Refined solution structure of the LpxC-TU-514 complex and pKa analysis of an active site histidine: insights into the mechanism and inhibitor design

Lipopolysaccharide, the major constituent of the outer monolayer of the outer membrane of Gram-negative bacteria, is anchored into the membrane through the hydrophobic moiety lipid A, a hexaacylated disaccharide. The zinc-dependent metalloamidase UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the second and committed step in the biosynthesis of lipid A. LpxC shows no homology to mammalian metalloamidases and is essential for cell viability, making it an important target for the development of novel antibacterial compounds. Recent NMR and X-ray studies of the LpxC from Aquifex aeolicus have provided the first structural information about this family of proteins. Insight into the catalytic mechanism and the design of effective inhibitors could be facilitated by more detailed structural and biochemical studies that define substrate-protein interactions and the roles of specific residues in the active site. Here, we report the synthesis of the (13)C-labeled substrate-analogue inhibitor TU-514, and the subsequent refinement of the solution structure of the A. aeolicus LpxC-TU-514 complex using residual dipolar couplings. We also reevaluate the catalytic role of an active site histidine, H253, on the basis of both its pK(a) as determined by NMR titration and pH-dependent kinetic analyses. These results provide a structural basis for the design of more potent LpxC inhibitors than those that are currently available.     

Mechanism of reversible (99m)Tc-sestamibi perfusion defects during pharmacologically induced vasodilatation

Reversible perfusion defects on (99m)Tc-sestamibi imaging during hyperemia are thought to occur due to myocardial blood flow (MBF) "mismatch" between regions with and without stenosis. We have recently shown that myocardial blood volume (MBV) distal to a stenosis decreases during hyperemia, resulting in a reversible perfusion defect on myocardial contrast echocardiography (MCE). In this study, we hypothesized that a reversible perfusion defect on (99m)Tc-sestamibi imaging during hyperemia results from the same mechanism. We tested our hypothesis under the following conditions: 1) increases in MBF in the absence of changes in MBV by using direct intracoronary infusion of adenosine (group I, n = 10 dogs); 2) decrease in MBV despite an increase in MBF by left main infusion of adenosine proximal to a noncritical coronary stenosis placed on either coronary artery (group II, n = 13 dogs); and 3) reduction in both resting MBF and MBV by placement of a severe stenosis (group III, n = 7 dogs). In group I dogs, no difference in MBV or (99m)Tc-sestamibi uptake was found between the two coronary beds despite an up to fourfold increase in MBF in one bed with adenosine. In group II dogs, MBV distal to the stenosis decreased during hyperemia despite a twofold increase in mean MBF. A good correlation was found between (99m)Tc-sestamibi uptake and MBV ratios from the stenosed versus normal bed (r = 0.91, P < 0.001). In group III dogs, both MBF and MBV were decreased in the stenosed bed at rest with a good correlation noted between (99m)Tc-sestamibi uptake and MBV ratios from the stenosed versus normal bed (r = 0.92, P = 0.004). We conclude that reversible defects on (99m)Tc-sestamibi during vasodilator stress imaging are related to decreases in MBV distal to a stenosis and not to "flow mismatch" between beds. The decrease in MBV results in reduced (99m)Tc-sestamibi uptake during hyperemia.     

Biochemical and X-ray crystallographic studies on shikimate kinase: the important structural role of the P-loop lysine

Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.     

Tickled to Death: Analysing Public Perceptions of ‘Cute’ Videos of Threatened Species (Slow Lorises – Nycticebus spp.) on Web 2.0 Sites

Background

The internet is gaining importance in global wildlife trade and changing perceptions of threatened species. There is little data available to examine the impact that popular Web 2.0 sites play on public perceptions of threatened species. YouTube videos portraying wildlife allow us to quantify these perceptions.

Methodology/Principal Findings

Focussing on a group of threatened and globally protected primates, slow lorises, we quantify public attitudes towards wildlife conservation by analysing 12,411 comments and associated data posted on a viral YouTube video ‘tickling slow loris’ over a 33-months period. In the initial months a quarter of commentators indicated wanting a loris as a pet, but as facts about their conservation and ecology became more prevalent this dropped significantly. Endorsements, where people were directed to the site by celebrities, resulted mostly in numerous neutral responses with few links to conservation or awareness. Two conservation-related events, linked to Wikipedia and the airing of a television documentary, led to an increase in awareness, and ultimately to the removal of the analysed video.

Conclusions/Significance

Slow loris videos that have gone viral have introduced these primates to a large cross-section of society that would not normally come into contact with them. Analyses of webometric data posted on the internet allow us quickly to gauge societal sentiments. We showed a clear temporal change in some views expressed but without an apparent increase in knowledge about the conservation plight of the species, or the illegal nature of slow loris trade. Celebrity endorsement of videos showing protected wildlife increases visits to such sites, but does not educate about conservation issues. The strong desire of commentators to express their want for one as a pet demonstrates the need for Web 2.0 sites to provide a mechanism via which illegal animal material can be identified and policed.     

Protective effects of agomelatine on testicular damage caused by bortezomib

Bortezomib is a chemotherapeutic agent used to treat several cancers; however, it exhibits severe side effects in testicular tissue. We investigated the use of agomelatine to prevent testicular tissue damage caused by bortezomib. We used 36 male Sprague-Dawley rats divided randomly into six equal groups: group 1, no treatment control; group 2, agomelatine treatment only; group 3, bortezomib treatment only for 48 h; group 4, bortezomib + agomelatine treatment for 48 h; group 5, bortezomib treatment only for 72 h; and group 6, bortezomib + agomelatine treatment for 72 h. After treatments, the rats were sacrificed and testicular tissue was harvested. Lipid oxidation (LPO) and superoxide dismutase (SOD) levels in the tissues were determined using biochemical methods. Tissue samples also were examined using histopathological and immunohistochemical techniques. The LPO level was increased, while the SOD level was decreased in the bortezomib treated groups. We found that agomelatine treatment normalized LPO and SOD activities in the bortezomib treated groups. In the spermatogonia and Sertoli cells, the staining density of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and caspase 3 were decreased in the bortezomib + agomelatine groups at both 48 and 72 h compared to bortezomib only treated groups. We observed maturation arrest, basal membrane thickening, increase in inflammatory cells and connective tissue, and edema between germ cells in the bortezomib only treated groups. By contrast, normal basal membrane, less edema and more normal maturation were observed in the bortezomib + agomelatine groups at 48 and 72 h. We found that agomelatine reduced the damaging effects of bortezomib. The use of agomelatine to prevent bortezomib induced testicular tissue damage in human patients should be investigated further.     

Crystallization of a type I 3-dehydroquinase from Salmonella typhi.

Crystals have been grown of a type I 3-dehydroquinase from both Escherichia coli and Salmonella typhi. However, only those from S. typhi diffract to a resolution of 2.3 A on a conventional X-ray source and are suitable for structure determination. The space group has been determined as P2(1)2(1)2 with unit cell dimensions a = 48.01 A, b = 114.29 A, c = 42.87 A. There is one subunit in the asymmetric unit.