Distributions of nutrients and avenanthramides within oat grain and effects on pearled kernel composition.

Hulled Reins and hulless Lamont oats were dehulled and/or sequentially abraded to produce ten pearling fines and corresponding pearled kernels. Contents of nutrients (protein, oil, starch, beta-glucan, ash and other carbohydrates) and avenanthramides (AVA) 2p, 2c, 2f, and 5p in processing fractions and starting grains were measured. Results show that distribution patterns of nutrients varied with individual nutrients, but those of AVAs varied with variety and individual AVAs. In both varieties, from the surface to inner endosperms, protein and oil increased then decreased; ash and other carbohydrates decreased; starch increased; and beta-glucan unchanged except for the surface area. In Lamont oat, the four AVAs decreased, but in Reins oat, AVA 2p decreased while 2c, 2f and 5p increased, then decreased. Compared to whole grain, pearled oats not only contained lower AVAs, protein, oil, ash, and other carbohydrates and higher beta-glucan and starch but also had a different AVA composition.

Lactate supports a metabolic-epigenetic link in macrophage polarization.

Lactate accumulation is a hallmark of solid cancers and is linked to the immune suppressive phenotypes of tumor-infiltrating immune cells. We report herein that interleukin-4 (IL-4)­induced M0 → M2 macrophage polarization is accompanied by interchangeable glucose- or lactate-dependent tricarboxylic acid (TCA) cycle metabolism that directly drives histone acetylation, M2 gene transcription, and functional immune suppression. Lactate-dependent M0 → M2 polarization requires both mitochondrial pyruvate uptake and adenosine triphosphate­citrate lyase (ACLY) enzymatic activity. Notably, exogenous acetate rescues defective M2 polarization and histone acetylation following mitochondrial pyruvate carrier 1 (MPC1) inhibition or ACLY deficiency. Lastly, M2 macrophage­dependent tumor progression is impaired by conditional macrophage ACLY deficiency, further supporting a dominant role for glucose/lactate mitochondrial metabolism and histone acetylation in driving immune evasion. This work adds to our understanding of how mitochondrial metabolism affects macrophage functional phenotypes and identifies a unique tumor microenvironment (TME)­driven metabolic-epigenetic link in M2 macrophages.

Avenanthramides inhibit proliferation of human colon cancer cell lines in vitro.

A high intake of whole grain foods is associated with reduced risk of colon cancer, but the mechanism underlying this protection has yet to be elucidated. Chronic inflammation and associated cyclooxygenase-2 (COX-2) expression in the colon epithelium are causally related to epithelial carcinogenesis, proliferation, and tumor growth. We examined the effect of avenanthramides (Avns), unique polyphenols from oats with anti-inflammatory properties, on COX-2 expression in macrophages, colon cancer cell lines, and on proliferation of human colon cancer cell lines. We found that Avns-enriched extract of oats (AvExO) had no effect on COX-2 expression, but it did inhibit COX enzyme activity and prostaglandin E(2) (PGE(2)) production in lipopolysaccharide-stimulated mouse peritoneal macrophages. Avns (AvExO, Avn-C, and the methylated form of Avn-C (CH3-Avn-C)) significantly inhibited cell proliferation of both COX-2-positive HT29, Caco-2, and LS174T, and COX-2-negative HCT116 human colon cancer cell lines, CH3-Avn-C being the most potent. However, Avns had no effect on COX-2 expression and PGE(2) production in Caco-2 and HT29 colon cancer cells. These results indicate that the inhibitory effect of Avns on colon cancer cell proliferation may be independent of COX-2 expression and PGE(2) production. Thus, Avns might reduce colon cancer risk through inhibition of macrophage PGE(2) production and non-COX-related antiproliferative effects in colon cancer cells. Interestingly, Avns had no effect on cell viability of confluence-induced differentiated Caco-2 cells, which display the characteristics of normal colonic epithelial cells. Our results suggest that the consumption of oats and oat bran may reduce the risk of colon cancer not only because of their high fiber content but also due to Avns, which attenuate proliferation of colonic cancer cells.

Avenanthramides, polyphenols from oats, inhibit IL-1beta-induced NF-kappaB activation in endothelial cells.

