TagSNP analyses of the PON gene cluster: effects on PON1 activity, LDL oxidative susceptibility, and vascular disease

Paraoxonase 1 (PON1) activity is consistently predictive of vascular disease, although the genotype at four functional PON1 polymorphisms is not. To address this inconsistency, we investigated the role of all common PON1 genetic variability, as measured by tagging single-nucleotide polymorphisms (tagSNPs), in predicting PON1 activity for phenylacetate hydrolysis, LDL susceptibility to oxidation ex vivo, plasma homocysteine (Hcy) levels, and carotid artery disease (CAAD) status. The biological goal was to establish whether additional common genetic variation beyond consideration of the four known functional SNPs improves prediction of these phenotypes. PON2 and PON3 tagSNPs were secondarily evaluated. Expanded analysis of an additional 26 tagSNPs found evidence of previously undescribed common PON1 polymorphisms that affect PON1 activity independently of the four known functional SNPs. PON1 activity was not significantly correlated with LDL oxidative susceptibility, but genotypes at the PON1(-108) promoter polymorphism and several other PON1 SNPs were. Neither PON1 activity nor PON1 genotype was significantly correlated with plasma Hcy levels. This study revealed previously undetected common functional PON1 polymorphisms that explain 4% of PON1 activity and a high rate of recombination in PON1, but the sum of the common PON1 locus variation does not explain the relationship between PON1 activity and CAAD.     

Computational sampling of a cryptic drug binding site in a protein receptor: explicit solvent molecular dynamics and inhibitor docking to p38 MAP kinase

An increasing number of structural studies reveal alternative binding sites in protein receptors that become apparent only when an inhibitor binds, and correct prediction of these situations presents a significant challenge to computer-aided drug design efforts. A striking example is provided by recent crystal structures of the p38 MAP kinase, where a 10A movement of the Phe169 side-chain creates a new binding site adjacent to the ATP binding site that is exploited by the diaryl urea inhibitor BIRB796. Here, we show that this binding site can be successfully and repeatedly identified in explicit-solvent molecular dynamics (MD) simulations of the protein that begin from an unliganded p38 crystal structure. Ligand-docking calculations performed on 5000 different structural snapshots generated during MD indicate that the conformations sampled are often surprisingly competent to bind the inhibitor BIRB796 in the crystallographically correct position and with docked energies that are generally more favorable than those of other positions. Similar docking studies with an ATP-binding site-directed inhibitor suggest that it may be possible to develop hybrid inhibitors that target both the ATP and cryptic binding sites simultaneously. Intriguingly, both inhibitors are occasionally found to dock correctly even with p38's "DFG" motif in the "wrong" conformation and BIRB796 can successfully dock, albeit infrequently, without significant displacement of the Phe169 side-chain; this suggests that the inhibitor might facilitate the latter's conformational change. Finally, two quite different conformations of p38's DFG motif are also sampled for extended periods of time during the simulations; these may provide new opportunities for inhibitor development. The MD simulations reported here, which total 390 ns in length, therefore demonstrate that existing computational methods may be of surprising utility in predicting cryptic binding sites in protein receptors prior to their experimental discovery.     

Nutrient enrichment weakens the stabilizing effect of species richness

With global freshwater biodiversity declining at an even faster rate than in the most disturbed terrestrial ecosystems, understanding the effects of changing environmental conditions on relationships between biodiversity and the variability of community and population processes in aquatic ecosystems is of significant interest. Evidence is accumulating that biodiversity loss results in more variable communities; however, the mechanisms underlying this effect have been the subject of considerable debate. We manipulated species richness and nutrients in outdoor aquatic microcosms composed of naturally occurring assemblages of zooplankton and benthic invertebrates to determine how the relationship between species richness and variability might change under different nutrient conditions. Temporal variability of populations and communities decreased with increasing species richness in low nutrient microcosms. In contrast, we found no relationship between species richness and either population or community variability in nutrient enriched microcosms. Of the different mechanisms we investigated (e.g. overyielding, statistical averaging, insurance effects, and the stabilizing effect of species richness on populations) the only one that was consistent with our results was that increases in species richness led to more stable community abundances through the stabilizing effect of species richness on the component populations. While we cannot conclusively determine the mechanism(s) by which species richness stabilized populations, our results suggest that more complete resource-use in the more species-rich low nutrient communities may have dampened population fluctuations.     

