Spatial patterns of gluten protein and polymer distribution in wheat grain

The starchy endosperm is the major storage tissue in the mature wheat grain and exhibits quantitative and qualitative gradients in composition, with the outermost cell layers being rich in protein, mainly gliadins, and the inner cells being low in protein but enriched in high-molecular-weight (HMW) subunits of glutenin. We have used sequential pearling to produce flour fractions enriched in particular cell layers to determine the protein gradients in four different cultivars grown at two nitrogen levels. The results show that the steepness of the protein gradient is determined by both genetic and nutritional factors, with three high-protein breadmaking cultivars being more responsive to the N treatment than a low-protein cultivar suitable for livestock feed. Nitrogen also affected the relative abundances of the three main classes of wheat prolamins: the sulfur-poor ω-gliadins showed the greatest response to nitrogen and increased evenly across the grain; the HMW subunits also increased in response to nitrogen but proportionally more in the outer layers of the starchy endosperm than near the core, while the sulfur-rich prolamins showed the opposite trend.

Trafficking of storage proteins in developing grain of wheat

The processing properties of the wheat flour are largely determined by the structures and interactions of the grain storage proteins (also called gluten proteins) which form a continuous visco-elastic network in dough. Wheat gluten proteins are classically divided into two groups, the monomeric gliadins and the polymeric glutenins, with the latter being further classified into low molecular weight (LMW) and high molecular weight (HMW) subunits. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. However, determination of the precise routes of trafficking and deposition of individual gluten proteins in developing wheat grain has been limited in the past by the difficulty of developing monospecific antibodies. To overcome this limitation, a single gluten protein (a LMW subunit) was expressed in transgenic wheat with a C-terminal epitope tag, allowing the protein to be located in the cells of the developing grain using highly specific antibodies. This approach was also combined with the use of wider specificity antibodies to compare the trafficking and deposition of different gluten protein groups within the same endosperm cells. These studies are in agreement with previous suggestions that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or accumulating directly within the lumen of the ER. They also suggest that the same individual protein could be trafficked by either pathway, possibly depending on the stage of development, and that segregation of gluten proteins both between and within protein bodies may occur.

Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers

The low-molecular-weight (LMW) glutenin subunits are components of the highly cross-linked glutenin polymers that confer viscoelastic properties to gluten and dough. They have both quantitative and qualitative effects on dough quality that may relate to differences in their ability to form the inter-chain disulphide bonds that stabilise the polymers. In order to determine the relationship between dough quality and the amounts and properties of the LMW subunits, we have transformed the pasta wheat cultivars Svevo and Ofanto with three genes encoding proteins, which differ in their numbers or positions of cysteine residues. The transgenes were delivered under control of the high-molecular-weight (HMW) subunit 1Dx5 gene promoter and terminator regions, and the encoded proteins were C-terminally tagged by the introduction of the c-myc epitope. Stable transformants were obtained with both cultivars, and the use of a specific antibody to the c-myc epitope tag allowed the transgene products to be readily detected in the complex mixture of LMW subunits. A range of transgene expression levels was observed. The addition of the epitope tag did not compromise the correct folding of the trangenic subunits and their incorporation into the glutenin polymers. Our results demonstrate that the ability to specifically epitope-tag LMW glutenin transgenes can greatly assist in the elucidation of their individual contributions to the functionality of the complex gluten system.

Relationship between condensed tannin structures and their ability to precipitate feed proteins in the rumen

Abstract BACKGROUND Tannins can bind to and precipitate protein by forming insoluble complexes resistant to fermentation and with a positive effect on protein utilisation by ruminants. Three protein types, Rubisco, rapeseed protein and bovine serum albumin (a single high-molecular weight protein), were used to test the effects of increasing concentrations of structurally different condensed tannins on protein solubility/precipitation. RESULTS Protein type (PT) influenced solubility after addition of condensed tannins (P < 0.001) in the order: Rubisco < rapeseed < BSA (P < 0.05). The type of condensed tannin (CT) affected protein solubility (P = 0.001) with a CT × PT interaction (P = 0.001). Mean degree of polymerisation, proportions of cis- versus trans-flavanol subunits or prodelphinidins versus procyanidins among CTs could not explain precipitation capacities. Increasing tannin concentration decreased protein solubility (P < 0.001) with a PT × CT concentration interaction. The proportion of low-molecular weight rapeseed proteins remaining in solution increased with CT concentration but not with Rubisco. CONCLUSIONS Results of this study suggest that PT and CT type are both of importance for protein precipitation but that the CT structures investigated did not allow identification of parameters that contribute most to precipitation. It is possible that the three-dimensional structures of tannins and proteins may be more important factors in tannin–protein interactions. © 2013 Society of Chemical Industry

A laminated polymer film formulation for enteric delivery of live vaccine and probiotic bacteria