The chronic inflammation of arterial walls is associated with the development of atherosclerosis. Earlier we reported that avenanthramide (Avn)s-enriched extract of oats (AvnsO) significantly suppressed interleukin (IL)-1beta-stimulated secretion of proinflammatory cytokines, such as IL-6, IL-8, and MCP-1, by human aortic endothelial cells (HAEC). The main objective of the current study was to determine if the mechanism of inhibitory effect of these polyphenols from oats on the expression of proinflammatory cytokines is mediated through modulation of nuclear factor kappaB (NF-kappaB)-dependent transcription. Confluent HAEC monolayers were treated for 24 h with AvnsO, and synthetically prepared Avn-c suppressed IL-beta-stimulated activation of NF-kappaB in a concentration-dependent manner. CH3-Avn-c, a synthetically prepared methyl ester derivative of Avn-c with a high biological potency, significantly and dose dependently decreased mRNA expression and secretion of IL-6, IL-8, and MCP-1 by HAEC as determined by real-time RT-PCR and ELISA, and it inhibited IL-1beta- and TNFalpha-stimulated NF-kappaB activation as determined by a NF-kappaB DNA binding assay and a NF-kappaB luciferase reporter assay. AvnsO and Avn-c as well as CH3-Avn-c also inhibited the NF-kappaB-dependent reporter gene expression activated by TNFR-associated factor 2 and 6 (TRAF2, TRAF6) and NFkappaB-inducing kinase (NIK). CH3-Avn-c also significantly and dose dependently decreased the phosphorylation level of IkappaB kinase (IKK) and IkappaB, and prevented IkappaB degradation as measured by Western blotting. In addition, CH3-Avn-c markedly increased the overall levels of high mass ubiquitin-conjugated protein levels while it mildly inhibited proteasome activity. These observations suggest that Avns, unique polyphenols from oats, decrease the expression of endothelial proinflammatory cytokines at least in part through inhibition of NF-kappaB activation by inhibiting the phosphorylation of IKK and IkappaB, and by suppressing proteasome activity.

High Levels of Avenanthramides in Oat-Based Diet Further Suppress High Fat Diet-Induced Atherosclerosis in Ldlr-/- Mice.

Oats, in addition to cholesterol-lowering properties, contain unique antioxidants called avenanthramides (Avns), which inhibit both inflammatory cytokines and adhesion molecules in endothelial cells in culture. This study evaluated the effects of Avns of oats on atherosclerosis in Ldlr-/- mice, one of the most commonly used atherosclerosis mouse models with their similar cholesterol distributions to humans. The Ldlr-/- mice were fed a low fat, high fat, high fat containing regular oat brans with low levels of Avns (HFLA), or high fat containing regular oat brans with high levels of Avns (HFHA) diet. After 16 weeks of intervention, blood cholesterol and extent of aortic lesions were evaluated. We found that both oat-based diets reduced high fat diet-induced atheroma lesions in the aortic valve (p < 0.01). Furthermore, the effects of oat-based diets are more profound in HFHA mice than mice fed HFLA. Total plasma cholesterol levels were similarly reduced in both oat-supplemented mice. We concluded that oat bran diets reduce atheroma lesions and higher levels of Avns further reduce aortic lesions compared to regular oat bran. These preliminary in vivo data indicate that consumption of oats bran, with high Avns, has demonstrable beneficial effects on prevention of cardiovascular disease.

Mechanism by which avenanthramide-c, a polyphenol of oats, blocks cell cycle progression in vascular smooth muscle cells.

Previously, we reported that avenanthramide-c (Avn-c), one of the major avenanthramides, polyphenols of oats, inhibited the serum-induced proliferation of vascular smooth muscle cells (SMC), which is an important process in the initiation and development of atherosclerosis. In the present study, we further investigated its cell cycle inhibitory mechanism. Rat embryonic aortic smooth muscle cell line A10 was used in this study. Flow cytometry analysis revealed that treatment of A10 cells with 80 muM Avn-c arrested the cell cycle in G1 phase as indicated by an increase in the number of cells in G1 phase and a decrease in the number of cells in S phase. This cell cycle arrest was associated with a decrease in the phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1 to S transition in the cell cycle. The inhibition of pRb phosphorylation with Avn-c was accompanied by a decrease in cyclin D1 expression and an increase in cyclin-dependent kinase inhibitor p21cip1 expression, without significant changes in p27kip1 expression. Furthermore, Avn-c treatment increased the expression level and stability of p53 protein, which could account for the increase of p21cip1 expression. Our results demonstrate for the first time that Avn-c, which is a unique polyphenol found in oats, arrests SMC proliferation at G1 phase by upregulating the p53-p21cip1 pathway and inhibiting pRB phosphorylation. This inhibitory effect of Avn-c on SMC proliferation is an additional indication for the potential health benefit of oat consumption in the prevention of coronary heart disease beyond its known effect through lowering blood cholesterol.

