The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo

Muthuramalingam M, Matros A, Scheibe R, Mock H-P, Dietz K-J. The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo. Frontiers in Plant Science. 2013;4:54-1-54-14.Hydrogen peroxide (H2O2) evolves during cellular metabolism and accumulates under various stresses causing serious redox imbalances. Many proteomics studies aiming to identify proteins sensitive to H2O2 used concentrations that were above the physiological range. Here the chloroplast proteins were subjected to partial oxidation by exogenous addition of H2O2 equivalent to 10% of available protein thiols which allowed for the identification of the primary targets of oxidation. The chosen redox proteomic approach employed differential labeling of non-oxidized and oxidized thiols using sequential alkylation with N-ethylmaleimide and biotin maleimide. The in vitro identified proteins are involved in carbohydrate metabolism, photosynthesis, redox homeostasis, and nitrogen assimilation. By using methyl viologen that induces oxidative stress in vivo, mostly the same primary targets of oxidation were identified and several oxidation sites were annotated. Ribulose-1,5-bisphosphate (RubisCO) was a primary oxidation target. Due to its high abundance, RubisCO is suggested to act as a chloroplast redox buffer to maintain a suitable redox state, even in the presence of increased reactive oxygen species release. 2-cysteine peroxiredoxins (2-Cys Prx) undergo redox-dependent modifications and play important roles in antioxidant defense and signaling. The identification of 2-Cys Prx was expected based on its high affinity to H2O2 and is considered as a proof of concept for the approach. Targets of Trx, such as phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, and sedoheptulose-1,7-bisphosphatase have at least one regulatory disulfide bridge which supports the conclusion that the identified proteins undergo reversible thiol oxidation. In conclusion, the presented approach enabled the identification of early targets of H2O2 oxidation within the cellular proteome under physiological experimental conditions

Comparative evaluation of extraction methods for apoplastic proteins from maize leaves

Proteins in the plant apoplast are essential for many physiological processes. We have analysed and compared six different infiltration solutions for proteins contained in the apoplast to recognize the most suitable method for leaves and to establish proteome maps for each extraction. The efficiency of protocols was evaluated by comparing the protein patterns resolved by 1-DE and 2-DE, and revealed distinct characteristics for each infiltration solution. Nano-LC-ESI-Q-TOF MS analysis of all fractions was applied to cover all proteins differentially extracted by infiltration solutions and led to the identification of 328 proteins in total in apoplast preparations. The predicted subcellular protein localisation distinguished the examined infiltration solutions in those with high or low amounts of intracellular protein contaminations, and with high or low quantities of secreted proteins. All tested infiltration solution extracted different subsets of proteins, and those implications on apoplast-specific studies are discussed

The sugar dataset:A multimodal hyperspectral dataset for classification and research

The sugar dataset is a multimodal hyperspectral dataset of sugar and sugar related substances. The substances that were used for the creation of dataset are: - Sugar Ester S170 - Sugar Ester S770 - Sugar Ester S1570 - Sugar Ester P1570 - D-Mannitol - D-Sorbitol - D-Glucose - D-Galactose - D-Fructose All of the substances were hyperspectrally recorded using different sensors, namely: - Canon EOS 70D - ASD FiledSpec 3 - Neo VNIR-1600 - Neo VNIR-1800 - Neo SWIR-320m-e - Neo SWIR-384 - Nuance Ex The different sensors cover different wavelength ranges as well as different wavelength resolutions. This creates a unique dataset, that not only takles the question of hyperspectral classification, but also enable the research on topics like high dimensional data exploration, sensor invariant classification and dimensionality reduction

Protein Composition and Baking Quality of Wheat Flour as Affected by Split Nitrogen Application

Baking quality of wheat flour is determined by grain protein concentration (GPC) and its composition and is highly influenced by environmental factors such as nitrogen (N) fertilization management. This study investigated the effect of split N application on grain protein composition and baking quality of two winter wheat cultivars, Tobak and JB Asano, belonging to different baking quality classes. Bread loaf volumes in both cultivars were enhanced by split N application. In contrast, GPC was only significantly increased in JB Asano. Comparative 2-DE revealed that the relative volumes of 21 and 28 unique protein spots were significantly changed by split N application in Tobak and JB Asano, respectively. Specifically, the alterations in relative abundance of certain proteins, i.e., globulins, LMW-GS, α-, and γ-gliadins as well as α-amylase/trypsin inhibitors were more sensitive to split N application. Furthermore, certain proteins identified as globulins and alpha-amylase inhibitors were changed in both wheat cultivars under split N application. These results implied that the functions of these unique proteins might have played important roles in affecting baking quality of wheat flour, especially for cultivars (i.e., Tobak in the present study) the baking quality of which is less dependent on GPC

