and remodel lipids. Endogenous lipid synthesis, in conjunction with uptake of exogenous cholesterol and certain fatty acids, leads to the formation of a plasma membrane that is especially rich in sphingomyelin, cholesterol, and long-chain saturated fatty acids. As a result of this unusual lipid composition, lens membranes have very low fluidity, which is restricted even further by lipid-protein interactions. The composition and metabolism of membrane lipids may affect the formation of various types of cataracts. Diets rich in vegetable oils offer some protection against the formation of osmotic cataracts and the hereditary cataract of the RCS rat, although the mechanism of this effect is not clear. Vitamin E also protects against the formation of several types of cataract in vivo and in vitro, suggesting that lipid peroxidation may play a role in cataractogenesis. Certain drugs which inhibit lipid synthesis or degradation are cataractogenic, and a deficiency in cataractogenic, and a deficiency in phosphatidylserine is associated with a loss of Na+/K+ ATPase activity in several types of cataract. Human senile cataracts show a marked loss of protein-lipid interactions, although the overall lipid composition is normal. This loss of protein-lipid interactions may be related to oxidative damage to membrane-associated proteins. Interestingly, the decrease in the fluidity of lens membranes with age would counteract the formation of aqueous pores in the membrane, which can result from the oxidative cross-linking of membrane-associated proteins. Certain pathways of lipid metabolism seem to have regulatory functions. Among these are phosphatidylinositol turnover, phosphatidylethanolamine methylation, and arachidonic acid metabolism. All of these pathways function in the lens. Phosphatidylinositol turnover is correlated with the rate of lens epithelial cell division, while phosphatidylethanolamine methylation seems to be related to the initiation of lens fiber cell formation. Both pathways are associated with the release and metabolism of arachidonic acid in other cell types. While it is not known whether phosphatidylinositol turnover or phosphatidylethanolamine methylation result in the release of arachidonic acid in the lens, recent work has shown that lens cells from a variety of species can metabolize arachidonic acid by both the cyclooxygenase and lipoxygenase pathways. The possible physiological significance of these metabolites to the lens is yet to be determined.

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation1[S

PubMed Central

Ikei, Kenneth N.; Yeung, Jennifer; Apopa, Patrick L.; Ceja, Jesús; Vesci, Joanne; Holinstat, Michael

2012-01-01

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane. PMID:22984144

Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase.

PubMed

Joshi, Vikram; Umashankara, M; Ramakrishnan, Chandrasekaran; Nanjaraj Urs, Ankanahalli N; Suvilesh, Kanve Nagaraj; Velmurugan, Devadasan; Rangappa, Kanchugarakoppal S; Vishwanath, Bannikuppe Sannanaik

2016-05-15

Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited ∼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance. Copyright © 2016 Elsevier Inc. All rights reserved.

15-Lipoxygenase-1 Production is Lost in Pancreatic Cancer and Overexpression of the Gene Inhibits Tumor Cell Growth1

PubMed Central

Hennig, René; Kehl, Timo; Noor, Seema; Ding, Xian-Zhong; Rao, Sambasiva M; Bergmann, Frank; Fürstenberger, Gerhard; Büchler, Markus W; Friess, Helmut; Krieg, Peter; Adrian, Thomas E

2007-01-01

Pancreatic cancer patients have an abysmal prognosis because of late diagnosis and lack of therapeutic options. Pancreatic intraepithelial neoplasias (PanINs), the precursor lesions, are a potential target for chemoprevention. Targeting eicosanoid pathways is an obvious choice because 5-lipoxygenase (5-LOX) has been suggested as a tumor promoter in pancreatic carcinogenesis. Here we provide evidence that 15-lipoxygenase-1 (15-LOX-1) expression and activity may exert antitumorigenic effects in pancreatic cancer. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis showed absence or very weak expression of 15-LOX-1 in all pancreatic cancer cell lines tested. 15-LOX-1 was strongly stained in normal ductal cells, tubular complexes, and centroacinar cells, but no staining was seen in islets, cancer cells, PanIN lesions, or in tumor cells in lymph node metastases, indicating that 15-LOX-1 expression is lost during tumor development in human pancreas. Overexpression of 15-LOX-1 in pancreatic tumor cells or treatment with its arachidonic acid-derived metabolite resulted in decreased cell growth. These findings provide evidence that loss of 15-LOX-1 may play an important role in pancreatic carcinogenesis, possibly as a tumor suppressor gene. Thus, induction of 15-LOX-1 expression may be an attractive option for the prevention and treatment of pancreatic cancer. PMID:18030360

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

PubMed

Pu, Shiyun; Ren, Lin; Liu, Qinhui; Kuang, Jiangying; Shen, Jing; Cheng, Shihai; Zhang, Yuwei; Jiang, Wei; Zhang, Zhiyong; Jiang, Changtao; He, Jinhan

2016-01-01

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. 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. 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. The activity of 5-LO may play a critical role in paracetamol-induced hepatic toxicity by regulating paracetamol metabolism and oxidative stress. © 2015 The British Pharmacological Society.

Molecular basis for TPR domain-mediated regulation of protein phosphatase 5.

PubMed

Yang, Jing; Roe, S Mark; Cliff, Matthew J; Williams, Mark A; Ladbury, John E; Cohen, Patricia T W; Barford, David

2005-01-12

Protein phosphatase 5 (Ppp5) is a serine/threonine protein phosphatase comprising a regulatory tetratricopeptide repeat (TPR) domain N-terminal to its phosphatase domain. Ppp5 functions in signalling pathways that control cellular responses to stress, glucocorticoids and DNA damage. Its phosphatase activity is suppressed by an autoinhibited conformation maintained by the TPR domain and a C-terminal subdomain. By interacting with the TPR domain, heat shock protein 90 (Hsp90) and fatty acids including arachidonic acid stimulate phosphatase activity. Here, we describe the structure of the autoinhibited state of Ppp5, revealing mechanisms of TPR-mediated phosphatase inhibition and Hsp90- and arachidonic acid-induced stimulation of phosphatase activity. The TPR domain engages with the catalytic channel of the phosphatase domain, restricting access to the catalytic site. This autoinhibited conformation of Ppp5 is stabilised by the C-terminal alphaJ helix that contacts a region of the Hsp90-binding groove on the TPR domain. Hsp90 activates Ppp5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the constitutively active phosphatase domain, whereas arachidonic acid prompts an alternate conformation of the TPR domain, destabilising the TPR-phosphatase domain interface.

Airway lipoxin A4 generation and lipoxin A4 receptor expression are decreased in severe asthma.

PubMed

Planagumà, Anna; Kazani, Shamsah; Marigowda, Gautham; Haworth, Oliver; Mariani, Thomas J; Israel, Elliot; Bleecker, Eugene R; Curran-Everett, Douglas; Erzurum, Serpil C; Calhoun, William J; Castro, Mario; Chung, Kian Fan; Gaston, Benjamin; Jarjour, Nizar N; Busse, William W; Wenzel, Sally E; Levy, Bruce D

2008-09-15

Airway inflammation is common in severe asthma despite antiinflammatory therapy with corticosteroids. Lipoxin A(4) (LXA(4)) is an arachidonic acid-derived mediator that serves as an agonist for resolution of inflammation. Airway levels of LXA(4), as well as the expression of lipoxin biosynthetic genes and receptors, in severe asthma. Samples of bronchoalveolar lavage fluid were obtained from subjects with asthma and levels of LXA(4) and related eicosanoids were measured. Expression of lipoxin biosynthetic genes was determined in whole blood, bronchoalveolar lavage cells, and endobronchial biopsies by quantitative polymerase chain reaction, and leukocyte LXA(4) receptors were monitored by flow cytometry. Individuals with severe asthma had significantly less LXA(4) in bronchoalveolar lavage fluids (11.2 +/- 2.1 pg/ml) than did subjects with nonsevere asthma (150.1 +/- 38.5 pg/ml; P < 0.05). In contrast, levels of cysteinyl leukotrienes were increased in both asthma cohorts compared with healthy individuals. In severe asthma, 15-lipoxygenase-1 mean expression was decreased fivefold in bronchoalveolar lavage cells. In contrast, 15-lipoxgenase-1 was increased threefold in endobronchial biopsies, but expression of both 5-lipoxygenase and 15-lipoxygenase-2 in these samples was decreased. Cyclooxygenase-2 expression was decreased in all anatomic compartments sampled in severe asthma. Moreover, LXA(4) receptor gene and protein expression were significantly decreased in severe asthma peripheral blood granulocytes. Mechanisms underlying pathological airway responses in severe asthma include lipoxin underproduction with decreased expression of lipoxin biosynthetic enzymes and receptors. Together, these results indicate that severe asthma is characterized, in part, by defective lipoxin counterregulatory signaling circuits.

Fenspiride inhibits histamine-induced responses in a lung epithelial cell line.

PubMed

Quartulli, F; Pinelli, E; Broué-Chabbert, A; Gossart, S; Girard, V; Pipy, B

1998-05-08

Using the human lung epithelial WI26VA4 cell line, we investigated the capacity of fenspiride, an anti-inflammatory drug with anti-bronchoconstrictor properties, to interfere with histamine-induced intracellular Ca2+ increase and eicosanoid formation. Histamine and a histamine H1 receptor agonist elicited a rapid and transient intracellular Ca2+ increase (0-60 s) in fluo 3-loaded WI26VA4 cells. This response was antagonized by the histamine H1 receptor antagonist, diphenhydramine, the histamine H2 receptor antagonist, cimetidine, having no effect. Fenspiride (10(-7)-10(-5) M) inhibited the histamine H1 receptor-induced Ca2+ increase. In addition, histamine induced a biphasic increase in arachidonic acid release. The initial rise (0-30 s), a rapid and transient arachidonic acid release, was responsible for the histamine-induced intracellular Ca2+ increase. In the second phase release (15-60 min), a sustained arachidonic acid release appeared to be associated with the formation of cyclooxygenase and lipoxygenase metabolites. Fenspiride (10(-5) M) abolished both phases of histamine-induced arachidonic acid release. These results suggest that anti-inflammatory and antibronchoconstrictor properties of fenspiride may result from the inhibition of these effects of histamine.

Formation of a Cyclopropyl Epoxide via a Leukotriene A Synthase-related Pathway in an Anaerobic Reaction of Soybean Lipoxygenase-1 with 15S-Hydroperoxyeicosatetraenoic Acid

PubMed Central

Zheng, Yuxiang; Brash, Alan R.

2010-01-01

The further conversion of an arachidonic acid hydroperoxide to a leukotriene A (LTA) type epoxide by specific lipoxygenase (LOX) enzymes constitutes a key step in inflammatory mediator biosynthesis. Whereas mammalian 5-LOX transforms its primary product (5S-hydroperoxyeicosatetraenoic acid; 5S-HPETE) almost exclusively to LTA4, the model enzyme, soybean LOX-1, normally produces no detectable leukotrienes and instead further oxygenates its primary product 15S-HPETE to 5,15- and 8,15-dihydroperoxides. Mammalian 15-LOX-1 displays both types of activity. We reasoned that availability of molecular oxygen within the LOX active site favors oxygenation, whereas lack of O2 promotes LTA epoxide synthesis. To test this, we reacted 15S-HPETE with soybean LOX-1 under anaerobic conditions and identified the products by high pressure liquid chromatography, UV, mass spectrometry, and NMR. Among the products, we identified a pair of 8,15-dihydroxy diastereomers with all-trans-conjugated trienes that incorporated 18O from H218O at C-8, indicative of the formation of 14,15-LTA4. A pair of 5,15-dihydroxy diastereomers containing two trans,trans-conjugated dienes (6E,8E,11E,13E) and that incorporated 18O from H218O at C-5 was deduced to arise from hydrolysis of a novel epoxide containing a cyclopropyl ring, 14,15-epoxy-[9,10,11-cyclopropyl]-eicosa-5Z,7E,13E-trienoic acid. Also identified was the δ-lactone of the 5,15-diol, a derivative that exhibited no 18O incorporation due to its formation by intramolecular reaction of the carboxyl anion with the proposed epoxide intermediate. Our results support a model in which access to molecular oxygen within the active site directs the outcome from competing pathways in the secondary reactions of lipoxygenases. PMID:20194505

Leukotriene B4 catabolism: quantitation of leukotriene B4 and its omega-oxidation products by reversed-phase high-performance liquid chromatography.

PubMed

Shak, S

1987-01-01

LTB4 and its omega-oxidation products may be rapidly, sensitively, and specifically quantitated by the methods of solid-phase extraction and reversed-phase high-performance liquid chromatography (HPLC), which are described in this chapter. Although other techniques, such as radioimmunoassay or gas chromatography-mass spectrometry, may be utilized for quantitative analysis of the lipoxygenase products of arachidonic acid, only the technique of reversed-phase HPLC can quantitate as many as 10 metabolites in a single analysis, without prior derivatization. In this chapter, we also reviewed the chromatographic theory which we utilized in order to optimize reversed-phase HPLC analysis of LTB4 and its omega-oxidation products. With this information and a gradient HPLC system, it is possible for any investigator to develop a powerful assay for the potent inflammatory mediator, LTB4, or for any other lipoxygenase product of arachidonic acid.

Adenosine up-regulates cyclooxygenase-2 in human granulocytes: impact on the balance of eicosanoid generation.

PubMed

Pouliot, Marc; Fiset, Marie-Elaine; Massé, Mireille; Naccache, Paul H; Borgeat, Pierre

2002-11-01

Polymorphonuclear neutrophils (granulocytes; PMNs) are often the first blood cells to migrate toward inflammatory lesions to perform host defense functions. PMNs respond to specific stimuli by releasing several factors and generate lipid mediators of inflammation from the 5-lipoxygenase and the inducible cyclooxygenase (COX)-2 pathways. In view of adenosine's anti-inflammatory properties and suppressive impact on the 5-lipoxygenase pathway, we addressed in this study the impact of this autacoid on the COX-2 pathway. We observed that adenosine up-regulates the expression of the COX-2 enzyme and mRNA. Production of PGE(2) in response to exogenous arachidonic acid was also increased by adenosine and correlated with COX-2 protein levels. The potentiating effect of adenosine on COX-2 could be mimicked by pharmacological increases of intracellular cAMP levels, involving the latter as a putative second messenger for the up-regulation of COX-2 by adenosine. Specific COX-2 inhibitors were used to confirm the predominant role of the COX-2 isoform in the formation of prostanoids by stimulated PMNs. Withdrawal of extracellular adenosine strikingly emphasized the inhibitory potential of PGE(2) on leukotriene B(4) formation and involved the EP(2) receptor subtype in this process. Thus, adenosine may promote a self-limiting regulatory process through the increase of PGE(2) generation, which may result in the inhibition of PMN functions. This study identifies a new aspect of the anti-inflammatory properties of adenosine in leukocytes, introducing the concept that this autacoid may exert its immunomodulatory activities in part by modifying the balance of lipid mediators generated by PMNs.

Cytochrome P450 and Lipoxygenase Metabolites on Renal Function

PubMed Central

Imig, John D.; Hye Khan, Md. Abdul

2018-01-01

Arachidonic acid metabolites have a myriad of biological actions including effects on the kidney to alter renal hemodynamics and tubular transport processes. Cyclooxygenase metabolites are products of an arachidonic acid enzymatic pathway that has been extensively studied in regards to renal function. Two lesser-known enzymatic pathways of arachidonic acid metabolism are the lipoxygenase (LO) and cytochrome P450 (CYP) pathways. The importance of LO and CYP metabolites to renal hemodynamics and tubular transport processes is now being recognized. LO and CYP metabolites have actions to alter renal blood flow and glomerular filtration rate. Proximal and distal tubular sodium transport and fluid and electrolyte homeostasis are also significantly influenced by renal CYP and LO levels. Metabolites of the LO and CYP pathways also have renal actions that influence renal inflammation, proliferation, and apoptotic processes at vascular and epithelial cells. These renal LO and CYP pathway actions occur through generation of specific metabolites and cell-signaling mechanisms. Even though the renal physiological importance and actions for LO and CYP metabolites are readily apparent, major gaps remain in our understanding of these lipid mediators to renal function. Future studies will be needed to fill these major gaps regarding LO and CYP metabolites on renal function. PMID:26756638

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

PubMed Central

Pu, Shiyun; Ren, Lin; Liu, Qinhui; Kuang, Jiangying; Shen, Jing; Cheng, Shihai; Zhang, Yuwei; Jiang, Wei; Zhang, Zhiyong; Jiang, Changtao

2015-01-01

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. PMID:26398229

Inhibition of 12/15 lipoxygenase by curcumin and an extract from Curcuma longa L.

PubMed

Bezáková, Lýdia; Košťálová, Daniela; Obložinský, Marek; Hoffman, Peter; Pekárová, Mária; Kollárová, Renáta; Holková, Ivana; Mošovská, Silvia; Sturdík, Ernest

2014-02-01

Curcumin (diferuloylmethane) is an orange-yellow secondary metabolic compound from the rhizome of turmeric (Curcuma longa L.), a spice often found in curry powder. It is one of the major curcuminoids of turmeric. For centuries, curcumin has been used in some medicinal preparations or as a food colouring agent. A variety of enzymes that are closely associated with inflammation and cancer were found to be modulated by curcumin. This paper summarized the results of the inhibitory effect of curcumin and a Curcuma longa L. ethanolic extract on lipoxygenase from the rat lung cytosolic fraction. The positional specificity determination of arachidonic acid dioxygenation by RP- and SP-HPLC methods showed that in a purified enzyme preparation from the rat lung cytosol the specific form of lipoxygenase (LOX) is present exhibiting 12/15-LOX dual specificity (with predominant 15-LOX activity). The inhibitory activity of curcumin and Curcuma longa extract on LOX from cytosolic fraction of rat lung was expressed in the percentage of inhibition and as IC50. Lineweaver-Burk plot analysis has indicated that curcumin is the competitive inhibitor of 12/15 LOX from the rat lung cytosolic fraction.

Flavocoxid Inhibits Phospholipase A2, Peroxidase Moieties of the Cyclooxygenases (COX), and 5-Lipoxygenase, Modifies COX-2 Gene Expression, and Acts as an Antioxidant

PubMed Central

Burnett, Bruce P.; Bitto, Alessandra; Altavilla, Domenica; Squadrito, Francesco; Levy, Robert M.; Pillai, Lakshmi

2011-01-01

The multiple mechanisms of action for flavocoxid relating to arachidonic acid (AA) formation and metabolism were studied in vitro. Flavocoxid titrated into rat peritoneal macrophage cultures inhibited cellular phospholipase A2 (PLA2) (IC50 = 60 μg/mL). In in vitro enzyme assays, flavocoxid showed little anti-cyclooxygenase (CO) activity on COX-1/-2 enzymes, but inhibited the COX-1 (IC50 = 12.3) and COX-2 (IC50 = 11.3 μg/mL) peroxidase (PO) moieties as well as 5-lipoxygenase (5-LOX) (IC50 = 110 μg/mL). No detectable 5-LOX inhibition was found for multiple traditional and COX-2 selective NSAIDs. Flavocoxid also exhibited strong and varied antioxidant capacities in vitro and decreased nitrite levels (IC50 = 38 μg/mL) in rat peritoneal macrophages. Finally, in contrast to celecoxib and ibuprofen, which upregulated the cox-2 gene, flavocoxid strongly decreased expression. This work suggests that clinically favourable effects of flavocoxid for management of osteoarthritis (OA) are achieved by simultaneous modification of multiple molecular pathways relating to AA metabolism, oxidative induction of inflammation, and neutralization of reactive oxygen species (ROS). PMID:21765617

Flavocoxid inhibits phospholipase A2, peroxidase moieties of the cyclooxygenases (COX), and 5-lipoxygenase, modifies COX-2 gene expression, and acts as an antioxidant.

PubMed

Burnett, Bruce P; Bitto, Alessandra; Altavilla, Domenica; Squadrito, Francesco; Levy, Robert M; Pillai, Lakshmi

2011-01-01

The multiple mechanisms of action for flavocoxid relating to arachidonic acid (AA) formation and metabolism were studied in vitro. Flavocoxid titrated into rat peritoneal macrophage cultures inhibited cellular phospholipase A2 (PLA(2)) (IC(50) = 60 μg/mL). In in vitro enzyme assays, flavocoxid showed little anti-cyclooxygenase (CO) activity on COX-1/-2 enzymes, but inhibited the COX-1 (IC(50) = 12.3) and COX-2 (IC(50) = 11.3 μg/mL) peroxidase (PO) moieties as well as 5-lipoxygenase (5-LOX) (IC(50) = 110 μg/mL). No detectable 5-LOX inhibition was found for multiple traditional and COX-2 selective NSAIDs. Flavocoxid also exhibited strong and varied antioxidant capacities in vitro and decreased nitrite levels (IC(50) = 38 μg/mL) in rat peritoneal macrophages. Finally, in contrast to celecoxib and ibuprofen, which upregulated the cox-2 gene, flavocoxid strongly decreased expression. This work suggests that clinically favourable effects of flavocoxid for management of osteoarthritis (OA) are achieved by simultaneous modification of multiple molecular pathways relating to AA metabolism, oxidative induction of inflammation, and neutralization of reactive oxygen species (ROS).

Molecular pharmacological profile of the nonredox-type 5-lipoxygenase inhibitor CJ-13,610.

PubMed

Fischer, Lutz; Steinhilber, Dieter; Werz, Oliver

2004-07-01

5-Lipoxygenase (5-LO) is a crucial enzyme in the synthesis of the bioactive leukotrienes (LTs) from arachidonic acid (AA), and inhibitors of 5-LO are thought to prevent the untowarded pathophysiological effects of LTs. In this study, we present the molecular pharmacological profile of the novel nonredox-type 5-LO inhibitor CJ-13,610 that was evaluated in various in vitro assays. In intact human polymorphonuclear leukocytes (PMNL), challenged with the Ca(2+)-ionophore A23187, CJ-13,610 potently suppressed 5-LO product formation with an IC(50)=0.07 microm. Supplementation of exogenous AA impaired the efficacy of CJ-13,610, implying a competitive mode of action. In analogy to ZM230487 and L-739.010, two closely related nonredox-type 5-LO inhibitors, CJ-13,610 up to 30 microm failed to inhibit 5-LO in cell-free assay systems under nonreducing conditions, but inclusion of peroxidase activity restored the efficacy of CJ-13,610 (IC(50)=0.3 microm). In contrast to ZM230487 and L-739.010, the potency of CJ-13,610 does not depend on the cell stimulus or the activation pathway of 5-LO. Thus, 5-LO product formation in PMNL induced by phosphorylation events was equally suppressed by CJ-13,610 as compared to Ca(2+)-mediated 5-LO activation. In transfected HeLa cells, CJ-13,610 only slightly discriminated between phosphorylatable wild-type 5-LO and a 5-LO mutant that lacks phosphorylation sites. In summary, CJ-13,610 may possess considerable potential as a potent orally active nonredox-type 5-LO inhibitor that lacks certain disadvantages of former representatives of this class of 5-LO inhibitors.

Cytidine 5′-Diphosphocholine (CDP-Choline) in Stroke and Other CNS Disorders

PubMed Central

Adibhatla, Rao Muralikrishna; Hatcher, J. F.

2007-01-01

Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1α/β) activate phospholipase A2 (PLA2) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA2 releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA2 stimulation and loss of CCT activity. TNF-α also stimulates proteolysis of CCT. TNF-α and IL-1β are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA2 and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF-α and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke. PMID:15756928

Role of arachidonic acid and protein kinase C during maturation-inducing hormone-dependent meiotic resumption and ovulation in ovarian follicles of Atlantic croaker

USGS Publications Warehouse

Patino, R.; Yoshizaki, G.; Bolamba, D.; Thomas, P.

2003-01-01

The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F2?? and PGE2, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquin- olinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 ??M) and PGF2?? (5 ??M) did not induce maturation, and NDGA (10 ??M) did not affect MIH-dependent maturation. However, IM (100 ??M) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 ??g/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 ??M; H7, 50??M) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 ??M) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF2?? restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role of COX products of AA during maturation is possible. A novel model of MIH-dependent ovulation is proposed in which 1) LOX and COX metabolites of AA are both required for ovulation, but at upstream and downstream sites of the pathway, respectively, relative to PKC, and 2) PKC is downstream of genomic activation.

A novel five-lipoxygenase activity protein inhibitor labeled with carbon-14 and deuterium.

PubMed

Latli, Bachir; Hrapchak, Matt; Gao, Joe J; Busacca, Carl A; Senanayake, Chris H

2015-07-01

2-[4-(3-{(1R)-1-[4-(2-Aminopyrimidin-5-yl)phenyl]-1-cyclopropylethyl}-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl]-N,N-dimethylacetamide (1), is a novel and selective five-lipoxygenase activity protein (FLAP) inhibitor with excellent pharmacokinetics properties. The availability of a key chiral intermediate allowed the synthesis of [(14) C]-(1) in six radiochemical steps and in 47% overall radiochemical yield with a specific activity of 51 mCi/mmol using carbon-14 zinc cyanide. 2-Chloro-N,N-dimethyl-(2)H6-acetamide was prepared and condensed with a penultimate intermediate to give [(2)H6]-(1) in very high yield and in more than 99% isotopic enrichment. Copyright © 2015 John Wiley & Sons, Ltd.

Characterization of two fungal lipoxygenases expressed in Aspergillus oryzae.

PubMed

Sugio, Akiko; Østergaard, Lars Henrik; Matsui, Kenji; Takagi, Shinobu

2018-05-24

Two fungal lipoxygenase genes were cloned from a rice pathogen, Magnaporthe salvinii, and the take-all fungus, Gaeumannomyces graminis var. tritici, and successfully expressed in Aspergillus oryzae in secreted form. The lipoxygenases expressed, termed MLOX and GLOX, were purified and characterized to evaluate suitability for industrial applications. Both enzymes were active broadly at pH 4-11 and had optimum temperatures around 60 °C, but they were largely different in substrate specificity. Where MLOX was active broadly on arachidonic acid, EPA and DHA, and even on derivatives of fatty acids, such as methyl linoleate or linoleoyl alcohol, GLOX was more specific to linoleic acid and linolenic acid. The most remarkable difference between the two fungal LOXs was the positional and stereo-specificity of oxygenation reactions on polyunsaturated fatty acids. When using linoleic acid as the substrate, the product of MLOX was 9S-hydroperoxy-(E,Z)-octadecadienoic acid (9S(E,Z)-HPODE), on the other hand, the product of GLOX was 13R(E,Z)-HPODE. The enzymes were evaluated for a couple of potential applications and found to be effective on bleaching colored compounds such as carotenoids. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells.

PubMed

Jiang, Jun-xia; Zhang, Shui-juan; Xiong, Yao-kang; Jia, Yong-liang; Sun, Yan-hong; Lin, Xi-xi; Shen, Hui-juan; Xie, Qiang-min; Yan, Xiao-feng

2015-01-01

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B4 (LTB4), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB4 production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB4 production and activity. We found that treatment with ox-LDL increased the production of LTB4 and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB4 receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB4 receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB4 production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.

EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells

PubMed Central

Xiong, Yao-kang; Jia, Yong-liang; Sun, Yan-hong; Lin, Xi-xi; Shen, Hui-juan; Xie, Qiang-min; Yan, Xiao-feng

2015-01-01

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B4 (LTB4), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB4 production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB4 production and activity. We found that treatment with ox-LDL increased the production of LTB4 and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB4 receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB4 receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB4 production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells. PMID:26035589

Role of the Cysteinyl Leukotrienes in the Pathogenesis and Progression of Cardiovascular Diseases

PubMed Central

Colazzo, Francesca; Gelosa, Paolo

2017-01-01

Cysteinyl leukotrienes (CysLTs) are potent lipid inflammatory mediators synthesized from arachidonic acid, through the 5-lipoxygenase (5-LO) pathway. Owing to their properties, CysLTs play a crucial role in the pathogenesis of inflammation; therefore, CysLT modifiers as synthesis inhibitors or receptor antagonists, central in asthma management, may become a potential target for the treatment of other inflammatory diseases such as the cardiovascular disorders. 5-LO pathway activation and increased expression of its mediators and receptors are found in cardiovascular diseases. Moreover, the cardioprotective effects observed by using CysLT modifiers are promising and contribute to elucidate the link between CysLTs and cardiovascular disease. The aim of this review is to summarize the state of present research about the role of the CysLTs in the pathogenesis and progression of atherosclerosis and myocardial infarction. PMID:28932020

Interleukin-1 inhibits the synthesis of collagen by fibroblasts.

PubMed

Bhatnagar, R; Penfornis, H; Mauviel, A; Loyau, G; Saklatvala, J; Pujol, J P

1986-10-01

Human dermal fibroblasts, exposed to human or porcine Interleukin-1, responded by an inhibition of collagen synthesis in a dose dependent manner. Incubation with Il-1 for more than 8 h was required to see an appreciable effect. The phenomenon was not dependent on the presence of serum in the culture medium. Since a stimulation of prostaglandin E2 secretion was also observed in presence of Il-1, we investigated the eventual role of arachidonic acid metabolites in the phenomenon. Inhibitors interfering with arachidonate metabolism, namely indomethacin, acetyl salicylic acid, BW 755 C and NDGA had no influence on the inhibition of collagen synthesis caused by Il-1. These data suggest that both cyclooxygenase and lipoxygenase derived metabolites of arachidonic acid are unlikely to play a role in the mechanism.

The influence of arachidonic acid metabolites on cell division in the intestinal epithelium and in colonic tumors.

PubMed

Petry, F M; Tutton, P J; Barkla, D H

1984-09-01

Various metabolites of arachidonic acid are now known to influence cell division. In this paper the effects on cell proliferation of arachidonic acid, some inhibitors of arachidonic acid metabolism and some analogs of arachidonic acid metabolites is described. The epithelial cell proliferation rate in the jejunum, in the descending colon and in dimethylhydrazine-induced tumors of rat colon was measured using a stathmokinetic technique. Administration of arachidonic acid resulted in retardation of cell proliferation in each of the tissues examined. A cyclooxygenase inhibitor (Flurbiprofen) prevented this effect of arachidonic acid in the jejunal crypts and in colonic tumors, but not in colonic crypts. In contrast, inhibitors of both cyclooxygenase and lipoxygenase (Benoxaprofen and BW755c) prevented the effect of arachidonic acid in the colonic crypts and reduced its effect on colonic tumours but did not alter its effect on the jejunum. An inhibitor of thromoboxane A2 synthetase (U51,605) was also able to prevent the inhibitory effect of arachidonic acid on colonic tumors. Treatment with 16,16-dimethyl PGE2 inhibited cell proliferation in jejunal crypts and in colonic tumors, as did a thromboxane A2 mimicking agent, U46619. Nafazatrom, an agent that stimulates prostacyclin synthesis and inhibits lypoxygenase, promoted cell proliferation in the jejunal crypts and colonic crypts, but inhibited cell proliferation in colonic tumours.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini.

PubMed

Tunaz, H; Bedick, J C.; Miller, J S.; Hoback, W W.; Rana, R L.; Stanley, D W.

1999-10-01

Nodulation is the first and quantitatively most important cellular defense reaction to bacterial infections in insects. Treating adults of the 17-year periodical cicadas, Magicicada septendecim and M. cassini, with eicosanoid biosynthesis inhibitors immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a multi-step process in which individual steps are separately mediated by lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent by 2 h after injection, and nodulation was significantly reduced, relative to control insects, over the following 14 h. The dexamethasone effects were reversed by treating bacteria-challenged insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids. These findings in adults of an exopterygote insect species with an unusual life history pattern broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

The Natural Protective Mechanism Against Hyperglycemia in Vascular Endothelial Cells

PubMed Central

Riahi, Yael; Sin-Malia, Yoav; Cohen, Guy; Alpert, Evgenia; Gruzman, Arie; Eckel, Juergen; Staels, Bart; Guichardant, Michel; Sasson, Shlomo

2010-01-01

OBJECTIVE Vascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid–metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator–activated receptor (PPAR) δ was investigated. RESEARCH DESIGN AND METHODS Effects of 4-HDDE and PPARδ on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions. RESULTS Using GW501516 (PPARδ agonist) and GSK0660 (PPARδ antagonist), we discovered that high-glucose–induced downregulation of the glucose transport system in VECs is mediated by PPARδ. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARδ in cells overexpressing human PPAR (hPPAR)δ but not hPPARα, -γ1, or -γ2. Moreover, silencing of PPARδ prevented high-glucose–dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARδ to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay. CONCLUSIONS Collectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARδ. PMID:20107107

Influence of experimental subarachnoid hemorrhage on nicotine-induced contraction of the rat basilar artery in relation to arachidonic acid metabolites signaling pathway.

PubMed

Ji, Xu; Wang, Aimin; Trandafir, Cristina C; Kurahashi, Kazuyoshi

2013-10-01

Smoking is one of the most important risk factors for cerebral circulatory disorders. The purpose of this study was to investigate the influence of experimental subarachnoid hemorrhage (SAH) on nicotine-induced contraction (arachidonic acid metabolites) in the basilar arteries of rats. Rats were killed at 1 hour and 1 week after blood injection, and the basilar artery was isolated and cut into a spiral strip. Testing of cyclooxygenase-1 (COX-1) and 5-lipoxygenase (5-LOX) inhibitors revealed no significant differences in their effects on normal and SAH (1 hour and 1 week). Phospholipase C (PLC) inhibitor (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17yl)amino)hexyl)-1H-pyrrole-2,5,-dione [U-73122]) slightly inhibited contraction of SAH (1 hour and 1 week) when compared to controls. Phospholipase A2 (PLA2) inhibitor (manoalide) and cytosolic PLA2 (cPLA2) inhibitor (arachidonyltrifluoromenthylketone [AACOCF3]) more strongly attenuated contraction in SAH (1 hour and 1 week) than in controls. Secreted PLA2 (sPLA2) inhibitor (indoxam), PLC inhibitor (2-nitro-4-carboxyphenyl N, N-diphenylcarbamate [NCDC]), and COX-2 inhibitors (nimesulide, (5-methanesulfonamido-6-(2,4-difluorothiophenyl)-1-indanone) [L-745337], and celecoxib) only slightly inhibited contraction of SAH (1 week) when compared to normal and SAH (1 hour). The calcium-independent PLA2 (iPLA2) inhibitor bromoenol lactone (BEL) showed greater inhibition of contraction in SAH (1 hour) when compared to normal and SAH (1 week). One week after exposure to SAH, PLC, sPLA2, and COX-2 activity were enhanced and cPLA2 activity was inhibited. One hour after exposure to SAH, PLC activity was enhanced and cPLA2 and iPLA2 activity was inhibited. Such changes of inflammatory arachidonic acid metabolites by smoking after SAH may play important roles in fatal cerebral circulatory disorders, suggesting important implications for the etiology and pathogenesis of SAH. Copyright © 2013 National Stroke Association. Published by Elsevier Inc. All rights reserved.

The natural dual cyclooxygenase and 5-lipoxygenase inhibitor flavocoxid is protective in EAE through effects on Th1/Th17 differentiation and macrophage/microglia activation.

PubMed

Kong, Weimin; Hooper, Kirsten M; Ganea, Doina

2016-03-01

Prostaglandins and leukotrienes, bioactive mediators generated by cyclooxygenases (COX) and 5-lipoxygenase (5-LO) from arachidonic acid, play an essential role in neuroinflammation. High levels of LTB4 and PGE2 and increased expression of COX and 5-LO, as well as high expression of PGE2 receptors were reported in multiple sclerosis (MS) patients and in experimental autoimmune encephalomyelitis (EAE). Prostaglandins and leukotrienes have an interdependent and compensatory role in EAE, which led to the concept of therapy using dual COX/5-LO inhibitors. The plant derived flavocoxid, a dual COX/5-LO inhibitor with anti-inflammatory and antioxidant properties, manufactured as a prescription pharmaconutrient, was reported to be neuroprotective in models of transient ischemic stroke and brain injury. The present study is the first report on prophylactic and therapeutic effects of flavocoxid in EAE. The beneficial effects correlate with reduced expression of proinflammatory cytokines and of COX2 and 5-LO in spinal cords and spleens of EAE mice. The protective mechanisms include: 1. reduction in expression of MHCII/costimulatory molecules and production of proinflammatory cytokines; 2. promotion of the M2 phenotype including IL-10 expression and release by macrophages and microglia; 3. inhibition of Th1 and Th17 differentiation through direct effects on T cells. The direct inhibitory effect on Th1/Th17 differentiation, and promoting the development of M2 macrophages and microglia, represent novel mechanisms for the flavocoxid anti-inflammatory activity. As a dual COX/5-LO inhibitor with antioxidant properties, flavocoxid might be useful as a potential therapeutic medical food agent in MS patients. Copyright © 2015. Published by Elsevier Inc.

Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of protein phosphatase 5 involved in autoinhibition and lipid activation.

PubMed

Kang, H; Sayner, S L; Gross, K L; Russell, L C; Chinkers, M

2001-09-04

Protein phosphatase 5 (PP5) exhibits low basal activity due to the autoinhibitory properties of its N-terminal and C-terminal domains but can be activated approximately 40-fold in vitro by polyunsaturated fatty acids. To identify residues involved in regulating PP5 activity, we performed scanning mutagenesis of its N-terminal tetratricopeptide repeat (TPR) domain and deletion mutagenesis of its C-terminal domain. Mutating residues in a groove of the TPR domain that binds to heat shock protein 90 had no effect on basal phosphatase activity. Mutation of Glu-76, however, whose side chain projects away from this groove, resulted in a 10-fold elevation of basal activity without affecting arachidonic acid-stimulated activity. Thus, the interface of the TPR domain involved in PP5 autoinhibition appears to be different from that involved in heat shock protein 90 binding. We also observed a 10-fold elevation of basal phosphatase activity upon removing the C-terminal 13 amino acids of PP5, with a concomitant 50% decrease in arachidonic acid-stimulated activity. These two effects were accounted for by two distinct amino acid deletions: deleting the four C-terminal residues (496-499) of PP5 had no effect on its activity, but removing Gln-495 elevated basal activity 10-fold. Removal of a further three amino acids had no additional effect, but deleting Asn-491 resulted in a 50% reduction in arachidonic acid-stimulated activity. Thus, Glu-76 in the TPR domain and Gln-495 at the C-terminus were implicated in maintaining the low basal activity of PP5. While the TPR domain alone has been thought to mediate fatty acid activation of PP5, our data suggest that Asn-491, near its C-terminus, may also be involved in this process.

Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

PubMed Central

Kaipainen, Arja; Greene, Emily R.; Huang, Sui

2010-01-01

Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

Effects of NSAIDs on the Inner Ear: Possible Involvement in Cochlear Protection

PubMed Central

Hoshino, Tomofumi; Tabuchi, Keiji; Hara, Akira

2010-01-01

Cyclooxygenase and lipoxygenase, two important enzymes involved in arachidonic acid metabolism, are major targets of non-steroidal anti-inflammatory drugs (NSAIDs). Recent investigations suggest that arachidonic cascades and their metabolites may be involved in maintaining inner ear functions. The excessive use of aspirin may cause tinnitus in humans and impairment of the outer hair cell functions in experimental animals. On the other hand, NSAIDs reportedly exhibit protective effects against various kinds of inner ear disorder. The present review summarizes the effects of NSAIDs on cochlear pathophysiology. NSAIDs are a useful ameliorative adjunct in the management of inner ear disorders. PMID:27713301

Lipoxygenases and their metabolites in formation of plant stress tolerance.

PubMed

Babenko, L M; Shcherbatiuk, M M; Skaterna, T D; Kosakivska, I V

2017-01-01

The review focuses on the analysis of new information concerning molecular enzymology of lipoxygenases – proteins involved in lipid peroxidation and found in animals and plants. Modern concept of structural features, catalytic characteristics and functions of lipoxygenase family enzymes as well as products of their catalytic activity in plants have been discussed and summarized. Issues of enzyme localization in plant cells and tissues, evolution and distribution of lipoxygenases, involvement in production of signaling substances involved in formation of adaptation response to abiotic and biotic stress factors and in regulation of lipoxygenase signal system activity are highlighted. Participants of the elements signaling of LOX-pathway reception and transduction into genome are considered. Special attention is given to jasmonates, metabolites of the allene oxide synthase branch of the lipoxygenase cascade, because these metabolites have high biological activity, are ubiquitously present in all plant organisms, and are involved in regulation of vitally important processes. Data concerning lipoxygenase phylogeny, possible occurrence of a common predecessor for modern isoforms of the enzyme in pro- and eukaryote have been examined. Some results of our studies that open up possibilities of using the lipoxygenase catalytic activity characteristics as biological markers in plant stress tolerance researches are given.

Cyclooxygenase and lipoxygenase gene expression in the inflammogenesis of breast cancer.

PubMed

Kennedy, Brian M; Harris, Randall E

2018-05-07

We examined the expression of major inflammatory genes, cyclooxygenase-1 and 2 (COX1, COX2) and arachidonate 5-lipoxygenase (ALOX5) in 1090 tumor samples of invasive breast cancer from The Cancer Genome Atlas (TCGA). Mean cyclooxygenase expression (COX1 + COX2) ranked in the upper 99th percentile of all 20,531 genes and surprisingly, the mean expression of COX1 was more than tenfold higher than COX2. Highly significant correlations were observed between COX2 with eight tumor-promoting genes (EGR2, IL6, RGS2, B3GNT5, SGK1, SLC2A3, SFRP1 and ETS2) and between ALOX5 and ten tumor promoter genes (CD33, MYOF1, NLRP1, GAB3, CD4, IFR8, CYTH4, BTK, FGR, CD37). Expression of CYP19A1 (aromatase) was significantly correlated with COX2, but only in tumors positive for ER, PR and HER2. Tumor-promoting genes correlated with the expression of COX1, COX2, and ALOX5 are known to effectively increase mitogenesis, mutagenesis, angiogenesis, cell survival, immunosuppression and metastasis in the pathogenesis of breast cancer.

On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid[S

PubMed Central

Robichaud, Philippe Pierre; Poirier, Samuel J.; Boudreau, Luc H.; Doiron, Jérémie A.; Barnett, David A.; Boilard, Eric; Surette, Marc E.

2016-01-01

Alkyne and azide analogs of natural compounds that can be coupled to sensitive tags by click chemistry are powerful tools to study biological processes. Arachidonic acid (AA) is a FA precursor to biologically active compounds. 19-Alkyne-AA (AA-alk) is a sensitive clickable AA analog; however, its use as a surrogate to study AA metabolism requires further evaluation. In this study, AA-alk metabolism was compared with that of AA in human cells. Jurkat cell uptake of AA was 2-fold greater than that of AA-alk, but significantly more AA-Alk was elongated to 22:4. AA and AA-alk incorporation into and remodeling between phospholipid (PL) classes was identical indicating equivalent CoA-independent AA-PL remodeling. Platelets stimulated in the pre­sence of AA-alk synthesized significantly less 12-lipoxygenase (12-LOX) and cyclooxygenase products than in the presence of AA. Ionophore-stimulated neutrophils produced significantly more 5-LOX products in the presence of AA-alk than AA. Neutrophils stimulated with only exogenous AA-alk produced significantly less 5-LOX products compared with AA, and leukotriene B4 (LTB4)-alk was 12-fold less potent at stimulating neutrophil migration than LTB4, collectively indicative of weaker leukotriene B4 receptor 1 agonist activity of LTB4-alk. Overall, these results suggest that the use of AA-alk as a surrogate for the study of AA metabolism should be carried out with caution. PMID:27538823

Thrombin-activated human platelets acutely generate oxidized docosahexaenoic-acid-containing phospholipids via 12-lipoxygenase.

PubMed

Morgan, Lloyd T; Thomas, Christopher P; Kühn, Hartmut; O'Donnell, Valerie B

2010-10-01

Arachidonate-containing oxidized phospholipids are acutely generated by 12-LOX (12-lipoxygenase) in agonist-activated platelets. In the present study, formation of structurally related lipids by oxidation of DHA (docosahexaenoic acid)-containing phospholipids is demonstrated using lipidomic approaches. Precursor scanning reverse-phase LC (liquid chromatography)-MS/MS (tandem MS) identified a new family of lipids that comprise phospholipid-esterified HDOHE (hydroxydocosahexaenoic acid). Two diacyl and two plasmalogen PEs (phosphatidylethanolamines) containing predominantly the 14-HDOHE positional isomer (18:0p/14-HDOHE-PE, 18:0a/14-HDOHE-PE, 16:0a/14-HDOHE-PE and 16:0p/14-HDOHE-PE) were structurally characterized using MS/MS and by comparison with biogenic standards. An involvement of 12-LOX was indicated as purified recombinant human 12-LOX also generated the 14-HDOHE isomer from DHA. Pharmacological studies using inhibitors and recombinant platelet 12-LOX indicate that they form via esterification of newly formed non-esterified HDOHE. HDOHE-PEs formed at significant rates (2-4 ng/4×10(7) cells) within 2-180 min of thrombin stimulation, and their formation was blocked by calcium chelation. In summary, a new family of oxidized phospholipid was identified in thrombin-activated human platelets.

Naproxen Induces Type X Collagen Expression in Human Bone-Marrow-Derived Mesenchymal Stem Cells Through the Upregulation of 5-Lipoxygenase

PubMed Central

Alaseem, Abdulrahman M.; Madiraju, Padma; Aldebeyan, Sultan A.; Noorwali, Hussain; Antoniou, John

2015-01-01

Several studies have shown that type X collagen (COL X), a marker of late-stage chondrocyte hypertrophy, is expressed in mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients. We recently found that Naproxen, but not other nonsteroidal anti-inflammatory drugs (NSAIDs) (Ibuprofen, Celebrex, Diclofenac), can induce type X collagen gene (COL10A1) expression in bone-marrow-derived MSCs from healthy and OA donors. In this study we determined the effect of Naproxen on COL X protein expression and investigated the intracellular signaling pathways that mediate Naproxen-induced COL10A1 expression in normal and OA hMSCs. MSCs of OA patients were isolated from aspirates from the intramedullary canal of donors (50–80 years of age) undergoing hip replacement surgery for OA and were treated with or without Naproxen (100 μg/mL). Protein expression and phosphorylation were determined by immunoblotting using specific antibodies (COL X, p38 mitogen-activated protein kinase [p38], phosphorylated-p38, c-Jun N-terminal kinase [JNK], phosphorylated-JNK, extracellular signal-regulated kinase [ERK], and phosphorylated-ERK). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of COL10A1 and Runt-related transcription factor 2 gene (Runx2). Our results show that Naproxen significantly stimulated COL X protein expression after 72 h of exposure both in normal and OA hMSCs. The basal phosphorylation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) in OA hMSCs was significantly higher than in normal. Naproxen significantly increased the MAPK phosphorylation in normal and OA hMSCs. NSAID cellular effects include cyclooxygenase, 5-lipoxygenase, and p38 MAPK signaling pathways. To investigate the involvement of these pathways in the Naproxen-induced COL10A1 expression, we incubated normal and OA hMSCs with Naproxen with and without inhibitors of ERK (U0126), JNK (BI-78D3), p38 (SB203580), and 5-lipoxygenase (MK-886). Our results showed that increased basal COL10A1 expression in OA hMSCs was significantly suppressed in the presence of JNK and p38 inhibitors, whereas Naproxen-induced COL10A1 expression was suppressed by 5-lipoxygenase inhibitor. This study shows that Naproxen induces COL X both at transcriptional and translational levels in normal and OA hMSCs. Elevated basal COL10A1 expression in OA hMSCs is probably through the activation of MAPK pathway and Naproxen-induced COL10A1 expression is through the increased 5-lipoxygenase signaling. PMID:25091567

THE 5-LIPOXYGENASE PATHWAY IS REQUIRED FOR ACUTE LUNG INJURY FOLLOWING HEMORRHAGIC SHOCK

PubMed Central

Eun, John C.; Moore, Ernest E.; Mauchley, David C.; Johnson, Chris A.; Meng, Xianzhong; Banerjee, Anirban; Wohlauer, Max V.; Zarini, Simona; Gijón, Miguel A.; Murphy, Robert C.

2012-01-01

The cellular and biochemical mechanisms leading to acute lung injury and subsequent multiple organ failure are only partially understood. In order to study the potential role of eicosanoids, particularly leukotrienes, as possible mediators of acute lung injury, we used a murine experimental model of acute lung injury induced by hemorrhagic shock after blood removal via cardiac puncture. Neutrophil sequestration as shown by immunofluorescence, and protein leakage into the alveolar space, were measured as markers of injury. We used liquid chromatography coupled to tandem mass spectrometry to unequivocally identify several eicosanoids in the bronchoalveolar lavage fluid of experimental animals. MK886, a specific inhibitor of the 5-lipoxygenase pathway, as well as transgenic mice deficient in 5-lipoxygenase, were used to determine the role of this enzymatic pathway in this model. Leukotriene B4 and leukotriene C4 were consistently elevated in shock-treated mice compared to sham-treated mice. MK886 attenuated neutrophil infiltration and protein extravasation induced by hemorrhagic shock. 5-lipoxygenase-deficient mice showed reduced neutrophil infiltration and protein extravasation after shock treatment, indicating greatly reduced lung injury. These results support the hypothesis that 5-lipoxygenase, most likely through the generation of leukotrienes, plays an important role in the pathogenesis of acute lung injury induced by hemorrhagic shock in mice. This pathway could represent a new target for pharmacological intervention to reduce lung damage following severe primary injury. PMID:22392149

The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation.

PubMed

Enyedi, Balázs; Jelcic, Mark; Niethammer, Philipp

2016-05-19

Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal. Osmotic swelling of cells and their nuclei activates cPla2 by translocating it from the nucleoplasm to the nuclear envelope. Elevated cytosolic Ca(2+) was necessary but not sufficient for cPla2 translocation, and nuclear swelling was required in parallel. cPla2 translocation upon nuclear swelling was reconstituted in isolated nuclei and appears to be a simple physical process mediated by tension in the nuclear envelope. Our data suggest that the nucleus plays a mechanosensory role in inflammation by transducing cell swelling and lysis into proinflammatory eicosanoid signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

The 5-lipoxygenase inhibitor tepoxalin induces oxidative damage and altered PTEN status prior to apoptosis in canine osteosarcoma cell lines.

PubMed

Loftus, J P; Cavatorta, D; Bushey, J J; Levine, C B; Sevier, C S; Wakshlag, J J

2016-06-01

The 5-lipoxygenase (5-LOX) inhibitor tepoxalin has been shown to slow canine osteosarcoma (OSA) tumour xenografts growth, yet the mechanisms are poorly elucidated. Further examination of tepoxalin in canine OSA cell lines shows that tepoxalin treated cells undergo apoptosis through caspase-3 activation and annexin staining. Interestingly, apoptosis is superseded by an increase in reactive oxygen species (ROS), as measured by activation of dihydrorhodamine 123 and mitosox. This increase in ROS appears to be related to the 5-LOX inhibitor regardless of cellular 5-LOX status, and was not observed after treatment with the tepoxalin metabolite RWJ20142. Additionally, 5-LOX inhibition by tepoxalin appears to increase phosphatase and tensin (PTEN) homolog activity by preventing its alkylation or oxidation. PTEN modification or inhibition allows phosphoinositide-3 (PI3) kinase activity thereby heightening activation of protein kinase B (AKT) phosphorylation. Our data suggest that off target oxidation and LOX inhibition play roles in the apoptotic response. © 2014 John Wiley & Sons Ltd.

Noise exposure alters cyclooxygenase 1 (COX-1) and 5-lipoxygenase (5-LO) expression in the guinea pig cochlea.

PubMed

Heinrich, Ulf-Rüdiger; Selivanova, Oxana; Schmidtmann, Irene; Feltens, Ralph; Brieger, Jürgen; Mann, Wolf J

2010-03-01

Changes in the metabolism of arachidonic acid (AA) might be part of a noise-induced compensatory mechanism with regional specificity. The released imbalance of prostaglandins and leukotrienes, both AA metabolites, might result in altered blood flow regulation in the inner ear and probably contributes to noise-induced hearing loss. The aim of this study was to gain further information about noise-dependent changes in AA metabolism in the mammalian cochlea. In this prospective animal study, 10 male guinea pigs were exposed to tone bursts for 1 h at 70 dB sound pressure level (SPL) (n = 5) or 90 dB SPL (n = 5). Five animals were used as controls. Alterations in cyclooxygenase 1 (COX-1) and 5-lipoxygenase (5-LO) expression were determined by quantitative immunohistochemical analysis in 11 cochlear regions. COX-1 expression was decreased after both 70 dB SPL and 90 dB SPL exposure in most cell types of the organ of Corti and increased in the nerve fibers of the osseous spiral lamina. 5-LO was lowered after 90 dB SPL exposure, preferentially in the third cochlear turn in the organ of Corti, in the first and second turn in spiral ganglion cells, and in all turns in the stria vascularis.

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.

PubMed

da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis

2014-07-01

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

Association of ALOX5AP with ischemic stroke: a population-based case-control study.

PubMed

Kaushal, Ritesh; Pal, Prodipto; Alwell, Kathleen; Haverbusch, Mary; Flaherty, Matthew; Moomaw, Charles; Sekar, Padmini; Kissela, Brett; Kleindorfer, Dawn; Chakraborty, Ranajit; Broderick, Joseph; Deka, Ranjan; Woo, Daniel

2007-06-01

Arachidonate 5-lipoxygenase activating protein (ALOX5AP) has been reported to demonstrate linkage and association with ischemic stroke and myocardial infarction. However, replication studies have been conflicting and to date, a significant proportion of blacks have not been studied. We prospectively recruited cases of ischemic stroke from all 16 hospitals in the Greater Cincinnati/Northern Kentucky region and demographically matched them to stroke-free population-based controls. Single nucleotide polymorphisms (SNPs) were selected based on association with ischemic stroke in prior studies. Allelic, genotypic and haplotypic association testing was performed using HAPLOVIEW. Multiple logistic regression was used to control for the presence of traditional risk factors including hypertension, diabetes, hypercholesterolemia and smoking. A total of 357 cases and 482 controls were genotyped. The SNPs, rs9579646 and rs4769874 were found to be significantly associated at both allelic (P=0.019 and P<10(-4), respectively) and genotypic level with ischemic stroke among whites after correction for multiple testing. Haplotype association was identified with ischemic stroke as well as ischemic stroke subtypes among whites. Although an overall haplotype association with ischemic stroke was identified among blacks no evidence of association among individual haplotypes, alleles or genotypes were observed. Allele frequencies for the SNPs examined were markedly different among whites and blacks. In conclusion, we report significant association of variants of ALOX5AP with ischemic stroke and ischemic stroke subtypes among whites. No significant association was identified among blacks.

Control of arachidonic acid release in chick muscle cultures

NASA Technical Reports Server (NTRS)

Templeton, G. H.; Padalino, M.; Wright, W.

1985-01-01

Cultures from thigh muscles of 12 day old embryonic chicks are utilized to examine arachidonic release, prostaglandin (PG) biosynthesis, and protein synthesis. The preparation of the cultures is described. It is observed that exogenous arachidonic acid is formed into photsphatidylethanolamine and phosphatidylcholine, is released by a calcium ionosphere or phospholiphase simulator, and is the substrate for the biosynthesis of PG; the epidermal growth factor and PGF do not stimulate protein synthesis over the basal levels. The relationship between arachidonate release and melittin is studied. The data reveal that a change in intracellular calcium stimulates phospholiphase activity, arachidonate release, and PG synthesis in chick muscle culture.

Inhibition of in vitro leukotriene B4 biosynthesis in human neutrophil granulocytes and docking studies of natural quinones.

PubMed

Landa, Premysl; Kutil, Zsofia; Temml, Veronika; Malik, Jan; Kokoska, Ladislav; Widowitz, Ute; Pribylova, Marie; Dvorakova, Marcela; Marsik, Petr; Schuster, Daniela; Bauer, Rudolf; Vanek, Tomas

2013-01-01

Quinones are compounds frequently contained in medicinal plants used for the treatment of inflammatory diseases. Therefore, the impact of plant-derived quinones on the arachidonic acid metabolic pathway is worthy of investigation. In this study, twenty-three quinone compounds of plant origin were tested in vitro for their potential to inhibit leukotriene B4 (LTB4) biosynthesis in activated human neutrophil granulocytes with 5-lipoxygenase (5-LOX) activity. The benzoquinones primin (3) and thymohydroquinone (4) (IC50 = 4.0 and 4.1 microM, respectively) showed activity comparable with the reference inhibitor zileuton (1C50 = 4.1 microM). Moderate activity was observed for the benzoquinone thymoquinone (2) (1C50 = 18.2 microM) and the naphthoquinone shikonin (1) (IC50 = 24.3 microM). The anthraquinone emodin and the naphthoquinone plumbagin (5) displayed only weak activities (IC50 > 50 microM). The binding modes of the active compounds were further evaluated in silico by molecular docking to the human 5-LOX crystal structure. This process supports the biological data and suggested that, although the redox potential is responsible for the quinone's activity on multiple targets, in the case of 5-LOX the molecular structure plays a vital role in the inhibition. The obtained results suggest primin as a promising compound for the development of dual COX-2/5-LOX inhibitors.

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

PubMed

Heshof, Ruud; van Schayck, J Paul; Tamayo-Ramos, Juan Antonio; de Graaff, Leo H

2014-01-01

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans

PubMed Central

2014-01-01

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans. PMID:25401068

NZB/NZW F1 mouse nephritis and immune response are not changed by treatment with a 15-lipoxygenase derivative.

PubMed

Aldigier, J C; Cook, J; Delebassée, S; Guibert, F; Touchard, G; Juzan, M; Gualde, N

1992-10-01

15-HETE is an arachidonic acid derivative issued from the 15 lipoxygenase pathway. This fatty acid possesses immunomodulatory capabilities since it was reported that it generates CD8 + suppressor T-cells either in vitro or ex vivo. The aim of the present report was to study if the suppressive capabilities of 15-HETE were able to influence the onset of the NZB/NZW Fl auto-immune disease. For that purpose we produced 15-HETE and injected the eicosanoid twice a week to NZB/WFI mice for 40 weeks. During the 15-HETE treatment of the animals it was observed an augmentation of the proliferative response of lectin-stimulated splenocytes (at weeks 20 and 30) then the thymidine uptake decreased (at week 40). In fact we observed that among 15-HETE treated mice the evolution of the nephropathy was not changed, the 'glomerular activity score' remained the same for the treated animals compared to controls. On the contrary antinuclear antibodies occurred earlier even if in some experiments the generation of CD8 + cells was demonstrated.

Effect of ω-3 and ω-9 fatty acid rich oils on lipoxygenases and cyclooxygenases enzymes and on the growth of a mammary adenocarcinoma model.

PubMed

Comba, Andrea; Maestri, Damian M; Berra, María A; Garcia, Carolina Paola; Das, Undurti N; Eynard, Aldo R; Pasqualini, María E

2010-10-08

Nutritional factors play a major role in cancer initiation and development. Dietary polyunsaturated fatty acids (PUFAs) have the ability to induce modifications in the activity of lipoxygenase (LOX) and cyclooxygenase (COX) enzymes that affect tumour growth. We studied the effect of two diets enriched in 6% Walnut and Peanut oils that are rich in ω-3 and ω9 PUFAs respectively on a murine mammary gland adenocarcinoma as compared with the control (C) that received commercial diet. Peanut oil enriched diet induced an increase in membrane arachidonic acid (AA) content and the cyclooxygenase enzyme derived 12-HHT (p < 0.05) and simultaneously showed decrease in 12-LOX, 15-LOX-2, 15-LOX-1 and PGE activities (p < 0.05) that corresponded to higher apoptosis and lower mitosis seen in this group (p < 0.05). Furthermore, Peanut oil group showed lower T-cell infiltration (p < 0.05), number of metastasis (p < 0.05) and tumour volume (p < 0.05) and longer survival rate compared to other groups. The results of the present study showed that Peanut oil-enriched diet protects against mammary cancer development by modulating tumour membrane fatty acids composition and LOX and COX enzyme activities.

Elastic network model of learned maintained contacts to predict protein motion

PubMed Central

Putz, Ines

2017-01-01

We present a novel elastic network model, lmcENM, to determine protein motion even for localized functional motions that involve substantial changes in the protein’s contact topology. Existing elastic network models assume that the contact topology remains unchanged throughout the motion and are thus most appropriate to simulate highly collective function-related movements. lmcENM uses machine learning to differentiate breaking from maintained contacts. We show that lmcENM accurately captures functional transitions unexplained by the classical ENM and three reference ENM variants, while preserving the simplicity of classical ENM. We demonstrate the effectiveness of our approach on a large set of proteins covering different motion types. Our results suggest that accurately predicting a “deformation-invariant” contact topology offers a promising route to increase the general applicability of ENMs. We also find that to correctly predict this contact topology a combination of several features seems to be relevant which may vary slightly depending on the protein. Additionally, we present case studies of two biologically interesting systems, Ferric Citrate membrane transporter FecA and Arachidonate 15-Lipoxygenase. PMID:28854238

Lipoxygenase-allene oxide synthase pathway in octocoral thermal stress response

NASA Astrophysics Data System (ADS)

Lõhelaid, H.; Teder, T.; Samel, N.

2015-03-01

Marine ecosystems are sensitive to elevated seawater temperature, with stony corals serving as model organisms for temperature-imposed declines in population viability and diversity. Several stress markers, including heat shock proteins, have been used for the detection and prediction of stress responses in stony corals. However, the stress indicators in soft corals remain elusive. In higher animals and plants, oxylipins synthesized by fatty acid di- and monooxygenases contribute to stress-induced signaling; however, the role of eicosanoid pathways in corals remains unclear. The eicosanoid gene specific to corals encodes for a natural fusion protein of allene oxide synthase and lipoxygenase ( AOS- LOX). In this work, using the easily cultivated soft coral Capnella imbricata as the stress response model, we monitored the expression of the AOS-LOX and the formation of arachidonic acid metabolites in response to an acute rise in water temperature. Gene expression profiles of two 70 kDa heat shock proteins ( Hsps: Hsp70 and Grp78) were used as a positive control for the stress response. In comparison with normal seawater temperature (23 °C), AOS- LOXa and Hsps were all up-regulated after modest (28 °C) and severe (31 °C) temperature elevation. While the up-regulation of AOS- LOXa and Grp78 was more sensitive to moderate temperature changes, Hsp70s were more responsive to severe heat shock. Concurrently, endogenous and exogenous AOS-LOXa-derived eicosanoids were up-regulated. Thus, together with the up-regulation of AOS- LOX by other abiotic and biotic stress stimuli, these data implicate AOS-LOX as part of the general stress response pathway in corals.

5-Lipoxygenase as an endogenous modulator of amyloid beta formation in vivo

PubMed Central

Chu, Jin; Praticò, Domenico

2010-01-01

Objective The 5-lipoxygenase (5-LO) enzymatic pathway is widely distributed within the central nervous system, and is up-regulated in Alzheimer's disease. However, the mechanism whereby it may influence the disease pathogenesis remains elusive. Methods We evaluated the molecular mechanism by which 5-LO regulates Amyloid β (Aβ) formation in vitro and in vivo by pharmacological and genetic approaches. Results Here we show that 5-LO regulates the formation of Aβ by activating the cAMP-response element binding protein (CREB), which in turn increases transcription of the γ-secretase complex. Preventing CREB activation by pharmacologic inhibition or dominant negative mutants blocks the 5-LO-dependent elevation of Aβ formation and the increase of γ-secretase mRNA and protein levels. Moreover, 5-LO targeted gene disruption or its in vivo selective pharmacological inhibition results in a significant reduction of Aβ, CREB and γ-secretase levels. Interpretation These data establish a novel functional role for 5-LO in regulating endogenous formation of Aβ levels in the central nervous system. Thus, 5-LO pharmacological inhibition may be beneficial in the treatment and prevention of Alzheimer's disease. PMID:21280074

Unraveling the Complex Relationship Triad between Lipids, Obesity, and Inflammation

PubMed Central

Khan, Shahida A.; Khan, Sarah A.; Zahran, Solafa A.; Damanhouri, Ghazi

2014-01-01

Obesity today stands at the intersection between inflammation and metabolic disorders causing an aberration of immune activity, and resulting in increased risk for diabetes, atherosclerosis, fatty liver, and pulmonary inflammation to name a few. Increases in mortality and morbidity in obesity related inflammation have initiated studies to explore different lipid mediated molecular pathways of attempting resolution that uncover newer therapeutic opportunities of anti-inflammatory components. Majorly the thromboxanes, prostaglandins, leukotrienes, lipoxins, and so forth form the group of lipid mediators influencing inflammation. Of special mention are the omega-6 and omega-3 fatty acids that regulate inflammatory mediators of interest in hepatocytes and adipocytes via the cyclooxygenase and lipoxygenase pathways. They also exhibit profound effects on eicosanoid production. The inflammatory cyclooxygenase pathway arising from arachidonic acid is a critical step in the progression of inflammatory responses. New oxygenated products of omega-3 metabolism, namely, resolvins and protectins, behave as endogenous mediators exhibiting powerful anti-inflammatory and immune-regulatory actions via the peroxisome proliferator-activated receptors (PPARs) and G protein coupled receptors (GPCRs). In this review we attempt to discuss the complex pathways and links between obesity and inflammation particularly in relation to different lipid mediators. PMID:25258478

Effect of lipoxygenase metabolites of arachidonic acid on proliferation of human T cells and T cell subsets.

PubMed

Gualde, N; Atluru, D; Goodwin, J S

1985-02-01

The lipoxygenase products LTB4 and 15 HPETE have been reported to stimulate T suppressor cell function and also to inhibit [3H]thymidine incorporation into mitogen-stimulated T cells. This present report documents that although these compounds do indeed inhibit [3H]thymidine incorporation into unfractionated T cells, they significantly enhance [3H]thymidine incorporation into T cell preparation enriched for cells bearing the cytotoxic suppressor cell phenotype identified by the OKT8 monoclonal antibody. The mitogen response of T cells enriched for OKT4+ helper-inducer cells is inhibited in manner similar to the response of unfractionated T cells. Thus, LTB4 and 15 HPETE stimulate both the function and the proliferation of the cytotoxic-suppressor T cell subset.

ALOX12 in human toxoplasmosis.

PubMed

Witola, William H; Liu, Susan Ruosu; Montpetit, Alexandre; Welti, Ruth; Hypolite, Magali; Roth, Mary; Zhou, Ying; Mui, Ernest; Cesbron-Delauw, Marie-France; Fournie, Gilbert J; Cavailles, Pierre; Bisanz, Cordelia; Boyer, Kenneth; Withers, Shawn; Noble, A Gwendolyn; Swisher, Charles N; Heydemann, Peter T; Rabiah, Peter; Muench, Stephen P; McLeod, Rima

2014-07-01

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [P, <0.000309], rs312462 [P, <0.028499], rs6502998 [P, <0.029794], and rs434473 [P, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12. In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

ALOX12 in Human Toxoplasmosis

PubMed Central

Witola, William H.; Liu, Susan Ruosu; Montpetit, Alexandre; Welti, Ruth; Hypolite, Magali; Roth, Mary; Zhou, Ying; Mui, Ernest; Cesbron-Delauw, Marie-France; Fournie, Gilbert J.; Cavailles, Pierre; Bisanz, Cordelia; Boyer, Kenneth; Withers, Shawn; Noble, A. Gwendolyn; Swisher, Charles N.; Heydemann, Peter T.; Rabiah, Peter; Muench, Stephen P.

2014-01-01

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [P, <0.000309], rs312462 [P, <0.028499], rs6502998 [P, <0.029794], and rs434473 [P, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12. In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans. PMID:24686056

Effect of a 5-lipoxygenase inhibitor and leukotriene antagonist (PF 5901) on antigen-induced airway responses in neonatally immunized rabbits.

PubMed Central

Herd, C. M.; Donigi-Gale, D.; Shoupe, T. S.; Burroughs, D. A.; Yeadon, M.; Page, C. P.

1994-01-01

1. The effect of a single intratracheal dose (10 mg) of PF 5901 (2-[3(1-hydroxyhexyl) phenoxymethyl] quinoline hydrochloride, a specific inhibitor of the 5-lipoxygenase pathway of arachidonic acid metabolism and a leukotriene D4 antagonist) on airway changes induced in response to Alternaria tenuis aerosol challenge was assessed in adult rabbits neonatally immunized. Leukotriene generation was determined in vivo by measuring leukotriene B4 (LTB4) levels in bronchoalveolar lavage (BAL) fluid and ex vivo by measuring calcium ionophore-stimulated production of LTB4 in whole blood. 2. While PF 5901 (10 mg) had no significant effect on the acute bronchoconstriction induced by antigen, this dose was sufficient to inhibit significantly the increase in airway responsiveness to inhaled histamine 24 h following antigen challenge (P < 0.05). 3. Total leucocyte infiltration into the airways induced by antigen, as assessed by bronchoalveolar lavage, was significantly inhibited by pretreatment with PF 5901 (10 mg). However, the pulmonary infiltration of neutrophils and eosinophils induced by antigen was unaltered by prior treatment with PF 5901 (10 mg). 4. PF 5901 (10 mg) had no effect on ex vivo LTB4 synthesis in whole blood. However, the antigen-induced increase in LTB4 levels in BAL 24 h following challenge was significantly inhibited (P < 0.05). 5. We suggest from the results of the present study that the antigen-induced airway hyperresponsiveness to inhaled histamine in immunized rabbits is mediated, at least in part, by products of the 5-lipoxygenase metabolic pathway, and is not dependent on the extent of eosinophil or neutrophil influx into the airway lumen. PMID:8032653

[Effect of lysine clonixinate and indomethacin on lipoxygenase and cyclooxygenase activity in colon isolated from cancer patients].

