Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat

RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms. The elucidation of the mechanism of RNA silencing has stimulated its use as a reverse genetics tool, because RNA silencing strongly down-regulates the expression of the target gene in a sequence-specific manner. The major protein fraction of wheat grain is gluten which is largely responsible for the functional properties of dough. Gliadins contribute mainly to the extensibility and viscosity of gluten and dough, with the polymeric glutenins being responsible for elasticity. The aim of this work was therefore to silence the expression of specific γ-gliadins by RNA interference, to demonstrate the feasibility of systematically silencing specific groups of gluten proteins. The sequence of a γ-gliadin gene was used to construct the pghp8.1 plasmid. The hpRNA silencing fragment was designed on the basis of 169 base pairs (bp) in sense and antisense orientation with the sequence of the Ubi1 intron as spacer region between the repeats. Two lines of bread wheat were transformed by particle bombardment. Gliadins were extracted from 30 mg of flour, separated by acid-PAGE and determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Seven transgenic lines were obtained and all of them showed reduced levels of γ-gliadins. All seven transgenic plants were fully fertile and their grain morphology and seed weight were comparable to the control lines. MALDI-TOF MS showed that six peaks, present in the untransformed line, were missing in transgenic lines of the BW208 genotype whereas three peaks were missing in the BW2003 genotypes. The proportion of γ-gliadins was reduced, by about 55–80% in the BW208 lines and by about 33–43% in the BW2003 lines. The ELISA assay based on the R5 antibody showed reductions in total gliadins (μg/mg flour) in three of the BW208 lines and in one BW2003 line, but an increase in one BW208 line (C613).     

Disease development and PR-protein activity in wheat (Triticum aestivum) seedlings treated with plant extracts prior to leaf rust (Puccinia triticina) infection

The potential to control leaf rust (Puccinia triticina; pathotype UVPt9) in vivo in susceptible (Thatcher) and resistant (Thatcher/Lr15) near-isogenic wheat (Triticum aestivum) lines by foliar applications of crude plant leaf extracts from Tulbaghia violacea and Agapanthus africanus, as well as a commercially available natural product, ComCat^®, and a Lupinus albus seed suspension (SS) was investigated. In vitro activities of the PR-proteins, β-1,3-glucanase, chitinase and peroxidase were quantified in these two wheat lines under both non-infected and infected conditions. Although foliar application of the different plant extracts accelerated the activities of all three enzymes in resistant infected wheat plants, it was only the A. africanus extract that increased the in vitro activities of the three apoplastic pathogenesis-related proteins significantly in both susceptible and resistant wheat cultivars, whether uninfected or infected. The latter resulted in the reduction of pustule and necrotic lesion formation in the susceptible and resistant wheat lines, respectively. Foliar treatment of wheat seedlings with either ComCat^® or SS had no effect on pustule and necrotic lesion formation. The A. africanus crude extract also had a significant in vitro inhibitory effect on both leaf rust spore germination and subsequent germ tube development.     

The combined use of hull-less barley flour and xylanase as a strategy for wheat/hull-less barley flour breads with increased arabinoxylan and (1→3,1→4)-β-D-glucan levels

Bread-making with a composite flour (CF) consisting of 60% wheat flour (WF) and 40% hull-less barley flour, increased the total and soluble (1→3,1→4)-β-D-glucan and total arabinoxylan (AX) contents of dough and bread samples, but decreased the specific bread loaf volume. A xylanase insensitive to inhibition by Triticum aestivum L. xylanase inhibitor (TAXI) and xylanase inhibiting protein (XIP), increased loaf volume by 8.8 and 20.1% for WF and CF breads, respectively. Xylanase addition not only markedly improved loaf volume of CF bread, but also increased the soluble AX content of the WF and CF dough and bread samples because of conversion of water-unextractable AX into soluble AX. The xylanase had no impact on the extractability and molecular weight of (1→3,1→4)-β-D-glucan, but (1→3,1→4)-β-D-glucan was degraded during bread-making probably because of endogenous β-glucanase activity. Taken together, the results clearly show that the combined use of hull-less barley flour and a xylanase active during bread making, lead to palatable breads with high total and soluble AX and (1→3,1→4)-β-D-glucan contents. The sum of total AX and (1→3,1→4)-β-D-glucan was 1.70% for WF bread and 3.06% for CF bread, while the sum of soluble AX and (1→3,1→4)-β-D-glucan was 0.49 and 1.41% for control WF and CF xylanase supplemented breads, respectively.     

Interactions of Globular and Ribbon [γ4E]GID with α4β2 Neuronal Nicotinic Acetylcholine Receptor

The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson’s and Alzheimer’s diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(−) or α4(+)α4(−) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)₃(β2)₂ nAChR was deduced using the information from the cryo-electron structure of (α4)₃(β2)₂ nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(−) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)₃(β2)₂ nAChR.     

