Determination of soluble wheat protein fractions using the Bradford assay

Background and objectives Determination of different grain protein fractions in wheat cultivars is an important task in analyzing bread baking quality. In many laboratories, the Bradford assay is used to determine protein concentrations in solutions. In any protein assay (including Bradford), the ideal protein to use as a standard is the purified protein being assayed. In the absence of such an absolute reference, protein another protein must be selected as a relative standard such as bovine serum albumin (BSA) which is widely used. The aim of this work was to find conversion factors for BSA to determine correct albumin–globulin, gliadin, and glutenin concentrations, because these purified wheat grain protein fractions are mostly not available to be used for calibration purposes. Findings In case of BSA calibration, gluten concentration was underestimated (50%–54%) compared to calibration with the respective purified wheat proteins (65%–70%) in extracts of wheat grain samples. This result is explained with the different amino acid composition of BSA and wheat protein fractions leading to a more intense signal with BSA in the Bradford assay. Calibration of the Bradford assay using BSA as well as purified wheat protein fractions allowed to calculate the conversion factors of 2.11 for BSA/albumin–globulin, 4.25 for BSA/gliadin, and 3.42 for BSA/glutenin. Application of these conversion factors proved to accurately adjust protein concentrations of wheat fractions originating from ten cultivars, determined with BSA calibration of the Bradford assay. Conclusions BSA calibration of the Bradford assay in combination with the conversion factors can be used to determine protein concentration of wheat grain fractions. Significance and novelty Findings of this study make a contribution toward the correction of a common method, to provide a basis for better comparability of research results from different laboratories

A Protein-Linger Strategy Keeps the Plant On-Hold After Rehydration of Drought-Stressed Beta vulgaris

Most crop plants are exposed to intermittent drought periods. To cope with these continuous changes, plants need strategies to prevent themselves from exhaustive adjustment maneuvers. Drought stress recovery has been shown to be an active process, possibly involved in a drought memory effect allowing plants to better cope with recurrent aridity. An integrated understanding of the molecular processes of enhanced drought tolerance is required to tailor key networks for improved crop protection. During summer, prolonged periods of drought are the major reason for economic yield losses of sugar beet (Beta vulgaris) in Europe. A drought stress and recovery time course experiment was carried out under controlled environmental conditions. In order to find regulatory key mechanisms enabling plants to rapidly react to periodic stress events, beets were either subjected to 11 days of progressive drought, or were drought stressed for 9 days followed by gradual rewatering for 14 days. Based on physiological measurements of leaf water relations and changes in different stress indicators, plants experienced a switch from moderate to severe water stress between day 9 and 11 of drought. The leaf proteome was analyzed, revealing induced protein pre-adjustment (prior to severe stress) and putative stress endurance processes. Three key protein targets, regulatory relevant during drought stress and with lingering levels of abundance upon rewatering were further exploited through their transcript performance. These three targets consist of a jasmonate induced, a salt-stress enhanced and a phosphatidylethanolamine-binding protein. The data demonstrate delayed protein responses to stress compared to their transcripts and indicate that the lingering mechanism is post-transcriptionally regulated. A set of lingering proteins is discussed with respect to a possible involvement in drought stress acclimation and memory effects

Pathogenic T cell responses against aquaporin 4

Inflammatory lesions in the central nervous system of patients with neuromyelitis optica are characterized by infiltration of T cells and deposition of aquaporin-4-specific antibodies and complement on astrocytes at the glia limitans. Although the contribution of aquaporin-4-specific autoantibodies to the disease process has been recently elucidated, a potential role of aquaporin-4-specific T cells in lesion formation is unresolved. To address this issue, we raised aquaporin-4-specific T cell lines in Lewis rats and characterized their pathogenic potential in the presence and absence of aquaporin-4-specific autoantibodies of neuromyelitis optica patients. We show that aquaporin-4-specific T cells induce brain inflammation with particular targeting of the astrocytic glia limitans and permit the entry of pathogenic anti-aquaporin-4-specific antibodies to induce NMO-like lesions in spinal cord and brain. In addition, transfer of aquaporin-4-specific T cells provoked mild (subclinical) myositis and interstitial nephritis. We further show that the expression of the conformational epitope, recognized by NMO patient-derived aquaporin-4-specific antibodies is induced in kidney cells by the pro-inflammatory cytokine gamma-interferon. Our data provide further support for the view that NMO lesions may be induced by a complex interplay of T cell mediated and humoral immune responses against aquaporin-4

Is a Change of Protein Composition after Late Application of Nitrogen Sufficient to Improve the Baking Quality of Winter Wheat?