PubMed

Franchi, A; Di Girolamo, G; de los Santos, A R; Marti, M L; Gimeno, M A

1998-01-01

The non-steroidal anti-inflammatory drugs (NSAIDS) induced ulcerations in the gastrointestinal tract are possibly associated with the reduction in prostaglandin (PGs) synthesis due to the inhibition of cyclooxygenase. On the other hand, it has been shown that 5-lipooxygenase products (5-LO) are ulcerogenic agents. In some cases, the utilization of NSAIDS stimulates 5-LO pathway to an excess of arachidonic acid because of cyclooxygenase inhibition. In these cases, the damage produced by NSAIDS is greater, since not only the cytoprotective PGs decrease but also the products of 5-LO are increased. The object of the present paper was to study the effects of lysine clonixinate (LC) and indomethacin (INDO) on PGs and 5-HETE synthesis. The concentrations used of LC (4 and 6 micrograms/ml) and INDO (0.035 micrograms/ml and 0.35 micrograms/ml) correspond to the plasmatic values reached with oral therapeutic doses for both drugs. The results show that in no case did LC reduce the production of PGE2. On the contrary INDO inhibited significantly the synthesis of PGe2. It is interesting to mention that LC 4 and 6 micrograms/ml inhibited drastically the production of 5-HETE. Only with the higher concentration of INDO did we observe a similar effect. These results may indicate an inhibitory action on 5-LO, the first enzyme in the metabolic pathway of arachidonic acid in the production of HETEs and LTS. We conclude that LC in therapeutic doses has a mechanism of action different from the classical NSAIDS. The data obtained in this study could explain the low incidence in gastrointestinal lesions with LC.

Teleost fish larvae adapt to dietary arachidonic acid supply through modulation of the expression of lipid metabolism and stress response genes.

PubMed

Alves Martins, Dulce; Rocha, Filipa; Martínez-Rodríguez, Gonzalo; Bell, Gordon; Morais, Sofia; Castanheira, Filipa; Bandarra, Narcisa; Coutinho, Joana; Yúfera, Manuel; Conceição, Luís E C

2012-09-01

Dietary fatty acid supply can affect stress response in fish during early development. Although knowledge on the mechanisms involved in fatty acid regulation of stress tolerance is scarce, it has often been hypothesised that eicosanoid profiles can influence cortisol production. Genomic cortisol actions are mediated by cytosolic receptors which may respond to cellular fatty acid signalling. An experiment was designed to test the effects of feeding gilthead sea-bream larvae with four microdiets, containing graded arachidonic acid (ARA) levels (0·4, 0·8, 1·5 and 3·0 %), on the expression of genes involved in stress response (steroidogenic acute regulatory protein, glucocorticoid receptor and phosphoenolpyruvate carboxykinase), lipid and, particularly, eicosanoid metabolism (hormone-sensitive lipase, PPARα, phospholipase A2, cyclo-oxygenase-2 and 5-lipoxygenase), as determined by real-time quantitative PCR. Fish fatty acid phenotypes reflected dietary fatty acid profiles. Growth performance, survival after acute stress and similar whole-body basal cortisol levels suggested that sea-bream larvae could tolerate a wide range of dietary ARA levels. Transcription of all genes analysed was significantly reduced at dietary ARA levels above 0·4 %. Nonetheless, despite practical suppression of phospholipase A2 transcription, higher leukotriene B4 levels were detected in larvae fed 3·0 % ARA, whereas a similar trend was observed regarding PGE2 production. The present study demonstrates that adaptation to a wide range of dietary ARA levels in gilthead sea-bream larvae involves the modulation of the expression of genes related to eicosanoid synthesis, lipid metabolism and stress response. The roles of ARA, other polyunsaturates and eicosanoids as signals in this process are discussed.

Human and mouse eLOX3 have distinct substrate specificities: implications for their linkage with lipoxygenases in skin

PubMed Central

Yu, Zheyong; Schneider, Claus; Boeglin, William E.; Brash, Alan R.

2008-01-01

Genetic and biochemical evidence suggests a functional link between human 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3) in normal differentiation of the epidermis; LOX-derived fatty acid hydroperoxide is isomerized by the atypical eLOX3 into a specific epoxyalcohol that is a potential mediator in the pathway. Mouse epidermis expresses a different complement of LOX enzymes, and therefore this metabolic linkage could differ. To test this concept, we compared the substrate specificities of recombinant mouse and human eLOX3 toward sixteen hydroperoxy stereoisomers of arachidonic and linoleic acids. Both enzymes metabolized R-hydroperoxides 2–3 times faster than the corresponding S enantiomers. Whereas 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) is the best substrate for human eLOX3 (2.4 sec−1; at 30 µM substrate), mouse eLOX3 shows the highest turnover with 8R-HPETE (2.9 sec−1) followed by 8S-HPETE (1.3 sec−1). Novel product structures were characterized from reactions of mouse eLOX3 with 5S-, 8R-, and 8S-HPETEs. 8S-HPETE is converted specifically to a single epoxyalcohol, identified as 10R-hydroxy-8S,9S-epoxyeicosa-5Z,11Z,14Z-trienoic acid. The substrate preference of mouse eLOX3 and the unique occurrence of an 8S-LOX enzyme in mouse skin point to a potential LOX pathway for the production of epoxyalcohol in murine epidermal differentiation. PMID:17045234

Effects of thyroid hormone status on metabolic pathways of arachidonic acid in mice and humans: A targeted metabolomic approach.

PubMed

Yao, Xuan; Sa, Rina; Ye, Cheng; Zhang, Duo; Zhang, Shengjie; Xia, Hongfeng; Wang, Yu-cheng; Jiang, Jingjing; Yin, Huiyong; Ying, Hao

2015-01-01

Symptoms of cardiovascular diseases are frequently found in patients with hypothyroidism and hyperthyroidism. However, it is unknown whether arachidonic acid metabolites, the potent mediators in cardiovascular system, are involved in cardiovascular disorders caused by hyperthyroidism and hypothyroidism. To answer this question, serum levels of arachidonic acid metabolites in human subjects with hypothyroidism, hyperthyroidism and mice with hypothyroidism or thyroid hormone treatment were determined by a mass spectrometry-based method. Over ten arachidonic acid metabolites belonging to three catalytic pathways: cyclooxygenases, lipoxygenases, and cytochrome P450, were quantified simultaneously and displayed characteristic profiles under different thyroid hormone status. The level of 20-hydroxyeicosatetraenoic acid, a cytochrome P450 metabolite, was positively correlated with thyroid hormone level and possibly contributed to the elevated blood pressured in hyperthyroidism. The increased prostanoid (PG) I2 and decreased PGE2 levels in hypothyroid patients might serve to alleviate atherosclerosis associated with dyslipidemia. The elevated level of thromboxane (TX) A2, as indicated by TXB2, in hyperthyroid patients and mice treated with thyroid hormone might bring about pulmonary hypertension frequently found in hyperthyroid patients. In conclusion, our prospective study revealed that arachidonic acid metabolites were differentially affected by thyroid hormone status. Certain metabolites may be involved in cardiovascular disorders associated with thyroid diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of MK-886, a 5-lipoxygenase activating protein (FLAP) inhibitor, and 5-lipoxygenase deficiency on the forced swimming behavior of mice

PubMed Central

Uz, Tolga; Dimitrijevic, Nikola; Imbesi, Marta; Manev, Hari; Manev, Radmila

2008-01-01

A common biological pathway may contribute to the comorbidity of atherosclerosis and depression. Increased activity of the enzymatic 5-lipoxygenase (5-LOX; 5LO) pathway is a contributing factor in atherosclerosis and a 5-LOX inhibitor, MK-886, is beneficial in animal models of atherosclerosis. In the brain, MK-886 increases phosphorylation of the glutamate receptor subunit GluR1, and the increased phosphorylation of this receptor has been associated with antidepressant treatment. In this work, we evaluated the behavioral effects of MK-886 in an automated assay of mouse forced swimming, which identifies antidepressant activity as increased climbing behavior and/or decreased rest time. Whereas a single injection of MK-886 (3 and 10 mg/kg) did not affect forced swimming behaviors assayed 30 min later, 6 daily injections of 3 mg/kg MK-886 slightly increased climbing and significantly reduced rest time in wild-type mice but not in 5-LOX-deficient mice. A diet delivery of MK-886, 4 μg per 100 mg body-weight per day, required three weeks to affect forced swimming; it increased climbing behavior. Climbing behavior was also increased in naive 5-LOX-deficient mice compared to naive wild-type controls. These results suggest that 5-LOX inhibition and deficiency may be associated with antidepressant activity. Increased climbing in a forced swimming assay is a typical outcome of antidepressants that increase noradrenergic and dopaminergic activity. Interestingly, 5-LOX deficiency and MK-886 treatment have been shown to be capable of increasing the behavioral effects of a noradrenaline/dopamine-potentiating drug, cocaine. Future research is needed to evaluate the clinical relevance of our findings. PMID:18403121

A new method to evaluate anti-allergic effect of food component by measuring leukotriene B4 from a mouse mast cell line.

PubMed

Takasugi, Mikako; Muta, Emi; Yamada, Koji; Arai, Hirofumi

2018-02-01

Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB 4 activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB 4 production by the cells was determined by HPLC with UV detection. LTB 4 was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB 4 production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB 4 detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB 4 production, but AACOCF 3 (phospholipase A 2 inhibitor) slightly increased LTB 4 production, suggesting that LTB 4 was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB 4 activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB 4 production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB 4 inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB 4 release suppression by food components such as flavonoids and the structure-activity relationship.

Hepatic transcriptome analysis and identification of differentially expressed genes response to dietary oxidized fish oil in loach Misgurnus anguillicaudatus.

PubMed

Zhang, Yin; Li, Yang; Liang, Xiao; Cao, Xiaojuan; Huang, Longfei; Yan, Jie; Wei, Yanxing; Gao, Jian

2017-01-01

RNA sequencing and short-read assembly were utilized to produce a transcriptome of livers from loaches (Misgurnus anguillicaudatus) fed with three different diets respectively containing fresh fish oil (FO group), medium oxidized fish oil (MO group) and high oxidized fish oil (HO group). A total of 60,663 unigenes were obtained in this study, with mean length 848.74 bp. 50,814, 49,584 and 49,814 unigenes were respectively obtained from FO, MO and HO groups. There were 2,343 differentially expressed genes between FO and MO, with 855 down- and 1,488 up-regulated genes in the MO group. 2,813 genes were differentially expressed between FO and HO, including 1,256 down- and 1,552 up-regulated genes in the HO group. 2,075 differentially expressed genes were found in the comparison of MO and HO, including 1,074 up- and 1,001 down-regulated genes in the MO group. Some differentially expressed genes, such as fatty acid transport protein (fatp), fatty acid binding protein (fabp), apolipoprotein (apo), peroxisome proliferator activated receptor-gamma (ppar-γ), acetyl-CoA synthetase (acs) and arachidonate 5-lipoxygenase (alox5), were involved in lipid metabolism, suggesting these genes in the loach were responsive to dietary oxidized fish oil. Results of transcriptome profilings here were validated using quantitative real time PCR in fourteen randomly selected unigenes. The present study provides insights into hepatic transcriptome profile of the loach, which is a valuable resource for studies of loach genomics. More importantly, this study identifies some important genes responsible for dietary oxidized fish oil, which will benefit researches of lipid metabolism in fish.

INTERPLAY OF SORBITOL PATHWAY OF GLUCOSE METABOLISM, 12/15-LIPOXYGENASE, AND MITOGEN-ACTIVATED PROTEIN KINASES IN THE PATHOGENESIS OF DIABETIC PERIPHERAL NEUROPATHY

PubMed Central

Stavniichuk, Roman; Shevalye, Hanna; Hirooka, Hiroko; Nadler, Jerry L.; Obrosova, Irina G.

2012-01-01

The interactions among multiple pathogenetic mechanisms of diabetic peripheral neuropathy largely remain unexplored. Increased activity of aldose reductase, the first enzyme of the sorbitol pathway, leads to accumulation of cytosolic Ca++, essentially required for 12/15-lipoxygenase activation. The latter, in turn, causes oxidative-nitrosative stress, an important trigger of MAPK phosphorylation. This study therefore evaluated the interplay of aldose reductase, 12/15-lipoxygenase, and MAPKs in diabetic peripheral neuropathy. In experiment 1, male control and streptozotocin-diabetic mice were maintained with or without the aldose reductase inhibitor fidarestat, 16 mg kg−1 d−1, for 12 weeks. In experiment 2, male control and streptozotocin-diabetic wild-type (C57Bl6/J) and 12/15-lipoxygenase-deficient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofluorometric assays) as well as 12(S) hydroxyeicosatetraenoic concentration (ELISA), a measure of 12/15-lipoxygenase activity, in the sciatic nerve and spinal cord. 12/15-lipoxygenase expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fidarestat-treated diabetic mice. 12/15-lipoxygenase gene deficiency prevented diabetesassociated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly activated in diabetic wild-type and 12/15-lipoxygenase−/− mice. These findings identify the nature and tissue specificity of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its management. PMID:22285226

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid.

PubMed

Maynard, Daniel; Müller, Sara Mareike; Hahmeier, Monika; Löwe, Jana; Feussner, Ivo; Gröger, Harald; Viehhauser, Andrea; Dietz, Karl-Josef

2018-04-01

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ 2 and PGA 2 , cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta. Copyright © 2017 Elsevier Ltd. All rights reserved.

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

PubMed Central

Kerjaschki, Dontscho; Bago-Horvath, Zsuzsanna; Rudas, Margaretha; Sexl, Veronika; Schneckenleithner, Christine; Wolbank, Susanne; Bartel, Gregor; Krieger, Sigurd; Kalt, Romana; Hantusch, Brigitte; Keller, Thomas; Nagy-Bojarszky, Katalin; Huttary, Nicole; Raab, Ingrid; Lackner, Karin; Krautgasser, Katharina; Schachner, Helga; Kaserer, Klaus; Rezar, Sandra; Madlener, Sybille; Vonach, Caroline; Davidovits, Agnes; Nosaka, Hitonari; Hämmerle, Monika; Viola, Katharina; Dolznig, Helmut; Schreiber, Martin; Nader, Alexander; Mikulits, Wolfgang; Gnant, Michael; Hirakawa, Satoshi; Detmar, Michael; Alitalo, Kari; Nijman, Sebastian; Offner, Felix; Maier, Thorsten J.; Steinhilber, Dieter; Krupitza, Georg

2011-01-01

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas. PMID:21540548

ON THE RELATIONSHIPS OF SUBSTRATE ORIENTATION, HYDROGEN ABSTRACTION AND PRODUCT STEREOCHEMISTRY IN SINGLE AND DOUBLE DIOXYGENATIONS BY SOYBEAN LIPOXYGENASE-1 AND ITS ALA542GLY MUTANT*

PubMed Central

Coffa, Gianguido; Imber, Ann N.; Maguire, Brendan C.; Laxmikanthan, Gurunathan; Schneider, Claus; Gaffney, Betty J.; Brash, Alan R.

2005-01-01

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-3H- and 11S-3H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective proS hydrogen abstraction from C-11 of the substrate (97–99% pro-S selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoyl-phosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S,15S- and 8S,15S-dihydroperoxides, a new product was formed and identified by HPLC, UV, GC-MS and NMR as 9R , 1 5 S -dihydroperoxy-eicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration “partner” of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site. PMID:16157595

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

PubMed Central

Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

1995-01-01

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves. PMID:7549487

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

PubMed

Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

1995-08-01

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

5-lipoxygenase activation is involved in the mechanisms of chronic hepatic injury in a rat model of chronic aluminum overload exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mai, Shaoshan

We previously confirmed that rats overloaded with aluminum exhibited hepatic function damage and increased susceptibility to hepatic inflammation. However, the mechanism of liver toxicity by chronic aluminum overload is poorly understood. In this study, we investigated changes in the 5-lipoxygenase (5-LO) signaling pathway and its effect on liver injury in aluminum-overloaded rats. A rat hepatic injury model of chronic aluminum injury was established via the intragastric administration of aluminum gluconate (Al{sup 3+} 200 mg/kg per day, 5 days a week for 20 weeks). The 5-LO inhibitor, caffeic acid (10 and 30 mg/kg), was intragastrically administered 1 h after aluminum administration.more » Hematoxylin and eosin staining was used to visualize pathological changes in rat liver tissue. A series of biochemical indicators were measured with biochemistry assay or ELISAs. Immunochemistry and RT-PCR methods were used to detect 5-LO protein and mRNA expression in the liver, respectively. Caffeic acid administration protected livers against histopathological injury, decreased plasma ALT, AST, and ALP levels, decreased TNF-α, IL-6, IL-1β and LTs levels, increased the reactive oxygen species content, and down-regulated the mRNA and protein expressions of 5-LO in aluminum overloaded rats. Our results indicate that 5-lipoxygenase activation is mechanistically involved in chronic hepatic injury in a rat model of chronic aluminum overload exposure and that the 5-LO signaling pathway, which associated with inflammation and oxidative stress, is a potential therapeutic target for chronic non-infection liver diseases. - Highlights: • 5-LO signaling contributes to mechanisms of hepatotoxicity of aluminum overload. • Oxidative and inflammatory reaction involve in chonic aluminum hepatotoxicity. • 5-LO inhibitor has a protective effect on aluminum-overload liver injury. • 5-LO signaling is a potential therapeutic target for non-infection liver diseases.« less

In vitro 5-lipoxygenase and anti-oxidant activities of South African medicinal plants commonly used topically for skin diseases.

PubMed

Frum, Y; Viljoen, A M

2006-01-01

An investigation was undertaken to determine the possible mechanisms of action of medicinal plants used for dermatological pathologies. A total of 14 plant species were selected from the readily available ethnobotanical literature. 5-Lipoxygenase and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays were used to determine the anti-inflammatory activity and the anti-oxidant activity of selected medicinal plants, respectively. Both aqueous and methanol extracts were tested. Among the plants screened, four species (Croton sylvaticus, Warburgia salutaris, Pentanisia prunelloides, and Melianthus comosus) displayed promising 5-lipoxygenase inhibitory activity with IC(50) values <61 ppm. A large number of plants exhibited significant anti-oxidant activities with IC(50) values between 5.27 and 83.36 ppm. Aqueous extracts of M. comosus exhibited the most potent anti-inflammatory and anti-oxidant activity.

Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ibα – 14-3-3ζ] association

PubMed Central

van der Wal, Dianne E.; Gitz, Eelo; Du, Vivian X.; Lo, Kimberly S.L.; Koekman, Cornelis A.; Versteeg, Sabine; Akkerman, Jan Willem N.

2012-01-01

Background Cold storage of platelets reduces bacterial growth and preserves their hemostatic properties better than current procedures do. However, storage at 0°C induces [14-3-3ζ-glycoprotein Ibα] association, 14-3-3ζ release from phospho-Bad, Bad activation and apoptosis. Design and Methods We investigated whether arachidonic acid, which also binds 14-3-3ζ, contributes to coldinduced apoptosis. Results Cold storage activated P38-mitogen-activated protein kinase and released arachidonic acid, which accumulated due to cold inactivation of cyclooxygenase-1/thromboxane synthase. Accumulated arachidonic acid released 14-3-3ζ from phospho-Bad and decreased the mitochondrial membrane potential, which are steps in the induction of apoptosis. Addition of arachidonic acid did the same and its depletion made platelets resistant to cold-induced apoptosis. Incubation with biotin-arachidonic acid revealed formation of an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex. Indomethacin promoted complex formation by accumulating arachidonic acid and released 14-3-3ζ from cyclo-oxygenase-1. Arachidonic acid depletion prevented the cold-induced reduction of platelet survival in mice. Conclusions We conclude that cold storage induced apoptosis through an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex, which released 14-3-3ζ from Bad in an arachidonic acid-dependent manner. Although arachidonic acid depletion reduced agonist-induced thromboxane A2 formation and aggregation, arachidonic acid repletion restored these functions, opening ways to reduce apoptosis during storage without compromising hemostatic functions post-transfusion. PMID:22371179

Effect of gamma irradiation on lipoxygenases, trypsin inhibitor, raffinose family oligosaccharides and nutritional factors of different seed coat colored soybean (Glycine max L.)

NASA Astrophysics Data System (ADS)

Kumar Dixit, Amit; Kumar, Vineet; Rani, Anita; Manjaya, J. G.; Bhatnagar, Deepak

2011-04-01

Three soybean genotypes Kalitur, Hara soya and NRC37 with black, green and yellow seed coat color, respectively, were gamma irradiated at 0.5, 2.0 and 5.0 kGy and tested for antinutritional and nutritional factors. Gamma irradiation at all doses reduced the level of lipoxygenase isomers, trypsin inhibitor (TI) and ascorbic acid in all the 3 soybean genotypes as compared to the unirradiated control. However, irradiation dose of 5.0 kGy increased the sucrose content of the soybean genotypes. No significant change was observed in oil, protein fatty acids and total tocopherol content of the 3 genotypes at any irradiation dose. It is suggested that inhibition of lipoxygenase, reduction in TI and ascorbic acid may be due to the breakage or oxidation of protein structure by the gamma irradiation. Similarly, gamma irradiation at higher doses may break glycosidic linkages in oligosaccharides to produce more sucrose and decrease the content of flatulence causing oligosaccharides.

Cylindol A, a novel biphenyl ether with 5-lipoxygenase inhibitory activity, and a related compound from Imperata Cylindrica.

PubMed

Matsunaga, K; Ikeda, M; Shibuya, M; Ohizumi, Y

1994-09-01

Cylindol A [1] and B [2], two novel substances, have been isolated from Imperata cylindrica, and their structures have been elucidated on the basis of their spectral data coupled with chemical evidence and total synthesis. Cylindol A [1] showed 5-lipoxygenase inhibitory activity.

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Shansky, Janet; Karlisch, Patricia; Solerssi, Rosa Lopez

1991-01-01

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins E2 and F2(alpha) which regulate protein turnover rates and muscle cell growth. Mechnical stimulation significantly increases the breakdown rate of (3)H-arachidonic acid labelled phospholipids, releasing free (3)H-arachidonic acid, and the rate-limiting precursor of prostaglandin synthesis. Mechanical stimulation also significantly increases (3)H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-2-(3)H inositol labelled phospholipids. Phospholipase A2, phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are activated by stretch. The lipase inhibitors bromophenacylbromide and RHC80267 together reduce stretch-induced prostaglandin production by 73-83 percent. The stretch-induced increases in prostaglandin production, (3)H-arachidonic acid labelled phospholipid breakdown, and (3)H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-2-(3)H inositol labelled phospholipids are dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and prostaglandins through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

Mechanical stimulation of skeletal muscle generates lipid-related second messengers by phospholipase activation

NASA Technical Reports Server (NTRS)

Vandenburgh, H. H.; Shansky, J.; Karlisch, P.; Solerssi, R. L.

1993-01-01

Repetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E2 and F2 alpha which regulate protein turnover rates and muscle cell growth. These stretch-induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of 3H-arachidonic acid labelled phospholipids, releasing free 3H-arachidonic acid, the rate-limiting precursor of PG synthesis. Mechanical stimulation also increases 3H-arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo-[2-3H]inositol labelled phospholipids. Phospholipase A2 (PLA2), phosphatidylinositol-specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch-induced increases in PG production, 3H-arachidonic acid labelled phospholipid breakdown, and 3H-arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitive) whereas the formation of inositol phosphates from myo-[2-3H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid-related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA2 and PLD, but not by activation of phosphatidylinositol-specific PLC.

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition[S

PubMed Central

Newie, Julia; Andreou, Alexandra; Neumann, Piotr; Einsle, Oliver; Feussner, Ivo; Ficner, Ralf

2016-01-01

In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The first step in their biosynthesis may be catalyzed by a lipoxygenase (LOX), which forms hydroperoxides by introducing dioxygen into PUFAs. Here we characterized CspLOX1, a phylogenetically distant LOX family member from Cyanothece sp. PCC 8801 and determined its crystal structure. In addition to the classical two domains found in plant, animal, and coral LOXs, we identified an N-terminal helical extension, reminiscent of the long α-helical insertion in Pseudomonas aeruginosa LOX. In liposome flotation studies, this helical extension, rather than the β-barrel domain, was crucial for a membrane binding function. Additionally, CspLOX1 could oxygenate 1,2-diarachidonyl-sn-glycero-3-phosphocholine, suggesting that the enzyme may act directly on membranes and that fatty acids bind to the active site in a tail-first orientation. This binding mode is further supported by the fact that CspLOX1 catalyzed oxygenation at the n-10 position of both linoleic and arachidonic acid, resulting in 9R- and 11R-hydroperoxides, respectively. Together these results reveal unifying structural features of LOXs and their function. While the core of the active site is important for lipoxygenation and thus highly conserved, peripheral domains functioning in membrane and substrate binding are more variable. PMID:26667668

Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Didolkar, A.K.; Sundaram, K.

1987-07-27

Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to insositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA) could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA augmented hCG induced T secretion, while at higher concentrations they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5,8,11,14 Eicosatetrayenoic acid, a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid, an inhibitor ofmore » the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. The authors conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclo-oxygenase products. 28 references, 4 figures, 2 tables.« less

A conserved π-cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability.

PubMed

Eek, Priit; Piht, Mari-Ann; Rätsep, Margus; Freiberg, Arvi; Järving, Ivar; Samel, Nigulas

2015-10-01

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that consist of a regulatory calcium- and membrane-binding PLAT (polycystin-1, lipoxygenase, α-toxin) domain and a catalytic domain. In a previous study, the crystal structure of an 11R-LOX revealed a conserved π-cation bridge connecting these two domains which could mediate the regulatory effect of the PLAT domain to the active site. Here we analyzed the role of residues Trp107 and Lys172 that constitute the π-cation bridge in 11R-LOX along with Arg106 and Asp173-a potential salt bridge, which could also contribute to the inter-domain communication. According to our kinetic assays and protein unfolding experiments conducted using differential scanning fluorimetry and circular dichroism spectroscopy, mutants with a disrupted link display diminished catalytic activity alongside reduced stability of the protein fold. The results demonstrate that both these bridges contribute to the two-domain interface, and are important for proper enzyme activation. Copyright © 2015 Elsevier B.V. All rights reserved.

Lipid Osteoclastokines Regulate Breast Cancer Bone Metastasis

PubMed Central

Krzeszinski, Jing Y.; Schwaid, Adam G.; Cheng, Wing Yin; Jin, Zixue; Gallegos, Zachary R.; Saghatelian, Alan

2017-01-01

Bone metastasis is a deadly consequence of cancers, in which osteoclast forms a vicious cycle with tumor cells. Bone metastasis attenuation by clinical usage of osteoclast inhibitors and in our osteopetrotic mouse genetic models with β-catenin constitutive activation or peroxisome proliferator-activated receptor γ deficiency fully support the important role of osteoclast in driving the bone metastatic niche. However, the mechanisms for this “partnership in crime” are underexplored. Here we show that osteoclasts reprogram their lipid secretion to support cancer cells. Metabolomic profiling reveals elevated prometastatic arachidonic acid (AA) but reduced antimetastatic lysophosphatidylcholines (LPCs). This shift in lipid osteoclastokines synergistically stimulates tumor cell proliferation, migration, survival, and expression of prometastatic genes. Pharmacologically, combined treatment with LPCs and BW-755C, an inhibitor of AA signaling via blocking lipoxygenase and cyclooxygenase, impedes breast cancer bone metastasis. Our findings elucidate key paracrine mechanisms for the osteoclast-cancer vicious cycle and uncover important therapeutic targets for bone metastasis. PMID:27967239

Effect of arachidonic acid metabolites on CR1 expression by B-lymphocytes.

PubMed

Cook, J M; Guibert, F; Delebassee, S; Gualde, N

1989-01-01

The effect of arachidonic acid metabolites on the expression of the receptor for the C3b/C4b fragment of complement (CR1) by human B-lymphocytes was investigated. Kinetic experiments to determine CR1 expression over time indicated that the maximal receptor number occurred at 2 h, followed by a return to baseline values. Addition of 10(-4) M puromycin to the cells suggested that the increase was due to the expression of an intracellular pool and not de novo synthesis of new receptor molecules. B-lymphocytes were incubated with arachidonic acid, 15-hydroxyeicosatetraenoic acid, leukotrienes B4 or C4 or prostaglandin E2 (PGE2). The quantity of membrane antigenic binding sites was determined before and after incubation. The lipoxygenase metabolites did not alter CR1 numbers. In contrast, PGE2 significantly decreased (P less than 0.05) the quantity of CR1 expressed. In kinetic experiments, PGE2 blocked the maximal expression of CR1 seen at 2 h, indicating that it prevents the appearance of an intracellular pool of receptor. These results show that CR1 number on B-lymphocytes can be altered by at least one arachidonic acid metabolite. This may offer a partial explanation for the inhibitory effects of PGE2 on B-cell proliferation and immunoglobulin secretion since CR1 is implicated in B-lymphocyte differentiation and specific antibody response.

Mechanisms of hypoxia-induced cerebrovascular dilation in the newborn pig.

PubMed

Leffler, C W; Smith, J S; Edrington, J L; Zuckerman, S L; Parfenova, H

1997-03-01

The hypothesis that endothelium-dependent components contribute to the cerebromicrovascular dilation to hypoxia in the newborn pig was addressed. Piglets anesthetized with ketamine-acepromazine and maintained on alpha-chloralose were equipped with closed cranial windows. Injury to the endothelium of pial arterioles was produced by light activation of fluorescein dye. Light/dye injury reduced the pial arteriolar dilation to hypoxia (5 min, arterial PO2 approximately 30 mmHg) from 57 +/- 9 to 19 +/- 5%. Light/dye injury abolished the pial arteriolar dilation to hypercapnia but did not affect dilation to sodium nitroprusside. The pial arteriolar dilation to hypoxia was not affected by tetrodotoxin, N(omega)-nitro-L-arginine, glibenclamide, iberiotoxin, charybdotoxin, tetraethylammonium, or 8-phenyltheophylline. Hypoxia caused increases in the cerebral cortical production of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. Cerebral vasodilation to hypoxia was inhibited by 5,8,11,14-eicosatetraynoic acid but was not greatly affected by cyclooxygenase or lipoxygenase inhibitors. In contrast, the cytochrome P-450 epoxygenase inhibitor miconazol decreased cerebral vasodilation to hypoxia from 45 +/- 5 to 17 +/- 4%. Therefore, the vascular endothelium appears to participate in cerebral microvascular dilation to hypoxia in newborn pigs. The mechanism may include cytochrome P-450 epoxygenase metabolites of arachidonic acid.

Cancer cells cause vascular endothelial cell (vEC) retraction via 12(S)HETE secretion; the possible role of cancer cell derived microparticle.

PubMed

Uchide, Keiji; Sakon, Masato; Ariyoshi, Hideo; Nakamori, Syouji; Tokunaga, Masaru; Monden, Morito

2007-02-01

Cancer cell mediated vascular endothelial cell (vEC) retraction plays a pivotal role in cancer metastasis. The aim of this study is to clarify the biochemical character of vEC retraction factor derived from human breast cancer cell line, MCF-7. In order to estimate vEC retracting activity, transwell chamber assay system was employed. We first tested the effects of trypsin digestion as well as lipid extraction of culture medium (CM). Trypsin digestion of CM resulted in approximately 40% loss of vEC retracting activity and lipid extraction of CM by Brigh and Dyer methods recovered approximately 60% of vEC retracting activity, suggesting that approximately 60% of vEC retracting activity in MCF-7 derived CM is due to lipid. Although Nordihydroguaiaretic acid (NDGA), the specific lipoxygenase inhibitor, suppressed vEC retracting activity in CM, Acetyl salicylic acid (ASA), a specific cyclooxygenase inhibitor, did not affect the activity, suggesting that lipid exerting vEC retracting activity in CM belongs to lipoxygenase mediated arachidonate metabolites. Thin layer chromatography clearly demonstrated that Rf value of lipid vEC retracting factor in CM is identical to 12HETE. Authentic 12(S)HETE, but not 12(R)HETE, showed vEC retracting activity. After the ultracentrifugation of CM, most lipid vEC retracting activity was recovered from the pellet fraction, and flow cytometric analysis using specific antibody against 12(S)HETE clearly showed the association of 12(S)HETE with small particle in CM. These findings suggested the principal involvement of 12(S)HETE in cancer cell derived microparticles in cancer cell mediated vEC retraction.

Myeloid Cell 5-Lipoxygenase Activating Protein Modulates the Response to Vascular Injury

PubMed Central

Yu, Zhou; Ricciotti, Emanuela; Miwa, Takashi; Liu, Shulin; Ihida-Stansbury, Kaori; Landersberg, Gavin; Jones, Peter L.; Scalia, Rosario; Song, Wenchao; Assoian, Richard K.; FitzGerald, Garret A.

2013-01-01

Rationale Human genetics have implicated the 5- lipoxygenase (5-LO) enzyme in the pathogenesis of cardiovascular disease and an inhibitor of the 5-LO activating protein (FLAP) is in clinical development for asthma. Objective Here we determined whether FLAP deletion modifies the response to vascular injury. Methods and Results Vascular remodeling was characterized 4 weeks after femoral arterial injury in FLAP knockout (FLAP KO) mice and wild type (WT) controls. Both neointimal hyperplasia and the intima/media ratio of the injured artery were significantly reduced in the FLAP KOs while endothelial integrity was preserved. Lesional myeloid cells were depleted and vascular smooth muscle cell (VSMC) proliferation, as reflected by bromodeoxyuridine (BrdU) incorporation, was markedly attenuated by FLAP deletion. Inflammatory cytokine release from FLAP KO macrophages was depressed and their restricted ability to induce VSMC migration ex vivo was rescued with leukotriene B4 (LTB4). FLAP deletion restrained injury and attenuated upregulation of the extracellular matrix protein, tenascin C (TNC), which affords a scaffold for VSMC migration. Correspondingly, the phenotypic modulation of VSMC to a more synthetic phenotype, reflected by morphological change, loss of α-smooth muscle cell actin and upregulation of vascular cell adhesion molecule (VCAM) -1 was also suppressed in FLAP KO mice. Transplantation of FLAP replete myeloid cells rescued the proliferative response to vascular injury. Conclusion Expression of lesional FLAP in myeloid cells promotes LTB4 dependent VSMC phenotypic modulation, intimal migration and proliferation. PMID:23250985

Outstanding Anti-inflammatory Potential of Selected Asteraceae Species through the Potent Dual Inhibition of Cyclooxygenase-1 and 5-Lipoxygenase.

PubMed

Chagas-Paula, Daniela Aparecida; Oliveira, Tiago Branquinho; Faleiro, Danniela Príscylla Vasconcelos; Oliveira, Rejane Barbosa; Costa, Fernando Batista Da

2015-09-01

Cyclooxygenase and 5-lipoxygenase are enzymes that catalyze important inflammatory pathways, suggesting that dual cyclooxygenase/lipoxygenase inhibitors should be more efficacious as anti-inflammatory medicines with lower side effects than the currently available nonsteroidal anti-inflammatory drugs. Many plants from the family Asteraceae have anti-inflammatory activities, which could be exerted by inhibiting the cyclooxygenase-1 and 5-lipoxygenase enzymes. Nevertheless, only a small number of compounds from this family have been directly evaluated for their ability to inhibit the enzymes in cell-free assays. Therefore, this study systematically evaluated 57 Asteraceae extracts in vitro in enzyme activity experiments to determine whether any of these extracts exhibit dual inhibition of cyclooxygenase-1 and 5-lipoxygenase. The chemical profiles of the extracts were obtained by the high-performance liquid chromatography-ultraviolet-diode array detector method, and their major constituents were dereplicated. Of the 57 tested extracts, 13 (26.6 %, IC50 range from 0.03-36.2 µg/mL) of them displayed dual inhibition. Extracts from known anti-inflammatory herbs, food plants, and previously uninvestigated species are among the most active. Additionally, the extract action was found to be specific with IC50 values close to or below those of the standard inhibitors. Thus, the active extracts and active substances of these species are potent inhibitors acting through the mechanism of dual inhibition of cyclooxygenase-1 and 5-lipoxygenase. The extracts were prepared for this study using nontoxic extraction solvents (EtOH-H2O), requiring only a small amount of plant material to carry out the bioassays and the phytochemical analyses. In summary, this study demonstrated the potential of the investigated species as dual inhibitors, revealing their potential as pharmaceuticals or nutraceuticals. Georg Thieme Verlag KG Stuttgart · New York.

Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

PubMed

Perales, J; Amorim, C Z; Rocha, S L; Domont, G B; Moussatché, H

1992-11-01

The pharmacological modulation of mice paw oedema produced by Bothrops jararaca venom (BJV) has been studied. Intraplantar injection of BJV (1-30 micrograms/paw) produced a dose- and time-related oedema, which was maximal 30 min after injection, reduced gradually thereafter and disappeared over 48 h. BJV heated at 100 degrees C for 5 or 15 min blocked local hemorrhage and caused partial inhibition of its oedematogenic activity. The BJV oedema was not inhibited by the anti-histamine meclizine, the inhibitor of histamine and serotonin, cyproheptadine, PAF-acether antagonist WEB 2170 or by the anti-leukotrienes C4/D4, LY 171883. Dexamethasone, aspirin, indomethacin, and the dual cyclooxygenase and lipoxygenase inhibitor BW 755C inhibited BJV-induced oedema indicating that arachidonic acid metabolism products via the cyclooxygenase pathway participate in its genesis and/or maintenance. The antibothropic fraction (ABF) (25-200 micrograms/paw) isolated from Didelphis marsupialis serum neutralized the oedema induced by the venom with and without heating, the hemorrhage induced by BJV and partially blocked the oedema induced by bradykinin and by cellulose sulphate. The oedema produced by histamine, serotonin, PAF-acether or leukotriene C4 was not inhibited.

Arachidonic acid, but not its metabolites, is essential for FcγR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

PubMed Central

Zheng, L; Zomerdijk, T P L; Van Den Barselaar, M T; Geertsma, M F; Van Furth, R; Nibbering, P H

1999-01-01

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcγ receptor (FcγR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcγR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcγR-mediated killing process. The production of O−2 by monocytes upon FcγR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcγR cross-linking. Furthermore, FcγR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcγR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcγR-stimulated intracellular killing of S. aureus by monocytes. PMID:10233682

A high-fat diet induces bone loss in mice lacking the Alox5 gene.

PubMed

Le, Phuong; Kawai, Masanobu; Bornstein, Sheila; DeMambro, Victoria E; Horowitz, Mark C; Rosen, Clifford J

2012-01-01

5-Lipoxygenase catalyzes leukotriene generation from arachidonic acid. The gene that encodes 5-lipoxygenase, Alox5, has been identified in genome-wide association and mouse Quantitative Trait Locus studies as a candidate gene for obesity and low bone mass. Thus, we tested the hypothesis that Alox5(-/-) mice would exhibit metabolic and skeletal changes when challenged by a high-fat diet (HFD). On a regular diet, Alox5(-/-) mice did not differ in total body weight, percent fat mass, or bone mineral density compared with wild-type (WT) controls (P < 0.05). However, when placed on a HFD, Alox5(-/-) gained more fat mass and lost greater areal bone mass vs. WT (P < 0.05). Microarchitectural analyses revealed that on a HFD, WT showed increases in cortical area (P < 0.01) and trabecular thickness (P < 0.01), whereas Alox5(-/-) showed no change in cortical parameters but a decrease in trabecular number (P < 0.05) and bone volume fraction compared with WT controls (P < 0.05). By histomorphometry, a HFD did not change bone formation rates of either strain but produced an increase in osteoclast number per bone perimeter in Alox5(-/-) mice (P < 0.03). In vitro, osteoclastogenesis of marrow stromal cells was enhanced in mutant but not WT mice fed a HFD. Gene expression for Rankl, Pparg, and Cox-2 was greater in the femur of Alox5(-/-) than WT mice on a HFD (P < 0.01), but these increases were suppressed in the Alox5(-/-) mice after 8 wk of treatment with celecoxib, a cyclooxygenase-2 inhibitor. In sum, there is a strong gene by environmental interaction for bone mass when mice lacking the Alox5 gene are fed a HFD.

Lipophilic flavonoids from Orthosiphon spicatus prevent oxidative inactivation of 15-lipoxygenase.

PubMed

Lyckander, I M; Malterud, K E

1996-04-01

15-Lipoxygenase from soybeans is inactivated by bubbling air through enzyme solutions. This inactivation is prevented by flavonoids from the East Asian medicinal plant Orthosiphon spicatus, previously found to be inhibitors of the enzyme. 5,7,4'-Trimethylapigenin, eupatorin and 5,7,3',4'-tetramethylluteolin show the strongest enzyme-stabilizing effects, decreasing loss of activity by 50% at concentrations of 2.0 +/- 0.04, 2.4 +/- 0.3 and 4.3 +/- 1.1 microM, respectively. There is no significant correlation between enzyme-inhibiting and enzyme-stabilizing effect. The Orthosiphon flavonoids show radical-scavenging activity towards the diphenylpicrylhydrazyl radical. This is correlated to their enzyme-stabilizing effect, but not to their inhibitory activity towards 15-lipoxygenase. When the enzyme is inactivated by air bubbling, a loss of sulfhydryl groups is observed. Sinensetin, a poor stabilizer of the enzyme, shows less efficiency in protecting sulfhydryl groups than tetramethylscutellarein, which stabilizes the enzyme more efficiently. Thus, oxidation of sulfhydryl groups may contribute to the observed air-induced inactivation of 15-lipoxygenase.

Effect of pepper lipoxygenase activity and its linked reactions on pigments of the pepper fruit.

PubMed

Jarén-Galán, M; Mínguez-Mosquera, M I

1999-11-01

The products formed during the enzymatic reaction catalyzed by the lipoxygenase of pepper (variety Agridulce) have in vitro a strong destructive action on the carotenoid pigments of the fruit. When conditions and proportions of enzyme and pigments are similar to those found in the fruit, and at a reaction temperature of 20 degrees C, almost 30% of the pigments are destroyed after 24 h of reaction. Of this amount, 2.5% is due to autoxidation of pigments, 4. 5% to oxidation induced by the presence of linoleic under saturating conditions, and the remaining 22% to the presence in the medium of reaction products of the lipoxygenase-catalyzed reaction. When the enzyme acts under substrate-saturating conditions, the rate of pigment destruction by lipoxygenase can be considered maximal at the experimental temperature. The fact that in vitro pepper lipoxygenase induces a heavy destruction of pigments and that, in vivo, its activity remains almost constant during over-ripening could explain why up to 40% of the pigment content in some varieties is lost during the postharvest period.

Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor.

PubMed

Maddipati, Krishna Rao; Romero, Roberto; Chaiworapongsa, Tinnakorn; Zhou, Sen-Lin; Xu, Zhonghui; Tarca, Adi L; Kusanovic, Juan Pedro; Munoz, Hernan; Honn, Kenneth V

2014-11-01

Lipid mediators play an important role in reproductive biology, especially, in parturition. Enhanced biosynthesis of eicosanoids, such as prostaglandin E2 (PGE2) and PGF2α, precedes the onset of labor as a result of increased expression of inducible cyclooxygenase 2 (COX-2) in placental tissues. Metabolism of arachidonic acid results in bioactive lipid mediators beyond prostaglandins that could significantly influence myometrial activity. Therefore, an unbiased lipidomic approach was used to profile the arachidonic acid metabolome of amniotic fluid. In this study, liquid chromatography-mass spectrometry was used for the first time to quantitate these metabolites in human amniotic fluid by comparing patients at midtrimester, at term but not in labor, and at term and in spontaneous labor. In addition to exposing novel aspects of COX pathway metabolism, this lipidomic study revealed a dramatic increase in epoxygenase- and lipoxygenase-pathway-derived lipid mediators in spontaneous labor with remarkable product selectivity. Despite their recognition as anti-inflammatory lipid mediators and regulators of ion channels, little is known about the epoxygenase pathway in labor. Epoxygenase pathway metabolites are established regulators of vascular homeostasis in cardiovascular and renal physiology. Their presence as the dominant lipid mediators in spontaneous labor at term portends a yet undiscovered physiological function in parturition. © FASEB.

Altered macrophage arachidonic acid metabolism induced by endotoxin tolerance: characterization and mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogers, T.S.

Altered macrophage arachidonic acid (AA) metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase and cyclooxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e., endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT) C/sub 4/D/sub 4/ and prostaglandin (PG) E/sub 2/ production by tolerant cells was greater than that by non-tolerant controls (p <0.001). However, A23187-stimulated i6-keto PGF/sub 1a/more » levels were lower in tolerant macrophages compared to controls (P < 0.05). iL TC/sub 4/D/sub 4/ production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in non-tolerant cells. Synthesis of iLTB/sub 4/ by control macrophages was stimulated by endotoxin (p <0.01). The effect of tolerance on factors that affect AA release was investigated by measuring /sup 14/C-AA incorporation and release and phospholipase A/sub 2/ activity« less

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed

Husain, S; Abdel-Latif, A A

1999-08-15

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle.

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed Central

Husain, S; Abdel-Latif, A A

1999-01-01

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle. PMID:10432304

Antiinflammatory and lipoxygenase inhibitory compounds from Vitex agnus-castus.

PubMed

Choudhary, M Iqbal; Jalil, Saima; Nawaz, Sarfraz Ahmad; Khan, Khalid Mohammed; Tareen, Rasool Bakhsh

2009-09-01

Several secondary metabolites, artemetin (1), casticin (2), 3,3'-dihydroxy-5,6,7,4'-tetramethoxy flavon (3), penduletin (4), methyl 4-hydroxybenzoate (5), p-hydroxybenzoic acid (6), methyl 3,4-dihydroxybenzoate (7), 5-hydroxy-2-methoxybenzoic acid (8), vanillic acid (9) and 3,4-dihydroxybenzoic acid (10) were isolated from a folkloric medicinal plant, Vitex agnus-castus. The structures of compounds 1-10 were identified with the help of spectroscopic techniques. Compounds 3-10 were isolated for the first time from this plant. These compounds were screened for their antiinflammatory and lipoxygenase inhibitory activities. Compounds 6, 7 and 10 were found to have significant antiinflammatory activity in a cell-based contemporary assay, whereas compounds 1 and 2 exhibited a potent lipoxygenase inhibition.

A non-capacitative pathway activated by arachidonic acid is the major Ca2+ entry mechanism in rat A7r5 smooth muscle cells stimulated with low concentrations of vasopressin

PubMed Central

Broad, Lisa M; Cannon, Toby R; Taylor, Colin W

1999-01-01

Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 μM Gd3+, but not by 1 μM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase. PMID:10226154

Antioxidant and Anti-Inflammatory Activities of Hydrolysates and Peptide Fractions Obtained by Enzymatic Hydrolysis of Selected Heat-Treated Edible Insects.

PubMed

Zielińska, Ewelina; Baraniak, Barbara; Karaś, Monika

2017-09-02

This study investigated the effect of heat treatment of edible insects on antioxidant and anti-inflammatory activities of peptides obtained by in vitro gastrointestinal digestion and absorption process thereof. The antioxidant potential of edible insect hydrolysates was determined as free radical-scavenging activity, ion chelating activity, and reducing power, whereas the anti-inflammatory activity was expressed as lipoxygenase and cyclooxygenase-2 inhibitory activity. The highest antiradical activity against DPPH • (2,2-diphenyl-1-picrylhydrazyl radical) was noted for a peptide fraction from baked cricket Gryllodes sigillatus hydrolysate (IC 50 value 10.9 µg/mL) and that against ABTS •+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical) was the highest for raw mealworm Tenebrio molitor hydrolysate (inhibitory concentration (IC 50 value) 5.3 µg/mL). The peptides obtained from boiled locust Schistocerca gregaria hydrolysate showed the highest Fe 2+ chelation ability (IC 50 value 2.57 µg/mL); furthermore, the highest reducing power was observed for raw G. sigillatus hydrolysate (0.771). The peptide fraction from a protein preparation from the locust S. gregaria exhibited the most significant lipoxygenase and cyclooxygenase-2 inhibitory activity (IC 50 value 3.13 µg/mL and 5.05 µg/mL, respectively).

Antioxidant and Anti-Inflammatory Activities of Hydrolysates and Peptide Fractions Obtained by Enzymatic Hydrolysis of Selected Heat-Treated Edible Insects

PubMed Central

Zielińska, Ewelina; Baraniak, Barbara; Karaś, Monika

2017-01-01

This study investigated the effect of heat treatment of edible insects on antioxidant and anti-inflammatory activities of peptides obtained by in vitro gastrointestinal digestion and absorption process thereof. The antioxidant potential of edible insect hydrolysates was determined as free radical-scavenging activity, ion chelating activity, and reducing power, whereas the anti-inflammatory activity was expressed as lipoxygenase and cyclooxygenase-2 inhibitory activity. The highest antiradical activity against DPPH• (2,2-diphenyl-1-picrylhydrazyl radical) was noted for a peptide fraction from baked cricket Gryllodes sigillatus hydrolysate (IC50 value 10.9 µg/mL) and that against ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical) was the highest for raw mealworm Tenebrio molitor hydrolysate (inhibitory concentration (IC50 value) 5.3 µg/mL). The peptides obtained from boiled locust Schistocerca gregaria hydrolysate showed the highest Fe2+ chelation ability (IC50 value 2.57 µg/mL); furthermore, the highest reducing power was observed for raw G. sigillatus hydrolysate (0.771). The peptide fraction from a protein preparation from the locust S. gregaria exhibited the most significant lipoxygenase and cyclooxygenase-2 inhibitory activity (IC50 value 3.13 µg/mL and 5.05 µg/mL, respectively). PMID:28869499

Lipoxygenase activity in different species of sweet lupin (Lupinus L.) seeds and flakes.

PubMed

Stephany, Michael; Bader-Mittermaier, Stephanie; Schweiggert-Weisz, Ute; Carle, Reinhold

2015-05-01

Lipoxygenase (LOX)-catalysed degradation of polyunsaturated fatty acids is supposed to be a major cause of undesirable off-flavour development in legumes. In the present study, a photometric LOX assay including adequate sample workup was adapted to lupin seeds, kernels and flakes, respectively. Optimum reaction conditions were at pH 7.5 using a phosphate buffer concentration of 150 mmol l(-1) without the addition of sodium chloride. The LOX activities of different lupin species and varieties were compared. Significant variations among the species and varieties ranging from 50 to 1004 units mg(-1) protein were determined, being significantly lower than soybean LOX activity. Hulling and flaking of the seeds resulted in a 15% increase of LOX activity. In contrast to soy and other legumes, LOX from lupin only converted free fatty acids, whereas trilinolein and β-carotene were not oxidised. Consequently, according to the established classification, lupin LOX activity may be assigned to the LOX type-1, which, to the best of our knowledge, was demonstrated for the first time. Copyright © 2014 Elsevier Ltd. All rights reserved.

Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo.

PubMed

Kundu, Suman; Roome, Talat; Bhattacharjee, Ashish; Carnevale, Kevin A; Yakubenko, Valentin P; Zhang, Renliang; Hwang, Sung Hee; Hammock, Bruce D; Cathcart, Martha K

2013-02-01

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A(2) (cPLA(2)). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA(2)-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA(2) activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.

PYRIDOXAMINE ANALOGS SCAVENGE LIPID-DERIVED γ-KETOALDEHYDES AND PROTECT AGAINST H2O2-MEDIATED CYTOTOXICITY†

PubMed Central

Davies, Sean S.; Brantley, Eric J.; Voziyan, Paul A.; Amarnath, Venkataraman; Zagol-Ikapitte, Irene; Boutaud, Olivier; Hudson, Billy G.; Oates, John A.; Jackson Roberts, L.

2008-01-01

Isoketals and levuglandins are highly reactive γ-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge α-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive γ-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic γ-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogs of pyridoxamine, salicylamine and 5’O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogs, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by α-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogs appear to preferentially scavenge γ-ketoaldehydes. Both pyridoxamine and its lipophilic analogs inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogs provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogs to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions. PMID:17176098

5-Lipoxygenase Inhibition of the Fructus of Foeniculum vulgare and Its Constituents

PubMed Central

Lee, Je Hyeong; Lee, Dong Ung; Kim, Yeong Shik; Kim, Hyun Pyo

2012-01-01

The fruits of Foeniculum vulgare (Foeniculi Fructus) have been widely used in Chinese medicine as an antiemetic, ameliorating stomach ailments and as an analgesic. In order to establish its potential for antiallergic use, inhibitory actions of the fruiton 5-lipoxgenase (5-LOX) and β-hexosaminidase release were evaluated. The 70% ethanol extract of this plant material (FR) considerably inhibited 5-LOX-catalyzed leukotriene production from A23187-induced rat basophilic leukemia (RBL)-1 cells. The IC50 was 3.2 μg/ml. From this extract, 12 major compounds including sabinene, fenchone, γ-terpinene, α-pinene, limonene, p-anisylacetone, p-anisylaldehyde, estragole (4-allylanisole), trans-anethole, scopoletin, bergapten and umbelliferone were isolated. And it was found that several terpene derivatives including γ-terpinene and fenchone as well as phenylpropanoid, trans-anethole, showed considerable inhibitory action of 5-LOX. In particular, the IC50 of trans-anethole was 51.6 μ M. In contrast, FR and the isolated compounds did not show considerable inhibitory activity on the degranulation reaction of β-hexosaminidase release from antigen-treated RBL-2H3 cells. Against arachidonic acid-induced ear edema in mice, FR and trans-anethole showed significant inhibition by oral administration at doses of 100-400 mg/kg. In conclusion, FR and several major constituents are 5-LOX inhibitors and they may have potential for treating 5-LOX-related disorders. PMID:24116283

Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis.

PubMed

Creelman, R A; Bell, E; Mullet, J E

1992-07-01

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9'-cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.

HPLC based activity profiling for 5-lipoxygenase inhibitory activity in Isatis tinctoria leaf extracts.

PubMed

Oberthür, C; Jäggi, R; Hamburger, M

2005-06-01

In the pursuit of the anti-inflammatory constituents in lipophilic woad extracts, the 5-lipoxygenase (5-LOX) inhibitory activity was investigated by HPLC-based activity profiling. In a low-resolution profiling, two time windows with peaks of activity were found. The first coincided with tryptanthrin, a known dual inhibitor of cyclooxygenase-2 (COX-2) and 5-LOX, whereas the major inhibitory fraction was towards the end of the HPLC run. The active fractions were profiled in a peak-resolved manner, and the compounds analyzed by LC-MS, GC and TLC. The activity in the lipophilic fractions of the Isatis extract could be linked to an unsaturated fatty acid, alpha-linolenic acid.

Preferential Generation of 15-HETE-PE Induced by IL-13 Regulates Goblet Cell Differentiation in Human Airway Epithelial Cells.

PubMed

Zhao, Jinming; Minami, Yoshinori; Etling, Emily; Coleman, John M; Lauder, Sarah N; Tyrrell, Victoria; Aldrovandi, Maceler; O'Donnell, Valerie; Claesson, Hans-Erik; Kagan, Valerian; Wenzel, Sally

2017-12-01

Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 μM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

Prediabetic nephropathy as an early consequence of the high-calorie/high-fat diet: relation to oxidative stress.

PubMed

Shevalye, Hanna; Lupachyk, Sergey; Watcho, Pierre; Stavniichuk, Roman; Khazim, Khaled; Abboud, Hanna E; Obrosova, Irina G

2012-03-01

This study evaluated early renal functional, structural, and biochemical changes in high-calorie/high-fat diet fed mice, a model of prediabetes and alimentary obesity. Male C57BL6/J mice were fed normal (11 kcal% fat) or high-fat (58 kcal% fat) diets for 16 wk. Renal changes were evaluated by histochemistry and immunohistochemistry, Western blot analysis, ELISA, enzymatic assays, and chemiluminometry. High-fat diet consumption led to increased body and kidney weights, impaired glucose tolerance, hyperinsulinemia, polyuria, a 2.7-fold increase in 24-h urinary albumin excretion, 20% increase in renal glomerular volume, 18% increase in renal collagen deposition, and 8% drop of glomerular podocytes. It also resulted in a 5.3-fold increase in urinary 8-isoprostane excretion and a 38% increase in renal cortex 4-hydroxynonenal adduct accumulation. 4-hydroxynonenal adduct level and immunoreactivity or Sirtuin 1 expression in renal medulla were not affected. Studies of potential mechanisms of the high-fat diet induced renal cortex oxidative injury revealed that whereas nicotinamide adenine dinucleotide phosphate reduced form oxidase activity only tended to increase, 12/15-lipoxygenase was significantly up-regulated, with approximately 12% increase in the enzyme protein expression and approximately 2-fold accumulation of 12(S)-hydroxyeicosatetraenoic acid, a marker of 12/15-lipoxygenase activity. Accumulation of periodic acid-Schiff -positive material, concentrations of TGF-β, sorbitol pathway intermediates, and expression of nephrin, CAAT/enhancer-binding protein homologous protein, phosphoeukaryotic initiation factor-α, and total eukaryotic initiation factor-α in the renal cortex were indistinguishable between experimental groups. Vascular endothelial growth factor concentrations were reduced in high-fat diet fed mice. In conclusion, systemic and renal cortex oxidative stress associated with 12/15-lipoxygenase overexpression and activation is an early phenomenon caused by high-calorie/high-fat diet consumption and a likely contributor to kidney disease associated with prediabetes and alimentary obesity.

Inhibitors of 15-lipoxygenase from orange peel.

PubMed

Malterud, K E; Rydland, K M

2000-11-01

A series of polymethoxylated flavonoids has been isolated from orange peel, and their inhibitory activity toward soybean 15-lipoxygenase was determined. The strongest inhibition was shown by 3,5,6,7,3',4'-hexamethoxyflavone (IC(50) = 49 +/- 5 microM). Sinensetin, nobiletin, tangeretin, tetramethylscutellarein, and 3,5, 6,7,8,3',4'-heptamethoxyflavone were somewhat less active, with IC(50) values of 70-86 microM, comparable to the positive control quercetin (IC(50) = 68 +/- 5 microM). Demethylation apparently results in less active compounds, with 5-O-demethylsinensetin having an IC(50) value of 144 +/- 10 microM. Some other orange peel constituents were isolated and tested as well, hesperidin (IC(50) = 180 +/- 10 microM) and ferulic acid (111 +/- 2 microM), showing moderate activity. The polymethoxylated flavonoids were virtually inactive as scavengers of the diphenylpicrylhydrazyl radical. Hesperidin was only slightly active (24.2 +/- 0.7% scavenged at a concentration of 2 mM), and ferulic acid showed good activity (IC(50) = 86.4 +/- 0.7 microM). From this, it appears that orange peel constituents may counteract enzymatic lipid peroxidation processes catalyzed by 15-lipoxygenase in vitro. The radical scavenging activity of orange peel extracts is only modest.

Synthesis and biological evaluation of novel 5-hydroxylaminoisoxazole derivatives as lipoxygenase inhibitors and metabolism enhancing agents.

PubMed

Averina, Elena B; Vasilenko, Dmitry A; Gracheva, Yulia A; Grishin, Yuri K; Radchenko, Eugene V; Burmistrov, Vladimir V; Butov, Gennady M; Neganova, Margarita E; Serkova, Tatyana P; Redkozubova, Olga M; Shevtsova, Elena F; Milaeva, Elena R; Kuznetsova, Tamara S; Zefirov, Nikolay S

2016-02-15

A versatile synthesis of novel 5-hydroxylaminoisoxazoles bearing adamantane moieties has been accomplished using the heterocyclization reactions of readily available unsaturated esters by the treatment with tetranitromethane in the presence of triethylamine and subsequent reduction of resulting 5-nitroisoxazoles by SnCl2 with the participation of THF. A number of obtained isoxazole derivatives were evaluated for their antioxidative activity, inhibition of lipoxygenases and impact on the rat liver mitochondria. The majority of tested compounds demonstrated moderate antiradical activity in DPPH test (up to EC50 16μM). The same compounds strongly inhibited soybean lipoxygenase (up to IC50 0.4μM) and Fe(2+)- and Fe(3+)-induced lipid peroxidation (LP) of rat brain cortex homogenate (up to IC50 0.3μM). All tested isoxazole derivatives promoted the phosphorylating respiratory activity simultaneously with maximal stimulated respiratory activity of mitochondria and do not reveal any toxicity towards the primary culture of rat cortex neurons. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study.

PubMed

Chlapek, Petr; Redova, Martina; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-05-11

We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4

PubMed Central

Maddipati, Krishna Rao; Romero, Roberto; Chaiworapongsa, Tinnakorn; Chaemsaithong, Piya; Zhou, Sen-Lin; Xu, Zhonghui; Tarca, Adi L.; Kusanovic, Juan Pedro; Gomez, Ricardo; Chaiyasit, Noppadol; Honn, Kenneth V.

2016-01-01

Bioactive lipids derived from the metabolism of polyunsaturated fatty acids are important mediators of the inflammatory response. Labor per se is considered a sterile inflammatory process. Intra-amniotic inflammation (IAI) due to microorganisms (i.e., intra-amniotic infection) or danger signals (i.e., sterile IAI) has been implicated in the pathogenesis of preterm labor and clinical chorioamnionitis at term. Early and accurate diagnosis of microbial invasion of the amniotic cavity (MIAC) requires analysis of amniotic fluid (AF). It is possible that IAI caused by microorganisms is associated with a stereotypic lipidomic profile, and that analysis of AF may help in the identification of patients with this condition. To test this hypothesis, we analyzed the fatty acyl lipidome of AF by liquid chromatography—mass spectrometry from patients in spontaneous labor at term and preterm gestations. We report that the AF concentrations of proinflammatory lipid mediators of the 5-lipoxygenase pathway are significantly higher in MIAC than in cases of sterile IAI. These results suggest that the concentrations of 5-lipoxygenase metabolites of arachidonic acid, 5-hydroxyeicosatetraenoic acid, and leukotriene B4 in particular could serve as potential biomarkers of MIAC. This finding could have important implications for the rapid identification of patients who may benefit from anti-microbial treatment.—Maddipati, K. R., Romero, R., Chaiworapongsa ,T., Chaemsaithong, P., Zhou, S.-L., Xu, Z., Tarca, A. L., Kusanovic, J. P., Gomez, R., Chaiyasit, N., Honn, K. V. Lipidomic analysis of patients with microbial invasion of the amniotic cavity reveals up-regulation of leukotriene B4. PMID:27312808

Fluorescence spectroscopic study on the interaction of resveratrol with lipoxygenase

NASA Astrophysics Data System (ADS)

Pinto, María del Carmen; Duque, Antonio Luis; Macías, Pedro

2010-09-01

The interaction of lipoxygenase with (E)-resveratrol was investigated by fluorescence spectroscopy. The data obtained revealed that the quenching of intrinsic fluorescence of lipoxygenase is produced by the formation of a complex lipoxygenase-(E)-resveratrol. From the value obtained for the binding constant, according to the Stern-Volmer modified equation, was deduced the existence of static quenching mechanism and, as consequence, the existence of a strong interaction between (E)-resveratrol and lipoxygenase. The values obtained for the thermodynamic parameter Δ H (-3.58 kJ mol -1) and Δ S (87.97 J mol -1K -1) suggested the participation of hydrophobic interactions and hydrogen bonds in the stabilization of the complex ligand-protein. From the static quenching we determined that only exist one independent binding site. Based on the Förster energy transfer theory, the distance between the acceptor ((E)-resveratrol) and the donor (Trp residues of lipoxygenase) was calculated to be 3.42 nm. Finally, based on the information obtained from the evaluation of synchronous and three-dimensional fluorescence spectroscopy, we deduced that the interaction of (E)-resveratrol with lipoxygenase produces micro-environmental and conformational alterations of protein in the binding region.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Insook; Park, Sujin; Cho, Jin Won

12/15-lipoxygenase (12/15-LOX) is involved in organelle homeostasis by degrading mitochondria in maturing red blood cells and by eliminating excess peroxisomes in liver. Furthermore, 12/15-LOX contributes to diseases by exacerbating oxidative stress-related injury, notably in stroke. Nonetheless, it is unclear what the consequences are of abolishing 12/15-LOX activity. Mice in which the alox15 gene has been ablated do not show an obvious phenotype, and LOX enzyme inhibition is not overtly detrimental. We show here that liver histology is also unremarkable. However, electron microscopy demonstrated that 12/15-LOX knockout surprisingly leads to increased macroautophagy in the liver. Not only macroautophagy but also mitophagymore » and pexophagy were increased in hepatocytes, which otherwise showed unaltered fine structure and organelle morphology. These findings were substantiated by immunofluorescence showing significantly increased number of LC3 puncta and by Western blotting demonstrating a significant increase for LC3-II protein in both liver and brain homogenates of 12/15-LOX knockout mice. Inhibition of 12/15-LOX activity by treatment with four structurally different inhibitors had similar effects in cultured HepG2 hepatoma cells and SH-SY5Y neuroblastoma cells with significantly increased autophagy discernable already after 2 hours. Hence, our study reveals a link between ablation or inhibition of 12/15-LOX and stimulation of macroautophagy. The enhanced macroautophagy may be related to the known tissue-protective effects of LOX ablation or inhibition under various diseased conditions caused by oxidative stress and ischemia. This could provide an important cleaning mechanism of cells and tissues to prevent accumulation of damaged mitochondria and other cellular components. - Highlights: • A relationship between lipoxygenases and autophagy is disclosed. • 12/15-lipoxygenase knockout increases autophagy in mice liver and brain. • Lipoxygenase inhibition boosts autophagy in human hepatoma and neuroblastoma cells. • Lipoxygenase knockout or inhibition triggers selective autophagy.« less

Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages.

PubMed

Altavilla, D; Squadrito, F; Bitto, A; Polito, F; Burnett, B P; Di Stefano, V; Minutoli, L

2009-08-01

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. LPS-stimulated (1 microg.mL(-1)) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32-128 microg.mL(-1)) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein kappaB-alpha (IkappaB-alpha) levels were evaluated by Western blot analysis. Nuclear factor kappaB (NF-kappaB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-alpha (TNF-alpha) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 microg.mL(-1)) significantly inhibited COX-2 (LPS = 18 +/- 2.1; flavocoxid = 3.8 +/- 0.9 integrated intensity), 5-LOX (LPS = 20 +/- 3.8; flavocoxid = 3.1 +/- 0.8 integrated intensity) and iNOS expression (LPS = 15 +/- 1.1; flavocoxid = 4.1 +/- 0.4 integrated intensity), but did not modify COX-1 expression. PGE(2) and LTB(4) levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IkappaB-alpha protein (LPS = 1.9 +/- 0.2; flavocoxid = 7.2 +/- 1.6 integrated intensity), blunted increased NF-kappaB binding activity (LPS = 9.2 +/- 2; flavocoxid = 2.4 +/- 0.7 integrated intensity) and the enhanced TNF-alpha mRNA levels (LPS = 8 +/- 0.9; flavocoxid = 1.9 +/- 0.8 n-fold/beta-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages. Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.

Reduced dietary omega-6 to omega-3 fatty acid ratio and 12/15-lipoxygenase deficiency are protective against chronic high fat diet-induced steatohepatitis.

PubMed

Lazic, Milos; Inzaugarat, Maria Eugenia; Povero, Davide; Zhao, Iris C; Chen, Mark; Nalbandian, Madlena; Miller, Yury I; Cherñavsky, Alejandra C; Feldstein, Ariel E; Sears, Dorothy D

2014-01-01

Obesity is associated with metabolic perturbations including liver and adipose tissue inflammation, insulin resistance, and type 2 diabetes. Omega-6 fatty acids (ω6) promote and omega-3 fatty acids (ω3) reduce inflammation as they can be metabolized to pro- and anti-inflammatory eicosanoids, respectively. 12/15-lipoxygenase (12/15-LO) enzymatically produces some of these metabolites and is induced by high fat (HF) diet. We investigated the effects of altering dietary ω6/ω3 ratio and 12/15-LO deficiency on HF diet-induced tissue inflammation and insulin resistance. We examined how these conditions affect circulating concentrations of oxidized metabolites of ω6 arachidonic and linoleic acids and innate and adaptive immune system activity in the liver. For 15 weeks, wild-type (WT) mice were fed either a soybean oil-enriched HF diet with high dietary ω6/ω3 ratio (11∶1, HFH), similar to Western-style diet, or a fat Kcal-matched, fish oil-enriched HF diet with a low dietary ω6/ω3 ratio of 2.7∶1 (HFL). Importantly, the total saturated, monounsaturated and polyunsaturated fat content was matched in the two HF diets, which is unlike most published fish oil studies in mice. Despite modestly increased food intake, WT mice fed HFL were protected from HFH-diet induced steatohepatitis, evidenced by decreased hepatic mRNA expression of pro-inflammatory genes and genes involved in lymphocyte homing, and reduced deposition of hepatic triglyceride. Furthermore, oxidized metabolites of ω6 arachidonic acid were decreased in the plasma of WT HFL compared to WT HFH-fed mice. 12/15-LO knockout (KO) mice were also protected from HFH-induced fatty liver and elevated mRNA markers of inflammation and lymphocyte homing. 12/15-LOKO mice were protected from HFH-induced insulin resistance but reducing dietary ω6/ω3 ratio in WT mice did not ameliorate insulin resistance or adipose tissue inflammation. In conclusion, lowering dietary ω6/ω3 ratio in HF diet significantly reduces steatohepatitis.

