Down-regulation of neprilysin (EC3.4.24.11) expression in vascular endothelial cells by laminar shear stress involves NADPH oxidase-dependent ROS production

Neprilysin (NEP, neutral endopeptidase, EC3.4.24.11), a zinc metallopeptidase expressed on the surface of endothelial cells, influences vascular homeostasis primarily through regulated inactivation of natriuretic peptides and bradykinin. Earlier in vivo studies reporting on the anti-atherosclerotic effects of NEP inhibition and on the atheroprotective effects of flow-associated laminar shear stress (LSS) have lead us to hypothesize that the latter hemodynamic stimulus may serve to down-regulate NEP levels within the vascular endothelium. To address this hypothesis, we have undertaken an investigation of the effects of LSS on NEP expression in vitro in bovine aortic endothelial cells (BAECs), coupled with an examination of the signalling mechanism putatively mediating these effects. BAECs were exposed to physiological levels of LSS (10 dynes/cm², 24 h) and harvested for analysis of NEP expression using real-time PCR, Western blotting, and immunocytochemistry. Relative to unsheared controls, NEP mRNA and protein were substantially down-regulated by LSS (≥50%), events which could be prevented by treatment of BAECs with either N-acetylcysteine, superoxide dismutase, or catalase, implicating reactive oxygen species (ROS) involvement. Employing pharmacological and molecular inhibition strategies, the signal transduction pathway mediating shear-dependent NEP suppression was also examined, and roles implicated for Gβγ, Rac1, and NADPH oxidase activation in these events. Treatment of static BAECs with angiotensin-II, a potent stimulus for NADPH oxidase activation, mimicked the suppressive effects of shear on NEP expression, further supporting a role for NADPH oxidase-dependent ROS production. Interestingly, inhibition of receptor tyrosine kinase signalling had no effect. In conclusion, we confirm for the first time that NEP expression is down-regulated in vascular endothelial cells by physiological laminar shear, possibly via a mechanotransduction mechanism involving NADPH oxidase-induced ROS production.     

Isoxazoline GPIIb/IIIa antagonists bearing a phosphoramidate

Isoxazolinylacetamides bearing a phosphoramidate group alpha- to the carboxylate moiety (3) were prepared and evaluated for in vitro antiplatelet efficacy. They were found to bind GPIIb/IIIa with high affinity and were potent antagonists of ADP mediated platelet aggregation.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts

Ammonia assimilation in chloroplasts occurs via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. To determine the extent to which these enzymes contribute to the control of ammonia assimilation, a metabolic control analysis was performed on isolated barley (Hordeum vulgare L.) leaf chloroplasts. Pathway flux was measured polarographically as ammonium-plus-2-oxoglutarate-plus-glutamine-dependent O2 evolution in illuminated chloroplasts. Enzyme activity was modulated by titration with specific, irreversible inhibitors of GS (phosphinothricin) and GOGAT (azaserine). Flux control coefficients (CJ0E0) were determined (a) by differentiation of best-fit hyperbolic curves of the data sets (flux versus enzyme activity), and (b) from estimates of the deviation indices (D/[prime]E0). Both analyses gave similar values for the coefficients. The control coefficient for GS was relatively high and the value did not change significantly with changes in 2-oxoglutarate concentration (C/0E0 = 0.58 at 5 mM 2-oxoglutarate and 0.40 at 20 mM 2-oxoglutarate). The control coefficient for GOGAT decreased with decreasing glutamine concentrations, from 0.76 at 20 mM glutamine to 0.19 at 10 mM glutamine. Thus, at high concentrations of glutamine, GOGAT exerts a major control over flux with a significant contribution also from GS. At lower concentrations of glutamine, however, GOGAT exerts far less control over pathway flux.     

Visual anthropology and multivocal ethnography: A dialogical approach to Japanese preschool class size

Joseph Jay Tobin is Assistant Professor of Family Studies at The University of New Hampshire     

Molecular diversity and allergenic profiles of Alternaria spp. from desert environments in Arizona

This study examined the genetic diversity of small-spored Alternaria species in the southwest desert of the USA by sampling 552 isolates from different habitats (soil and plant debris) in different locations (urban and an undisturbed desert). To estimate the genetic diversity, Amplified Fragment Length Polymorphism (AFLP) fingerprinting analysis was performed for all isolates. Strains representative of the sampled genotypic diversity (n = 125) were further characterized according their sporulation pattern and the capability to produce allergens. Morphological characterization assigned the majority of the strains to the Alternaria alternata and Alternaria tenuissima morpho-groups with only two isolates assigned to the Alternaria arborescens morpho-group. AFLP fingerprinting differentiated the A. arborescens morpho-groups, but could not distinguish between the A. alternata and A. tenuissima morpho-groups. Western blot analysis showed that a large number of allergenic proteins were produced by strains. These proteins were not specific for any morpho-group nor source of isolation. A hierarchical analysis of molecular variance was performed on the AFLP data to quantify molecular variation and partition this variation among sampled locations and habitat. No statistically significant differentiation among locations and habitat was detected indicating a lack of population structure across environments.     

Structure-activity studies of a series of dipyrazolo[3,4-b:3',4'-d]pyridin-3-ones binding to the immune regulatory protein B7.1

The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. Herein, we report the identification of potent small molecule inhibitors of the B7.1-CD28 interaction. In a high-throughput screen we identified several leads that prevented the interaction of B7.1 with CD28 with activities in the nanomolar to low micromolar range. One of these, the dihydrodipyrazolopyridinone 1, was subsequently shown to bind the V-like domain of human B7.1 at equimolar stoichiometry. With this as a starting point, we report here the synthesis and initial in vitro structure-activity relationships of a series of these compounds.     

Investigations into sequence and conformational dependence of backbone entropy,inter-basin dynamics and the Flory isolated-pair hypothesis for peptides

The populations and transitions between Ramachandran basins are studied for combinations of the standard 20 amino acids in monomers, dimers and trimers using an implicit solvent Langevin dynamics algorithm and employing seven commonly used force-fields. Both the basin populations and inter-conversion rates are influenced by the nearest neighbor's conformation and identity, contrary to the Flory isolated-pair hypothesis. This conclusion is robust to the choice of force-field, even though the use of different force-fields produces large variations in the populations and inter-conversion rates between the dominant helical, extended beta, and polyproline II basins. The computed variation of conformational and dynamical properties with different force-fields exceeds the difference between explicit and implicit solvent calculations using the same force-field. For all force-fields, the inter-basin transitions exhibit a directional dependence, with most transitions going through extended beta conformation, even when it is the least populated basin. The implications of these results are discussed in the context of estimates for the backbone entropy of single residues, and for the ability of all-atom simulations to reproduce experimental protein folding data.     

Lipid diffusion in the thylakoid membranes of the cyanobacterium Synechococcus sp.: effect of fatty acid desaturation

Thylakoid membranes are crucial to photosynthesis in cyanobacteria and plants. In cyanobacteria, genetic modification of membrane lipid composition strongly influences cold tolerance and susceptibility to photoinhibition. We have used fluorescence recovery after photobleaching to measure the diffusion of a lipid-soluble fluorescent marker in cells of the cyanobacterium Synechococcus sp. PCC 7942. We have compared the wild-type strain with a transformant with an increased level of fatty acid unsaturation. The transformant showed a six-fold increase in the diffusion coefficient for the fluorescent marker at growth temperature. This is the first direct measurement of lipid diffusion in a photosynthetic membrane.