Concentration and composition of storage proteins affect the baking quality of wheat. Although both are influenced by late nitrogen fertilization, it is not clear whether compositional changes are sufficient to improve the baking quality, and whether such effects are cultivar specific. In a pot experiment, two winter wheat cultivars belonging to different quality classes were supplied with two levels of late N fertilizer. Protein subunits were analysed by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). Late N supply increased grain yield and protein content in both cultivars, but improved baking quality only in Discus, correlated with stronger changes in glutenin and gliadin fractions. Where baking quality was improved, this occurred at the lower late N level. Overall, the composition rather than the amount of gluten proteins was decisive for flour quality. Measures for enhancing grain protein concentration and composition are less necessary for cultivars such as Rumor in order to achieve optimum baking quality. These results open up an opportunity to reduce N fertilization in wheat production systems

2-D clinorotation alters the uptake of some nutrients in Arabidopsis thaliana

Future long-term spaceflight missions rely on bioregenerative life support systems (BLSS) in order to provide the required resources for crew survival. Higher plants provide an essential part since they supply food and oxygen and recycle carbon dioxide. There are indications that under space conditions plants might be inefficient regarding the uptake, transport and distribution of nutrients, which in turn affects growth and metabolism. Therefore, Arabidopsis thaliana (Col-0) seeds were germinated and grown for five days under fast clinorotation (2-D clinostat, 60rpm) in order to simulate microgravity. Concentrations of ten different nutrients (potassium, sulfur, phosphorus, calcium, sodium, magnesium, manganese, iron, zinc, and boron) in shoots of plants grown under reduced and normal (1g) gravity conditions were compared. A protocol was developed for the determination of different nutrients by means of inductively coupled plasma optical emission spectrometry (ICPOES), flame emission spectrometry and spectrophotometry. The concentrations of boron and sulfur were significantly decreased in clinorotated shoots, while the concentration of sodium was elevated, suggesting that altered gravity conditions differentially affected nutrient uptake. Possible mechanisms for such effects include reduced transpiration, altered expression of channels or transporters and direct effects on nutrient assimilation. The observed nutrient imbalances might have a negative impact on plant growth and nutritional quality during prolonged space missions

Genomic profiling of acquired resistance to apoptosis in cells derived from human atherosclerotic lesions: Potential role of STATs, cyclinD1, BAD, and Bcl-X\u3csub\u3eL\u3c/sub\u3e

Current theories suggest that atherosclerosis, plaque rupture, stroke, and restenosis after angioplasty may involve defective apoptotic mechanisms in vascular cells. Prior work has demonstrated that cells from human atherosclerotic lesions, and cells from the aorta of aged rats, exhibit functional resistance to apoptosis induced by TGF-β and glucocorticoids. The present studies demonstrate that human lesion-derived cells (LDC) are also resistant to apoptosis induced by fas ligation compared to cells derived from the adjacent media, and that in vitro expansion of LDC causes acquired resistance to apoptosis. Microarray profiling of fas-resistant versus sensitive cells identified a set of genes including STATs, caspase 1, cyclin D1, Bcl-xL, VDAC2, and BAD. The STAT proteins have been implicated in resistance to apoptosis, potentially via their ability to modulate caspase 1 (ICE), Bcl-xL, and cyclin D1 expression. Western blot analysis of sensitive and resistant LDC clonal lines confirmed increases in cyclin D1, STAT6, Bcl-xL, and BAD, with decreased expression of caspase 1. Thus, transcript profiling has identified a potential pathway of apoptotic regulation in subsets of lesion cells. The resistant phenotype may contribute to plaque stability and excessive vascular repair, while sensitive cells may be involved in plaque rupture and infarction. The data suggests both genetic interventions and novel small-molecule inhibitors that may be effective modulators of apoptosis in atherosclerosis, angina, and in-stent restenosis. © 2005 Elsevier Ltd. All rights reserved

1H-NMR metabolomic profiling reveals a distinct metabolic recovery response in shoots and roots of temporarily drought-stressed sugar beets.

Yield formation in regions with intermittent drought periods depends on the plant's ability to recover after cessation of the stress. The present work assessed differences in metabolic recovery of leaves and roots of drought-stressed sugar beets with high temporal resolution. Plants were subjected to drought for 13 days, and rewatered for 12 days. At one to two-day intervals, plant material was harvested for untargeted 1H-NMR metabolomic profiling, targeted analyses of hexose-phosphates, starch, amino acids, nitrate and proteins, and physiological measurements including relative water content, osmotic potential, electrolyte leakage and malondialdehyde concentrations. Drought triggered changes in primary metabolism, especially increases in amino acids in both organs, but leaves and roots responded with different dynamics to rewatering. After a transient normalization of most metabolites within 8 days, a second accumulation of amino acids in leaves might indicate a stress imprint beneficial in upcoming drought events. Repair mechanisms seemed important during initial recovery and occurred at the expense of growth for at least 12 days. These results indicate that organ specific metabolic recovery responses might be related to distinct functions and concomitant disparate stress levels in above- and belowground organs. With respect to metabolism, recovery was not simply a reversal of the stress responses