Involvement of a Lipoxygenase-Like Enzyme in Abscisic Acid Biosynthesis 1

PubMed Central

Creelman, Robert A.; Bell, Erin; Mullet, John E.

1992-01-01

Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. 18O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties. PMID:16668998

Oral gold compound auranofin triggers arachidonate release and cyclooxygenase metabolism in the alveolar macrophage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peters-Golden, M.; Shelly, C.

1988-12-01

We examined the effect of in vitro incubation with the oral gold compound auranofin (AF) on arachidonic acid (AA) release and metabolism by rat alveolar macrophages (AMs). AF stimulated dose- and time-dependent release of /sup 14/C-AA from prelabeled AMs, which reached 4.7 +/- 0.3% (mean +/- SEM) of incorporated radioactivity at 10 micrograms/ml for 90 min, as compared to 0.5 +/- 0.1% release following control incubation for 90 min (p less than 0.001). Similar dose- and time-dependent synthesis of thromboxane (Tx) A2 (measured as TxB2) and prostaglandin (PG) E2 was demonstrated by radioimmunoassay of medium from unlabeled cultures, reaching 18-foldmore » and 9-fold, respectively, of the control values at 10 micrograms/ml AF for 90 min (p less than 0.001 for both). AF-induced TxB2 and PGE2 synthesis was inhibited by indomethacin as well as by pretreatment with methylprednisolone. No increase in the synthesis of immunoreactive leukotrienes (LT) B4 or C4 was noted at any dose or time of AF. High performance liquid chromatographic separation of /sup 14/C-eicosanoids synthesized by prelabeled AMs confirmed that AF induced the release of free AA and its metabolism to cyclooxygenase, but not 5-lipoxygenase, metabolites. The ability of AF to trigger macrophage AA metabolism may be relevant to the exacerbation of certain inflammatory processes which sometimes accompany gold therapy.« less

Using the medical food flavocoxid [corrected] in managing osteoarthritis.

PubMed

Gottlieb, Daniel; Kuritzky, Louis

2011-01-01

Osteoarthritis (OA) is the most common debilitating joint disease worldwide. Clinicians have many therapeutic modalities and prescription medications in their arsenals to treat chronic inflammatory pain. However, as patients age, and develop numerous comorbidities, the most common, and often most effective pharmacologic treatment for OA, nonsteroidal anti-inflammatory drugs (NSAIDs), becomes problematic in that it may exacerbate or even cause cardiovascular, renal, and/or gastrointestinal pathology. This paper reviews the metabolism of arachidonic acid as it relates to the clinical treatment of inflammation, and explores a novel botanical therapy, flavocoxid, that has shown equal efficacy to naproxen in treating pain associated with mild to moderate OA of the knee. Flavocoxid has demonstrated balanced inhibition of the three primary enzymes responsible for processing AA, cyclooxygenases 1 and 2, and 5-lipoxygenase. Researchers have proposed that balanced inhibition of AA metabolism offers the promise of analgesia similar to NSAIDs without the associated cardiovascular, renal, or gastrointestinal side effects.

Alternative products to treat allergic rhinitis and alternative routes for allergy immunotherapy.

PubMed

Ipci, Kagan; Oktemer, Tugba; Muluk, Nuray Bayar; Şahin, Ethem; Altıntoprak, Niyazi; Bafaqeeh, Sameer Ali; Kurt, Yasemin; Mladina, Ranko; Šubarić, Marin; Cingi, Cemal

2016-09-01

Some alternative products instead of immunotherapy are used in patients with allergic rhinitis (AR). In this paper, alternative products to treat allergic rhinitis and alternative routes for allergy immunotherapy are reviewed. Alternative products and methods used instead of immunotherapy are tea therapy, acupuncture, Nigella sativa, cinnamon bark, Spanish needle, acerola, capsaicin (Capsicum annum), allergen-absorbing ointment, and cellulose powder. N. sativa has been used in AR treatment due to its anti-inflammatory effects. N. sativa oil also inhibits the cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism. The beneficial effects of N. sativa seed supplementation on the symptoms of AR may be due to its antihistaminic properties. To improve the efficacy of immunotherapy, some measures are taken regarding known immunotherapy applications and alternative routes of intralymphatic immunotherapy and epicutaneous immunotherapy are used. There are alternative routes and products to improve the efficacy of immunotherapy.

Mast cell granules modulate alveolar macrophage respiratory-burst activity and eicosanoid metabolism.

PubMed

Rock, M J; Despot, J; Lemanske, R F

1990-10-01

Alveolar macrophages (AMs) and mast cells reside in the airway, and both have been demonstrated to contribute independently to allergic inflammatory responses through the generation of respiratory-burst metabolites and the release of biologically active mediators, respectively. Since mast cell granules (MCGs) contain mediators that could potentially interact with the AM respiratory burst, we investigated the effects of isolated MCGs on this important inflammatory pathway of the AM. MCGs and AMs were obtained by peritoneal and tracheoalveolar lavage, respectively, of Sprague-Dawley rats. First, the overall respiratory-burst activity was measured by luminal-enhanced chemiluminescence (CL), and second, the individual oxygen species contributing to CL (superoxide anion [O2-], hydrogen peroxide [H2O2], and hypochlorous acid) were measured. MCGs alone enhanced AM CL responses to an equivalent degree compared to zymosan-stimulated AMs. However, AMs preincubated with MCGs followed by zymosan stimulation significantly and synergistically enhanced the CL responses. This enhanced CL was not due to an increased production of O2-, H2O2, or hypochlorous acid; in fact, there were decreased measured amounts of O2- and H2O2 from zymosan-stimulated AMs in the presence of MCGs, most likely caused by the content of granules of superoxide dismutase and peroxidase, respectively. The lipoxygenase inhibitor, nordihydroguaiaretic acid, completely abolished the enhanced CL of AM preincubated with MCGs and subsequently stimulated by zymosan, but O2- production was not affected by nordihydroguaiaretic acid. Taken together, these results suggest that derivatives of arachidonic acid metabolism, most likely those of the lipoxygenase pathway, are responsible for the enhanced AM CL response observed in the presence of MCGs. Thus, mast cell-macrophage interactions may be important within the airway in enhancing the generation of mediators that contribute to tissue inflammation and bronchospasm.

Dipetalodipin, a Novel Multifunctional Salivary Lipocalin That Inhibits Platelet Aggregation, Vasoconstriction, and Angiogenesis through Unique Binding Specificity for TXA2, PGF2α, and 15(S)-HETE*

PubMed Central

Assumpção, Teresa C. F.; Alvarenga, Patricia H.; Ribeiro, José M. C.; Andersen, John F.; Francischetti, Ivo M. B.

2010-01-01

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA2, TXA2 mimetic (U-46619), TXB2, PGH2 mimetic (U-51605), PGD2, PGJ2, and PGF2α. It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE1, PGE2, 8-iso-PGF2α, prostacyclin), leukotrienes (e.g,. LTB4, LTC4, LTD4, LTE4), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF2α and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA2 antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg39 and Gln135 in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation. PMID:20889972

Defense Activated by 9-Lipoxygenase-Derived Oxylipins Requires Specific Mitochondrial Proteins1[W

PubMed Central

Vellosillo, Tamara; Aguilera, Verónica; Marcos, Ruth; Bartsch, Michael; Vicente, Jorge; Cascón, Tomas; Hamberg, Mats; Castresana, Carmen

2013-01-01

9-Lipoxygenases (9-LOXs) initiate fatty acid oxygenation, resulting in the formation of oxylipins activating plant defense against hemibiotrophic pathogenic bacteria. Previous studies using nonresponding to oxylipins (noxy), a series of Arabidopsis (Arabidopsis thaliana) mutants insensitive to the 9-LOX product 9-hydroxy-10,12,15-octadecatrienoic acid (9-HOT), have demonstrated the importance of cell wall modifications as a component of 9-LOX-induced defense. Here, we show that a majority (71%) of 41 studied noxy mutants have an added insensitivity to isoxaben, an herbicide inhibiting cellulose synthesis and altering the cell wall. The specific mutants noxy2, noxy15, and noxy38, insensitive to both 9-HOT and isoxaben, displayed enhanced susceptibility to Pseudomonas syringae DC3000 as well as reduced activation of salicylic acid-responding genes. Map-based cloning identified the mutation in noxy2 as At5g11630 encoding an uncharacterized mitochondrial protein, designated NOXY2. Moreover, noxy15 and noxy38 were mapped at the DYNAMIN RELATED PROTEIN3A and FRIENDLY MITOCHONDRIA loci, respectively. Fluorescence microscopy and molecular analyses revealed that the three noxy mutants characterized exhibit mitochondrial dysfunction and that 9-HOT added to wild-type Arabidopsis causes mitochondrial aggregation and loss of mitochondrial membrane potential. The results suggest that the defensive responses and cell wall modifications caused by 9-HOT are under mitochondrial retrograde control and that mitochondria play a fundamental role in innate immunity signaling. PMID:23370715

The lipoxygenase pathway in tulip (Tulipa gesneriana): detection of the ketol route.

PubMed

Grechkin, A N; Mukhtarova, L S; Hamberg, M

2000-12-01

The in vitro metabolism of [1-(14)C]linoleate, [1-(14)C]linolenate and their 9(S)-hydroperoxides was studied in cell-free preparations from tulip (Tulipa gesneriana) bulbs, leaves and flowers. Linoleate and its 9-hydroperoxide were converted by bulb and leaf preparations into three ketols: (12Z)-9-hydroxy-10-oxo-12-octadecadienoic acid (alpha-ketol), (11E)-10-oxo-13-hydroxy-11-octadecadienoic acid (gamma-ketol) and a novel compound, (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol), in the approximate molar proportions of 10:3:1. The corresponding 15, 16-dehydro alpha- and gamma-ketols were the main metabolites of [1-(14)C]linolenate and its 9-hydroperoxide. Thus bulbs and leaves possessed 9-lipoxygenase and allene oxide synthase activities. Incubations with flower preparations gave alpha-ketol hydro(pero)xides as predominant metabolites. Bulb and leaf preparations possessed a novel enzyme activity, gamma-ketol reductase, which reduces gamma-ketol to 10-oxo-13-hydroxyoctadecanoic acid (dihydro-gamma-ketol) in the presence of NADH. Exogenous linolenate 13(S)-hydroperoxide was converted mostly into chiral (9S,13S)-12-oxo-10-phytodienoate (99.5% optical purity) by bulb preparations, while [1-(14)C]linolenate was a precursor for ketols only. Thus tulip bulbs possess abundant allene oxide cyclase activity, the substrate for which is linolenate 13(S)-hydroperoxide, even though 13(S)-lipoxygenase products were not detectable in the bulbs. The majority of the cyclase activity was found in the microsomes (10(5) g pellet). Cyclase activity was not found in the other tissues examined, but only in the bulbs. The ketol route of the lipoxygenase pathway, mediated by 9-lipoxygenase and allene oxide synthase activities, has not been detected previously in the vegetative organs of any plant species.

The lipoxygenase pathway in tulip (Tulipa gesneriana): detection of the ketol route.

PubMed Central

Grechkin, A N; Mukhtarova, L S; Hamberg, M

2000-01-01

The in vitro metabolism of [1-(14)C]linoleate, [1-(14)C]linolenate and their 9(S)-hydroperoxides was studied in cell-free preparations from tulip (Tulipa gesneriana) bulbs, leaves and flowers. Linoleate and its 9-hydroperoxide were converted by bulb and leaf preparations into three ketols: (12Z)-9-hydroxy-10-oxo-12-octadecadienoic acid (alpha-ketol), (11E)-10-oxo-13-hydroxy-11-octadecadienoic acid (gamma-ketol) and a novel compound, (12Z)-10-oxo-11-hydroxy-12-octadecadienoic acid (10,11-ketol), in the approximate molar proportions of 10:3:1. The corresponding 15, 16-dehydro alpha- and gamma-ketols were the main metabolites of [1-(14)C]linolenate and its 9-hydroperoxide. Thus bulbs and leaves possessed 9-lipoxygenase and allene oxide synthase activities. Incubations with flower preparations gave alpha-ketol hydro(pero)xides as predominant metabolites. Bulb and leaf preparations possessed a novel enzyme activity, gamma-ketol reductase, which reduces gamma-ketol to 10-oxo-13-hydroxyoctadecanoic acid (dihydro-gamma-ketol) in the presence of NADH. Exogenous linolenate 13(S)-hydroperoxide was converted mostly into chiral (9S,13S)-12-oxo-10-phytodienoate (99.5% optical purity) by bulb preparations, while [1-(14)C]linolenate was a precursor for ketols only. Thus tulip bulbs possess abundant allene oxide cyclase activity, the substrate for which is linolenate 13(S)-hydroperoxide, even though 13(S)-lipoxygenase products were not detectable in the bulbs. The majority of the cyclase activity was found in the microsomes (10(5) g pellet). Cyclase activity was not found in the other tissues examined, but only in the bulbs. The ketol route of the lipoxygenase pathway, mediated by 9-lipoxygenase and allene oxide synthase activities, has not been detected previously in the vegetative organs of any plant species. PMID:11085944

Lipoxygenase activity accelerates programmed spore germination in Aspergillus fumigatus

Treesearch

Gregory J. Fischer; William Bacon; Jun Yang; Jonathan M. Palmer; Taylor Dagenais; Bruce D. Hammock; Nancy P. Keller

2017-01-01

The opportunistic human pathogen Aspergillus fumigatus initiates invasive growth through a programmed germination process that progresses from dormant spore to swollen spore (SS) to germling (GL) and ultimately invasive hyphal growth. We find a lipoxygenase with considerable homology to human Alox5 and Alox15, LoxB, that impacts the transitions of...

Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters.

PubMed

Thangavel, Samikkannu; Mulet, Carmen T; Atluri, Venkata S R; Agudelo, Marisela; Rosenberg, Rhonda; Devieux, Jessy G; Nair, Madhavan P N

2018-02-01

Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction. In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGE 2 ) in plasma when compared with either HIV or alcohol alone. This article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users. These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to immune toxicity, and further exacerbation with alcohol use. Antioxid. Redox Signal. 28, 324-337.

Influence of lipid content and lipoxygenase on flavor volatiles in the tomato peel and flesh.

PubMed

Ties, Paige; Barringer, Sheryl

2012-07-01

Ten different varieties of tomatoes were separated into peel and flesh and each portion was measured separately. Headspace volatiles were measured in real time using selected ion flow tube mass spectrometry. Lipoxygenase activity was measured using the adsorption of conjugated dienes formed by lipoxygenase. Lipid was extracted and fatty acids were quantified using a gas chromatograph. Volatiles were significantly greater in the peel than flesh when there was a significant difference. The lipoxygenase activity of flesh and peel correlated with the volatiles produced by the lipoxygenase pathway. There was no correlation with other volatiles, which are not dependent on lipid oxidation by lipoxygenase. The lipoxygenase activity, total fatty acid content, and linolenic acid of the peel were greater than the flesh, which is directly related to an increase in fresh, green volatiles. Addition of exogenous lipoxygenase had no effect on lipoxygenase-derived volatiles formed. The addition of linoleic acid caused an increase in hexanal, 1-hexanol, and (E)-2-heptenal in the flesh and (E)-2-heptenal in the peel. Stored unrefrigerated peel had higher volatile concentrations, whereas refrigerated peel had significantly lower concentration than day 0. Storage decreased lipoxygenase activity in the unrefrigerated and refrigerated peel, but had no effect on the fatty acid content. Overall, linolenic acid was the most important to the formation of headspace volatiles, but lipoxygenase activity and unknown factors are also important. The peel of a tomato is most beneficial to the production of volatiles associated with the fresh aroma of tomatoes; therefore, it should be used in the processing of tomato products to produce a fresh, green aroma rather than being removed. Knowledge of the effects of lipoxygenase activity, total fatty acid content, and fatty acid profile on flavor volatiles will allow for better selection of a variety for raw consumption. © 2012 Institute of Food Technologists®

A role for 9-lipoxygenases in maize defense against insect herbivory.

PubMed

Woldemariam, Melkamu G; Ahern, Kevin; Jander, Georg; Tzin, Vered

2018-01-02

Feeding by Spodoptera exigua (beet armyworm) larvae on Zea mays (maize) induces expression of 9-lipoxygenases to a greater extent than 13-lipoxygenases. Whereas 13-lipoxygenases have an established role in the synthesis of jasmonates that serve as defense signaling molecules in many plant species, relatively little is known about the role of 9-lipoxygenases in herbivore defense. Phylogenetic analysis of lipoxygenases from maize inbred lines B73 and W22 shows that, although most Lox genes are present in both lines, Lox12, a 9-lipoxygenase that has been implicated in fungal defense, is truncated and unlikely to encode a functional protein in W22. Two independent Mutator transposon insertions in another 9-lipoxygenase, Lox4, caused improved S. exigua growth on the mutant lines relative to wildtype W22. This observation suggests a function in herbivore defense for metabolic products downstream of maize Lox4, either through direct toxicity or a perhaps an as yet unknown signaling function.

Michael acceptor containing drugs are a novel class of 5-lipoxygenase inhibitor targeting the surface cysteines C416 and C418.

PubMed

Maucher, Isabelle V; Rühl, Michael; Kretschmer, Simon B M; Hofmann, Bettina; Kühn, Benjamin; Fettel, Jasmin; Vogel, Anja; Flügel, Karsten T; Manolikakes, Georg; Hellmuth, Nadine; Häfner, Ann-Kathrin; Golghalyani, Vahid; Ball, Ann-Katrin; Piesche, Matthias; Matrone, Carmela; Geisslinger, Gerd; Parnham, Michael J; Karas, Michael; Steinhilber, Dieter; Roos, Jessica; Maier, Thorsten J

2017-02-01

Recently, we published that nitro-fatty acids (NFA) are potent electrophilic molecules which inhibit 5-lipoxygenase (5-LO) by interacting catalytically with cysteine residues next to a substrate entry channel. The electrophilicity is derived from an intramolecular Michael acceptor moiety consisting of an electron-withdrawing group in close proximity to a double bond. The potential of the Michael acceptor moiety to interact with functionally relevant cysteines of proteins potentially renders them effective and sustained enzyme activity modulators. We screened a large library of naturally derived and synthetic electrophilic compounds to investigate whether other types of Michael acceptor containing drugs suppress 5-LO enzyme activity. The activity was measured by assessing the effect on the 5-LO product formation of intact human polymorphonuclear leukocytes. We demonstrated that a number of structurally different compounds were suppressive in the activity assays and showed that Michael acceptors of the quinone and nitro-alkene group produced the strongest inhibition of 5-LO product formation. Reactivity with the catalytically relevant cysteines 416 and 418 was confirmed using mutated recombinant 5-LO and mass spectrometric analysis (MALDI-MS). In the present study, we show for the first time that a number of well-recognized naturally occurring or synthetic anti-inflammatory compounds carrying a Michael acceptor, such as thymoquinone (TQ), the paracetamol metabolite NAPQI, the 5-LO inhibitor AA-861, and bardoxolone methyl (also known as RTA 402 or CDDO-methyl ester) are direct covalent 5-LO enzyme inhibitors that target the catalytically relevant cysteines 416 and 418. Copyright © 2016 Elsevier Inc. All rights reserved.

Increases in levels of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EETs and DHETs) in liver and heart in vivo by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and in hepatic EET:DHET ratios by cotreatment with TCDD and the soluble epoxide hydrolase inhibitor AUDA.

PubMed

Diani-Moore, Silvia; Ma, Yuliang; Gross, Steven S; Rifkind, Arleen B

2014-02-01

The environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) binds and activates the transcription factor aryl hydrocarbon receptor (AHR), inducing CYP1 family cytochrome P450 enzymes. CYP1A2 and its avian ortholog CYP1A5 are highly active arachidonic acid epoxygenases. Epoxygenases metabolize arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) and selected monohydroxyeicosatetraenoic acids (HETEs). EETs can be further metabolized by epoxide hydrolases to dihydroxyeicosatrienoic acids (DHETs). As P450-arachidonic acid metabolites affect vasoregulation, responses to ischemia, inflammation, and metabolic disorders, identification of their production in vivo is needed to understand their contribution to biologic effects of TCDD and other AHR activators. Here we report use of an acetonitrile-based extraction procedure that markedly increased the yield of arachidonic acid products by lipidomic analysis over a standard solid-phase extraction protocol. We show that TCDD increased all four EETs (5,6-, 8,9-, 11,12-, and 14,15-), their corresponding DHETs, and 18- and 20-HETE in liver in vivo and increased 5,6-EET, the four DHETs, and 18-HETE in heart, in a chick embryo model. As the chick embryo heart lacks arachidonic acid-metabolizing activity, the latter findings suggest that arachidonic acid metabolites may travel from their site of production to a distal organ, i.e., heart. To determine if the TCDD-arachidonic acid-metabolite profile could be altered pharmacologically, chick embryos were treated with TCDD and the soluble epoxide hydrolase inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). Cotreatment with AUDA increased hepatic EET-to-DHET ratios, indicating that the in vivo profile of P450-arachidonic acid metabolites can be modified for potential therapeutic intervention.

A High-Fat Diet Induces Bone Loss in Mice Lacking the Alox5 Gene

PubMed Central

Le, Phuong; Kawai, Masanobu; Bornstein, Sheila; DeMambro, Victoria E.; Horowitz, Mark C.

2012-01-01

5-Lipoxygenase catalyzes leukotriene generation from arachidonic acid. The gene that encodes 5-lipoxygenase, Alox5, has been identified in genome-wide association and mouse Quantitative Trait Locus studies as a candidate gene for obesity and low bone mass. Thus, we tested the hypothesis that Alox5−/− mice would exhibit metabolic and skeletal changes when challenged by a high-fat diet (HFD). On a regular diet, Alox5−/− mice did not differ in total body weight, percent fat mass, or bone mineral density compared with wild-type (WT) controls (P < 0.05). However, when placed on a HFD, Alox5−/− gained more fat mass and lost greater areal bone mass vs. WT (P < 0.05). Microarchitectural analyses revealed that on a HFD, WT showed increases in cortical area (P < 0.01) and trabecular thickness (P < 0.01), whereas Alox5−/− showed no change in cortical parameters but a decrease in trabecular number (P < 0.05) and bone volume fraction compared with WT controls (P < 0.05). By histomorphometry, a HFD did not change bone formation rates of either strain but produced an increase in osteoclast number per bone perimeter in Alox5−/− mice (P < 0.03). In vitro, osteoclastogenesis of marrow stromal cells was enhanced in mutant but not WT mice fed a HFD. Gene expression for Rankl, Pparg, and Cox-2 was greater in the femur of Alox5−/− than WT mice on a HFD (P < 0.01), but these increases were suppressed in the Alox5−/− mice after 8 wk of treatment with celecoxib, a cyclooxygenase-2 inhibitor. In sum, there is a strong gene by environmental interaction for bone mass when mice lacking the Alox5 gene are fed a HFD. PMID:22128029

Synthesis and biological activities of fluorinated chalcone derivatives.

PubMed

Nakamura, Chika; Kawasaki, Nobuhide; Miyataka, Hideki; Jayachandran, Ezhuthachan; Kim, In Ho; Kirk, Kenneth L; Taguchi, Takeo; Takeuchi, Yoshio; Hori, Hitoshi; Satoh, Toshio

2002-03-01

We have designed and synthesized new 5-lipoxygenase inhibitors, fluorinated 3,4-dihydroxychalcones, and evaluated their biological activities with respect to antiperoxidation activity and in vitro antitumor activities. All fluorinated chalcones tested showed 5-lipoxygenase inhibition on rat basophilic leukemia-1 (RBL-1) cells and inhibitory action on Fe(3+)-ADP induced NADPH-dependent lipid peroxidation in rat liver microsomes. The potencies were comparable or better to that of the lead 3,4-dihydroxychalcone. 6-Fluoro-3,4-dihydroxy-2',4'-dimethoxy chalcone (7) was the most effective compound in the in vitro assay using a human cancer cell line panel (HCC panel) consisting of 39 systems.

Transient postnatal fluoxetine leads to decreased brain arachidonic acid metabolism and cytochrome P450 4A in adult mice.

PubMed

Ramadan, Epolia; Blanchard, Helene; Cheon, Yewon; Fox, Meredith A; Chang, Lisa; Chen, Mei; Ma, Kaizong; Rapoport, Stanley I; Basselin, Mireille

2014-05-01

Fetal and perinatal exposure to selective serotonin (5-HT) reuptake inhibitors (SSRIs) has been reported to alter childhood behavior, while transient early exposure in rodents is reported to alter their behavior and decrease brain extracellular 5-HT in adulthood. Since 5-HT2A/2C receptor-mediated neurotransmission can involve G-protein coupled activation of cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (ARA) from synaptic membrane phospholipid, we hypothesized that transient postnatal exposure to fluoxetine would alter brain ARA metabolism in adult mice. Brain ARA incorporation coefficients k* and rates Jin were quantitatively imaged following intravenous [1-(14)C]ARA infusion of unanesthetized adult mice that had been injected daily with fluoxetine (10mg/kg i.p.) or saline during postnatal days P4-P21. Expression of brain ARA metabolic enzymes and other relevant markers also was measured. On neuroimaging, k* and Jin was decreased widely in early fluoxetine- compared to saline-treated adult mice. Of the enzymes measured, cPLA2 activity was unchanged, while Ca(2+)-independent iPLA2 activity was increased. There was a significant 74% reduced protein level of cytochrome P450 (CYP) 4A, which can convert ARA to 20-HETE. Reduced brain ARA metabolism in adult mice transiently exposed to postnatal fluoxetine, and a 74% reduction in CYP4A protein, suggest long-term effects independent of drug presence in brain ARA metabolism, and in CYP4A metabolites. These changes might contribute to reported altered behavior following early SSRI in rodents. Published by Elsevier Ltd.

Impact of wines and wine constituents on cyclooxygenase-1, cyclooxygenase-2, and 5-lipoxygenase catalytic activity.

PubMed

Kutil, Zsofia; Temml, Veronika; Maghradze, David; Pribylova, Marie; Dvorakova, Marcela; Schuster, Daniela; Vanek, Tomas; Landa, Premysl

2014-01-01

Cyclooxygenases and lipoxygenases are proinflammatory enzymes; the former affects platelet aggregation, vasoconstriction, vasodilatation and later the development of atherosclerosis. Red wines from Georgia and central and western Europe inhibited cyclooxygenase-1 (COX-1) activity in the range of 63-94%, cyclooxygenase-2 (COX-2) activity in the range of 20-44% (tested at a concentration of 5 mL/L), and 5-lipoxygenase (5-LOX) activity in the range of 72-84% (at a concentration of 18.87 mL/L). White wines inhibited 5-LOX in the range of 41-68% at a concentration of 18.87 mL/L and did not inhibit COX-1 and COX-2. Piceatannol (IC50 = 0.76 μM) was identified as a strong inhibitor of 5-LOX followed by luteolin (IC50 = 2.25 μM), quercetin (IC50 = 3.29 μM), and myricetin (IC50 = 4.02 μM). trans-Resveratrol was identified as an inhibitor of COX-1 (IC50 = 2.27 μM) and COX-2 (IC50 = 3.40 μM). Red wine as a complex mixture is a powerful inhibitor of COX-1, COX-2, and 5-LOX, the enzymes involved in eicosanoid biosynthetic pathway.

Impact of Wines and Wine Constituents on Cyclooxygenase-1, Cyclooxygenase-2, and 5-Lipoxygenase Catalytic Activity

PubMed Central

Temml, Veronika; Maghradze, David; Vanek, Tomas

2014-01-01

Cyclooxygenases and lipoxygenases are proinflammatory enzymes; the former affects platelet aggregation, vasoconstriction, vasodilatation and later the development of atherosclerosis. Red wines from Georgia and central and western Europe inhibited cyclooxygenase-1 (COX-1) activity in the range of 63–94%, cyclooxygenase-2 (COX-2) activity in the range of 20–44% (tested at a concentration of 5 mL/L), and 5-lipoxygenase (5-LOX) activity in the range of 72–84% (at a concentration of 18.87 mL/L). White wines inhibited 5-LOX in the range of 41–68% at a concentration of 18.87 mL/L and did not inhibit COX-1 and COX-2. Piceatannol (IC50 = 0.76 μM) was identified as a strong inhibitor of 5-LOX followed by luteolin (IC50 = 2.25 μM), quercetin (IC50 = 3.29 μM), and myricetin (IC50 = 4.02 μM). trans-Resveratrol was identified as an inhibitor of COX-1 (IC50 = 2.27 μM) and COX-2 (IC50 = 3.40 μM). Red wine as a complex mixture is a powerful inhibitor of COX-1, COX-2, and 5-LOX, the enzymes involved in eicosanoid biosynthetic pathway. PMID:24976682

Specific Caleosin/Peroxygenase and Lipoxygenase Activities Are Tissue-Differentially Expressed in Date Palm (Phoenix dactylifera L.) Seedlings and Are Further Induced Following Exposure to the Toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin.

PubMed

Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Rahman, Farzana; Hassan, Mehedi; Murphy, Denis J

2016-01-01

Two caleosin/peroxygenase isoforms from date palm, Phoenix dactylifera L., PdCLO2 and PdCLO4, were characterized with respect to their tissue expression, subcellular localization, and oxylipin pathway substrate specificities in developing seedlings. Both PdCLO2 and PdCLO4 had peroxygenase activities that peaked at the mid-stage (radicle length of 2.5 cm) of seedling growth and were associated with the lipid droplet (LD) and microsomal fractions. Recombinant PdCLO2 and PdCLO4 proteins heterologously expressed in yeast cells were localized in both LD and microsomal fractions. Each of the purified recombinant proteins exhibited peroxygenase activity but they were catalytically distinct with respect to their specificity and product formation from fatty acid epoxide and hydroxide substrates. We recently showed that date palm CLO genes were upregulated following exposure to the potent toxin, 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) (Hanano et al., 2016), and we show here that transcripts of 9- and 13-lipoxygenase (LOX) genes were also induced by TCDD exposure. At the enzyme level, 9-LOX and 13-LOX activities were present in a range of seedling tissues and responded differently to TCDD exposure, as did the 9- and 13-fatty acid hydroperoxide reductase activities. This demonstrates that at least two branches of the oxylipin pathway are involved in responses to the environmental organic toxin, TCDD in date palm.

Alveolar Macrophages Drive Hepatocellular Carcinoma Lung Metastasis by Generating Leukotriene B4.

PubMed

Nosaka, Takuto; Baba, Tomohisa; Tanabe, Yamato; Sasaki, Soichiro; Nishimura, Tatsunori; Imamura, Yoshiaki; Yurino, Hideaki; Hashimoto, Shinichi; Arita, Makoto; Nakamoto, Yasunari; Mukaida, Naofumi

2018-03-01

Macrophages in lungs can be classified into two subpopulations, alveolar macrophages (AMs) and interstitial macrophages (IMs), which reside in the alveolar and interstitial spaces, respectively. Accumulating evidence indicates the involvement of IMs in lung metastasis, but the roles of AMs in lung metastasis still remain elusive. An i.v. injection of a mouse hepatocellular carcinoma (HCC) cell line, BNL, caused lung metastasis foci with infiltration of AMs and IMs. Comprehensive determination of arachidonic acid metabolite levels revealed increases in leukotrienes and PGs in lungs in this metastasis model. A 5-lipoxygenase (LOX) inhibitor but not a cyclooxygenase inhibitor reduced the numbers of metastatic foci, particularly those of a larger size. A major 5-LOX metabolite, LTB 4 , augmented in vitro cell proliferation of human HCC cell lines as well as BNL cells. Moreover, in this lung metastasis course, AMs exhibited higher expression levels of the 5-LOX and LTB 4 than IMs. Consistently, 5-LOX-expressing AMs increased in the lungs of human HCC patients with lung metastasis, compared with those without lung metastasis. Furthermore, intratracheal clodronate liposome injection selectively depleted AMs but not IMs, together with reduced LTB 4 content and metastatic foci numbers in this lung metastasis process. Finally, IMs in mouse metastatic foci produced CCL2, thereby recruiting blood-borne, CCR2-expressing AMs into lungs. Thus, AMs can be recruited under the guidance of IM-derived CCL2 into metastatic lungs and can eventually contribute to the progression of lung metastasis by providing a potent arachidonic acid-derived tumor growth promoting mediator, LTB 4 . Copyright © 2018 by The American Association of Immunologists, Inc.

Phospholipid-esterified eicosanoids are generated in agonist-activated human platelets and enhance tissue factor-dependent thrombin generation.

PubMed

Thomas, Christopher P; Morgan, Lloyd T; Maskrey, Benjamin H; Murphy, Robert C; Kühn, Hartmut; Hazen, Stanley L; Goodall, Alison H; Hamali, Hassan A; Collins, Peter W; O'Donnell, Valerie B

2010-03-05

Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 +/- 1.42 (PE) or 18.35 +/- 4.61 (PC), whereas free was 65.5 +/- 17.6 ng/4 x 10(7) cells (n = 5 separate donors, mean +/- S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca(2+) mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.

5,7-dihydroxy-2-(3-hydroxy-4, 5-dimethoxy-phenyl)-chromen-4-one-a flavone from Bruguiera gymnorrhiza displaying anti-inflammatory properties.

PubMed

Barik, Rajib; Sarkar, Ratul; Biswas, Prova; Bera, Rammohan; Sharma, Soma; Nath, Suvadeep; Karmakar, Sanmoy; Sen, Tuhinadri

2016-01-01

Bruguiera gymnorrhiza (BRG) (L.) Lamk (Rhizophoraceae), a mangrove species, is widely distributed in the Pacific region, eastern Africa, Indian subcontinent, and subtropical Australia. The leaves of this plant are traditionally used for treating burns and inflammatory lesions. This study isolates the bioactive compound from the methanol extract of BRG leaves and evaluates the possible mechanisms of anti-inflammatory activity involved. Bioassay-guided fractionation of BRG was performed to identify the bioactive fraction (displaying inhibition of cyclooxygenase 2 [COX2] - 5-lipoxygenase (5-LOX) activities and tumor necrosis factor-alpha (TNF-α) production at the tested concentrations of 100 and 10 μg/ml). The fractionation was performed by solvent extraction and preparative high-performance liquid chromatography. The bioactive compound was characterized by ultraviolet-visible, liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The antioxidant potential was evaluated by electron spin resonance spectrum of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 250 μM. The effect of the compound was also studied on TNF-α converting enzyme and nuclear factor kappa B (NF-κB) activities at the concentrations 100, 10 and 1 μg/ml. Bioassay-guided purification of BRG revealed the presence of a flavone (5,7-dihydroxy-2- [3-hydroxy-4,5-dimethoxy-phenyl]-chromen-4-one) of molecular weight 330Da. It demonstrated more than 80% inhibition against COX2, 5-LOX activities and TNF-α production at 100 μg/ml. It also displayed 40% inhibition against DPPH radical at the tested concentration along with 23.1% inhibition of NF-κB activity at 100 μg/ml. The isolated methoxy-flavone may play a predominant role in the anti-inflammatory properties displayed by BRG leaves. Such activity may involve multiple mechanisms, namely (a) modulation of oxidative stress (b) inhibition of arachidonic acid metabolism and (c) downregulation of pro-inflammatory cytokines probably through NF-κB inhibition.

Pharmacological studies on the TXA2 synthetase inhibitor (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046).

PubMed

Hiraku, S; Taniguchi, K; Wakitani, K; Omawari, N; Kira, H; Miyamoto, T; Okegawa, T; Kawasaki, A; Ujiie, A

1986-07-01

The effects of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046) on thromboxane A2 (TXA2) synthetase in vitro and on experimental animal models of sudden death and cerebral infarction were studied. IC50 values of OKY-046 for the TXA2 synthetase of human, rabbit, dog and guinea pig washed platelets were 0.004, 0.004, 0.26 and 2.4 microM, respectively. OKY-046 at concentrations up to 1 mM, however, did not inhibit prostacyclin (PGI2) synthetase from bovine aorta microsomes or cyclooxygenase and PGE2 isomerase from sheep seminal vesicle microsomes. Similarly, platelet 12-lipoxygenase was not affected by OKY-046. Evidence for a re-direction of arachidonate metabolism from thromboxane synthesis toward PGI2 synthesis was obtained using rat peritoneal cells. Namely, OKY-046 increased PGI2 production accompanied by an inhibition of TXA2 production at a concentration of more than 1 microM. OKY-046 at a dose of 0.1 mg/kg (i.v.) in dogs inhibited the aortic and mesenteric arterial contraction of rabbit induced by the addition of arachidonate to extracorporated blood of the dogs. OKY-046 at a dose of 0.3 mg/kg (i.v.) prevented the arachidonate-induced sudden death and also decreased the incidence of cerebral infarction induced by injection of arachidonate into the internal carotid artery in rabbits. Aspirin also decreased the incidence of cerebral infarction at a dose of 30 mg/kg (i.v.). These results suggest that OKY-046 may be valuable for the treatment of cerebrovascular and cardiovascular diseases associated with vasoconstriction and thrombosis due to TXA2.

A natural protective mechanism against hyperglycaemia in vascular endothelial and smooth-muscle cells: role of glucose and 12-hydroxyeicosatetraenoic acid.

PubMed Central

Alpert, Evgenia; Gruzman, Arie; Totary, Hanan; Kaiser, Nurit; Reich, Reuven; Sasson, Shlomo

2002-01-01

Bovine aortic endothelial and smooth-muscle cells down-regulate the rate of glucose transport in the face of hyperglycaemia, thus providing protection against deleterious effects of increased intracellular glucose levels. When exposed to high glucose concentrations these cells reduced the mRNA and protein content of their typical glucose transporter, GLUT-1, as well as its plasma-membrane abundance. Inhibition of the lipoxygenase (LO) pathway, and particularly 12-LO, reversed this glucose-induced down-regulatory process and restored the rate of hexose transport to the level seen in vascular cells exposed to normal glucose levels. This reversal was accompanied by increased levels of GLUT-1 mRNA and protein, as well as of its plasma-membrane content. Exposure of the vascular cells to elevated glucose concentrations increased by 2-3-fold the levels of cell-associated and secreted 12-hydroxyeicosatetraenoic acid (12-HETE), the product of 12-LO. Inhibition of 15- and 5-LO, cyclo-oxygenases 1 and 2, and eicosanoid-producing cytochrome P450 did not modify the hexose-transport system in vascular cells. These results suggest a role for HETEs in the autoregulation of hexose transport in vascular cells. 8-Iso prostaglandin F(2alpha), a non-enzymic oxidation product of arachidonic acid, had no effect on the hexose-transport system in vascular cells exposed to hyperglycaemic conditions. Taken together, these findings show that hyperglycaemia increases the production rate of 12-HETE, which in turn mediates the down-regulation of GLUT-1 expression and the glucose-transport system in vascular endothelial and smooth-muscle cells. PMID:11853550

Effects of chalcone derivatives on lipoxygenase and cyclooxygenase activities of mouse epidermis.

PubMed

Nakadate, T; Aizu, E; Yamamoto, S; Kato, R

1985-09-01

The effects of chalcone derivatives on 12-lipoxygenase and cyclooxygenase of mouse epidermis were investigated. The chalcone derivatives which have 3,4-dihydroxycinnamoyl structure in the molecule, such as 3,4-dihydroxychalcone, 3,4,2'-trihydroxychalcone, 3,4,4'-trihydroxychalcone and 3,4,2'4'-tetrahydroxychalcone, potently inhibited epidermal 12-lipoxygenase activity. Although some of them also inhibited cyclooxygenase activity at relatively high concentrations, the inhibitory effects of these chalcone derivatives on 12-lipoxygenase were 10 times or more potent than their effects on cyclooxygenase. The chalcone derivatives which have cinnamoyl or 4-hydroxycinnamoyl structure, instead of 3,4-dihydroxycinnamoyl structure, in the molecule, showed little or no inhibitory effects on either 12-lipoxygenase or cyclooxygenase activities. The inhibitory effects of chalcone derivatives on 12-lipoxygenase and cyclooxygenase of mouse epidermis are dependent on the particular structure, i.e. 3,4-dihydroxycinnamoyl structure, of the chalcone derivatives.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

PubMed Central

In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

1997-01-01

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation. PMID:9062372

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

PubMed

In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

1997-03-01

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

5-Lipoxygenase-dependent apoptosis of human lymphocytes in the International Space Station: data from the ROALD experiment.

PubMed

Battista, Natalia; Meloni, Maria A; Bari, Monica; Mastrangelo, Nicolina; Galleri, Grazia; Rapino, Cinzia; Dainese, Enrico; Agrò, Alessandro Finazzi; Pippia, Proto; Maccarrone, Mauro

2012-05-01

The functional adaptation of the immune system to the surrounding environment is also a fundamental issue in space. It has been suggested that a decreased number of lymphocytes might be a cause of immunosuppression, possibly due to the induction of apoptosis. Early activation of 5-lipoxygenase (5-LOX) might play a central role in the initiation of the apoptotic program. The goal of the role of apoptosis in lymphocyte depression (ROALD) experiment, flown on the International Space Station as part of the BIO-4 mission of the European Space Agency, was to ascertain the induction of apoptosis in human lymphocytes under authentic microgravity, and to elucidate the possible involvement of 5-LOX. Our results demonstrate that exposure of human lymphocytes to microgravity for 48 h onboard the ISS remarkably increased apoptotic hallmarks such as DNA fragmentation (∼3-fold compared to ground-based controls) and cleaved-poly (ADP-ribose) polymerase (PARP) protein expression (∼3-fold), as well as mRNA levels of apoptosis-related markers such as p53 (∼3-fold) and calpain (∼4-fold); these changes were paralleled by an early increase of 5-LOX activity (∼2-fold). Our findings provide a molecular background for the immune dysfunction observed in astronauts during space missions, and reveal potential new markers to monitor health status of ISS crew members.

Structural Basis for Ligand Regulation of the Fatty Acid-binding Protein 5, Peroxisome Proliferator-activated Receptor β/δ (FABP5-PPARβ/δ) Signaling Pathway*

PubMed Central

Armstrong, Eric H.; Goswami, Devrishi; Griffin, Patrick R.; Noy, Noa; Ortlund, Eric A.

2014-01-01

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain “activating” fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. PMID:24692551

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein.

PubMed

Maxis, Kelitha; Delalandre, Aline; Martel-Pelletier, Johanne; Pelletier, Jean-Pierre; Duval, Nicolas; Lajeunesse, Daniel

2006-01-01

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-beta (TGF-beta), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-beta also modulated PGE2 production. TGF-beta stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-beta in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)2D3 and TGF-beta depending on their endogenous low and high PGE2 levels.

Role of thymus-eicosanoids in the immune response.

PubMed

Juzan, M; Hostein, I; Gualde, N

1992-08-01

The present review deals with the role(s) of thymus-eicosanoids in the immune response. It reports the production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by cells of the thymus microenvironment and the role(s) of these eicosanoids in the differentiation and the maturation of immature T-cells. The possibility that these products may be involved in tolerance to self is discussed. Briefly, it is likely that cells from the monocyte-macrophage lineage which constitute a part of the thymus microenvironment could contribute to the education of immature thymocytes by both presenting self-antigens and producing eicosanoids. Tolerance to self might result from PGE2-driven apoptosis and/or LTB4-induced generation of suppressor cells.

Effect of arachidonic acid metabolites on thymocyte proliferation.

PubMed

Delebassée, S; Gualde, N

1988-01-01

The influences of prostaglandin E2 (PGE2), 15-hydroxyeicosatetraenoic acid (15-HETE) and leukotrienes (LT) on the proliferative response of mature (PNA-) and immature (PNA+) mouse thymocytes was investigated. Both PNA+ and PNA- thymocytes proliferated when cultured with concanavalin A plus interleukin-2. PGE2 in concentrations of 10(-6) to 10(-9) M caused significant inhibition of proliferation of both PNA+ and PNA- thymocytes in these cultures. In contrast, the lipoxygenase products 15-HETE, LTB4, LTC4 and LTD4 caused marked increases in proliferation of PNA+ thymocytes while having no effect on PNA- cells. Therefore, the effect of leukotrienes on thymocyte proliferation depends upon the level of cell maturation and mainly affects immature PNA+ thymocytes.

Arachidonic acid metabonomics study for understanding therapeutic mechanism of Huo Luo Xiao Ling Dan on rat model of rheumatoid arthritis.

PubMed

Wang, Nannan; Zhao, Xiaoning; Huai, Jiaxin; Li, Yiran; Cheng, Congcong; Bi, Kaishun; Dai, Ronghua

2018-05-10

Huo Luo Xiao Ling Dan (HLXLD), a traditional Chinese medicine (TCM), is commonly used for the treatment of rheumatoid arthritis (RA). To explore the potential therapeutic mechanism of HLXLD on anti-inflammatory activity. A metabolomic approach based on UFLC-MS/MS to profile arachidonic acid (AA) metabolic changes was used. The cyclooxygenase (COX) and lipoxygenase (LOX) catalyzed metabolites in plasma were quantified on 7, 14, 21, and 28 days after the rats injected with Complete Freund's adjuvant and orally administrated with HLXLD, methotrexate and dexamethasone in parallel as the positive control drugs. Nineteen metabolites involved in COX and LOX pathways in RA model group were significant increased compared with normal group (P < 0.05), including 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, 8-HETE, leukotriene B 4 (LTB 4 ), prostaglandin E 2 (PGE 2 ), PGI 2 , PGD 2 , PGF 2α , thromboxane B 2 (TXB 2 ), etc. From day 7 to day 28, the trajectory direction of HLXLD group and positive control groups gradually moved towards the initial space, and the concentrations of AA and its metabolites after HLXLD treatment were significantly reduced in dual pathways compared to control groups. HLXLD induced a substantial change in the AA metabolic profiles through refrain the expression of COX and LOX. The present investigation also highlights that distinct ingredients of this formula tend to inhibit different target to achieve a therapeutic effect. Copyright © 2018. Published by Elsevier B.V.

The effect of trinitrobenzene sulfonic acid (TNB) on colonocyte arachidonic acid metabolism.

PubMed

Stratton, M D; Sexe, R; Peterson, B; Kaminski, D L; Li, A P; Longo, W E

1996-02-01

In previous studies we found that luminal perfusion of the isolated left colon of the rabbit with the hapten, trinitrobenzene, resulted in the production of an acute inflammatory process associated with alterations in eicosanoid metabolism. As the colitis was attenuated by cyclooxygenase inhibitors it is possible that the inflammation was mediated by arachidonic acid metabolites. In the present study it was intended to evaluate the effect of trinitrobenzene on eicosanoid metabolism in transformed human colonic cells by exposing Caco-2++ cells to various doses of trinitrobenzene. Cell injury was evaluated by measuring lactate dehydrogenase levels and cyclooxygenase and lipoxygenase activity was evaluated by measuring prostanoid and leukotriene production. In separate experiments resting and trinitrobenzene stimulated cells were treated with indomethacin and dexamethasone. Trinitrobenzene produced increased prostaglandin E2 and 6-keto prostaglandin F1alpha++ and increased lactate dehydrogenase levels. Leukotriene B4 was significantly increased compared to control values at the highest TNB concentration administered. Indomethacin inhibited the lactate dehydrogenase and prostanoid changes, suggesting that the inflammatory changes produced were mediated by the prostanoids. Dexamethasone administered for 1 hr prior to trinitrobenzene decreased the 6-keto prostaglandin F1alpha but did not alter trinitrobenzene produced changes in lactate dehydrogenase concentrations. Exposure of Caco-2 cells to dexamethasone for 24 hr decreased the trinitrobenzene produced lactate dehydrogenase and eicosanoid changes. The results suggest that trinitrobenzene produces an acute injury to Caco-2 cells that may be mediated by the cyclooxygenase enzymes.

The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils.

PubMed

Doussiere, Jacques; Bouzidi, Farid; Vignais, Pierre V

2002-07-01

In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2- generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac (Doussière J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun.285, 1317-1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A-Sepharose bound to anti-p67phox antibodies, the recovered immunoprecipitate contained the S100 protein, p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose, followed by isoelectric focusing. The S100A8/A9 heterodimeric protein comigrated with the cytosolic phox proteins, and more particularly with p67phox and Rac2, whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell-free system of oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b, but not to a change of affinity for NADPH or O2. In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration of cytosolic phox proteins. Addition of S100A8/A9 to the cell-free system rendered the kinetics fully michaelian. The propensity of S100A8/A9 to bind the cytosolic phox proteins, and the effects of S100A8/A9 on the kinetics of oxidase activation, suggest that S100A8/A9 might be a scaffold protein for the cytosolic phox proteins or might help to deliver arachidonic acid to the oxidase, thus favoring the productive interaction of the cytosolic phox proteins with the membrane-bound flavocytochrome b.

Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

PubMed Central

Altavilla, D; Squadrito, F; Bitto, A; Polito, F; Burnett, BP; Di Stefano, V; Minutoli, L

2009-01-01

Background and purpose: The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. Experimental approach: LPS-stimulated (1 µg·mL−1) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL−1) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. Key results: LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL−1) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE2 and LTB4 levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages. Conclusion and implications: Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression. PMID:19681869

Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content.

PubMed

Koshiishi, Ichiro; Tsuchida, Kazunori; Takajo, Tokuko; Komatsu, Makiko

2006-04-15

Lipoxygenases have been proposed to be a possible factor that is responsible for the pathology of certain diseases, including ischaemic injury. In the peroxidation process of linoleic acid by lipoxygenase, the E,Z-linoleate allyl radical-lipoxygenase complex seems to be generated as an intermediate. In the present study, we evaluated whether E,Z-linoleate allyl radicals on the enzyme are scavenged by radical scavengers. Linoleic acid, the content of which was greater than the dissolved oxygen content, was treated with soya bean lipoxygenase-1 (ferric form) in the presence of radical scavenger, CmP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl). The reaction rate between oxygen and lipid allyl radical is comparatively faster than that between CmP and lipid allyl radical. Therefore a reaction between linoleate allyl radical and CmP was not observed while the dioxygenation of linoleic acid was ongoing. After the dissolved oxygen was depleted, CmP stoichiometrically trapped linoleate-allyl radicals. Accompanied by this one-electron redox reaction, the resulting ferrous lipoxygenase was re-oxidized to the ferric form by hydroperoxylinoleate. Through the adduct assay via LC (liquid chromatography)-MS/MS (tandem MS), four E,Z-linoleate allyl radical-CmP adducts corresponding to regio- and diastereo-isomers were detected in the linoleate/lipoxygenase system, whereas E,E-linoleate allyl radical-CmP adducts were not detected at all. If E,Z-linoleate allyl radical is liberated from the enzyme, the E/Z-isomer has to reach equilibrium with the thermodynamically favoured E/E-isomer. These data suggested that the E,Z-linoleate allyl radicals were not liberated from the active site of lipoxygenase before being trapped by CmP. Consequently, we concluded that the lipid allyl radicals complexed with lipoxygenase could be scavenged by radical scavengers at lower oxygen content.

Radical scavenger can scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content

PubMed Central

Koshiishi, Ichiro; Tsuchida, Kazunori; Takajo, Tokuko; Komatsu, Makiko

2006-01-01

Lipoxygenases have been proposed to be a possible factor that is responsible for the pathology of certain diseases, including ischaemic injury. In the peroxidation process of linoleic acid by lipoxygenase, the E,Z-linoleate allyl radical–lipoxygenase complex seems to be generated as an intermediate. In the present study, we evaluated whether E,Z-linoleate allyl radicals on the enzyme are scavenged by radical scavengers. Linoleic acid, the content of which was greater than the dissolved oxygen content, was treated with soya bean lipoxygenase-1 (ferric form) in the presence of radical scavenger, CmP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl). The reaction rate between oxygen and lipid allyl radical is comparatively faster than that between CmP and lipid allyl radical. Therefore a reaction between linoleate allyl radical and CmP was not observed while the dioxygenation of linoleic acid was ongoing. After the dissolved oxygen was depleted, CmP stoichiometrically trapped linoleate-allyl radicals. Accompanied by this one-electron redox reaction, the resulting ferrous lipoxygenase was re-oxidized to the ferric form by hydroperoxylinoleate. Through the adduct assay via LC (liquid chromatography)–MS/MS (tandem MS), four E,Z-linoleate allyl radical–CmP adducts corresponding to regio- and diastereo-isomers were detected in the linoleate/lipoxygenase system, whereas E,E-linoleate allyl radical–CmP adducts were not detected at all. If E,Z-linoleate allyl radical is liberated from the enzyme, the E/Z-isomer has to reach equilibrium with the thermodynamically favoured E/E-isomer. These data suggested that the E,Z-linoleate allyl radicals were not liberated from the active site of lipoxygenase before being trapped by CmP. Consequently, we concluded that the lipid allyl radicals complexed with lipoxygenase could be scavenged by radical scavengers at lower oxygen content. PMID:16396633

Arachidonic acid metabolites follow the preferential course of cyclooxygenase pathway for the basal tone in the internal anal sphincter.

PubMed

de Godoy, Márcio A F; Rattan, Neeru; Rattan, Satish

2009-04-01

Present studies determined the roles of the cyclooxygenase (COX) versus the lipoxygenase (LO) pathways in the metabolic pathway of arachidonic acid (AA) in the internal anal sphincter (IAS) tone. Studies were performed in the rat IAS versus the nontonic rectal smooth muscle (RSM). Indomethacin, the dual COX inhibitor, but not nordihydroguaiaretic acid (NDGA), the LO inhibitor, produced a precipitous decrease in the IAS tone. However, when added in the background of indomethacin, NDGA caused significant reversal of the IAS tone. These inhibitors had no significant effect on the RSM. To follow the significance of COX versus LO pathways, we examined the effects of AA and its metabolites. In the IAS, AA caused an increase in the IAS tone (Emax=38.8+/-3.0% and pEC50=3.4+/-0.1). In the RSM, AA was significantly less efficacious and potent (Emax=11.3+/-3.5% and pEC50=2.2+/-0.3). The AA metabolites (via COXs) PGF2alpha and U-46619 (a stable analog of thromboxane A2) produced increases in the IAS tone and force in the RSM. Conversely, AA metabolites (via 5-LO) lipoxin A4, 5-HETE, and leukotriene C4 decreased the IAS tone. Finally, the contractile effects of AA in the IAS were selectively attenuated by the COX-1 but not the COX-2 inhibitor. Collectively, the specific effects of AA and the COX inhibitor, the Western blot and RT-PCR analyses showing specifically higher levels of COX-1, suggest a preferential role of the COX (specifically COX-1) pathway versus the LO in the regulation of the IAS tone.

BIOLOGICAL AND PHYTOCHEMICAL INVESTIGATIONS OF EXTRACTS FROM PTEROCARPUS ERINACEUS POIR (FABACEAE) ROOT BARKS

PubMed Central

Noufou, Ouédraogo; Anne-Emmanuelle, Hay; Claude W, Ouédraogo Jean; Richard, Sawadogo W; André, Tibiri; Marius, Lompo; Jean-baptiste, Nikiema; Jean, Koudou; Marie-Genevieve, Dijoux-Franca; Pierre, Guissou Innocent

2017-01-01

Background: Pterocarpus erinaceus Poir. belonging to Fabacae familly is used as medicinal plant in Burkina Faso’s folk medicine. Roots of P. erinaceus are used to treat ulcer, stomach ache and inflammatory diseases. The objective of the present study was to carry out phytochemical composition of methanol (MeOH) and dichloromethane (DCM) extracts from Pterocarpus erinaceus roots, to isolate pure compounds, and to evaluate their pharmacological activities. Methods: Chromatographic fractionation led to the isolation of active components of the extracts. The structures were established by NMR analysis and comparison with data from literature. The anti-inflammatory activity was evaluated using croton oil-induced edema of mice ear as well as the effect of extracts against lipoxygenase and lipid peroxidation was evaluated. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Cupric-reducing antioxidant capacity (CUPRAC) methods were used to evaluate the antioxidant activity of the extracts. Results: Friedelin (1), 3a-hydroxyfriedelan-2-one (2), a-sophoradiol (3) and stigmasterol (4) were isolated from DCM extract and maltol-6-O-apiofuranoside-glucopyranoside (5) isolated from MeOH. DCM extract and friedelin, 3a-hydroxyfriedelan-2-one, a-sophoradiol showed a significant anti-inflammatory effect against ear edema. Friedelin (1), α-sophoradiol (3) and maltol-6-O-apiofuranoside-glucopyranoside (5) exhibited lipoxygenase inhibition. MeOH extract (100 μg/mL) inhibited lipoxygenase and lipid peroxidation activities at 45.1 ± 3% and 30.7 ± 0.5% respectively. MeOH extract, ethyl acetate fraction and butanol fraction exhibited antioxidant property with both two methods used. Conclusion: The results suggested that the extracts and compounds from roots of Pterocarpus erinaceus possessed local anti-inflammatory effect, antioxidant properties and inhibitor effect against lipoxygenase and lipid peroxidation activities. PMID:28480397

Effects of a garlic-derived principle (ajoene) on aggregation and arachidonic acid metabolism in human blood platelets.

PubMed

Srivastava, K C; Tyagi, O D

1993-08-01

When garlic cloves are chopped or crushed several dialkyl thiosulfinates are rapidly formed by the action of the enzyme alliin lyase or alliinase (EC 4.4.1.4) on S(+)-alkyl-L-cysteine sulfoxides. Allicin (diallyl thiosulfinate or allyl 2-propene thiosulfinate) is the dominant thiosulfinate released. A variety of sulfur containing compounds are formed from allicin and other thiosulfinates depending on the way in which garlic is handled. One such compound identified recently is ajoene which has been reported to possess antithrombotic properties. We present here data on the antiplatelet properties of ajoene together with its effects on the metabolism of arachidonic acid (AA) in intact platelets. Thus, ajoene was found to inhibit platelet aggregation induced by AA, adrenaline, collagen, adenosine diphosphate (ADP) and calcium ionophore A23187; the nature of the inhibition was irreversible. In washed platelets stimulated by labelled arachidonate, ajoene inhibited the formation of thromboxane A2; 12-lipoxygenase product(s) were reduced at higher ajoene concentrations. This garlic-derived substance inhibited the incorporation of labelled AA into platelet phospholipids at higher concentration. In labelled platelets, on stimulation with either calcium ionophore A23187 or collagen, reduced amounts of thromboxane and 12-HETE (12-hydroxyeicosatetraenoic acid) were produced in ajoene-treated platelets compared to control platelets. This substance had no effect on the deacylation of platelet phospholipids. The results suggest that at least one of the mechanisms by which ajoene shows antiplatelet effects could be related to altered metabolism of AA.

Structural basis for ligand regulation of the fatty acid-binding protein 5, peroxisome proliferator-activated receptor β/δ (FABP5-PPARβ/δ) signaling pathway.

PubMed

Armstrong, Eric H; Goswami, Devrishi; Griffin, Patrick R; Noy, Noa; Ortlund, Eric A

2014-05-23

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain "activating" fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Specific Caleosin/Peroxygenase and Lipoxygenase Activities Are Tissue-Differentially Expressed in Date Palm (Phoenix dactylifera L.) Seedlings and Are Further Induced Following Exposure to the Toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin

PubMed Central

Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Rahman, Farzana; Hassan, Mehedi; Murphy, Denis J.

2017-01-01

Two caleosin/peroxygenase isoforms from date palm, Phoenix dactylifera L., PdCLO2 and PdCLO4, were characterized with respect to their tissue expression, subcellular localization, and oxylipin pathway substrate specificities in developing seedlings. Both PdCLO2 and PdCLO4 had peroxygenase activities that peaked at the mid-stage (radicle length of 2.5 cm) of seedling growth and were associated with the lipid droplet (LD) and microsomal fractions. Recombinant PdCLO2 and PdCLO4 proteins heterologously expressed in yeast cells were localized in both LD and microsomal fractions. Each of the purified recombinant proteins exhibited peroxygenase activity but they were catalytically distinct with respect to their specificity and product formation from fatty acid epoxide and hydroxide substrates. We recently showed that date palm CLO genes were upregulated following exposure to the potent toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Hanano et al., 2016), and we show here that transcripts of 9- and 13-lipoxygenase (LOX) genes were also induced by TCDD exposure. At the enzyme level, 9-LOX and 13-LOX activities were present in a range of seedling tissues and responded differently to TCDD exposure, as did the 9- and 13-fatty acid hydroperoxide reductase activities. This demonstrates that at least two branches of the oxylipin pathway are involved in responses to the environmental organic toxin, TCDD in date palm. PMID:28111588

Prevalence of 5-lipoxygenase expression in canine osteosarcoma and the effects of a dual 5-lipoxygenase/cyclooxygenase inhibitor on osteosarcoma cells in vitro and in vivo.

PubMed

Goupil, R C; Bushey, J J; Peters-Kennedy, J; Wakshlag, J J

2012-09-01

Canine osteosarcoma is an insidious disease with few effective treatment modalities; therefore, use of pharmacologic intervention to improve mortality or morbidity is constantly sought. The use of cyclooxygenase enzyme inhibitors has been an area of interest with limited efficacy based on retrospective examination of tumor expression and in vivo cell proliferation models. Recently, examination of dual cyclooxygenase and 5-lipoxygenase inhibitors in human and canine oncology suggests that 5-lipoxygenase inhibitors may be an effective approach in vitro and during tumor induction in rodent models. Therefore, the authors decided to examine 5-lipoxygenase expression in primary canine osteosarcoma samples and have shown that approximately 65% of osteosarcomas label positive for cytoplasmic 5-lipoxygenase. Further examination of a cell culture and xenograft model shows similar 5-lipoxygenase expression. Surprisingly, a canine 5-lipoxygenase inhibitor (tepoxalin) significantly reduced cell proliferation at physiologic doses in vitro and diminished xenograft tumor growth in nude mice, suggesting that further investigation is needed. Traditionally, 5-lipoxygense leads to production of lipid mediators, such as leukotriene B(4) and 5-oxo-eicosatetraenoic acid, which, when added back to the media of tepoxalin-treated cells, did not recover cell proliferation. The lack of nuclear staining in primary and xenografted tumors and the lack of response to eicoasanoids suggest that lipid mediator production is not the primary means by which tepoxalin acts to alter proliferation. Regardless of the mechanisms involved in retarding cell proliferation, future investigation is warranted.

Obesity superimposed on aging magnifies inflammation and delays the resolving response after myocardial infarction

PubMed Central

Lopez, Elizabeth F.; Kabarowski, Janusz H.; Ingle, Kevin A.; Kain, Vasundhara; Barnes, Stephen; Crossman, David K.; Lindsey, Merry L.

2014-01-01

Polyunsaturated fatty acid (PUFA) intake has increased over the last 100 yr, contributing to the current obesogenic environment. Obesity and aging are prominent risk factors for myocardial infarction (MI). How obesity interacts with aging to alter the post-MI response, however, is unclear. We tested the hypothesis that obesity in aging mice would impair the resolution of post-MI inflammation. PUFA diet (PUFA aging group) feeding to 12-mo-old C57BL/6J mice for 5 mo showed higher fat mass compared with standard lab chow (LC)-fed young (LC young group; 3–5 mo old) or aging alone control mice (LC aging group). LC young, LC aging, and PUFA aging mice were subjected to coronary artery ligation to induce MI. Despite similar infarct areas post-MI, plasma proteomic profiling revealed higher VCAM-1 in the PUFA aging group compared with LC young and LC aging groups, leading to increased neutrophil infiltration in the PUFA aging group (P < 0.05). Macrophage inflammatory protein-1γ and CD40 were also increased at day 1, and myeloperoxidase remained elevated at day 5, an observation consistent with delayed wound healing in the PUFA aging group. Lipidomic analysis showed higher levels of arachidonic acid and 12(S)-hydroxyeicosatetraenoic acid at day 1 post-MI in the PUFA aging group compared with the LC aging group (all P < 0.05), thereby mediating neutrophil extravasation in the PUFA aging group. The inflammation-resolving enzymes 5-lipoxygenase, cyclooxygenase-2, and heme oxyegnase-1 were altered to delay wound healing post-MI in the PUFA aging group compared with LC young and LC aging groups. PUFA aging magnifies the post-MI inflammatory response and impairs the healing response by stimulating prolonged neutrophil trafficking and proinflammatory lipid mediators. PMID:25485899

Group X Phospholipase A2 Stimulates the Proliferation of Colon Cancer Cells by Producing Various Lipid Mediators

PubMed Central

Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G.; Payré, Christine; Mounier, Carine M.; Talvinen, Kati A.; Laine, Veli J. O.; Nevalainen, Timo J.; Gelb, Michael H.

2009-01-01

Among mammalian secreted phospholipases A2 (sPLA2s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA2 [mouse (m)GX] is one of the most highly expressed PLA2 in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA2s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA2 inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA2α and M-type sPLA2 receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA2 mitogenic effects. Together, our results indicate that group X sPLA2 may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression. PMID:19602573

Group X phospholipase A2 stimulates the proliferation of colon cancer cells by producing various lipid mediators.

PubMed

Surrel, Fanny; Jemel, Ikram; Boilard, Eric; Bollinger, James G; Payré, Christine; Mounier, Carine M; Talvinen, Kati A; Laine, Veli J O; Nevalainen, Timo J; Gelb, Michael H; Lambeau, Gérard

2009-10-01

Among mammalian secreted phospholipases A2 (sPLA(2)s), the group X enzyme has the most potent hydrolyzing capacity toward phosphatidylcholine, the major phospholipid of cell membrane and lipoproteins. This enzyme has recently been implicated in chronic inflammatory diseases such as atherosclerosis and asthma and may also play a role in colon tumorigenesis. We show here that group X sPLA(2) [mouse (m)GX] is one of the most highly expressed PLA(2) in the mouse colon and that recombinant mouse and human enzymes stimulate proliferation and mitogen-activated protein kinase activation of various colon cell lines, including Colon-26 cancer cells. Among various recombinant sPLA(2)s, mGX is the most potent enzyme to stimulate cell proliferation. Based on the use of sPLA(2) inhibitors, catalytic site mutants, and small interfering RNA silencing of cytosolic PLA(2)alpha and M-type sPLA(2) receptor, we demonstrate that mGX promotes cell proliferation independently of the receptor and via its intrinsic catalytic activity and production of free arachidonic acid and lysophospholipids, which are mitogenic by themselves. mGX can also elicit the production of large amounts of prostaglandin E2 and other eicosanoids from Colon-26 cells, but these lipid mediators do not play a role in mGX-induced cell proliferation because inhibitors of cyclooxygenases and lipoxygenases do not prevent sPLA(2) mitogenic effects. Together, our results indicate that group X sPLA(2) may play an important role in colon tumorigenesis by promoting cancer cell proliferation and releasing various lipid mediators involved in other key events in cancer progression.

Structure and Ligand Based Drug Design Strategies in the Development of Novel 5-LOX Inhibitors

PubMed Central

Aparoy, Polamarasetty; Kumar Reddy, Kakularam; Reddanna, Pallu

2012-01-01

Lipoxygenases (LOXs) are non-heme iron containing dioxygenases involved in the oxygenation of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA). Depending on the position of insertion of oxygen, LOXs are classified into 5-, 8-, 9-, 12- and 15-LOX. Among these, 5-LOX is the most predominant isoform associated with the formation of 5-hydroperoxyeicosatetraenoic acid (5-HpETE), the precursor of non-peptido (LTB4) and peptido (LTC4, LTD4, and LTE4) leukotrienes. LTs are involved in inflammatory and allergic diseases like asthma, ulcerative colitis, rhinitis and also in cancer. Consequently 5-LOX has become target for the development of therapeutic molecules for treatment of various inflammatory disorders. Zileuton is one such inhibitor of 5-LOX approved for the treatment of asthma. In the recent times, computer aided drug design (CADD) strategies have been applied successfully in drug development processes. A comprehensive review on structure based drug design strategies in the development of novel 5-LOX inhibitors is presented in this article. Since the crystal structure of 5-LOX has been recently solved, efforts to develop 5-LOX inhibitors have mostly relied on ligand based rational approaches. The present review provides a comprehensive survey on these strategies in the development of 5-LOX inhibitors. PMID:22680930

[Inhibiting properties of stable nitroxyl radicals in reactions of linoleic acid and linoleyl alcohol oxidation catalyzed by 5-lipoxygenase].