Copper(II) complexes of N-heteroaromatic hydrazones: Synthesis, X-ray structure, magnetic behavior, and antibacterial activity

Three novel copper(II) complexes as condensation derivatives of 2-pyridinecarboxaldehyde, 2-acetylpyridine or 2-quinolinecarboxaldehyde with ethyl hydrazinoacetate hydrochloride were synthesized. X-ray crystal structures of all three complexes were determined. Crystallographic data indicate a common basis of the structures with some major differences. Both pyridine derivatives form complexes of the type [CuCl₂L] with distorted square-planar geometry around copper(II), but show a decisive difference in the side chain orientation. The quinoline derivative forms a dimeric complex of type [Cu₂Cl₄L₂] with distorted trigonal bipyramidal geometry around copper(II), but a rather short Cu···Cu contact, which is reflected in its magnetic properties. All three complexes showed antibacterial activity.     

Mechanism of Epac Activation: STRUCTURAL AND FUNCTIONAL ANALYSES OF Epac2 HINGE MUTANTS WITH CONSTITUTIVE AND REDUCED ACTIVITIES*

Epac2 is a member of the family of exchange proteins directly activated by cAMP (Epac). Our previous studies suggest a model of Epac activation in which cAMP binding to the enzyme induces a localized “hinge” motion that reorients the regulatory lobe relative to the catalytic lobe without inducing large conformational changes within individual lobes. In this study, we identified the location of the major hinge in Epac2 by normal mode motion correlation and structural alignment analyses. Targeted mutagenesis was then performed to test the functional importance of hinge bending for Epac activation. We show that substitution of the conserved residue phenylalanine 435 with glycine (F435G) facilitates the hinge bending and leads to a constitutively active Epac2 capable of stimulating nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan (F435W), impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters determined by small angle x-ray scattering further reveal that whereas the F435G mutant assumes a more extended conformation in the absence of cAMP, the F435W mutant is incapable of adopting the fully extended and active conformation in the presence of cAMP. These findings demonstrate the importance of hinge motion in Epac activation. Our study also suggests that phenylalanine at position 435 is the optimal size side chain to keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac extension and activation in the presence of cAMP.Exchange proteins directly activated by cAMP (Epac)² are a family of novel intracellular sensors for the second messenger cAMP (1, 2). Unlike the classic eukaryotic cAMP receptor, cAMP-dependent protein kinase (protein kinase A; PKA), Epac proteins do not function as protein kinases that phosphorylate downstream substrates. Instead, they act as guanine nucleotide exchange factors and exert their functions by activating small GTP-binding proteins, Rap1 and Rap2. At the cellular level, Epac proteins assume distinct subcellular localization (3), and depending upon the specific cellular environment, Epac and PKA may act independently, converge synergistically, or oppose each other in regulating a specific cellular function (4, 5).Both Epac and PKA share a common cyclic nucleotide binding domain (CBD), a compact and evolutionarily conserved structural motif found in a variety of proteins with diverse cellular functions (6). CBDs act as molecular switches for sensing intracellular second messenger cAMP levels and regulate the functionality of the domain(s) to which they are linked (6, 7). In depth structural and biophysical analyses of CBDs in several CBD-containing families, including cAMP receptor proteins, PKAs, and cyclic nucleotide-gated ion channels, have revealed a conserved structural core as well as functional motifs important for cyclic nucleotide-induced activation (8–11). The CBD is composed of an eight-strand β-barrel core that forms the base of the nucleotide binding pocket and a lateral α-helical bundle subdomain. Although the β-barrel core remains relatively constant between the ligand-free and nucleotide-bound states, binding of cAMP to a CBD leads to significant conformational changes in the overall arrangement of the α-helical bundle subdomain. A general mechanism of cyclic nucleotide-induced activation of CBD-containing proteins has been recently proposed (12). In this model, binding of cAMP leads to the retraction of the phosphate-binding cassette toward the cyclic nucleotide binding pocket and consequently releases the steric hindrance imposed on the α-helix C-terminal to the β-barrel, termed the CBD lid, by a conserved hydrophobic residue within the phosphate-binding cassette. These structural changes result in a hinge motion that allows the lid to move closer to the β-barrel core and to interact with the base of the cyclic nucleotide.The recently solved crystal structure of Epac2 reveals that, unlike other CBD-containing proteins, the corresponding lid region in Epac points away from the cAMP binding pocket as a two-strand β-sheet that forms the first part of the five-strand β-sheet “switchboard” structure (13). Although this major structural difference suggests that the detailed mechanisms of PKA and Epac activation by cAMP will most likely be different at the atomic level, it is not clear if the aforementioned general mechanism, namely the hinge motion, is conserved during Epac activation. To address this important question, we determined the effects of targeted hinge perturbations on Epac activation using site-directed mutagenesis that specifically targeted a key residue in the hinge region of Epac2.     

