Analysis of the thyrotropin-releasing hormone-degrading ectoenzyme by site-directed mutagenesis of cysteine residues. Cys68 is involved in disulfide-linked dimerization.

Thyrotropin-releasing hormone-degrading ectoenzyme is a member of the M1 family of Zn-dependent aminopeptidases and catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2). Cloning of the cDNA of this enzyme and biochemical studies revealed that the large extracellular domain of the enzyme with the catalytically active site contains nine cysteine residues that are highly conserved among species. To investigate the functional role of these cysteines in TRH-DE we used a site-directed mutagenesis approach and replaced individually each cysteine by a serine residue. The results revealed that the proteolytically truncated and enzymatically fully active enzyme consists of two identical subunits that are associated noncovalently by protein-protein interactions but not via interchain S-S bridges. The eight cysteines contained within this region are all important for the structure of the individual subunit and the enzymatic activity, which is dramatically reduced in all mutant enzymes. This is even true for the four cysteines that are clustered within the C-terminal domain remote from the Zn-binding consensus sequence HEICH. In contrast, Cys68, which resides within the stalk region seven residues from the end of the hydrophobic membrane-spanning domain, can be replaced by serine without a significant change in the enzymatic activity. Interestingly, this residue is involved in the formation of an interchain disulfide bridge. Covalent dimerization of the subunits, however, does not seem to be essential for efficient biosynthesis, enzymatic activity and trafficking to the cell surface.     

Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility,Gating Residues and Multiple Binding Pockets

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.     

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

The Carbon Footprint of Games Distribution

This research investigates the carbon footprint of the lifecycle of console games, using the example of PlayStation®3 distribution in the UK. We estimate total carbon equivalent emissions for an average 8.8‐gigabyte (GB) game based on data for 2010. The bulk of emissions are accounted for by game play, followed by production and distribution. Two delivery scenarios are compared: The first examines Blu‐ray discs (BDs) delivered by retail stores, and the second, games files downloaded over broadband Internet. Contrary to findings in previous research on music distribution, distribution of games by physical BDs results in lower greenhouse gas emissions than by Internet download. The estimated carbon emissions from downloading only fall definitively below that of BDs for games smaller than 1.3 GB. Sensitivity analysis indicates that as average game file sizes increase, and the energy intensity of the Internet falls, the file size at which BDs would result in lower emissions than downloads could shift either up‐ or downward over the next few years. Overall, the results appear to be broadly applicable to title games within the European Union (EU), and for larger‐than‐average sized games in the United States. Further research would be needed to confirm whether similar findings would apply in future years with changes in game size and Internet efficiency. The study findings serve to illustrate why it is not always true that digital distribution of media will have lower carbon emissions than distribution by physical means when file sizes are large.     

Armeniaspiroles, a new class of antibacterials: antibacterial activities and total synthesis of 5-chloro-Armeniaspirole A

Armeniaspiroles, a novel class of natural products isolated from Streptomyces armeniacus, are characterized by a novel spiro[4.4]non-8-ene scaffold. Various derivatives of Armeniaspiroles could be obtained by halogenation, alkylation, addition/elimination or reductions. A total synthesis of the 5-chloro analog of Armeniaspirole A has been accomplished in a linear six-step sequence. 5-Chloro-Armeniaspirole A exhibits good activity against a range of multidrug-resistant, Gram-positive bacterial pathogens.     

A comparison of the nitrogen metabolic networks of Kluyveromyces marxianus and Saccharomyces cerevisiae

In grape must, nitrogen is available as a complex mixture of various compounds (ammonium and amino acids). Wine yeasts assimilate these multiple sources in order to suitably fulfil their anabolic requirements during alcoholic fermentation. Nevertheless, the order of uptake and the intracellular fate of these sources are likely to differ between strains and species. Using a two-pronged strategy of isotopic filiation and RNA sequencing, the metabolic network of nitrogen utilization and its regulation in Kluyveromyces marxianus were described, in comparison with those of Saccharomyces cerevisiae. The data highlighted differences in the assimilation of ammonium and arginine between the two species. The data also revealed that the metabolic fate of certain nitrogen sources differed, thereby resulting in the production of various amounts of key wine aroma compounds. These observations were corroborated by the gene expression analysis.     

The development of cephalic armor in the tokay gecko (Squamata: Gekkonidae: Gekko gecko)

Armored skin resulting from the presence of bony dermal structures, osteoderms, is an exceptional phenotype in gekkotans (geckos and flap-footed lizards) only known to occur in three genera: Geckolepis, Gekko, and Tarentola. The Tokay gecko (Gekko gecko LINNAEUS 1758) is among the best-studied geckos due to its large size and wide range of occurrence, and although cranial dermal bone development has previously been investigated, details of osteoderm development along a size gradient remain less well-known. Likewise, a comparative survey of additional species within the broader Gekko clade to determine the uniqueness of this trait has not yet been completed. Here, we studied a large sample of gekkotans (38 spp.), including 18 specimens of G. gecko, using X-rays and high-resolution computed tomography for visualizing and quantifying the dermal armor in situ. Results from this survey confirm the presence of osteoderms in a second species within this genus, Gekko reevesii GRAY 1831, which exhibits discordance in timing and pattern of osteoderm development when compared with its sister taxon, G. gecko. We discuss the developmental sequence of osteoderms in these two species and explore in detail the formation and functionality of these enigmatic dermal ossifications. Finally, we conducted a comparative analysis of endolymphatic sacs in a wide array of gekkotans to explore previous ideas regarding the role of osteoderms as calcium reservoirs. We found that G. gecko and other gecko species with osteoderms have highly enlarged endolymphatic sacs relative to their body size, when compared to species without osteoderms, which implies that these membranous structures might fulfill a major role of calcium storage even in species with osteoderms.     

Variability in benthic diazotrophy and cyanobacterial diversity in a tropical intertidal lagoon

Benthic nitrogen fixation has been estimated to contribute 15 Tg N year(-1) to the marine nitrogen budget. With benthic marine nitrogen fixation being largely overlooked in more recent surveys, a refocus on benthic diazotrophy was considered important. Variations in nitrogenase activity (acetylene reduction-gas chromatography) in a tropical lagoon in the western Indian Ocean (Zanzibar, Tanzania) were monitored over a 3-year period (2003-2005) and related to cyanobacterial and diazotrophic microbial diversity using a polyphasic approach. Different nitrogenase activity patterns were discerned, with the predominant pattern being high daytime activities combined with low nighttime activities. Analyses of the morphological and 16S rRNA gene diversity among cyanobacteria revealed filamentous nonheterocystous (Oscillatoriales) and unicellular (Chroococcales) representatives to be predominant. Analyses of the nifH gene diversity showed that the major phylotypes belonged to noncyanobacterial prokaryotes. However, as shown by cyanobacterial selective nifH-denaturing gradient gel electrophoresis analysis, cyanobacterial nifH gene sequences were present at all sites. Several nifH and 16S rRNA gene phylotypes were related to uncultured cyanobacteria or bacteria of geographically distant habitats, stressing the widespread occurrence of still poorly characterized microorganisms in tropical benthic marine communities.