PubMed

Kharchenko, O V; Kharitonenko, A I; Vovk, A I; Kukhar', V P; Babiĭ, L V; Khil'chevskiĭ, A N; Mel'nik, A K

2005-01-01

The inhibiting effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and its 4-substituted derivatives in reactions of linoleyl acid or linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase were investigated. Inhibiting properties of stable nitroxyl radicals in presence of lubrol and SDS were reduced at the transition from TEMPO to 4-hydroxy-TEMPO or 4-amino-TEMPO and increased at use of adamantane-1-carboxylic or 3-methyladamantane-1-carboxylic acid 1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl esters. Enzyme activity at saturating concentrations of inhibitor was not suppressed completely, and decreased up to the certain level determined by the substrate nature. The dependence of partial inhibition efficiency on rotational correlation time of stable nitroxides in model micellar systems were analysed. It was supposed that 5-lipoxygenase inhibition includes the interaction of hydrophobic nitroxide with radical intermediate formed in enzymatic process.

Morusinol extracted from Morus alba inhibits arterial thrombosis and modulates platelet activation for the treatment of cardiovascular disease.

PubMed

Lee, Jung-Jin; Yang, Hyun; Yoo, Yeong-Min; Hong, Seong Su; Lee, Dongho; Lee, Hyun-Jung; Lee, Hak-Ju; Myung, Chang-Seon; Choi, Kyung-Chul; Jeung, Eui-Bae

2012-01-01

Morus alba (white mulberry) has been used in traditional Chinese medicine as an anti-headache, diuretic, expectorant, and anti-diabetic agent. In previous studies, extracts of Morus alba demonstrated favorable biological properties, such as antioxidant activity, suppression of lipoxygenase (LOX)-1, cytotoxicity against cancer cells, and inhibition of the invasion and migration of cancer cells. This study further evaluated the effects of morusinol, a flavonoid derived from Morus alba root bark, on platelet aggregation and thromboxane B(2) (TXB(2) formation in vitro and thrombus formation in vivo. The antiplatelet potential of morusinol was measured using in vitro rabbit platelet aggregation and TXB(2) formation assays. Arterial thrombus formation was investigated using an in vivo ferric chloride (FeCl(3)-induced thrombosis model. Morusinol significantly inhibited collagen- and arachidonic acid-induced platelet aggregation and TXB(2) formation in cultured platelets in a concentration-dependent manner. Thrombus formation was reduced by 32.1, 42.0, and 99.0% for collagen-induced TXB(2) formation, and 8.0, 24.1, and 29.2% for arachadonic acid-induced TXB(2) formation, with 5, 10, and 30 µg/mL morusinol, respectively. Moreover, oral morusinol (20 mg/kg) or aspirin (20 mg/kg) for three days significantly increased the time to occlusion in vivo by 20.3±5.0 or 6.8±2.9 min, respectively, compared with the control (1% CMC, carboxymethyl cellulose). Taken together, these results indicate that morusinol may significantly inhibit arterial thrombosis in vivo due to antiplatelet activity. Thus, morusinol may exert beneficial effects on transient ischemic attacks or stroke via the modulation of platelet activation.

Evaluation of the sensitizing potential of food proteins using two mouse models.

PubMed

Smit, Joost; Zeeuw-Brouwer, Mary-Lène de; van Roest, Manon; de Jong, Govardus; van Bilsen, Jolanda

2016-11-16

The current methodology to identify allergenic food proteins is effective in identifying those that are likely to cross-react with known allergens. However, most assays show false positive results for low/non-allergens. Therefore, an ex vivo/in vitro DC-T cell assay and an in vivo mouse model were used to distinguish known allergenic food proteins (Ara h 1, β-Lactoglobulin, Pan b 1, bovine serum albumin, whey protein isolate) from low/non allergenic food proteins (soy lipoxygenase, gelatin, beef tropomyosin, rubisco, Sola t 1). CD4+ T cells from protein/alum-immunized mice were incubated with corresponding protein-pulsed bone marrow-derived DC and analyzed for cytokine release. All known allergens induced Th2 responses in vitro, whereas soy lipoxygenase, gelatin or beef tropomyosin did not. Sola t 1 and rubisco induced a more generalized T cell response due to endotoxin contamination, indicating the endotoxin-sensitivity of the DC-T assay. To analyze responses in vivo, mice were orally sensitized on days 0 and 7. Known allergens induced IgE and mMCP-1 release upon oral challenge at day 16, whereas the low/non-allergens did not. Both the DC-T cell assay and the mouse model were able to distinguish 5 known allergens from 5 low/non-allergens and may be useful to identify novel allergenic food proteins. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

5-Lipoxygenase-Dependent Recruitment of Neutrophils and Macrophages by Eotaxin-Stimulated Murine Eosinophils

PubMed Central

Luz, Ricardo Alves; Xavier-Elsas, Pedro; de Luca, Bianca; Masid-de-Brito, Daniela; Cauduro, Priscila Soares; Gondar Arcanjo, Luiz Carlos; Cordeiro Faria dos Santos, Ana Carolina; de Oliveira, Ivi Cristina Maria; Gaspar-Elsas, Maria Ignez Capella

2014-01-01

The roles of eosinophils in antimicrobial defense remain incompletely understood. In ovalbumin-sensitized mice, eosinophils are selectively recruited to the peritoneal cavity by antigen, eotaxin, or leukotriene(LT)B4, a 5-lipoxygenase (5-LO) metabolite. 5-LO blockade prevents responses to both antigen and eotaxin. We examined responses to eotaxin in the absence of sensitization and their dependence on 5-LO. BALB/c or PAS mice and their mutants (5-LO-deficient ALOX; eosinophil-deficient GATA-1) were injected i.p. with eotaxin, eosinophils, or both, and leukocyte accumulation was quantified up to 24 h. Significant recruitment of eosinophils by eotaxin in BALB/c, up to 24 h, was accompanied by much larger numbers of recruited neutrophils and monocytes/macrophages. These effects were abolished by eotaxin neutralization and 5-LO-activating protein inhibitor MK886. In ALOX (but not PAS) mice, eotaxin recruitment was abolished for eosinophils and halved for neutrophils. In GATA-1 mutants, eotaxin recruited neither neutrophils nor macrophages. Transfer of eosinophils cultured from bone-marrow of BALB/c donors, or from ALOX donors, into GATA-1 mutant recipients, i.p., restored eotaxin recruitment of neutrophils and showed that the critical step dependent on 5-LO is the initial recruitment of eosinophils by eotaxin, not the secondary neutrophil accumulation. Eosinophil-dependent recruitment of neutrophils in naive BALB/c mice was associated with increased binding of bacteria. PMID:24723744

Corneal reepithelialization and anti-inflammatory agents.

PubMed Central

Srinivasan, B D

1982-01-01

These studies have demonstrated that nonsteroidal anti-inflammatory agents (cyclooxygenase and lipoxygenase inhibitors) can inhibit PMN arrival in the tear fluid following corneal injury but do not inhibit the reepithelialization either by corneal epithelial cells or by conjunctival epithelial cells. Therefore, they can be used safely in ocular inflammatory conditions even when corneal epithelial defects are present. Corticosteroids, on the other hand, inhibit reepithelialization by conjunctival epithelial cells and not by corneal epithelial cells in the doses tested. This inhibition does not occur with pretreatment prior to injury, suggesting that corticosteroids can be used clinically in conditions that have intact corneal epithelium without fear of slowing down wound healing should epithelial defects occur when not on steroid therapy. Furthermore, the steroid inhibition is temporary since there is a breakthrough in steroid inhibition with time, and occurs only if the steroids have been used shortly after deepithelialization. The steroid inhibition can be reversed by specific steroid antagonist, indicating that the steroid effect is mediated through specific receptors. An exciting and new hypothesis proposes that corticosteroids induce the formation of an inhibitory protein that inhibits the phospholipase enzyme to cause a block in arachidonic acid release from cell membranes. This mechanism of action may also be prevalent in the steroid effect on corneal reepithelialization, and experiments are under way to isolate this inhibitory protein from steroid-treated conjunctival epithelium. This isolation and pharmacologic characterization of this inhibitory protein is of obvious advantage to the field of ophthalmic therapeutics since this protein may have the anti-inflammatory potential of the steroids without their steroid sideeffects. Images FIGURE 3 a FIGURE 3 b PMID:6763806

Melatonin suppresses activation of hepatic stellate cells through RORα-mediated inhibition of 5-lipoxygenase.

PubMed

Shajari, Shiva; Laliena, Almudena; Heegsma, Janette; Tuñón, María Jesús; Moshage, Han; Faber, Klaas Nico

2015-10-01

Liver fibrosis is scar tissue resulting from an uncontrolled wound-healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture-activated rat HSC. Melatonin dose-dependently suppressed the expression of HSC activation markers Col1a1 and alpha-smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor-related orphan receptor-alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5-lipoxygenase (5-LO). The pharmacological 5-LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα-mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor

PubMed Central

Maddipati, Krishna Rao; Romero, Roberto; Chaiworapongsa, Tinnakorn; Zhou, Sen-Lin; Xu, Zhonghui; Tarca, Adi L.; Kusanovic, Juan Pedro; Munoz, Hernan; Honn, Kenneth V.

2014-01-01

Lipid mediators play an important role in reproductive biology, especially, in parturition. Enhanced biosynthesis of eicosanoids, such as prostaglandin E2 (PGE2) and PGF2α, precedes the onset of labor as a result of increased expression of inducible cyclooxygenase 2 (COX-2) in placental tissues. Metabolism of arachidonic acid results in bioactive lipid mediators beyond prostaglandins that could significantly influence myometrial activity. Therefore, an unbiased lipidomic approach was used to profile the arachidonic acid metabolome of amniotic fluid. In this study, liquid chromatography–mass spectrometry was used for the first time to quantitate these metabolites in human amniotic fluid by comparing patients at midtrimester, at term but not in labor, and at term and in spontaneous labor. In addition to exposing novel aspects of COX pathway metabolism, this lipidomic study revealed a dramatic increase in epoxygenase- and lipoxygenase-pathway-derived lipid mediators in spontaneous labor with remarkable product selectivity. Despite their recognition as anti-inflammatory lipid mediators and regulators of ion channels, little is known about the epoxygenase pathway in labor. Epoxygenase pathway metabolites are established regulators of vascular homeostasis in cardiovascular and renal physiology. Their presence as the dominant lipid mediators in spontaneous labor at term portends a yet undiscovered physiological function in parturition.—Maddipati, K. R., Romero, R., Chaiworapongsa, T., Zhou, S.-L., Xu, Z., Tarca, A. L., Kusanovic, J. P., Munoz, H., Honn, K. V. Eicosanomic profiling reveals dominance of the epoxygenase pathway in human amniotic fluid at term in spontaneous labor. PMID:25059230

DOE Office of Scientific and Technical Information (OSTI.GOV)

G Deb; K Boeshanes; W Idler

Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34 kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-LOX resulted in increased solubility of 12R-LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16 kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusionmore » method using 1.5 M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2{sub 1}, with unit-cell parameters a = 138.97, b = 266.11, c = 152.26 {angstrom}, {beta} = 101.07{sup o}. Based on the calculated Matthews coefficient (3.1 {angstrom}3 Da{sup -1}), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4 {angstrom} resolution using synchrotron radiation.« less

Evidence for differential activation of arachidonic acid metabolism in formylpeptide- and macrophage-activation-factor-stimulated guinea-pig macrophages.

PubMed Central

Homma, Y; Hashimoto, T; Nagai, Y; Takenawa, T

1985-01-01

Alterations of phospholipid and arachidonic acid metabolism were studied by treatment of guinea-pig peritoneal-exudate macrophages with chemotactic peptide, formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) and macrophage activation factor (MAF). The chemotactic peptide caused a rapid rearrangement in inositol phospholipids, including a breakdown of polyphosphoinositides within 30s, followed by a resultant formation of phosphatidylinositol (PI), diacylglycerol, phosphatidic acid and non-esterified arachidonic acid within 5 min. In addition to these sequential alterations, arachidonic acid was released mainly from PI. On the other hand, MAF induced a slow liberation of arachidonic acid, mainly from phosphatidylethanolamine (PE) and phosphatidylcholine (PC) by phospholipase A2 after the incubation period of 30 min, but not any rapid changes in phospholipids. Treatment of macrophages for 15 min with fMet-Leu-Phe produced the leukotrienes (LTs) B4, C4 and D4, prostaglandins (PG) E2 and F2 alpha and thromboxane (TX) B2. In contrast, MAF could not stimulate the production of arachidonic acid metabolites during the incubation period of 15 min, but could enhance that of PGE2, PGF2 alpha, TXB2 and hydroxyeicosatetraenoic acids at 6 h. However, the stimulated formation of LTs was not detected at any time. These results indicate that the effects of fMet-Leu-Phe on both phospholipid and arachidonic acid metabolism are very different from those mediated by MAF. PMID:3931627

Isomer distribution of hydroxyoctadecadienoates (HODE) and hydroxyeicosatetraenoates (HETE) produced in the plasma oxidation mediated by peroxyl radical, peroxynitrite, hypochlorite, 15-lipoxygenase, and singlet oxygen.

PubMed

Umeno, Aya; Morita, Mayuko; Yoshida, Yasukazu; Naito, Yuji; Niki, Etsuo

2017-12-01

Free and ester forms of unsaturated fatty acids and cholesterol are oxidized in vivo by multiple oxidants to give diverse products. Some lipid oxidation is mediated by enzymes to selectively give specific products, while others proceed randomly to produce mixtures of many kinds of regioisomers and stereoisomers. The efficacy of antioxidants against lipid oxidation depends on the nature of the oxidants and therefore the identification of oxidant is important for understanding the roles and effects of lipid oxidation and antioxidants in vivo. In the present study, the isomer distribution of hydro(pero)xyoctadecadienoates (H(p)ODEs) and hydro(pero)xyeicosatetraenoates (H(p)ETEs), the most abundant lipid oxidation products found in human plasma, produced in the oxidation of plasma by peroxyl radicals, peroxynitrite, hypochlorite, 15-lipoxygenase, and singlet oxygen were examined. It was shown that 9- and 13-(E,E)-HODEs, 13(S)-(Z,E)-HODE, and 10- and 12-(Z,E)-HODEs were specific lipid oxidation products by free radical, 15-lipoxygenase, and singlet oxygen, respectively. The isomer distribution of HODEs produced by peroxynitrite was similar to that by peroxyl radical, suggesting that the peroxynitrite mediated lipid oxidation proceeds by free radical mechanisms. The production of HODEs and HETEs by hypochlorite was very small. HODEs may be a better biomarker than HETEs since linoleates are oxidized by simpler mechanisms than arachidonates and all the HODEs isomers can be quantified more easily. These products may be used as specific biomarkers for the identification of responsible oxidants and for the assessment of oxidant-specific lipid oxidation levels and effects of antioxidants in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular basis of activation of the arachidonate-regulated Ca2+ (ARC) channel, a store-independent Orai channel, by plasma membrane STIM1

PubMed Central

Thompson, Jill L; Shuttleworth, Trevor J

2013-01-01

Currently, Orai proteins are known to encode two distinct agonist-activated, highly calcium-selective channels: the store-operated Ca2+ release-activated Ca2+ (CRAC) channels, and the store-independent, arachidonic acid-activated ARC channels. Surprisingly, whilst the trigger for activation of these channels is entirely different, both depend on stromal interacting molecule 1 (STIM1). However, whilst STIM1 in the endoplasmic reticulum membrane is the critical sensor for the depletion of this calcium store that triggers CRAC channel activation, it is the pool of STIM1 constitutively resident in the plasma membrane that is essential for activation of the ARC channels. Here, using a variety of approaches, we show that the key domains within the cytosolic part of STIM1 identified as critical for the activation of CRAC channels are also key for activation of the ARC channels. However, examination of the actual steps involved in such activation reveal marked differences between these two Orai channel types. Specifically, loss of calcium from the EF-hand of STIM1 that forms the key initiation point for activation of the CRAC channels has no effect on ARC channel activity. Secondly, in marked contrast to the dynamic and labile nature of interactions between STIM1 and the CRAC channels, STIM1 in the plasma membrane appears to be constitutively associated with the ARC channels. Finally, specific mutations in STIM1 that induce an extended, constitutively active, conformation for the CRAC channels actually prevent activation of the ARC channels by arachidonic acid. Based on these findings, we propose that the likely role of arachidonic acid lies in inducing the actual gating of the channel. PMID:23690558

Molecular characterization of lipoxygenase genes on chromosome 4BS in Chinese bread wheat (Triticum aestivum L.).

PubMed

Zhang, Fuyan; Chen, Feng; Wu, Peipei; Zhang, Ning; Cui, Dangqun

2015-08-01

This study cloned two novel TaLox genes on chromosome of 4BS and developed a co-dominant marker, Lox-B23, in bread wheat that showed highly significant association with lipoxygenase activity. Lipoxygenase (Lox), a critical enzyme in the carotenoid biosynthetic pathway, significantly influences the color and processing quality of wheat-based products. Two novel Lox genes, designated TaLox-B2 and TaLox-B3, were cloned on chromosome 4BS of Chinese bread wheat. The deduced amino acid sequence showed that both TaLox-B2 and TaLox-B3 genes encoded an 861-aa protein and possessed a lipoxygenase superfamily domain at the 170-838 interval. Two different TaLox-B2 alleles, designated TaLox-B2a and TaLox-B2b, were subsequently discovered. A co-dominant marker, Lox-B23, was developed based on sequences of TaLox-B2a, TaLox-B2b, and TaLox-B3 genes to precisely distinguish these three alleles in Chinese bread cultivars. Among five allelic combinations of Lox genes at Lox-B1, Lox-B2, and Lox-B3 loci, wheat cultivars with TaLox-B1a/TaLox-B2a/TaLox-B3a combination exhibited the highest Lox activity, whereas those with TaLox-B1a/TaLox-B2b/TaLox-B3b combination significantly showed the lowest Lox activity. A RIL population was used to evaluate the influence of TaLox-B3a gene on Lox activity. Results showed that TaLox-B3a gene could significantly increase the Lox activity in bread wheat. Physical mapping indicated that both TaLox-B2 and TaLox-B3 genes were located on chromosome 4BS in bread wheat. This study provides useful information to further understand the molecular and genetic bases of Lox activity in bread wheat.

Chronic Arachidonic Acid Administration Decreases Docosahexaenoic Acid- and Eicosapentaenoic Acid-Derived Metabolites in Kidneys of Aged Rats.

PubMed

Katakura, Masanori; Hashimoto, Michio; Inoue, Takayuki; Mamun, Abdullah Al; Tanabe, Yoko; Arita, Makoto; Shido, Osamu

2015-01-01

Arachidonic acid (ARA) metabolites produced by cyclo-oxygenase and lipoxygenase are important mediators maintaining physiological renal function. However, the effects of exogenous ARA on kidney function in vivo remain unknown. This study examined the effects of long-term oral ARA administration on normal renal function as well as inflammation and oxidative stress in aged rats. In addition, we measured levels of renal eicosanoids and docosanoids using liquid chromatography-tandem mass spectrometry. Control or ARA oil (240 mg/kg body weight/day) was orally administered to 21-month-old Wistar rats for 13 weeks. Levels of plasma creatinine, blood urea nitrogen, inflammatory and anti-inflammatory cytokines, reactive oxygen species, and lipid peroxidation were not significantly different between the two groups. The ARA concentration in the plasma, kidney, and liver increased in the ARA-administered group. In addition, levels of free-form ARA, prostaglandin E2, and 12- and 15-hydroxyeicosatetraenoic acid increased in the ARA-administered group, whereas renal concentration of docosahexaenoic acid and eicosapentaenoic acid decreased in the ARA-administered group. Levels of docosahexaenoic acid-derived protectin D1, eicosapentaenoic acid-derived 5-, and 18-hydroxyeicosapentaenoic acids, and resolvin E2 and E3 decreased in the ARA-administered group. Our results indicate that long-term ARA administration led to no serious adverse reactions under normal conditions and to a decrease in anti-inflammatory docosahexaenoic acid- and eicosapentaenoic acid-derived metabolites in the kidneys of aged rats. These results indicate that there is a possibility of ARA administration having a reducing anti-inflammatory effect on the kidney.

Chronic Arachidonic Acid Administration Decreases Docosahexaenoic Acid- and Eicosapentaenoic Acid-Derived Metabolites in Kidneys of Aged Rats

PubMed Central

Katakura, Masanori; Hashimoto, Michio; Inoue, Takayuki; Mamun, Abdullah Al; Tanabe, Yoko; Arita, Makoto; Shido, Osamu

2015-01-01

Arachidonic acid (ARA) metabolites produced by cyclo-oxygenase and lipoxygenase are important mediators maintaining physiological renal function. However, the effects of exogenous ARA on kidney function in vivo remain unknown. This study examined the effects of long-term oral ARA administration on normal renal function as well as inflammation and oxidative stress in aged rats. In addition, we measured levels of renal eicosanoids and docosanoids using liquid chromatography–tandem mass spectrometry. Control or ARA oil (240 mg/kg body weight/day) was orally administered to 21-month-old Wistar rats for 13 weeks. Levels of plasma creatinine, blood urea nitrogen, inflammatory and anti-inflammatory cytokines, reactive oxygen species, and lipid peroxidation were not significantly different between the two groups. The ARA concentration in the plasma, kidney, and liver increased in the ARA-administered group. In addition, levels of free-form ARA, prostaglandin E2, and 12- and 15-hydroxyeicosatetraenoic acid increased in the ARA-administered group, whereas renal concentration of docosahexaenoic acid and eicosapentaenoic acid decreased in the ARA-administered group. Levels of docosahexaenoic acid-derived protectin D1, eicosapentaenoic acid-derived 5-, and 18-hydroxyeicosapentaenoic acids, and resolvin E2 and E3 decreased in the ARA-administered group. Our results indicate that long-term ARA administration led to no serious adverse reactions under normal conditions and to a decrease in anti-inflammatory docosahexaenoic acid- and eicosapentaenoic acid-derived metabolites in the kidneys of aged rats. These results indicate that there is a possibility of ARA administration having a reducing anti-inflammatory effect on the kidney. PMID:26485038

Arachidonic acid metabolomic study of BPH in rats and the interventional effects of Zishen pill, a traditional Chinese medicine.

PubMed

Bian, Qiaoxia; Wang, Weihui; Wang, Nannan; Peng, Yan; Ma, Wen; Dai, Ronghua

2016-09-05

Zishen pill (ZSP) is a traditional Chinese medicine (TCM) used to treat benign prostatic hyperplasia (BPH). The study used a metabolomic approach based on UHPLC-MS/MS to profile arachidonic acid (AA) metabolic changes and to investigate the interventional mechanisms of ZSP in testosterone- induced BPH rats. In order to explore the potential therapeutic effect of ZSP, rat models were constructed and orally administrated with ZSP. Plasma and urine samples were collected after four weeks and then eleven potential biomarkers (15-HETE, 12-HETE, TXA2, 5-HETE, AA, PGI2, PGF2α, 8-HETE, PGD2, PGE2 and LTB4) were identified and quantified by UHPLC-MS/MS. The chromatographic separation was carried out with gradient elution using a mobile phase comprised of 0.05% formic acid aqueous solution (pH=3.3) (A) and acetonitrile: methanol (80:20, V/V) (B), and each AA metabolites was measured using electrospray ionization source with negative mode and multiple reaction monitoring. The eleven biomarkers in BPH group rat plasma and urine were significant higher than those in sham group rats. Using the potential biomarkers as a screening index, the results suggest that ZSP can potentially reverse the process of BPH by partially regulating AA metabolism through refrain the expression of cyclooxygenase (COX) and lipoxygenase (LOX). This study demonstrates that a metabolomic strategy is useful for identifying potential BPH biomarkers and investigating the underlying mechanisms of a TCM in BPH treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

[Effect of nonsteroidal antiinflammatory drugs on colonic lipoxygenase and cyclooxygenase activities from patients with colonic neoplasia].

PubMed

Di Girolamo, G; Franchi, A; De Los Santos, A R; Martí, M L; Farina, M; Fernández de Gimeno, M A

2001-01-01

Lysine clonixinate (LC) is a nonsteroidal anti-inflammatory drug (NSAID) with good gastrointestinal tolerance. Treatment with LC at levels equivalent to those found in plasma following therapeutic doses resulted in significant inhibition of both cyclooxygenase 2 (COX-2) and production of 5 hydroxy-eicosatetraeonic acid (5-HETE) and slightly affected levels of cyclooxygenase 1 (COX-1) in in vitro studies carried out on human tissues. This study deals with the in vivo effect of the drug on human colon segments. Experiment 1: Five patients about to undergo hemicholectomy due to colon neoplasia were treated preoperatively with a continuous infusion of LC, to achieve a steady-state concentration between 4 and 6 mg/ml. Human colon segments from the five patients and from another five control patients receiving no treatment with [14C]-arachidonic acid were incubated. Human colon segments treated with LC showed significant inhibition of PGE2, the only prostaglandin (PG) synthesised by the tissue, as well as of 5-HETE. Experiment 2: Fifteen patients received an i.v. bolus of LC 100 mg (n1 = 5); LC 200 mg (n2 = 5) or indomethacin (INDO) 50 mg (n3 = 5). Both doses of LC showed greater inhibition of PGE2 synthesis than the INDO bolus. Both NSAIDs studied proved to have different effects on the production of 5-HETE; while treatment with LC elicited significant inhibition, levels with INDO remained unchanged. Western blotting analysis showed expression of both COX isoforms in colon segments, COX-2 levels being 20% higher. Both types of in vivo studies conducted continuous infusion and i.v. bolus, revealed that LC exerted significant inhibition of basal synthesis of PGE2 and 5-HETE.

From Molecular Docking to 3D-Quantitative Structure-Activity Relationships (3D-QSAR): Insights into the Binding Mode of 5-Lipoxygenase Inhibitors.

PubMed

Eren, Gokcen; Macchiarulo, Antonio; Banoglu, Erden

2012-02-01

Pharmacological intervention with 5-Lipoxygenase (5-LO) is a promising strategy for treatment of inflammatory and allergic ailments, including asthma. With the aim of developing predictive models of 5-LO affinity and gaining insights into the molecular basis of ligand-target interaction, we herein describe QSAR studies of 59 diverse nonredox-competitive 5-LO inhibitors based on the use of molecular shape descriptors and docking experiments. These studies have successfully yielded a predictive model able to explain much of the variance in the activity of the training set compounds while predicting satisfactorily the 5-LO inhibitory activity of an external test set of compounds. The inspection of the selected variables in the QSAR equation unveils the importance of specific interactions which are observed from docking experiments. Collectively, these results may be used to design novel potent and selective nonredox 5-LO inhibitors. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

PubMed Central

Bell, E; Creelman, R A; Mullet, J E

1995-01-01

Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7567995

Anti-thrombotic and anti-inflammatory activities of protopine.

PubMed

Saeed, S A; Gilani, A H; Majoo, R U; Shah, B H

1997-07-01

The effects of protopine on human platelet aggregation and arachidonic acid (AA) metabolism via cyclooxygenase (COX) and lipoxygenase (LOP) enzymes were examined. Platelet aggregation induced by various platelet agonists (AA, ADP, collagen and PAF) was strongly inhibited by protopine in a concentration-related manner. The IC50 values (microM) of protopine (mean +/- SEM) against: AA; 12 +/- 2: ADP; 9 +/- 2: collagen; 16 +/- 2 and PAF; 11 +/- 1, were much less than those observed for aspirin. In addition, protopine selectively inhibited the synthesis of thromboxane A2 (TXA2) via COX pathway and had no effect on the LOP pathway in platelets. In vivo, pretreatment with protopine (50-100 mg kg-1) protected rabbits from the lethal effects of AA (2 mg kg-1) or PAF (11 micrograms kg-1) in dose-dependent fashion. Protopine (50-100 mg kg-1) also inhibited carrageenan-induced rat paw oedema with a potency of three-fold as compared to aspirin. These results are suggestive that protopine acts as a potent inhibitor of thromboxane synthesis and PAF with anti-inflammatory properties.

Inhibition of leukocyte-type 12-lipoxygenase by guava tea leaves prevents development of atherosclerosis.

PubMed

Takahashi, Yoshitaka; Otsuki, Akemi; Mori, Yoshiko; Kawakami, Yuki; Ito, Hideyuki

2015-11-01

Oxidation of low-density lipoprotein (LDL) is one of the crucial steps for atherosclerosis development, and an essential role of leukocyte-type 12-lipoxygenase expressed in macrophages in this process has been demonstrated. The biochemical mechanism of the oxidation of circulating LDL by leukocyte-type 12-lipoxygenase in macrophages has been proposed. The major ingredients in guava tea leaves which inhibited the catalytic activity of leukocyte-type 12-lipoxygenase were quercetin and ethyl gallate. Administration of extracts from guava tea leaves to apoE-deficient mice significantly attenuated atherogenic lesions in the aorta and aortic sinus. We recently showed that Qing Shan Lu Shui inhibited the catalytic activity of leukocyte-type 12-lipoxygenase. The major components inhibiting the enzyme contained in Qing Shan Lu Shui were identified to be novel monoterpene glycosides. The anti-atherogenic effect of the tea leaves might be attributed to the inhibition of leukocyte-type 12-lipoxygenase by these components. Copyright © 2015 Elsevier Ltd. All rights reserved.

Flavocoxid, a Nutraceutical Approach to Blunt Inflammatory Conditions

PubMed Central

Squadrito, Francesco; Mecchio, Anna

2014-01-01

Flavonoids, from Scutellaria baicalensis (Chinese skullcap) and Acacia catechu (black catechu), have been shown to exert a variety of therapeutic effects, including anti-inflammatory, antiviral, antibacterial, and anticancer activities. Flavocoxid is a mixed extract containing baicalin and catechin and it acts as a dual balanced inhibitor of cyclooxygenase-1 (COX-1) and COX-2 peroxidase enzyme activities with a significant inhibition of 5-lipoxygenase (5-LOX) enzyme activity in vitro. Flavocoxid downregulates gene or protein expression of several inflammatory markers and exerts also strong antioxidant activity in several experimental models. Controlled clinical trials and a postmarketing study have clearly shown that flavocoxid is as effective as naproxen in managing the signs and symptoms of osteoarthritis of the knee and it has better upper gastrointestinal, renal, and respiratory safety profile than naproxen. Flavocoxid may therefore provide a potential therapeutic approach to the treatment of chronic inflammatory conditions. PMID:25242871

Flavocoxid, a nutraceutical approach to blunt inflammatory conditions.

PubMed

Bitto, Alessandra; Squadrito, Francesco; Irrera, Natasha; Pizzino, Gabriele; Pallio, Giovanni; Mecchio, Anna; Galfo, Federica; Altavilla, Domenica

2014-01-01

Flavonoids, from Scutellaria baicalensis (Chinese skullcap) and Acacia catechu (black catechu), have been shown to exert a variety of therapeutic effects, including anti-inflammatory, antiviral, antibacterial, and anticancer activities. Flavocoxid is a mixed extract containing baicalin and catechin and it acts as a dual balanced inhibitor of cyclooxygenase-1 (COX-1) and COX-2 peroxidase enzyme activities with a significant inhibition of 5-lipoxygenase (5-LOX) enzyme activity in vitro. Flavocoxid downregulates gene or protein expression of several inflammatory markers and exerts also strong antioxidant activity in several experimental models. Controlled clinical trials and a postmarketing study have clearly shown that flavocoxid is as effective as naproxen in managing the signs and symptoms of osteoarthritis of the knee and it has better upper gastrointestinal, renal, and respiratory safety profile than naproxen. Flavocoxid may therefore provide a potential therapeutic approach to the treatment of chronic inflammatory conditions.

[Discover potential inhibitors of 5-LOX and LTA4H from Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix based on molecular simulation methods].

PubMed

Gu, Yu; Zhang, Xu; Chen, Yan-Kun; Zhao, Bo-Wen; Zhang, Yan-Ling

2017-12-01

5-lipoxygenase (5-LOX) and leukotriene A4 hydrolase (LTA4H), as the major targets of 5-LOX branch in the arachidonic acid (AA) metabolic pathway, play an important role in the treatment of inflammation. Rhei Radix et Rhizoma, Notopterygii Rhizoma et Radix and Genitana Macrophyllae Radix have clear anti-inflammation activities. In this paper, the targets of 5-LOX and LTA4H were used as the research carrier, and Hiphop module in DS4.0 (Discovery studio) was used to construct ingredients database for preliminary screening of three traditional Chinese medicines based on target inhibitor pharmacophore, so as to obtain 5-LOX and LTA4H potential active ingredients. The ingredients obtained in initial pharmacophore screening were further screened by using CDOCKER module, and the screening rules were established based on the score of initial compound and the key amino acids to obtain 12 potential 5-LOX inhibitors and 7 potential LTA4H inhibitors. To be more specific, the potential 5-LOX inhibitors included 6 ingredients in Rhei Radix et Rhizoma, such as procyanidins B2-3,3'-O-double gallate and revandchinone 2; four ingredients in notopterygium, such as dodecanoic acid and so on. On the other hand, potential LTA4H inhibitors included revandchinone 1, revandchinone 4 in Rhei Radix et Rhizoma, tridecanoic acid, tetracosanoic acid and methyl eicosanoate in Notopterygii Rhizoma et Radix, montanic acid methyl ester and N-docosanoyl-O-aminobenzoate in Genitana Macrophyllae Radix and so on. The molecular simulation methods were highly efficient and time-saving to obtain the potential inhibitors of 5-LOX and LTA4H, which could provide assistance for discovering the chemical quality indicators of anti-inflammatory efficacy of three Chinese herbs, and may be helpful to promote the whole-process quality control of three Chinese herbs. Copyright© by the Chinese Pharmaceutical Association.

Antioxidant lipoxygenase inhibitors from the leaf extracts of Simmondsia chinensis.

PubMed

Abdel-Mageed, Wael Mostafa; Bayoumi, Soad Abdel Latief Hassan; Salama, Awwad Abdoh Radwan; Salem-Bekhit, Mounir Mohamed; Abd-Alrahman, Sherif Hussein; Sayed, Hanaa Mohamed

2014-09-01

To isolate and identify chemical constituents with antioxidant and lipoxygenase inhibitory effects of the ethanolic extract of Simmondsia chinensis (Jojoba) leaves. The alcoholic extract was subjected to successive solvent fractionation. The antioxidant active fractions (chloroform, ethyl acetate and aqueous fractions) were subjected to a combination of different chromatographic techniques guided by the antioxidant assay with DPPH. The structures of the isolated compounds were elucidated on the basis of spectroscopic evidences and correlated with known compounds. The antioxidant activity was assessed quantitively using DPPH and β-carotene methods. The inhibitory potential against enzyme lipoxygenase was assessed on soybean lipoxygenase enzyme. Ten flavonoids and four lignans were isolated. Flavonoid aglycones showed stronger antioxidant and lipoxygenase inhibitory effects than their glycosides. Lignoid glycosides showed moderate to weak antioxidant and lipoxygenase inhibitory effects. A total of 14 compounds were isolated and identified from Simmondsia chinensis; 12 of them were isolated for the first time. This is the first report that highlights deeply on the phenolic content of jojoba and their potential biological activities and shows the importance of this plant as a good source of phenolics in particular the flavonoid content. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Effect of alpha lipoic acid on leukotriene A4 hydrolase.

PubMed

Torres, María José; Fierro, Angélica; Pessoa-Mahana, C David; Romero-Parra, Javier; Cabrera, Gonzalo; Faúndez, Mario

2017-03-15

Leukotriene A 4 hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A 4 to leukotriene B 4 and the hydrolysis of the peptide proline-glycine-proline. Imbalances in leukotriene B 4 synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B 4 or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A 4 Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10μM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme. Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A 4 hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells. Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme. Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A 4 hydrolase using in vivo models. Copyright © 2017 Elsevier B.V. All rights reserved.

Insights using the molecular model of Lipoxygenase from Finger millet (Eleusine coracana (L.)).

PubMed

Tiwari, Apoorv; Avashthi, Himanshu; Jha, Richa; Srivastava, Ambuj; Kumar Garg, Vijay; Wasudev Ramteke, Pramod; Kumar, Anil

2016-01-01

Lipoxygenase-1 (LOX-1) protein provides defense against pests and pathogens and its presence have been positively correlated with plant resistance against pathogens. Linoleate is a known substrate of lipoxygenase and it induces necrosis leading to the accumulation of isoflavonoid phytoalexins in plant leaves. Therefore, it is of interest to study the structural features of LOX-1 from Finger millet. However, the structure ofLOX-1 from Finger millet is not yet known. A homology model of LOX-1 from Finger millet is described. Domain architecture study suggested the presence of two domains namely PLAT (Phospho Lipid Acyl Transferase) and lipoxygenase. Molecular docking models of linoleate with lipoxygenase from finger millet, rice and sorghum are reported. The features of docked models showed that finger millet have higher pathogen resistance in comparison to other cereal crops. This data is useful for the molecular cloning of fulllength LOX-1 gene for validating its role in improving plant defense against pathogen infection and for various other biological processes.

Chronic administration of docosahexaenoic acid or eicosapentaenoic acid, but not arachidonic acid, alone or in combination with uridine, increases brain phosphatide and synaptic protein levels in gerbils.

PubMed

Cansev, M; Wurtman, R J

2007-08-24

Synthesis of phosphatidylcholine, the most abundant brain membrane phosphatide, requires three circulating precursors: choline; a pyrimidine (e.g. uridine); and a polyunsaturated fatty acid. Supplementing a choline-containing diet with the uridine source uridine-5'-monophosphate (UMP) or, especially, with UMP plus the omega-3 fatty acid docosahexaenoic acid (given by gavage), produces substantial increases in membrane phosphatide and synaptic protein levels within gerbil brain. We now compare the effects of various polyunsaturated fatty acids, given alone or with UMP, on these synaptic membrane constituents. Gerbils received, daily for 4 weeks, a diet containing choline chloride with or without UMP and/or, by gavage, an omega-3 (docosahexaenoic or eicosapentaenoic acid) or omega-6 (arachidonic acid) fatty acid. Both of the omega-3 fatty acids elevated major brain phosphatide levels (by 18-28%, and 21-27%) and giving UMP along with them enhanced their effects significantly. Arachidonic acid, given alone or with UMP, was without effect. After UMP plus docosahexaenoic acid treatment, total brain phospholipid levels and those of each individual phosphatide increased significantly in all brain regions examined (cortex, striatum, hippocampus, brain stem, and cerebellum). The increases in brain phosphatides in gerbils receiving an omega-3 (but not omega-6) fatty acid, with or without UMP, were accompanied by parallel elevations in levels of pre- and post-synaptic proteins (syntaxin-3, PSD-95 and synapsin-1) but not in those of a ubiquitous structural protein, beta-tubulin. Hence administering omega-3 polyunsaturated fatty acids can enhance synaptic membrane levels in gerbils, and may do so in patients with neurodegenerative diseases, especially when given with a uridine source, while the omega-6 polyunsaturated fatty acid arachidonic acid is ineffective.

Chronic administration of docosahexaenoic acid or eicosapentaenoic acid, but not arachidonic acid, alone or in combination with uridine, increases brain phosphatide and synaptic protein levels in gerbils

PubMed Central

Cansev, M.; Wurtman, R. J.

2007-01-01

Synthesis of phosphatidylcholine, the most abundant brain membrane phosphatide, requires three circulating precursors: choline; a pyrimidine (e.g., uridine); and a polyunsaturated fatty acid. Supplementing a choline-containing diet with the uridine source uridine-5′-monophosphate (UMP) or, especially, with UMP plus the omega-3 fatty acid docosahexaenoic acid (given by gavage), produces substantial increases in membrane phosphatide and synaptic protein levels within gerbil brain. We now compare the effects of various polyunsaturated fatty acids, given alone or with UMP, on these synaptic membrane constituents. Gerbils received, daily for 4 weeks, a diet containing choline chloride with or without UMP and/or, by gavage, an omega-3 (docosahexaenoic or eicosapentaenoic acid) or omega-6 (arachidonic acid) fatty acid. Both of the omega-3 fatty acids elevated major brain phosphatide levels (by 18-28%, and 21-27%) and giving UMP along with them enhanced their effects significantly. Arachidonic acid, given alone or with UMP, was without effect. After UMP plus docosahexaenoic acid treatment, total brain phospholipids levels and those of each individual phosphatide increased significantly in all brain regions examined (cortex, striatum, hippocampus, brain stem, and cerebellum). The increases in brain phosphatides in gerbils receiving an omega-3 (but not omega-6) fatty acid, with or without UMP, were accompanied by parallel elevations in levels of pre- and post-synaptic proteins (syntaxin-3, PSD-95 and Synapsin-1) but not in those of a ubiquitous structural protein, β-tubulin. Hence administering omega-3 polyunsaturated fatty acids can enhance synaptic membrane levels in gerbils, and may do so in patients with neurodegenerative diseases, especially when given with a uridine source, while the omega-6 polyunsaturated fatty acid arachidonic acid is ineffective. PMID:17683870

Control of polyclonal immunoglobulin production from human lymphocytes by leukotrienes; leukotriene B4 induces an OKT8(+), radiosensitive suppressor cell from resting, human OKT8(-) T cells.

PubMed Central

Atluru, D; Goodwin, J S

1984-01-01

We report that leukotriene B4 (LTB4), a 5-lipoxygenase metabolite of arachidonic acid, is a potent suppressor of polyclonal Ig production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood lymphocytes, while LTC4 and LTD4 have little activity in this system. Preincubation of T cells with LTB4 in nanomolar to picomolar concentrations rendered these cells suppressive of Ig production in subsequent PWM-stimulated cultures of fresh, autologous B + T cells. This LTB4-induced suppressor cell was radiosensitive, and its generation could be blocked by cyclohexamide but not by mitomycin C. The LTB4-induced suppressor cell was OKT8(+), while the precursor for the cell could be OKT8(-). The incubation of OKT8(-) T cells with LTB4 for 18 h resulted in the appearance of the OKT8(+) on 10-20% of the cells, and this could be blocked by cyclohexamide but not by mitomycin C. Thus, LTB4 in very low concentrations induces a radiosensitive OKT8(+) suppressor cell from OKT8(-) cells. In this regard, LTB4 is three to six orders of magnitude more potent than any endogenous hormonal inducer of suppressor cells previously described. Glucocorticosteroids, which block suppressor cell induction in many systems, may act by inhibiting endogenous production of LTB4. Images PMID:6090503

Effects of sexually dimorphic growth hormone secretory patterns on arachidonic acid metabolizing enzymes in rodent heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Furong; Yu, Xuming; He, Chunyan

The arachidonic acid (AA) metabolizing enzymes are the potential therapeutic targets of cardiovascular diseases (CVDs). As sex differences have been shown in the risk and outcome of CVDs, we investigated the regulation of heart AA metabolizing enzymes (COXs, LOXs, and CYPs) by sex-dependent growth hormone (GH) secretory patterns. The pulsatile (masculine) GH secretion at a physiological concentration decreased CYP1A1 and CYP2J3 mRNA levels more efficiently in the H9c2 cells compared with the constant (feminine) GH secretion; however, CYP1B1 mRNA levels were higher following the pulsatile GH secretion. Sex differences in CYP1A1, CYP1B1, and CYP2J11 mRNA levels were observed in bothmore » the wild-type and GHR deficient mice. No sex differences in the mRNA levels of COXs, LOXs, or CYP2E1 were observed in the wild-type mice. The constant GH infusion induced heart CYP1A1 and CYP2J11, and decreased CYP1B1 in the male C57/B6 mice constantly infused with GH (0.4 μg/h, 7 days). The activity of rat Cyp2j3 promoter was inhibited by the STAT5B protein, but was activated by C/EBPα (CEBPA). Compared with the constant GH administration, the levels of the nuclear phosphorylated STAT5B protein and its binding to the rat Cyp2j3 promoter were higher following the pulsatile GH administration. The constant GH infusion decreased the binding of the nuclear phosphorylated STAT5B protein to the mouse Cyp2j11 promoter. The data suggest the sexually dimorphic transcription of heart AA metabolizing enzymes, which might alter the risk and outcome of CVDs. GHR-STAT5B signal transduction pathway may be involved in the sex difference in heart CYP2J levels. - Highlights: • The transcription of heart Cyp1a1, Cyp1b1 and Cyp2j genes is sexually dimorphic. • There are no sex differences in the mRNA levels of heart COXs, LOXs, or CYP2E1. • GHR-STAT5B pathway is involved in sexually dimorphic transcription of heart Cpy2j genes. • Heart CYPs-mediated metabolism pathway of arachidonic acid may be sex different.« less

ALOX12 polymorphisms are associated with fat mass but not peak bone mineral density in Chinese nuclear families.

PubMed

Xiao, W-J; He, J-W; Zhang, H; Hu, W-W; Gu, J-M; Yue, H; Gao, G; Yu, J-B; Wang, C; Ke, Y-H; Fu, W-Z; Zhang, Z-L

2011-03-01

Arachidonate 12-lipoxygenase (ALOX12) is a member of the lipoxygenase superfamily, which catalyzes the incorporation of molecular oxygen into polyunsaturated fatty acids. The products of ALOX12 reactions serve as endogenous ligands for peroxisome proliferator-activated receptor γ (PPARG). The activation of the PPARG pathway in marrow-derived mesenchymal progenitors stimulates adipogenesis and inhibits osteoblastogenesis. Our objective was to determine whether polymorphisms in the ALOX12 gene were associated with variations in peak bone mineral density (BMD) and obesity phenotypes in young Chinese men. All six tagging single-nucleotide polymorphisms (SNPs) in the ALOX12 gene were genotyped in a total of 1215 subjects from 400 Chinese nuclear families by allele-specific polymerase chain reaction. The BMD at the lumbar spine and hip, total fat mass (TFM) and total lean mass (TLM) were measured using dual-energy X-ray absorptiometry. The pairwise linkage disequilibrium among SNPs was measured, and the haplotype blocks were inferred. Both the individual SNP markers and the haplotypes were tested for an association with the peak BMD, body mass index, TFM, TLM and percentage fat mass (PFM) using the quantitative transmission disequilibrium test (QTDT). Using the QTDT, significant within-family association was found between the rs2073438 polymorphism in the ALOX12 gene and the TFM and PFM (P=0.007 and 0.012, respectively). Haplotype analyses were combined with our individual SNP results and remained significant even after correction for multiple testing. However, we failed to find significant within-family associations between ALOX12 SNPs and the BMD at any bone site in young Chinese men. Our present results suggest that the rs2073438 polymorphism of ALOX12 contributes to the variation of obesity phenotypes in young Chinese men, although we failed to replicate the association with the peak BMD variation in this sample. Further independent studies are needed to confirm our findings.

Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis.

PubMed

Singh, Soudamani; Arthur, Subha; Sundaram, Uma

2018-03-01

The only Na-nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na-nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2-week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used. Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

High expression of arachidonate 15-lipoxygenase and proinflammatory markers in human ischemic heart tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magnusson, Lisa U.; Lundqvist, Annika; Asp, Julia

Highlights: Black-Right-Pointing-Pointer We found a 17-fold upregulation of ALOX15 in the ischemic heart. Black-Right-Pointing-Pointer Incubation of human muscle cells in hypoxia showed a 22-fold upregulation of ALOX15. Black-Right-Pointing-Pointer We observed increased levels of proinflammatory markers in ischemic heart tissue. Black-Right-Pointing-Pointer Suggesting a link between ischemia and inflammation in ischemic heart biopsies. -- Abstract: A common feature of the ischemic heart and atherosclerotic plaques is the presence of hypoxia (insufficient levels of oxygen in the tissue). Hypoxia has pronounced effects on almost every aspect of cell physiology, and the nuclear transcription factor hypoxia inducible factor-1{alpha} (HIF-1{alpha}) regulates adaptive responses to lowmore » concentrations of oxygen in mammalian cells. In our recent work, we observed that hypoxia increases the proinflammatory enzyme arachidonate 15-lipoxygenase (ALOX15B) in human carotid plaques. ALOX15 has recently been shown to be present in the human myocardium, but the effect of ischemia on its expression has not been investigated. Here we test the hypothesis that ischemia of the heart leads to increased expression of ALOX15, and found an almost 2-fold increase in HIF-1{alpha} mRNA expression and a 17-fold upregulation of ALOX15 mRNA expression in the ischemic heart biopsies from patients undergoing coronary bypass surgery compared with non ischemic heart tissue. To investigate the effect of low oxygen concentration on ALOX15 we incubated human vascular muscle cells in hypoxia and showed that expression of ALOX15 increased 22-fold compared with cells incubated in normoxic conditions. We also observed increased mRNA levels of proinflammatory markers in ischemic heart tissue compared with non-ischemic controls. In summary, we demonstrate increased ALOX15 in human ischemic heart biopsies. Furthermore we demonstrate that hypoxia increases ALOX15 in human muscle cells. Our results yield important insights into the underlying association between hypoxia and inflammation in the human ischemic heart disease.« less

The hidden mechanism beyond ginger (Zingiber officinale Rosc.) potent in vivo and in vitro anti-inflammatory activity.

PubMed

Ezzat, Shahira M; Ezzat, Marwa I; Okba, Mona M; Menze, Esther T; Abdel-Naim, Ashraf B

2018-03-25

Ginger (Zingiber officinale Roscoe) is a well known anti-inflammatory drug in the Egyptian, Indian and Chinese folk medicines, yet its mechanism of action is unclear. To explore its mechanism of action and to correlate it to its biophytochemicals. Various extracts viz. water, 50%, 70%, 80%, and 90% ethanol were prepared from ginger rhizomes. Fractionation of the aqueous extract (AE) was accomplished using Diaion HP-20. In vitro anti-inflammatory activity of the different extracts and isolated compounds was evaluated using protein denaturation inhibition, membrane stabilization, protease inhibition, and anti-lipoxygenase assays. In vivo anti-inflammatory activity of AE was estimated using carrageenan-induced rat paw edema in rats at doses 25, 50, 100 and 200mg/kg b.wt. All the tested extracts showed significant (p< 0.1) in vitro anti-inflammatory activities. The strongest anti-lipoxygenase activity was observed for AE that was more significant than that of diclofenac (58% and 52%, respectively) at the same concentration (125μg/ml). Purification of AE led to the isolation of 6-poradol (G1), 6-shogaol (G2); methyl 6- gingerol (G3), 5-gingerol (G4), 6-gingerol (G5), 8-gingerol (G6), 10-gingerol (G7), and 1-dehydro-6-gingerol (G8). G1, G2 and G8 exhibited potent activity in all the studied assays, while G4 and G5 exhibited moderate activity. In vivo administration of AE ameliorated rat paw edema in a dose-dependent manner. AE (at 200mg/kg) showed significant reduction in production of PGE2, TNF-α, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T-cell expressed and secreted (RANTES), myeloperoxidase (MPO) activity by 60%, 57%, 60%, 41%, 32% and 67%, respectively. AE at 100 and 200mg/kg was equipotent to indomethacin in reduction of NO x level and in increasing the total antioxidant capacity (TAC). Histopathological examination revealed very few inflammatory cells infiltration and edema after administration of AE (200mg/kg) prior to carrageenan. Ginger anti-inflammatory activity is mediated by inhibiting macrophage and neutrophils activation as well as negatively affecting monocyte and leukocyte migration. This was evidenced by the dose-dependent decrease in pro-inflammatory cytokines and chemokines and replenishment the total antioxidant capacity. Copyright © 2017 Elsevier B.V. All rights reserved.

Eicosanoid signalling pathways in the development and progression of colorectal cancer: novel approaches for prevention/intervention.

PubMed

Cathcart, Mary-Clare; Lysaght, Joanne; Pidgeon, Graham P

2011-12-01

Arachidonic acid metabolism through cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P-450 epoxygenase (EPOX) pathways leads to the generation of biologically active eicosanoids, including prostanoids, leukotrienes, hydroxyeicosatetraenoic acid, epoxyeicosatrienoic acid and hydroperoxyeicosatetraenoic acids. Eicosanoid expression levels vary during tumor development and progression of a range of malignancies, including colorectal cancer. The actions of these autocoids are also directly influenced by diet, as demonstrated by recent evidence for omega-3 fatty acids in colorectal cancer (CRC) prevention and/or treatment. Eicosanoids regulate CRC development and progression, while inhibition of these pathways has generally been shown to inhibit tumor growth/progression. A progressive sequence of colorectal cancer development has been identified, ranging from normal colon, to colitis, dysplasia, and carcinoma. While both COX and LOX inhibition are both promising candidates for colorectal cancer prevention and/or treatment, there is an urgent need to understand the mechanisms through which these signalling pathways mediate their effects on tumorigenesis. This will allow identification of safer, more effective strategies for colorectal cancer prevention and/or treatment. In particular, binding to/signalling through prostanoid receptors have recently been the subject of considerable interest in this area. In this review, we discuss the role of the eicosanoid signalling pathways in the development and progression of colorectal cancer. We discuss the effects of the eicosanoids on tumor cell proliferation, their roles in cell death induction, effects on angiogenesis, migration, invasion and their regulation of the immune response. Signal transduction pathways involved in these processes are also discussed. Finally, novel approaches targeting these arachidonic acid-derived eicosanoids (using pharmacological or natural agents) for chemoprevention and/or treatment of colorectal cancer are outlined.

Arachidonic acid metabolites and endothelial dysfunction of portal hypertension.

PubMed

Sacerdoti, David; Pesce, Paola; Di Pascoli, Marco; Brocco, Silvia; Cecchetto, Lara; Bolognesi, Massimo

2015-07-01

Increased resistance to portal flow and increased portal inflow due to mesenteric vasodilatation represent the main factors causing portal hypertension in cirrhosis. Endothelial cell dysfunction, defined as an imbalance between the synthesis, release, and effect of endothelial mediators of vascular tone, inflammation, thrombosis, and angiogenesis, plays a major role in the increase of resistance in portal circulation, in the decrease in the mesenteric one, in the development of collateral circulation. Reduced response to vasodilators in liver sinusoids and increased response in the mesenteric arterioles, and, viceversa, increased response to vasoconstrictors in the portal-sinusoidal circulation and decreased response in the mesenteric arterioles are also relevant to the pathophysiology of portal hypertension. Arachidonic acid (AA) metabolites through the three pathways, cyclooxygenase (COX), lipoxygenase, and cytochrome P450 monooxygenase and epoxygenase, are involved in endothelial dysfunction of portal hypertension. Increased thromboxane-A2 production by liver sinusoidal endothelial cells (LSECs) via increased COX-1 activity/expression, increased leukotriens, increased epoxyeicosatrienoic acids (EETs) (dilators of the peripheral arterial circulation, but vasoconstrictors of the portal-sinusoidal circulation), represent a major component in the increased portal resistance, in the decreased portal response to vasodilators and in the hyper-response to vasoconstrictors. Increased prostacyclin (PGI2) via COX-1 and COX-2 overexpression, and increased EETs/heme-oxygenase-1/K channels/gap junctions (endothelial derived hyperpolarizing factor system) play a major role in mesenteric vasodilatation, hyporeactivity to vasoconstrictors, and hyper-response to vasodilators. EETs, mediators of liver regeneration after hepatectomy and of angiogenesis, may play a role in the development of regenerative nodules and collateral circulation, through stimulation of vascular endothelial growth factor (VEGF) inside the liver and in the portal circulation. Pharmacological manipulation of AA metabolites may be beneficial for cirrhotic portal hypertension. Copyright © 2015 Elsevier Inc. All rights reserved.

[Characteristics of the aggregative state of the substrate in the reaction of 5-lipoxygenase oxidation of linoleic acid].

PubMed

Butovich, I A; Kharchenko, O V; Paboka, Iu N; Kazachkov, M G

2001-01-01

5-lipoxygenase (EC 1.13.11.12) oxidizes polyunsaturated fatty acids by molecular oxygen. The enzyme acts in close contact with the cell membranes, which main components are ionic and non-ionic lipids. In order to investigate the kinetic parameters of 5-lipoxygenase reaction in vitro, extremely hydrophobic fatty acid substrate (linoleic acid) should be solubilized in the reaction mixture. We used Lubrol PX ("Sigma" Chem. Co), as a non-ionic detergent consisted of oligoethylene glycol and fatty alcohol. Linoleic acid and Lubrol PX formed mixed micelles thus solubilizing the fatty acid substrate in a buffer with appropriate pH. We have studied the sizes and shapes of mixed micelles Lubrol PX/linoleic acid (aggregates type 1) and Lubrol PX/linoleic acid/SDS (aggregates type 2; SDS was an effective activator of potato tuber 5-lipoxygenase) by means of gel-filtration and laser light scattering techniques. The parameters under investigation were molecular weights, Stocks radii and shapes of the mixed micelles. The average molecular weights and Stocks radii of the mixed micelles type 1 determined by mean of gel-filtration on Sephadex G-200 were 95,142 +/- 5184 Da and 3.45 +/- 0.11 nm, respectively. The same parameters for the mixed micelles type 2 were 73,694 +/- 893 Da and 3.02 +/- 0.02 nm, respectively. The strong similarity in physicochemical parameters for both types of mixed micelles indicated that SDS did not influence the size and shape of mixed micelles of Lubrol PX and linoleic acid. The activatory action of SDS on potato tuber lipoxygenase may be a result of electrostatic effect or direct participation of SDS in enzymatic catalysis. The laser light scattering technique allowed to determine two main fraction of particles in type 1 system with hydrodynamic diameters 2.6 and 5.7 nm and relative contribution to light scattering 13 and 87%, respectively. The particles with d = 5.7 nm were interpreted as the mixed micelles. The particles with d = 2.6 nm were interpreted as isolated molecules of Lubrol PX, linoleic acid and (or) their premicellar aggregates. The data obtained are to be used in creation of reliable physical and mathematical models of 5-lipoxygenase.

Psoralidin, a dual inhibitor of COX-2 and 5-LOX, regulates ionizing radiation (IR)-induced pulmonary inflammation.

PubMed

Yang, Hee Jung; Youn, HyeSook; Seong, Ki Moon; Yun, Young Ju; Kim, Wanyeon; Kim, Young Ha; Lee, Ji Young; Kim, Cha Soon; Jin, Young-Woo; Youn, BuHyun

2011-09-01

Radiotherapy is the most significant non-surgical cure for the elimination of tumor, however it is restricted by two major problems: radioresistance and normal tissue damage. Efficiency improvement on radiotherapy is demanded to achieve cancer treatment. We focused on radiation-induced normal cell damage, and are concerned about inflammation reported to act as a main limiting factor in the radiotherapy. Psoralidin, a coumestan derivative isolated from the seed of Psoralea corylifolia, has been studied for anti-cancer and anti-bacterial properties. However, little is known regarding its effects on IR-induced pulmonary inflammation. The aim of this study is to investigate mechanisms of IR-induced inflammation and to examine therapeutic mechanisms of psoralidin in human normal lung fibroblasts and mice. Here, we demonstrated that IR-induced ROS activated cyclooxygenases-2 (COX-2) and 5-lipoxygenase (5-LOX) pathway in HFL-1 and MRC-5 cells. Psoralidin inhibited the IR-induced COX-2 expression and PGE(2) production through regulation of PI3K/Akt and NF-κB pathway. Also, psoralidin blocked IR-induced LTB(4) production, and it was due to direct interaction of psoralidin and 5-lipoxygenase activating protein (FLAP) in 5-LOX pathway. IR-induced fibroblast migration was notably attenuated in the presence of psoralidin. Moreover, in vivo results from mouse lung indicate that psoralidin suppresses IR-induced expression of pro-inflammatory cytokines (TNF-α, TGF-β, IL-6 and IL-1 α/β) and ICAM-1. Taken together, our findings reveal a regulatory mechanism of IR-induced pulmonary inflammation in human normal lung fibroblast and mice, and suggest that psoralidin may be useful as a potential lead compound for development of a better radiopreventive agent against radiation-induced normal tissue injury. Copyright © 2011 Elsevier Inc. All rights reserved.

COX-2, aspirin and metabolism of arachidonic, eicosapentaenoic and docosahexaenoic acids and their physiological and clinical significance.

PubMed

Poorani, R; Bhatt, Anant N; Dwarakanath, B S; Das, Undurti N

2016-08-15

Polyunsaturated fatty acids (PUFAs) are vital for normal growth and development and physiological function of various tissues in humans. PUFAs have immunomodulatory actions in addition to their ability to modulate inflammation, vascular reactivity, neurotransmission and stem cell biology. PUFAs and their metabolites possess both pro- and anti-inflammatory properties that underlie their actions and involvement in several diseases. Aspirin, a non-steroidal anti-inflammatory drug (NSAID), possesses both cyclo-oxygenase (COX) and lipoxygenase (LOX) inhibitory action and enhances the production of anti-inflammatory lipoxin A4 {(called as epi-lipoxin A4, aspirin-triggered lipoxins (ATLs))}. In addition, at low doses aspirin may not interfere with the production of prostacyclin (PGI2). Both lipoxin A4 and PGI2 have vasodilator, platelet anti-aggregator and anti-inflammatory actions that may underlie the beneficial actions of aspirin. Paradoxically, other NSAIDs may not have the same actions as that of aspirin on PUFA metabolism. Similar anti-inflammatory compounds are formed from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by the action of aspirin termed as resolvins (from EPA and DHA) and protectins and maresins from DHA. PUFAs: arachidonic acid (AA), EPA and DHA and their various products modulate not only inflammation and immune response but also possess actions on various genes, nuclear factors, cyclic AMP and GMP, G-protein coupled receptors (GPRs), hypothalamic neurotransmitters, hormones, cytokines and enzymes, and interact with nitric oxide, carbon monoxide, and hydrogen sulfide to regulate their formation and action and to form new compounds that have several biological actions. These pleiotropic actions of PUFAs and their metabolites may explain their ability to play a role in several physiological actions and diseases. The big challenge is to harness these actions to prevent and manage clinical conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

A single active site residue directs oxygenation stereospecificity in lipoxygenases: Stereocontrol is linked to the position of oxygenation

PubMed Central

Coffa, Gianguido; Brash, Alan R.

2004-01-01

Lipoxygenases are a class of dioxygenases that form hydroperoxy fatty acids with distinct positional and stereo configurations. Several amino acid residues influencing regiospecificity have been identified, whereas the basis of stereocontrol is not understood. We have now identified a single residue in the lipoxygenase catalytic domain that is important for stereocontrol; it is conserved as an Ala in S lipoxygenases and a Gly in R lipoxygenases. Our results with mutation of the conserved Ala to Gly in two S lipoxygenases (mouse 8S-LOX and human 15-LOX-2) and the corresponding Gly–Ala substitution in two R lipoxygenases (human 12R-LOX and coral 8R-LOX) reveal that the basis for R or S stereo-control also involves a switch in the position of oxygenation on the substrate. After the initial hydrogen abstraction, antarafacial oxygenation at one end or the other of the activated pair of double bonds (pentadiene) gives, for example, 8S or 12R product. The Ala residue promotes oxygenation on the reactive pentadiene at the end deep in the substrate binding pocket and S stereochemistry of the product hydroperoxide, and a Gly residue promotes oxygenation at the proximal end of the reactive pentadiene resulting in R stereochemistry. A model of lipoxygenase reaction specificity is proposed in which product regiochemistry and stereochemistry are determined by fixed relationships between substrate orientation, hydrogen abstraction, and the Gly or Ala residue we have identified. PMID:15496467

Relationship between proportion and composition of albumins, and in vitro protein digestibility of raw and cooked pea seeds (Pisum sativum L.).

PubMed

Park, Sei Joon; Kim, Tae Wan; Baik, Byung-Kee

2010-08-15

Peas provide an excellent plant protein resource for human diets, but their proteins are less readily digestible than animal proteins. To identify the relationship between composition and in vitro digestibility of pea protein, eight pea varieties with a wide range of protein content (157.3-272.7 g kg(-1)) were determined for the proportion of albumins and globulins, their compositions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in vitro protein digestibility (IVPD) before and after heat treatment using a multi-enzyme (trypsin, chymotrypsin and peptidase) method. The proportion of albumins based on total seed protein content decreased from 229 to 147 g kg(-1) as seed protein content increased from 157.3 to 272.7 g kg(-1), while the proportion of globulins increased from 483 to 590 g kg(-1). The IVPDs of eight raw pea seeds were 79.9-83.5%, with significant varietal variations, and those were improved to 85.9-86.8% by cooking. Albumins, including (pea albumins 2) PA2, trypsin inhibitor, lectin and lipoxygenase, were identified as proteolytic resistant proteins. Globulins were mostly digested by protease treatment after heating. The quantitative ratio of albumins and globulins, and the quantitative variations of albumin protein components, including lipoxygenase, PA2, lectins and trypsin inhibitors, appear to influence the protein digestibility of both raw and cooked pea seeds. Copyright (c) 2010 Society of Chemical Industry.

Use of microwaves to improve nutritional value of soybeans for future space inhabitants

NASA Technical Reports Server (NTRS)

Singh, G.

1983-01-01

Whole soybeans from four different varieties at different moisture contents were microwaved for varying times to determine the conditions for maximum destruction of trypsin inhibitor and lipoxygenase activities, and optimal growth of chicks. Microwaving 150 gm samples of soybeans (at 14 to 28% moisture) for 1.5 min was found optimal for reduction of trypsin inhibitor and lipoxygenase activities. Microwaving 1 kgm samples of soybeans for 9 minutes destroyed 82% of the trypsin inhibitor activity and gave optimal chick growth. It should be pointed out that the microwaving time would vary according to the weight of the sample and the power of the microwave oven. The microwave oven used in the above experiments was rated at 650 watts 2450 MHz.

Membrane-derived second messenger regulates x-ray-mediated tumor necrosis factor alpha gene induction.

PubMed Central

Hallahan, D E; Virudachalam, S; Kuchibhotla, J; Kufe, D W; Weichselbaum, R R

1994-01-01

Cells adapt to adverse environmental conditions through a wide range of responses that are conserved throughout evolution. Physical agents such as ionizing radiation are known to initiate a stress response that is triggered by the recognition of DNA damage. We have identified a signaling pathway involving the activation of phospholipase A2 and protein kinase C in human cells that confers x-ray induction of the tumor necrosis factor alpha gene. Treatment of human cells with ionizing radiation or H2O2 was associated with the production of arachidonic acid. Inhibition of phospholipase A2 abolished radiation-mediated arachidonate production as well as the subsequent activation of protein kinase C and tumor necrosis factor alpha gene expression. These findings demonstrate that ionizing radiation-mediated gene expression in human cells is regulated in part by extranuclear signal transduction. One practical application of phospholipase A2 inhibitors is to ameliorate the adverse effects of radiotherapy associated with tumor necrosis factor alpha production. Images PMID:8197153

Expression of aquaporin 1 and 5 and their regulation by ovarian hormones, arachidonic acid, forskolin and cAMP during implantation in pigs.

PubMed

Skowronska, A; Mlotkowska, P; Majewski, M; Nielsen, S; Skowronski, M T

2016-11-08

Aquaporin proteins (AQPs) are a family of channels expressed in numerous mammalian tissues, where they play a fundamental role in regulating water transport across cell membranes. Based on reports that AQPs are present in the reproductive system and participate in reproductive processes, our aim was to investigate the effect of progesterone (P(4)), estradiol (E(2)), oxytocin (OT), arachidonic acid (AA), forskolin (FSK) and cyclic adenosine monophosphate (cAMP) on AQP1 and AQP5 expression at mRNA and protein levels in porcine uterine explants from Days 14-16 of gestation in order to determine if they play a role in implantation period in pigs. Quantitative real time PCR and Western-blot analysis revealed that the uterine explants treated with FSK and cAMP produce delayed, but long-term effects on AQP1 abundance (24 h) while AQP5 had a rapid and sustained response to FSK and cAMP in protein content (3 and 24 h). AA increases gene and protein content of AQP1 after longer exposition whereas AQP5 increases after 3 h only at the protein level. Both AQPs potentially remains under control of steroid hormones. OT has been shown to increase AQP1, and decrease AQP5 mRNA, without visible changes in protein content. P(4), E(2), AA, FSK and cAMP caused the appearance of AQP5 expression in the basolateral plasma membrane of the epithelial cells. The staining represents most likely AQP5 functioning mechanism for both absorption and reabsorption across the glandular epithelium.

[Differential action of non-steroidal antiinflammatory drugs on human gallbladder cyclooxygenase and lipoxygenase].

