BioSystem (Germany)

Understanding sorghum response to water stress at different developmental stages is important for developing sorghum varieties with improved tolerance to drought. This study set out to finely characterize key agro-physiological responses and water use of sorghum subjected to different drought scenarios. A greenhouse experiment was conducted using a DroughtSpotter facility that enables real-time quantification of water use by gravimetric tracking. Four different water treatments were assessed: (i) early vegetative drought stress (DS1), (ii) DS1 plus recovery (DS1R), (iii) late vegetative drought stress (DS2), and (iv) well-watered (WW). Plant pheno-morphology and yield data were recorded. Leaf fluorescence and photosynthesis were continuously recorded throughout the experiment. Our results showed that green leaf number and plant leaf area were more affected by DS2 than DS1 and delayed plant flowering. Nevertheless, plants in DS2 were taller and produced higher stem and leaf biomass compared to DS1. No significant difference was recorded in grain yield between DS1 and DS2 but were outperformed by DS1R and WW. The transpiration and photosynthetic rates were shown to decrease at the same time as the stomatal conductance. This can therefore be assimilated to a stomatal down-regulation limiting CO2 uptake. However, the increase in intercellular CO2 concentration is likely to indicate the presence of CO2 in the substomatal cavity that was not conveyed to the carboxylation sites. This suggests a non-stomatal limitation of the photosynthesis. Moreover, the plants recovered quite well from DS1, and this was more prominent for physiological parameters than morphological ones. Globally, water use efficiency (WUE) for DS2 was higher compared to WW and DS1 treatments, confirming the growing point differentiation as a critical stage where drought stress should be avoided to ensure yield and better WUE. Adaptation responses were related to the reduction of transpiration through plant leaf area reduction, the reduction of stomatal conductance, and the increase of intercellular CO2 limiting photosynthesis. Further studies focusing on the biomarkers of stress and transcriptomic analyses are needed to provide further insight into the drought adaptation mechanisms of this line.

Over the years a large body of information has accumulated for the amino acid glycine, which prove that this amino acid is critical to animal nutrition in order for feeding to be more economic, eco-sustainable and supportive of animal health and food safety. Underpinning these glycine efficacies are its multifaceted metabolic interrelationships with other essential amino acids, and the resultant physiological responses. Key among these interactions is the inter-convertibility between glycine and serine, the participation of glycine in sulphur amino acid metabolism, and its biochemical linkages with arginine. Crucial to this is the metabolic release of glycine in threonine catabolism and cellular application in N detoxification- all linked to body amino acid homoeostasis. This is the first of a two-paper series reviewing the biochemistry and multifunctional metabolic involvements of glycine and the impacts on physiological efficiency of sulphur amino acids, threonine, arginine and total crude protein, mainly in broilers.

Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.

Introduction: Protein phosphorylation is one of the most important post-translational modifications that regulate the function, localization, and binding specificity of target proteins. Specifically, protein phosphorylation works as a switch to turn “on” and “off” protein activity or a cellular pathway. Protein phosphorylation is also known to be both spatial and dynamic in its distribution. Therefore, a work-flow that provides the detailed information of phosphorylation site location, site occupancy under specific conditions, etc., is necessary. Current strategies such as ELISA, radio isotope workflow, and Western blotting lack the ability to ascertain independent information of each phosphorylation site. Our mass spectrometry–based method using a chemical labeling strategy provides dynamic information about phosphorylation on each phosphorylation site. Method: A model system using JNK1 kinase and the substrate protein myelin basic protein (MBP) was selected. JNK1 phosphorylates serine and threonine residues just before the proline residue, typical of this MAP kinase family. The MBP substrate was incubated with JNK1 in vitro, and samples were removed at four different time points (0, 5, 30, and 60 min). These samples were digested and labeled with iTRAQ reagent in accordance with the standard procedure. A nano-LC-MS/MS experiment was performed using the 4000 Q TRAP System, and the data were analyzed by ProteinPilot Software. Results: All of the peptides possessing a possible phosphorylation site motif in MBP were detected. For each identified peptide, the iTRAQ reagent reporter ions clearly showed the change in abundance of specific MBP peptides as they become phosphorylated by JNK1 over the time course of the assay. These results indicate that this workflow is a robust strategy for routinely characterizing the variation of phosphorylation through the interaction between protein substrate and kinase.

BigDye XTerminator is a new single-tube method for purifying DNA sequencing reactions prior to electrophoretic analysis. Sequencing reactions purified using this method display very few artifacts from residual dye-labeled nucleotides (dye blobs), as well as excellent recovery of the smallest extension products. BigDye XTerminator also has unique workflow advantages over conventional purification schemes such as ethanol precipitation or spin column purifications. BigDye XTerminator is added directly to finished DNA sequencing reactions and mixed. After mixing, the samples purified with BigDye XTerminator can be directly injected from the reaction tube without additional transfer steps. Performance of BigDye XTerminator with a variety of DNA sequencing reactions will be demonstrated. Workflow and automation issues will also be described.

Nearly one in eight US women will develop breast cancer in their lifetime. Most breast cancer is not associated with a hereditary syndrome, occurs in postmenopausal women, and is estrogen and progesterone receptor-positive. Estrogen exposure is an epidemiologic risk factor for breast cancer and estrogen is a potent mammary mitogen. We studied single nucleotide polymorphisms (SNPs) in estrogen receptors in 615 healthy subjects and 1011 individuals with histologically confirmed breast cancer, all from New York City. We analyzed 13 SNPs in the progesterone receptor gene (PGR), 17 SNPs in estrogen receptor 1 gene (ESR1), and 8 SNPs in the estrogen receptor 2 gene (ESR2). We observed three common haplotypes in ESR1 that were associated with a decreased risk for breast cancer [odds ratio (OR), approximately O.4; 95% confidence interval (CI), 0.2-0.8; P < 0.01]. Another haplotype was associated with an increased risk of breast cancer (OR, 2.1; 95% CI, 1.2-3.8; P < 0.05). A unique risk haplotype was present in approximately 7% of older Ashkenazi Jewish study subjects (OR, 1.7; 95% CI, 1.2-2.4; P < 0.003). We narrowed the ESR1 risk haplotypes to the promoter region and first exon. We define several other haplotypes in Ashkenazi Jews in both ESR1 and ESR2 that may elevate susceptibility to breast cancer. In contrast, we found no association between any PGR variant or haplotype and breast cancer. Genetic epidemiology study replication and functional assays of the haplotypes should permit a better understanding of the role of steroid receptor genetic variants and breast cancer risk.