Isolation of Scalimides A–L: β-Alanine-Bearing Scalarane Analogs from the Marine Sponge Spongia sp.

A chemical investigation of a methanol extract of Spongia sp., a marine sponge collected from the Philippines, identified 12 unreported scalarane-type alkaloids—scalimides A–L (1–12)—together with two previously described scalarin derivatives. The elucidation of the structure of the scalaranes based on the interpretation of their NMR and HRMS data revealed that 1–12 featured a β-alanine-substituted E-ring but differed from each other through variations in their oxidation states and substitutions occurring at C16, C24, and C25. Evaluation of the antimicrobial activity of 1–12 against several Gram-positive and Gram-negative bacteria showed that 10 and 11 were active against Micrococcus luteus and Bacillus subtilis, respectively, with MIC values ranging from 4 to 16 μg/mL.     

Yield dilution of grain Zn in wheat grown in open-top chamber experiments with elevated CO2 and O3 exposure

Wheat (Triticum aestivum L.) grain Zn data from six open-top chamber experiments performed in south-west Sweden were combined to study the relationship between Zn accumulation and grain yield, grain protein, and yield components. Treatments included, in addition to open-top chamber controls, elevated CO₂, elevated O₃, combined CO₂ and O₃ exposure, combined elevated CO₂ and supplemental irrigation, supplemental irrigation, and ambient air comparison plots. The grain Zn concentration was strongly correlated with grain protein (R² = 0.90) over the range of the experimental treatments, representing non-soil factors. A significant yield dilution effect was found for Zn. For a 10% increase in grain yield, Zn yield was increased by 6.8% on average. Effects on Zn yield correlated strongly with effects on grain protein yield, with a slope close to unity, showing that yield dilution effects for grain Zn and grain protein were similar. Treatment effects on grain Zn concentration were related to effects on grain weight (P < 0.01) and grain number (P < 0.05), but not to harvest index. It was concluded that yield stimulation caused by rising CO₂ concentrations is likely to lead to reduced Zn concentrations of wheat grain, thus reducing the nutritional quality.     

Mechanism of Action and Therapeutic Potential of the β-Hairpin Antimicrobial Peptide Capitellacin from the Marine Polychaeta Capitella teleta

Among the most potent and proteolytically resistant antimicrobial peptides (AMPs) of animal origin are molecules forming a β-hairpin structure stabilized by disulfide bonds. In this study, we investigated the mechanism of action and therapeutic potential of the β-hairpin AMP from the marine polychaeta Capitella teleta, named capitellacin. The peptide exhibits a low cytotoxicity toward mammalian cells and a pronounced activity against a wide range of bacterial pathogens including multi-resistant bacteria, but the mechanism of its antibacterial action is still obscure. In view of this, we obtained analogs of capitellacin and tachyplesin-inspired chimeric variants to identify amino acid residues important for biological activities. A low hydrophobicity of the β-turn region in capitellacin determines its modest membranotropic activity and slow membrane permeabilization. Electrochemical measurements in planar lipid bilayers mimicking the E. coli membrane were consistent with the detergent-like mechanism of action rather than with binding to a specific molecular target in the cell. The peptide did not induce bacterial resistance after a 21-day selection experiment, which also pointed at a membranotropic mechanism of action. We also found that capitellacin can both prevent E. coli biofilm formation and destroy preformed mature biofilms. The marked antibacterial and antibiofilm activity of capitellacin along with its moderate adverse effects on mammalian cells make this peptide a promising scaffold for the development of drugs for the treatment of chronic E. coli infections, in particular those caused by the formation of biofilms.     

α-Conotoxin TxID and [S9K]TxID, α3β4 nAChR Antagonists,Attenuate Expression and Reinstatement of Nicotine-Induced Conditioned Place Preference in Mice

Tobacco smoking has become a prominent health problem faced around the world. The α3β4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3β4 nAChR antagonist, which has weak inhibition activity of α6/α3β4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3β4 nAChR (IC₅₀ = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3β4 nAChR antagonists (TxID and [S9K]TxID) on the expression and reinstatement of nicotine-induced conditioned place preference (CPP) and explores the behaviors of acute nicotine in mice. The animal experimental results showed that TxID and [S9K] TxID could inhibit the expression and reinstatement of CPP, respectively. Moreover, both had no effect in acute nicotine experiment and the locomotor activity in mice. Therefore, these findings reveal that the α3β4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3β4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.     

