Simultaneous analysis of multiple aminothiols in human plasma by high performance liquid chromatography with fluorescence detection

Aminothiols serve numerous vital functions in biochemistry, including detoxification and regulation of cellular metabolism, enzymatic activity, and protein trafficking and degradation. Plasma aminothiol concentrations are frequently measured for clinical and translational research investigating oxidative stress, and for routine clinical diagnosis and monitoring of vascular injury. Although a variety of techniques are available to measure aminothiol concentrations in plasma, high performance liquid chromatography with fluorescence detection (HPLC–FD) is the most widely used. This review summarizes HPLC–FD methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and chromatographic separation of the primary biological aminothiols cysteine, homocysteine, cysteinylglycine, and glutathione in human plasma.     

REPRODUCTIVE OUTPUT AND BIOMASS ALLOCATION IN SESBANIA EMERUS IN A TROPICAL SWAMP

This study examined the correlation of moisture, reproductive phenology, density of mature plants, and herbivory of apical meristems with the morphology and reproductive output of Sesbania emerus, an annual legume growing along a moisture gradient in a swamp in Guanacaste Province, Costa Rica. It also determined how biomass allocation varied and how it was related to reproductive output of plants growing along the moisture gradient within the swamp. Morphological changes included production of more stems and branches in response to herbivory, more and higher prop roots and more aerenchyma as water depth increased, and greater stem diameter in lower densities. Plant height varied greatly within a site, but not among habitats. Plants began to produce flowers and fruits later in wet sites than in dry sites. Reproductive output was generally more sensitive to environmental variables than was plant size. Fruit number and plant height were positively correlated for almost every treatment. Greater fruit and seed production were correlated with drier sites, earlier phenology, and lower density, but not with herbivory. Total biomass accumulation did not vary among moisture sites, but root production appeared to occur at the expense of reproductive output in the wetter sites. Plants in the wetter sites had both a greater percent and a greater absolute amount of biomass in roots, and a lower percent and a lower absolute amount of biomass in fruits and seeds than plants in drier sites. The root: shoot ratio was nearly five times higher in the wet than the dry site. Seed number per plant ranged from a mean of 6,800 at the wet site to a mean of 16,878 at the dry site. If this striking phenotypic variation in reproductive output and biomass accumulation has a genetic basis, the possibility of ecotypic differentiation exists in S. emerus.     

Arabinan Metabolism during Seed Development and Germination in Arabidopsis

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan I. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non- cellulosic polysaccharides of embryos. Arabinose levels decline to -15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using 14C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth.     

Engineered bromodomains to explore the acetylproteome

MS‐based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti‐acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine‐containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot‐to‐lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.