PubMed

Franchi, A; Di Girolamo, G; Farina, M; de los Santos, A R; Martí, M L; Gimeno, M A

2000-01-01

Lysine clonixinate (LC) is a non-steroidal antiinflammatory agent (NSAID) with only few adverse effects. This characteristic has prompted us to suggest that its administration, at levels equivalent to those found in human plasma following therapeutic doses, slightly inhibits cyclooxygenase I (COX I). Three experiments were performed. Experiment 1: to study the in vitro effect of LC at concentrations of 4 and 6 micrograms/ml, comparable with those found in plasma following an oral therapeutic dose of 125 mg. Gallbladder tissue segments were incubated with 0.25 microCi of 14C-arachidonic acid and the production of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) was measured. LC did not affect basal production of any of the 3 prostaglandins (PGs) but at 6 micrograms/ml slightly reduced the levels of 5-hidroxyeicosatetraenoic acid (5-HETE). Experiment 2: LC was administered preoperatively to 6 patients by continuous perfusion, to achieve a steady-state concentration between 4 and 6 micrograms/ml. Gallbladder segments from the 6 treated and another 6 control patients were incubated in 14C-arachidonic acid. Gallbladder segments treated with LC did not show a decreased production of any of the three PGs whereas 5-HETE released to the medium was significantly lower. Experiment 3: 18 patients received an i.v. bolus of LC 100 mg (n1 = 6) or LC 200 mg (n2 = 6) or indomethacin (INDO) 50 mg (n3 = 6). Unlike the administration of INDO bolus, LC in the above doses did not inhibit PG synthesis. Both NSAIDs showed different effects when the production of 5-HETE synthesis was assessed. Treatment with INDO did not alter the production of 5-HETE while LC elicited significant inhibition. The three studies conducted, namely in vitro and in vivo continuous perfusion and i.v. bolus, revealed that LC had no effect on prostaglandin synthesis while reducing significantly the levels of 5-HETE.

Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

PubMed Central

Hsuan, S. L.; Kannan, M. S.; Jeyaseelan, S.; Prakash, Y. S.; Sieck, G. C.; Maheswaran, S. K.

1998-01-01

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation. PMID:9596757

Pasteurella haemolytica A1-derived leukotoxin and endotoxin induce intracellular calcium elevation in bovine alveolar macrophages by different signaling pathways.

PubMed

Hsuan, S L; Kannan, M S; Jeyaseelan, S; Prakash, Y S; Sieck, G C; Maheswaran, S K

1998-06-01

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381-388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237-252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

PubMed Central

Adams, A F; Haslam, R J

1978-01-01

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors. PMID:29607

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

PubMed

Adams, A F; Haslam, R J

1978-07-15

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.

In vitro extinction learning in Hermissenda: involvement of conditioned inhibition molecules

PubMed Central

Cavallo, Joel S.; Hamilton, Brittany N.; Farley, Joseph

2014-01-01

Extinction of a conditioned association is typically viewed as the establishment of new learning rather than the erasure of the original memory. However, recent research in the nudibranch, Hermissenda crassicornis (H.c.) demonstrated that extinction training (using repeated light-alone presentations) given 15 min, but not 23 h, after memory acquisition reversed both the cellular correlates of learning (enhanced Type B cell excitability) and the behavioral changes (reduced phototaxis) produced by associative conditioning (pairings of light, CS, and rotation, US). Here, we investigated the putative molecular signaling pathways that underlie this extinction in H.c. by using a novel in vitro protocol combined with pharmacological manipulations. After intact H.c. received either light-rotation pairings (Paired), random presentations of light and rotation (Random), or no stimulation (Untrained), B cells from isolated CNSs were recorded from during exposure to extinction training consisting of two series of 15 consecutive light-steps (LSs). When in vitro extinction was administered shortly (2 h, but not 24 h) after paired training, B cells from Paired animals showed progressive and robust declines in spike frequency by the 30th LS, while control cells (Random and Untrained) did not. We found that several molecules implicated in H.c. conditioned inhibitory (CI) learning, protein phosphatase 1 (PP1) and arachidonic acid (AA)/12-lipoxygenase (12-LOX) metabolites, also contributed to the spike frequency decreases produced by in vitro extinction. Protein phosphatase 2B (PP2B) also appeared to play a role. Calyculin A (PP1 inhibitor), cyclosporin A (PP2B inhibitor), and baicalein (a 12-LOX inhibitor) all blocked the spike frequency declines in Paired B cells produced by 30 LSs. Conversely, injection of catalytically-active PP1 (caPP1) or PP2B (caPP2B) into Untrained B cells partially mimicked the spike frequency declines observed in Paired cells, as did bath-applied AA, and occluded additional LS-produced reductions in spiking in Paired cells. PMID:25374517

High efficiency capillary column-gas chromatography mass spectrometry: analysis of the lipoxygenase pathway in eukaryot cells.

PubMed

Rabinovitch-Chable, H; Durand, J; Aldigier, J C; Chebroux, P; Gualde, N; Beneytout, J L; Rigaud, M

1984-01-01

Lipoxygenases are ubiquitous enzymes able to oxygenate polyunsaturated fatty acids. This metabolic pathway leads to hydroperoxides, hydroxyepoxyene compounds and leukotrienes. Using high performance gas chromatography prior to mass spectrometry, we studied the activity of the lipoxygenases from mouse peritoneal macrophages. Further studies on mechanism of biosynthesis of hydroxyepoxyene compounds were successfully carried out using 18O2 labelled precursors.

Hyperforin is a novel type of 5-lipoxygenase inhibitor with high efficacy in vivo.

PubMed

Feisst, Christian; Pergola, Carlo; Rakonjac, Marija; Rossi, Antonietta; Koeberle, Andreas; Dodt, Gabriele; Hoffmann, Marika; Hoernig, Christina; Fischer, Lutz; Steinhilber, Dieter; Franke, Lutz; Schneider, Gisbert; Rådmark, Olof; Sautebin, Lidia; Werz, Oliver

2009-08-01

We previously showed that, in vitro, hyperforin from St. John's wort (Hypericum perforatum) inhibits 5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis. Here, we demonstrate that hyperforin possesses a novel and unique molecular pharmacological profile as a 5-LO inhibitor with remarkable efficacy in vivo. Hyperforin (4 mg/kg, i.p.) significantly suppressed leukotriene B(4) formation in pleural exudates of carrageenan-treated rats associated with potent anti-inflammatory effectiveness. Inhibition of 5-LO by hyperforin, but not by the iron-ligand type 5-LO inhibitor BWA4C or the nonredox-type inhibitor ZM230487, was abolished in the presence of phosphatidylcholine and strongly reduced by mutation (W13A-W75A-W102A) of the 5-LO C2-like domain. Moreover, hyperforin impaired the interaction of 5-LO with coactosin-like protein and abrogated 5-LO nuclear membrane translocation in ionomycin-stimulated neutrophils, processes that are typically mediated via the regulatory 5-LO C2-like domain. Together, hyperforin is a novel type of 5-LO inhibitor apparently acting by interference with the C2-like domain, with high effectiveness in vivo.

Identification of a Specific Isoform of Tomato Lipoxygenase (TomloxC) Involved in the Generation of Fatty Acid-Derived Flavor Compounds1

PubMed Central

Chen, Guoping; Hackett, Rachel; Walker, David; Taylor, Andy; Lin, Zhefeng; Grierson, Donald

2004-01-01

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed. PMID:15347800

Zyflamend reduces LTB4 formation and prevents oral carcinogenesis in a 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced hamster cheek pouch model.

PubMed

Yang, Peiying; Sun, Zheng; Chan, Diana; Cartwright, Carrie A; Vijjeswarapu, Mary; Ding, Jibin; Chen, Xiaoxin; Newman, Robert A

2008-11-01

Aberrant arachidonic acid metabolism, especially altered cyclooxygenase and 5-lipoxygenase (LOX) activities, has been associated with chronic inflammation as well as carcinogenesis in human oral cavity tissues. Here, we examined the effect of Zyflamend, a product containing 10 concentrated herbal extracts, on development of 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced inflammation and oral squamous cell carcinoma (SCC). A hamster cheek pouch model was used in which 0.5% DMBA was applied topically onto the left cheek pouch of male Syrian golden hamsters either three times per week for 3 weeks (short term) or 6 weeks (long term). Zyflamend was then applied topically at one of three different doses (25, 50 and 100 microl) onto the left cheek pouch three times for 1 week (short-term study) or chronically for 18 weeks. Zyflamend significantly reduced infiltration of inflammatory cells, incidence of hyperplasia and dysplastic lesions, bromodeoxyuridine-labeling index as well as number of SCC in a concentration-dependent manner. Application of Zyflamend (100 microl) reduced formation of leukotriene B(4) (LTB(4)) by 50% compared with DMBA-treated tissues. The reduction of LTB(4) was concentration dependent. The effect of Zyflamend on inhibition of LTB(4) formation was further confirmed with in vitro cell-based assay. Adding LTB(4) to RBL-1 cells, a rat leukemia cell line expressing high levels of 5-LOX and LTA(4) hydrolase, partially blocked antiproliferative effect of Zyflamend. This study demonstrates that Zyflamend inhibited LTB(4) formation and modulated adverse histopathological changes in the DMBA-induced hamster cheek pouch model. The study suggests that Zyflamend might prevent oral carcinogenesis at the post-initiation stage.

Lipoxygenase and Hydroperoxide Lyase in Germinating Watermelon Seedlings 1

PubMed Central

Vick, Brady A.; Zimmerman, Don C.

1976-01-01

Lipoxygenase (EC 1.13.1.13) was found in seedlings of Citrullus lanatus (Thunb.) Matsum. and Nakai (watermelon). The enzyme has pH optima of 4.4 and 5.5 and is inhibited by 0.2 mM nordihydroguaiaretic acid. It is present in two functional units with estimated molecular weights of 120,000 and 240,000, respectively. A new enzyme, tentatively termed hydroperoxide lyase, has been partially purified from watermelon seedlings. The enzyme, located principally in the region of the hypocotyl-root junction, catalyzes the conversion of 13-l-hydroperoxy-cis-9-trans-11-octadecadienoic acid to 12-oxo-trans-10-dodecenoic acid and hexanal. The hydroperoxide lyase enzyme from watermelon has a molecular weight in excess of 250,000, a pH optimum in the range of 6 to 6.5, and is inhibited by p-chloromercuribenzoic acid. Its presence has also been demonstrated in other cucurbits. The maximum activity of both enzymes occurs on the 6th day of germination. The identification of the products of the hydroperoxide lyase reaction suggests that lipoxygenase and hydroperoxide lyase may be involved in the conversion of certain polyunsaturated fatty acids to traumatic acid (trans-2-dodecenedioic acid). PMID:16659569

An Iron 13S-Lipoxygenase with an α-Linolenic Acid Specific Hydroperoxidase Activity from Fusarium oxysporum

PubMed Central

Brodhun, Florian; Cristobal-Sarramian, Alvaro; Zabel, Sebastian; Newie, Julia; Hamberg, Mats; Feussner, Ivo

2013-01-01

Jasmonates constitute a family of lipid-derived signaling molecules that are abundant in higher plants. The biosynthetic pathway leading to plant jasmonates is initiated by 13-lipoxygenase-catalyzed oxygenation of α-linolenic acid into its 13-hydroperoxide derivative. A number of plant pathogenic fungi (e.g. Fusarium oxysporum) are also capable of producing jasmonates, however, by a yet unknown biosynthetic pathway. In a search for lipoxygenase in F. oxysporum, a reverse genetic approach was used and one of two from the genome predicted lipoxygenases (FoxLOX) was cloned. The enzyme was heterologously expressed in E. coli, purified via affinity chromatography, and its reaction mechanism characterized. FoxLOX was found to be a non-heme iron lipoxygenase, which oxidizes C18-polyunsaturated fatty acids to 13S-hydroperoxy derivatives by an antarafacial reaction mechanism where the bis-allylic hydrogen abstraction is the rate-limiting step. With α-linolenic acid as substrate FoxLOX was found to exhibit a multifunctional activity, because the hydroperoxy derivatives formed are further converted to dihydroxy-, keto-, and epoxy alcohol derivatives. PMID:23741422

Stretch-induced ERK2 phosphorylation requires PLA2 activity in skeletal myotubes.

PubMed

Burkholder, Thomas J

2009-08-14

Mechanical stretch rapidly activates multiple signaling cascades, including phospholipases and kinases, to stimulate protein synthesis and growth. The purpose of this study was to determine whether PLA2 activation contributes to stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes. Myotubes derived from neonatal C57 mice were cultured on silicone membranes and subjected to brief cyclic stretch. Inhibition of PLA2 prevented ERK2 phosphorylation, while inhibition of prostaglandin or leukotriene synthesis did not. ERK2 phosphorylation was also blocked by genistein and PD98059, implicating the canonical raf-MEK-ERK cassette. It appears that PLA2, but not further metabolism of arachidonic acid, is required for stretch-induced activation of ERK2. Exposure to exogenous arachidonic acid had no effect on ERK2 phosphorylation, but exposure to lysophosphatidylcholine, the other metabolite of PLA2, caused a dose-dependent increase in ERK2 phosphorylation. These results suggest that stretch-induced activation of ERK2 may result from an interaction between PLA2 derived lysophosphatidylcholine and membrane receptors.

Stretch-induced ERK2 phosphorylation requires PLA2 activity in skeletal myotubes

PubMed Central

Burkholder, Thomas J.

2009-01-01

Mechanical stretch rapidly activates multiple signaling cascades, including phospholipases and kinases, to stimulate protein synthesis and growth. The purpose of this study was to determine whether PLA2 activation contributes to stretch-induced phosphorylation of ERK2 in skeletal muscle myotubes. Myotubes derived from neonatal C57 mice were cultured on silicone membranes and subjected to brief cyclic stretch. Inhibition of PLA2 prevented ERK2 phosphorylation, while inhibition of prostaglandin or leukotriene synthesis did not. ERK2 phosphorylation was also blocked by genistein and PD98059, implicating the canonical raf-MEK-ERK cassette. It appears that PLA2, but not further metabolism of arachidonic acid, is required for stretch-induced activation of ERK2. Exposure to exogenous arachidonic acid had no effect on ERK2 phosphorylation, but exposure to lysophosphatidylcholine, the other metabolite of PLA2, caused a dose-dependent increase in ERK2 phosphorylation. These results suggest that stretch-induced activation of ERK2 may result from an interaction between PLA2 derived lysophosphatidylcholine and membrane receptors. PMID:19524551

Effect of extrusion conditions and lipoxygenase inactivation treatment on the physical and nutritional properties of corn/cowpea (Vigna unguiculata) blends.

PubMed

Sosa-Moguel, Odri; Ruiz-Ruiz, Jorge; Martínez-Ayala, Alma; González, Rolando; Drago, Silvina; Betancur-Ancona, David; Chel-Guerrero, Luis

2009-01-01

The influence of lipoxygenase inactivation and extrusion cooking on the physical and nutritional properties of corn/cowpea (Vigna unguiculata) blends was studied. Corn was blended in an 80:15 proportion with cowpea flour treated to inactivate lipoxygenase (CI) or non-inactivated cowpea flour (CNI). Extrusion variables were temperature (150 degrees C, 165 degrees C and 180 degrees C) and moisture (15%, 17% and 19%). Based on their physical properties, the 165 degrees C/15% corn:CNI, and 165 degrees C/15% corn:CI, and 150 degrees C/15% corn:CI blends were chosen for nutritional quality analysis. Extrudate chemical composition indicated high crude protein levels compared with standard corn-based products. With the exception of lysine, essential amino acids content in the three treatments met FAO requirements. Extrusion and lipoxygenase inactivation are promising options for developing corn/cowpea extruded snack products with good physical properties and nutritional quality.

Physalis alkekengi Carotenoidic Extract Inhibitor of Soybean Lipoxygenase-1 Activity

PubMed Central

Chedea, Veronica Sanda; Pintea, Adela; Bunea, Andrea; Braicu, Cornelia; Stanila, Andreea; Socaciu, Carmen

2014-01-01

The aim of this study was to evaluate the effect of the carotenoidic saponified extract of Physalis alkekengi sepals (PA) towards the lipoxygenase (LOX) oxidation of linoleic acid. Lipoxygenase activity in the presence of carotenoids, standard and from extract, was followed by its kinetic behaviour determining the changes in absorption at 234 nm. The standard carotenoids used were β-carotene (β-car), lutein (Lut), and zeaxanthin (Zea). The calculated enzymatic specific activity (ESA) after 600 s of reaction proves that PA carotenoidic extract has inhibitory effect on LOX oxidation of linoleic acid. A longer polyenic chain of carotenoid structure gives a higher ESA during the first reaction seconds. This situation is not available after 600 s of reaction and may be due to a destruction of this structure by cooxidation of carotenoids, besides the classical LOX reaction. The PA carotenoidic extract inhibiting the LOX-1 reaction can be considered a source of lipoxygenase inhibitors. PMID:24511537

Anti-inflammatory activity of 6-hydroxy-2,7-dimethoxy-1,4-henanthraquinone from tuberous roots of yam (Dioscorea batatas) through inhibition of prostaglandin D₂ and leukotriene C₄ production in mouse bone marrow-derived mast cells.

PubMed

Jin, Meihua; Lu, Yue; Yang, Ju Hye; Jo, Tae Hyung; Park, Young In; Lee, Chong-Kil; Park, Sang-Jo; Son, Kun Ho; Chang, Hyeun Wook

2011-09-01

6-Hydroxy-2,7-dimethoxy-1,4-phenanthraquinone (PAQ) isolated from the tuberous roots of Yam (Dioscorea batatas) inhibited cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) dependent prostaglandin D(2) (PGD(2)) generation in mouse bone marrow-derived mast cells in a concentration-dependent manner with IC(50) values of 0.08 μM and 0.27 μM, respectively. In the Western blotting with specific anti-COX-2 antibodies, the decrease of the quantity of PGD(2) was accompanied by a decrease in the COX-2 protein level. But PAQ did not affect COX-1 protein level. In addition, this compound inhibited 5-lipoxygenase (5-LOX) dependent production of leukotriene C(4) in a dose-dependent manner, with an IC(50) of 0.032 μM. These results demonstrate that PAQ has a dual COX-2/5-LOX inhibitory activity. This compound also inhibited the degranulation reaction in a dose-dependent manner with an IC(50) of 2.7 μM. Thus, these results suggest that PAQ may be useful in regulating mast cell-mediated inflammatory diseases.

Arachidonic acid and lipoxinA4 attenuate streptozotocin-induced cytotoxicity to RIN5 F cells in vitro and type 1 and type 2 diabetes mellitus in vivo.

PubMed

Gundala, Naveen K V; Naidu, Vegi G M; Das, Undurti N

2017-03-01

The aim of this study was to observe whether polyunsaturated fatty acids (PUFAs) can protect rat insulinoma (RIN5 F) cells against streptozotocin (STZ)-induced apoptosis in vitro and type 1 diabetes mellitus (T1DM) and type 2 DM (T2DM) in vivo and if so, what would be the mechanism of this action. RIN5 F cells were used for the in vitro study, whereas the in vivo study was performed in Wistar rats. STZ was used to induce apoptosis of RIN5 F cells in vitro and T1- and T2DM in vivo. The effect of PUFAs: γ-linolenic acid (GLA), arachidonic acid (AA) of ω-6 series, and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) of ω-3 series; cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors and antiinflammatory metabolite of AA and DHA, lipoxin A4 (LXA4), and resolvin D2 and protectin, respectively against STZ-induced cytotoxicity to RIN5 F cells in vitro and LXA4 against T1- and T2DM in vivo was studied. Changes in the antioxidant content, lipid peroxides, nitric oxide, and expression of PDX1, P65, nuclear factor-κb (NF-κb), and IKB genes in STZ-treated RIN5 F cells in vitro and Nrf2, GLUT2, COX2, iNOS protein levels in the pancreatic tissue of T1- and T2DM and LPCLN2 (lipocalin 2), NF-κb, IKB I in adipose tissue of T2DM after LXA4 treatment were studied. Plasma glucose, insulin, and tumor necrosis factor (TNF)-α levels also were measured in STZ-induced T1- and T2DM Wistar rats. Among all PUFAs tested, AA and EPA are the most effective against STZ-induced cytotoxicity to RIN5 F cells in vitro. Neither COX nor LOX inhibitors blocked the cytoprotective action of AA in vitro and T1- and T2DM by STZ. LXA4 production by RIN5 F cells in vitro and plasma LXA4 levels in STZ-induced T1- and T2DM animals were decreased by STZ that reverted to normal after AA treatment. AA prevented both T1- and T2DM induced by STZ. Antiinflammatory metabolite of AA and LXA4 prevented both T1- and T2DM induced by STZ. The expression of Pdx1, NF-κb, IKB genes in the pancreas and plasma TNF-α levels in T1- and T2DM; Nrf2, Glut2, COX2, and iNOS proteins in pancreatic tissue of T1DM and LPCLN2, NF-κb, IKB I in adipose tissue of T2DM reverted to normal in LXA4-treated animals. Both AA and LXA4 prevented STZ-induced cytotoxicity to RIN5 F cells in vitro and T1- and T2DM in vivo, suggesting that these two bioactive lipids may function as antidiabetic molecules. AA is beneficial against STZ-induced cytotoxicity and T1- and T2DM by enhancing the production of LXA4. Copyright © 2016 Elsevier Inc. All rights reserved.

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-κB Pathways.

PubMed

Lee, Seung Jin; Seo, Kyo Won; Kim, Chi Dae

2015-05-01

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 µg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-κB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-κB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-κB pathways in monocytes.

Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murayama, T.; Ui, M.

1985-06-25

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separatemore » effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.« less

The 15-lipoxygenase inhibitory, antioxidant, antimycobacterial activity and cytotoxicity of fourteen ethnomedicinally used African spices and culinary herbs.

PubMed

Dzoyem, Jean Paul; Kuete, Victor; McGaw, Lyndy J; Eloff, Jacobus N

2014-10-28

Culinary herbs and spices are widely used ethnomedically across Africa. They are traditionally employed in the treatment of several ailments including inflammation disorders, pain alleviation and infectious diseases. Pharmacological studies are necessary to provide a scientific basis to substantiate their traditional use and safety. In this study, the 15-lipoxygenase inhibitory, antioxidant, antimycobacterial and the cytotoxic activities, total phenolic and flavonoid contents of fourteen edible plants were investigated. The 15-lipoxygenase inhibitory activity was evaluated by the ferrous oxidation-xylenol orange (FOX) assay method. The antioxidant activity was determined using free-radical scavenging assays. The antimycobacterial activity was determined by a broth microdilution method against three species of mycobacteria: Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium fortuitum using tetrazolium violet as growth indicator. The cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero monkey kidney cells. All the extracts tested had some 15-lipoxygenase inhibitory activity ranging from 32.9 to 78.64%. Adansonia digitata (fruit) had the highest antioxidant capacity (IC₅₀ values of 8.15 μg/mL and 9.16 μg/mL in the DPPH and ABTS assays respectively; TEAC of 0.75 in the FRAP assay) along with the highest amount of total phenolics (237.68 mg GAE/g) and total flavonoids (16.14 mg E/g). There were good correlations between DPPH and ABTS values (R(2) 0.98) and between total phenolics and total flavonoids (R(2) 0.94). Tamarindus indica had significant antimycobacterial activity against Mycobacterium aurum (MIC 78 μg/mL). As could be expected with edible plants, all the extracts had a relatively low cytotoxicity with LC₅₀ values higher than 102 μg/mL with the exception of the two Aframomum species (33 and 74 μg/mL). This study provides scientific support for some of the the traditional uses and the pharmacological activities of the culinary herbs and spices investigated. The results suggest that increasing intake of some of these herbs may be useful in preventing or reducing the progression of lifestyle-related diseases. The diversity of the pharmacological activities of the extract from the fruit of Adansonia digitata suggested that this plant might be valuable for application in human and animal health. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Analysis of leukotrienes in cerebrospinal fluid of a reference population and patients with inborn errors of metabolism: further evidence for a pathognomonic profile in LTC(4)-synthesis deficiency.

PubMed

Mayatepek, E; Zelezny, R; Hoffmann, G F

2000-02-25

Cysteinyl leukotrienes (LTC(4), LTD(4), LTE(4)) are potent lipid mediators derived from arachidonate in the 5-lipoxygenase pathway. Recently, the first inborn error of leukotriene synthesis, LTC(4)-synthesis deficiency, has been identified in association with a fatal developmental syndrome. The absence of leukotrienes in cerebrospinal fluid was one of the most striking biochemical findings in this disorder. We analysed leukotrienes in cerebrospinal fluid of patients with a broad spectrum of other well-defined inborn errors of metabolism, including glutathione synthetase deficiency (n=2), Zellweger syndrome (n=3), mitochondrial disorders (n=8), fatty acid oxidation defects (n=7), organic acidurias (n=7), neurotransmitter defects (n=5) and patients with non-specific neurological symptoms, as a reference population (n=120). The concentrations of leukotrienes were not related to age. Representative percentiles were calculated as reference intervals of each leukotriene. In all patients with an inborn error of metabolism concentration of cysteinyl leukotrienes and LTB(4) did not differ from the reference group. Our results indicate that absence of cysteinyl leukotrienes (<5 pg/ml) in association with normal or increased LTB(4) (50.0-67.3 pg/ml) is pathognomonic for LTC(4)-synthesis deficiency. The unique profile of leukotrienes in cerebrospinal fluid in this new disorder is primarily related to the defect and represents a new diagnostic approach.

In vitro antioxidant, lipoxygenase and xanthine oxidase inhibitory activities of fractions from Cienfuegosia digitata Cav., Sida alba L. and Sida acuta Burn f. (Malvaceae).

PubMed

Konaté, K; Souza, A; Coulibaly, A Y; Meda, N T R; Kiendrebeogo, M; Lamien-Meda, A; Millogo-Rasolodimby, J; Lamidi, M; Nacoulma, O G

2010-11-15

In this study polyphenol content, antioxidant activity, lipoxygenase (LOX) and Xanthine Oxidase (XO) inhibitory effects of n-hexane, dichloromethane, ethyl acetate and n-butanol fractions of aqueous acetone extracts from S. alba L., S. acuta Burn f and Cienfuegosia digitata Cav. were investigated. The total phenolics, flavonoids, flavonols and total tannins were determined by spectrophotometric methods using Folin-ciocalteu, AlCl3 reagents and tannic acid, respectively. The antioxidant potential was evaluated using three methods: inhibition of free radical 2,2-diphenyl-1-picrylhydramzyl (DPPH), ABTS radical cation decolorization assay and Iron (III) to iron (II) reduction activity (FRAP). For enzymatic activity, lipoxygenase and xanthine oxidase inhibitory activities were used. This study shows a relationship between polyphenol contents, antioxidant and enzymatic activities. Present results showed that ethyl acetate and dichloromethane fractions elicit the highest polyphenol content, antioxidant and enzymatic activities.

Lipoxygenase in Caragana jubata responds to low temperature, abscisic acid, methyl jasmonate and salicylic acid.

PubMed

Bhardwaj, Pardeep Kumar; Kaur, Jagdeep; Sobti, Ranbir Chander; Ahuja, Paramvir Singh; Kumar, Sanjay

2011-09-01

Lipoxygenase (LOX) catalyses oxygenation of free polyunsaturated fatty acids into oxylipins, and is a critical enzyme of the jasmonate signaling pathway. LOX has been shown to be associated with biotic and abiotic stress responses in diverse plant species, though limited data is available with respect to low temperature and the associated cues. Using rapid amplification of cDNA ends, a full-length cDNA (CjLOX) encoding lipoxygenase was cloned from apical buds of Caragana jubata, a temperate plant species that grows under extreme cold. The cDNA obtained was 2952bp long consisting of an open reading frame of 2610bp encoding 869 amino acids protein. Multiple alignment of the deduced amino acid sequence with those of other plants demonstrated putative LH2/ PLAT domain, lipoxygenase iron binding catalytic domain and lipoxygenase_2 signature sequences. CjLOX exhibited up- and down-regulation of gene expression pattern in response to low temperature (LT), abscisic acid (ABA), methyl jasmonate (MJ) and salicylic acid (SA). Among all the treatments, a strong up-regulation was observed in response to MJ. Data suggests an important role of jasmonate signaling pathway in response to LT in C. jubata. Copyright © 2011 Elsevier B.V. All rights reserved.

Spice phenolics inhibit human PMNL 5-lipoxygenase.

PubMed

Prasad, N Satya; Raghavendra, R; Lokesh, B R; Naidu, K Akhilender

2004-06-01

A wide variety of phenolic compounds and flavonoids present in spices possess potent antioxidant, antimutagenic and anticarcinogenic activities. We examined whether 5-lipoxygenase (5-LO), the key enzyme involved in biosynthesis of leukotrienes is a possible target for the spices. Effect of aqueous extracts of turmeric, cloves, pepper, chili, cinnamon, onion and also their respective active principles viz., curcumin, eugenol, piperine, capsaicin, cinnamaldehyde, quercetin, and allyl sulfide were tested on human PMNL 5-LO activity by spectrophotomeric and HPLC methods. The formation of 5-LO product 5-HETE was significantly inhibited in a concentration-dependent manner with IC(50) values of 0.122-1.44 mg for aqueous extracts of spices and 25-83 microM for active principles, respectively. The order of inhibitory activity was of quercetin>eugenol>curcumin>cinnamaldehyde>piperine>capsaicin>allyl sulfide. Quercetin, eugenol and curcumin with one or more phenolic ring and methoxy groups in their structure showed high inhibitory effect, while the non-phenolic spice principle allyl sulfide showed least inhibitory effect on 5-LO. The inhibitory effect of quercetin, curcumin and eugenol was similar to that of synthetic 5-LO inhibitors-phenidone and NDGA. Moreover, the inhibitory potency of aqueous extracts of spice correlated with the active principles of their respective spices. The synergistic or antagonistic effect of mixtures of spice active principles and spice extracts were investigated and all the combinations of spice active principles/extracts exerted synergistic effect in inhibiting 5-LO activity. These findings clearly suggest that phenolic compounds present in spices might have physiological role in modulating 5-LO pathway.

5-Lipoxygenase contributes to PPARγ activation in macrophages in response to apoptotic cells.

PubMed

von Knethen, Andreas; Sha, Lisa K; Kuchler, Laura; Heeg, Annika K; Fuhrmann, Dominik; Heide, Heinrich; Wittig, Ilka; Maier, Thorsten J; Steinhilber, Dieter; Brüne, Bernhard

2013-12-01

Macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells is a contributing hallmark to immune suppression during the late phase of sepsis. Although the peroxisome proliferator-activated receptor γ (PPARγ) supports this macrophage phenotype switch, it remains elusive how apoptotic cells activate PPARγ. Assuming that a molecule causing PPARγ activation in macrophages originates in the cell membrane of apoptotic cells we analyzed lipid rafts from apoptotic, necrotic, and living human Jurkat T cells which showed the presence of 5-lipoxygenase (5-LO) in lipid rafts of apoptotic cells only. Incubating macrophages with lipid rafts of apoptotic, but not necrotic or living cells, induced PPAR responsive element (PPRE)-driven mRuby reporter gene expression in RAW 264.7 macrophages stably transduced with a 4xPPRE containing vector. Experiments with lipid rafts of apoptotic murine EL4 T cells revealed similar results. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated with the 5-LO inhibitor MK-866 prior to induction of apoptosis, which failed to induce mRuby expression. Similar results were obtained with lipid rafts of apoptotic EL4 T cells preexposed to the 5-LO inhibitors zileuton and CJ-13610. Interestingly, Jurkat T cells overexpressing 5-LO failed to activate PPARγ in macrophages, while their 5-LO overexpressing apoptotic counterparts did. Our results suggest that during apoptosis 5-LO gets associated with lipid rafts and synthesizes ligands that in turn stimulate PPARγ in macrophages. © 2013.

Prostaglandins mediate the stimulatory effects of endothelin-1 on cAMP accumulation and inositol-1,4,5-trisphosphate production and contraction in cat iris sphincter.

PubMed

Yousufzai, S Y; Ye, Z; Abdel-Latif, A A

1995-12-01

We previously reported that in the iris sphincter smooth muscle, endothelin-1 (ET-1) activates both adenylyl cyclase and the phosphoinositide cascade and that the changes in the levels of cAMP and inositol-1,4,5-trisphosphate (IP3) produced are species specific. In the present study, we examined the mechanism of the ET-1 effects in cat iris sphincter. In general, we found that ET-1 (0.1 microM) increased prostaglandin E2 (PGE2) release by 156%, cAMP accumulation by 310%, IP3 production by 88% and induced contraction; that PGE2 increased cAMP accumulation, IP3 production and contraction; and that the effects of ET-1 are inhibited by indomethacin (Indo), suggesting that arachidonic acid metabolites may mediate the responses to the peptide. Kinetic studies revealed the following: (1) The effect of ET-1 on cAMP accumulation is rapid (within 30 sec), dose dependent (EC50 = 5.8 nM) and completely abolished by Indo (Ki = 0.16 microM), a cyclooxygenase inhibitor, but not by nordihydroguairetic acid, a lipoxygenase inhibitor, implying the involvement of PGs. (2) ET-1 dose-dependently evoked PGe2 release (EC50 = 1.8 nM), IP3 production (EC50 = 4.5 nM) and contraction (EC50 = 5 nM) and that all of these responses were inhibited by Indo. (3) PGE2 increased cAMP accumulation in a dose-dependent manner with an EC50 of 1.5 x 10(-7) M, and PGD2 and PGF2 alpha had little effect on the cyclic nucleotide. (4) PGE2 (1 microM), increased IP3 production by 55% and induced muscle contraction in a dose-dependent manner (EC50 = 40 nM). We conclude from these data that in cat iris sphincter PGs may mediate ET-1-induced cAMP accumulation, IP3 production and smooth muscle contraction.

Role of Ca2+-independent phospholipase A2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts.

PubMed

Martínez, Javier; Moreno, Juan J

2005-09-01

Store-operated calcium (SOC) channels and capacitative Ca2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [3H] arachidonic acid (AA) release, 45Ca2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca2+]i. Thapsigargin-induced elevation of [Ca2+]i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca2+ influx was sensible to receptor-operated Ca2+ channel blockers (NiCl2, LaCl3) but not to voltage-dependent Ca2+ channel blocker as verapamil. Thus, AA released by Ca2+-independent phospholipase A2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

The role of metabolic activation of analgesics and non-steroidal anti-inflammatory drugs in the development of renal papillary necrosis and upper urothelial carcinoma.

PubMed

Bach, P H; Bridges, J W

1984-08-01

There has been no cogent hypothesis to explain the molecular basis of analgesic and non-steroidal anti-inflammatory drug (NSAID) associated renal papillary necrosis (RPN) and upper urothelial carcinoma (UUC). The microsomal cytochrome P-450 enzyme system may generate reactive intermediates which promote pathophysiological effects in the lung, liver and renal cortex, but the absence of P-450 activity in the medulla suggests that it is unlikely that similar events lead to RPN and UUC. Other enzymes (eg. peroxidases) convert substituted aromatics into benzoquinoneimines (an intermediate that has previously been defined in P-450-mediated toxicity). The medulla is rich in fatty acid peroxidases involved in the metabolism of arachidonic acid. NSAID and analgesics interact with key enzymes in this pathway, which could lead to the co-oxygenation of exogenous and endogenous compounds via the peroxidase, lipoxygenase, or prostaglandin hydroperoxidase enzymes. The generation of reactive molecules in the medulla could explain both RPN and UUC via the alkylation of macromolecules. The formation of free radicals would give rise to extensive lipid peroxidation, (there are large quantities of free polyunsaturated fatty acids in the medullary interstitial cells), an event of major potential importance to local cell destruction and genotoxic effects. At present this proposed mechanism of co-oxygenation offers the most attractive working hypothesis to explain the molecular pathogenesis of both RPN and UUC.

Influence of (9Z)-12-hydroxy-9-dodecenoic acid and methyl jasmonate on plant protein phosphorylation.

PubMed

Tarchevsky, I A; Karimova, F G; Grechkin, A N; Moukhametchina, N U

2000-12-01

The products of the lipoxygenase pathway, methyl jasmonic acid (MeJA) and (9Z)-12-hydroxy-9-dodecenoic acid (HDA), hardly changed the relative level of phosphorylated polypeptides (RLPPs) during 2 h of incubation: 15 and 17 kDa RLPPs were enhanced by HDA, but decreased by MeJA. RLPPs of 73 and 82 kDa were increased by both compounds. MeJA and HDA treatment induced specific and unspecific effects in some RLPPs. It was shown that HDA and MeJA increased protein kinase activity in the presence of 1 microM cAMP.

A dual inhibitor of cyclooxygenase and 5-lipoxygenase protects against kainic acid-induced brain injury.

PubMed

Minutoli, Letteria; Marini, Herbert; Rinaldi, Mariagrazia; Bitto, Alessandra; Irrera, Natasha; Pizzino, Gabriele; Pallio, Giovanni; Calò, Margherita; Adamo, Elena Bianca; Trichilo, Vincenzo; Interdonato, Monica; Galfo, Federica; Squadrito, Francesco; Altavilla, Domenica

2015-06-01

Systemic administration of kainic acid causes inflammation and apoptosis in the brain, resulting in neuronal loss. Dual cyclooxygenase/5-lipoxygenase (COX/5-LOX) inhibitors could represent a possible neuroprotective approach in preventing glutamate excitotoxicity. Consequently, we investigated the effects of a dual inhibitor of COX/5-LOX following intraperitoneal administration of kainic acid (KA, 10 mg/kg) in rats. Animals were randomized to receive either the dual inhibitor of COX/5-LOX (flavocoxid, 20 mg/kg i.p.) or its vehicle (1 ml/kg i.p.) 30 min after KA administration. Sham brain injury rats were used as controls. We evaluated protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK1/2) and tumor necrosis factor alpha (TNF-α) as well as levels of malondialdehyde (MDA), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) in the hippocampus. Animals were also observed for monitoring behavioral changes according to Racine Scale. Finally, histological analysis and brain edema evaluation were carried out. Treatment with the dual inhibitor of COX/5-LOX decreased protein expression of p-ERK1/2 and TNF-α in hippocampus, markedly reduced MDA, LTB4 and PGE2 hippocampal levels, and also ameliorated brain edema. Histological analysis showed a reduction in cell damage in rats treated with the dual inhibitor of COX/5-LOX, particularly in hippocampal subregion CA3c. Moreover, flavocoxid significantly improved behavioral signs following kainic acid administration. Our results suggest that dual inhibition of COX/5-LOX by flavocoxid has neuroprotective effects during kainic acid-induced excitotoxicity.

Omega-oxidation is the major pathway for the catabolism of leukotriene B4 in human polymorphonuclear leukocytes.

PubMed

Shak, S; Goldstein, I M

1984-08-25

Leukotriene B4 (LTB4), formed by the 5-lipoxygenase pathway in human polymorphonuclear leukocytes (PMN), may be an important mediator of inflammation. Recent studies suggest that human leukocytes can convert LTB4 to products that are less biologically active. To examine the catabolism of LTB4, we developed (using high performance liquid chromatography) a sensitive, reproducible assay for this mediator and its omega-oxidation products (20-OH- and 20-COOH-LTB4). With this assay, we have found that human PMN (but not human monocytes, lymphocytes, or platelets) convert exogenous LTB4 almost exclusively to 20-OH- and 20-COOH-LTB4 (identified by gas chromatography-mass spectrometry). Catabolism of exogenous LTB4 by omega-oxidation is rapid (t1/2 approximately 4 min at 37 degrees C in reaction mixtures containing 1.0 microM LTB4 and 20 X 10(6) PMN/ml), temperature-dependent (negligible at 0 degrees C), and varies with cell number as well as with initial substrate concentration. The pathway for omega-oxidation in PMN is specific for LTB4 and 5(S),12(S)-dihydroxy-6,8,10,14-eicosatetraenoic acid (only small amounts of other dihydroxylated-derivatives of arachidonic acid are converted to omega-oxidation products). Even PMN that are stimulated by phorbol myristate acetate to produce large amounts of superoxide anion radicals catabolize exogenous leukotriene B4 primarily by omega-oxidation. Finally, LTB4 that is generated when PMN are stimulated with the calcium ionophore, A23187, is rapidly catabolized by omega-oxidation. Thus, human PMN not only generate and respond to LTB4, but also rapidly and specifically catabolize this mediator by omega-oxidation.

Impact of EPA ingestion on COX- and LOX-mediated eicosanoid synthesis in skin with and without a pro-inflammatory UVR challenge – Report of a randomised controlled study in humans

PubMed Central

Pilkington, Suzanne M; Rhodes, Lesley E; Al-Aasswad, Naser M I; Massey, Karen A; Nicolaou, Anna

2014-01-01

Scope Eicosapentaenoic acid (EPA), abundant in oily fish, is reported to reduce skin inflammation and provide photoprotection, potential mechanisms include competition with arachidonic acid (AA) for metabolism by cyclooxygenases/lipoxygenases to less pro-inflammatory mediators. We thus examine impact of EPA intake on levels of AA, EPA and their resulting eicosanoids in human skin with or without ultraviolet radiation (UVR) challenge. Methods and results In a double-blind randomised controlled study, 79 females took 5 g EPA-rich or control lipid for 12 wk. Pre- and post-supplementation, red blood cell and skin polyunsaturated fatty acids were assessed by GC, and eicosanoids from unexposed and UVR-exposed skin by LC-MS/MS. Active supplementation increased red blood cell and dermal EPA versus control (both p < 0.001), lowering relative AA:EPA content (4:1 versus 15:1 and 5:1 versus 11:1, respectively; both p < 0.001). Pre-supplementation, UVR increased PGE2, 12-hydroxyeicosatetraenoic acids, 12-HEPE (all p < 0.001) and PGE3 (p < 0.05). Post-EPA, PGE2 was reduced in unchallenged skin (p < 0.05) while EPA-derived PGE3 (non-sign) and 12-HEPE (p < 0.01) were elevated post-UVR. Thus, post-EPA, PGE2:PGE3 was lower in unchallenged (12:1 versus 28:1; p < 0.05) and UVR exposed (12:1 versus 54:1; p < 0.01) skin; 12-hydroxyeicosatetraenoic acids:12-HEPE was lower in UVR-exposed skin (3:1 versus 11:1; p < 0.001). Conclusion Dietary EPA augments skin EPA:AA content, shifting eicosanoid synthesis towards less pro-inflammatory species, and promoting a regulatory milieu under basal conditions and in response to inflammatory insult. PMID:24311515

A novel neutrophil-activating factor released by Trichomonas vaginalis.

PubMed Central

Shaio, M F; Lin, P R; Lee, C S; Hou, S C; Tang, P; Yang, K D

1992-01-01

We have investigated the effects of a novel neutrophil-activating factor released by Trichomonas vaginalis (TV-NAF) on neutrophil chemotaxis. TV-NAF was present in the supernatant from 10(7) T. vaginalis (STV) cultured in 1 ml of serum-free Hanks' balanced salt solution (HBSS) at 37 degrees C for 30 min. With a multichamber chemotactic assay, we found that there were 112 +/- 15 migrated neutrophils (mean +/- standard deviation, n = 7) for STV and 11 +/- 4 for HBSS per high-power field (x 400). STV was also able to induce neutrophil actin assembly (increased 1.5-fold), enhance expression of complement receptor type 3 (increased 5-fold), and promote intracellular calcium mobilization (increased 2.5-fold). There was no chemotactic activity in the preparation of STV from killed trichomonads. The fact that heating up to 100 degrees C or deproteinization by treatment with proteinase K at 65 degrees C for 1 h did not abolish its chemotactic activity suggests that the TV-NAF involved was not a protein. The chemotactic activity of TV-NAF was associated with the fraction containing small molecules of less than 3,000 Da. Therefore, the possibility that eicosanoid production by trichomonads is responsible for neutrophil activation was investigated. Leukotriene B4 (LTB4; 500 pg/ml) but not thromboxane B2 (< 20 pg/ml) or prostaglandin E2 (< 8 pg/ml) was found in the STV by radioimmunoassay. Production of LTB4 by trichomonads was time dependent and increased twofold when arachidonic acid (100 microM) was added but was not decreased when eicosanoid inhibitors were present. Evidence for the presence of LTB4 in STV was further provided by the fact that rabbit anti-LTB4 antiserum could abolish the chemotactic activity of STV. These studies suggest that the spontaneous release of TV-NAF, which is most likely LTB4, may activate neutrophils, presumably through a different arachidonate metabolic pathway than that in mammalian cells. PMID:1398962

Enhancement of bradykinin and resensitization of its B2 receptor.

PubMed

Marcic, B; Deddish, P A; Jackman, H L; Erdös, E G

1999-03-01

We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

Alterations of Na,K-ATPase isoenzymes in the rat diabetic neuropathy: protective effect of dietary supplementation with n-3 fatty acids.

PubMed

Gerbi, A; Maixent, J M; Barbey, O; Jamme, I; Pierlovisi, M; Coste, T; Pieroni, G; Nouvelot, A; Vague, P; Raccah, D

1998-08-01

Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase alpha subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of alpha1- and alpha3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.

Antioxidant and anti-inflammatory effects of Scoparia dulcis L.

PubMed

Coulibaly, Ahmed Y; Kiendrebeogo, Martin; Kehoe, Patrick G; Sombie, Pierre A E D; Lamien, Charles E; Millogo, Jeanne F; Nacoulma, Odile G

2011-12-01

Different extracts were obtained from Scoparia dulcis L. (Scrophulariaceae) by successive extraction with hexane, chloroform, and methanol. These extracts exhibited significant antioxidant capacity in various antioxidant models mediated (xantine oxidase and lipoxygenase) or not mediated (2,2-diphenyl-picrylhydrazyl, ferric-reducing antioxidant power, β-carotene bleaching, lipid peroxidation) by enzymes. The antioxidant activity of the extracts was related to their phytochemical composition in terms of polyphenol and carotenoid contents. The chloroform extract was richest in phytochemicals and had the highest antioxidant activity in the different antioxidant systems. All the extracts exhibited less than 50% inhibition on xanthine oxidase but more than 50% inhibition on lipid peroxidation and lipoxygenase. The extracts strongly inhibited lipid peroxidation mediated by lipoxygenase.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Askari, Ara A.; Thomson, Scott; Edin, Matthew L.

Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stablemore » commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.« less

Individual Variation in Lipidomic Profiles of Healthy Subjects in Response to Omega-3 Fatty Acids

PubMed Central

Nording, Malin L.; Yang, Jun; Georgi, Katrin; Hegedus Karbowski, Christine; German, J. Bruce; Weiss, Robert H.; Hogg, Ronald J.; Trygg, Johan; Hammock, Bruce D.; Zivkovic, Angela M.

2013-01-01

Introduction Conflicting findings in both interventional and observational studies have resulted in a lack of consensus on the benefits of ω3 fatty acids in reducing disease risk. This may be due to individual variability in response. We used a multi-platform lipidomic approach to investigate both the consistent and inconsistent responses of individuals comprehensively to a defined ω3 intervention. Methods The lipidomic profile including fatty acids, lipid classes, lipoprotein distribution, and oxylipins was examined multi- and uni-variately in 12 healthy subjects pre vs. post six weeks of ω3 fatty acids (1.9 g/d eicosapentaenoic acid [EPA] and 1.5 g/d docosahexaenoic acid [DHA]). Results Total lipidomic and oxylipin profiles were significantly different pre vs. post treatment across all subjects (p=0.00007 and p=0.00002 respectively). There was a strong correlation between oxylipin profiles and EPA and DHA incorporated into different lipid classes (r2=0.93). However, strikingly divergent responses among individuals were also observed. Both ω3 and ω6 fatty acid metabolites displayed a large degree of variation among the subjects. For example, in half of the subjects, two arachidonic acid cyclooxygenase products, prostaglandin E2 (PGE2) and thromboxane B2 (TXB2), and a lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) significantly decreased post intervention, whereas in the other half they either did not change or increased. The EPA lipoxygenase metabolite 12-hydroxyeicosapentaenoic acid (12-HEPE) varied among subjects from an 82% decrease to a 5,000% increase. Conclusions Our results show that certain defined responses to ω3 fatty acid intervention were consistent across all subjects. However, there was also a high degree of inter-individual variability in certain aspects of lipid metabolism. This lipidomic based phenotyping approach demonstrated that individual responsiveness to ω3 fatty acids is highly variable and measurable, and could be used as a means to assess the effectiveness of ω3 interventions in modifying disease risk and determining metabolic phenotype. PMID:24204640

Antioxidant/anti-inflammatory activities and total phenolic content of extracts obtained from plants grown in Vietnam.

PubMed

Sone, Yasuko; Moon, Joon-Kwan; Mai, Truong Tuyet; Thu, Nghiem Nguyet; Asano, Eri; Yamaguchi, Keiko; Otsuka, Yuzuru; Shibamoto, Takayuki

2011-09-01

Medicinal plants have been used to treat various diseases since ancient times. Their specific activities, such as antioxidant, anti-inflammatory and anti-cancer, have been studied intensively. In particular, plants grown in Vietnam have attracted considerable attention among food chemists as ideal sources of natural medicinal chemicals. The methanol extracts from three edible Vietnamese-grown plants, Tram, Voi and Gac, tested with the DPPH assay showed antioxidant activities of 91.7 ± 0.4, 63.4 ± 0.7 and 3.7 ± 0.1% respectively. The malonaldehyde/gas chromatography assay also revealed strong antioxidant activity in Tram and Voi at a level of 25 µg mL(-1) (95.5 ± 0.3 and 78.5 ± 1.4% respectively). These results were confirmed by the thiobarbituric acid assay. The antioxidant activities correlated positively with the level of total phenolics in all plants. Tram exhibited dose response-related lipoxygenase-inhibitory activity, with values of 74.2 ± 3.1% at 5 µg mL(-1) , 62.0 ± 0% at 0.5 µg mL(-1) and 3.0 ± 1.5% at 0.05 µg mL(-1) . Conversely, Voi and Gac showed negative anti-lipoxygenase activity. The antioxidant/anti-inflammatory activities and total phenolic contents of the three edible plants grown in Vietnam revealed that they are good sources of supplements for human health. Copyright © 2011 Society of Chemical Industry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kikuno, Shota; Taguchi, Keiko; Iwamoto, Noriko

1,2-Naphthoquinone (1,2-NQ) has recently been identified as an environmental quinone in diesel exhaust particles (DEP) and atmospheric PM{sub 2.5}. We have found that this quinone is capable of causing a concentration-dependent contraction of tracheal smooth muscle in guinea pigs with EC{sub 5} value of 18.7 {mu}M. The contraction required extracellular calcium and was suppressed by L-type calcium channel blockers nifedipine and diltiazem. It was found that 1,2-NQ activated phospholipase A2 (PLA2)/lipoxygenase (LO)/vanilloid receptor (VR1) signaling. Additionally, 1,2-NQ was capable of transactivating protein tyrosine kinases (PTKs) such as epidermal growth factor receptor (EGFR) in guinea pig trachea, suggesting that phosphorylation ofmore » PTKs contributes to 1,2-NQ-induced tracheal contraction. Consistent with this notion, this action was blocked by the PTKs inhibitor genistein and the EGFR antagonist PD153035, indicating that contraction was, at least in part, attributable to PTKs phosphorylation that activates VR1, resulting in increased intracellular calcium content in the smooth muscle cells.« less

Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice.

PubMed

Oda, Masataka; Domon, Hisanori; Kurosawa, Mie; Isono, Toshihito; Maekawa, Tomoki; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

2017-01-01

The Streptococcus pyogenes phospholipase A 2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes , which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the Δ slaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.

Progesterone inhibits contraction and increases TREK-1 potassium channel expression in late pregnant rat uterus

PubMed Central

Yin, Zongzhi; Li, Yun; He, Wenzhu; Li, Dan; Li, Hongyan; Yang, Yuanyuan; Shen, Bing; Wang, Xi; Cao, Yunxia; Khalil, Raouf A.

2018-01-01

Objective The aim of this study was to investigate the effect and mechanism by which progesterone regulates uterine contraction in late pregnant rats Results Progesterone caused concentration-dependent relaxation of uterine strips that was enhanced compared with control nontreated uterine strips. Uterine strips incubated with progesterone showed a significant increase in TREK-1 mRNA expression and protein level. TREK-1 inhibitor L-methionine partly reversed uterine relaxation caused by the progesterone, while TREK-1 activator arachidonic acid did not cause significant change in progesterone-induced relaxation. Conclusions Progesterone inhibits uterine contraction and induces uterine relaxation in late pregnancy. The progesterone-induced inhibition of uterine contraction appears to partly involve increased potassium channel TREK-1 expression/activity. Materials and Methods Uterus from late-pregnant rats (gestational day 19) was isolated, and uterine strips were prepared for isometric contraction measurement. Oxytocin-induced contraction was compared in uterine strips pretreated with different concentration of progesterone. TREK-1 potassium channel inhibitor L-methionine and TREK-1 agonist arachidonic acid were used to determine whether the changes caused by progesterone involve changes in TREK-1 activity. The mRNA and protein expression of TREK-1 in uterine tissues were measured using qPCR and Western blot. PMID:29416642

Role of phospholipase A2 pathway in regulating activation of Bufo arenarum oocytes.

PubMed

Ajmat, M T; Bonilla, F; Hermosilla, P C; Zelarayán, L; Bühler, M I

2013-08-01

Transient increases in the concentration of cytosolic Ca(2+) are essential for triggering egg activation events. Increased Ca(2+) results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A(2) (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A(2) (PLA(2)) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA(2) activator, and arachidonic acid, the main PLA(2) reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.

Activation of Protein Kinase C by Mycobacterial Cord Factor, Trehalose 6‐Monomycolate, Resulting in Tumor Necrosis Factor‐α Release in Mouse Lung Tissues

PubMed Central

Sueoka, Eisaburo; Nishiwaki, Shinji; Okabe, Sachiko; Iida, Naoyuki; Suganuma, Masami; Yano, Ikuya; Aoki, Kunio

1995-01-01

Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage‐derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6‐monomycolate (mTMM) and trehalose 6,6′‐dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKCα more strongly than PKCβ or γ under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor‐α (TNF‐α) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF‐α is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed. PMID:7559098

Activation of protein kinase C by mycobacterial cord factor, trehalose 6-monomycolate, resulting in tumor necrosis factor-alpha release in mouse lung tissues.

PubMed

Sueoka, E; Nishiwaki, S; Okabe, S; Iida, N; Suganuma, M; Yano, I; Aoki, K; Fujiki, H

1995-08-01

Cord factors are mycoloyl glycolipids in cell walls of bacteria belonging to Actinomycetales, such as Mycobacterium, Nocardia and Rhodococcus. They induce granuloma formation in the lung and interstitial pneumonitis, associated with production of macrophage-derived cytokines. We studied how cord factors induce biological activities in the cells. Cord factors isolated from M. tuberculosis, trehalose 6-monomycolate (mTMM) and trehalose 6,6'-dimycolate (mTDM), enhanced protein kinase C (PKC) activation in the presence of phosphatidylserine (PtdSer), diacylglycerol and Ca2+, and mTMM activated PKC alpha more strongly than PKC beta or gamma under the same assay conditions. Kinetic studies of mTMM in response to PKC activation revealed that mTMM increased the apparent affinity of PKC to Ca2+ in the presence of both PtdSer and diolein. Although this is similar to observations with unsaturated fatty acids, such as arachidonic acid, mTMM was synergistic with PtdSer for PKC activation, but arachidonic acid was not. mTMM was also different as regards PKC activation, as phorbol ester was. A single i.p. administration of mTMM to mouse induced tumor necrosis factor-alpha (TNF-alpha) in serum and in the lung, which is a unique target tissue of cord factors. Based on our recent finding that TNF-alpha is an endogenous tumor promoter, the correlation between lung cancer and pulmonary tuberculosis is discussed.

Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis: an in vitro study.

PubMed

Skowronska, Agnieszka; Młotkowska, Patrycja; Wojciechowicz, Bartosz; Okrasa, Stanisław; Nielsen, Soren; Skowronski, Mariusz T

2015-02-18

The cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression. Uterine tissues were collected on Days 10-12 and 14-16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry. The results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P4, E2, AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus. This study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs.

Pharmacological manipulation of the chronic granulomatous reactions in the livers of mice infected with schistosomiasis.

PubMed

Rainsford, K D

1985-01-01

The severe granulomatous reactions in the liver which occur following infestation by adult Schistosoma mansonii are largely initiated by invading eosinophils and monocytes. The present studies were designed to investigate the possibility that (a) anti-inflammatory drugs could be employed beneficially to attenuate the liver granulomatous reactions in schistosomiasis, and (b) that part of the therapeutic effects of anti-schistosomal (AS) drugs might be due to possible influences on arachidonate metabolism. Therefore the effects were determined of (a) AS as compared with NSAI drugs on eicosanoid metabolism in isolated human peripheral leucocyte populations, and (b) electron-microscopic changes in the livers of mice infected with Schistosoma mansoni in response to AS and/or NSAI drugs. Of the AS drugs only praziquantel (10-100 microM) inhibited 5-HETE production by the 5-lipoxygenase pathway. No effects were observed of this or the other AS drugs on prostaglandin production. In S. mansoni infected mice, praziquantel (250 mg/kg/d), given orally with indomethacin (5 mg/kg/d) for 5 days did not improve the inflammatory reactions around worms or eggs of schistosomes. Furthermore, both indomethacin (5 mg/kg/d) alone and benoxaprofen (20 mg/kg/d for 5 days) elicited liver changes suggestive of specific liver damage by these drugs. These results suggest that liver pathology may be enhanced by NSAI drugs perhaps as a consequence of the liver metabolism of these drugs being compromised. Their use to modify inflammatory reactions at the peak of liver schistosome infections may thus be contraindicated.

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males

PubMed Central

Pace, Simona; Pergola, Carlo; Dehm, Friederike; Rossi, Antonietta; Gerstmeier, Jana; Troisi, Fabiana; Pein, Helmut; Schaible, Anja M.; Weinigel, Christina; Rummler, Silke; Northoff, Hinnak; Laufer, Stefan; Maier, Thorsten J.; Rådmark, Olof; Samuelsson, Bengt; Sautebin, Lidia

2017-01-01

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO–activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor–induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development. PMID:28737505

Role of the Lipoxygenase Pathway in RSV-induced Alternatively Activated Macrophages Leading to Resolution of Lung Pathology

PubMed Central

Shirey, Kari Ann; Lai, Wendy; Pletneva, Lioubov M.; Karp, Christopher L.; Divanovic, Senad; Blanco, Jorge C. G.; Vogel, Stefanie N.

2013-01-01

Resolution of severe RSV-induced bronchiolitis is mediated by alternatively activated macrophages (AA-Mϕ) that counteract cyclooxygenase (COX)-2-induced lung pathology. Herein, we report that RSV infection of 5-lipoxygenase (LO)−/− and 15-LO−/− macrophages or mice failed to elicit AA-Mϕ differentiation and concomitantly exhibited increased COX-2 expression. Further, RSV infection of 5-LO−/− mice resulted in enhanced lung pathology. Pharmacologic inhibition of 5-LO or 15-LO also blocked differentiation of RSV-induced AA-Mϕ in vitro and, conversely, treatment of 5-LO−/− macrophages with downstream products, lipoxin A4 (LXA4) and resolvin E1 (RvE1), but not leukotriene B4 (LTB4) or LTD4, partially restored expression of AA-Mϕ markers. Indomethacin blockade of COX activity in RSV-infected macrophages increased 5-LO, and 15-LO, as well as arginase-1 mRNA expression. Treatment of RSV-infected mice with indomethacin also resulted not only in enhanced lung arginase-1 mRNA expression and decreased COX-2, but also, decreased lung pathology in RSV-infected 5-LO−/− mice. Treatment of RSV-infected cotton rats with a COX-2-specific inhibitor resulted in enhanced lung 5-LO mRNA and AA-Mϕ marker expression. Together, these data suggest a novel therapeutic approach for RSV that promotes AA-Mϕ differentiation by activating the 5-LO pathway. PMID:24064666

Monocyte 15-lipoxygenase gene expression requires ERK1/2 MAPK activity.

PubMed

Bhattacharjee, Ashish; Mulya, Anny; Pal, Srabani; Roy, Biswajit; Feldman, Gerald M; Cathcart, Martha K

2010-11-01

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13-mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13-induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB.

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain.

PubMed

Hörnig, Christina; Albert, Dana; Fischer, Lutz; Hörnig, Michael; Rådmark, Olof; Steinhilber, Dieter; Werz, Oliver

2005-07-22

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.

Enteroaggregative Escherichia coli Promotes Transepithelial Migration of Neutrophils Through a Conserved 12-Lipoxygenase Pathway

PubMed Central

Boll, Erik J.; Struve, Carsten; Sander, Anja; Demma, Zachary; Krogfelt, Karen A.; McCormick, Beth A.

2014-01-01

Summary Enteroaggregative Escherichia coli (EAEC) induces release of pro-inflammatory markers and disruption of intestinal epithelial barriers in vitro suggesting an inflammatory aspect to EAEC infection. However, the mechanisms underlying EAEC-induced mucosal inflammatory responses and the extent to which these events contribute to pathogenesis is not well characterized. Employing an established in vitro model we demonstrated that EAEC prototype strain 042 induces migration of polymorphonuclear neutrophils (PMNs) across polarized T84 cell monolayers. This event was mediated through a conserved host cell signaling cascade involving the 12/15-LOX pathway and led to apical secretion of an arachidonic acid-derived lipid PMN chemoattractant, guiding PMNs across the epithelia to the site of infection. Moreover, supporting the hypothesis that inflammatory responses may contribute to EAEC pathogenesis, we found that PMN transepithelial migration promoted enhanced attachment of EAEC 042 to T84 cells. These findings suggest that EAEC-induced PMN infiltration may favor colonization and thus pathogenesis of EAEC. PMID:21951973

A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

PubMed

Bell, E; Creelman, R A; Mullet, J E

1995-09-12

Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene.

Ligand-independent activation of EphA2 by arachidonic acid induces metastasis-like behaviour in prostate cancer cells

PubMed Central

Tawadros, T; Brown, M D; Hart, C A; Clarke, N W

2012-01-01

Background: High intake of omega-6 polyunsaturated fatty acids (PUFA) has been associated with clinical progression in prostate cancer (CaP). This study investigates the signalling mechanism by which the omega-6 PUFA arachidonic acid (AA) induces prostatic cellular migration to bone marrow stroma. Methods: Western blot analysis of the PC-3, PC3-GFP, DU 145 and LNCaP cells or their lipid raft (LR) components post AA stimulation was conducted in association with assays for adhesion and invasion through the bone marrow endothelial monolayers. Results: Arachidonic acid increased transendothelial migration of PC3-GFP cells (adhesion 37%±0.08, P=0.0124; transmigration 270%±0.145, P=0.0008). Akt, Src and focal adhesion kinase (FAK) pathways were induced by AA and integrally involved in transendothelial migration. LR were critical in AA uptake and induced Akt activity. Ephrin receptor A2 (EphA2), localised in LR, is expressed in DU 145 and PC-3 cells. Arachidonic acid induced a rapid increase of EphA2 Akt-dependent/ligand-independent activation, while knockdown of the EphrinA1 ligand decreased AA induced transendothelial migration, with an associated decrease in Src and FAK activity. Arachidonic acid activated Akt in EphA2− LNCaP cells but failed to induce BMEC transendothelial invasion. Conclusion: Arachidonic acid induced stimulation of EphA2 in vitro is associated fundamentally with CaP epithelial migration across the endothelial barrier. PMID:23037715

Maize death acids, 9-lipoxygenase derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators

USDA-ARS?s Scientific Manuscript database

Plant damage promotes the interaction of lipoxygenases (LOX) with fatty acids yielding 9-hydroperoxides, 13-hydroperoxides and complex arrays of oxylipins. The action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downstream products, termed jasmonates. ...

A Circadian Rhythm-Regulated Tomato Gene Is Induced by Arachidonic Acid and Phythophthora infestans Infection1[W

PubMed Central

Weyman, Philip D.; Pan, Zhiqiang; Feng, Qin; Gilchrist, David G.; Bostock, Richard M.

2006-01-01

A cDNA clone of unknown function, DEA1, was isolated from arachidonic acid-treated tomato (Solanum lycopersicum) leaves by differential display PCR. The gene, DEA1, is expressed in response to the programmed cell death-inducing arachidonic acid within 8 h following treatment of a tomato leaflet, 16 h prior to the development of visible cell death. DEA1 transcript levels were also affected by the late blight pathogen, Phytophthora infestans. To gain further insight into the transcriptional regulation of DEA1, the promoter region was cloned by inverse PCR and was found to contain putative stress-, signaling-, and circadian-response elements. DEA1 is highly expressed in roots, stems, and leaves, but not in flowers. Leaf expression of DEA1 is regulated by circadian rhythms during long days with the peak occurring at midday and the low point midway through the dark period. During short days, the rhythm is lost and DEA1 expression becomes constitutive. The predicted DEA1 protein has a conserved domain shared by the eight-cysteine motif superfamily of protease inhibitors, α-amylase inhibitors, seed storage proteins, and lipid transfer proteins. A DEA1-green fluorescent protein fusion protein localized to the plasma membrane in protoplasts and plasmolysis experiments, suggesting that the native protein is associated with the plasmalemma in intact cells. PMID:16361525

5-Lypoxygenase products are involved in renal tubulointerstitial injury induced by albumin overload in proximal tubules in mice.

PubMed

Landgraf, Sharon Schilling; Silva, Leandro Souza; Peruchetti, Diogo Barros; Sirtoli, Gabriela Modenesi; Moraes-Santos, Felipe; Portella, Viviane Gomes; Silva-Filho, João Luiz; Pinheiro, Carla Silva; Abreu, Thiago Pereira; Takiya, Christina Maeda; Benjamin, Claudia Farias; Pinheiro, Ana Acacia Sá; Canetti, Claudio; Caruso-Neves, Celso

2014-01-01

The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO(-/-)). The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO(-/-) mice. The levels of urinary protein observed in the 5-LO(-/-) mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO(-/-) mice. LTB4 and LTD4, 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-α and interleukin (IL)-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO(-/-) mice. However, 5-LO(-/-) mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload.

5-Lypoxygenase Products Are Involved in Renal Tubulointerstitial Injury Induced by Albumin Overload in Proximal Tubules in Mice

PubMed Central

Landgraf, Sharon Schilling; Silva, Leandro Souza; Peruchetti, Diogo Barros; Sirtoli, Gabriela Modenesi; Moraes-Santos, Felipe; Portella, Viviane Gomes; Silva-Filho, João Luiz; Pinheiro, Carla Silva; Abreu, Thiago Pereira; Takiya, Christina Maeda; Benjamin, Claudia Farias; Pinheiro, Ana Acacia Sá; Canetti, Claudio; Caruso-Neves, Celso

2014-01-01

The role of albumin overload in proximal tubules (PT) in the development of tubulointerstitial injury and, consequently, in the progression of renal disease has become more relevant in recent years. Despite the importance of leukotrienes (LTs) in renal disease, little is known about their role in tubulointerstitial injury. The aim of the present work was to investigate the possible role of LTs on tubulointerstitial injury induced by albumin overload. An animal model of tubulointerstitial injury challenged by bovine serum albumin was developed in SV129 mice (wild-type) and 5-lipoxygenase-deficient mice (5-LO–/–). The changes in glomerular morphology and nestin expression observed in wild-type mice subjected to kidney insult were also observed in 5-LO–/– mice. The levels of urinary protein observed in the 5-LO–/– mice subjected or not to kidney insult were lower than those observed in respective wild-type mice. Furthermore, the increase in lactate dehydrogenase activity, a marker of tubule damage, observed in wild-type mice subjected to kidney insult did not occur in 5-LO–/– mice. LTB4 and LTD4, 5-LO products, decreased the uptake of albumin in LLC-PK1 cells, a well-characterized porcine PT cell line. This effect correlated with activation of protein kinase C and inhibition of protein kinase B. The level of proinflammatory cytokines, tumor necrosis factor-α and interleukin (IL)-6, increased in mice subjected to kidney insult but this effect was not modified in 5-LO–/– mice. However, 5-LO–/– mice subjected to kidney insult presented lower macrophage infiltration and higher levels of IL-10 than wild-type mice. Our results reveal that LTs have an important role in tubulointerstitial disease induced by albumin overload. PMID:25302946

Role of protein kinase C alpha and mitogen-activated protein kinases in endothelin-1-stimulation of cytosolic phospholipase A2 in iris sphincter smooth muscle.

PubMed

Abdel-Latif, A A; Husain, S; Yousufzai, S Y

2000-11-01

We have investigated the roles of protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) in the phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in endothelin-1- (ET-1) stimulated cat iris sphincter smooth muscle (CISM) cells. We found that in these cells both PKC and p38 MAP kinases play a critical role in ET-1-induced cPLA, phosphorylation and arachidonic acid (AA) release. Our findings indicate that stimulation of the endothelin-A- (ET(A)) receptor leads to: (1) activation of Gq protein which stimulates phospholipase C to hydrolyze the polyphosphoinositide PIP, into diacylglycerol (DAG) and inositol trisphosphate (IP3), the DAG may then activate PKC to phosphorylate and activate cPLA2; and (2) activation of Gi protein, which, through a series of kinases, leads to the stimulation of p38 MAPK and subsequently to phosphorylation and activation of cPLA2. The ability of the activated ET(A)-receptor, which is coupled to both Gq and Gi proteins, to recruit and activate this complex signal transduction mechanism remains to be clarified.

