BackgroundWheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences.ResultsGrain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%.ConclusionsThis is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome the difficulties of matching peptides to gene sequences for members of highly similar, rapidly evolving storage protein families. Prospects for simplifying this process for routine analyses are discussed. The ability to measure expression levels for individual flour protein genes complements information gained from efforts to sequence the wheat genome and is essential for studies of effects of environment on gene expression.

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The preparation of a reconstructed human epidermis is described with examples of its utilization in in vitro studies. The model was obtained by culturing normal human keratinocytes at high cell density for 14 days in serum-free and high calcium (1.5 m M) medium on an inert polycarbonate filter at the air-liquid interface. These stratified cultures showed histological features similar to those observed in vivo in the epidermis: a proliferating basal layer and differentiating spinous, granular, and cornified layers. Electron microscopy illustrated lamellar bodies, junctions and keratohyalin granules. Immunofluorescent localization of epidermal markers (keratins 14 and 10, involucrin and filaggrin) revealed typical differentiation. This in vitro reconstructed tissue was used in studies of toxic effects of chemicals. The modelled tissue showed progressive cytotoxicity of a skin irritant (benzalkonium chloride) and a sensitizer (dinitrochlorobenzene) as assessed by MTT assay. Moreover, differential release of interleukin-1alpha and interleukin-8 were measured after 20 h of incubation allowing the irritant to be distinguished from the sensitizer. Permeation studies indicated efficient barrier function of the reconstructed epidermis, as well as metabolizing properties towards hormones. This model can be custom-made and is potentially useful for studies involving keratinocytes in the epidermis, in basic science, dermatology or toxicology.

Evidence was found for a Na/H antiport in tonoplast vesicles isolated from barley (Hordeum vulgare L. cv California Mariout 72) roots. The activity of the antiport was observed only in membranes from roots that were grown in NaCl. Measurements of acridine orange fluorescence were used to estimate relative proton influx and efflux from the vesicles. Addition of MgATP to vesicles from a tonoplast-enriched fraction caused the formation of a pH gradient, interior acid, across the vesicle membranes. EDTA was added to inhibit the ATPase, by chelating Mg2, and the pH gradient gradually dissipated. When 50 millimolar K' or Na' was added along with the EDTA to vesicles from control roots, the salts caused a slight increase in the rate of dissipation of the pH gradient, as did the addition of 50 millimolar K to vesicles from salt-rown roots. However, when 50 millimolar Na' was added to vesicles from salt-grown roots it caused a 7-fold increase in the proton efflux. Inclusion of 20 millimolar K' and 1 micromolar valinomycin in the assay buffer did not affect this rapid Na/H' exchange. The NaefH exchange rate for vesicles from salt-rown roots showed saturation kinetics with respect to Na concentration, with an apparent K. for Na of 9 millimolar. The rate of Na+/H+ exchange with 10 millimolar Na+ was inhibited 97% by 0.1 millimolar dodecyltriethylammonium.

By contrast to chloroplasts, our knowledge of amyloplasts--organelles that synthesize and store starch in heterotrophic plant tissues--is in a formative stage. While our understanding of what is considered their primary function, i.e. the biosynthesis and degradation of starch, has increased dramatically in recent years, relatively little is known about other biochemical processes taking place in these organelles. To help fill this gap, a proteomic analysis of amyloplasts isolated from the starchy endosperm of wheat seeds (10 d post-anthesis) has been conducted. The study has led to the identification of 289 proteins that function in a range of processes, including carbohydrate metabolism, cytoskeleton/plastid division, energetics, nitrogen and sulphur metabolism, nucleic acid-related reactions, synthesis of various building blocks, protein-related reactions, transport, signalling, stress, and a variety of other activities grouped under 'miscellaneous'. The function of 12% of the proteins was unknown. The results highlight the role of the amyloplast as a starch-storing organelle that fulfills a spectrum of biosynthetic needs of the parent tissue. When compared with a recent proteomic analysis of whole endosperm, the current study demonstrates the advantage of using isolated organelles in proteomic studies.