Crystal structures of E. coli nicotinate mononucleotide adenylyltransferase and its complex with deamido-NAD.

Nicotinamide/Nicotinate mononucleotide (NMN/NaMN) adenylyltransferase is an indispensable enzyme in both de novo biosynthesis and salvage of NAD+ and NADP+. In prokaryotes, it is absolutely required for cell survival, thus representing an attractive target for the development of new broad-spectrum antibacteria inhibitors. The crystal structures of E. coli NaMN adenylyltransferase (NMNAT) and its complex with deamido-NAD (NaAD) revealed that ligand binding causes large conformational changes in several loop regions around the active site. The enzyme specifically recognizes the deamidated pyridine nucleotide through interactions between nicotinate carboxylate with several protein main chain amides and a positive helix dipole. Comparison of E. coli NMNAT with those from archaeal organisms revealed extensive differences in the active site architecture, enzyme-ligand interaction mode, and bound dinucleotide conformations. The bacterial NaMN adenylyltransferase structures described here provide a foundation for structure-based design of specific inhibitors that may have therapeutic potential.     

Membrane insertion of the FYVE domain is modulated by pH

The FYVE domain associates with phosphatidylinositol 3‐phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we detail principles of membrane anchoring and show that the FYVE domain insertion into PtdIns(3)P‐enriched membranes and membrane‐mimetics is substantially increased in acidic conditions. The EEA1 FYVE domain binds to POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however associates ～24 fold weaker at pH 8.0. The decrease in the affinity is primarily due to much faster dissociation of the protein from the bilayers in basic media. Lowering the pH enhances the interaction of the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains with PtdIns(3)P‐containing membranes in vitro and in vivo, indicating that pH‐dependency is a general function of the FYVE finger family. The PtdIns(3)P binding and membrane insertion of the FYVE domain is modulated by the two adjacent His residues of the R(R/K)HHCRXCG signature motif. Mutation of either His residue abolishes the pH‐sensitivity. Both protonation of the His residues and nonspecific electrostatic contacts stabilize the FYVE domain in the lipid‐bound form, promoting its penetration and increasing the membrane residence time. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Structural and genetic characterization of Escherichia coli O99 antigen

The influence of antibiotic exposure in the early postnatal period on the development of intestinal microbiota was monitored in 26 infants including five antibiotic-treated (AT) subjects orally administered a broad-spectrum antibiotic for the first 4 days of life and three caesarean-delivered (CD) subjects whose mothers were intravenously injected by the similar type of antibiotics in the same period. The faecal bacterial composition was analysed daily for the first 5 days and monthly for the first 2 months. Terminal restriction fragment length polymor-phisms in the AT subjects showed less diversity with the attenuation of the colonization of some bacterial groups, especially in Bifidobacterium and unusual colonization of Enterococcus in the first week than the control antibiotic-free infants (AF, n =18). Quantitative real-time PCR showed overgrowth of enterococci (day 3, P =0.01; day 5, P =0.003; month 1, P =0.01) and arrested growth of Bifidobacterium (day 3, P =0.03) in the AT group. Furthermore, after 1 month, the Enterobacteriaceae population was markedly higher in the AT group than in the AF group (month 1, P =0.02; month 2, P =0.02). CD infants sustained similar, although relatively weaker, alteration in the developing microbiota. These results indicate that antibiotic exposure at the beginning of life greatly influences the development of neonatal intestinal microbiota.     

Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates

The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa⁺ phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri⁺ phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants’ responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri⁺ mutant BK759.G gave rise to stable Swa⁺⁺ derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa⁺⁺ derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates.     

2′-Fluorosugar analogues of the highly potent anti-varicella-zoster virus bicyclic nucleoside analogue (BCNA) Cf 1743

We report the preparation of 2′-α-F, 2′-β-F and 2′,2′-difluoro analogues of the leading anti-varicella zoster virus (VZV) pentylphenyl BCNA Cf 1743. VZV thymidine kinase showed the highest phosphorylating capacity for the β-fluoro derivative, that retained equal antiviral potency as the parent compound. In contrast, the α-fluoro- and 2′,2′-difluoro BCNA derivatives were markedly less (100-fold) antivirally active.     

Design,synthesis, and structure–activity relationship of novel orally efficacious pyrazole/sulfonamide based dihydroquinoline γ-secretase inhibitors

In this Letter, we report our strategy to design potent and metabolically stable γ-secretase inhibitors that are efficacious in reducing the cortical Aβx-40 levels in FVB mice via a single PO dose.     

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

Background  

Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.     

Density-dependent regulation in populations of potato-colonizing aphids

Scarcity of long-term (over 30 years) data series represents a major challenge for an accurate estimation of the role of density-dependent processes in population regulation. We analyzed population densities of the wingless parthenogenic morphs of buckthorn aphid (BA), Aphis nasturtii Kaltenbach, potato aphid (PA), Macrosiphum euphorbiae (Thomas), and green peach aphid (GPA), Myzus persicae (Sulzer) from 1949 to 2003 for signs of density-dependent regulation. We found strong evidence of density-dependent regulation, with detection of density dependence being fairly consistent among the different statistical techniques. Direct density dependence was detected for the populations of all three species. There was also evidence of delayed density dependence for PA. The periodicity of population fluctuations for BA and GPA was 6.1 years and 3.9 years, respectively. The periodicity for PA was not explicit, being highly variable throughout the time series. Effects of density-independent weather factors were relatively minor compared to density-dependent regulation. The BA populations experienced a significant reduction in both density and annual oscillations starting in 1995, while GPA populations experienced a similar reduction in 1991. No such change was apparent for PA. The most likely explanation for the observed phenomenon is a change in the composition of the lady beetle community following the establishment of two alien coccinellid species, and/or changes in insecticide use by commercial growers in the area of the study.