A potential use of wild pea as a source of lower trypsin inhibitor activity

The aim of this work was to estimate the trypsin inhibitor activity (TIA) in seeds of cultivated pea varieties, wild varieties and selected crosses between varieties from the first group and wild pea varieties and to study the variation in genes coding trypsin inhibitors. Mean TIA in field pea varieties ranged from 3.12 TIU/mg of sample in field pea variety from Czech Republic to 12.90 TIU/mg of sample in field pea variety FP S4 of Serbian origin. Wild field pea varieties showed TIA between 0.98 TIU/mg of sample in Pisum elatius and 9.79 TIU/mg of sample in Pisum abyssinicum. Selected crosses between cultivated field pea varieties and Pisum elatius showed a decrease in TIA in comparison with a parent line that has higher TIA content. The PCR amplification resulted in variety-specific amplification. Varieties with low TIA activity showed amplification with At13/At5 primer pair, while varieties with higher TIA activity showed amplification with primer pairs At12/At5, At14/At5 and At14/At8. Thus, At13/At5 primer pair could be sufficient to distinguish most varieties. These markers can be applied during an early screening of the valuable materials for future breeding programs of pea cultivars with the low level of tripsin inhibitor.     

Interpopulation hybridization generates meiotically stable rDNA epigenetic variants in allotetraploid Tragopogon mirus

Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d‐rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p‐rDNA dominant progenitor were meiotically unstable, frequently switching to co‐dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d‐rDNA dominance, indicating immediate suppression of p‐homeologs in F₁ hybrids. Original p‐rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p‐rDNA and d‐rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co‐dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids.     

Glutathione–ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (Say)

In view of the antioxidant role of glutathione (GSH) and ascorbic acid (AA), we have examined capacity of the GSH–AA redox cycle in relation to oxidative stress effects in the midgut of the Colorado potato beetle Leptinotarsa decemlineata. Adult gut harbors a higher capacity to cope with oxidative stress than the larval gut. Protein carbonylation was pronounced in the wall of anterior larval midgut and was generally lower in the food digest than in the gut wall. Restriction of oxidative stress effects in anterior gut lumen manifested by lipid peroxidation and protein carbonylation is interpreted as a mechanism favoring digestion and absorption in the posterior midgut. Presence of high GSH in the posterior midgut and AA in both posterior and anterior midguts of adults points to higher utility of the GSH–AA redox system in limiting oxidative stress to manageable levels. The presence, gene expression and activity of thioredoxin reductase (TrxR) were demonstrated for the first time in L. decemlineata which was markedly higher in the anterior than in the posterior midgut in both stages. It is probably central to the maintenance of reduced GSH levels in the whole gut, despite a GSSG/2GSH redox potential tending towards oxidizing ranging from ?183.5 to ?124.4 mV. Glutathione-dehydroascorbate reductase (G_DHAR) activity was markedly augmented in adult gut compared with larva, pointing to a more efficient conversion of dehydroascorbate (DHA) to AA. Also, ascorbate peroxidase (APOX) activity was significantly elevated in all gut compartments of adult except the wall of posterior midgut. The results emphasize the potential importance and role of the GSH–AA redox cycle as a defense strategy against oxidative stress in the gut of L. decemlineata.     

Computational analysis of lung deformation after murine pneumonectomy

In many mammalian species, the removal of one lung (pneumonectomy) is associated with the compensatory growth of the remaining lung. To investigate the hypothesis that parenchymal deformation may trigger lung regeneration, we used microCT scanning to create 3D finite element geometric models of the murine lung pre- and post-pneumonectomy (24 h). The structural correspondence between models was established using anatomic landmarks and an iterative computational algorithm. When compared with the pre-pneumonectomy lung, the post-pneumonectomy models demonstrated significant translation and rotation of the cardiac lobe into the post-pneumonectomy pleural space. 2D maps of lung deformation demonstrated significant heterogeneity; the areas of greatest deformation were present in the subpleural regions of the lobe. Consistent with the previously identified growth patterns, subpleural regions of enhanced deformation are compatible with a mechanical signal – likely involving parenchymal stretch – triggering lung growth.     

Biophysical and Structural Characterization of the Thioredoxin-binding Domain of Protein Kinase ASK1 and Its Interaction with Reduced Thioredoxin

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp³¹ being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.     

Role of the EF-hand-like Motif in the 14-3-3 Protein-mediated Activation of Yeast Neutral Trehalase Nth1

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.