Lipoxygenases during Brassica napus seed germination

The peroxidation of polyunsaturated fatty acids is mostly catalyzed by members of the lipoxygenase enzyme family. Lipoxygenase products can be metabolized further in the oxylipin pathway and are known as signalling substances that play a role in plant development as well as in plant responses to wounding and pathogen attack. Apart from accumulating data in model plants like Arabidopsis, information on the relevance of lipid peroxide metabolism in the crop plant oilseed rape is scarce. Thus we aimed to analyze lipoxygenases and oxylipin patterns in seedlings of oilseed rape. RNA isolated from 3 day etiolated seedlings contains mRNAs for at least two different lipoxygenases. These have been cloned as cDNAs and named Bn-Lox-1fl and Bn-Lox-2fl. The protein encoded by Bn-Lox-2fl was identified as a 13-lipoxygenase by expression in Escherichia coli. The Bn-Lox-1fl yielded an inactive protein when expressed in E. coli. Based on Bn-Lox-1fl active site determinants and on sequence homology the Bn-Lox-1fl is most likely a 9-lipoxygenase. Both genes are expressed in light-grown and etiolated cotyledons as well as in leaves. Bn-Lox-2fl protein is more abundant in cotyledons of etiolated seedlings than in cotyledons of green seedlings. Both 13- and 9-lipoxygenase-derived hydroperoxides can be detected during germination. Etiolated seedlings contain more lipoxygenase-derived hydroperoxides in non esterified fatty acids than green seedlings. The 13-lipoxygenase derivatives are 6-8-fold more abundant than the 9-derivatives. Lipoxygenase-derived hydroperoxides in esterified lipids are almost not present during germination. These results suggest that 13-lipoxygenases acting on free fatty acids dominate during B. napus seed germination.     

Functional and pathological roles of the 12- and 15-lipoxygenases

The 12/15-lipoxygenase enzymes react with fatty acids producing active lipid metabolites that are involved in a number of significant disease states. The latter include type 1 and type 2 diabetes (and associated complications), cardiovascular disease, hypertension, renal disease, and the neurological conditions Alzheimer’s disease and Parkinson’s disease. A number of elegant studies over the last thirty years have contributed to unraveling the role that lipoxygenases play in chronic inflammation. The development of animal models with targeted gene deletions has led to a better understanding of the role that lipoxygenases play in various conditions. Selective inhibitors of the different lipoxygenase isoforms are an active area of investigation, and will be both an important research tool and a promising therapeutic target for treating a wide spectrum of human diseases.     

An Ancient Relative of Cyclooxygenase in Cyanobacteria Is a Linoleate 10S-Dioxygenase That Works in Tandem with a Catalase-related Protein with Specific 10S-Hydroperoxide Lyase Activity

In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as “cyclooxygenase-2,” appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate.     

Olive phenols efficiently inhibit the oxidation of serum albumin-bound linoleic acid and butyrylcholine esterase

Background

Olive phenols are widely consumed in the Mediterranean diet and can be detected in human plasma. Here, the capacity of olive phenols and plasma metabolites to inhibit lipid and protein oxidations is investigated in two plasma models.

Methods

The accumulation of lipid oxidation products issued from the oxidation of linoleic acid bound to human serum albumin (HSA) by AAPH-derived peroxyl radicals is evaluated in the presence and absence of phenolic antioxidants. Phenol binding to HSA is addressed by quenching of the Trp214 fluorescence and displacement of probes (quercetin, dansylsarcosine and dansylamide). Next, the esterase activity of HSA-bound butyrylcholine esterase (BChE) is used as a marker of protein oxidative degradation.

Results

Hydroxytyrosol, oleuropein, caffeic and chlorogenic acids inhibit lipid peroxidation as well as HSA-bound BChE as efficiently as the potent flavonol quercetin. Hydroxycinnamic derivatives bind noncompetitively HSA subdomain IIA whereas no clear site could be identified for hydroxytyrosol derivatives.

General significance

In both models, olive phenols and their metabolites are much more efficient inhibitors of lipid and protein oxidations compared to vitamins C and E. Low postprandial concentrations of olive phenols may help to preserve the integrity of functional proteins and delay the appearance of toxic lipid oxidation products.     

Cholesterol is more susceptible to oxidation than linoleates in cultured cells under oxidative stress induced by selenium deficiency and free radicals

Polyunsaturated fatty acids and their esters are known to be susceptible to free-radical mediated oxidation, while cholesterol is more resistant to oxidation. The present study focused on the relative susceptibilities of linoleates and cholesterol in Jurkat cells under oxidative stress induced by selenium deficiency and free radical insult, as assessed by total hydroxyoctadecadienoic acids (tHODE) and total 7-hydroxycholesterol (t7-OHCh) measured after reduction and saponification. It was observed that the levels of tHODE and t7-OHCh significantly increased by both oxidative insults. The increased amounts of t7-OHCh were higher than those of tHODE in both selenium-deficient and free radical-treated cells. These results suggest that, in contrast to plasma oxidation where cholesterol is much more resistant to oxidation than linoleates, cellular cholesterol is more susceptible to oxidation than cellular linoleates.     

7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: Difference between cellular and plasma lipid peroxidation

Polyunsaturated fatty acids and their esters are known to be susceptible to free radical-mediated oxidation, whereas cholesterol is thought to be more resistant to oxidation. In fact, it has been observed that in the case of plasma lipid peroxidation, the amount of oxidation products of polyunsaturated fatty acids such as linoleic acid was higher than that of cholesterol. In contrast, during oxidative stress-induced cellular lipid peroxidation, oxidation products of cholesterol such as 7-hydroxycholesterol (7-OHCh) were detected in greater amounts than those of linoleates such as hydroxyoctadecadienoic acid (HODE). There are several forms of oxidation products of cholesterol and linoleates in vivo, namely, hydroperoxides, as well as the hydroxides of both the free and ester forms of cholesterol and linoleates. To evaluate these oxidation products, a method used to determine the lipid oxidation products after reduction and saponification was developed. With this method, several forms of oxidation products of cholesterol and linoleates are measured as total 7-OHCh (t7-OHCh) and total HODE (tHODE), respectively. During free radical-mediated lipid peroxidation in plasma, the amount of tHODE was 6.3-fold higher than that of t7-OHCh. In contrast, when Jurkat cells were exposed to free radicals, the increased amount of cellular t7-OHCh was 5.7-fold higher than that of tHODE. Higher levels of t7-OHCh than those of tHODE have also been observed in selenium-deficient Jurkat cells and glutamate-treated neuronal cells. These results suggest that, in contrast to plasma oxidation, cellular cholesterol is more susceptible to oxidation than cellular linoleates. Collectively, cholesterol oxidation products at the 7-position may be a biomarker of cellular lipid peroxidation.