Targeting arachidonic acid pathway by natural products for cancer prevention and therapy.

PubMed

Yarla, Nagendra Sastry; Bishayee, Anupam; Sethi, Gautam; Reddanna, Pallu; Kalle, Arunasree M; Dhananjaya, Bhadrapura Lakkappa; Dowluru, Kaladhar S V G K; Chintala, Ramakrishna; Duddukuri, Govinda Rao

2016-10-01

Arachidonic acid (AA) pathway, a metabolic process, plays a key role in carcinogenesis. Hence, AA pathway metabolic enzymes phospholipase A 2 s (PLA 2 s), cyclooxygenases (COXs) and lipoxygenases (LOXs) and their metabolic products, such as prostaglandins and leukotrienes, have been considered novel preventive and therapeutic targets in cancer. Bioactive natural products are a good source for development of novel cancer preventive and therapeutic drugs, which have been widely used in clinical practice due to their safety profiles. AA pathway inhibitory natural products have been developed as chemopreventive and therapeutic agents against several cancers. Curcumin, resveratrol, apigenin, anthocyans, berberine, ellagic acid, eugenol, fisetin, ursolic acid, [6]-gingerol, guggulsteone, lycopene and genistein are well known cancer chemopreventive agents which act by targeting multiple pathways, including COX-2. Nordihydroguaiaretic acid and baicalein can be chemopreventive molecules against various cancers by inhibiting LOXs. Several PLA 2 s inhibitory natural products have been identified with chemopreventive and therapeutic potentials against various cancers. In this review, we critically discuss the possible utility of natural products as preventive and therapeutic agents against various oncologic diseases, including prostate, pancreatic, lung, skin, gastric, oral, blood, head and neck, colorectal, liver, cervical and breast cancers, by targeting AA pathway. Further, the current status of clinical studies evaluating AA pathway inhibitory natural products in cancer is reviewed. In addition, various emerging issues, including bioavailability, toxicity and explorability of combination therapy, for the development of AA pathway inhibitory natural products as chemopreventive and therapeutic agents against human malignancy are also discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

In vitro biological activity and essential oil composition of four indigenous South African Helichrysum species.

PubMed

Lourens, A C U; Reddy, D; Başer, K H C; Viljoen, A M; Van Vuuren, S F

2004-12-01

Helichrysum species are used widely to treat various medical conditions. In this study, the anti-microbial, anti-oxidant (DPPH assay) and anti-inflammatory activity (5-lipoxygenase assay) of Helichrysum dasyanthum, Helichrysum felinum, Helichrysum excisum and Helichrysum petiolare were investigated. The essential oil compositions of these species were determined. The acetone and methanol extracts as well as the essential oils exhibited activity against Gram-positive bacteria, while both the methanol and acetone extracts of all four species were active in the anti-oxidant assay. The essential oils, on the other hand, displayed activity in the 5-lipoxygenase assay, which was used as an indication of anti-inflammatory activity. Two extracts exhibited promising activity in the anti-microbial assay, the acetone extract of Helichrysum dasyanthum with a MIC value of 15.63 microg/ml and the methanol extract of Helichrysum excisum with a MIC value of 62.5 microg/ml. The acetone extract of Helichrysum dasyanthum was the most active free radical scavenger in the DPPH assay (IC(50) of 9.53 microg/ml) while values for the anti-inflammatory activity of the essential oils ranged between 25 and 32 microg/ml. The essential oil compositions of three species (Helichrysum dasyanthum, Helichrysum excisum and Helichrysum petiolare) were dominated by the presence of monoterpenes such as alpha-pinene, 1,8-cineole and p-cymene. In the oil of Helichrysum felinum, monoterpenes were largely absent. Its profile consisted of a variety of sesquiterpenes in low concentrations with beta-caryophyllene dominating.

Altered Arachidonate Distribution in Macrophages from Caveolin-1 Null Mice Leading to Reduced Eicosanoid Synthesis*

PubMed Central

Astudillo, Alma M.; Pérez-Chacón, Gema; Meana, Clara; Balgoma, David; Pol, Albert; del Pozo, Miguel A.; Balboa, María A.; Balsinde, Jesús

2011-01-01

In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E2 and LTB4 production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response. PMID:21852231

Role of ion channels and subcellular Ca2+ signaling in arachidonic acid-induced dilation of pressurized retinal arterioles.

PubMed

Kur, Joanna; McGahon, Mary K; Fernández, Jose A; Scholfield, C Norman; McGeown, J Graham; Curtis, Tim M

2014-05-02

To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacologic agents inhibiting Ca(2+) sparks and oscillations and K(+) channels. Subcellular Ca(2+) signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Arachidonic acid dilated pressurized retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. Arachidonic acid-induced dilation was associated with an inhibition of subcellular Ca(2+) signals. Interventions known to block Ca(2+) sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. Arachidonic acid accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous large-conductance calcium-activated K(+) (BK) currents, but only at positive membrane potentials. Pharmacologic inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. Arachidonic acid suppressed L-type Ca(2+) currents. These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca(2+)-signaling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca(2+)-channel activity. Arachidonic acid actions on K(+) currents are inconsistent with a model in which K(+) channels contribute to the vasodilator effects of AA.

Physiological inhibitory effect of ocs in arachidonic acid-rich Parietochloris incisa (trebouxiophyceae, chlorophyta)

NASA Astrophysics Data System (ADS)

Liu, Jian-Guo; Zhang, Cheng-Wu; Cohen, Zvi; Richmond, Amos

2002-09-01

Parietochloris incisa is an arachidonic acid-rich snow green alga. The main physiological profiles, such as ash free dry weight (AFDW), chlorophyll, carotenoid, protein and total fatty acids (TFA), in this alga exposed to old culture supernatant (OCS) at the decline phase or its crude ethyl acetate extracts (CEAE) were investigated by using tubular photobioreactors of different diameters. Results showed that both OCS and CEAE had strong inhibitory effect on the above physiological parameters. The longer the culture was exposed to OCS and the more CEAE were added into the algal culture, the more the above physiological properties were inhibited. Arachidonic acid (AA), the dominant component of fatty acids in this alga, was also seriously inhibited with respect to total TFA, AFDW of cell mass, or culture volume, due to a probable reduction of enzymes activities catalyzing chain elongation from C18; 1ω9 to AA. These results incontestably evidenced that some CEAE dissolving substances existing in OCS. like auto-inhibitors, inhibited P. incisa growth through feedback. Hence, any efficient removal of auto-inhibitors from algal culture to decrease their bioactivity could be good for maximal production of desired products like AA.

Effects of flavocoxid, a dual inhibitor of COX and 5-lipoxygenase enzymes, on benign prostatic hyperplasia

PubMed Central

Altavilla, D; Minutoli, L; Polito, F; Irrera, N; Arena, S; Magno, C; Rinaldi, M; Burnett, BP; Squadrito, F; Bitto, A

2012-01-01

BACKGROUND AND PURPOSE Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5-lipoxygenase (5-LOX) pathways are elevated in the enlarging prostate. Flavocoxid is a novel flavonoid–based ‘dual inhibitor’ of the COX and 5-LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH. EXPERIMENTAL APPROACH Rats were treated daily with testosterone propionate (3 mg·kg−1 s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg−1, i.p.) or flavocoxid (20 mg·kg−1, i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis-related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells. KEY RESULTS Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX-2 and 5-LOX as well as the increased production of PGE2 and leukotriene B4 (LTB4), enhanced pro-apoptotic Bax and caspase-9 and decreased the anti-apoptotic Bcl-2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid-mediated induction of apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid-mediated apoptosis during prostatic growth. CONCLUSION AND IMPLICATIONS Our results show that a ‘dual inhibitor’ of the COX and 5-LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase-induced apoptotic mechanism. PMID:22471974

Effects of flavocoxid, a dual inhibitor of COX and 5-lipoxygenase enzymes, on benign prostatic hyperplasia.

PubMed

Altavilla, D; Minutoli, L; Polito, F; Irrera, N; Arena, S; Magno, C; Rinaldi, M; Burnett, B P; Squadrito, F; Bitto, A

2012-09-01

Inflammation plays a key role in the development of benign prostatic hyperplasia (BPH). Eicosanoids derived from the COX and 5-lipoxygenase (5-LOX) pathways are elevated in the enlarging prostate. Flavocoxid is a novel flavonoid-based 'dual inhibitor' of the COX and 5-LOX enzymes. This study evaluated the effects of flavocoxid in experimental BPH. Rats were treated daily with testosterone propionate (3 mg·kg(-1)  s.c.) or its vehicle for 14 days to induce BPH. Animals receiving testosterone were randomized to receive vehicle (1 mL·kg(-1) , i.p.) or flavocoxid (20 mg·kg(-1) , i.p.) for 14 days. Histological changes, eicosanoid content and mRNA and protein levels for apoptosis-related proteins and growth factors were assayed in prostate tissue. The effects of flavocoxid were also tested on human prostate carcinoma PC3 cells. Flavocoxid reduced prostate weight and hyperplasia, blunted inducible expression of COX-2 and 5-LOX as well as the increased production of PGE(2) and leukotriene B(4) (LTB(4) ), enhanced pro-apoptotic Bax and caspase-9 and decreased the anti-apoptotic Bcl-2 mRNA. Flavocoxid also reduced EGF and VEGF expression. In PC3 cells, flavocoxid stimulated apoptosis and inhibited growth factor expression. Flavocoxid-mediated induction of apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, in PC3 cells, suggesting an essential role of caspases in flavocoxid-mediated apoptosis during prostatic growth. Our results show that a 'dual inhibitor' of the COX and 5-LOX enzymes, such as flavocoxid, might represent a rational approach to reduce BPH through modulation of eicosanoid production and a caspase-induced apoptotic mechanism. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Mixed-ligand Cu II complexes with Me 5dien and heterocyclic acids. Synthesis, antioxidant and anti-inflammatory activity. Crystal structure of [Cu(Me 5dien)(tpaa)(H 2O)](ClO 4)

NASA Astrophysics Data System (ADS)

Christidis, Panayiotis C.; Georgousis, Zacharias D.; Hadjipavlou-Litina, Dimitra; Bolos, Christos A.

2008-01-01

The reaction of sodium salt of 2-thiophenecarboxylic acid (tpca), 2-thiopheneacetic acid (tpaa), 2-furoic acid (fa) and picolinic acid (pica), with [Cu(Me 5dien)(ClO 4) 2] ( 1) (Me 5dien = N, N, N', N″ N″-pentamethyldiethylenetriamine) in a 1:1 molar ratio, afforded new mixed-ligand compounds of the type [Cu(Me 5dien)(tpca)(H 2O)](ClO 4) ( 2), [Cu(Me 5dien)(tpaa)(H 2O)](ClO 4) ( 3), [Cu(Me 5dien)(fa)](BPh 4) ( 4) and [Cu(Me 5dien)(pica)](ClO 4) ( 5). The new mixed-ligand complexes are mononuclear, paramagnetic, conductive compounds with a distorted square pyramidal geometry. The square pyramidal stereochemistry proposed by spectroscopic (IR, UV-vis) data was further confirmed by the X-ray structure analysis of the compound ( 3) in which the Cu atom is coordinated by the three N atoms from the Me 5dien ligand, one O atom from the mono-carboxylate anion, lying on the equatorial square plane, and one O atom from the water molecule, occupying the axial position. The two Cu sbnd O bond distances are 1.955(2) and 2.212(2) Ǻ, respectively. The complexes were tested for antioxidant/anti-inflammatory activity. Complex 4 is the most active against soybean lipoxygenase with IC 50 = 100 μM. The presence of a furoic ring leads to higher lipoxygenase inhibition, whereas the picolinyl-ring supports scavenging activity.

FABP-1 gene ablation impacts brain endocannabinoid system in male mice.

PubMed

Martin, Gregory G; Chung, Sarah; Landrock, Danilo; Landrock, Kerstin K; Huang, Huan; Dangott, Lawrence J; Peng, Xiaoxue; Kaczocha, Martin; Seeger, Drew R; Murphy, Eric J; Golovko, Mikhail Y; Kier, Ann B; Schroeder, Friedhelm

2016-08-01

Liver fatty acid-binding protein (FABP1, L-FABP) has high affinity for and enhances uptake of arachidonic acid (ARA, C20:4, n-6) which, when esterified to phospholipids, is the requisite precursor for synthesis of endocannabinoids (EC) such as arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG). The brain derives most of its ARA from plasma, taking up ARA and transporting it intracellularly via cytosolic fatty acid-binding proteins (FABPs 3,5, and 7) localized within the brain. In contrast, the much more prevalent cytosolic FABP1 is not detectable in the brain but is instead highly expressed in the liver. Therefore, the possibility that FABP1 outside the central nervous system may regulate brain AEA and 2-AG was examined in wild-type (WT) and FABP1 null (LKO) male mice. LKO increased brain levels of AA-containing EC (AEA, 2-AG), correlating with increased free and total ARA in brain and serum. LKO also increased brain levels of non-ARA that contain potentiating endocannabinoids (EC*) such as oleoyl ethanolamide (OEA), PEA, 2-OG, and 2-PG. Concomitantly, LKO decreased serum total ARA-containing EC, but not non-ARA endocannabinoids. LKO did not elicit these changes in the brain EC and EC* as a result of compensatory up-regulation of brain protein levels of enzymes in EC synthesis (NAPEPLD, DAGLα) or cytosolic EC chaperone proteins (FABPs 3, 5, 7, SCP-2, HSP70), or cannabinoid receptors (CB1, TRVP1). These data show for the first time that the non-CNS fatty acid-binding protein FABP1 markedly affected brain levels of both ARA-containing endocannabinoids (AEA, 2-AG) as well as their non-ARA potentiating endocannabinoids. Fatty acid-binding protein-1 (FABP-1) is not detectable in brain but instead is highly expressed in liver. The possibility that FABP1 outside the central nervous system may regulate brain endocannabinoids arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) was examined in wild-type (WT) and FABP-1 null (LKO) male mice. LKO increased brain levels of arachidonic acid-containing endocannabinoids (AEA, 2-AG), correlating with increased free and total arachidonic acid in brain and serum. Read the Editorial Highlight for this article on page 371. © 2016 International Society for Neurochemistry.

Maize death acids, 9-lipoxygenase-derived cyclopente(a)nones display activity as cytotoxic phytoalexins and transcriptional mediators

USDA-ARS?s Scientific Manuscript database

Plant cellular damage promotes the interaction of lipoxygenases (LOX) with free fatty acids to yield 9- and 13-hydroperoxides which are further metabolized into diverse oxylipins. The enzymatic action of 13-LOX on linolenic acid enables production of 12-oxo-phytodienoic acid (12-OPDA) and its downst...

HPLC PROFILING OF PHENOLIC ACIDS AND FLAVONOIDS AND EVALUATION OF ANTI-LIPOXYGENASE AND ANTIOXIDANT ACTIVITIES OF AQUATIC VEGETABLE LIMNOCHARIS FLAVA.

PubMed

Ooh, Keng-fei; Ong, Hean-Chooi; Wong, Fai-Chu; Chai, Tsun-Thai

2015-01-01

Limnocharis flava is an edible wetland plant, whose phenolic acid and flavonoid compositions as well as bioactivities were underexplored. This study analyzed the profiles of selected hydroxybenzoic acids, hydroxycinnamic acids and flavonoids in the aqueous extracts of L. flava leaf, rhizome and root by high performance liquid chromatography (HPLC). Anti-lipoxygenase and antioxidant (iron chelating, 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical scavenging, and nitric oxide (NO) scavenging) activities of the extracts were also evaluated. Leaf extract had the highest phenolic contents, being most abundant in p-hydroxybenzoic acid (3861.2 nmol/g dry matter), ferulic acid (648.8 nmol/g dry matter), and rutin (4110.7 nmol/g dry matter). Leaf extract exhibited the strongest anti-lipoxygenase (EC50 6.47 mg/mL), iron chelating (EC50 6.65 mg/mL), DPPH scavenging (EC50 15.82 mg/mL) and NO scavenging (EC50 3.80 mg/mL) activities. Leaf extract also had the highest ferric reducing ability. This is the most extensive HPLC profiling of phenolic acids and flavonoids in L.flava to date. In conclusion, L. flava leaf is a source of health-promoting phenolics, anti-lipoxygenase agents and antioxidants.

Peroxygenase-Catalyzed Fatty Acid Epoxidation in Cereal Seeds (Sequential Oxidation of Linoleic Acid into 9(S),12(S),13(S)-Trihydroxy-10(E)-Octadecenoic Acid).

PubMed Central

Hamberg, M.; Hamberg, G.

1996-01-01

Peroxygenase-catalyzed epoxidation of oleic acid in preparations of cereal seeds was investigated. The 105,000g particle fraction of oat (Avena sativa) seed homogenate showed high peroxygenase activity, i.e. 3034 [plus or minus] 288 and 2441 [plus or minus] 168 nmol (10 min)-1 mg-1 protein in two cultivars, whereas the corresponding fraction obtained from barley (Hordeum vulgare and Hordeum distichum), rye (Secale cereale), and wheat (Triticum aestivum) showed only weak activity, i.e. 13 to 138 nmol (10 min)-1 mg-1 protein. In subcellular fractions of oat seed homogenate, peroxygenase specific activity was highest in the 105,000g particle fraction, whereas lipoxygenase activity was more evenly distributed and highest in the 105,000g supernatant fraction. Incubation of [1-14C]linoleic acid with the 105,000g supernatant of oat seed homogenate led to the formation of several metabolites, i.e. in order of decreasing abundance, 9(S)-hydroxy-10(E),12(Z)-octadecadienoic acid, 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, cis-9,10-epoxy-12(Z)-octadecenoic acid [mainly the 9(R),10(S) enantiomer], cis-12,13-epoxy-9(Z)-octadecenoic acid [mainly the 12(R),13(S) enantiomer], threo-12,13-dihydroxy-9(Z)-octadecenoic acid, and 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. Incubation of linoleic acid with the 105,000g particle fraction gave a similar, but not identical, pattern of metabolites. Conversion of linoleic acid into 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, a naturally occurring oxylipin with antifungal properties, took place by a pathway involving sequential catalysis by lipoxygenase, peroxygenase, and epoxide hydrolase. PMID:12226220

Effects of COX1-2/5-LOX blockade in Alzheimer transgenic 3xTg-AD mice.

PubMed

Bitto, Alessandra; Giuliani, Daniela; Pallio, Giovanni; Irrera, Natasha; Vandini, Eleonora; Canalini, Fabrizio; Zaffe, Davide; Ottani, Alessandra; Minutoli, Letteria; Rinaldi, Mariagrazia; Guarini, Salvatore; Squadrito, Francesco; Altavilla, Domenica

2017-05-01

Alzheimer's disease (AD) is associated with amyloid plaques (Aβ) and hyperphosphorylated tau protein tangles in the brain. We investigated the possible neuroprotective role of flavocoxid, a dual inhibitor of cyclooxygenases-1/2 (COX-1/2) and 5-Lipoxygenase (5-LOX), in triple-transgenic (3xTg-AD) mice. Mice were 3 months at the beginning of the study. Animals received once daily for 3-month saline solution or flavocoxid (20 mg/kg/ip). Morris water maze was used to assess learning and memory. Histology was performed to evidence Aβ plaques and neuronal loss, while inflammatory proteins were determined by western blot analysis. Saline-treated 3xTg-AD mice showed an impairment in spatial learning and memory (assessed at 6 months of age), and increased expression of inflammatory and apoptotic molecules. Treatment of 3xTg-AD mice with flavocoxid reduced: (1) learning and memory loss; (2) the increased eicosanoid production and the phosphorylation level of amyloid precursor protein (APP-pThr668), Aβ 1-42, p-tau (pThr181), pERK, and the activation of the NLRP3 inflammasome; (3) Aβ plaques; and (4) neuronal loss, compared to saline-treated animals. Pharmacological blockade of both COX-1/2 and 5-LOX was able to counteract the progression of AD by targeting pathophysiological mechanisms up- and downstream of Aβ and tau.

Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta.

PubMed Central

Kent, R S; Diedrich, S L; Whorton, A R

1983-01-01

To address the hypothesis that metabolites of arachidonic acid are important regulators of prostaglandin (PG) synthesis in intact vascular tissue, we studied arachidonate metabolism in rabbit aortas in response to a continuous infusion of arachidonic acid, 10 micrograms/ml. Prostacyclin (PGI2; measured as 6-keto-PGF1 alpha) production rate accelerated during the first 2 min, reached peak velocity at 2 min, and then progressively decelerated. The velocity profile of PGI2 production was similar to that previously reported for cyclooxygenase holoenzyme assayed in vitro, and was consistent with progressive inactivation of the enzymes leading to PGI2 synthesis. We determined the specific inhibition of cyclooxygenase and prostacyclin synthetase by measuring PGI2 and PGE2 production rates and by infusing cyclic endoperoxides. Our results indicate preferential inactivation of cyclooxygenase during arachidonate metabolism, most likely due to cyclooxygenase-derived oxidative intermediates. This was a dose-dependent response and resulted in a progressive decrease in the 6-keto-PGF1 alpha/PGE2 ratio. Exogenously added 15-hydroperoxy eicosatetraenoic acid, on the other hand, actually stimulated cyclooxygenase activity at low doses, while markedly inhibiting prostacyclin synthetase. This finding, along with the accelerating nature of arachidonate metabolism, is consistent with the concept of "peroxide tone" as a mediator of cyclooxygenase activity in this system. These results demonstrate that arachidonate metabolites regulate PG synthesis in intact blood vessels. The progressive enzymatic inhibition intrinsic to arachidonate metabolism may be a model for similar changes occurring in states of enhanced lipid peroxidation. These metabolic alterations might greatly influence the numerous vascular functions known to involve arachidonic acid metabolism. PMID:6409932

PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes.

PubMed

Li, Qing; Subbulakshmi, Venkita; Oldfield, Claudine M; Aamir, Rozina; Weyman, Crystal M; Wolfman, Alan; Cathcart, Martha K

2007-02-01

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCalpha and cPLA(2) in O(2)(-) generation are solely due to their seminal role in generating arachidonic acid.

Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages

PubMed Central

Allegra, M.; D’Acquisto, F.; Tesoriere, L.; Attanzio, A.; Livrea, M.A.

2014-01-01

Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50–100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5–3 h) modest inhibition, followed by a progressive (3–12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5–3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages. PMID:25180166

Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages.

PubMed

Allegra, M; D'Acquisto, F; Tesoriere, L; Attanzio, A; Livrea, M A

2014-01-01

Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50-100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5-3 h) modest inhibition, followed by a progressive (3-12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5-3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

Biological activities of xanthatin from Xanthium strumarium leaves.

PubMed

Nibret, Endalkachew; Youns, Mahamoud; Krauth-Siegel, R Luise; Wink, Michael

2011-12-01

The objective of the present work was to evaluate the biological activities of the major bioactive compound, xanthatin, and other compounds from Xanthium strumarium (Asteraceae) leaves. Inhibition of bloodstream forms of Trypanosoma brucei brucei and leukaemia HL-60 cell proliferation was assessed using resazurin as a vital stain. Xanthatin was found to be the major and most active compound against T. b. brucei with an IC(50) value of 2.63 µg/mL and a selectivity index of 20. The possible mode of action of xanthatin was further evaluated. Xanthatin showed antiinflammatory activity by inhibiting both PGE(2) synthesis (24% inhibition) and 5-lipoxygenase activity (92% inhibition) at concentrations of 100 µg/mL and 97 µg/mL, respectively. Xanthatin exhibited weak irreversible inhibition of parasite specific trypanothione reductase. Unlike xanthatin, diminazene aceturate and ethidium bromide showed strong DNA intercalation with IC(50) values of 26.04 µg/mL and 44.70 µg/mL, respectively. Substantial induction of caspase 3/7 activity in MIA PaCa-2 cells was observed after 6 h of treatment with 100 µg/mL of xanthatin. All these data taken together suggest that xanthatin exerts its biological activity by inducing apoptosis and inhibiting both PGE(2) synthesis and 5-lipoxygenase activity thereby avoiding unwanted inflammation commonly observed in diseases such as trypanosomiasis. Copyright © 2011 John Wiley & Sons, Ltd.

Role of Inflammatory Reponse in Experimental Decompression Sickness

NASA Technical Reports Server (NTRS)

Butler, B. D.; Little, T.

1999-01-01

Decompression to altitude can result in gas bubble formation both in tissues and in the systemic veins. The venous gas emboli (VGE) are often monitored during decompression exposures to assess risk for decompression sickness (DCS). Astronauts are at risk for DCS during extravehicular activities (EVA), where decompression occurs from the Space Shuttle or Space Station atmospheric pressure of 14.7 pounds per square inch (PSI) to that of the space suit pressure of 4.3 PSI. DCS symptoms include diffuse pain, especially around joints, inflammation and edema. Pathophysiological effects include interstitial inflammatory responses and recurring injury to the vascular endothelium. Such responses can result in vasoconstriction and associated hemodynamic changes.The granulocyte cell activation and chemotaxin release results in the formation of vasoactive and microvascular permeability altering mediators, especially from the lungs which are the principal target organ for the venous bubbles, and from activated cells (neutrophils, platelets, macrophages). Such mediators include free arachidonic acid and the byproducts of its metabolism via the cyclooxygenase and lipoxygenase pathways (see figure). The cyclooxygenase pathway results in formation of prostacyclin and other prostaglandins and thromboxanes that cause vasoconstriction, bronchoconstriction and platelet aggregation. Leukotrienes produced by the alternate pathway cause pulmonary and bronchial smooth muscle contraction and edema. Substances directly affecting vascular tone such as nitric oxide may also play a role in the respose to DCS. We are studying the role and consequent effects of the release inflammatory bioactive mediators as a result of DCS and VGE. More recent efforts are focused on identifying the effects of the body's circadian rhythm on these physiological consequences to decompression stress. al

Imbricaric Acid and Perlatolic Acid: Multi-Targeting Anti-Inflammatory Depsides from Cetrelia monachorum

PubMed Central

Oettl, Sarah K.; Gerstmeier, Jana; Khan, Shafaat Y.; Wiechmann, Katja; Bauer, Julia; Atanasov, Atanas G.; Malainer, Clemens; Awad, Ezzat M.; Uhrin, Pavel; Heiss, Elke H.; Waltenberger, Birgit; Remias, Daniel; Breuss, Johannes M.; Boustie, Joel; Dirsch, Verena M.; Stuppner, Hermann; Werz, Oliver; Rollinger, Judith M.

2013-01-01

In vitro screening of 17 Alpine lichen species for their inhibitory activity against 5-lipoxygenase, microsomal prostaglandin E2 synthase-1 and nuclear factor kappa B revealed Cetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb. As conceivable source for novel anti-inflammatory compounds. Phytochemical investigation of the ethanolic crude extract resulted in the isolation and identification of 11 constituents, belonging to depsides and derivatives of orsellinic acid, olivetolic acid and olivetol. The two depsides imbricaric acid (4) and perlatolic acid (5) approved dual inhibitory activities on microsomal prostaglandin E2 synthase-1 (IC50 = 1.9 and 0.4 µM, resp.) and on 5-lipoxygenase tested in a cell-based assay (IC50 = 5.3 and 1.8 µM, resp.) and on purified enzyme (IC50 = 3.5 and 0.4 µM, resp.). Additionally, these two main constituents quantified in the extract with 15.22% (4) and 9.10% (5) showed significant inhibition of tumor necrosis factor alpha-induced nuclear factor kappa B activation in luciferase reporter cells with IC50 values of 2.0 and 7.0 µM, respectively. In a murine in vivo model of inflammation, 5 impaired the inflammatory, thioglycollate-induced recruitment of leukocytes to the peritoneum. The potent inhibitory effects on the three identified targets attest 4 and 5 a pronounced multi-target anti-inflammatory profile which warrants further investigation on their pharmacokinetics and in vivo efficacy. PMID:24130812

Activation of phospholipase activity during Semliki Forest virus infection.

PubMed

Pérez, L; Irurzun, A; Carrasco, L

1993-05-01

Infection of animal cells by a number of cytolytic viruses leads to increased membrane permeability. Thus, Semliki Forest virus (SFV) infection of susceptible cells modifies the permeability of the membrane for a number of cations and metabolites (Muñoz et al. (1985), Virology 146, 203-212). The molecular basis of this modification of the cell membrane has not been investigated in detail. We report that during the infection of HeLa cells with SFV, or BHK cells with vesicular stomatitis virus, there is a significant increase in the release of choline and arachidonic acid into the culture medium, suggesting that both phospholipases (PLases) C and A2 become activated during infection. Both choline and phosphorylcholine are released into the medium as expected when PLase C is activated. Cells prelabeled with arachidonic acid release a significant amount of radioactivity from the third hour postinfection. Most of this radioactivity is present in the medium of SFV-infected cells in the form of free fatty acid, suggesting that phospholipid hydrolysis has occurred; no intact phospholipids are detected in the culture medium. Finally, the action of several inhibitors of PLases, such as zinc and cadmium ions, chloroquine, chlorpromazine, amantadine, and dansylcadaverine were assayed. Our findings indicate that the release of choline or arachidonic acid is potently blocked by some of these lipase inhibitors. Following infection by SFV HeLa cells become susceptible to the inhibition of protein synthesis by hygromycin B due to increased uptake of this antibiotic. Entry of hygromycin B was prevented by zinc ions or chloroquine, suggesting that the increase in membrane permeability in SFV-infected cells may be mediated in part by lipase activation.

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

PubMed Central

Benistant, C; Rubin, R

1990-01-01

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization. Images p495-a PMID:2117442

Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl.

PubMed

Stamm, Anne; Strauß, Sarah; Vogt, Peter; Scheper, Thomas; Pepelanova, Iliyana

2018-04-07

AmbLOXe is a lipoxygenase, which is up-regulated during limb-redevelopment in the Mexican axolotl, Ambystoma mexicanum, an animal with remarkable regeneration capacity. Previous studies have shown that mammalian cells transformed with the gene of this epidermal lipoxygenase display faster migration and wound closure rate during in vitro wound healing experiments. In this study, the gene of AmbLOXe was codon-optimized for expression in Escherichia coli and was produced in the insoluble fraction as protein aggregates. These inclusion bodies or nanopills were shown to be reservoirs containing functional protein during in vitro wound healing assays. For this purpose, functional inclusion bodies were used to coat cell culture surfaces prior cell seeding or were added directly to the medium after cells reached confluence. In both scenarios, AmbLOXe inclusion bodies led to faster migration rate and wound closure, in comparison to controls containing either no AmbLOXe or GFP inclusion bodies. Our results demonstrate that AmbLOXe inclusion bodies are functional and may serve as stable reservoirs of this enzyme. Nevertheless, further studies with soluble enzyme are also necessary in order to start elucidating the exact molecular substrates of AmbLOXe and the biochemical pathways involved in the wound healing effect.

Modulatory effects of steroid hormones, oxytocin, arachidonic acid, forskolin and cyclic AMP on the expression of aquaporin 1 and aquaporin 5 in the porcine uterus during placentation.

PubMed

Skowronska, A; Mlotkowska, P; Okrasa, S; Nielsen, S; Skowronski, M T

2016-04-01

Aquaporins (AQPs) are proteins forming trans-membrane channels responsible for water transport. AQP1 and AQP5 are present in structures of the female reproductive system. In the uterus, these AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages. Thus, the study aimed to assess the influence of P4 (progesterone), E2 (estradiol), OT (oxytocin), AA (arachidonic acid), cAMP and FSK (forskolin) on the AQP1 and AQP5 mRNA and protein expression in the uterine tissue of gilts on Days 30 - 32 of gestation (the placentation period), following short (3 h) and long (24 h) incubations. Steroid hormones influenced the expression of AQP1 and AQP5; E2 up-regulated, but P4 down-regulated mRNAs of these AQPs, whereas the protein level of studied AQPs was increased by both steroids. OT treatment decreased AQP1 (after 24 h), but increased AQP5 (after 3 h) mRNA expression. Treatment with AA significantly reduced the AQP1 expression at the mRNA level, but stimulated at the protein level. The expression of AQP5 mRNA and protein was stimulated by AA. FSK markedly decreased AQP1 mRNA, but increased of AQP5 after 3-h incubation. In turn, cAMP stimulated and inhibited transcription of AQP5 after 3- and 24-h incubations, respectively. Immunohistochemical analysis confirmed the uterine localization of AQP1 in the apical and basal membranes of endothelial cells and AQP5 in the apical membranes of epithelial cells under control condition. Treatments with P4, E2, AA, cAMP or FSK have caused additional appearance of AQP5 labeling in the basolateral membranes of epithelial cells. These results suggest a participation of steroid hormones (P4 and E2), AA derivatives and cAMP in controlling the expression of AQP1 and AQP5 as well as the distribution of AQP5 in the uterine tissue of pregnant gilts during placentation (Days 30 - 32 of gestation).

Simvastatin Attenuates Endothelial Activation through 15-Epi-Lipoxin A4 Production in Murine Chronic Chagas Cardiomyopathy.

PubMed

González-Herrera, Fabiola; Cramer, Allysson; Pimentel, Pollyana; Castillo, Christian; Liempi, Ana; Kemmerling, Ulrike; Machado, Fabiana S; Maya, Juan D

2017-03-01

Current treatments for chronic Chagas cardiomyopathy, a disease with high mortality rates and caused by the protozoan Trypanosoma cruzi , are unsatisfactory. Myocardial inflammation, including endothelial activation, is responsible for the structural and functional damage seen in the chronic phase. The clinical efficacy of benznidazole could be improved by decreasing chronic inflammation. Statins, which have anti-inflammatory properties, may improve the action of benznidazole. Here, the action of simvastatin in a murine model of chronic Chagas cardiomyopathy and the link with the production of the proresolving eicosanoid 15-epi-lipoxin A4, produced by 5-lipoxygenase, are evaluated. Simvastatin decreased the expression of the adhesion molecules E-selectin, intracellular adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule type 1 (VCAM-1) in T. cruzi -infected mice. However, when this drug was administered to 5-lipoxygenase-deficient mice, the anti-inflammatory effect was not observed unless exogenous 15-epi-lipoxin A4 was administered. Thus, in chronic Chagas disease, 5-epi-lipoxin A4 induced by simvastatin treatment could improve the pathophysiological condition of patients by increasing the trypanocidal action of benznidazole. Copyright © 2017 American Society for Microbiology.

A proof for substitution of endogenous iron (II) in lipoxygenase by exogenous Cu2+.

PubMed

Cai, Yan; Xu, Hong; Xia, Yongmei; Su, Yafen; Fang, Yun

2010-10-01

Soybean lipoxygenase (LOX) contains endogenous iron (II) at the active site, which is important for the enzyme activity. The activity of LOX can be accelerated by some exogenous metal ions including Cu2+. However, the mechanism of the activity improvement caused by exogenous metal ions remains unclear, not only for LOX but for most other metalloenzymes. Meanwhile, the possibility that exogenous metal ions can displace endogenous iron (II) is still in discussion for a lack of a direct and quantitative proof. In this paper, a quantitative proof of replacing iron (II) inside LOX by exogenous Cu2+ was provided, simply using UV-Vis spectrometry with two indicators p-carboxylantipyrylazo and 9-(4-carboxyphenyl)-2,3,7-trihydroxyl-6-fluorine. A 0.56 microM free iron (II) was observed in the bulk solution after incubating 9.45 microM Cu2+ with 16.10 microM LOX at 20 degrees C for 5 min, which is in coincidence with the decrement of Cu2+ in the bulk solution (0.53 microM), implying that iron (II) was replaced by Cu2+.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

PubMed

Zhang, Huidan; Lu, Dong; Li, Xin; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2018-05-02

Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L - 1 , which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Structure/Function Relationships of Adipose Phospholipase A2 Containing a Cys-His-His Catalytic Triad*

PubMed Central

Pang, Xiao-Yan; Cao, Jian; Addington, Linsee; Lovell, Scott; Battaile, Kevin P.; Zhang, Na; Rao, J. L. Uma Maheswar; Dennis, Edward A.; Moise, Alexander R.

2012-01-01

Adipose phospholipase A2 (AdPLA or Group XVI PLA2) plays an important role in the onset of obesity by suppressing adipose tissue lipolysis. As a consequence, AdPLA-deficient mice are resistant to obesity induced by a high fat diet or leptin deficiency. It has been proposed that AdPLA mediates its antilipolytic effects by catalyzing the release of arachidonic acid. Based on sequence homology, AdPLA is part of a small family of acyltransferases and phospholipases related to lecithin:retinol acyltransferase (LRAT). To better understand the enzymatic mechanism of AdPLA and LRAT-related proteins, we solved the crystal structure of AdPLA. Our model indicates that AdPLA bears structural similarity to proteins from the NlpC/P60 family of cysteine proteases, having its secondary structure elements configured in a circular permutation of the classic papain fold. Using both structural and biochemical evidence, we demonstrate that the enzymatic activity of AdPLA is mediated by a distinctive Cys-His-His catalytic triad and that the C-terminal transmembrane domain of AdPLA is required for the interfacial catalysis. Analysis of the enzymatic activity of AdPLA toward synthetic and natural substrates indicates that AdPLA displays PLA1 in addition to PLA2 activity. Thus, our results provide insight into the enzymatic mechanism and biochemical properties of AdPLA and LRAT-related proteins and lead us to propose an alternate mechanism for AdPLA in promoting adipose tissue lipolysis that is not contingent on the release of arachidonic acid and that is compatible with its combined PLA1/A2 activity. PMID:22923616

Antioxidant activity of Citrus paradisi seeds glyceric extract.

PubMed

Giamperi, Laura; Fraternale, Daniele; Bucchini, Anahi; Ricci, Donata

2004-03-01

The antioxidant activity of Citrus paradisi (grapefruit) seeds glyceric extract dissolved in ethanol and in aqueous media was evaluated using three different methods: evaluation by DPPH assay, by 5-lipoxygenase assay and by luminol/xanthine/xanthine oxidase chemiluminescence assay. The total phenolic content was determined by the Prussian Blue method opportunely modified. The grapefruit seeds glyceric extract utilized as aqueous solutions demonstrated antioxidant properties better than those displayed by alcoholic solutions.

Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells

NASA Technical Reports Server (NTRS)

Chen, Y.; Hughes-Fulford, M.

2000-01-01

Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

A role for 12/15-lipoxygenase-derived proresolving mediators in postoperative ileus: protectin DX-regulated neutrophil extravasation.

PubMed

Stein, Kathy; Stoffels, Melissa; Lysson, Mariola; Schneiker, Bianca; Dewald, Oliver; Krönke, Gerhard; Kalff, Jörg C; Wehner, Sven

2016-02-01

Resolution of inflammation is an active counter-regulatory mechanism involving polyunsaturated fatty acid-derived proresolving lipid mediators. Postoperative intestinal motility disturbances, clinically known as postoperative ileus, occur frequently after abdominal surgery and are mediated by a complex inflammation of the intestinal muscularis externa. Herein, we tested the hypothesis that proresolving lipid mediators are involved in the resolution of postoperative ileus. In a standardized experimental model of postoperative ileus, we detected strong expression of 12/15-lipoxygenase within the postoperative muscularis externa of C57BL/6 mice, predominately located within CX3CR1(+)/Ly6C(+) infiltrating monocytes rather than Ly6G(+) neutrophils. Mass spectrometry analyses demonstrated that a 12/15-lipoxygenase increase was accompanied by production of docosahexaenoic acid-derived lipid mediators, particularly protectin DX and resolvin D2, and their common precursor 17-hydroxy docosahexaenoic acid. Perioperative administration of protectin DX, but not resolvin D2 diminished blood-derived leukocyte infiltration into the surgically manipulated muscularis externa and improved the gastrointestinal motility. Flow cytometry analyses showed impaired Ly6G(+)/Ly6C(+) neutrophil extravasation after protectin DX treatment, whereas Ly6G(-)/Ly6C(+) monocyte numbers were not affected. 12/15-lipoxygenase-deficient mice, lacking endogenous protectin DX synthesis, demonstrated increased postoperative leukocyte levels. Preoperative intravenous administration of a docosahexaenoic acid-rich lipid emulsion reduced postoperative leukocyte infiltration in wild-type mice but failed in 12/15-lipoxygenase-deficient mice mice. Protectin DX application reduced leukocyte influx and rescued 12/15-lipoxygenase-deficient mice mice from postoperative ileus. In conclusion, our results show that 12/15-lipoxygenase mediates postoperative ileus resolution via production of proresolving docosahexaenoic acid-derived protectin DX. Perioperative, parenteral protectin DX or docosahexaenoic acid supplementation, as well as modulation of the 12/15-lipoxygenase pathway, may be instrumental in prevention of postoperative ileus. © Society for Leukocyte Biology.

A phosphatidylinositol species acutely generated by activated macrophages regulates innate immune responses.

PubMed

Gil-de-Gómez, Luis; Astudillo, Alma M; Meana, Clara; Rubio, Julio M; Guijas, Carlos; Balboa, María A; Balsinde, Jesús

2013-05-15

Activation of macrophages with stimuli of the innate immune response results in the intense remodeling of arachidonate-containing phospholipids, leading to the mobilization of large quantities of this fatty acid for conversion into biologically active eicosanoids. As a consequence of this process, the arachidonate levels in membrane phospholipids markedly decrease. We have applied mass spectrometry-based lipid profiling to study the levels of arachidonate-containing phospholipids under inflammatory activation of macrophages. We identify an unusual inositol phospholipid molecule, PI(20:4/20:4), the levels of which do not decrease but actually increase by 300% after activation of the macrophages. PI(20:4/20:4) is formed and degraded rapidly, suggesting a role for this molecule in regulating cell signaling events. Using a metabolipidomic approach consisting in exposing the cells to deuterium-labeled arachidonate at the time they are exposed to stimuli, we show that PI(20:4/20:4) biosynthesis occurs via the sequential incorporation of arachidonate, first into the sn-2 position of a preformed phosphatidylinositol (PI) molecule, followed by the rapid introduction of a second arachidonate moiety into the sn-1 position. Generation requires the participation of cytosolic phospholipase A2α and CoA-dependent acyltransferases. PI(20:4/20:4) formation is also detected in vivo in murine peritonitis exudates. Elevating the intracellular concentration of PI(20:4/20:4) by introducing the lipid into the cells results in enhancement of the microbicidal capacity of macrophages, as measured by reactive oxygen metabolite production and lysozyme release. These findings suggest that PI(20:4/20:4) is a novel bioactive inositol phospholipid molecule that regulates innate immune responses in macrophages.

The increased level of COX-dependent arachidonic acid metabolism in blood platelets from secondary progressive multiple sclerosis patients.

PubMed

Morel, Agnieszka; Miller, Elzbieta; Bijak, Michal; Saluk, Joanna

2016-09-01

Platelet activation is increasingly postulated as a possible component of the pathogenesis of multiple sclerosis (MS), especially due to the increased risk of cardiovascular events in MS. Arachidonic acid cascade metabolized by cyclooxygenase (COX) is a key pathway of platelet activation. The aim of our study was to investigate the COX-dependent arachidonic acid metabolic pathway in blood platelets from secondary progressive multiple sclerosis (SP MS) patients. The blood samples were obtained from 50 patients (man n = 22; female n = 28), suffering from SP MS, diagnosed according to the revised McDonald criteria. Platelet aggregation was measured in platelet-rich plasma after arachidonic acid stimulation. The level of COX activity and thromboxane B2 concentration were determined by ELISA method. Lipid peroxidation was assessed by measuring the level of malondialdehyde. The results were compared with a control group of healthy volunteers. We found that blood platelets obtained from SP MS patients were more sensitive to arachidonic acid and their response measured as platelet aggregation was stronger (about 14 %) relative to control. We also observed a significantly increased activity of COX (about 40 %) and synthesis of thromboxane B2 (about 113 %). The generation of malondialdehyde as a marker of lipid peroxidation was about 10 % higher in SP MS than in control. Cyclooxygenase-dependent arachidonic acid metabolism is significantly increased in blood platelets of patients with SP MS. Future clinical studies are required to recommend the use of low-dose aspirin, and possibly other COX inhibitors in the prevention of cardiovascular risk in MS.

CJ-13610, an orally active inhibitor of 5-lipoxygenase is efficacious in preclinical models of pain.

PubMed

Cortes-Burgos, Luz A; Zweifel, Ben S; Settle, Steven L; Pufahl, Robert A; Anderson, Gary D; Hardy, Medora M; Weir, Dana E; Hu, George; Happa, Fernando A; Stewart, Zachary; Muthian, Shanmugam; Graneto, Matthew J; Masferrer, Jaime L

2009-09-01

Zileuton, a redox and iron chelator 5-lipoxygenase (5-LOX) inhibitor and, leukotriene receptor antagonists are presently used clinically in the long term treatment of asthma. Recent data implicate 5-LOX pathway in pain signaling. We report 5-LOX expression in the central nervous system (CNS) and analyze the pain efficacy of a new class of non redox, non iron chelating 5-LOX inhibitor. CJ-13610, 4-(3-(4-(2-methyl-1H-imidazol-1-yl) phenylthio) phenyl)-tetrahydro-2H-pyran-4-carboxamide, demonstrated antihyperalgesic activity in inflammatory pain models including the acute carrageenan model and the chronic inflammatory model using complete Freund's adjuvant. Following complete Freund's adjuvant stimulus leukotrieneB(4) concentration in the brain was elevated (9+/-1 ng/g, mean+/-S.E.M.) by about 3 times that of the control group (3+/-0.11, mean+/-S.E.M.). Hyperalgesia and leukotrieneB(4) concentration were both reversed following CJ-13610 treatment. Furthermore, we demonstrate CJ-13610 efficacy against osteoarthritis like pain using the rat medial meniscal transection model. CJ-13610 at oral doses of 0.6, 2 and 6 mg/kg/day reversed two modalities of pain in this model; tactile allodynia and weight bearing differential. Taken together, these data suggest that 5-LOX pathway and the leukotriene products are important mediators of pain.

Cyclooxygenase and lipoxygenase-like activity in Drosophila melanogaster.

PubMed

Pagés, M; Roselló, J; Casas, J; Gelpí, E; Gualde, N; Rigaud, M

1986-11-01

To determine the possible activity of cyclooxygenase and lipoxygenase like enzymes in Drosophila melanogaster, we have investigated whether fly homogenates can biosynthesize prostaglandins and HETEs. Incubation of fly extracts with AA yields a mixture of 15- 12- 9- and 8-HETE as detected by selected ion monitoring GC-MS. Also the combination of HPLC-RIA using a PGE antibody shows the presence of endogenous PGE2 immunoreactivity in the extracts (405 pg/g in males and 165 pg/g in females). We have also detected the presence of lipoxygenase like immunoreactivity in the reproductive male system by using immunocytochemical techniques in whole body sections of the fly as well as reactivity in the digestive system of both males and females. Finally, we have not been able to detect endogenous AA in the fly by GC-MS methods. However, estimates by GC-MS of the total body fatty acids indicate substantial amounts of potential AA precursors.

Beyond DNA binding - a review of the potential mechanisms mediating quinacrine's therapeutic activities in parasitic infections, inflammation, and cancers

PubMed Central

2011-01-01

This is an in-depth review of the history of quinacrine as well as its pharmacokinetic properties and established record of safety as an FDA-approved drug. The potential uses of quinacrine as an anti-cancer agent are discussed with particular attention to its actions on nuclear proteins, the arachidonic acid pathway, and multi-drug resistance, as well as its actions on signaling proteins in the cytoplasm. In particular, quinacrine's role on the NF-κB, p53, and AKT pathways are summarized. PMID:21569639

Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase.

PubMed

Mittal, Monica; Hasan, Mahmudul; Balagunaseelan, Navisraj; Fauland, Alexander; Wheelock, Craig; Rådmark, Olof; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

2017-08-01

A 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. A soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. Here we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca 2+ -dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. The study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general. Copyright © 2017 Elsevier B.V. All rights reserved.

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography

PubMed Central

1985-01-01

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors. PMID:3926781

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

PubMed Central

Sato, Hiroyasu; Taketomi, Yoshitaka; Isogai, Yuki; Miki, Yoshimi; Yamamoto, Kei; Masuda, Seiko; Hosono, Tomohiko; Arata, Satoru; Ishikawa, Yukio; Ishii, Toshiharu; Kobayashi, Tetsuyuki; Nakanishi, Hiroki; Ikeda, Kazutaka; Taguchi, Ryo; Hara, Shuntaro; Kudo, Ichiro; Murakami, Makoto

2010-01-01

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction. PMID:20424323

The discovery and early structural studies of arachidonic acid

PubMed Central

Martin, Sarah A.; Brash, Alan R.; Murphy, Robert C.

2016-01-01

Arachidonic acid and esterified arachidonate are ubiquitous components of every mammalian cell. This polyunsaturated fatty acid serves very important biochemical roles, including being the direct precursor of bioactive lipid mediators such as prostaglandin and leukotrienes. This 20 carbon fatty acid with four double bonds was first isolated and identified from mammalian tissues in 1909 by Percival Hartley. This was accomplished prior to the advent of chromatography or any spectroscopic methodology (MS, infrared, UV, or NMR). The name, arachidonic, was suggested in 1913 based on its relationship to the well-known arachidic acid (C20:0). It took until 1940 before the positions of the four double bonds were defined at 5,8,11,14 of the 20-carbon chain. Total synthesis was reported in 1961 and, finally, the configuration of the double bonds was confirmed as all-cis-5,8,11,14. By the 1930s, the relationship of arachidonic acid within the family of essential fatty acids helped cue an understanding of its structure and the biosynthetic pathway. Herein, we review the findings leading up to the discovery of arachidonic acid and the progress toward its complete structural elucidation. PMID:27142391

Are there attacking points in the eicosanoid cascade for chemotherapeutic options in benign meningiomas?

PubMed

Pfister, Christina; Ritz, Rainer; Pfrommer, Heike; Bornemann, Antje; Tatagiba, Marcos S; Roser, Florian

2007-01-01

The current treatment for recurrent or malignant meningiomas with adjuvant therapies has not been satisfactory, and there is an intense interest in evaluating new molecular markers to act as therapeutic targets. Enzymes of the arachidonic acid (AA) cascade such as cyclooxygenase (COX)-2 or 5-lipoxygenase (5-LO) are upregulated in a number of epithelial tumors, but to date there are hardly any data about the expression of these markers in meningiomas. To find possible targets for chemotherapeutic intervention, the authors evaluated the expression of AA derivatives at different molecular levels in meningiomas. One hundred and twenty-four meningioma surgical specimens and normal human cortical tissue samples were immunohistochemically and cytochemically stained for COX-2, COX-1, 5-LO, and prostaglandin E receptor 4 (PTGER4). In addition, Western blot and polymerase chain reaction (PCR) analyses were performed to detect the presence of eicosanoids in vivo and in vitro. Sixty (63%) of 95 benign meningiomas, 21 (88%) of 24 atypical meningiomas, all five malignant meningiomas, and all normal human cortex samples displayed high COX-2 immunoreactivity. All cultured specimens and IOMM-Lee cells stained positive for COX-2, COX-1, 5-LO, and PTGER4. The PCR analysis demonstrated no changes in eicosanoid expression among meningiomas of different World Health Organization grades and in normal human cortical and dura mater tissue. Eicosanoid derivatives COX-1, COX-2, 5-LO, and PTGER4 enzymes show a high universal expression in meningiomas but are not upregulated in normal human cortex and dura tissue. This finding of the ubiquitous presence of these enzymes in meningiomas offers an excellent baseline for testing upcoming chemotherapeutic treatments.

Diverse ways of perturbing the human arachidonic acid metabolic network to control inflammation.

PubMed

Meng, Hu; Liu, Ying; Lai, Luhua

2015-08-18

Inflammation and other common disorders including diabetes, cardiovascular disease, and cancer are often the result of several molecular abnormalities and are not likely to be resolved by a traditional single-target drug discovery approach. Though inflammation is a normal bodily reaction, uncontrolled and misdirected inflammation can cause inflammatory diseases such as rheumatoid arthritis and asthma. Nonsteroidal anti-inflammatory drugs including aspirin, ibuprofen, naproxen, or celecoxib are commonly used to relieve aches and pains, but often these drugs have undesirable and sometimes even fatal side effects. To facilitate safer and more effective anti-inflammatory drug discovery, a balanced treatment strategy should be developed at the biological network level. In this Account, we focus on our recent progress in modeling the inflammation-related arachidonic acid (AA) metabolic network and subsequent multiple drug design. We first constructed a mathematical model of inflammation based on experimental data and then applied the model to simulate the effects of commonly used anti-inflammatory drugs. Our results indicated that the model correctly reproduced the established bleeding and cardiovascular side effects. Multitarget optimal intervention (MTOI), a Monte Carlo simulated annealing based computational scheme, was then developed to identify key targets and optimal solutions for controlling inflammation. A number of optimal multitarget strategies were discovered that were both effective and safe and had minimal associated side effects. Experimental studies were performed to evaluate these multitarget control solutions further using different combinations of inhibitors to perturb the network. Consequently, simultaneous control of cyclooxygenase-1 and -2 and leukotriene A4 hydrolase, as well as 5-lipoxygenase and prostaglandin E2 synthase were found to be among the best solutions. A single compound that can bind multiple targets presents advantages including low risk of drug-drug interactions and robustness regarding concentration fluctuations. Thus, we developed strategies for multiple-target drug design and successfully discovered several series of multiple-target inhibitors. Optimal solutions for a disease network often involve mild but simultaneous interventions of multiple targets, which is in accord with the philosophy of traditional Chinese medicine (TCM). To this end, our AA network model can aptly explain TCM anti-inflammatory herbs and formulas at the molecular level. We also aimed to identify activators for several enzymes that appeared to have increased activity based on MTOI outcomes. Strategies were then developed to predict potential allosteric sites and to discover enzyme activators based on our hypothesis that combined treatment with the projected activators and inhibitors could balance different AA network pathways, control inflammation, and reduce associated adverse effects. Our work demonstrates that the integration of network modeling and drug discovery can provide novel solutions for disease control, which also calls for new developments in drug design concepts and methodologies. With the rapid accumulation of quantitative data and knowledge of the molecular networks of disease, we can expect an increase in the development and use of quantitative disease models to facilitate efficient and safe drug discovery.

Inhibition of soybean and potato lipoxygenases by bhilawanols from bhilawan (Semecarpus anacardium) nut shell liquid and some synthetic salicylic acid analogues.

PubMed

Nagabhushana, Kyatanahalli S; Umamaheshwari, S; Tocoli, Felismino E; Prabhu, Sandeep K; Green, Ivan R; Ramadoss, Candadai S

2002-08-01

Bhilawanol diene (3) isolated from bhilawan nut shell liquid was found to be a potent inhibitor of both soybean and potato lipoxygenases with IC50 values of 0.85 microM and 1.1 microM, respectively. However, the monoene (2) and saturated (1) bhilawanols exhibited relatively lower inhibitory activity. In addition, inhibition studies with synthetic analogues of salicylic acid (4-8) suggested that the unsaturated lipophilic side chain may be an absolute requirement for inhibitory activity.

A New Model to Study the Role of Arachidonic Acid in Colon Cancer Pathophysiology.

PubMed

Fan, Yang-Yi; Callaway, Evelyn; M Monk, Jennifer; S Goldsby, Jennifer; Yang, Peiying; Vincent, Logan; S Chapkin, Robert

2016-09-01

A significant increase in cyclooxygenase 2 (COX2) gene expression has been shown to promote cylcooxygenase-dependent colon cancer development. Controversy associated with the role of COX2 inhibitors indicates that additional work is needed to elucidate the effects of arachidonic acid (AA)-derived (cyclooxygenase and lipoxygenase) eicosanoids in cancer initiation, progression, and metastasis. We have recently developed a novel Fads1 knockout mouse model that allows for the investigation of AA-dependent eicosanoid deficiency without the complication of essential fatty acid deficiency. Interestingly, the survival rate of Fads1-null mice is severely compromised after 2 months on a semi-purified AA-free diet, which precludes long-term chemoprevention studies. Therefore, in this study, dietary AA levels were titrated to determine the minimal level required for survival, while maintaining a distinct AA-deficient phenotype. Null mice supplemented with AA (0.1%, 0.4%, 0.6%, 2.0%, w/w) in the diet exhibited a dose-dependent increase (P < 0.05) in AA, PGE2, 6-keto PGF1α, TXB2, and EdU-positive proliferative cells in the colon. In subsequent experiments, null mice supplemented with 0.6% AA diet were injected with a colon-specific carcinogen (azoxymethane) in order to assess cancer susceptibility. Null mice exhibited significantly (P < 0.05) reduced levels/multiplicity of aberrant crypt foci (ACF) as compared with wild-type sibling littermate control mice. These data indicate that (i) basal/minimal dietary AA supplementation (0.6%) expands the utility of the Fads1-null mouse model for long-term cancer prevention studies and (ii) that AA content in the colonic epithelium modulates colon cancer risk. Cancer Prev Res; 9(9); 750-7. ©2016 AACR. ©2016 American Association for Cancer Research.

Resolution of Inflammation by Resolvin D1 Is Essential for Peroxisome Proliferator-activated Receptor-γ-mediated Analgesia during Postincisional Pain Development in Type 2 Diabetes.

PubMed

Saito, Takayuki; Hasegawa-Moriyama, Maiko; Kurimoto, Tae; Yamada, Tomotsugu; Inada, Eichi; Kanmura, Yuichi

2015-12-01

The wound healing process following acute inflammation after surgery is impaired in diabetes. Altered macrophage functions are linked to delayed tissue repair and pain development in diabetes. Although peroxisome proliferator-activated receptor (PPAR)-γ agonists are used to treat diabetes, their postoperative analgesic effects in diabetes have not been evaluated. The PPARγ agonist rosiglitazone (rosi) was injected at the incision site of diabetic (db/db) mice with resolvin (Rv) D1, a lipid mediator involved in resolution of inflammation. Pain-related behavior, neutrophil infiltration, phagocytosis, and macrophage polarity were assessed for 7 days postoperatively. Rosiglitazone and RvD1 alleviated mechanical hyperalgesia in db/db (db) mice, whereas rosiglitazone alone did not alter mechanical thresholds on days 4 (db rosi + RvD1 vs. db rosi: 0.506 ± 0.106 vs. 0.068 ± 0.12) and 7 (0.529 ± 0.184 vs. 0.153 ± 0.183) after incision (n = 10 per group). In control m/m mice, the rosiglitazone-induced analgesic effects were reversed by knockdown with arachidonate 5-lipoxygenase small interfering RNA, but these were restored by addition of RvD1. In db/db mice treated with rosiglitazone and RvD1, local infiltration of neutrophils was markedly reduced, with an associated decrease in total TdT-mediated dUTP nick-end labeling cells. Acceleration of rosiglitazone-induced phenotype conversion of infiltrated macrophages from M1 to M2 was impaired in db/db mice, but it was effectively restored by RvD1 in db/db wounds. In diabetes, exogenous administration of RvD1 is essential for PPARγ-mediated analgesia during development of postincisional pain. Resolution of inflammation accelerated by RvD1 might promote PPARγ-mediated macrophage polarization to the M2 phenotype.

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Yuting; Zhou, Lubing; Gunnet, Joseph W.

2006-06-23

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A{sub 2} (PLA{sub 2})/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeuticmore » effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca{sup 2+}-mobilization and enhanced bradykinin-promoted Ca{sup 2+}-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPAR{gamma} agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.« less

Preeclampsia and reproductive performance in a community of vegans.

PubMed

Carter, J P; Furman, T; Hutcheson, H R

1987-06-01

Studies at "the Farm," a community of spiritually gathered young people in Summertown, Tenn, have shown that it is possible to sustain a normal pregnancy on a vegan diet. The source of dietary protein (ie, animal or vegetable) does not seem to affect birth weight, as long as vegans are health conscious, receive continuous prenatal care, supplement their diets with prenatal vitamins, calcium, and iron, and apply protein-complementing nutritional principles. Preeclampsia may be caused by a relative prostacyclin deficiency in the face of excessive production of thromboxane A2. A vegan diet (one low in arachidonic acid) might provide protection against this condition, especially if the conversion of linoleic acid to arachidonic acid is inhibited by decreased activity of the enzyme delta-6-desaturase. We examined the maternity care records of 775 vegan mothers for symptoms of preeclampsia, and only one case met the clinical criteria. Since preeclampsia in our culture is frequently associated with unrestrained consumption of "fast foods" (foods having high levels of saturated fat) and rapid weight gain, it is possible that a vegan diet could alleviate most, if not all, of the signs and symptoms of preeclampsia.

Impact of EPA ingestion on COX- and LOX-mediated eicosanoid synthesis in skin with and without a pro-inflammatory UVR challenge--report of a randomised controlled study in humans.

PubMed

Pilkington, Suzanne M; Rhodes, Lesley E; Al-Aasswad, Naser M I; Massey, Karen A; Nicolaou, Anna

2014-03-01

Eicosapentaenoic acid (EPA), abundant in oily fish, is reported to reduce skin inflammation and provide photoprotection, potential mechanisms include competition with arachidonic acid (AA) for metabolism by cyclooxygenases/lipoxygenases to less pro-inflammatory mediators. We thus examine impact of EPA intake on levels of AA, EPA and their resulting eicosanoids in human skin with or without ultraviolet radiation (UVR) challenge. In a double-blind randomised controlled study, 79 females took 5 g EPA-rich or control lipid for 12 wk. Pre- and post-supplementation, red blood cell and skin polyunsaturated fatty acids were assessed by GC, and eicosanoids from unexposed and UVR-exposed skin by LC-MS/MS. Active supplementation increased red blood cell and dermal EPA versus control (both p < 0.001), lowering relative AA:EPA content (4:1 versus 15:1 and 5:1 versus 11:1, respectively; both p < 0.001). Pre-supplementation, UVR increased PGE2, 12-hydroxyeicosatetraenoic acids, 12-HEPE (all p < 0.001) and PGE3 (p < 0.05). Post-EPA, PGE2 was reduced in unchallenged skin (p < 0.05) while EPA-derived PGE3 (non-sign) and 12-HEPE (p < 0.01) were elevated post-UVR. Thus, post-EPA, PGE2 :PGE3 was lower in unchallenged (12:1 versus 28:1; p < 0.05) and UVR exposed (12:1 versus 54:1; p < 0.01) skin; 12-hydroxyeicosatetraenoic acids:12-HEPE was lower in UVR-exposed skin (3:1 versus 11:1; p < 0.001). Dietary EPA augments skin EPA:AA content, shifting eicosanoid synthesis towards less pro-inflammatory species, and promoting a regulatory milieu under basal conditions and in response to inflammatory insult. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Chae E.; Lee, Seung J.; Seo, Kyo W.

2010-05-15

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B{sub 4} (LTB{sub 4}) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB{sub 4} production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK amongmore » these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB{sub 4}. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB{sub 4}, subsequent MMP-9 production and plaque rupture.« less

The anti-inflammatory effects of Yunnan Baiyao are involved in regulation of the phospholipase A2/arachidonic acid metabolites pathways in acute inflammation rat model.

PubMed

Ren, Xiaobin; Zhang, Mingzhu; Chen, Lingxiang; Zhang, Wanli; Huang, Yu; Luo, Huazhen; Li, Ling; He, Hongbing

2017-10-01

The traditional Chinese medicine Yunnan Baiyao (YNB) has been reported to possess anti‑inflammatory properties, however its mechanism of action remains unclear. It was previously reported that YNB ameliorated depression of arachidonic acid (AA) levels in a rat model of collagen-induced arthritis. In the current study, the capacity of YNB to ameliorate inflammation was compared in carrageenan‑induced and AA‑induced acute inflammation of the rat paw with celecoxib and mizolastine, respectively (n=24 per group). The capacity of YNB to affect the phospholipase A2 (PLA2)/AA pathway (using reverse transcription‑quantitative polymerase chain reaction) and release of inflammatory lipid mediators (by ELISA) were investigated. Celecoxib ameliorated carrageenan‑induced paw edema, and mizolastine ameliorated AA‑induced rat paw edema. YNB alleviated paw edema and inhibited inflammatory cell infiltration in the two models. YNB inhibited production of 5‑LOX AA metabolite leukotriene B4 (LTB4), and suppressed expression of 5‑LOX, cytosolic PLA2 (cPLA2), 5‑LOX‑activating protein, and LTB4 receptor mRNA in the AA‑induced inflammation model (P<0.05). YNB Inhibited the production of the COX‑2 AA metabolite prostaglandin E2 (PGE2) and suppressed expression of COX‑2, cPLA2, PGE2 mRNA in the carrageenan‑induced inflammation mode (P<0.05). Taken together, the data suggest that modulation of COX and LOX pathways in AA metabolism represent a novel anti-inflammatory mechanism of YNB.

Natural Forms of Vitamin E and 13′-Carboxychromanol, a Long-Chain Vitamin E Metabolite, Inhibit Leukotriene Generation from Stimulated Neutrophils by Blocking Calcium Influx and Suppressing 5-Lipoxygenase Activity, Respectively

PubMed Central

Jiang, Ziying; Yin, Xinmin; Jiang, Qing

2014-01-01

Leukotrienes generated by 5-lipoxygenase (5-LOX)–catalyzed reaction are key regulators of inflammation. In ionophore-stimulated (A23187; 1–2.5 μM) human blood neutrophils or differentiated HL-60 cells, vitamin E forms differentially inhibited leukotriene B4 (LTB4) with an IC50 of 5–20 μM for γ-tocopherol, δ-tocopherol (δT), and γ-tocotrienol, but a much higher IC50 for α-tocopherol. 13′-Carboxychromanol, a long-chain metabolite of δT, suppressed neutrophil- and HL-60 cell-generated LTB4 with an IC50 of 4–7 μM and potently inhibited human recombinant 5-LOX activity with an IC50 of 0.5–1 μM. In contrast, vitamin E forms had no effect on human 5-LOX activity but impaired ionophore-induced intracellular calcium increase and calcium influx as well as the subsequent signaling including ERK1/2 phosphorylation and 5-LOX translocation from cytosol to the nucleus, a key event for 5-LOX activation. Further investigation showed that δT suppressed cytosolic Ca2+ increase and/or LTB4 formation triggered by ionophores, sphingosine 1-phosphate, and lysophosphatidic acid but not by fMLP or thapsigargin, whereas 13′-carboxychromanol decreased cellular production of LTB4 regardless of different stimuli, consistent with its strong inhibition of the 5-LOX activity. These observations suggest that δT does not likely affect fMLP receptor-mediated signaling or store depletion-induced calcium entry. Instead, we found that δT prevented ionophore-caused cytoplasmic membrane disruption, which may account for its blocking of calcium influx. These activities by vitamin E forms and long-chain carboxychromanol provide potential molecular bases for the differential anti-inflammatory effects of vitamin E forms in vivo. PMID:21169551

Induction of suppressor cells from peripheral blood T cells by 15-hydroperoxyeicosatetraenoic acid (15-HPETE).

PubMed

Gualde, N; Rigaud, M; Goodwin, J S

1985-11-01

15-hydroperoxyeicosetetraenoic acid (15-HPETE), a lipoxygenase metabolite of arachidonic acid, inhibited polyclonal IgG and IgM production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood mononuclear cells, whereas 15-hydroxyeicosetetraenoic acid (15-HETE) had little effect in this system. T cells preincubated for 18 hr with 15-HPETE caused substantial inhibition of IgG and IgM production of fresh, autologous B and T cells stimulated by PWM. The suppressive effect of the 15-HPETE-treated cells was lost if the cells were irradiated before the PWM culture, but not by treatment with mitomycin C. The suppressive effect was also lost if OKT8+ T cells were removed after, but not before, preincubation of the T cells with 15-HPETE. OKT8- T cells incubated with 15-HPETE for 18 hr showed a large increase in the percentage of cells staining with directly fluoresceinated Leu-2, another marker for suppressor cells. Thus, 15-HPETE induces functional and phenotypic suppressor cells from resting human peripheral blood T cells.

Anti-inflammatory and anticholinesterase activity of six flavonoids isolated from Polygonum and Dorstenia species.

PubMed

Dzoyem, Jean Paul; Nkuete, Antoine H L; Ngameni, Barthelemy; Eloff, Jacobus N

2017-10-01

This study was aimed at investigating the anti-inflammatory and anticholinesterase activity of six naturally occurring flavonoids: (-) pinostrobin (1), 2',4'-dihydroxy-3',6'-dimethoxychalcone (2), 6-8-diprenyleriodictyol (3), isobavachalcone (4), 4-hydroxylonchocarpin (5) and 6-prenylapigenin (6). These compounds were isolated from Dorstenia and Polygonum species used traditionally to treat pain. The anti-inflammatory activity was determined by using the Griess assay and the 15-lipoxygenase inhibitory activity was determined with the ferrous oxidation-xylenol orange assay. Acetylcholinesterase inhibition was determined by the Ellman's method. At the lowest concentration tested (3.12 µg/ml), compounds 2, 3 and 4 had significant NO inhibitory activity with 90.71, 84.65 and 79.57 % inhibition respectively compared to the positive control quercetin (67.93 %). At this concentration there was no significant cytotoxicity against macrophages with 91.67, 72.86 and 70.86 % cell viability respectively, compared to 73.1 % for quercetin. Compound 4 had the most potent lipoxygenase inhibitory activity (IC 50 of 25.92 µg/ml). With the exception of (-) pinostrobin (1), all the flavonoids had selective anticholinesterase activity with IC 50 values ranging between 5.93 and 8.76 µg/ml compared to the IC 50 4.94 µg/ml of eserine the positive control. These results indicate that the studied flavonoids especially isobavachalcone are potential anti-inflammatory natural products that may have the potential to be developed as therapeutic agents against inflammatory conditions and even Alzheimer's disease.

Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis.

PubMed

Coffey, Marcus J; Jarvis, Gavin E; Gibbins, Jonathan M; Coles, Barbara; Barrett, Natasha E; Wylie, Oliver R E; O'Donnell, Valerie B

2004-06-25

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 microg/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Monocyte 15-Lipoxygenase Gene Expression Requires ERK1/2 MAPK Activity

PubMed Central

Bhattacharjee, Ashish; Mulya, Anny; Pal, Srabani; Roy, Biswajit; Feldman, Gerald M.; Cathcart, Martha K.

2011-01-01

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13–mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13–induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB. PMID:20861348

Dual inhibitory activities of tannins from Hamamelis virginiana and related polyphenols on 5-lipoxygenase and lyso-PAF: acetyl-CoA acetyltransferase.

PubMed

Hartisch, C; Kolodziej, H; von Bruchhausen, F

1997-04-01

In the present study, the effects of tannins obtained from various plant sources on the activity of 5-LOX and AT were examined. With IC50 values ranging from 1.0 to 18.7 muM, hamamelitannin and the galloylated proanthocyanidins isolated from Hamamelis virginiana L. were found to be most potent inhibitors of 5-LOX. Unlike the 5-LOX study, hamamelitannin proved to be ineffective in the AT assay. Potent candidates are represented by the group of B-type proanthocyanidins. Structure-activity relationships regarding the in vitro inhibitory potency of the polyphenols in the biological assays are discussed.

Metabolism of endocannabinoids.

PubMed

Biernacki, Michał; Skrzydlewska, Elżbieta

2016-08-11

Endocannabinoids belong to a group of ester, ether and amide derivatives of fatty acids, which are endogenous ligands of receptors CB1, CB2, TRPV1 and GPR55 that are included in the endocannabinoid system of the animal organism. The best known endocannabinoids are: N-arachidonylethanolamide called anandamide (AEA) and 2-arachidonoylglycerol (2-AG). They occur in all organisms, and their highest level is observed in the brain. In this review the mechanisms of synthesis and degradation of both AEA and 2-AG are shown. Endocannabinoids are synthesized from phospholipids (mainly phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) located in the cell membrane. As a result of arachidonic acid transfer from phosphatidylcholine to phosphatidylethanolamine, N-arachidonoyl phosphatidylethanolamine is formed, which is hydrolyzed to AEA by phospholipase D, C and A2. However, 2-AG is formed during the hydrolysis of phosphatidylinositol catalyzed mainly by DAGL. The primary role of endocannabinoids is the activation of cannabinoid receptors. Both AEA and 2-AG are primarily agonists of the CB1 receptor and to a lower degree CB2 and TRPV1r eceptors, but 2-AG has stronger affinity for these receptors. Through activation of receptors, endocannabinoids affect cellular metabolism and participate in the metabolic processes by receptor-independent pathways. Endocannabinoids which are not bound to the receptors are degraded. The main enzymes responsible for the hydrolysis of AEA and 2-AG are FAAH and MAGL, respectively. Apart from hydrolytic degradation, endocannabinoids may also be oxidized by cyclooxygenase-2, lipoxygenases, and cytochrome P450. It has been shown that the metabolites of both endocannabinoids also have biological significance.

Dietary rice bran component γ-oryzanol inhibits tumor growth in tumor-bearing mice.

PubMed

Kim, Sung Phil; Kang, Mi Young; Nam, Seok Hyun; Friedman, Mendel

2012-06-01

We investigated the effects of rice bran and components on tumor growth in mice. Mice fed standard diets supplemented with rice bran, γ-oryzanol, Ricetrienol®, ferulic acid, or phytic acid for 2 weeks were inoculated with CT-26 colon cancer cells and fed the same diet for two additional weeks. Tumor mass was significantly lower in the γ-oryzanol and less so in the phytic acid group. Tumor inhibition was associated with the following biomarkers: increases in cytolytic activity of splenic natural killer (NK) cells; partial restoration of nitric oxide production and phagocytosis in peritoneal macrophages increases in released the pro-inflammatory cytokines tumor necrosis factor-α, IL-1β, and IL-6 from macrophages; and reductions in the number of blood vessels inside the tumor. Pro-angiogenic biomarkers vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), and 5-lipoxygenase-5 (5-LOX) were also significantly reduced in mRNA and protein expression by tumor genes. ELISA of tumor cells confirmed reduced expression of COX-2 and 5-LOX up to 30%. Reduced COX-2 and 5-LOX expression downregulated VEGF and inhibited neoangiogenesis inside the tumors. Induction of NK activity, activation of macrophages, and inhibition of angiogenesis seem to contribute to the inhibitory mechanism of tumor regression by γ-oryzanol. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of Membrane-Bound Kallikrein and Renin in the Kidney.

DTIC Science & Technology

1980-05-23

included repeated washings with hypotonic buffer. Kallikrein activity in the PM fraction (PM-kallikrein) averaged 1.81 nmol of S-2266 hydrolyzed per min...thousand Fig. 1 times more active than lysolecithin on a molar basis. Lecithin and arachidonic acid were active only at a much higher concentration...taglandin E2 (11), arachidonic acid or lecithin . However, melittin, on a molar basis, was about three orders of magnitude more potent than

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells.

PubMed

Surin, A M; Bolshakov, A P; Mikhailova, M M; Sorokina, E G; Senilova, Ya E; Pinelis, V G; Khodorov, B I

2006-08-01

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.

Arachidonate-Regulated Ca2+ Influx in Human Airway Smooth Muscle

PubMed Central

Thompson, Michael A.; Prakash, Y. S.

2014-01-01

Plasma membrane Ca2+ influx, especially store-operated Ca2+ entry triggered by sarcoplasmic reticulum (SR) Ca2+ release, is a key component of intracellular calcium concentration ([Ca2+]i) regulation in airway smooth muscle (ASM). Agonist-induced Ca2+ oscillations in ASM that involve both influx and SR mechanisms have been previously demonstrated. In nonexcitable cells, [Ca2+]i oscillations involve Ca2+ influx via arachidonic acid (AA) –stimulated channels, which show similarities to store-operated Ca2+ entry, although their molecular identity remains undetermined. Little is known about AA-regulated Ca2+ channels or their regulation in ASM. In enzymatically dissociated human ASM cells loaded with the Ca2+ indicator, fura-2, AA (1–10 μM) triggered [Ca2+]i oscillations that were inhibited by removal of extracellular Ca2+. Other fatty acids, such as the diacylglycerol analog, 1-oleoyl-2-acetyl-SN-glycerol, oleic acid, and palmitic acid (10 μM each), failed to elicit similar [Ca2+]i responses. Preincubation with LaCl3 (1 μM or 1 mM) inhibited AA-induced oscillations. Inhibition of receptor-operated channels (SKF96,365 [10 μM]), lipoxygenase (zileuton [10 μM]), or cyclooxygenase (indomethacin [10 μM]) did not affect oscillation parameters. Inhibition of SR Ca2+ release (ryanodine [10 μM] or inositol 1,4,5-trisphosphate receptor inhibitor, xestospongin C [1 μM]) decreased [Ca2+]i oscillation frequency and amplitude. Small interfering RNA against caveolin-1, stromal interaction molecule 1, or Orai3 (20 nM each) reduced the frequency and amplitude of AA-induced [Ca2+]i oscillations. In ASM cells derived from individuals with asthma, AA increased oscillation amplitude, but not frequency. These results are highly suggestive of a novel AA-mediated Ca2+–regulatory mechanism in human ASM, reminiscent of agonist-induced oscillations. Given the role of AA in ASM intracellular signaling, especially with inflammation, AA-regulated Ca2+ channels could potentially contribute to increased [Ca2+]i in diseases such asthma. PMID:24471656

Cytosolic phospholipase A2α is critical for angiotensin II-induced hypertension and associated cardiovascular pathophysiology.

PubMed

Khan, Nayaab S; Song, Chi Young; Jennings, Brett L; Estes, Anne M; Fang, Xiao R; Bonventre, Joseph V; Malik, Kafait U

2015-04-01

Angiotensin II activates cytosolic phospholipase A(2)α (cPLA2α) and releases arachidonic acid from tissue phospholipids, which mediate or modulate ≥1 cardiovascular effects of angiotensin II and has been implicated in hypertension. Because arachidonic acid release is the rate limiting step in eicosanoid production, cPLA2α might play a central role in the development of angiotensin II-induced hypertension. To test this hypothesis, we investigated the effect of angiotensin II infusion for 13 days by micro-osmotic pumps on systolic blood pressure and associated pathogenesis in wild type (cPLA2α(+/+)) and cPLA2α(-/-) mice. Angiotensin II-induced increase in systolic blood pressure in cPLA2α(+/+) mice was abolished in cPLA2α(-/-) mice; increased systolic blood pressure was also abolished by the arachidonic acid metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid in cPLA2α(+/+) mice. Angiotensin II in cPLA2α(+/+) mice increased cardiac cPLA2 activity and urinary eicosanoid excretion, decreased cardiac output, caused cardiovascular remodeling with endothelial dysfunction, and increased vascular reactivity in cPLA2α(+/+) mice; these changes were diminished in cPLA2α(-/-) mice. Angiotensin II also increased cardiac infiltration of F4/80(+) macrophages and CD3(+) T lymphocytes, cardiovascular oxidative stress, expression of endoplasmic reticulum stress markers p58(IPK), and CHOP in cPLA2α(+/+) but not cPLA2α(-/-) mice. Angiotensin II increased cardiac activity of ERK1/2 and cSrc in cPLA2α(+/+) but not cPLA2α(-/-) mice. These data suggest that angiotensin II-induced hypertension and associated cardiovascular pathophysiological changes are mediated by cPLA2α activation, most likely through the release of arachidonic acid and generation of eicosanoids with predominant prohypertensive effects and activation of ≥1 signaling molecules, including ERK1/2 and cSrc. © 2015 American Heart Association, Inc.

Protectin DX, a double lipoxygenase product of DHA, inhibits both ROS production in human neutrophils and cyclooxygenase activities

PubMed Central

Liu, Miao; Boussetta, Tarek; Makni-Maalej, Karama; Fay, Michèle; Driss, Fathi; El-Benna, Jamel; Lagarde, Michel; Guichardant, Michel

2014-01-01

Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits with an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX) - a docosahexaenoic acid (DHA) di-hydroxylated product which inhibits blood platelet aggregation - on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. 1.14.99.1) as well as COX-2 in lipopolysaccharides (LPS)-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B4 (LTB4). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases. PMID:24254970

Quantitative structure-activity relationship of organosulphur compounds as soybean 15-lipoxygenase inhibitors using CoMFA and CoMSIA.

PubMed

Caballero, Julio; Fernández, Michael; Coll, Deysma

2010-12-01

Three-dimensional quantitative structure-activity relationship studies were carried out on a series of 28 organosulphur compounds as 15-lipoxygenase inhibitors using comparative molecular field analysis and comparative molecular similarity indices analysis. Quantitative information on structure-activity relationships is provided for further rational development and direction of selective synthesis. All models were carried out over a training set including 22 compounds. The best comparative molecular field analysis model only included steric field and had a good Q² = 0.789. Comparative molecular similarity indices analysis overcame the comparative molecular field analysis results: the best comparative molecular similarity indices analysis model also only included steric field and had a Q² = 0.894. In addition, this model predicted adequately the compounds contained in the test set. Furthermore, plots of steric comparative molecular similarity indices analysis field allowed conclusions to be drawn for the choice of suitable inhibitors. In this sense, our model should prove useful in future 15-lipoxygenase inhibitor design studies. © 2010 John Wiley & Sons A/S.

Chemical Composition and Immuno-Modulatory Effects of Urtica dioica L. (Stinging Nettle) Extracts.

PubMed

Francišković, Marina; Gonzalez-Pérez, Raquel; Orčić, Dejan; Sánchez de Medina, Fermín; Martínez-Augustin, Olga; Svirčev, Emilija; Simin, Nataša; Mimica-Dukić, Neda

2017-08-01

The purpose of this work was to determine the chemical profile of stinging nettle and to provide an insight into the mechanisms by which it ameliorates the immune response. Qualitative and quantitative liquid chromatography tandem mass spectrometry analyses indicated that phenolic acids (5-O-caffeoylquinic acid as dominant) and flavonol glycosides (rutin, isoquercitrin, and kaempferol 3-O-glucoside) are present in the aerial parts, while lignans (secoisolariciresinol, 9,9'-bisacetyl-neo-olivil and their glucosides) were detected in the root. Herb and root extracts expressed selective inhibition toward cyclooxygenase and lipoxygenase branches in human platelets: root extracts were better at inhibiting thromboxane production, while herb extracts were more specific toward inhibition of 12-lipoxygenase pathway. Stinging nettle extracts mildly increased monocyte chemoattractant protein-1 and growth-related oncogene release from nonstimulated intestinal epithelial cells, stimulating MyD88/NF-κB/p38 signaling, hence preserving the epithelial integrity and enhancing intestinal steady-state defense. Additionally, root extract reduced lipopolysaccharide-induced monocyte chemoattractant protein-1/growth-related oncogene secretion and cyclooxygenase-2 expression in intestinal epithelial cells, thus showing the potential protective effect against tissue damage caused by inflammation processes. These observations suggest that stinging nettle is an interesting candidate for the development of phytopharmaceuticals or dietary supplements for cotreatment of various inflammatory diseases, particularly inflammatory bowel diseases. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Phospholipase A2 activation as a therapeutic approach for cognitive enhancement in early-stage Alzheimer disease.

PubMed

Schaeffer, Evelin L; Forlenza, Orestes V; Gattaz, Wagner F

2009-01-01

Alzheimer disease (AD) is the leading cause of dementia in the elderly and has no known cure. Evidence suggests that reduced activity of specific subtypes of intracellular phospholipases A2 (cPLA2 and iPLA2) is an early event in AD and may contribute to memory impairment and neuropathology in the disease. The objective of this study was to review the literature focusing on the therapeutic role of PLA2 stimulation by cognitive training and positive modulators, or of supplementation with arachidonic acid (PLA2 product) in facilitating memory function and synaptic transmission and plasticity in either research animals or human subjects. MEDLINE database was searched (no date restrictions) for published articles using the keywords Alzheimer disease (mild, moderate, severe), mild cognitive impairment, healthy elderly, rats, mice, phospholipase A(2), phospholipid metabolism, phosphatidylcholine, arachidonic acid, cognitive training, learning, memory, long-term potentiation, protein kinases, dietary lipid compounds, cell proliferation, neurogenesis, and neuritogenesis. Reference lists of the identified articles were checked to select additional studies of interest. Overall, the data suggest that PLA2 activation is induced in the healthy brain during learning and memory. Furthermore, learning seems to regulate endogenous neurogenesis, which has been observed in AD brains. Finally, PLA2 appears to be implicated in homeostatic processes related to neurite outgrowth and differentiation in both neurodevelopmental processes and response to neuronal injury. The use of positive modulators of PLA2 (especially of cPLA2 and iPLA2) or supplementation with dietary lipid compounds (e.g., arachidonic acid) in combination with cognitive training could be a valuable therapeutic strategy for cognitive enhancement in early-stage AD.

Differential Induction of Lipoxygenase Isoforms in Wheat upon Treatment with Rust Fungus Elicitor, Chitin Oligosaccharides, Chitosan, and Methyl Jasmonate.

PubMed Central

Bohland, C.; Balkenhohl, T.; Loers, G.; Feussner, I.; Grambow, H. J.

1997-01-01

A glycopeptide elicitor prepared from germ tubes of the rust fungus Puccinia graminis Pers. f. sp. tritici Erikss. & Henn (Pgt), as well as chitin oligosaccharides, chitosan, and methyl jasmonate (MJ) stimulated lipoxygenase (LOX) activity (E.C. 1.13.11.12) in wheat (Triticum aestivum) leaves. Immunoblot analysis using anti-LOX antibodies revealed the induction of 92- and 103-kD LOX species after Pgt elicitor treatment. In contrast, MJ treatment led to a significant increase of a 100-kD LOX species, which was also detected at lower levels in control plants. The effects of chitin oligomers and chitosan resembled those caused by MJ. In conjunction with other observations the results suggest that separate reaction cascades exist, and that jasmonates may not be involved in Pgt elicitor action. LOX-92 appears to be mainly responsible for the increase in LOX activity after Pgt elicitor treatment because its appearance on western blots coincided with high LOX activity in distinct anion-exchange chromatography fractions. It is most active at pH 5.5 to 6.0, and product formation from linoleic and [alpha]-linolenic acid is clearly in favor of the 9-LOOHs. It is interesting that a 92-kD LOX species, which seems to correspond to the Pgt elicitor-induced LOX species, was also detected in rust-inoculated leaves. PMID:12223735

In vitro glycoxidized low-density lipoproteins and low-density lipoproteins isolated from type 2 diabetic patients activate platelets via p38 mitogen-activated protein kinase.

PubMed

Calzada, Catherine; Coulon, Laurent; Halimi, Déborah; Le Coquil, Elodie; Pruneta-Deloche, Valérie; Moulin, Philippe; Ponsin, Gabriel; Véricel, Evelyne; Lagarde, Michel

2007-05-01

Platelet hyperactivation contributes to the increased risk for atherothrombosis in type 2 diabetes and is associated with oxidative stress. Plasma low-density lipoproteins (LDLs) are exposed to both hyperglycemia and oxidative stress, and their role in platelet activation remains to be ascertained. The aim of this study was to investigate the effects of LDLs modified by both glycation and oxidation in vitro or in vivo on platelet arachidonic acid signaling cascade. The activation of platelet p38 MAPK, the stress kinase responsible for the activation of cytosolic phospholipase A(2), and the concentration of thromboxane B(2), the stable catabolite of the proaggregatory arachidonic acid metabolite thromboxane A(2), were assessed. First, in vitro-glycoxidized LDLs increased the phosphorylation of platelet p38 MAPK as well as the concentration of thromboxane B(2). Second, LDLs isolated from plasma of poorly controlled type 2 diabetic patients stimulated both platelet p38 MAPK phosphorylation and thromboxane B(2) production and possessed high levels of malondialdehyde but normal alpha-tocopherol concentrations. By contrast, LDLs from sex- and age-matched healthy volunteers had no activating effects on platelets. Our results indicate that LDLs modified by glycoxidation may play an important contributing role in platelet hyperactivation observed in type 2 diabetes via activation of p38 MAPK.

Higher Activities of Defense-Associated Enzymes may Contribute to Greater Resistance of Manchurian Ash to Emerald Ash Borer Than A closely Related and Susceptible Congener.

PubMed

Rigsby, Chad M; Herms, Daniel A; Bonello, Pierluigi; Cipollini, Don

2016-08-01

Emerald ash borer (EAB) is an invasive beetle native to Asia that infests and kills ash (Fraxinus spp.) in North America. Previous experiments indicated that larvae feeding on co-evolved, resistant Manchurian ash (F. mandshurica) have increased antioxidant and quinone-protective enzyme activities compared to larvae feeding on susceptible North American species. Here, we examined mechanisms of host-generated oxidative and quinone-based stress and other putative defenses in Manchurian ash and the closely related and chemically similar, but susceptible, black ash (F. nigra), with and without exogenous application of methyl jasmonate (MeJA) to induce resistance mechanisms. Peroxidase activities were 4.6-13.3 times higher in Manchurian than black ash, although both species appeared to express the same three peroxidase isozymes. Additionally, peroxidase-mediated protein cross-linking activity was stronger in Manchurian ash. Polyphenol oxidase, β-glucosidase, chitinase, and lipoxygenase activities also were greater in Manchurian ash, but only lipoxygenase activity increased with MeJA application. Phloem H 2 O 2 levels were similar and were increased by MeJA application in both species. Lastly, trypsin inhibitor activity was detected in methanol and water extracts that were not allowed to oxidize, indicating the presence of phenolic-based trypsin inhibitors. However, no proteinaceous trypsin inhibitor activity was detected in either species. In response to MeJA application, Manchurian ash had higher trypsin inhibitor activity than black ash using the unoxidized water extracts, but no treatment effects were detected using methanol extracts. Based on these results we hypothesize that peroxidases, lignin polymerization, and quinone generation contribute to the greater resistance to EAB displayed by Manchurian ash.

Difference in expression between AQP1 and AQP5 in porcine endometrium and myometrium in response to steroid hormones, oxytocin, arachidonic acid, forskolin and cAMP during the mid-luteal phase of the estrous cycle and luteolysis.

PubMed

Skowronska, Agnieszka; Mlotkowska, Patrycja; Nielsen, Soren; Skowronski, Mariusz T

2015-12-01

Recently, we demonstrated in vitro that AQP1 and AQP5 in the porcine uterus are regulated by steroid hormones (P4, E2), arachidonic acid (AA), forskolin (FSK) and cAMP during the estrous cycle. However, the potential of the porcine separated uterine tissues, the endometrium and myometrium, to express these AQPs remains unknown. Thus, in this study, the responses of AQP1 and AQP5 to P4, E2 oxytocin (OT), AA, FSK and cAMP in the porcine endometrium and myometrium were examined during the mid-luteal phase of the estrous cycle and luteolysis. Real-time PCR and western blot analysis. Progesterone up-regulated the expression of AQP1/AQP5 mRNAs and proteins in the endometrium and myometrium, especially during luteolysis. Similarly, E2 also stimulated the expression of both AQPs, but only in the endometrium. AA led to the upregulation of AQP1/AQP5 in the endometrium during luteolysis. In turn, OT increased the expression of AQP1/AQP5 mRNAs and proteins in the myometrium during mid-luteal phase. Moreover, a stimulatory effect of forskolin and cAMP on the expression of AQP1/AQP5 mRNAs and proteins in the endometrium and myometrium dominated during luteolysis, but during the mid-luteal phase their influence on the expression of these AQPs was differentiated depending on the type of tissue and the incubation duration. These results seem to indicate that uterine tissues; endometrium and myometrium, exhibit their own AQP expression profiles in response to examined factors. Moreover, the responses of AQP1/AQP5 at mRNA and protein levels to the studied factors in the endometrium and myometrium are more pronounced during luteolysis. This suggests that the above effects of the studied factors are connected with morphological and physiological changes taking place in the pig uterus during the estrous cycle.

Transient postnatal fluoxetine decreases brain concentrations of 20-HETE and 15-epi-LXA4, arachidonic acid metabolites in adult mice.

PubMed

Yuan, Zhi-Xin; Rapoport, Stanley I

2015-10-01

Transient postnatal exposure of rodents to the selective serotonin (5-HT) reuptake inhibitor (SSRI) fluoxetine alters behavior and brain 5-HT neurotransmission during adulthood, and also reduces brain arachidonic (ARA) metabolic consumption and protein level of the ARA metabolizing enzyme, cytochrome P4504A (CYP4A). Brain 20-hydroxyeicosatetraenoic acid (20-HETE), converted by CYP4A from ARA, will be reduced in adult mice treated transiently and postnatally with fluoxetine. Male mice pups were injected i.p. daily with fluoxetine (10mg/kg) or saline during P4-P21. At P90 their brain was high-energy microwaved and analyzed for 20-HETE and six other ARA metabolites by enzyme immunoassay. Postnatal fluoxetine vs. saline significantly decreased brain concentrations of 20-HETE (-70.3%) and 15-epi-lipoxin A4 (-60%) in adult mice, but did not change other eicosanoid concentrations. Behavioral changes in adult mice treated postnatally with fluoxetine may be related to reduced brain ARA metabolism involving CYP4A and 20-HETE formation. Published by Elsevier Ltd.

Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities.

PubMed

Castro, D P; Figueiredo, M B; Genta, F A; Ribeiro, I M; Tomassini, T C B; Azambuja, P; Garcia, E S

2009-06-01

The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A(2) (PLA(2)) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1mug/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10microg/insect) or PAF (1microg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA(2) activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.

Effects of protopine on blood platelet aggregation. III. Effect of protopine on the metabolic system of arachidonic acid in platelets.

PubMed

Shiomoto, H; Matsuda, H; Kubo, M

1991-02-01

The mode of action of protopine on blood platelet aggregation was investigated in the metabolic system of arachidonic acid and in liberation of platelet activating factor using in vitro experimental models. Protopine inhibited the releases of arachidonic acid and platelet activating factor from platelet membrane phospholipids. Protopine also inhibited the conversion of prostaglandin G2 to thromboxane A2, as well as carboxyheptyl imidazole, a thromboxane synthetase inhibitor. These results indicated that protopine functions both as a phospholipase inhibitor and a thromboxane synthetase inhibitor. It is expected that protopine can be applied for treatment of thrombosis as an antiplatelet drug.

A cell wall extract from Piriformospora indica promotes tuberization in potato (Solanum tuberosum L.) via enhanced expression of Ca(+2) signaling pathway and lipoxygenase gene.

PubMed

Upadhyaya, Chandrama Prakash; Gururani, Mayank Anand; Prasad, Ram; Verma, Ajit

2013-06-01

Piriformospora indica is an axenically cultivable phytopromotional endosymbiont that mimics capabilities of arbuscular mycorrhizal fungi. This is a basidiomycete of the Sebacinaceae family, which promotes growth, development, and seed production in a variety of plant species. We report that the cell wall extract (CWE) from P. indica induces tuberization in vitro and promotes tuber growth and yield in potato. The CWE altered the calcium signaling pathway that regulates tuberization process. An increase in tuber number and size was correlated with increased transcript expression of the two Ca(2+)-dependant proteins (CaM1 and St-CDPK1) and the lipoxygenase (LOX) mRNA, which are known to play distinct roles in potato tuberization. External supplementation of Ca(2+) ions induced a similar set of tuberization pathway genes, indicating presence of an active Ca(2+) in the CWE of P. indica. Since potato tuberization is directly influenced by the presence of microflora in nature, the present study provides an insight into the novel mechanism of potato tuberization in relation to plant-microbe association. Ours is the first report on an in vitro tuber-inducing beneficial fungus.

Mechanism of Arachidonic Acid Accumulation during Aging in Mortierella alpina: A Large-Scale Label-Free Comparative Proteomics Study.

PubMed

Yu, Yadong; Li, Tao; Wu, Na; Ren, Lujing; Jiang, Ling; Ji, Xiaojun; Huang, He

2016-11-30

Arachidonic acid (ARA) is an important polyunsaturated fatty acid having various beneficial physiological effects on the human body. The aging of Mortierella alpina has long been known to significantly improve ARA yield, but the exact mechanism is still elusive. Herein, multiple approaches including large-scale label-free comparative proteomics were employed to systematically investigate the mechanism mentioned above. Upon ultrastructural observation, abnormal mitochondria were found to aggregate around shrunken lipid droplets. Proteomics analysis revealed a total of 171 proteins with significant alterations of expression during aging. Pathway analysis suggested that reactive oxygen species (ROS) were accumulated and stimulated the activation of the malate/pyruvate cycle and isocitrate dehydrogenase, which might provide additional NADPH for ARA synthesis. EC 4.2.1.17-hydratase might be a key player in ARA accumulation during aging. These findings provide a valuable resource for efforts to further improve the ARA content in the oil produced by aging M. alpina.

Irradiation of human skin by short wavelength ultraviolet radiation (100--290 nm) (u.v.C): increased concentrations of arachidonic acid and prostaglandines E2 and F2alpha.

PubMed Central

Camp, R D; Greaves, M W; Hensby, C N; Plummer, N A; Warin, A P

1978-01-01

1. Human abdominal skin was irradiated with six times the minimal erythema dose of ultraviolet C (100--290 nm) radiation. Erythema appeared at 3 h, was of moderate degree by 6 h and was maximal at 12--24 h. It was reduced at 48 h and by 72 h had disappeared. 2. A suction bulla technique was used for the recovery of exudate from normal and inflamed skin at 6, 18, 24 and 48 h after irradiation. 3. Prostaglandin-like activity, estimated by bioassay, showed maximum increase at 18 h, when erythema was also maximum. PGF 2alpha, measured by both radioimmunoassay and by combined gas-liquid chromatography--gas spectrometry, followed a similar time course then fell to normal, or near normal, levels at 48 h. 4. Prostaglandin E2 and arachidonic acid concentrations, measured by gas chromatography--mass spectrometry, were maximally raised at 18--24 h. At 48 h, when some erythema was still present, though reduced, prostaglandin E2 concentrations were still raised above control values. 5. The results provide direct evidence in support of the view that the erythma following irradiation of human skin by u.v.C involves activation of arachidonic acid metabolism. However, the relationship between the erythema and increased prostaglandin activity is not fully understood. PMID:678391

Aging dysregulates D- and E-series resolvins to modulate cardiosplenic and cardiorenal network following myocardial infarction.

PubMed

Halade, Ganesh V; Kain, Vasundhara; Black, Laurence M; Prabhu, Sumanth D; Ingle, Kevin A

2016-10-18

Post-myocardial infarction (MI), overactive inflammation is the hallmark of aging, however, the mechanism is unclear. We hypothesized that excess influx of omega 6 fatty acids may impair resolution, thus impacting the cardiosplenic and cardiorenal network post-MI. Young and aging mice were fed on standard lab chow (LC) and excess fatty acid (safflower oil; SO)-enriched diet for 2 months and were then subjected to MI surgery. Despite similar infarct areas and left ventricle (LV) dysfunction post-MI, splenic mass spectrometry data revealed higher levels of arachidonic acid (AA) derived pro-inflammatory metabolites in young-SO, but minimal formation of docosanoids, D- and E- series resolvins in SO-fed aged mice. The aged mice receiving excess intake of fatty acids exhibit; 1) decreased lipoxygenases (5-,12-, and 15) in the infarcted LV; 2) lower levels of 14HDHA, RvD1, RvD5, protectin D1, 7(S)maresin1, 8-,11-,18-HEPE and RvE3 with high levels of tetranor-12-HETEs; 3) dual population of macrophages (CD11b low /F480 high and CD11b high /F480 high ) with increased pro-inflammatory (CD11bp + F4/80 + Ly6C hi ) phenotype and; 4) increased kidney injury marker NGAL with increased expression of TNF-α and IL-1β indicating MI-induced non-resolving response compared with LC-group. Thus, excess fatty acid intake magnifies the post-MI chemokine signaling and inflames the cardiosplenic and cardiorenal network towards a non-resolving microenvironment in aging.

Aging dysregulates D- and E-series resolvins to modulate cardiosplenic and cardiorenal network following myocardial infarction

PubMed Central

Halade, Ganesh V.; Kain, Vasundhara; Black, Laurence M.; Prabhu, Sumanth D.; Ingle, Kevin A.

2016-01-01

Post-myocardial infarction (MI), overactive inflammation is the hallmark of aging, however, the mechanism is unclear. We hypothesized that excess influx of omega 6 fatty acids may impair resolution, thus impacting the cardiosplenic and cardiorenal network post-MI. Young and aging mice were fed on standard lab chow (LC) and excess fatty acid (safflower oil; SO)-enriched diet for 2 months and were then subjected to MI surgery. Despite similar infarct areas and left ventricle (LV) dysfunction post-MI, splenic mass spectrometry data revealed higher levels of arachidonic acid (AA) derived pro-inflammatory metabolites in young-SO, but minimal formation of docosanoids, D- and E- series resolvins in SO-fed aged mice. The aged mice receiving excess intake of fatty acids exhibit; 1) decreased lipoxygenases (5-,12-, and 15) in the infarcted LV; 2) lower levels of 14HDHA, RvD1, RvD5, protectin D1, 7(S)maresin1, 8-,11-,18-HEPE and RvE3 with high levels of tetranor-12-HETEs; 3) dual population of macrophages (CD11blow/F480high and CD11bhigh/F480high) with increased pro-inflammatory (CD11b+F4/80+Ly6Chi) phenotype and; 4) increased kidney injury marker NGAL with increased expression of TNF-ɑ and IL-1β indicating MI-induced non-resolving response compared with LC-group. Thus, excess fatty acid intake magnifies the post-MI chemokine signaling and inflames the cardiosplenic and cardiorenal network towards a non-resolving microenvironment in aging. PMID:27777380

Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis

PubMed Central

Yoo, Sang-Hun; Chang, Yoon Hyuk

2016-01-01

The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G′, G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities. PMID:28078256

Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis.

PubMed

Yoo, Sang-Hun; Chang, Yoon Hyuk

2016-12-01

The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G', G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities.

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

PubMed Central

Kausch, K D; Handa, A K

1997-01-01

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation. PMID:9112767

Expression of inactive glutathione peroxidase 4 leads to embryonic lethality, and inactivation of the Alox15 gene does not rescue such knock-in mice.

PubMed

Brütsch, Simone Hanna; Wang, Chi Chiu; Li, Lu; Stender, Hannelore; Neziroglu, Nilgün; Richter, Constanze; Kuhn, Hartmut; Borchert, Astrid

2015-02-01

Glutathione peroxidases (Gpx) and lipoxygenases (Alox) are functional counterplayers in the metabolism of hydroperoxy lipids that regulate cellular redox homeostasis. Gpx4 is a moonlighting protein that has been implicated not only as an enzyme in anti-oxidative defense, gene expression regulation, and programmed cell death, but also as a structural protein in spermatogenesis. Homozygous Gpx4 knock-out mice are not viable, but molecular reasons for intrauterine lethality are not completely understood. This study was aimed at investigating whether the lack of catalytic activity or the impaired function as structural protein is the dominant reason for embryonic lethality. We further explored whether the pro-oxidative enzyme mouse 12/15 lipoxygenase (Alox15) plays a major role in embryonic lethality of Gpx4-deficient mice. To achieve these goals, we first created knock-in mice, which express a catalytically inactive Gpx4 mutant (Sec46Ala). As homozygous Gpx4-knock-out mice Sec46Ala-Gpx4(+/+) knock-in animals are not viable but undergo intrauterine resorption between embryonic day 6 and 7 (E6-7). In contrast, heterozygous knock-in mice (Sec46Ala-Gpx4(-/+)) are viable, fertile and do not show major phenotypic alterations. Interestingly, homozygous Alox15 deficiency did not rescue the U46A-Gpx4(+/+) mice from embryonic lethality. In fact, when heterozygous U46A-Gpx4(-/+) mice were stepwise crossed into an Alox15-deficent background, no viable U46A-Gpx4(+/+)+Alox15(-/-) individuals were obtained. However, we were able to identify U46A-Gpx4(+/+)+Alox15(-/-) embryos in the state of resorption around E7. These data suggest that the lack of catalytic activity is the major reason for the embryonic lethality of Gpx4(-/-) mice and that systemic inactivation of the Alox15 gene does not rescue homozygous knock-in mice expressing catalytically silent Gpx4.

Up-regulation of 5-lipoxygenase by inhibition of cathepsin G enhances TRAIL-induced apoptosis through down-regulation of survivin

PubMed Central

Woo, Seon Min; Min, Kyoung-Jin; Seo, Seung Un; Kim, Shin; Park, Jong-Wook; Song, Dae Kyu; Lee, Hyun-Shik; Kim, Sang Hyun; Kwon, Taeg Kyu

2017-01-01

Cathepsin G is a serine protease secreted from activated neutrophils, it has important roles in inflammation and immune response. Moreover, cathepsin G promotes tumor cell-cell adhesion and migration in cancer cells. In this study, we investigated whether inhibition of cathepsin G could sensitize TRAIL-mediated apoptosis in cancer cells. An inhibitor of cathepsin G [Cathepsin G inhibitor I (Cat GI); CAS 429676-93-7] markedly induced TRAIL-mediated apoptosis in human renal carcinoma (Caki, ACHN, and A498), lung cancer (A549) and cervical cancer (Hela) cells. In contrast, combined treatment with Cat GI and TRAIL had no effect on apoptosis in normal cells [mesangial cell (MC) and human skin fibroblast (HSF)]. Cat GI induced down-regulation of survivin expression at the post-translational level, and overexpression of survivin markedly blocked apoptosis induced by combined treatment with Cat GI plus TRAIL. Interestingly, Cat GI induced down-regulation of survivin via 5-lipoxygenase (5-LOX)-mediated reactive oxygen species (ROS) production. Inhibition of 5-LOX by gene silencing (siRNA) or a pharmacological inhibitor of 5-LOX (zileuton) markedly attenuated combined treatment-induced apoptosis. Taken together, our results indicate that inhibition of cathepsin G sensitizes TRAIL-induced apoptosis through 5-LOX-mediated down-regulation of survivin expression. PMID:29290980

In vitro study on the antioxidant activity of a polyphenol-rich extract from Pinus brutia bark and its fractions.

PubMed

Cretu, Elena; Karonen, Maarit; Salminen, Juha-Pekka; Mircea, Cornelia; Trifan, Adriana; Charalambous, Christiana; Constantinou, Andreas I; Miron, Anca

2013-11-01

A crude hydromethanolic extract from Pinus brutia bark and its fractions (diethyl ether, ethyl acetate, n-butanol, and aqueous fractions) were studied with regard to their phenolic content and antioxidant activities. The total phenolics and proanthocyanidins in each extract were quantified by spectrophotometric methods; the polyphenolic profile was analyzed by RP-HPLC-DAD-ESI-MS. All extracts were tested with regard to their ability to scavenge free radicals (ABTS radical cation, superoxide and hydroxyl radicals), reduce ferric ions, and inhibit 15-lipoxygenase. P. brutia bark extracts had high phenolic contents (303.79±7.34-448.90±1.39 mg/g). Except diethyl ether extract, all other extracts contained proanthocyanidins ranging from 225.79±3.94 to 250.40±1.44 mg/g. Several polyphenols were identified by RP-HPLC-DAD-ESI-MS: taxifolin in diethyl ether extract, a taxifolin-O-hexoside, catechin, procyanidin dimers, and trimers in ethyl acetate extract. Except diethyl ether extract, all other extracts were effective scavengers of superoxide and hydroxyl radicals (EC₅₀=33.5±1.1-54.93±2.85 μg/mL and 0.47±0.06-0.6±0.0 mg/mL, respectively). All extracts had noticeable 15-lipoxygenase inhibitory effects (EC₅₀=22.47±0.75-34.43±2.25 μg/mL). We conclude that P. brutia bark is very promising for the dietary supplements industry due to its high free radical scavenging and 15-lipoxygenase inhibitory effects.

Effects of prostaglandin F(2alpha)and carbachol on MAP kinases, cytosolic phospholipase A(2)and arachidonic acid release in cat iris sphincter smooth muscle cells.

PubMed

Husain, S; Abdel-Latif, A A

2001-05-01

The signal transduction pathways initiated by Ca(2+)-mobilizing agonists, such as prostaglandin F(2alpha)(PGF(2alpha)) and carbachol (CCh), leading to activation of cytosolic phospholipase A(2)(cPLA(2)) and arachidonic acid (AA) release in a wide variety of tissues remain obscure. To further define the role of protein kinases in receptor mediated stimulation of cPLA(2)and consequently AA release we have investigated the role of mitogen-activated protein (MAP) kinases and protein kinase C (PKC) in PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation and AA release in cat iris sphincter smooth muscle (CISM) cells. The cells were prelabeled with [(3)H]AA for 24 hr and incubated in the absence or presence of the agonist for 5-10 min as indicated. MAP kinases activities and cPLA(2)phosphorylation were determined in immunoprecipitates obtained by using anti-p38 MAP kinase and anti-cPLA(2)antibodies. We found that: (a) PGF(2alpha)and CCh increased p38 MAP kinase activity by 197 and 215%, respectively, and increased p42/p44 MAP kinase activity by 200 and 125%, respectively. (b) SB202190, a p38 MAP kinase specific inhibitor, inhibited PGF(2alpha)- and CCh-induced cPLA(2)phosphorylation by 92 and 85%, respectively, and AA release by 62 and 78%, respectively. (c) PD98059, a p42/p44 MAP kinase inhibitor, inhibited CCh-induced cPLA(2)phosphorylation by 70% and AA release by 71%, but had no effect on that of PGF(2alpha). (d) Inhibition of PKC activity by RO 31-8220 inhibited both PGF(2alpha)- and CCh-stimulation of p38 MAP kinase, p42/p44 MAP kinases and cPLA(2)phosphorylation. We conclude from these results that in CISM cells PGF(2alpha)-induced cPLA(2)phosphorylation and AA release is mediated through p38 MAP kinase, but not through p42/p44 MAP kinases, whereas that of CCh is mediated through both p38 MAP kinase and p42/p44 MAP kinases. These effects of PGF(2alpha)and CCh are regulated by the MAP kinases in a PKC-dependent manner. Studies aimed at elucidating the role of protein kinases in the coupling mechanism between the activation of PGF(2alpha)and muscarinic receptors, and the stimulation of cPLA(2)and AA release in the smooth muscles of the iris-ciliary body will provide important information about the role of protein kinases signaling pathways in smooth muscle function, as well as about the mechanism of the intraocular pressure-lowering effects of PGF(2alpha)and its analog, latanoprost, in glaucoma therapy. Copyright 2001 Academic Press.

Effect of a 5-lipoxygenase inhibitor on endotoxin-induced pulmonary dysfunction in awake sheep.

PubMed

Kuratomi, Y; Lefferts, P L; Christman, B W; Parker, R E; Smith, W G; Mueller, R A; Snapper, J R

1993-02-01

We studied the effects of a 5-lipoxygenase inhibitor, SC-45662, on endotoxin-induced pulmonary dysfunction in chronically instrumented unanesthetized sheep. Each sheep was studied with endotoxin alone, SC-45662 alone, and endotoxin after SC-45662 pretreatment. Endotoxin did not cause consistent increases in plasma or lung lymph concentrations of leukotriene B4 (LTB4). Ex vivo stimulation of whole blood from sheep before and after treatment with SC-45662 demonstrated no inhibition of cyclooxygenase metabolism but an approximately 80% inhibition of LTB4 production. At drug concentrations obtained in vivo, SC-45662 did not significantly inhibit in vitro A23187-stimulated whole blood thromboxane B2 production but did inhibit LTB4 production from ionophore-stimulated sheep granulocytes. SC-45662 attenuated the early changes in lung mechanics and pulmonary hypertension but did not attenuate the later increase in lung fluid and solute exchange observed after endotoxemia. We conclude that 5-lipoxygenase products are not measurably involved in the later increase in lung fluid and solute exchange observed after endotoxemia in sheep.

Gaultheria trichophylla (Royle): a source of minerals and biologically active molecules, its antioxidant and anti-lipoxygenase activities.

PubMed

Alam, Fiaz; Saqib, Qazi Najam Us; Ashraf, Mohammad

2017-01-03

Gaultheria trichophylla (Royle) is used as food and for treating many ailments in folk medicine especially against inflammation. The purpose of this in vitro study was to evaluate the ability of extracts of G. trichophylla as anti-oxidant and anti-inflammatory agent and for its mineral contents. Powdered plant material (100 g) was extracted with 100 ml each of methanol, chloroform, and n-hexane using soxhlet extractor. Antioxidant activity of methanol extract was assessed by DPPH radical scavenging and FRAP assays. Determination of enzyme inhibition activity was determined using 5-LOX inhibitory activity. Total phenolic and flavonoids contents were measured by Folin-Chicalteu and colorimeteric methods respectively. Minerals and heavy metals contents were determined using Atomic absorption spectrophotometer. Qualitative HPLC analysis were performed using some standard phenolic compounds. The highest phenolic (17.5 ± 2.5 mg GA equivalent/g) and flavonoids (41.3 ± 0.1 mg QE equivalent/g) concentrations were found in methanol extract, which also showed more scavenging activity of 1, 1-diphenyl-2-picrylhydrazyl and ferrous reducing power with IC 50  = 81.2 ± 0.2 and IC 50  = 11.2 ± 0.1 μg/ml, respectively. The methanol and chloroform extracts showed best inhibition of 5-lipoxygenase enzyme with 90.5 ± 0.7% and 66.9 ± 0.1% at 0.5 mg/ml, respectively. G. trichophylla extract was also evaluated for mineral contents (K, Na, Ca, Mg, Fe, and Cu), and for chemical profiling of heavy metals (Cr, Pb, Cd, Co, Zn, Ni and Hg). Our current findings suggest that this plant is good source of minerals and concentration of all heavy metals were within permissible limits. The results revealed that this ignored plant has great pharmaceutical and nutraceutical potential.

Involvement of Reactive Oxygen Species and Mitochondrial Proteins in Biophoton Emission in Roots of Soybean Plants under Flooding Stress.

PubMed

Kamal, Abu Hena Mostafa; Komatsu, Setsuko

2015-05-01

To understand the mechanism of biophoton emission, ROS and mitochondrial proteins were analyzed in soybean plants under flooding stress. Enzyme activity and biophoton emission were increased in the flooding stress samples when assayed in reaction mixes specific for antioxidant enzymes and reactive oxygen species; although the level of the hydroxyl radicals was increased at day 4 (2 days of flooding) compared to nonflooding at day 4, the emission of biophotons did not change. Mitochondria were isolated and purified from the roots of soybean plants grown under flooding stress by using a Percoll gradient, and proteins were analyzed by a gel-free proteomic technique. Out of the 98 mitochondrial proteins that significantly changed abundance under flooding stress, 47 increased and 51 decreased at day 4. The mitochondrial enzymes fumarase, glutathione-S-transferase, and aldehyde dehydrogenase increased at day 4 in protein abundance and enzyme activity. Enzyme activity and biophoton emission decreased at day 4 by the assay of lipoxygenase under stress. Aconitase, acyl CoA oxidase, succinate dehydrogenase, and NADH ubiquinone dehydrogenase were up-regulated at the transcription level. These results indicate that oxidation and peroxide scavenging might lead to biophoton emission and oxidative damage in the roots of soybean plants under flooding stress.

Characterization and Anti-Inflammatory Potential of an Exopolysaccharide from Submerged Mycelial Culture of Schizophyllum commune

PubMed Central

Du, Bin; Yang, Yuedong; Bian, Zhaoxiang; Xu, Baojun

2017-01-01

Background and Purpose: Mushroom polysaccharides have attracted attention in food and pharmacology fields because of their many biological activities. The structure characterization and anti-inflammatory activity of exopolysaccharide from Schizophyllum commune were evaluated in present study. Methods: An exopolysaccharide from a submerged mycelial fermentation of S. commune was obtained using DEAE-52 cellulose and Sephadex G-150 chromatography. The molecular weight (MW), monosaccharide compositions, chemical compositions, methylation analysis, circular dichroism studies, Fourier transform infrared spectroscopy, nuclear magnetic resonance (NMR) spectra, scanning electron microscopy (SEM), and atomic force microscopy were investigated. Results: It was a homogeneous protein-bound heteropolysaccharide with MW of 2,900 kDa. The exopolysaccharide contained a β-(1→3) glycosidic backbone, (1→4)- and (1→6)- glycosidic side chain, and high amount of glucose. The anti-inflammatory activity of exopolysaccharide was assessed by inhibiting the production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and 5- lipoxygenase (5-LOX) from macrophages. This exopolysaccharide significantly (p < 0.05) inhibited lipopolysaccharides-induced iNOS expression levels in the cells in a dose-dependent manner. Conclusion: It indicated significant anti-inflammatory effects, which showed that exopolysaccharide might be exploited as an effective anti-inflammatory agent for application in NO-related disorders such as inflammation and cancer. PMID:28555107

Pharmacokinetic and pharmacodynamic interaction between the lipoxygenase inhibitor MK-0591 and the cyclooxygenase inhibitor ibuprofen in man.

PubMed

Depré, M; Van Hecken, A; Verbesselt, R; De Lepeleire, I; Schwartz, J; Porras, A; Larson, P; Lin, C; De Schepper, P J

1998-01-01

Twelve healthy male subjects participated in a double-blind, placebo-controlled, randomized, three-period, crossover study to investigate the safety, tolerability, biochemical activity and pharmacokinetics of ibuprofen, a cyclooxygenase inhibitor and MK-0591, a 5-lipoxygenase inhibitor, given as single entities and in combination. Each subject received for three consecutive 8-day periods, separated by 1 week washout, each of the following treatments: ibuprofen 600 mg three times a day with 125 mg MK-0591 twice a day, ibuprofen 600 mg three times a day with placebo for MK-0591 and MK-0591 125 mg twice a day with placebo for ibuprofen. Cyclooxygenase inhibition was measured by platelet thromboxane (TxB2) generation test, and 5-lipoxygenase inhibition was measured by urinary leukotriene E4 excretion and ex vivo LTB4 generation in calcium-ionophore-stimulated blood. TxB2 suppression on day 8 by ibuprofen was not affected by concomitant treatment with MK-0591. MK-0591 alone had no effect on TxB2 generation. Leukotriene biosynthesis was inhibited by more than 90% by MK-0591 alone and by combined treatment, while ibuprofen alone had no effect. Coadministration appears to affect the pharmacokinetics of MK-0591 (decrease of area under the plasma concentration-vs-time curve [AUC] and maximum plasma concentrations [Cmax]) and of ibuprofen (increase of AUC and half-lives of elimination (t1/2) of the (S)-enantiomer, increase of t1/2 the (R)-enantiomer). Combined treatment had no effect on creatinine clearance nor on the number and intensity of the reported adverse experiences.

Composition and Bioactivity of Essential Oil from Citrus grandis (L.) Osbeck 'Mato Peiyu' Leaf.

PubMed

Tsai, Mei-Lin; Lin, Cai-Di; Khoo, Keh Ai; Wang, Mei-Ying; Kuan, Tsang-Kuei; Lin, Wei-Chao; Zhang, Ya-Nan; Wang, Ya-Ying

2017-12-05

'Mato Peiyu' pomelo ( Citrus grandis (L.) Osbeck 'Mato Peiyu') leaves from pruning are currently an agricultural waste. The aim of this study was to isolate essential oils from these leaves through steam distillation (SD) and solvent-free microwave extraction (SFME) and to evaluate their applicability to skin care by analyzing their antimicrobial, antioxidant (diphenyl-1-picrylhydrazyl scavenging assay, β-carotene/linoleic acid assay, and nitric oxide scavenging assay), anti-inflammatory (5-lipoxygenase inhibition assay), and antityrosinase activities. The gas chromatography-mass spectrometry results indicated that the main components of 'Mato Peiyu' leaf essential oils were citronellal and citronellol, with a total percentage of 50.71% and 59.82% for SD and SFME, respectively. The highest bioactivity among all assays was obtained for 5-lipoxygenase inhibition, with an IC 50 value of 0.034% ( v / v ). The MIC 90 of the antimicrobial activity of essential oils against Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , and Candida albicans ranged from 0.086% to 0.121% ( v / v ). Citronellal and citronellol were the main contributors, accounting for at least 54.58% of the essential oil's bioactivity. This paper is the first to report the compositions and bioactivities of 'Mato Peiyu' leaf essential oil, and the results imply that the pomelo leaf essential oil may be applied in skin care.

Investigations on the role of leukotrienes in remote hind limb preconditioning-induced cardioprotection in rats.

PubMed

Singh, Baljeet; Randhawa, Puneet Kaur; Singh, Nirmal; Jaggi, Amteshwar Singh

2016-05-01

The cardioprotective effects of remote hind limb preconditioning (RIPC) are well established, but its mechanisms still remain to be explored. Therefore, the present study was aimed to explore the possible involvement of 5-lipoxygenase-derived leukotrienes in RIPC. The hind limb was tied by a pressure cuff and was subjected to four episodes of inflation and deflation (5min each) to induce remote hind-limb preconditioning. Thereafter, the hearts were isolated and were subjected to global ischemia (30min) followed by reperfusion (120min) on the Langendorff apparatus. The extent of myocardial injury was assessed by measuring lactate dehydrogenase (LDH) and creatine kinase (CK) levels in the coronary effluent; the infarct size using TTC staining, and the hemodynamic parameters including LVDP, dp/dtmax and dp/dtmin. RIPC significantly decreased ischemia and reperfusion-induced increase in LDH, CK release, infarct size and improved LVDP, dp/dtmax and dp/dtmin. Administration of montelukast, leukotriene receptor antagonist (10 and 20mg/kg) and zileuton, 5-lipoxygenase inhibitor, (2.5 and 5mg/kg) abolished RIPC-induced cardioprotection. It may be concluded that hind limb ischemia stimulates 5-lipoxygenase to release leukotrienes which may elicit cardioprotection by humoral or neurogenic pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of a novel monoclonal antibody against 4-hydroxy-2E,6Z-dodecadienal (4-HDDE)-protein adducts: Immunochemical application in quantitative and qualitative analyses of lipid peroxidation in vitro and ex vivo.

PubMed

Uchida, Koji; Shibata, Takahiro; Toyokuni, Shinya; Daniel, Bareket; Zarkovic, Kamelija; Zarkovic, Neven; Sasson, Shlomo

2018-05-25

Non-enzymatic peroxidation of polyunsaturated fatty acids (PUFA) results in the formation of various α,β-unsaturated aldehydes, of which 4-hydroxyalkenals are abundant. The propensity of n-6 PUFA, such as linoleic acid, γ-linolenic acid and arachidonic acid, to undergo radical-induced peroxidation and generate 4-hydroxy-2E-nonenal (4-HNE) has been widely demonstrated. The ability of the latter to form covalent adducts with macromolecules and modify cellular functions has been linked to numerous pathological processes. Concomitantly, evidence has accumulated on specific signaling properties of low concentrations of 4-HNE that may induce hormetic and protective responses to peroxidation stress in cells. It has long been known that peroxidation of PUFA, and particularly arachidonic acid, also give rise to 4-hydroxy-2E,6Z-dodecadienal (4-HDDE), which is more chemically reactive than 4-HNE. Few studies on 4-HDDE revealed its ability to avidly interact covalently with electronegative moieties in macromolecules and to its ability to selectively activate the transcriptional regulator Peroxisome Proliferator-Activated Receptor (PPAR)-β/δ. The research on 4-HDDE has been impeded due to the lack of available pure 4-HDDE and antibodies that recognize 4-HDDE-modified epitopes in proteins. The purpose of this study was to employ an established procedure to synthesize 4-HDDE and use it to create and characterize a monoclonal antibody against 4-HDDE-modified proteins and establish its application for ELISA and immunohistochemical analysis of cells and tissues and further expand lipid peroxidation research. Copyright © 2018 Elsevier Inc. All rights reserved.

The pearl millet mitogen-activated protein kinase PgMPK4 is involved in responses to downy mildew infection and in jasmonic- and salicylic acid-mediated defense.

PubMed

Melvin, Prasad; Prabhu, S Ashok; Veena, Mariswamy; Shailasree, Sekhar; Petersen, Morten; Mundy, John; Shetty, Shekar H; Kini, K Ramachandra

2015-02-01

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor β-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

Fabp1 gene ablation inhibits high-fat diet-induced increase in brain endocannabinoids.

PubMed

Martin, Gregory G; Landrock, Danilo; Chung, Sarah; Dangott, Lawrence J; Seeger, Drew R; Murphy, Eric J; Golovko, Mikhail Y; Kier, Ann B; Schroeder, Friedhelm

2017-01-01

The endocannabinoid system shifts energy balance toward storage and fat accumulation, especially in the context of diet-induced obesity. Relatively little is known about factors outside the central nervous system that may mediate the effect of high-fat diet (HFD) on brain endocannabinoid levels. One candidate is the liver fatty acid binding protein (FABP1), a cytosolic protein highly prevalent in liver, but not detected in brain, which facilitates hepatic clearance of fatty acids. The impact of Fabp1 gene ablation (LKO) on the effect of high-fat diet (HFD) on brain and plasma endocannabinoid levels was examined and data expressed for each parameter as the ratio of high-fat diet/control diet. In male wild-type mice, HFD markedly increased brain N-acylethanolamides, but not 2-monoacylglycerols. LKO blocked these effects of HFD in male mice. In female wild-type mice, HFD slightly decreased or did not alter these endocannabinoids as compared with male wild type. LKO did not block the HFD effects in female mice. The HFD-induced increase in brain arachidonic acid-derived arachidonoylethanolamide in males correlated with increased brain-free and total arachidonic acid. The ability of LKO to block the HFD-induced increase in brain arachidonoylethanolamide correlated with reduced ability of HFD to increase brain-free and total arachidonic acid in males. In females, brain-free and total arachidonic acid levels were much less affected by either HFD or LKO in the context of HFD. These data showed that LKO markedly diminished the impact of HFD on brain endocannabinoid levels, especially in male mice. © 2016 International Society for Neurochemistry.

Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

PubMed Central

Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

2008-01-01

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792

Lipoxygenase-modified Ru-bpy/graphene oxide: Electrochemical biosensor for on-farm monitoring of non-esterified fatty acid.

PubMed

Veerapandian, Murugan; Hunter, Robert; Neethirajan, Suresh

2016-04-15

Elevated concentrations of non-esterified fatty acids (NEFA) in biological fluids are recognized as critical biomarkers for early diagnosis of dairy cow metabolic diseases. Herein, a cost-effective, electrochemically active, and bio-friendly sensor element based on ruthenium bipyridyl complex-modified graphene oxide nanosheets ([Ru(bpy)3](2+)-GO) is proposed as a biosensor platform for NEFA detection. Electrochemical analysis demonstrates that the [Ru(bpy)3](2+)-GO electrodes exhibit superior and durable redox properties compared to the pristine carbon and GO electrodes. Target specificity is accomplished through immobilization of the enzyme, lipoxygenase, which catalyzes the production of redox active species from NEFA. Lipoxygenases retain their catalytic ability upon immobilization and exhibit changes to amperometric signals upon interaction with various concentrations of standard NEFA and serum samples. Our study demonstrates that the [Ru(bpy)3](2+)-GO electrode has the potential to serve as a biosensor platform for developing a field deployable, rapid, and user-friendly detection tool for on-farm monitoring of dairy cow metabolic diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

CALCIUM-INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME-BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN.

PubMed

Paes, Marcia C; Silveira, Alan B; Ventura-Martins, Guilherme; Luciano, Monalisa; Coelho, Marsen G P; Todeschini, Adriane R; Bianconi, M Lucia; Atella, Georgia C; Silva-Neto, Mário A C

2015-10-01

Lipid peroxidation is promoted by the quasi-lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme-binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium-dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation. © 2015 Wiley Periodicals, Inc.

Monocyte migration explains the changes in macrophage arachidonate metabolism during the immune response.

PubMed Central

Tripp, C S; Unanue, E R; Needleman, P

1986-01-01

The profile of arachidonic acid metabolites in resident peritoneal macrophages is distinctly different from the profile of macrophages isolated after an acute bacterial infection. The latter produce decreased prostaglandins E2 and I2 and leukotriene C4 while conserving the synthesis of thromboxane A2. We show here that the initial changes in peritoneal macrophage arachidonate metabolism during the immune response appear to be the result of the large influx of blood monocytes, which have a characteristic metabolism distinct from resident macrophages. We demonstrate that the initial decrease in peritoneal macrophage arachidonate metabolism and the increase in macrophage numbers occur simultaneously after infection with Listeria monocytogenes. Also the macrophage arachidonate metabolism seen at the height of the peritoneal cellular influx is the same as that of purified blood monocytes. Both Listeria peritoneal macrophages and blood monocytes produce equal or greater quantities of thromboxane A2 relative to prostaglandins I2 and E2 or leukotriene C4 whereas resident cells produce 1/10 to 1/25 as much thromboxane A2 compared to the other products. Furthermore, the changes in peritoneal macrophage arachidonate metabolism in response to Listeria infection do not occur if the influx of blood monocytes is stopped by irradiating the mice prior to infection implying that the cellular influx is necessary to see the changes in arachidonate metabolism. Finally, activation of peritoneal macrophages, measured as an increase in Ia expression, occurs 36 hr after the influx of monocytes from the blood and the resultant shift in arachidonate metabolism during Listeria infection. PMID:3099288

Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

PubMed

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

PubMed Central

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders. PMID:24868545

A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.

PubMed

Maccarrone, M; Bari, M; Agrò, A F

1999-02-15

A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low. Copyright 1999 Academic Press.

Effects of two-months balanced diet in metabolically healthy obesity: lipid correlations with gender and BMI-related differences.

PubMed

Rondanelli, Mariangela; Klersy, Chaterine; Perna, Simone; Faliva, Milena Anna; Montorfano, Gigliola; Roderi, Paola; Colombo, Irma; Corsetto, Paola Antonia; Fioravanti, Marisa; Solerte, Sebastiano Bruno; Rizzo, Angela Maria

2015-10-29

Nowadays no researches has been performed on fatty acid profile (FA) and desaturase activity in metabolically healthy obesity (MHO). The aim of this study was to assessed gender and BMI-related difference in FA, estimated desaturase activities and the efficacy on metabolic changes produced by 2-months well-balance diet in MHO subjects. In 103 MHO subjects (30/73 M/F; age:42.2 ± 9.5) FA, estimated desaturase activity, body composition (by DXA), Body Mass Index (BMI), lipid profile, adipokines (leptin, adiponectin, grelin, glucagon-like peptide-1), insulin resistence (by Homestasis metabolic assessment), C-reactive proteine, Atherogenic index of plasma (AIP) and Body Shape Index (ABSI) have been assessed. Gender and BMI related difference have been evaluated and the efficacy produced by 2-months well-balance diet has been considered. At baseline, obese subjects, compared to overweight, show a significantly higher oleic (p <0.050), monounsaturated fatty acids (p <0.040), C18:0 delta-9 desaturase activity (D9D) (p <0.040) and lower linoleic acid (p <0.020), polyunsaturated fatty acids (p <0.020) and n-6 LCPUFA (p <0.010). Concerning gender-related difference, women show a significantly higher arachidonic acid (p <0.001), polyunsaturated fatty acids (p <0.001), n-6 LCPUFA (p <0.002), and lower monounsaturated fatty acids (p <0.001), D6D activity (p <0.030), C18:0 D9D (0.000) and C16:0 D9D (p <0.030). The 2-months diet was associated with a significantly increase in arachidonic acid (p = 0.007), eicosapentaenoic acid (p = 0.030), docosahexaenoic acid (p <0.001), long chain omega 3 polyunsaturated fatty acids (n-3 LCPUFA) (p <0.001), delta-5 desaturase activity (D5D) (p = 0.002), glucagon like peptide-1 (p <0.001) and a significant decrease in palmitoleic acid (p = <0.030), n-6/n-3 LCPUFA (p <0.001), insulin resistance (p = 0.006), leptin (p = 0.006), adiponectin (p <0.001), grelin (p = 0.030), CRP (p = 0.004), BMI (p <0.001) and android fat mass (p <0.001). The balanced diet intervention was effective in improving metabolic indices.

Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamiana for biotechnological application

PubMed Central

2011-01-01

Background Plant lipoxygenases (LOXs) have been proposed to form biologically active compounds both during normal developmental stages such as germination or growth as well as during responses to environmental stress such as wounding or pathogen attack. In our previous study, we found that enzyme activity of endogenous 9-LOX in Nicotiana benthamiana was highly induced by agroinfiltration using a tobacco mosaic virus (TMV) based vector system. Results A LOX gene which is expressed after treatment of the viral vectors was isolated from Nicotiana benthamiana. As the encoded LOX has a high amino acid identity to other 9-LOX proteins, the gene was named as Nb-9-LOX. It was heterologously expressed in yeast cells and its enzymatic activity was characterized. The yeast cells expressed large quantities of stable 9-LOX (0.9 U ml-1 cell cultures) which can oxygenate linoleic acid resulting in high yields (18 μmol ml-1 cell cultures) of hydroperoxy fatty acid. The product specificity of Nb-9-LOX was examined by incubation of linoleic acid and Nb-9-LOX in combination with a 13-hydroperoxide lyase from watermelon (Cl-13-HPL) or a 9/13-hydroperoxide lyase from melon (Cm-9/13-HPL) and by LC-MS analysis. The result showed that Nb-9-LOX possesses both 9- and 13-LOX specificity, with high predominance for the 9-LOX function. The combination of recombinant Nb-9-LOX and recombinant Cm-9/13-HPL produced large amounts of C9-aldehydes (3.3 μmol mg-1 crude protein). The yield of C9-aldehydes from linoleic acid was 64%. Conclusion The yeast expressed Nb-9-LOX can be used to produce C9-aldehydes on a large scale in combination with a HPL gene with 9-HPL function, or to effectively produce 9-hydroxy-10(E),12(Z)-octadecadienoic acid in a biocatalytic process in combination with cysteine as a mild reducing agent. PMID:21450085

Advances in Our Understanding of Oxylipins Derived from Dietary PUFAs12

PubMed Central

Gabbs, Melissa; Leng, Shan; Devassy, Jessay G; Monirujjaman, Md; Aukema, Harold M

2015-01-01

Oxylipins formed from polyunsaturated fatty acids (PUFAs) are the main mediators of PUFA effects in the body. They are formed via cyclooxygenase, lipoxygenase, and cytochrome P450 pathways, resulting in the formation of prostaglandins, thromboxanes, mono-, di-, and tri-hydroxy fatty acids (FAs), epoxy FAs, lipoxins, eoxins, hepoxilins, resolvins, protectins (also called neuroprotectins in the brain), and maresins. In addition to the well-known eicosanoids derived from arachidonic acid, recent developments in lipidomic methodologies have raised awareness of and interest in the large number of oxylipins formed from other PUFAs, including those from the essential FAs and the longer-chain n–3 (ω-3) PUFAs. Oxylipins have essential roles in normal physiology and function, but can also have detrimental effects. Compared with the oxylipins derived from n–3 PUFAs, oxylipins from n–6 PUFAs generally have greater activity and more inflammatory, vasoconstrictory, and proliferative effects, although there are notable exceptions. Because PUFA composition does not necessarily reflect oxylipin composition, comprehensive analysis of the oxylipin profile is necessary to understand the overall physiologic effects of PUFAs mediated through their oxylipins. These analyses should include oxylipins derived from linoleic and α-linolenic acids, because these largely unexplored bioactive oxylipins constitute more than one-half of oxylipins present in tissues. Because collated information on oxylipins formed from different PUFAs is currently unavailable, this review provides a detailed compilation of the main oxylipins formed from PUFAs and describes their functions. Much remains to be elucidated in this emerging field, including the discovery of more oxylipins, and the understanding of the differing biological potencies, kinetics, and isomer-specific activities of these novel PUFA metabolites. PMID:26374175

Secretion of leukotriene C and other arachidonic acid metabolites by macrophages challenged with immunoglobulin E immune complexes

PubMed Central

1982-01-01

Resident mouse peritoneal macrophages release the slow-reacting substance leukotriene C (LTC) on exposure to particulate IgE immune complexes. Because these cells lose their responsiveness to an IgE stimulus after 4 h in culture, maximum release of 20:4 metabolites is observed before this time. However, a similar diminution in 20:4 metabolism was not observed with a zymosan stimulus. Freshly explanted cells are deficient in intracellular glutathione (GSH) (12.4 +/- 0.4 pmol/micrograms cell protein), but GSH increases to a steady state value of 30-35 pmol/micrograms of cell protein between 3 and 9 h of culture. Because GSH is required for the synthesis of LTC and prostaglandin (PG)E2, cultures challenged immediately after explanation have a diminished capacity to synthesize these 20:4 metabolites and release prostacyclin as the major product. By 4-5 h in culture, macrophages form significant amounts of LTC and PGE2. Under optimum conditions of maximum responsiveness to an IgE stimulus and GSH content (after 4 h of culture), macrophages challenged with latex beads coated with IgE immune complexes synthesize 1.0 +/- 0.3 pmol of LTC/microgram cell protein (60 +/- 18 pmol/10(6) cells) in addition to prostacyclin (8.2 +/- 0.8 pmol/micrograms cell protein) and PGE2 (4.7 +/- 1.5 pmol/micrograms cell protein). These amounts are quantitatively similar to the arachidonic acid metabolites produced by macrophages challenged with IgG immune complex-coated latex beads or zymosan. These data demonstrate that macrophages produce large quantities of LTC and other 20:4 metabolites in response to particle-bound IgE and antigen, provided that the appropriate in vitro conditions are met. The macrophage might, therefore, be a major source of slow-reacting substance and other 20:4 metabolites generated during IgE-mediated reactions in vivo. PMID:6759607

Differential response to sulfur nutrition of two common bean genotypes differing in storage protein composition.