Live bacterial cells (LBC) are administered orally as attenuated vaccines, to deliver biopharmaceutical agents, and as probiotics to improve gastrointestinal health. However, LBC present unique formulation challenges and must survive gastrointestinal antimicrobial defenses including gastric acid after administration. We present a simple new formulation concept, termed Polymer Film Laminate (PFL). LBC are ambient dried onto cast acid-resistant enteric polymer films that are then laminated together to produce a solid oral dosage form. LBC of a model live bacterial vaccine and a probiotic were dried directly onto a cast film of enteric polymer. The effectiveness at protecting dried cells in a simulated gastric fluid (pH 2.0) depended on the composition of enteric polymer film used, with a blend of ethylcellulose plus Eudragit L100 55 providing greater protection from acid than Eudragit alone. However, although PFL made from blended polymers films completely released low molecular weight dye into intestinal conditions (pH 7.0), they failed to release LBC. In contrast, PFL made from Eudragit alone successfully protected dried probiotic or vaccine LBC from simulated gastric fluid for 2h, and subsequently released all viable cells within 60min of transfer into simulated intestinal fluid. Release kinetics could be controlled by modifying the lamination method.

The trafficking pathway of a wheat storage protein in transgenic rice endosperm

Background and Aims The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these. Methods The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies. Key results The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells. Conclusions The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.

Tripodal molecules for the promotion of phosphoester hydrolysis

A series of low molecular weight tripodal amide/histidine-containing compounds (1–2) have been synthesised and shown to increase the rate of bis-(p-nitrophenyl) phosphate (BNPP) and soman (GD) breakdown in buffered aqueous solution.

GTP binding controls complex formation by the human ROCO protein MASL1

The human ROCO proteins are a family of multi-domain proteins sharing a conserved ROC-COR supra-domain. The family has four members: leu- cine-rich repeat kinase 1 (LRRK1), leucine-rich repeat kinase 2 (LRRK2), death-associated protein kinase 1 (DAPK1) and malignant fibrous histiocy- toma amplified sequences with leucine-rich tandem repeats 1 (MASL1). Previous studies of LRRK1/2 and DAPK1 have shown that the ROC (Ras of complex proteins) domain can bind and hydrolyse GTP, but the cellular consequences of this activity are still unclear. Here, the first biochemical characterization of MASL1 and the impact of GTP binding on MASL1 complex formation are reported. The results demonstrate that MASL1, similar to other ROCO proteins, can bind guanosine nucleotides via its ROC domain. Furthermore, MASL1 exists in two distinct cellular com- plexes associated with heat shock protein 60, and the formation of a low molecular weight pool of MASL1 is modulated by GTP binding. Finally, loss of GTP enhances MASL1 toxicity in cells. Taken together, these data point to a central role for the ROC/GTPase domain of MASL1 in the reg- ulation of its cellular function.

Drug permeability in 16HBE14o- airway cell layers correlates with absorption from the isolated perfused rat lung

The permeability of the lung is critical in determining the disposition of inhaled drugs and the respiratory epithelium provides the main physical barrier to drug absorption. The 16HBE14o- human bronchial epithelial cell line has been developed recently as a model of the airway epithelium. In this study, the transport of 10 low molecular weight compounds was measured in the 16HBE14o- cell layers, with apical to basolateral (absorptive) apparent permeability coefficients (P(app)) ranging from 0.4 x 10(-6)cms(-1) for Tyr-D-Arg-Phe-Phe-NH(2) to 25.2x10(-6)cms(-1) for metoprolol. Permeability in 16HBE14o- cells was found to correlate with previously reported P(app) in Caco-2 cells and absorption rates in the isolated perfused rat lung (k(a,lung)) and the rat lung in vivo (k(a,in vivo)). Log linear relationships were established between P(app) in 16HBE14o- cells and P(app) in Caco-2 cells (r(2)=0.82), k(a,lung) (r(2)=0.78) and k(a,in vivo) (r(2)=0.68). The findings suggest that permeability in 16HBE14o- cells may be useful to predict the permeability of compounds in the lung, although no advantage of using the organ-specific cell line 16HBE14o- compared to Caco-2 cells was found in this study.

On the barrier properties of the cornea: a microscopy study of the penetration of fluorescently labeled nanoparticles, polymers, and sodium fluorescein

Overcoming the natural defensive barrier functions of the eye remains one of the greatest challenges of ocular drug delivery. Cornea is a chemical and mechanical barrier preventing the passage of any foreign bodies including drugs into the eye, but the factors limiting penetration of permeants and nanoparticulate drug delivery systems through the cornea are still not fully understood. In this study, we investigate these barrier properties of the cornea using thiolated and PEGylated (750 and 5000 Da) nanoparticles, sodium fluorescein, and two linear polymers (dextran and polyethylene glycol). Experiments used intact bovine cornea in addition to bovine cornea de-epithelialized or tissues pretreated with cyclodextrin. It was shown that corneal epithelium is the major barrier for permeation; pretreatment of the cornea with β-cyclodextrin provides higher permeation of low molecular weight compounds, such as sodium fluorescein, but does not enhance penetration of nanoparticles and larger molecules. Studying penetration of thiolated and PEGylated (750 and 5000 Da) nanoparticles into the de-epithelialized ocular tissue revealed that interactions between corneal surface and thiol groups of nanoparticles were more significant determinants of penetration than particle size (for the sizes used here). PEGylation with polyethylene glycol of a higher molecular weight (5000 Da) allows penetration of nanoparticles into the stroma, which proceeds gradually, after an initial 1 h lag phase.

