Understanding the regulatory relationship of abscisic acid and bZIP transcription factors towards amylose biosynthesis in wheat

Molecular Biology Reports - Starch is biosynthesized during seed development and this process is regulated by many bZIP proteins in bread wheat. Abscisic acid (ABA), an important phyto-hormone...     

Quaternary variations in the structural assembly of N‐acetylglucosamine‐6‐phosphate deacetylase from Pasteurella multocida

N‐acetylglucosamine 6‐phosphate deacetylase (NagA) catalyzes the conversion of N‐acetylglucosamine‐6‐phosphate to glucosamine‐6‐phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 Å. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artifact using single particle electron cryo‐microscopy. Analysis of the determined crystal structure reveals a set of hot‐spot residues involved in novel interactions at the dimer‐dimer interface. PmNagA binds to one Zn²⁺ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10‐fold the K_M), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.     

Siglec-E Promotes β2-Integrin-dependent NADPH Oxidase Activation to Suppress Neutrophil Recruitment to the Lung

Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury.     

Effect of a short-term diet and exercise intervention on oxidative stress, inflammation, MMP-9, and monocyte chemotactic activity in men with metabolic syndrome factors.

The present study was designed to examine the effects of lifestyle modification on key contributing factors to atherogenesis, including oxidative stress, inflammation, chemotaxis, and cell adhesion. Obese men (n = 31), 15 of whom had metabolic syndrome, were placed on a high-fiber, low-fat diet in a 3-wk residential program where food was provided ad libitum and daily aerobic exercise was performed. In each subject, pre- and postintervention fasting blood was drawn for circulating levels of serum lipids, glucose and insulin (for estimation of insulin sensitivity), oxidative stress-generating enzyme myeloperoxidase and marker 8-isoprostaglandin F2alpha, the inflammatory protein C-reactive protein, soluble ICAM-1 as an indicator of endothelial activation, sP-selectin as a marker of platelet activation, the chemokine macrophage inflammatory protein-1alpha, and total matrix metalloproteinase-9. Using subject sera and human aortic endothelial cell culture systems, we measured VCAM-1 cell surface abundance and monocyte chemotactic protein-1, nitric oxide, superoxide, and hydrogen peroxide production in vitro by fluorometric detection. Also determined in vitro was serum-induced, monocyte adhesion and monocyte chemotactic activity. After 3 wk, significant reductions (P < 0.05) in body mass index, all serum lipids and lipid ratios, fasting glucose, insulin, homeostasis model assessment for insulin resistance, myeloperoxidase, 8-isoprostaglandin F2alpha, C-reactive protein, soluble ICAM-1, soluble P-selectin, macrophage inflammatory protein-1alpha, and matrix metalloproteinase-9 were noted. In vitro, serum-stimulated cellular VCAM-1 expression, monocyte chemotactic protein-1 production, and fluorometric detection of superoxide and hydrogen peroxide production decreased, whereas a concomitant increase in NO production was noted (all P < 0.01). Additionally, both monocyte adhesion (P < 0.05) and MCA (P < 0.01) decreased. Nine of 15 were no longer positive for metabolic syndrome postintervention. Intensive lifestyle modification may ameliorate novel coronary artery disease risk factors in men with metabolic syndrome factors before reversal of obesity.     

Soluble endoglin contributes to the pathogenesis of preeclampsia

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Maternal endothelial dysfunction mediated by excess placenta-derived soluble VEGF receptor 1 (sVEGFR1 or sFlt1) is emerging as a prominent component in disease pathogenesis. We report a novel placenta-derived soluble TGF-beta coreceptor, endoglin (sEng), which is elevated in the sera of preeclamptic individuals, correlates with disease severity and falls after delivery. sEng inhibits formation of capillary tubes in vitro and induces vascular permeability and hypertension in vivo. Its effects in pregnant rats are amplified by coadministration of sFlt1, leading to severe preeclampsia including the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and restriction of fetal growth. sEng impairs binding of TGF-beta1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-beta signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.     

Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma

Stress can alter immunological, neurochemical and endocrinological functions, but its role in cancer progression is not well understood. Here, we show that chronic behavioral stress results in higher levels of tissue catecholamines, greater tumor burden and more invasive growth of ovarian carcinoma cells in an orthotopic mouse model. These effects are mediated primarily through activation of the tumor cell cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway by the beta(2) adrenergic receptor (encoded by ADRB2). Tumors in stressed animals showed markedly increased vascularization and enhanced expression of VEGF, MMP2 and MMP9, and we found that angiogenic processes mediated the effects of stress on tumor growth in vivo. These data identify beta-adrenergic activation of the cAMP-PKA signaling pathway as a major mechanism by which behavioral stress can enhance tumor angiogenesis in vivo and thereby promote malignant cell growth. These data also suggest that blocking ADRB-mediated angiogenesis could have therapeutic implications for the management of ovarian cancer.     

dGPredictor: Automated fragmentation method for metabolic reaction free energy prediction and de novo pathway design

Group contribution (GC) methods are conventionally used in thermodynamics analysis of metabolic pathways to estimate the standard Gibbs energy change (Δ_rG′^o) of enzymatic reactions from limited experimental measurements. However, these methods are limited by their dependence on manually curated groups and inability to capture stereochemical information, leading to low reaction coverage. Herein, we introduce an automated molecular fingerprint-based thermodynamic analysis tool called dGPredictor that enables the consideration of stereochemistry within metabolite structures and thus increases reaction coverage. dGPredictor has comparable prediction accuracy compared to existing GC methods and can capture Gibbs energy changes for isomerase and transferase reactions, which exhibit no overall group changes. We also demonstrate dGPredictor’s ability to predict the Gibbs energy change for novel reactions and seamless integration within de novo metabolic pathway design tools such as novoStoic for safeguarding against the inclusion of reaction steps with infeasible directionalities. To facilitate easy access to dGPredictor, we developed a graphical user interface to predict the standard Gibbs energy change for reactions at various pH and ionic strengths. The tool allows customized user input of known metabolites as KEGG IDs and novel metabolites as InChI strings (https://github.com/maranasgroup/dGPredictor).