Discovery and Characterization of HemQ: AN ESSENTIAL HEME BIOSYNTHETIC PATHWAY COMPONENT*

Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. We have characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. The presence of HemQ along with the terminal two pathway enzymes, protoporphyrinogen oxidase (HemY) and ferrochelatase, is required to synthesize heme in vivo and in vitro. Although the exact role played by HemQ remains to be characterized, to be fully functional in vitro it requires the presence of a bound heme. HemQ possesses minimal peroxidase activity, but as a catalase it has a turnover of over 10⁴ min⁻¹. We propose that this activity may be required to eliminate hydrogen peroxide that is generated by each turnover of HemY. Given the essential nature of heme synthesis and the restricted distribution of HemQ, this protein is a potential antimicrobial target for pathogens such as Mycobacterium tuberculosis.     

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.     

Opportunities and challenges for managing nitrogen in urban stormwater: A review and synthesis

Although nitrogen (N) is prevalent in urban stormwater, regulation of this pollutant has occurred only more recently. This paper reviews the concerns over N in urban stormwater, mechanisms and design enhancements for N uptake and denitrification through various stormwater control measures (SCMs), and presents opportunities to integrate this current knowledge into the regulatory framework. A survey of personnel directly involved in various aspects of US state and territory NPDES programs revealed that the top three pollutants of concern were total suspended solids (TSS), pathogens and bacteria, and total phosphorus (TP). Surprisingly, nitrate (NO₃^?) was of little concern among the survey respondents, with 3.9% giving it the highest level of concern, 2.0% ranking it second, and 6.0% ranking it third. When asked which strategies were currently used in their geographic area for stormwater management, the most common results were wet ponds and dry ponds. At the same time, wet ponds and dry ponds were recognized as less effective practices to manage stormwater.A review of current literature reveals that several alternative SCMs, such as bioretention, filters, and wetlands, show greater promise in their ability to remove N from stormwater than more conventional practices such as dry ponds and wet ponds. Enhanced N removal via denitrification and plant uptake is often observed under the combination of aerobic followed by sustained anoxic conditions, the presence of a carbon source (organic material), and the presence of mature, dense vegetation.Given the lack of concern or awareness of local officials related to N loading from urban stormwater, and variation in the efficacy of various SCMs, it is not surprising that regulators remain focused on conventional dry pond and wet pond control measures. More needs to be done to quantify the impact of urban sources of N on water quality and aquatic ecosystems. In addition, greater focus needs to be placed on the development of design criteria for SCMs, such as bioretention, filters, and constructed wetlands, which show more promise for N removal.     

Synthesis and antituberculosis activity of new fatty acid amides

This work reports the synthesis of new fatty acid amides from C16:0, 18:0, 18:1, 18:1 (OH), and 18:2 fatty acids families with cyclic and acyclic amines and demonstrate for the first time the activity of these compounds as antituberculosis agents against Mycobacterium tuberculosis H₃₇Rv, M. tuberculosis rifampicin resistance (ATCC 35338), and M. tuberculosis isoniazid resistance (ATCC 35822). The fatty acid amides derivate from ricinoleic acid were the most potent one among a series of tested compounds, with a MIC 6.25 μg/mL for resistance strains.