Arxula adeninivorans Recombinant Guanine Deaminase and Its Application in the Production of Food with Low Purine Content

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods

Arxula adeninivorans Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food

Bilateral Mastectomy versus Breast-Conserving Surgery for Early-Stage Breast Cancer: The Role of Breast Reconstruction

BACKGROUND: Although breast-conserving surgery is oncologically safe for women with early-stage breast cancer, mastectomy rates are increasing. The objective of this study was to examine the role of breast reconstruction in the surgical management of unilateral early-stage breast cancer. METHODS: A retrospective cohort study of women diagnosed with unilateral early-stage breast cancer (1998 to 2011) identified in the National Cancer Data Base was conducted. Rates of breast-conserving surgery, unilateral and bilateral mastectomy with contralateral prophylactic procedures (per 1000 early-stage breast cancer cases) were measured in relation to breast reconstruction. The association between breast reconstruction and surgical treatment was evaluated using a multinomial logistic regression, controlling for patient and disease characteristics. RESULTS: A total of 1,856,702 patients were included. Mastectomy rates decreased from 459 to 360 per 1000 from 1998 to 2005 (p < 0.01), increasing to 403 per 1000 in 2011 (p < 0.01). The mastectomy rates rise after 2005 reflects a 14 percent annual increase in contralateral prophylactic mastectomies (p < 0.01), as unilateral mastectomy rates did not change significantly. Each percentage point of increase in reconstruction rates was associated with a 7 percent increase in the probability of contralateral prophylactic mastectomies, with the greatest variation explained by young age(32 percent), breast reconstruction (29 percent), and stage 0 (5 percent). CONCLUSIONS: Since 2005, an increasing proportion of early-stage breast cancer patients have chosen mastectomy instead of breast-conserving surgery. This trend reflects a shift toward bilateral mastectomy with contralateral prophylactic procedures that may be facilitated by breast reconstruction availability

Greining á próteinsamsetningu kuldaaðlagaðs vetrarhveitis í útiræktun

A proteomics analysis was used to investigate how a winter wheat (Triticum aestivum L. cv Pishgam) adapted to LT during the autumn season and then initiated reproductive growth under field conditions. Cross-comparisons of leaf proteome were performed for consecutive stages including T1 (before the beginning of cold acclimation; zero-tolerance), T2 (initiation of the cold acclimation; LT tolerance=~-6 °C), T3 (vernalization fulfillment stage; LT tolerance=~-15 °C) and T4 (first weeks of reproductive stage; LT tolerance=~- 10 °C). Approximately 400 protein spots were reproducibly detected on each gel. However, only 56 differentially abundant protein spots showed any considerable change (p≤0.05) between experimental stages. Proteomic analysis indicated a substantial decrease in the abundance of several proteins such as oxygen-evolving enhancer protein, NADH dehydrogenase and dehydroascorbate reductase during the initiation of cold acclimation. However, the abundance of some metabolic regulator, ion transporter, redox and photosynthetic proteins increased by achieving maximum LT tolerance (T3). By initiation of the reproductive phase (T4) the abundance of some proteins that mainly participate in photosynthesis and carbon metabolism significantly increased.Heildar próteingreining á vetrarhveiti (Triticum aestivum L. cv Pishgam) var gerð í þeim tilgangi að skoða þær breytingar sem verða í próteinsamsetningu plantnanna á mismunandi stigum kuldaaðlögunar og herslu. Sýni til greiningar og samanburðar voru tekin á eftirfarandi stigum þroska og kuldaaðlögunar: T1 (áður en hersluferill gegn lækkuðum útihita hefst; án kuldaþols), T2 (í upphafi hersluferils; kuldaþol LT=~-6 °C), T3 (við fulla herslu; kuldaþol LT=~-15 °C) ogT4 (á fyrstu viku kynvaxtar; kuldaþol LT =~-10 °C). Aðgreindir voru 400 próteinblettir með rafdrætti en af þeim sýndu aðeins 56 blettir marktækan mun (p≤0.05) á milli ofangreindra herslu- /þroskastiga. Við upphaf hersluferils minnkaði framboð nokkurrra próteina þar á meðal ljóstíllífunarpróteinsins oxygen-evolving enhancer, NADH dehydrogenasa og dehydroascorbate afoxunarensímsins. Við fulla herslu jókst magn nokkurra próteina sem tengjast stýrihlutverkum í efnaskiptum, jónaflutningi yfir himnur, afoxun og ljóstillífun. Við upphaf kynvaxtar kom fram marktæk aukning í nokkrum próteinum sem tengjast ljóstillífun og sykruefnaskiptum