Suitability of tef varieties in mixed wheat flour bread matrices: A physico-chemical and nutritional approach

Wheat flour replacement from 0 to 40% by single tef flours from three Ethiopian varieties DZ-01-99 (brown grain tef), DZ-Cr-37 (white grain tef) and DZ-Cr-387 (Quncho, white grain tef) yielded a technologically viable ciabatta type composite bread with acceptable sensory properties and enhanced nutritional value, as compared to 100% refined wheat flour. Incorporation of tef flour from 30% to 40% imparted discreet negative effects in terms of decreased loaf volume and crumb resilience, and increase of crumb hardness in brown tef blended breads. Increment of crumb hardness on aging was in general much lower in tef blended breads compared to wheat bread counterparts, revealing slower firming kinetics, especially for brown tef blended breads. Blended breads with 40% white tef exhibited similar extent and variable rate of retrogradation kinetics along storage, while brown tef-blended breads retrograded slower but in higher extent than control wheat flour breads. Breads that contains 40% tef grain flour were found to contain five folds (DZ-01-99, DZ-Cr-387) to 10 folds (DZ-Cr-37) Fe, three folds Mn, twice Cu, Zn and Mg, and 1.5 times Ca, K, and P contents as compared to the contents found in 100% refined wheat grain flour breads. In addition, suitable dietary trends for lower rapidly digestible starch and starch digestion rate index were met from tef grain flour fortified breads.     

Dough quality of bread wheat lacking α-gliadins with celiac disease epitopes and addition of celiac-safe avenins to improve dough quality

Celiac disease is a T-cell mediated immune response in the small intestine of genetically susceptible individuals caused by ingested gluten proteins from wheat, rye, and barley. In the allohexaploid bread wheat (Triticum aestivum), gluten proteins are encoded by multigene loci present on the homoeologous chromosomes 1 and 6 of the three homoeologous genomes A, B, and D. The effect of deleting individual gluten loci was analyzed in a set of deletion lines of T. aestivum cv. Chinese Spring with regard to the level of T-cell stimulatory epitopes (Glia-α9 and Glia-α20) and to technological properties of the dough including mixing, stress relaxation, and extensibility.     

β-Elimination reactions and formation of covalent cross-links in gliadin during heating at alkaline pH

The aim of this study was to increase insight into gluten polymerisation. While previous research on this topic focused on disulfide (SS) bonds, the present paper focuses on cross-links based on dehydro-protein formation through β-elimination reactions. Gliadin, the monomeric fraction of gluten containing no free sulfhydryl (SH) groups, was heated for 120 min at pH 8.0 and 130 °C, and cross-link formation was evaluated by determining extractability in sodium dodecyl sulfate containing buffer, reaction products of β-elimination reactions, and cross-links involving the latter. Heating decreased gliadin extractability. Reduction of SS bonds increased extractability of heated gliadin, but did not restore it to that of non-heated gliadin, suggesting contribution of both SS and non-SS bonds to gliadin cross-linking. Decreased SS levels and the presence of dehydroalanine and SH groups in heated gliadin samples indicated cleavage of SS bonds by β-elimination reactions. Some of the formed free SH groups were then involved in oxidation and/or SH–SS interchange reactions leading to intermolecular SS bonds. In addition, amino acid analysis revealed formation of an irreversible non-SS cross-link between dehydroalanine and the free SH group of cysteine, namely lanthionine. In conclusion, non-SS bonds may well contribute to the gluten network under specified reaction conditions.     

Alpha‐tocopherol and β‐carotene in legume–grass mixtures as influenced by wilting,ensiling and type of silage additive

Effects of wilting, ensiling and type of additive on α‐tocopherol and β‐carotene contents in legume–grass mixtures were examined. Swards of birdsfoot trefoil + timothy (Bft + Ti), red clover + timothy (Rc + Ti) and red clover + meadow fescue (Rc + Mf) were harvested as a first regrowth in August 2005. Forage was wilted to a dry‐matter (DM) content of 273 g kg^?1 and ensiled without additive or with an inoculant or acid. Wilting decreased α‐tocopherol concentration by 30% in the Bft + Ti mixture (P = 0·015). Untreated Bft + Ti silage had higher α‐tocopherol content than red clover silages (56·9 vs. 34·2 mg kg^?1 DM; P = 0·015). The α‐tocopherol concentration of Bft + Ti forages increased during ensiling from 41·1 mg kg^?1 DM in wilted herbage to 56·9, 65·2 and 56·8 mg kg^?1 DM in untreated, inoculated and acid‐treated silage respectively (P = 0·015). The inoculant increased α‐tocopherol content in the red clover silages (50·1 vs. 34·2 mg kg^?1 DM; P = 0·015) compared with untreated red clover silages. Red clover mixtures had lower β‐carotene content than Bft + Ti (32·3 vs. 46·2 mg kg^?1 DM; P = 0·016), averaged over treatments. In conclusion, wilting had small effects but the use of bacterial inoculant as an additive and a Bft + Ti mixture increased α‐tocopherol concentration in the silage.     