Avenanthramide, a polyphenol from oats, inhibits vascular smooth muscle cell proliferation and enhances nitric oxide production.

The proliferation of vascular smooth muscle cells (SMC) and impaired nitric oxide (NO) production are both crucial pathophysiological processes in the initiation and development of atherosclerosis. Epidemiological data have indicated that diets rich in whole grain foods are associated with a reduced risk of developing atherosclerosis. Avenanthramides are polyphenols found exclusively in oats (Avena sativa L.). The present study was conducted to examine the effect of synthetically prepared avenanthramide-2c on the proliferation of SMC and NO production by SMC and human aortic endothelial cells (HAEC). Avenanthramide-2c significantly inhibited serum-induced SMC proliferation. At a concentration of 120 microM, avenanthramide-2c inhibited more than 50% of SMC proliferation, as measured by [3H] thymidine incorporation, and increased the doubling time of rat SMC line (A10) from 28 to 48 h. Treatment of human SMC with 40, 80, and 120 microM avenanthramide-2c inhibited cell number increase by 41, 62, and 73%, respectively. In addition, avenanthramide-2c treatment significantly and dose-dependently increased NO production in both SMC and HAEC. At a concentration of 120 microM, avenanthramide-2c increased NO production by three-fold in SMC, and by nine-fold in HAEC. These increases were in parallel with the up-regulation of mRNA expression for endothelial NO synthase (eNOS) in both vascular SMC and HAEC. These results suggest that the avenanthramides of oats may contribute to the prevention of atherosclerosis through inhibition of SMC proliferation and increasing NO production.

Oat avenanthramides induce heme oxygenase-1 expression via Nrf2-mediated signaling in HK-2 cells.

SCOPE: Numerous studies have shown that avenanthramides (AVAs), unique compounds found in oats, are strong antioxidants, though the mechanism of action remains unclear. Here, we investigated whether AVAs affect heme oxygenase-1 (HO-1) expression through the activation of Nrf2 translocation. METHODS AND RESULTS: We investigated the effects AVA 2c, 2f, and 2p on HK-2 cells, and found that AVAs could significantly increase HO-1 expression in both a dose- and time-dependent manner. Furthermore, we found that AVA-induced HO-1 expression is mediated by Nrf2 translocation. The addition of N-acetylcysteine (NAC), but not specific inhibitors of p38 (SB202190), PI3K (LY294002), and MEK1 (PD098059) attenuated AVA-induced HO-1 expression, demonstrating an important role for reactive oxygen species, but not PI3K or MAPK activation, in activating the HO-1 pathway. Moreover, hydrogenation of the double bond of the functional α,ß-unsaturated carbonyl group of AVAs eliminated their effects on HO-1 expression, suggesting that this group is crucial for the antioxidant activity of AVAs. CONCLUSION: Our results suggest a novel mechanism whereby AVAs exert an antioxidant function on human health. Further investigation of these markers in human is warranted to explore the beneficial health effects of whole grain oat intake.

Quantitative Trait Loci Associated with the Tocochromanol (Vitamin E) Pathway in Barley.

The Genome-Wide Association Studies approach was used to detect Quantitative Trait Loci associated with tocochromanol concentrations using a panel of 1,466 barley accessions. All major tocochromanol types- α-, ß-, δ-, γ-tocopherol and tocotrienol- were assayed. We found 13 single nucleotide polymorphisms associated with the concentration of one or more of these tocochromanol forms in barley, seven of which were within 2 cM of sequences homologous to cloned genes associated with tocochromanol production in barley and/or other plants. These associations confirmed a prior report based on bi-parental QTL mapping. This knowledge will aid future efforts to better understand the role of tocochromanols in barley, with specific reference to abiotic stress resistance. It will also be useful in developing barley varieties with higher tocochromanol concentrations, although at current recommended daily consumption amounts, barley would not be an effective sole source of vitamin E. However, it could be an important contributor in the context of whole grains in a balanced diet.

In vitro total antioxidant capacity and anti-inflammatory activity of three common oat-derived avenanthramides.

