A high-grain protein content locus on barley (Hordeum vulgare) chromosome 6 is associated with increased flag leaf proteolysis and nitrogen remobilization

Leaf senescence and nitrogen remobilization from senescing tissues are two important factors determining grain protein content (GPC) in cereals. We compared near-isogenic barley ( Hordeum vulgare L.) germplasm varying in the allelic state of a major GPC quantitative trait locus on chromosome 6, delineated by molecular markers HVM74 and ABG458 and explaining approximately 46% of the variability in this trait. High GPC was consistently associated with earlier whole-plant senescence. SDS–PAGE and immunoblot analysis of flag leaf proteins indicated earlier leaf protein [including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)] degradation in high-GPC germplasm. This was accompanied by enhanced availability of ammonium and glutamine in developing kernels, suggesting increased phloem retranslocation of nitrogen. Based on previous microarray analysis, we performed a detailed expression study of six leaf genes, tentatively involved in plastidial proteolysis, vacuolar proteolysis, intermediary N metabolism and N transport. All of these were upregulated in high-GPC barley, mostly around 21 to 28 days past anthesis, prior to or around the time demonstrating maximal differences in leaf protein (including Rubisco) levels. Therefore, these genes represent potential targets to manipulate grain protein accumulation. It appears likely that their functional analysis will enhance our understanding of whole-plant N recycling. Additionally, earlier leaf (photosynthetic) protein degradation may lead to reduced N carbon assimilation in high-GPC germplasm, explaining past studies demonstrating a negative correlation between GPC and yield.     

Insilico Structural 3D Modelling of Novel Cry1Ib9 and Cry3A Toxins from Local Isolates of Bacillus thuringiensis

Three-dimensional (3D) models for the 79.2 kDa activated Cry1Ib9 and 77.4 kDa activated Cry3A δ-endotoxins from Bacillus thuringiensis (Bt) native isolates that are specifically toxic to Coleopteran insect pests were constructed by utilizing homology modeling online tool. Evidences presented here, based on the identification of structural equivalent residues of Cry1Ib9 and Cry3A toxin through homology modelling indicate that, they share a common Bt toxin tridimensional structure. The main differences observed in Cry1I9 domain I at positions α2b (S56-I60), α4 (F78-l93) and additionally β0 (Q10-L12), α8a (T280-V282) were observed, in domain II at positions α9b (P333-L339), β6(T390-Q393), β7(V398-W404), β8 (V418-W425), β9 (E453-N454), β10 (S470-I479) where as in domain III the changes were observed at positions β19 (R601-F607), β20 (609-L613), β21 (S618-F627) and α11a (K655-F664), α13, α14 components present at downstream sites, where as in Cry3A main differences observed in domain I is at the position of α4 (P105-I152), α5 (Q163-A185), β1A(E190-L192), α6 (F193-Y217), Domain II is not consevered and main variations were observed at β2 (E292-L295), β3(V299-L308), β4(I340-F347), β5(D356-P368), β6(I375-T377), β7(V389-F394), β8(K398-N405), β9(Y416-Y427), β10 (T436-Y439), β12(G476-H495), β12A (M503-I504) where as in domain III main variations observed at positions of β18 (P583-I593), β19(F604-S610), β20(P611-L615), β21(N619-G626). Cry1Ib9 and Cry3A contain the most variable regions in the loops of domain II, which determine the specificity of these toxins. These are the first models of Coleopteran-active protein from native isolates of Bt and its importance can be perceived since members of this group of toxins are potentially important candidates for coleoptera insect pest control programs.     

Down-regulated Peroxisome Proliferator-activated Receptor γ (PPARγ) in Lung Epithelial Cells Promotes a PPARγ Agonist-reversible Proinflammatory Phenotype in Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition and a leading cause of death, with no available cure. We assessed the actions in pulmonary epithelial cells of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor with anti-inflammatory effects, whose role in COPD is largely unknown. We found that PPARγ was down-regulated in lung tissue and epithelial cells of COPD patients, via both reduced expression and phosphorylation-mediated inhibition, whereas pro-inflammatory nuclear factor-κB (NF-κB) activity was increased. Cigarette smoking is the main risk factor for COPD, and exposing airway epithelial cells to cigarette smoke extract (CSE) likewise down-regulated PPARγ and activated NF-κB. CSE also down-regulated and post-translationally inhibited the glucocorticoid receptor (GR-α) and histone deacetylase 2 (HDAC2), a corepressor important for glucocorticoid action and whose down-regulation is thought to cause glucocorticoid insensitivity in COPD. Treating epithelial cells with synthetic (rosiglitazone) or endogenous (10-nitro-oleic acid) PPARγ agonists strongly up-regulated PPARγ expression and activity, suppressed CSE-induced production and secretion of inflammatory cytokines, and reversed its activation of NF-κB by inhibiting the IκB kinase pathway and by promoting direct inhibitory binding of PPARγ to NF-κB. In contrast, PPARγ knockdown via siRNA augmented CSE-induced chemokine release and decreases in HDAC activity, suggesting a potential anti-inflammatory role of endogenous PPARγ. The results imply that down-regulation of pulmonary epithelial PPARγ by cigarette smoke promotes inflammatory pathways and diminishes glucocorticoid responsiveness, thereby contributing to COPD pathogenesis, and further suggest that PPARγ agonists may be useful for COPD treatment.     

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit.     

Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology

The effect of microwave irradiation on the simultaneous extraction and transesterification (in situ transesterification) of dry algal biomass to biodiesel was investigated. A high degree of oil/lipid extraction from dry algal biomass and an efficient conversion of the oils/lipids to biodiesel were demonstrated in a set of well-designed experimental runs. A response surface methodology (RSM) was used to analyze the influence of the process variables (dry algae to methanol (wt/vol) ratio, catalyst concentration, and reaction time) on the fatty acid methyl ester conversion. Based on the experimental results and RSM analysis, the optimal conditions for this process were determined as: dry algae to methanol (wt/vol) ratio of around 1:12, catalyst concentration about 2 wt.%, and reaction time of 4 min. The algal biodiesel samples were analyzed with GC-MS and thin layer chromatography (TLC) methods. Transmission electron microscopy (TEM) images of the algal biomass samples before and after the extraction/transesterification reaction are also presented.     

DOMON: an ancient extracellular domain in dopamine beta-monooxygenase and other proteins

A previously uncharacterized 110-125 residue-long domain was identified both in the physiologically important enzyme dopamine beta-monooxygenase and in several other secreted and transmembrane proteins such as SDR2 and CG-6. This domain was predicted to adopt an all-beta fold with seven or eight strands and might function as a module mediating a range of extracellular adhesive interactions.     

Novel eukaryotic enzymes modifying cell-surface biopolymers

Background  

Eukaryotic extracellular matrices such as proteoglycans, sclerotinized structures, mucus, external tests, capsules, cell walls and waxes contain highly modified proteins, glycans and other composite biopolymers. Using comparative genomics and sequence profile analysis we identify several novel enzymes that could be potentially involved in the modification of cell-surface glycans or glycoproteins.