PubMed

Pandurangan, Sudhakar; Sandercock, Mark; Beyaert, Ronald; Conn, Kenneth L; Hou, Anfu; Marsolais, Frédéric

2015-01-01

It has been hypothesized that the relatively low concentration of sulfur amino acids in legume seeds might be an ecological adaptation to nutrient poor, marginal soils. SARC1 and SMARC1N-PN1 are genetically related lines of common bean (dry bean, Phaseolus vulgaris) differing in seed storage protein composition. In SMARC1N-PN1, the lack of phaseolin and major lectins is compensated by increased levels of sulfur-rich proteins, resulting in an enhanced concentration of cysteine and methionine, mostly at the expense of the abundant non-protein amino acid, S-methylcysteine. To identify potential effects associated with an increased concentration of sulfur amino acids in the protein pool, the response of the two genotypes to low and high sulfur nutrition was evaluated under controlled conditions. Seed yield was increased by the high sulfate treatment in SMARC1N-PN1. The seed concentrations of sulfur, sulfate, and S-methylcysteine were altered by the sulfur treatment in both genotypes. The concentration of total cysteine and extractible globulins was increased specifically in SMARC1N-PN1. Proteomic analysis identified arcelin-like protein 4, lipoxygenase-3, albumin-2, and alpha amylase inhibitor beta chain as having increased levels under high sulfur conditions. Lipoxygenase-3 accumulation was sensitive to sulfur nutrition only in SMARC1N-PN1. Under field conditions, both SARC1 and SMARC1N-PN1 exhibited a slight increase in yield in response to sulfur treatment, typical for common bean.

The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro

PubMed Central

Carlsson, Johan A.; Wold, Agnes E.; Sandberg, Ann-Sofie; Östman, Sofia M.

2015-01-01

Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c+ CD11b+ and CD11c+ CD11bneg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IAd remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violetlow) and expressed CD69 or CD25, while more were necrotic (7AAD+). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4+ FoxP3+ CTLA-4+, CD4+ FoxP3+ Helios+ or CD4+ FoxP3+ PD-1+, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E2 while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells. PMID:26619195

Persisting eicosanoid pathways in rheumatic diseases.

PubMed

Korotkova, Marina; Jakobsson, Per-Johan

2014-04-01

An unmet clinical need exists for early treatment of rheumatic diseases and improved treatment strategies that can better maintain remission with reduced ongoing subclinical inflammation and bone destruction. Eicosanoids form one of the most complex networks in the body controlling many physiological and pathophysiological processes, including inflammation, autoimmunity and cancer. Persisting eicosanoid pathways are thought to be involved in the development of rheumatic diseases, and targeting this pathway might enable improved treatment strategies. Several enzymes of the arachidonic acid cascade as well as eicosanoid receptors (all part of the eicosanoid pathway) are today well-recognized targets for anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases. In this Review, we outline the evidence supporting pivotal roles of eicosanoid signalling in the pathogenesis of rheumatic diseases and discuss findings from studies in animals and humans. We focus first on rheumatoid arthritis and discuss the upregulation of the cyclooxygenase and lipoxygenase pathways as most data are available in this condition. Research into the roles of eicosanoids in other rheumatic diseases (osteoarthritis, idiopathic inflammatory myopathies, systemic lupus erythematosus and gout) is also progressing rapidly and is discussed. Finally, we summarize the prospects of targeting eicosanoid pathways as anti-inflammatory treatment strategies for patients with rheumatic diseases.

Fatty acid binding proteins and the nervous system: Their impact on mental conditions.

PubMed

Matsumata, Miho; Inada, Hitoshi; Osumi, Noriko

2016-01-01

The brain is rich in lipid and fatty molecules. In this review article, we focus on fatty acid binding proteins (Fabps) that bind to fatty acids such as arachidonic acid and docosahexianoic acid and transfer these lipid ligands within the cytoplasm. Among Fabp family molecules, Fabp3, Fabp5, and Fabp7 are specifically localized in neural stem/progenitor cells, neurons and glia in a cell-type specific manner. Quantitative trait locus analysis has revealed that Fabp7 is related with performance of prepulse inhibition (PPI) that is used as an endophenotype of psychiatric diseases such as schizophrenia. Fabp5 and Fabp7 play important roles on neurogenesis and differentially regulate acoustic startle response and PPI. However, other behavior performances including spatial memory, anxiety-like behavior, and diurnal changes in general activity were not different in mice deficient for Fabp7 or Fabp5. Considering the importance of fatty acids in neurogenesis, we would like to emphasize that lipid nutrition and its dynamism via Fabps play significant roles in mental conditions. This might provide a good example of how nutritional environment can affect psychiatric conditions at the molecular level. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Comparative proteomic analyses reveal the proteome response to short-term drought in Italian ryegrass (Lolium multiflorum)

PubMed Central

Wang, Jianping; Wang, Pengxi; Ma, Xiao; Zhou, Meiliang; Li, Ji; Gang, Nie; Feng, Guangyan; Zhao, Junming

2017-01-01

Drought is a major abiotic stress that impairs growth and productivity of Italian ryegrass. Comparative analysis of drought responsive proteins will provide insight into molecular mechanism in Lolium multiflorum drought tolerance. Using the iTRAQ-based approach, proteomic changes in tolerant and susceptible lines were examined in response to drought condition. A total of 950 differentially accumulated proteins was found to be involved in carbohydrate metabolism, amino acid metabolism, biosynthesis of secondary metabolites, and signal transduction pathway, such as β-D-xylosidase, β-D-glucan glucohydrolase, glycerate dehydrogenase, Cobalamin-independent methionine synthase, glutamine synthetase 1a, Farnesyl pyrophosphate synthase, diacylglycerol, and inositol 1, 4, 5-trisphosphate, which might contributed to enhance drought tolerance or adaption in Lolium multiflorum. Interestingly, the two specific metabolic pathways, arachidonic acid and inositol phosphate metabolism including differentially accumulated proteins, were observed only in the tolerant lines. Cysteine protease cathepsin B, Cysteine proteinase, lipid transfer protein and Aquaporin were observed as drought-regulated proteins participating in hydrolysis and transmembrane transport. The activities of phospholipid hydroperoxide glutathione peroxidase, peroxiredoxin, dehydroascorbate reductase, peroxisomal ascorbate peroxidase and monodehydroascorbate reductase associated with alleviating the accumulation of reactive oxygen species in stress inducing environments. Our results showed that drought-responsive proteins were closely related to metabolic processes including signal transduction, antioxidant defenses, hydrolysis, and transmembrane transport. PMID:28910323

Comparative Physiological and Proteomic Analysis Reveal Distinct Regulation of Peach Skin Quality Traits by Altitude

PubMed Central

Karagiannis, Evangelos; Tanou, Georgia; Samiotaki, Martina; Michailidis, Michail; Diamantidis, Grigorios; Minas, Ioannis S.; Molassiotis, Athanassios

2016-01-01

The role of environment in fruit physiology has been established; however, knowledge regarding the effect of altitude in fruit quality traits is still lacking. Here, skin tissue quality characters were analyzed in peach fruit (cv. June Gold), harvested in 16 orchards located in low (71.5 m mean), or high (495 m mean) altitutes sites. Data indicated that soluble solids concentration and fruit firmness at commercial harvest stage were unaffected by alitute. Peach grown at high-altitude environment displayed higher levels of pigmentation and specific antioxidant-related activity in their skin at the commercial harvest stage. Skin extracts from distinct developmental stages and growing altitudes exhibited different antioxidant ability against DNA strand-scission. The effects of altitude on skin tissue were further studied using a proteomic approach. Protein expression analysis of the mature fruits depicted altered expression of 42 proteins that are mainly involved in the metabolic pathways of defense, primary metabolism, destination/storage and energy. The majority of these proteins were up-regulated at the low-altitude region. High-altitude environment increased the accumulation of several proteins, including chaperone ClpC, chaperone ClpB, pyruvate dehydrogenase E1, TCP domain class transcription factor, and lipoxygenase. We also discuss the altitude-affected protein variations, taking into account their potential role in peach ripening process. This study provides the first characterization of the peach skin proteome and helps to improve our understanding of peach's response to altitude. PMID:27891143

Indirubin core structure of glycogen synthase kinase-3 inhibitors as novel chemotype for intervention with 5-lipoxygenase.

PubMed

Pergola, Carlo; Gaboriaud-Kolar, Nicolas; Jestädt, Nadine; König, Stefanie; Kritsanida, Marina; Schaible, Anja M; Li, Haokun; Garscha, Ulrike; Weinigel, Christina; Barz, Dagmar; Albring, Kai F; Huber, Otmar; Skaltsounis, Alexios L; Werz, Oliver

2014-05-08

The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3Z)-6-bromo-3-[(3E)-3-hydroxyiminoindolin-2-ylidene]indolin-2-one (15) as a potent, direct, and reversible 5-LO inhibitor (IC50 = 1.5 μM), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention.

Basic Aspects of Tumor Cell Fatty Acid-Regulated Signaling and Transcription Factors

PubMed Central

Comba, Andrea; Lin, Yi-Hui; Eynard, Aldo Renato; Valentich, Mirta Ana; Fernandez-Zapico, Martin Ernesto; Pasqualini, Marìa Eugenia

2012-01-01

This article reviews the current knowledge and experimental research about the mechanisms by which fatty acids and their derivatives control specific gene expression involved during carcinogenesis. Changes in dietary fatty acids, specifically the polyunsaturated fatty acids (PUFAs) of the ω-3 and ω-6 families and some derived eicosanoids from lipoxygenases (LOXs), cyclooxygenases (COXs), and cytochrome P-450 (CYP-450), seem to control the activity of transcription factor families involved in cancer cell proliferation or cell death. Their regulation may be carried out either through direct binding to DNA as peroxisome proliferator–activated receptors (PPARs) or via modulation in an indirect manner of signaling pathway molecules (e.g., protein kinase C [PKC]) and other transcription factors (nuclear factor kappa B [NFκB] and sterol regulatory element binding protein [SREBP]). Knowledge of the mechanisms by which fatty acids control specific gene expression may identify important risk factors for cancer, and provide insight into the development of new therapeutic strategies for a better management of whole-body lipid metabolism. PMID:22048864

Basic aspects of tumor cell fatty acid-regulated signaling and transcription factors.

PubMed

Comba, Andrea; Lin, Yi-Hui; Eynard, Aldo Renato; Valentich, Mirta Ana; Fernandez-Zapico, Martín Ernesto; Pasqualini, Marìa Eugenia

2011-12-01

This article reviews the current knowledge and experimental research about the mechanisms by which fatty acids and their derivatives control specific gene expression involved during carcinogenesis. Changes in dietary fatty acids, specifically the polyunsaturated fatty acids of the ω-3 and ω-6 families and some derived eicosanoids from lipoxygenases, cyclooxygenases, and cytochrome P-450, seem to control the activity of transcription factor families involved in cancer cell proliferation or cell death. Their regulation may be carried out either through direct binding to DNA as peroxisome proliferator-activated receptors or via modulation in an indirect manner of signaling pathway molecules (e.g., protein kinase C) and other transcription factors (nuclear factor kappa B and sterol regulatory element binding protein). Knowledge of the mechanisms by which fatty acids control specific gene expression may identify important risk factors for cancer and provide insight into the development of new therapeutic strategies for a better management of whole body lipid metabolism.

BIOSYNTHESIS AND ACTION OF JASMONATES IN PLANTS.

PubMed

Creelman, Robert A.; Mullet, John E.

1997-06-01

Jasmonic acid and its derivatives can modulate aspects of fruit ripening, production of viable pollen, root growth, tendril coiling, and plant resistance to insects and pathogens. Jasmonate activates genes involved in pathogen and insect resistance, and genes encoding vegetative storage proteins, but represses genes encoding proteins involved in photosynthesis. Jasmonic acid is derived from linolenic acid, and most of the enzymes in the biosynthetic pathway have been extensively characterized. Modulation of lipoxygenase and allene oxide synthase gene expression in transgenic plants raises new questions about the compartmentation of the biosynthetic pathway and its regulation. The activation of jasmonic acid biosynthesis by cell wall elicitors, the peptide systemin, and other compounds will be related to the function of jasmonates in plants. Jasmonate modulates gene expression at the level of translation, RNA processing, and transcription. Promoter elements that mediate responses to jasmonate have been isolated. This review covers recent advances in our understanding of how jasmonate biosynthesis is regulated and relates this information to knowledge of jasmonate modulated gene expression.

Computational Approaches for Designing Protein/Inhibitor Complexes and Membrane Protein Variants

NASA Astrophysics Data System (ADS)

Vijayendran, Krishna Gajan

Drug discovery of small-molecule protein inhibitors is a vast enterprise that involves several scientific disciplines (i.e. genomics, cell biology, x-ray crystallography, chemistry, computer science, statistics), with each discipline focusing on a particular aspect of the process. In this thesis, I use computational and experimental approaches to explore the most fundamental aspect of drug discovery: the molecular interactions of small-molecules inhibitors with proteins. In Part I (Chapters I and II), I describe how computational docking approaches can be used to identify structurally diverse molecules that can inhibit multiple protein targets in the brain. I illustrate this approach using the examples of microtubule-stabilizing agents and inhibitors of cyclooxygenase(COX)-I and 5-lipoxygenase (5-LOX). In Part II (Chapters III and IV), I focus on membrane proteins, which are notoriously difficult to work with due to their low natural abundances, low yields for heterologous over expression, and propensities toward aggregation. I describe a general approach for designing water-soluble variants of membrane proteins, for the purpose of developing cell-free, label-free, detergent-free, solution-phase studies of protein structure and small-molecule binding. I illustrate this approach through the design of a water-soluble variant of the membrane protein Smoothened, wsSMO. This wsSMO stands to serve as a first-step towards developing membrane protein analogs of this important signaling protein and drug target.

Bacopa monniera (L.) wettst inhibits type II collagen-induced arthritis in rats.

PubMed

Viji, V; Kavitha, S K; Helen, A

2010-09-01

Bacopa monniera (L.) Wettst is an Ayurvedic herb with antirheumatic potential. This study investigated the therapeutic efficacy of Bacopa monniera in treating rheumatoid arthritis using a type II collagen-induced arthritis rat model. Arthritis was induced in male Wistar rats by immunization with bovine type II collagen in complete Freund's adjuvant. Bacopa monniera extract (BME) was administered after the development of arthritis from day 14 onwards. The total duration of experiment was 60 days. Paw swelling, arthritic index, inflammatory mediators such as cyclooxygenase, lipoxygenase, myeloperoxidase and serum anti-collagen IgG and IgM levels were analysed in control and experimental rats. Arthritic induction significantly increased paw edema and other classical signs of arthritis coupled to upregulation of inflammatory mediators such as cyclooxygenase, lipoxygenase, neutrophil infiltration and increased anti-collagen IgM and IgG levels in serum. BME significantly inhibited the footpad swelling and arthritic symptoms. BME was effective in inhibiting cyclooxygenase and lipoxygenase activities in arthritic rats. Decreased neutrophil infiltration was evident from decreased myeloperoxidase activity and histopathological data where an improvement in joint architecture was also observed. Serum anti-collagen IgM and IgG levels were consistently decreased. Thus the study demonstrates the potential antiarthritic effect of Bacopa monniera for treating arthritis which might confer its antirheumatic activity. Copyright 2010 John Wiley & Sons, Ltd.

Inhibition of lipoxygenases and cyclooxygenase-2 enzymes by extracts isolated from Bacopa monniera (L.) Wettst.

PubMed

Viji, V; Helen, A

2008-07-23

Bacopa monniera Linn is described in the Ayurvedic Materia Medica, as a therapeutically useful herb for the treatment of inflammation. In the current study, we investigated the anti-inflammatory activity of methanolic extract of Bacopa monniera (BME). For some experiments EtOAc and bacoside fractions were prepared from BME. The effect of these extracts in modulating key mediators of inflammation was evaluated. Carrageenan-induced rat paw edema, rat mononuclear cells and human whole blood assay were employed as in vivo and in vitro models. In carrageenan-induced rat paw edema, BME brought about 82% edema inhibition at a dose of 100mg/kg i.p. when compared to indomethacin (INDO) (3mg/kg) that showed 70% edema inhibition. BME also significantly inhibited 5-lipoxygenase (5-LOX), 15-LOX and cyclooxygenase-2 (COX-2) activities in rat monocytes in vivo. Among the fractions tested in vitro, EtOAc fraction possessed significant 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with IC(50) value of 30 microg/ml compared to butylated hydroxyl toluene (IC(50) = 13 microg/ml). This fraction also exerted significant hydroxyl radical scavenging activity with IC(50) value of 25 microg/ml in comparison with quercetin (IC(50) = 5 microg/ml). Inhibitory effects of EtOAc and bacoside fractions on LOX and COX activities in Ca-A23187 stimulated rat mononuclear cells were also assessed. 5-LOX IC(50) values were 25 microg/ml for EtOAc, 68 microg/ml for bacosides and 2 microg/ml for nordihydroguaiaretic acid (NDGA) where as COX-2 IC(50) values were 1.32 microg/ml for EtOAc, 1.19 microg/ml for bacoside fraction and 0.23 microg/ml for indomethacin. EtOAc and bacoside fractions also brought about significant decrease in TNF-alpha release ex vivo. Bacopa monniera possesses anti-inflammatory activity through inhibition of COX and LOX and downregulation of TNF-alpha.

On the Role of Molecular Oxygen in Lipoxygenase Activation

PubMed Central

Zheng, Yuxiang; Brash, Alan R.

2010-01-01

The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O2 concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O2 in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O2 with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O2 within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity. PMID:20923767

Omega-6 polyunsaturated fatty acids, serum zinc, delta-5- and delta-6-desaturase activities and incident metabolic syndrome.

PubMed

Yary, T; Voutilainen, S; Tuomainen, T-P; Ruusunen, A; Nurmi, T; Virtanen, J K

2017-08-01

The associations of n-6 polyunsaturated fatty acids (PUFA) with metabolic syndrome have been poorly explored. We investigated the associations of the serum n-6 PUFA and the activities of enzymes involved in the PUFA metabolism, delta-5-desaturase (D5D) and delta-6-desaturase (D6D) with risk of incident metabolic syndrome. We also investigated whether zinc, a cofactor for these enzymes, modifies these associations. A prospective follow-up study was conducted on 661 men who were aged 42-60 years old at baseline in 1984-1989 and who were re-examined in 1998-2001. Men in the highest versus the lowest serum total omega-6 PUFA tertile had a 70% lower multivariate-adjusted risk of incident metabolic syndrome [odds ratio (OR) = 0.30; 95% confidence interval (CI) = 0.18-0.51, P trend < 0.001]. Inverse associations were also observed for linoleic acid, arachidonic acid and D5D activity. By contrast, men in the highest tertile of D6D activity had an 84% higher risk (OR = 1.84; 95% CI = 1.15-2.94, P trend = 0.008). Similar associations were observed with many of the metabolic syndrome components at the re-examinations. Most associations were attenuated after adjustment for body mass index. Finally, the associations of D6D and LA were stronger among those with a higher serum zinc concentration. Higher serum total n-6 PUFA, linoleic acid and arachidonic acid concentrations and D5D activity were associated with a lower risk of developing metabolic syndrome and higher D6D activity was associated with a higher risk. The role of zinc also needs to be investigated in other populations. © 2016 The British Dietetic Association Ltd.

Activity-Based Protein Profiling of Organophosphorus and Thiocarbamate Pesticides Reveals Multiple Serine Hydrolase Targets in Mouse Brain

PubMed Central

NOMURA, DANIEL K.; CASIDA, JOHN E.

2010-01-01

Organophosphorus (OP) and thiocarbamate (TC) agrochemicals are used worldwide as insecticides, herbicides, and fungicides, but their safety assessment in terms of potential off-targets remains incomplete. In this study, we used a chemoproteomic platform, termed activity-based protein profiling, to broadly define serine hydrolase targets in mouse brain of a panel of 29 OP and TC pesticides. Among the secondary targets identified, enzymes involved in degradation of endocannabinoid signaling lipids, monoacylglycerol lipase and fatty acid amide hydrolase, were inhibited by several OP and TC pesticides. Blockade of these two enzymes led to elevations in brain endocannabinoid levels and dysregulated brain arachidonate metabolism. Other secondary targets include enzymes thought to also play important roles in the nervous system and unannotated proteins. This study reveals a multitude of secondary targets for OP and TC pesticides and underscores the utility of chemoproteomic platforms in gaining insights into biochemical pathways that are perturbed by these toxicants. PMID:21341672

The Bacteroid Periplasm in Soybean Nodules Is an Interkingdom Symbiotic Space.

PubMed

Strodtman, Kent N; Stevenson, Severin E; Waters, James K; Mawhinney, Thomas P; Thelen, Jay J; Polacco, Joseph C; Emerich, David W

2017-12-01

The functional role of the periplasm of nitrogen-fixing bacteroids has not been determined. Proteins were isolated from the periplasm and cytoplasm of Bradyrhizobium diazoefficiens bacteroids and were analyzed using liquid chromatography tandem mass spectrometry proteomics. Identification of bacteroid periplasmic proteins was aided by periplasm prediction programs. Approximately 40% of all the proteins identified as periplasmic in the B. diazoefficiens genome were found expressed in the bacteroid form of the bacteria, indicating the periplasm is a metabolically active symbiotic space. The bacteroid periplasm possesses many fatty acid metabolic enzymes, which was in contrast to the bacteroid cytoplasm. Amino acid analysis of the periplasm revealed an abundance of phosphoserine, phosphoethanolamine, and glycine, which are metabolites of phospholipid metabolism. These results suggest the periplasm is a unique space and not a continuum with the peribacteroid space. A number of plant proteins were found in the periplasm fraction, which suggested contamination. However, antibodies to two of the identified plant proteins, histone H2A and lipoxygenase, yielded immunogold labeling that demonstrated the plant proteins were specifically targeted to the bacteroids. This suggests that the periplasm is an interkingdom symbiotic space containing proteins from both the bacteroid and the plant.

Epoxy Fatty Acids and Inhibition of the Soluble Epoxide Hydrolase Selectively Modulate GABA Mediated Neurotransmission to Delay Onset of Seizures

PubMed Central

Inceoglu, Bora; Zolkowska, Dorota; Yoo, Hyun Ju; Wagner, Karen M.; Yang, Jun; Hackett, Edward; Hwang, Sung Hee; Lee, Kin Sing Stephen; Rogawski, Michael A.; Morisseau, Christophe; Hammock, Bruce D.

2013-01-01

In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders. PMID:24349022

Cyclic strain is a weak inducer of prostacyclin synthase expression in bovine aortic endothelial cells

NASA Technical Reports Server (NTRS)

Segurola, R. J. Jr; Oluwole, B.; Mills, I.; Yokoyama, C.; Tanabe, T.; Kito, H.; Nakajima, N.; Sumpio, B. E.

1997-01-01

Recent studies indicate that hemodynamic forces such as cyclic strain and shear stress can increase prostacyclin (PGI2) secretion by endothelial cells (EC) but the effect of these forces on prostacyclin synthase (PGIS) gene expression remains unclear and is the focus of this study. Bovine aortic EC were seeded onto type I collagen coated flexible membranes and grown to confluence. The membranes and attached EC were subjected to 10% average strain at 60 cpm (0.5 sec deformation alternating with 0.5 sec relaxation) for up to 5 days. PGIS gene expression was determined by Northern blot analysis and protein level by Western blot analysis. The effect of cyclic strain on the PGIS promoter was determined by the transfection of a 1-kb human PGIS gene promoter construct coupled to a luciferase reporter gene into EC, followed by determination of luciferase activity. PGIS gene expression increased 1.7-fold in EC subjected to cyclic strain for 24 hr. Likewise, EC transfected with a pGL3B-PGIS (-1070/-10) construct showed an approximate 1.3-fold elevation in luciferase activity in EC subjected to cyclic strain for 3, 4, 8, and 12 hr. The weak stimulation of PGIS gene expression by cyclic strain was reflected in an inability to detect alterations in PGIS protein levels in EC subjected to cyclic strain for as long as 5 days. These data suggest that strain-induced stimulation of PGIS gene expression plays only a minor role in the ability of cyclic strain to stimulate PGI2 release in EC. These findings coupled with our earlier demonstration of a requisite addition of exogenous arachidonate in order to observe strain-induced PGI2 release, implicates a mechanism that more likely involves strain-induced stimulation of PGIS activity.

Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology.

PubMed

Harizi, Hedi; Corcuff, Jean-Benoît; Gualde, Norbert

2008-10-01

Arachidonic acid (AA)-derived eicosanoids belong to a complex family of lipid mediators that regulate a wide variety of physiological responses and pathological processes. They are produced by various cell types through distinct enzymatic pathways and act on target cells via specific G-protein-coupled receptors. Although originally recognized for their capacity to elicit biological responses such as vascular homeostasis, protection of the gastric mucosa and platelet aggregation, eicosanoids are now understood to regulate immunopathological processes ranging from inflammatory responses to chronic tissue remodelling, cancer, asthma, rheumatoid arthritis and autoimmune disorders. Here, we review the major properties of eicosanoids and their expanding roles in biology and medicine.

Butyrylcholinesterase, lipoxygenase inhibiting and antifungal alkaloids from Isatis tinctoria.

PubMed

Ahmad, Ijaz; Fatima, Itrat

2008-06-01

Phytochemical investigations on the alkaloidal fraction of the whole plant of the Isatis tinctoria led to the isolation of the alkaloids 1-6. Compounds 3, 2 were found to be potent butyrylcholinesterase and lipoxygenase enzymes inhibitors in a concentration-dependent manner with the IC(50) values 16.3 +/- 0.06 and 19.7 +/- 0.03 microM against BChE and 30.6 +/- 0.02 and 33.7 +/- 0.05 microM against LOX, respectively. The compounds (1-6) showed significant antifungal activity against Trichophyton schoen leinii, Aspergillus niger, Candida albicans, Trichophyton simii, and Macrophomina phaseolina.

Attenuation of cysteamine-induced duodenal ulcer with Cochinchina momordica seed extract through inhibiting cytoplasmic phospholipase A2/5-lipoxygenase and activating γ-glutamylcysteine synthetase.

PubMed

Choi, Ki-Seok; Kim, Eun-Hee; Hong, Hua; Ock, Chan Young; Lee, Jeong Sang; Kim, Joo-Hyun; Hahm, Ki-Baik

2012-04-01

Cysteamine is a reducing aminothiol used for inducing duodenal ulcer through mechanisms of oxidative stress related to thiol-derived H(2)O(2) reaction. Cochinchina momordica saponins have been suggested to be protective against various gastric diseases based on their cytoprotective and anti-inflammatory mechanisms. This study was aimed to document the preventive effects of Cochinchina momordica seed extract against cysteamine-induced duodenal ulcer as well as the elucidation of its pharmacological mechanisms. Cochinchina momordica seed extract (50, 100, 200 mg/kg) was administrated intragastrically before cysteamine administration, after which the incidence of the duodenal ulcer, ulcer size, serum gastrin level, and the ratio of reduced glutathione (GSH)/oxidized glutathione disulfide (GSSG) as well as biochemical and molecular measurements of cytoplasmic phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), 5-lipoxygenase and the expression of proinflammatory genes including IL-1β, IL-6, COX-2 were measured in rat model. Additional experiments of electron spin resonance measurement and the changes of glutathione were performed. Cochinchina momordica seed extract effectively prevented cysteamine-induced duodenal ulcer in a dose-dependent manner as reflected with significant decreases in either duodenal ulcerogenesis or perforation accompanied with significantly decreased in serum gastrin in addition to inflammatory mediators including cPLA(2), COX-2, and 5-lipoxygenase. Cochinchina momordica seed extract induced the expression of γ-glutamylcysteine synthetase (γ-GCS)-related glutathione synthesis as well as significantly reduced the expression of cPLA(2). Cochinchina momordica seed extract preserved reduced glutathione through increased expressions of γ-GCS. Cochinchina momordica seed extracts exerted significantly protective effect against cysteamine-induced duodenal ulcer by either cPLA2 inhibition or glutathione preservation. © 2012 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.

Bidirectional Control of Blood Flow by Astrocytes: A Role for Tissue Oxygen and Other Metabolic Factors.

PubMed

Gordon, Grant R J; Howarth, Clare; MacVicar, Brian A

2016-01-01

Altering cerebral blood flow through the control of cerebral vessel diameter is critical so that the delivery of molecules important for proper brain functioning is matched to the activity level of neurons. Although the close relationship of brain glia known as astrocytes with cerebral blood vessels has long been recognized, it is only recently that these cells have been demonstrated to translate information on the activity level and energy demands of neurons to the vasculature. In particular, astrocytes respond to elevations in extracellular glutamate as a consequence of synaptic transmission through the activation of group 1 metabotropic glutamate receptors. These Gq-protein coupled receptors elevate intracellular calcium via IP3 signaling. A close examination of astrocyte endfeet calcium signals has been shown to cause either vasoconstriction or vasodilation. Common to both vasomotor responses is the generation of arachidonic acid in astrocytes by calcium sensitive phospholipase A2. Vasoconstriction ensues from the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid, while vasodilation ensues from the production of epoxyeicosatrienoic acids or prostaglandins. Factors that determine whether constrictor or dilatory pathways predominate include brain oxygen, lactate, adenosine as well as nitric oxide. Changing the oxygen level itself leads to many downstream changes that facilitate the switch from vasoconstriction at high oxygen to vasodilation at low oxygen. These findings highlight the importance of astrocytes as sensors of neural activity and metabolism to coordinate the delivery of essential nutrients via the blood to the working cells.

Identification of a novel SPLIT-HULL (SPH) gene associated with hull splitting in rice (Oryza sativa L.).

PubMed

Lee, Gileung; Lee, Kang-Ie; Lee, Yunjoo; Kim, Backki; Lee, Dongryung; Seo, Jeonghwan; Jang, Su; Chin, Joong Hyoun; Koh, Hee-Jong

2018-07-01

The split-hull phenotype caused by reduced lemma width and low lignin content is under control of SPH encoding a type-2 13-lipoxygenase and contributes to high dehulling efficiency. Rice hulls consist of two bract-like structures, the lemma and palea. The hull is an important organ that helps to protect seeds from environmental stress, determines seed shape, and ensures grain filling. Achieving optimal hull size and morphology is beneficial for seed development. We characterized the split-hull (sph) mutant in rice, which exhibits hull splitting in the interlocking part between lemma and palea and/or the folded part of the lemma during the grain filling stage. Morphological and chemical analysis revealed that reduction in the width of the lemma and lignin content of the hull in the sph mutant might be the cause of hull splitting. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive gene, sph (Os04g0447100), which encodes a type-2 13-lipoxygenase. SPH knockout and knockdown transgenic plants displayed the same split-hull phenotype as in the mutant. The sph mutant showed significantly higher linoleic and linolenic acid (substrates of lipoxygenase) contents in spikelets compared to the wild type. It is probably due to the genetic defect of SPH and subsequent decrease in lipoxygenase activity. In dehulling experiment, the sph mutant showed high dehulling efficiency even by a weak tearing force in a dehulling machine. Collectively, the results provide a basis for understanding of the functional role of lipoxygenase in structure and maintenance of hulls, and would facilitate breeding of easy-dehulling rice.

Synthesis and Structure–Activity Relationship Studies of 4-((2-Hydroxy-3-methoxybenzyl)amino)benzenesulfonamide Derivatives as Potent and Selective Inhibitors of 12-Lipoxygenase

PubMed Central

Luci, Diane K.; Jameson, J. Brian; Yasgar, Adam; Diaz, Giovanni; Joshi, Netra; Kantz, Auric; Markham, Kate; Perry, Steve; Kuhn, Norine; Yeung, Jennifer; Kerns, Edward H.; Schultz, Lena; Holinstat, Michael; Nadler, Jerry L.; Taylor-Fishwick, David A.; Jadhav, Ajit; Simeonov, Anton; Holman, Theodore R.; Maloney, David J.

2014-01-01

Human lipoxygenases (LOXs) are a family of iron-containing enzymes which catalyze the oxidation of polyunsaturated fatty acids to provide the corresponding bioactive hydroxyeicosatetraenoic acid (HETE) metabolites. These eicosanoid signaling molecules are involved in a number of physiologic responses such as platelet aggregation, inflammation, and cell proliferation. Our group has taken a particular interest in platelet-type 12-(S)-LOX (12-LOX) because of its demonstrated role in skin diseases, diabetes, platelet hemostasis, thrombosis, and cancer. Herein, we report the identification and medicinal chemistry optimization of a 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide-based scaffold. Top compounds, exemplified by 35 and 36, display nM potency against 12-LOX, excellent selectivity over related lipoxygenases and cyclooxygenases, and possess favorable ADME properties. In addition, both compounds inhibit PAR-4 induced aggregation and calcium mobilization in human platelets and reduce 12-HETE in β-cells. PMID:24393039

Ginsenoside Rb1 inhibits autophagy through regulation of Rho/ROCK and PI3K/mTOR pathways in a pressure-overload heart failure rat model.

PubMed

Yang, Tianrui; Miao, Yunbo; Zhang, Tong; Mu, Ninghui; Ruan, Libo; Duan, Jinlan; Zhu, Ying; Zhang, Rongping

2018-06-01

This study was designed to explore the relationship between ginsenoside Rb1 (Grb1) and high-load heart failure (HF) in rats. The parameters of cardiac systolic function (left ventricular posterior wall thickness (LVPWT), left ventricular internal diastolic diameter (LVID), fraction shortening (FS) and mitral valves (MVs)) of rat hearts in each group were inspected by echocardiogram. The expressions of rat myocardial contractile proteins, autophagy-related proteins and the activation of Rho/ROCK and PI3K/mTOR pathways were detected by Western blot. LVPWT, FS, MVs and the expression of myocardial contractile proteins α-MHC, apoptosis-related proteins Bcl-2 and signalling pathway involved proteins pAkt and mTOR were significantly reduced in the HF, HF+5 mg/kg Grb1 (HF+Grb1-5) and HF+Grb1+arachidonic acid (AA) groups with LVID, β-MHC, cell apoptosis, cell autophagy and Rho/ROCK significantly increased compared with the control group, of which the tendency was contrary to the HF+20 mg/kg Grb1 (HF+Grb1-20) group compared with the HF group (P < 0.05). In the HF+Grb1+AA group, there was no significant change in the above indexes compared with the HF group. The results indicated that Grb1 can exert anti-HF function by inhibiting cardiomyocyte autophagy of rats through regulation of Rho/ROCK and PI3K/mTOR pathways. © 2018 Royal Pharmaceutical Society.

A simple in vitro test to evaluate biocompatibility of dialysis membranes.

PubMed

Vincent, D; Charmes, J P; Benzakour, M; Gualde, N; Rigaud, M; Leroux-Robert, C

1989-01-01

As arachidonic acid metabolites are implicated in hypersensitivity reactions, we measured arachidonic acid metabolites of dialysed patient's granulocytes, preincubated with different dialysis membranes. Results indicate that cuprophan and cellulose acetate membranes partially inhibit in vitro production of 15-HETE and 5-HETE, whereas polyacrylonitrile membrane does not. This suggests that polyacrylonitrile is a more biocompatible membrane.

Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity.

PubMed

Chalbi, Najla; Martínez-Ballesta, Ma Carmen; Youssef, Nabil Ben; Carvajal, Micaela

2015-03-01

Changes in plasma membrane lipids, such as sterols and fatty acids, have been observed as a result of salt stress. These alterations, together with modification of the plasma membrane protein profile, confer changes in the physical properties of the membrane to be taken into account for biotechnological uses. In our experiments, the relationship between lipids and proteins in three different Brassicaceae species differing in salinity tolerance (Brassica oleracea, B. napus and Cakile maritima) and the final plasma membrane stability were studied. The observed changes in the sterol (mainly an increase in sitosterol) and fatty acid composition (increase in RUFA) in each species led to physical adaptation of the plasma membrane to salt stress. The in vitro vesicles stability was higher in the less tolerant (B. oleracea) plants together with low lipoxygenase activity. These results indicate that the proteins/lipids ratio and lipid composition is an important aspect to take into account for the use of natural vesicles in plant biotechnology. Copyright © 2014 Elsevier GmbH. All rights reserved.

Involvement of PLA2, COX and LOX in Rhinella arenarum oocyte maturation.

PubMed

Ortiz, Maria Eugenia; Bühler, Marta Inés; Zelarayán, Liliana Isabel

2014-11-01

In Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism - phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 μM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation.

Effect of Different Elicitors and Preharvest Day Application on the Content of Phytochemicals and Antioxidant Activity of Butterhead Lettuce (Lactuca sativa var. capitata) Produced under Hydroponic Conditions.

PubMed

Moreno-Escamilla, Jesús Omar; Alvarez-Parrilla, Emilio; de la Rosa, Laura A; Núñez-Gastélum, José Alberto; González-Aguilar, Gustavo A; Rodrigo-García, Joaquín

2017-07-05

The effect of four elicitors on phytochemical content in two varieties of lettuce was evaluated. The best preharvest day for application of each elicitor was chosen. Solutions of arachidonic acid (AA), salicylic acid (SA), methyl jasmonate (MJ), and Harpin protein (HP) were applied by foliar aspersion on lettuce leaves while cultivating under hydroponic conditions. Application of elicitors was done at 15, 7, 5, 3, or 1 day before harvest. Green lettuce showed the highest increase in phytochemical content when elicitors (AA, SA, and HP) were applied on day 7 before harvest. Similarly, antioxidant activity rose in all treatments on day 7. In red lettuce, the highest content of bioactive molecules occurred in samples treated on day 15. AA, SA, and HP were the elicitors with the highest effect on phytochemical content for both varieties, mainly on polyphenol content. Antioxidant activity also increased in response to elicitation. HPLC-MS showed an increase in the content of phenolic acids in green and red lettuce, especially after elicitation with SA, suggesting activation of the caffeic acid pathway due to elicitation.

Metabolism of Linoleic Acid by Barley Lipoxygenase and Hydroperoxide Isomerase 1

PubMed Central

Lulai, Edward C.; Baker, Charles W.; Zimmerman, Don C.

1981-01-01

The oxidation of linoleic acid in incubation mixtures containing extracts of barley lipoxygenase and hydroperoxide isomerase, and the production of these enzymes in quiescent and germinated barley, were investigated. The ratio of 9-hydroperoxylinoleic acid to 13-hydroperoxylinoleic acid was higher for incubation mixtures containing extracts of quiescent barley than for mixtures containing extracts of germinated barley; production of 13-hydroperoxylinoleic acid from germinated barley exceeded that of quiescent barley. Hydroperoxy metabolites of linoleic acid were converted to 9-hydroxy-10-oxo-cis-12-octadecenoic acid, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, and small amounts of 11-hydroxy-12,13-epoxy-cis-9-octadecenoic acid and 11-hydroxy-9,10-epoxy-cis-13-octadecenoic acid whether quiescent or germinated barley was the enzyme source; a fifth product, 13-hydroxy-12-oxo-cis-9-octadecenoic acid was formed only when germinated barley was the enzyme source. Lipoxygenase was readily extracted by buffer, but hydroperoxide isomerase was bound in a catalytically active state to the insoluble barley grist and was efficiently extracted only when Triton X-100 was included in the extraction buffer. Hydroperoxide isomerase was localized in the embryo of quiescent barley, but it was present in the embryo, acrospire, and in small but concentrated amounts in the rootlet of germinating barley. The levels of both lipoxygenase and hydroperoxide isomerase increased through the thirteenth day of germination. Images PMID:16662032

Natural products isolated from Tetragonula carbonaria cerumen modulate free radical-scavenging and 5-lipoxygenase activities in vitro.

PubMed

Hamilton, Karina D; Brooks, Peter R; Ogbourne, Steven M; Russell, Fraser D

2017-04-26

Propolis and cerumen are plant-derived products found in honeybees and stingless bees, respectively. Although propolis is an ancient folk medicine, the bioactivities of cerumen obtained from Australian native stingless bees (Tetragonula carbonaria) have not been widely studied. Therefore, we investigated selected anti-oxidant and anti-inflammatory properties of T. carbonaria cerumen. A methanolic extract was prepared from the combined cerumen of 40 T. carbonaria hives, and HPLC was used to screen for chemical constituents that scavenged 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH). The ability of cerumen extracts to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and to interfere with leukotriene B 4 (LTB 4 ) production in ionomycin-stimulated human neutrophils was also examined. The extract dose-dependently scavenged DPPH (EC 50  = 27.0 ± 2.3 μg/mL); and inhibited the 5-lipoxygenase (5-LOX)-mediated oxidation of linoleic acid (IC 50  = 67.1 ± 9.6 μg/mL). Pre-treatment of isolated human neutrophils with the methanolic cerumen extract additionally inhibited the ionomycin-stimulated production of LTB 4 from these cells (IC 50  = 13.3 ± 5.3 μg/mL). Following multi-solvent extraction, the free radical-scavenging and 5-LOX-inhibiting activities of the initial cerumen extract were retained in a polar, methanol-water extract, which contained gallic acid and a range of flavonone and phenolic natural products. The findings identify free radical scavenging activity, and interference by extracts of T. carbonaria cerumen in 5-LOX-LTB 4 signaling. Further investigation is needed to determine whether the extracts will provide therapeutic benefits for medical conditions in which oxidative stress and inflammation are implicated, including cardiovascular disease and impaired wound healing.

ARACHIDONIC ACID PRODUCTS IN AIRWAY NOCICEPTOR ACTIVATION DURING ACUTE LUNG INJURY

PubMed Central

Lin, Shuxin; Li, Huafeng; Xu, Ling; Moldoveanu, Bogdan; Guardiola, Juan; Yu, Jerry

2011-01-01

We have reported that airway nociceptors [C fiber receptors (CFRs) and high threshold A-delta fiber receptors (HTARs)] are activated during oleic acid (OA) induced acute lung injury. In the current studies, we tested the hypothesis that this nociceptor activation is mediated by arachidonic acid products. In anesthetized, open chest, and mechanically ventilated rabbits, we examined the response of the nociceptors to intravenous injection of OA before and after blocking the cyclo-oxygenase pathways by indomethacin. Pre-treatment with indomethacin (20 mg/kg) decreased the background activities of both CFRs (from 0.48±0.12 to 0.25±0.08, n=7, p<0.05) and HTARs (from 0.54±0.14 to 0.23±0.08, n=10, p<0.01). It also blocked the nociceptors’ response to OA. Similarly, pre-treatment with thromboxane synthase inhibitor (ketoconazole) also blocked the nociceptor response to OA. In addition, local microinjection or intravenous injection of a thromboxane mimetic stimulated CFRs and HTARs. The current results clearly indicate that arachidonic acid metabolites mediate airway nociceptor activation during OA-induced acute lung injury and suggest that thromboxane may be a key mediator. PMID:21622966

Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators.

PubMed Central

Lau, L F; Pumiglia, K; Côté, Y P; Feinstein, M B

1994-01-01

Synthetic thrombin receptor peptides (TRPs), comprising the first 6-14 amino acids of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity, were reported to activate platelets equally with thrombin itself and are considered to be full agonists [Vu et al. (1991) Cell 64, 1057-1068]. Using aspirin plus ADP-scavengers or the ADP-receptor antagonist adenosine 5'-[alpha-thio]triphosphate to prevent the secondary effects of the potent agonists that are normally released from stimulated platelets (i.e. ADP and thromboxane A2), we assessed the direct actions of thrombin and TRPs (i.e. TRP42-47 and TRP42-55). Compared with thrombin, under these conditions, TRPs: (1) failed to aggregate platelets completely; (2) produced less activation of glycoprotein (GP)IIb-IIIa; (3) did not cause association of GPIIb and pp60c-src with the cytoskeleton; and (4) caused less alpha-granule secretion, phosphorylation of cytoplasmic phospholipase A2, arachidonic acid release and phosphatidyl inositol (PtdOH) production. Furthermore, TRPs induced transient increases in protein phosphorylation mediated by protein kinase C and protein tyrosine phosphorylation, whereas these same responses to thrombin were greater and more sustained. Hirudin added after thrombin accelerated protein dephosphorylation, thereby mimicking the rate of spontaneous dephosphorylation seen after stimulation by TRPs. Platelets totally desensitized to very high concentrations of TRPs, by prior exposure to maximally effective concentrations of the peptides, remained responsive to alpha- and gamma-thrombins. Thrombin-stimulated PtdOH production in permeabilized platelets desensitized to TRPs was abolished by guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), as in normal platelets. These results are discussed in terms of the allosteric Ternary Complex Model for G-protein linked receptors [Samama et al. (1993) J. Biol. Chem. 268, 4625-4636]. We conclude that: (1) TRPs are partial agonists for the thrombin receptor and produce incomplete receptor desensitization in keeping with their lower intrinsic activity; (2) thrombin's effects in platelets, even in TRP-desensitized platelets, are entirely mediated through the recently cloned G-protein linked receptor, and (3) thrombin's ability to produce sustained signals, compared with TRPs, may require the continued progressive proteolytic activation of naive thrombin receptors. Images Figure 3 PMID:7526841

Flavocoxid, a dual inhibitor of COX-2 and 5-LOX of natural origin, attenuates the inflammatory response and protects mice from sepsis

PubMed Central

2012-01-01

Introduction Cecal ligation and puncture (CLP) is an inflammatory condition that leads to multisystemic organ failure. Flavocoxid, a dual inhibitor of cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX), has been shown in vitro to possess antiinflammatory activity in lipopolysaccharide (LPS)-stimulated rat macrophages by reducing nuclear factor (NF)-κB activity and COX-2, 5-LOX and inducible nitric oxide synthase (iNOS) expression. The aim of this study was to evaluate the effects of flavocoxid in a murine model of CLP-induced polymicrobial sepsis. Methods C57BL/6J mice were subjected to CLP or sham operation. In a first set of experiments, an intraperitoneal injection of flavocoxid (20 mg/kg) or vehicle was administered 1 hour after surgery and repeated every 12 hours. Survival rate was monitored every 24 hours throughout 120 hours. Furthermore, additional groups of sham and CLP mice were killed 18 hours after surgical procedures for blood-sample collection and the lung and liver were collected for biomolecular, biochemical and histopathologic studies. Results COX-2, 5-LOX, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, extracellular-regulated-kinase 1/2 (ERK), JunN-terminal kinase (JNK), NF-κB, and β-arrestin 2 protein expression were evaluated in lung and liver with Western blot analysis. In addition, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), cytokines, and lipoxin A4 serum content were measured with an enzyme-linked immunosorbent assay (ELISA). Flavocoxid administration improved survival, reduced the expression of NF-κB, COX-2, 5-LOX, TNF-α and IL-6 and increased IL-10 production. Moreover, flavocoxid inhibited the mitogen-activated protein kinases (MAPKs) pathway, preserved β-arrestin 2 expression, reduced blood LTB4, PGE2, TNF-α and IL-6, and increased IL-10 and lipoxin A4 serum levels. The treatment with flavocoxid also protected against the histologic damage induced by CLP and reduced the myeloperoxidase (MPO) activity in the lung and liver. Conclusions Flavocoxid protects mice from sepsis, suggesting that this dual inhibitor may represent a promising approach in such a life-threatening condition. PMID:22356547

Flavocoxid, a dual inhibitor of COX-2 and 5-LOX of natural origin, attenuates the inflammatory response and protects mice from sepsis.

PubMed

Bitto, Alessandra; Minutoli, Letteria; David, Antonio; Irrera, Natasha; Rinaldi, Mariagrazia; Venuti, Francesco S; Squadrito, Francesco; Altavilla, Domenica

2012-02-22

Cecal ligation and puncture (CLP) is an inflammatory condition that leads to multisystemic organ failure. Flavocoxid, a dual inhibitor of cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX), has been shown in vitro to possess antiinflammatory activity in lipopolysaccharide (LPS)-stimulated rat macrophages by reducing nuclear factor (NF)-κB activity and COX-2, 5-LOX and inducible nitric oxide synthase (iNOS) expression. The aim of this study was to evaluate the effects of flavocoxid in a murine model of CLP-induced polymicrobial sepsis. C57BL/6J mice were subjected to CLP or sham operation. In a first set of experiments, an intraperitoneal injection of flavocoxid (20 mg/kg) or vehicle was administered 1 hour after surgery and repeated every 12 hours. Survival rate was monitored every 24 hours throughout 120 hours. Furthermore, additional groups of sham and CLP mice were killed 18 hours after surgical procedures for blood-sample collection and the lung and liver were collected for biomolecular, biochemical and histopathologic studies. COX-2, 5-LOX, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10, extracellular-regulated-kinase 1/2 (ERK), JunN-terminal kinase (JNK), NF-κB, and β-arrestin 2 protein expression were evaluated in lung and liver with Western blot analysis. In addition, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), cytokines, and lipoxin A4 serum content were measured with an enzyme-linked immunosorbent assay (ELISA). Flavocoxid administration improved survival, reduced the expression of NF-κB, COX-2, 5-LOX, TNF-α and IL-6 and increased IL-10 production. Moreover, flavocoxid inhibited the mitogen-activated protein kinases (MAPKs) pathway, preserved β-arrestin 2 expression, reduced blood LTB4, PGE2, TNF-α and IL-6, and increased IL-10 and lipoxin A4 serum levels. The treatment with flavocoxid also protected against the histologic damage induced by CLP and reduced the myeloperoxidase (MPO) activity in the lung and liver. Flavocoxid protects mice from sepsis, suggesting that this dual inhibitor may represent a promising approach in such a life-threatening condition.

A dual positional specific lipoxygenase functions in the generation of flavor compounds during climacteric ripening of apple

PubMed Central

Schiller, Doreen; Contreras, Carolina; Vogt, Jörg; Dunemann, Frank; Defilippi, Bruno G; Beaudry, Randolph; Schwab, Wilfried

2015-01-01

Lipoxygenase (LOX) is an important contributor to the formation of aroma-active C6 aldehydes in apple (Malus × domestica) fruit upon tissue disruption but little is known about its role in autonomously produced aroma volatiles from intact tissue. We explored the expression of 22 putative LOX genes in apple throughout ripening, but only six LOXs were expressed in a ripening-dependent manner. Recombinant LOX1:Md:1a, LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b proteins showed 13/9-LOX, 9-LOX, 13/9-LOX and 13-LOX activity with linoleic acid, respectively. While products of LOX1:Md:1c and LOX2:Md:2b were S-configured, LOX1:Md:1a and LOX2:Md:2a formed 13(R)-hydroperoxides as major products. Site-directed mutagenesis of Gly567 to an alanine converted the dual positional specific LOX1:Md:1a to an enzyme with a high specificity for 9(S)-hydroperoxide formation. The high expression level of the corresponding MdLOX1a gene in stored apple fruit, the genetic association with a quantitative trait locus for fruit ester and the remarkable agreement in regio- and stereoselectivity of the LOX1:Md:1a reaction with the overall LOX activity found in mature apple fruits, suggest a major physiological function of LOX1:Md:1a during climacteric ripening of apples. While LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b may contribute to aldehyde production in immature fruit upon cell disruption our results furnish additional evidence that LOX1:Md:1a probably regulates the availability of precursors for ester production in intact fruit tissue. PMID:26504564

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guardiola, John J.

Background: Occupational vinyl chloride (VC) exposures have been associated with toxicant-associated steatohepatitis and liver cancer. Metabolomics has been used to clarify mode of action in drug-induced liver injury but has not been performed following VC exposures. Methods: Plasma samples from 17 highly exposed VC workers without liver cancer and 27 unexposed healthy volunteers were obtained for metabolite extraction and GC/MS and LC/MS{sup 2} analysis. Following ion identification/quantification, Ingenuity pathway analysis was performed. Results: 613 unique named metabolites were identified. Of these, 189 metabolites were increased in the VC exposure group while 94 metabolites were decreased. Random Forest analysis indicated thatmore » the metabolite signature could separate the groups with 94% accuracy. VC exposures were associated with increased long chain (including arachidonic acid) and essential (including linoleic acid) fatty acids. Occupational exposure increased lipid peroxidation products including monohydroxy fatty acids (including 13-HODE); fatty acid dicarboxylates; and oxidized arachidonic acid products (including 5,9, and 15-HETE). Carnitine and carnitine esters were decreased, suggesting peroxisomal/mitochondrial dysfunction and alternate modes of lipid oxidation. Differentially regulated metabolites were shown to interact with extracellular-signal-regulated kinase 1/2 (ERK1/2), Akt, AMP-activated protein kinase (AMPK), and the N-Methyl-D-aspartate (NMDA) receptor. The top canonical pathways affected by occupational exposure included tRNA charging, nucleotide degradation, amino acid synthesis/degradation and urea cycle. Methionine and homocysteine was increased with decreased cysteine, suggesting altered 1-carbon metabolism. Conclusions: Occupational exposure generated a distinct plasma metabolome with markedly altered lipid and amino acid metabolites. ERK1/2, Akt, AMPK, and NMDA were identified as protein targets for vinyl chloride toxicity. - Highlights: • Occupational vinyl chloride exposure is linked to toxicant-associated steatohepatitis, liver cancer, and other diseases. • Vinyl chloride exposure led to a distinct plasma metabolome with markedly altered lipid and amino acid metabolites. • A metabolomics approach can provide useful information regarding exposure in chemical workers.« less

Cucumber Cotyledon Lipoxygenase during Postgerminative Growth. Its Expression and Action on Lipid Bodies

PubMed Central

Matsui, Kenji; Hijiya, Kohko; Tabuchi, Yutaka; Kajiwara, Tadahiko

1999-01-01

In cucumber (Cucumis sativus), high lipoxygenase-1 (LOX-1) activity has been detected in the soluble fraction prepared from cotyledons of germinating seeds, and the involvement of this enzyme in lipid turnover has been suggested (K. Matsui, M. Irie, T. Kajiwara, A. Hatanaka [1992] Plant Sci 85: 23–32; I. Fuessner, C. Wasternack, H. Kindl, H. Kühn [1995] Proc Natl Acad Sci USA 92: 11849–11853). In this study we have investigated the expression of the gene lox-1, corresponding to the LOX-1 enzyme. LOX-1 expression is highly coordinated with that of a typical glyoxysomal enzyme, isocitrate lyase, during the postgerminative stage of cotyledon development. In contrast, although icl transcripts accumulated in tissue during in vitro senescence, no accumulation of lox-1 mRNA could be observed, suggesting that lox-1 plays a specialized role in fat mobilization. LOX-1 is also known to be a major lipid body protein. The partial peptide sequences of purified LOX-1 and lipid body LOX-1 entirely coincided with that deduced from the lox-1 cDNA sequence. The data strongly suggest that LOX-1 and lipid body LOX-1 are derived from a single gene and that LOX-1 can exist both in the cytosol and on the lipid bodies. We constructed an in vitro oxygenation system to address the mechanism of this dual localization and to investigate the action of LOX-1 on lipids in the lipid bodies. LOX-1 cannot act on the lipids in intact lipid bodies, although degradation of lipid body proteins, either during seedling growth or by treatment with trypsin, allows lipid bodies to become susceptible to LOX-1. We discuss the role of LOX-1 in fat mobilization and its mechanism of action. PMID:10198086

Triterpene derivatives as inhibitors of protein involved in the inflammatory process: molecules interfering with phospholipase A2, cycloxygenase, and lipoxygenase.

PubMed

Braca, Alessandra; Dal Piaz, Fabrizio; Marzocco, Stefania; Autore, Giuseppina; Vassallo, Antonio; De Tommasi, Nunziatina

2011-03-01

Over the past years, there was an explosion in the knowledge of the protein target and molecular mechanism associated with various disease types and in the new research of drugs of natural origin. The key idea is to evaluate bioactive natural products interacting with protein domains of different genetic origin but structurally preserved to develop libraries of compounds biologically validated and selected from an evolutionistic point of view. Compared with synthetic compounds, natural products have a major number of unused scaffolds and not comparable to the libraries of synthetic compounds, and could represent a promising starting points for the discovery of new bioactive compounds. Many natural products are reported to interact with proteins involved in serious diseases, such as inflammation and cancer. Recently various chemical classes of plant secondary metabolites have emerged as potential therapeutic compounds in several inflammatory diseases. Owing to the findings that triterpenoids, a common class of plant secondary metabolites, have anti-inflammatory and anti-cancer effects on humans, the interest in their potential application in human health and disease is increasing. The present review describes anti-inflammatory triterpenes derivatives from plant and fungi reported during the last two decades in order to provide an account of this field of investigation, sorting compounds according to their targets, phospholipase A(2) (PLA(2)), cycloxygenase (COX), and lipoxygenase (LOX). The attempt is also being made to enumerate the possible leads for further synthetic and drug discovery program development.

The targets of curcumin.

PubMed

Zhou, Hongyu; Beevers, Christopher S; Huang, Shile

2011-03-01

Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-kB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimer's disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here.

Targets of curcumin

PubMed Central

Zhou, Hongyu; Beevers, Christopher S.; Huang, Shile

2010-01-01

Curcumin (diferuloylmethane), an orange-yellow component of turmeric or curry powder, is a polyphenol natural product isolated from the rhizome of the plant Curcuma longa. For centuries, curcumin has been used in some medicinal preparation or used as a food-coloring agent. In recent years, extensive in vitro and in vivo studies suggested curcumin has anticancer, antiviral, antiarthritic, anti-amyloid, antioxidant, and anti-inflammatory properties. The underlying mechanisms of these effects are diverse and appear to involve the regulation of various molecular targets, including transcription factors (such as nuclear factor-κB), growth factors (such as vascular endothelial cell growth factor), inflammatory cytokines (such as tumor necrosis factor, interleukin 1 and interleukin 6), protein kinases (such as mammalian target of rapamycin, mitogen-activated protein kinases, and Akt) and other enzymes (such as cyclooxygenase 2 and 5 lipoxygenase). Thus, due to its efficacy and regulation of multiple targets, as well as its safety for human use, curcumin has received considerable interest as a potential therapeutic agent for the prevention and/or treatment of various malignant diseases, arthritis, allergies, Alzheimer’s disease, and other inflammatory illnesses. This review summarizes various in vitro and in vivo pharmacological aspects of curcumin as well as the underlying action mechanisms. The recently identified molecular targets and signaling pathways modulated by curcumin are also discussed here. PMID:20955148

A Salmon Protein Hydrolysate Exerts Lipid-Independent Anti-Atherosclerotic Activity in ApoE-Deficient Mice

PubMed Central

Busnelli, Marco; Bjørndal, Bodil; Holm, Sverre; Brattelid, Trond; Manzini, Stefano; Ganzetti, Giulia S.; Dellera, Federica; Halvorsen, Bente; Aukrust, Pål; Sirtori, Cesare R.; Nordrehaug, Jan E.; Skorve, Jon; Berge, Rolf K.; Chiesa, Giulia

2014-01-01

Fish consumption is considered health beneficial as it decreases cardiovascular disease (CVD)-risk through effects on plasma lipids and inflammation. We investigated a salmon protein hydrolysate (SPH) that is hypothesized to influence lipid metabolism and to have anti-atherosclerotic and anti-inflammatory properties. 24 female apolipoprotein (apo) E−/− mice were divided into two groups and fed a high-fat diet with or without 5% (w/w) SPH for 12 weeks. The atherosclerotic plaque area in aortic sinus and arch, plasma lipid profile, fatty acid composition, hepatic enzyme activities and gene expression were determined. A significantly reduced atherosclerotic plaque area in the aortic arch and aortic sinus was found in the 12 apoE−/− mice fed 5% SPH for 12 weeks compared to the 12 casein-fed control mice. Immunohistochemical characterization of atherosclerotic lesions in aortic sinus displayed no differences in plaque composition between mice fed SPH compared to controls. However, reduced mRNA level of Icam1 in the aortic arch was found. The plasma content of arachidonic acid (C20∶4n-6) and oleic acid (C18∶1n-9) were increased and decreased, respectively. SPH-feeding decreased the plasma concentration of IL-1β, IL-6, TNF-α and GM-CSF, whereas plasma cholesterol and triacylglycerols (TAG) were unchanged, accompanied by unchanged mitochondrial fatty acid oxidation and acyl-CoA:cholesterol acyltransferase (ACAT)-activity. These data show that a 5% (w/w) SPH diet reduces atherosclerosis in apoE−/− mice and attenuate risk factors related to atherosclerotic disorders by acting both at vascular and systemic levels, and not directly related to changes in plasma lipids or fatty acids. PMID:24840793

Identification of 12/15-lipoxygenase as a suppressor of myeloproliferative disease

PubMed Central

Middleton, Melissa Kristine; Zukas, Alicia Marie; Rubinstein, Tanya; Jacob, Michele; Zhu, Peijuan; Zhao, Liang; Blair, Ian; Puré, Ellen

2006-01-01

Though Abl inhibitors are often successful therapies for the initial stages of chronic myelogenous leukemia (CML), refractory cases highlight the need for novel molecular insights. We demonstrate that mice deficient in the enzyme 12/15-lipoxygenase (12/15-LO) develop a myeloproliferative disorder (MPD) that progresses to transplantable leukemia. Although not associated with dysregulation of Abl, cells isolated from chronic stage 12/15-LO–deficient (Alox15) mice exhibit increased activation of the phosphatidylinositol 3–kinase (PI3-K) pathway, as indicated by enhanced phosphorylation of Akt. Furthermore, the transcription factor interferon consensus sequence binding protein (ICSBP) is hyperphosphorylated and displays decreased nuclear accumulation, translating into increased levels of expression of the oncoprotein Bcl-2. The ICSBP defect, exaggerated levels of Bcl-2, and prolonged leukemic cell survival associated with chronic stage Alox15 MPD are all reversible upon treatment with a PI3-K inhibitor. Remarkably, the evolution of Alox15 MPD to leukemia is associated with additional regulation of ICSBP on an RNA level, highlighting the potential usefulness of the Alox15 model for understanding the transition of CML to crisis. Finally, 12/15-LO expression suppresses the growth of a human CML–derived cell line. These data identify 12/15-LO as an important suppressor of MPD via its role as a critical upstream effector in the regulation of PI3-K–dependent ICSBP phosphorylation. PMID:17043146

Evidences for Chlorogenic Acid — A Major Endogenous Polyphenol Involved in Regulation of Ripening and Senescence of Apple Fruit

PubMed Central

Xi, Yu; Cheng, Dai; Zeng, Xiangquan; Cao, Jiankang; Jiang, Weibo

2016-01-01

To learn how the endogenous polyphenols may play a role in fruit ripening and senescence, apple pulp discs were used as a model to study the influences of chlorogenic acid (CHA, a major polyphenol in apple pulp) on fruit ripening and senescence. Apple (‘Golden Delicious’) pulp discs prepared from pre-climacteric fruit were treated with 50 mg L-1 CHA and incubated in flasks with 10 mM MES buffer (pH 6.0, 11% sorbitol). Compared to the control samples, treatment with CHA significantly reduced ethylene production and respiration rate, and enhanced levels of firmness and soluble solids content of the pulp discs during incubation at 25°C. These results suggested that CHA could retard senescence of the apple pulp discs. Proteomics analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry (MALDI-TOF/TOF) revealed that the expressions of several key proteins correlated to fruit ripening and senescence were affected by the treatment with CHA. Further study showed that treating the pulp discs with CHA remarkably reduced levels of lipoxygenase, β-galactosidase, NADP-malic enzyme, and enzymatic activities of lipoxygenase and UDP-glucose pyrophosphorylase, all of which are known as promoters of fruit ripening and senescence. These results could provide new insights into the functions of endogenous phenolic compounds in fruit ripening and senescence. PMID:26756813

OsLOX2, a rice type I lipoxygenase, confers opposite effects on seed germination and longevity.

PubMed

Huang, Jiexue; Cai, Maohong; Long, Qizhang; Liu, Linglong; Lin, Qiuyun; Jiang, Ling; Chen, Saihua; Wan, Jianmin

2014-08-01

Rice production and seed storage are confronted with grain deterioration and loss of seed viability. Some members of the lipoxygenase (LOX) family function in degradation of storage lipids during the seed germination, but little is known about their influence on seed longevity during storage. We characterized the role of rice OsLOX2 gene in seed germination and longevity via over-expression and knock-down approaches. Abundant expression of OsLOX2 was detected in panicles, roots, and stems, but not in leaves. Moreover, OsLOX2 was highly induced during germination. OsLOX2 protein, located in the cytoplasm, showed a wide range of temperature adaptation (20-50 °C) and a substrate preference to linoleic acid. Lines over-expressing OsLOX2 showed accelerated seed germination under normal condition and lower seed viability after accelerated aging. RNA interference (RNAi) of OsLOX2 caused delayed germination and enhanced seed longevity. RNAi lines with strongly repressed OsLOX2 activity completely lost the capability of germination after accelerated aging. More lipid hydroperoxide were found in OE15 than the control, but less in RNAi lines than in the WT Nipponbare. Therefore, OsLOX2 acts in opposite directions during seed germination and longevity during storage. Appropriate repression of the OsLOX2 gene may delay the aging process during the storage without compromising germination under normal conditions.

12- and 15-lipoxygenases in human carotid atherosclerotic lesions: Associations with cerebrovascular symptoms

USDA-ARS?s Scientific Manuscript database

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovasc...

PGE2 is a UVR-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation

PubMed Central

Starner, Renny J.; McClelland, Lindy; Abdel-Malek, Zalfa; Fricke, Alex; Scott, Glynis

2013-01-01

Melanocyte proliferation, dendrite formation, and pigmentation are controlled by paracrine factors, particularly following exposure to ultraviolet radiation (UVR). Little is known about autocrine factors for melanocytes. Prostaglandins activate signaling pathways involved in growth, differentiation and apoptosis. Prostaglandin E2 (PGE2) is the most abundant prostaglandin released by keratinocytes following UVR, and stimulates the formation of dendrites in melanocytes. Synthesis of PGE2 is controlled by cPLA2, which releases arachidonic acid from membranes, and COX-2 and prostaglandin E2 synthases (PGES), which convert arachidonic acid to PGH2 and PGH2 to PGE2, respectively. In this report we show that multiple irradiations of human melanocytes with UVR stimulates tyrosinase activity, independent of expression of a functional melanocortin 1 receptor, suggesting the presence of a non-melanocortin autocrine factor. Irradiation of melanocytes activated cPLA2, the rate-limiting step in eicosanoid synthesis, and stimulated PGE2 secretion. PGE2 increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE2 binds to four distinct G-protein coupled receptors (EP1–4). We show that EP4 receptor signaling stimulates cAMP production in melanocytes. Conversely, stimulation of the EP3 receptor lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE2 on cAMP in melanocytes. The data are the first to identify PGE2 as an UVR-inducible autocrine factor for melanocytes that stimulates tyrosinase activity and proliferation, and to show that EP3 and EP4 receptor signaling have opposing effects on cAMP production, a critical signaling pathway that regulates proliferation and melanogenesis in melanocytes. PMID:20500768

Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK.

PubMed

Garcia, Bibian; Martinez-de-Mena, Raquel; Obregon, Maria-Jesus

2012-10-01

Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat. Copyright © 2012 Elsevier B.V. All rights reserved.

Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins.

PubMed

Markoutsa, Stavroula; Bahr, Ute; Papasotiriou, Dimitrios G; Häfner, Ann-Kathrin; Karas, Michael; Sorg, Bernd L

2014-03-01

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prostaglandin Actions in Established Insect Cell Lines

USDA-ARS?s Scientific Manuscript database

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals that mediate a wide range of physiological functions in animal cells. For example, PGs influence protein expression in establish insect cell lines ...

[Modeling of linoleyl hydroxamic acid influence on lipoxygenases in vitro].

PubMed

Skaterna, T D; Kopich, V M; Tserniuk, V M; Kharchenko, O V

2009-01-01

5-Lipoxygenase (5-LO) (1.13.11.12) demonstrates its activity in membrane-associated state. A system in vitro with increasing quantity of mixed micelle of nonionic detergent Lubrol PX and substrate--linoleic acid (LA) was used for understanding of 5-LO catalytic activity mechanism, which depends on the membrane environment. Physical parameters of micelles with molar ratio LA-Lubrol PX = 0.3:1 and micelles with 5-LO inhibitor--linoleyl hydroxamic acid (LHA), LA and Lubrol PX (0.03:0.3:1) were characterized by gel-filtration method on Sephadex G-200. It was determined, that Stock's radii were 4.83-5.79 nm for micelles with total LA--50-2000 microM and average molecular mass--177 000-212 000 Da. The presence of 10 microM LHA has no influence on physical parameters of the system. Influence of LHA on kinetic parameters of LA oxidation reaction catalized by potato tubers 5-LO in characterized mixed micelle system was also studied. Substrate dependences curves of 5-LO LA oxidation steady-state rates under conditions of the mixed micelle with ratio LA-lubrol PX = 0.3:1, LHA-LA-Lubrol PX = 0.03:0.3:1 and LHA-LA-Lubrol PX = 0.12:0.3:1 were typical of the substrate inhibition. The presence of inhibitor had no effect on the number of additional substrate molecules--LA which contact with enzyme-substrate complex and decreased V(max) essentially. To predict further inhibitor transformation in the cell the influence of 13-hydroperoxy- and 13-hydroxy LHA on potato tubers 5-LO and porcine leucocyte 12-LO was investigated. It was established that LHA oxidized forms displayed as no less effective inhibitors of the analyzed enzymes; 13-hydroperoxy LHA efficiency increased by an order (IC50 was 0.7 microM) for 12-LO. The possibility of 5-LO to oxidize inhibitor LHA under 50 microM phosphatidic acid at pH 5.0 was demonstrated.