The contribution of fiber components to water absorption of wheat grown in the UK.

BACKGROUND AND OBJECTIVES: The water absorption (WA) of white wheat flour is a major factor affecting processing quality, and millers, therefore, process their wheat to achieve the required level. Although it is likely that WA is determined by the amounts and compositions of three major grain components, starch, protein, and arabinoxylan, the contribution of the latter is not agreed and not recognized in the widely used Farrand equation. FINDINGS: We have measured a range of parameters related to fiber amount and composition and tested the ability of these to improve the prediction of WA using a modified Farrand equation. The addition of a range of single fiber traits improved the prediction of WA from a baseline of 82.98% to a maximum of 86.78%, but inclusion of all fiber traits as PCs resulted in a further improvement to 90%. Inclusion of the PCs also accounted for variation in WA between harvest years. The greatest improvement from inclusion of a single trait was observed with ß-glucan, the inclusion of arabinogalactan peptide (AGP) also resulted in improved prediction of WA. CONCLUSIONS: The study shows that fiber components contribute to variation in WA, including differences between harvest years, but that ß-glucan and AGP have similar or greater impacts than AX. SIGNIFICANCE AND NOVELTY: The study dissects the contributions of AX amount and composition to WA and demonstrates a contribution of b-glucan for the first time.

Cyclic-AMP initiates protein tyrosine phosphorylation independent of cholesterol efflux during ram sperm capacitation.

Unlike most other species, ram spermatozoa are difficult to capacitate in vitro. Bicarbonate and Ca(2+) are necessary, whereas bovine serum albumin does not appear to be obligatory. In the present investigation we have assessed (1) the ability of the cholesterol-sequestering agent, methyl-beta-cyclodextrin (M-beta-CD), to initiate protein tyrosine phosphorylation, and (2) the importance of phosphodiesterases (PDEs) in controlling the levels of cAMP. Results show that despite removing significant amounts of membrane cholesterol, as assessed by filipin staining, M-beta-CD treatment did not stimulate major increases in protein tyrosine phosphorylation. Addition of a cocktail of PDE inhibitors (theophylline and caffeine), a phosphatase inhibitor (okadaic acid) and dibutyryl-cAMP (db-cAMP), however, stimulated specific tyrosine phosphorylation of several proteins between 30 and 120 kDa. On their own, none of the above reagents were effective but a combination of db-cAMP + PDE inhibitors was sufficient to achieve a maximal response. H-89, a protein kinase-A inhibitor, suppressed tyrosine phosphorylation significantly. Immunofluorescence revealed that the newly-phosphorylated proteins localised mainly in the sperm tail. These findings suggest that in ram spermatozoa cAMP levels are too low to initiate tyrosine phosphorylation of flagellar proteins that are indicative of the capacitation state and that this is caused by unusually high levels of intracellular PDEs.

Supramolecular organization of the sperm plasma membrane during maturation and capacitation.

AIM: In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm's plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. METHODS: Atomic force microscopy identified a novel region (EqSS) within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. RESULTS: Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. CONCLUSION: A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg.

Interactions between zona pellucida glycoproteins and sperm proacrosin/acrosin during fertilization.

Fertilization is one of the most specific and carefully regulated cell-cell interactions in the animal body and is determined to a large extent by compatibility between ligand and receptor molecules on the surface of each gamete. On the zona pellucida (ZP), sperm receptor activity is associated with glycoproteins ZP3 (primary receptor for acrosome-intact sperm) and ZP2 (secondary receptor for acrosome-reacted sperm) but their complementary binding proteins on sperm are less well defined. In this communication we review the evidence for proacrosin as a secondary ZP binding protein. Proacrosin/acrosin binds non-enzymically to ZP glycoproteins. Binding is a strong ionic interaction between polysulphate groups on ZP glycoproteins (probably on their carbohydrate moieties) and basic residues on the surface of proacrosin. The stereochemistry of the reactants is crucial and determines to a large extent the affinity of binding. Site-directed mutagenesis and a 3D-structural analysis of boar and ram acrosin have identified 2 clusters of basic residues potentially involved in binding. A polysulphonated anticancer drug, suramin, has been shown to bind strongly to proacrosin/acrosin and to inhibit sperm-egg binding in vitro. In the mouse model, 125I-ZP2 and 3H-suramin bind approximately 65% less effectively to acrosin 'null' sperm than to wild-type sperm. Neither ZP2 nor suramin bind to acrosome intact sperm and can, therefore, only exert their effects after exposure of the acrosomal contents. Overall, this combination of biochemical, genetic and functional data supports the hypothesis that proacrosin is a multifunctional protein with a significant role in retaining acrosome-reacted sperm on the ZP surface long enough to enable ZP penetration to begin.