An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.

Effect of breadmaking process on In Vitro gut microbiota parameters in irritable bowel syndrome

A variety of foods have been implicated in symptoms of patients with Irritable Bowel Syndrome (IBS) but wheat products are most frequently cited by patients as a trigger. Our aim was to investigate the effects of breads, which were fermented for different lengths of time, on the colonic microbiota using in vitro batch culture experiments. A set of in vitro anaerobic culture systems were run over a period of 24 h using faeces from 3 different IBS donors (Rome Criteria–mainly constipated) and 3 healthy donors. Changes in gut microbiota during a time course were identified by fluorescence in situ hybridisation (FISH), whilst the small -molecular weight metabolomic profile was determined by NMR analysis. Gas production was separately investigated in non pH-controlled, 36 h batch culture experiments. Numbers of bifidobacteria were higher in healthy subjects compared to IBS donors. In addition, the healthy donors showed a significant increase in bifidobacteria (P<0.005) after 8 h of fermentation of a bread produced using a sourdough process (type C) compared to breads produced with commercial yeasted dough (type B) and no time fermentation (Chorleywood Breadmaking process) (type A). A significant decrease of δ-Proteobacteria and most Gemmatimonadetes species was observed after 24 h fermentation of type C bread in both IBS and healthy donors. In general, IBS donors showed higher rates of gas production compared to healthy donors. Rates of gas production for type A and conventional long fermentation (type B) breads were almost identical in IBS and healthy donors. Sourdough bread produced significantly lower cumulative gas after 15 h fermentation as compared to type A and B breads in IBS donors but not in the healthy controls. In conclusion, breads fermented by the traditional long fermentation and sourdough are less likely to lead to IBS symptoms compared to bread made using the Chorleywood Breadmaking Process.

Effects of high hydrostatic pressure and chemical reduction on the emulsification properties of gum arabic

Gum arabic is widely used in the food industry as an additive, both as a thickener and an emulsifier. This study has compared the emulsification properties of two types of gums, KLTA (Acacia senegal) and GCA (Acacia seyal), both in their native/untreated forms and after exposure to high pressure (800 MPa). Further studies were undertaken to chemically modify the disulphide linkages present and to investigate the effects of their reduction on the diffusion of the carbohydrate materials. The emulsification properties of the gum samples were examined by determining the droplet size distribution in a ‘‘model’’ oil-in-water system. Results showed that high pressure treatment and chemical reduction of gums changed the emulsification properties of both gums. The high molecular weight component in arabinogalactanproteins (AGP/GP), and more ‘‘branched’’ carbohydrates present in gum arabic, may be responsible for the emulsification properties of GCA gum, indicating that the emulsification mechanisms for KLTA and GCA were different.

Tuning thermal properties and microphase separation in aliphatic polyester ABA copolymers

Four alkyl substituted β-lactones were investigated as monomers in ring opening polymerisation to produce a family of poly(3-hydroxyalkanoate)s. Homopolymers were synthesised using a robust aluminium salen catalyst, resulting in polymers with low dispersity (Đ < 1.1) and predictable molecular weights. ABA triblock copolymers were prepared using poly(L-lactic acid) as the A block and the aforementioned poly(3-hydroxyalkanoate) as the B block via a sequential addition method. Characterisation of these copolymers determined they were well controlled with low dispersities and predictable molecular weight. DSC analysis determined copolymers prepared from β-butyrolactone or β-valerolactone yielded polymers with tunable and predictable thermal properties. Copolymers prepared from β-heptanolactone yielded a microphase separated material as indicated by SAXS, with two distinct Tgs. The polymers could be readily cast into flexible films and their improved tensile properties were explored.

Influence of temperature during grain filling on gluten viscoelastic properties and gluten protein composition

BACKGROUND The aim of this study was to investigate the effects of low to moderate temperatures on gluten functionality and gluten protein composition. Four spring wheat cultivars were grown in climate chambers with three temperature regimes (day/night temperatures of 13/10, 18/15 and 23/20 °C) during grain filling. RESULTS The temperature strongly influenced grain weight and protein content. Gluten quality measured by maximum resistance to extension (Rmax) was highest in three cultivars grown at 13 °C. Rmax was positively correlated with the proportion of sodium dodecyl sulfate-unextractable polymeric proteins (%UPP). The proportions of ω-gliadins and D-type low-molecular-weight glutenin subunits (LMW-GS) increased and the proportions of α- and γ-gliadins and B-type LMW-GS decreased with higher temperature, while the proportion of high-molecular-weight glutenin subunits (HMW-GS) was constant between temperatures. The cultivar Berserk had strong and constant Rmax between the different temperatures. CONCLUSION Constant low temperature, even as low as 13 °C, had no negative effects on gluten quality. The observed variation in Rmax related to temperature could be explained more by %UPP than by changes in the proportions of HMW-GS or other gluten proteins. The four cultivars responded differently to temperature, as gluten from Berserk was stronger and more stable over a wide range of temperature