Detection, isolation and characterisation of cyclolinopeptides J and K in ageing flax.

Methionine sulfone containing peptides CLs J (11) and K (12) may be produced from their reduced forms by oxidation but it is not known if these compounds occur in foods that contain flax. These compounds have been reported to possess greater immunosuppressive activity than their reduced methionine sulfoxide peptide forms 4 and 6, respectively. Since 11 and 12 have not been detected in commercial flax oil and milled flax seed, we tested for their presence in flax food products. Here we report that 11 and 12 accumulate in ground flaxseed that is exposed to air and heat (100°C) for more than 4h. Standards of 11 and 12 were prepared, isolated and extensively characterised using HPLC-MS/MS, 1D and 2D NMR methods. We also report the excellent thermal and oxidative stability of these peptides. Due to the harsh conditions required to produce 11 and 12, it is expected that their levels in flax based foods would be low and therefore their presence could serve as an indicative measure of severe oxidation of a food product.

Human serum-derived hydroxy long-chain fatty acids exhibit anti-inflammatory and anti-proliferative activity.

BACKGROUND: Circulating levels of novel long-chain hydroxy fatty acids (called GTAs) were recently discovered in the serum of healthy subjects which were shown to be reduced in subjects with colorectal cancer (CRC), independent of tumor burden or disease stage. The levels of GTAs were subsequently observed to exhibit an inverse association with age in the general population. The current work investigates the biological activity of these fatty acids by evaluating the effects of enriched human serum extracts on cell growth and inflammation. METHODS: GTAs were extracted from commercially available bulk human serum and then chromatographically separated into enriched (GTA-positive) and depleted (GTA-negative) fractions. SW620, MCF7 and LPS stimulated RAW264.7 cells were treated with various concentrations of the GTA-positive and GTA-negative extracts, and the effects on cell growth and inflammation determined. RESULTS: Enriched fractions resulted in poly-ADP ribose polymerase (PARP) cleavage, suppression of NFκB, induction of IκBα, and reduction in NOS2 mRNA transcript levels. In RAW264.7 mouse macrophage cells, incubation with enriched fractions prior to treatment with LPS blocked the induction of several pro-inflammatory markers including nitric oxide, TNFα, IL-1ß, NOS2 and COX2. CONCLUSIONS: Our results show that human serum extracts enriched with endogenous long-chain hydroxy fatty acids possess anti-inflammatory and anti-proliferative activity. These findings support a hypothesis that the reduction of these metabolites with age may result in a compromised ability to defend against uncontrolled cell growth and inflammation, and could therefore represent a significant risk for the development of CRC.

Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study.

BACKGROUND: Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimer's disease (AD), and cancer. In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer. RESULTS: Using plasmalogen deficient (NRel-4) and plasmalogen sufficient (HEK293) cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA)-containing ethanolamine plasmalogen (PlsEtn) present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1) levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition. CONCLUSION: The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.

Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection.

BACKGROUND: There are currently no accurate serum markers for detecting early risk of colorectal cancer (CRC). We therefore developed a non-targeted metabolomics technology to analyse the serum of pre-treatment CRC patients in order to discover putative metabolic markers associated with CRC. Using tandem-mass spectrometry (MS/MS) high throughput MS technology we evaluated the utility of selected markers and this technology for discriminating between CRC and healthy subjects. METHODS: Biomarker discovery was performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Comprehensive metabolic profiles of CRC patients and controls from three independent populations from different continents (USA and Japan; total n = 222) were obtained and the best inter-study biomarkers determined. The structural characterization of these and related markers was performed using liquid chromatography (LC) MS/MS and nuclear magnetic resonance technologies. Clinical utility evaluations were performed using a targeted high-throughput triple-quadrupole multiple reaction monitoring (TQ-MRM) method for three biomarkers in two further independent populations from the USA and Japan (total n = 220). RESULTS: Comprehensive metabolomic analyses revealed significantly reduced levels of 28-36 carbon-containing hydroxylated polyunsaturated ultra long-chain fatty-acids in all three independent cohorts of CRC patient samples relative to controls. Structure elucidation studies on the C28 molecules revealed two families harbouring specifically two or three hydroxyl substitutions and varying degrees of unsaturation. The TQ-MRM method successfully validated the FTICR-MS results in two further independent studies. In total, biomarkers in five independent populations across two continental regions were evaluated (three populations by FTICR-MS and two by TQ-MRM). The resultant receiver-operator characteristic curve AUCs ranged from 0.85 to 0.98 (average = 0.91 +/- 0.04). CONCLUSIONS: A novel comprehensive metabolomics technology was used to identify a systemic metabolic dysregulation comprising previously unknown hydroxylated polyunsaturated ultra-long chain fatty acid metabolites in CRC patients. These metabolites are easily measurable in serum and a decrease in their concentration appears to be highly sensitive and specific for the presence of CRC, regardless of ethnic or geographic background. The measurement of these metabolites may represent an additional tool for the early detection and screening of CRC.

Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia.