Near infrared spectra indicate specific mutant endosperm genes and reveal a new mechanism for substituting starch with (1→3,1→4)-β-glucan in barley

Near Infrared Reflectance spectroscopy was tested as a screening method to characterise high lysine mutants from a barley collection by classification through Principal Component Analysis (PCA). Mean spectra of the samples within each cluster identified gene-specific patterns in the 2270–2360 nm region. The characteristic spectral signatures representing the lys5 locus (Risø mutants 13 and 29) were found to be associated with large changes in percentage of starch and (1→3,1→4)-β-glucan. These alleles compensated for a low level of starch (down to 30%) by a high level of (1→3,1→4)-β-glucan (up to 15–20%), thus, maintaining a constant production of polysaccharides at 50–55%, within the range of normal barley.The spectral tool was tested by an independent data set with six mutants with unknown polysaccharide composition. Spectral data from four of these were classified within the high (1→3,1→4)-β-glucan BG lys5 cluster in a PCA. Their high (1→3,1→4)-β-glucan and low starch content was verified. It is concluded that genetic diversity such as from gene regulated polysaccharide and storage protein pathways in the endosperm tissue can be discovered directly from the phenotype by chemometric classification of a spectral library, representing the digitised phenome from a barley gene bank.     

Structure,chemical composition and enzymatic activities of pearlings and bran obtained from pearled wheat (Triticum aestivum L.) by roller milling

While abrasive pearling (also referred to as debranning) of wheat kernels prior to milling increases the quality of the resultant flour for producing bread, the potential applications of the co-products of pearling is largely unknown. We studied the impact of different degrees of pearling (0, 3, 6, 9 and 12% by weight) on the composition of pearlings and bran obtained when subsequently roller milling pearled wheat kernels. Pearling does not remove the kernel outer tissues homogeneously as abrasion affects especially the accessible parts of the kernels. Nevertheless, the first 3% removed consisted of mainly pericarp. With 6% or more removed, a significant amount of starchy endosperm ended up in the pearlings. The starting bran material and bran obtained by subsequent roller milling of pearled wheat kernels had similar compositions but the latter had a lower average particle size. Moreover, removal of the outermost kernel layers substantially decreased the enzyme activity levels in the bran.     

壳聚糖复合β-乳球蛋白负载EGCG纳米粒的制备及其对糖尿病小鼠血糖的影响

表没食子儿茶素没食子酸酯（EGCG）作为茶叶中主要生物活性成分,具有良好的生理功能,但低稳定性使其容易被氧化降解,生物利用率低。利用羧甲基壳聚糖（CMC）、壳聚糖盐酸盐（CHC）、β-乳球蛋白（β-LG）作为壁材,制备壳聚糖复合β-乳球蛋白负载EGCG纳米粒,通过透射电镜、结构表征（粒径、Zeta电位测定）对颗粒微观形态进行观察,利用高效液相色谱仪对颗粒包埋率、模拟胃肠液释放率进行测定,最后建立糖尿病小鼠模型,探究包埋后颗粒的降血糖活性。结果表明,CS-β-LG-EGCG纳米粒结构完整、粒径10~100 nm、粒子分散;包埋率大于50%,且在肠液和胃液中具有缓释作用;CS-β-LG-EGCG纳米粒与胰岛素无拮抗作用,与未包埋的EGCG相比,包埋后颗粒具备的缓释作用可减缓血糖的回升。     

Interspecific and Intraspecific Variation in Grain and Groat Characteristics of Wild Oat (Avena) Species: Very High Groat (1→3),(1→4)-β- -glucan in an Avena atlantica Genotype

Grain characteristics and groat composition have been evaluated in 35 genotypes from nine taxonomic species of Avena, including three species (A. agadiriana, A. atlantica, A. damascena) for which no previous data are available. There was substantial interspecific and intraspecific variation for all characteristics measured. The proportion of groat in the grain ranged from 32·7–62·1%, and mean groat weight from 2·4–37·4 mg. Groat protein concentrations ranged from 13·9–41·3%, and exceeded 32% in one A. atlantica, two A. damascena and one A. murphyi genotype. Groat β-glucan concentration showed very wide variation (2·2–11·3%) are there were substantial interspecific and intraspecific differences. The highest β-glucan concentrations were found in genotypes of A. atlantica. Although there were interspecific and intraspecific variations in groat oil concentration (4·2–10·6%), and in fatty acid composition, data were within previously reported ranges for A. sativa. Overall these data indicate that some of the genotypes of the wild species studied may be of value for breeding oats with improved levels of β-glucan and protein, and that further studies are warranted into both interspecific and intraspecific variations in grain quality factors in wild oat species.