To better understand mechanisms underlying the health benefits of oats, the free radical scavenging capacities of oat avenanthramides 2c, 2f, and 2p and their ability to inhibit NF-κB activation were evaluated. The antioxidant capacities of 2c, 2f, and 2p against peroxyl radicals, hydroxyl radicals, superoxide anion, singlet oxygen, and peroxynitrite were determined by using ORAC, HORAC, SORAC, SOAC, and NORAC assays, respectively. The total antioxidant capacity of 2c was approximately 1.5-fold those of 2f and 2p. Total antioxidant capacity was primarily attributable to SORAC and ORAC for 2c (>77%, p<0.05), and to ORAC and SOAC for 2f. ORAC accounted for approximately 32% of total antioxidant capacity in 2p. EC50 values for inhibiting TNF-α-induced NF-κB activation in C2C12 cells were 64.3, 29.3, and 9.10 µM for 2c, 2f, and 2p, respectively. Differences in antioxidant capacities and ability to inhibit NF-κB among the avenanthramides could be ascribed to structural variations.

Comparative systems biology reveals allelic variation modulating Tocochromanol profiles in Barley (Hordeum vulgare L.).

Tocochromanols are recognized for nutritional content, plant stress response, and seed longevity. Here we present a systems biological approach to characterize and develop predictive assays for genes affecting tocochromanol variation in barley. Major QTL, detected in three regions of a SNP linkage map, affected multiple tocochromanol forms. Candidate genes were identified through barley/rice orthology and sequenced in genotypes with disparate tocochromanol profiles. Gene-specific markers, designed based on observed polymorphism, mapped to the originating QTL, increasing R2 values at the respective loci. Polymorphism within promoter regions corresponded to motifs known to influence gene expression. Quantitative PCR analysis revealed a trend of increased expression in tissues grown at cold temperatures. These results demonstrate utility of a novel method for rapid gene identification and characterization, and provide a resource for efficient development of barley lines with improved tocochromanol profiles.

Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.

In vitro antioxidant capacity and anti-inflammatory activity of seven common oats.

Oats are gaining increasing scientific and public interest for their purported antioxidant-associated health benefits. Most reported studies focused on specific oat extracts or particular oat components, such as ß-glucans, tocols (vitamin E), or avenanthramides. Studies on whole oats with respect to antioxidant and anti-inflammatory activities are still lacking. Here the antioxidant and anti-inflammatory activities from whole oat groats of seven common varieties were evaluated. All oat varieties had very similar oxygen radical absorption capacity compared with other whole grains. In an anti-inflammatory assay, oat variety CDC Dancer inhibited tumor necrosis factor-α induced nuclear factor-kappa B activation by 27.5% at 2 mg/ml, whereas variety Deiter showed 13.7% inhibition at a comparable dose. Avenanthramide levels did not correlate with the observed antioxidant and anti-inflammatory activities. Further investigations are needed to pinpoint the specific antioxidant and anti-inflammatory compounds, and potential synergistic and/or matrix effects that may help explain the mechanisms of oat's anti-inflammatory actions.

Phytochemicals attenuating aberrant activation of ß-catenin in cancer cells.

Phytochemicals are a rich source of chemoprevention agents but their effects on modulating the Wnt/ß-catenin signaling pathway have remained largely uninvestigated. Aberrantly activated Wnt signaling can result in the abnormal stabilization of ß-catenin, a key causative step in a broad spectrum of cancers. Here we report the modulation of lithium chloride-activated canonical Wnt/ß-catenin signaling by phytochemicals that have antioxidant, anti-inflammatory or chemopreventive properties. The compounds were first screened with a cervical cancer-derived stable Wnt signaling reporter HeLa cell line. Positive hits were subsequently evaluated for ß-catenin degradation, suppression of ß-catenin nuclear localization and down-regulation of downstream oncogenic targets of Wnt/ß-catenin pathway. Our study shows a novel degradation path of ß-catenin protein in HeLa cells by Avenanthramide 2p (a polyphenol) and Triptolide (a diterpene triepoxide), respectively from oats and a Chinese medicinal plant. The findings present Avenanthramide 2p as a potential chemopreventive dietary compound that merits further study using in vivo models of cancers; they also provide a new perspective on the mechanism of action of Triptolide.

Effect of chemical systemic acquired resistance elicitors on avenanthramide biosynthesis in oat (Avena sativa).