Although dementia of the Alzheimer's type (DAT) is the most common form of dementia, the severity of dementia is only weakly correlated with DAT pathology. In contrast, postmortem measurements of cholinergic function and membrane ethanolamine plasmalogen (PlsEtn) content in the cortex and hippocampus correlate with the severity of dementia in DAT. Currently, the largest risk factor for DAT is age. Because the synthesis of PlsEtn occurs via a single nonredundant peroxisomal pathway that has been shown to decrease with age and PlsEtn is decreased in the DAT brain, we investigated potential relationships between serum PlsEtn levels, dementia severity, and DAT pathology. In total, serum PlsEtn levels were measured in five independent population collections comprising >400 clinically demented and >350 nondemented subjects. Circulating PlsEtn levels were observed to be significantly decreased in serum from clinically and pathologically diagnosed DAT subjects at all stages of dementia, and the severity of this decrease correlated with the severity of dementia. Furthermore, a linear regression model predicted that serum PlsEtn levels decrease years before clinical symptoms. The putative roles that PlsEtn biochemistry play in the etiology of cholinergic degeneration, amyloid accumulation, and dementia are discussed.

Triterpenoids from leaves of Elaeophorbia drupifera.

A survey of the hexane extract of the leaves of Elaeophorbia drupifera led to the isolation of a new triterpenoid named elaeophorbate [methyl 4,4,8,10,13,14-hexamethyl-1,17-di (prop-1-en-2-yl)hexadecahydro-1H-cyclo penta[a]phenanthrene-3-carboxylate, 5] and eight known triterpenoids. Based on spectroscopic methods, coupled with high resolution gas chromatography-mass spectrometry the structures of all the compounds were elucidated.

Unprecedented chemical structure and biomimetic synthesis of erucalexin, a phytoalexin from the wild crucifer Erucastrum gallicum.

The isolation, structure determination, total synthesis and antifungal activity of erucalexin, a novel phytoalexin produced by the wild crucifer dog mustard are described. Erucalexin is a structurally unique plant alkaloid, representing the first example of a spiro[2H-indole-2,5'(4'H)-thiazol]-3-one, likely derived from a C-3-C-2 carbon migration in a 3-substituted indolyl nucleus.

Metabolism and detoxification of phytoalexins and analogs by phytopathogenic fungi.

To date, the many examples reporting that fungal pathogens can efficiently detoxify phytoalexins provide strong evidence that the pathogenicity and/or virulence of some fungi is linked to their ability to detoxify their hosts' phytoalexins. The pathways used by plant pathogenic fungi to metabolize and detoxify phytoalexins are reviewed. Prospects for application of recent findings are discussed.

Detoxification of the cruciferous phytoalexin brassinin in Sclerotinia sclerotiorum requires an inducible glucosyltransferase.

The phytoalexins, brassinin, 1-methoxybrassinin and cyclobrassinin, were metabolized by the stem rot fungus Sclerotinia sclerotiorum into their corresponding glucosyl derivatives displaying no detectable antifungal activity. Importantly, co-incubation of S. sclerotiorum with camalexins, various phytoalexin analogs, and brassinin indicated that a synthetic camalexin derivative could slow down substantially the rate of brassinin detoxification. Furthermore, inducible brassinin glucosyltransferase (BGT) activity was detected in crude cell-free extracts of S. sclerotiorum. BGT activity was induced by the phytoalexin camalexin, and the brassinin analogs methyl tryptamine dithiocarbamate and methyl 1-methyltryptamine dithiocarbamate. The overall results suggest that the fungus S. sclerotiorum in its continuous adaptation and co-evolution with brassinin producing plants, has acquired efficient glucosyltransferase(s) that can disarm some of the most active plant chemical defenses.

Phytotoxin production and phytoalexin elicitation by the phytopathogenic fungus Sclerotinia sclerotiorum.

The fungus Sclerotinia sclerotiorum (Lib.) de Bary causes rot disease in a vast range of plant families, including Cruciferae (Brassicaceae). We investigated the production of phytotoxins by S. sclerotiorum by using a bioassay-guided isolation, as well as the phytoalexins produced by the resistant wild crucifer Erucastrum gallicum under elicitation by S. sclerotiorum and other agents. We established for the first time that S. sclerotiorum produces a somewhat selective phytotoxin, sclerin, which is phytotoxic to three cruciferous species (Brassica napus, B. juncea, and Sinapis alba) susceptible to Sclerotinia stem rot disease, causing severe necrosis and chlorosis, but not to a resistant species (Erucastrum gallicum). In addition, we have shown that oleic acid, the major fatty acid isolated from sclerotia of S. sclerotiorum is responsible for the toxic activity of extracts of sclerotia to brine shrimp larvae (Artemia salina). Phytoalexin elicitation in leaves of E. gallicum led to the isolation of three known phytoalexins: indole-3-acetonitrile, arvelexin, and 1-methoxyspirobrassinin. Considering that resistance of E. gallicum to S. sclerotiorum is potentially transferable to B. rapa, a susceptible canola species, and that arvelexin, and 1-methoxyspirobrassinin are not produced by B. rapa, these phytoalexins may become useful markers for resistance against S. sclerotiorum.

Probing the phytopathogenic stem rot fungus with phytoalexins and analogues: unprecedented glucosylation of camalexin and 6-methoxycamalexin.

The remarkable metabolism of the cruciferous phytoalexins camalexin and 6-methoxycamalexin by the stem rot phytopathogen Sclerotinia sclerotiorum is reported. The biotransformations yielded camalexins glucosylated at N-1 or C-6 of the indole ring, with substantially lower antifungal activity than camalexins. A camalexin analogue with the positions N-1 and C-6 blocked was metabolized but at a much slower rate than the natural phytoalexins. The chemistry involved in the metabolism of natural camalexins and two new analogues, as well as their novel metabolites and respective antifungal activities is described.