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1.
Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations'' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.  相似文献   
2.

Aims

This study investigated Cu uptake and accumulation as well as physiological and biochemical changes in grapevines grown in soils containing excess Cu.

Methods

The grapevines were collected during two productive cycles from three vineyards with increasing concentrations of Cu in the soil and at various growth stages, before and after the application of Cu-based fungicides. The Cu concentrations in the grapevine organs and the macronutrients and biochemical parameters in the leaf blades were analyzed.

Results

At close to the flowering stage of the grapevines, the concentration and content of Cu in the leaves were increased. However, the Cu concentrations in the roots, stem, shoots and bunches did not correlate with the metal concentrations in the soil. The application of Cu-based fungicides to the leaves increased the Cu concentrations in the shoots, leaves and rachis; however, the effect of the fungicides on the Cu concentration in the berries was not significant. The biochemical analyses of the leaf blades demonstrated symptoms of oxidative stress that correlated with the Cu concentrations in soil.

Conclusions

The increased availability of Cu in soil had a slight effect on the levels and accumulation of Cu in mature grapevines during the productive season and did not alter the nutritional status of the plant. However, increased Cu concentrations were observed in the leaves. The evidence of oxidative stress in the leaves correlated with the increased levels of Cu in soil.  相似文献   
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Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A2-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.  相似文献   
5.
The cholinergic system is critically involved in the modulation of cognitive functions, including learning and memory. Acetylcholine acts through muscarinic (mAChRs) and nicotinic receptors (nAChRs), which are both abundantly expressed in the hippocampus. Previous evidence indicates that choline, the precursor and degradation product of Acetylcholine, can itself activate nAChRs and thereby affects intrinsic and synaptic neuronal functions. Here, we asked whether the cellular actions of choline directly affect hippocampal network activity. Using mouse hippocampal slices we found that choline efficiently suppresses spontaneously occurring sharp wave–ripple complexes (SPW‐R) and can induce gamma oscillations. In addition, choline reduces synaptic transmission between hippocampal subfields CA3 and CA1. Surprisingly, these effects are mediated by activation of both mAChRs and α7‐containing nAChRs. Most nicotinic effects became only apparent after local, fast application of choline, indicating rapid desensitization kinetics of nAChRs. Effects were still present following block of choline uptake and are, therefore, likely because of direct actions of choline at the respective receptors. Together, choline turns out to be a potent regulator of patterned network activity within the hippocampus. These actions may be of importance for understanding state transitions in normal and pathologically altered neuronal networks.

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New insights into community-level responses at the urban fringe, and the mechanisms underlying them, are needed. In our study, we investigated the compositional distinctiveness and variability of a breeding bird community at both sides of established edges between suburban residential areas and woodland reserves in Canberra, Australia. Our goals were to determine if: (1) community-level responses were direct (differed with distance from the edge, independent of vegetation) or indirect (differed in response to edge-related changes in vegetation), and (2) if guild-level responses provided the mechanism underpinning community-level responses. We found that suburbs and reserves supported significantly distinct bird communities. The suburban bird community, characterised by urban-adapted native and exotic species, had a weak direct edge response, with decreasing compositional variability with distance from the edge. In comparison, the reserve bird community, characterised by woodland-dependent species, was related to local tree and shrub cover. This was not an indirect response, however, as tree and shrub cover was not related to edge distance. We found that the relative richness of nesting, foraging and body size guilds also displayed similar edge responses, indicating that they underpinned the observed community-level responses. Our study illustrates how community-level responses provide valuable insights into how communities respond to differences in resources between two contrasting habitats. Further, the effects of the suburban matrix penetrate into reserves for greater distances than previously thought. Suburbs and adjacent reserves, however, provided important habitat resources for many native species and the conservation of these areas should not be discounted from continued management strategies.  相似文献   
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