Oats produce a group of phenolic antioxidants termed avenanthramides. These metabolites are, among food crops, unique to oats and have shown, in experimental systems, certain desirable nutritional characteristics such as inhibiting atherosclerotic plaque formation and reducing inflammation. Avenanthramides occur in both the leaves and grain of oat. In the leaves they are expressed as phytoalexins in response to crown rust (Puccina coronata) infection. The experiments reported here demonstrate that avenanthramide levels in vegetative tissue can be enhanced by treatment with benzothiadiazole (BTH), an agrochemical formulated to elicit systemic acquired resistance (SAR). The response to BTH was dramatically stronger than those produced with salicylic acid treatment. The roots of BTH treated plants also showed a smaller but distinct increase in avenanthramides. The dynamics of the root avenanthramide increase was substantially slower than that observed in the leaves, suggesting that avenanthramides might be transported from the leaves.

Avenanthramides are bioavailable and accumulate in hepatic, cardiac, and skeletal muscle tissue following oral gavage in rats.

Avenanthramides (AVA), polyphenols found exclusively in oats ( Avena sativa L.), may play a role in the anti-inflammatory and antiatherogenic activity of oats. The bioavailability of AVA has been demonstrated previously, but its distribution at the organ and tissue level and the extent of conjugation following ingestion have been unexplored. Synthetic AVA was administered to 24 rats by oral gavage, whereas 6 control rats received saline. AVA concentrations were measured via HPLC in plasma, liver, heart, and gastrocnemius (GAS) obtained over a 12 h period (0, 2, 4, 12 h; n = 6 at each time point). Samples were extracted with and without glucuronidase-sulfatase to assess the level of conjugation. We conclude that AVA are bioavailable to the blood circulation following oral ingestion in the rat and reach peripheral tissues where they can be taken up by various organs differentially. With AVA remaining in the organs for up to 12 h, it seems possible to maintain an increased level of AVA in the rat via repeated feedings.

Biosynthesis of marine natural products: isolation and characterization of a myrcene synthase from cultured tissues of the marine red alga Ochtodes secundiramea.

The acyclic monoterpene myrcene is the likely progenitor of the unusual cytotoxic halogenated monoterpenes that are found in marine algae and that function as feeding deterrents to herbivores. Myrcene synthase was isolated from suspension cultures of the marine red alga Ochtodes secundiramea, representing the first enzyme of this type from a marine organism. The algal myrcene synthase produces exclusively myrcene from the natural substrate geranyl diphosphate (GDP), utilizes Mg(+2) as the required divalent metal ion cofactor, has a molecular mass of about 69 kDa, and exhibits a pH optimum near 7.2. These features are similar to those of monoterpene synthases from terrestrial organisms. When incubated with neryl diphosphate (the cis-isomer of GDP), the O. secundiramea myrcene synthase produces the cyclic monoterpene limonene, whereas incubation with (+/-)linalyl diphosphate (the tertiary allylic isomer of geranyl diphosphate) yields both acyclic and cyclic monoterpenes. These results suggest that the enzyme is incapable of isomerizing geranyl diphosphate to linalyl diphosphate, a feature common to all monoterpene cyclases from terrestrial sources. The limited catalytic capability of the myrcene synthase may reflect the ancient evolutionary origin of the producing organism. The ability to assay this enzyme in cultured algae, grown under strictly defined conditions, provides an unparalleled opportunity to delineate factors eliciting the biosynthesis of this class of secondary metabolites, to investigate the metabolic pathway leading to the halogenated monoterpenes, and to determine their role in the chemical ecology of marine algae.

Syn stereochemistry of cyclic ether formation in 1,8-cineole biosynthesis catalyzed by recombinant synthase from Salvia officinalis.

Regiospecifically labeled geranyl diphosphates ((2E,6E)-[1,1,8,8,8-(2)H(5)]- and (2E,6Z)-[1,1,9,9,9-(2)H(5)]-GDP) and D(2)O incorporation, in concert with NMR spectrometry, were employed to demonstrate a unique intramolecular syn-facial protonation-cyclization mechanism of action of 1,8-cineole synthase.

Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA.

The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and pIs of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues.

Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase.

The x-ray crystal structure of dimeric (+)-bornyl diphosphate synthase, a metal-requiring monoterpene cyclase from Salvia officinalis, is reported at 2.0-A resolution. Each monomer contains two alpha-helical domains: the C-terminal domain catalyzes the cyclization of geranyl diphosphate, orienting and stabilizing multiple reactive carbocation intermediates; the N-terminal domain has no clearly defined function, although its N terminus caps the active site in the C-terminal domain during catalysis. Structures of complexes with aza analogues of substrate and carbocation intermediates, as well as complexes with pyrophosphate and bornyl diphosphate, provide "snapshots" of the terpene cyclization cascade.