Structural basis for the transcriptional repressor NicR2 in nicotine degradation from Pseudomonas

Nicotine is an environmental toxicant in tobacco wastes, imposing severe hazards for the health of human and other mammalians. NicR2, a TetR‐like repressor from Pseudomonas putida S16, plays a critical role in regulating nicotine degradation. Here, we determined the crystal structures of NicR2 and its complex with the inducer 6‐hydroxy‐3‐succinoyl‐pyridine (HSP). The N‐terminal domain of NicR2 contains a conserved helix‐turn‐helix (HTH) DNA‐binding motif, while the C‐terminal domain contains a cleft for its selective recognition for HSP. Residues R91, Y114 and Q118 of NicR2 form hydrogen bonds with HSP, their indispensable roles in NicR2's recognition with HSP were confirmed by structure‐based mutagenesis combined with isothermal titration calorimetry analysis. Based on sequence alignment and structure comparison, Tyr67, Tyr68 and Lys72 of HTH motif were corroborated to take the major responsibility for DNA‐binding using site‐directed mutants. The 30‐residue N‐terminal extension of NicR2, especially residues 21–30 in the TFR arm, is required for the association with the operator DNA. Finally, we proposed that either NicR2 or the DNA would undergo a conformational change upon their association. Altogether, our structural and biochemical investigations unravel how NicR2 selectively recognizes HSP and DNA, and provide new insights into the TetR family of repressors.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.     

Virulence Determinants in the PB2 Gene of a Mouse-Adapted H9N2 Virus

The molecular bases of adaptation and pathogenicity of H9N2 influenza virus in mammals are largely unknown. Here, we show that a mouse-adapted PB2 gene with a phenylalanine-to-leucine mutation (F404L) mainly contributes to enhanced polymerase activity, replication, and pathogenicity of H9N2 in mice and also increases the virulence of the H5N1 and 2009 pandemic H1N1 influenza viruses. Therefore, we defined a novel pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals.     

Atrazine degradation by a simple consortium of <Emphasis Type="Italic">Klebsiella</Emphasis> sp. A1 and <Emphasis Type="Italic">Comamonas</Emphasis> sp. A2 in nitrogen enriched medium

A simple consortium consisted of two members of Klebsiella sp. A1 and Comamonas sp. A2 was isolated from the sewage of a pesticide mill in China. One member of Klebsiella sp. A1 is a novel strain that could use atrazine as the sole carbon and nitrogen source. The consortium showed high atrazine-mineralizing efficiency and about 83.3% of 5 g l⁻¹ atrazine could be mineralized after 24 h degradation. Contrary to many other reported microorganisms, the consortium was insensitive to some nitrogenous fertilizers commonly used, not only in presence of 200 mg l⁻¹ atrazine but also in 5 g l⁻¹ atrazine mediums. After 24 h incubation, 200 mg l⁻¹ atrazine was completely mineralized despite of the presence of urea, (NH₄)₂CO₃ and (NH₄)₂HPO₄ in the medium. Very minor influence was observed when NH₄Cl was added as additional nitrogen source. Advantages of the simple consortium, high mineralizing efficiency and insensitivity to most of exogenous nitrogen sources, all suggested application potential of the consortium for the bioremediation of atrazine-contaminated soils and waters.     

Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction

Suppression of inflammation is critical for effective therapy of many infectious diseases. However, the high rates of mortality caused by sepsis attest to the need to better understand the basis of the inflammatory sequelae of sepsis and to develop new options for its treatment. In mice, inflammatory responses to host danger-associated molecular patterns (DAMPs), but not to microbial pathogen-associated molecular patterns (PAMPs), are repressed by the interaction [corrected] of CD24 and SiglecG (SIGLEC10 in human). Here we use an intestinal perforation model of sepsis to show that microbial sialidases target the sialic acid-based recognition of CD24 by SiglecG/10 to exacerbate inflammation. Sialidase inhibitors protect mice against sepsis by a mechanism involving both CD24 and Siglecg, whereas mutation of either gene exacerbates sepsis. Analysis of sialidase-deficient bacterial mutants confirms the key contribution of disrupting sialic acid-based pattern recognition to microbial virulence and supports the clinical potential of sialidase inhibition for dampening inflammation caused by infection.     

Sodium Hydrosulphide Alleviates Remote Lung Injury Following Limb Traumatic Injury in Rats

Hydrogen sulphide (H₂S) was found to attenuate ventilator or oleic acid induced lung injury. The aim of this study was to explore the effects of exogenous H₂S donor, sodium Hydrosulphide (NaHS), on lung injury following blast limb trauma and the underlying mechanisms. For in vitro experiments, pulmonary micro-vessel endothelial cells (PMVECs) were cultured and treated with NaHS or vehicle in the presence of TNF-α. For in vivo, blast limb traumatic rats, induced by using chartaceous electricity detonators, were randomly treated with NaHS, cystathionine gamma-lyase inhibitor (PAG) or vehicle. In vitro, NaHS (100 µM) treatment increased PMVECs viability and decreased LDH release into culture media after tumor necrosis factor (TNF) α challenge. In addition, NaHS treatment prevented the increase of nitric oxide, Intercellular Adhesion Molecule 1(ICAM-1) and interleukin (IL)-6 production and inducible nitric oxide synthase activation induced by TNF-α. Knock-down of NF-E2-Related Factor 2 (Nrf2) partially abolished the protective effect of NaHS. In vivo, NaHS treatment significantly alleviated lung injury following blast limb trauma, demonstrated by a decreased histopathological score and lung water content. Furthermore, NaHS treatment reversed the decrease of H₂S concentration in plasma, prevented the increase of TNF-α, IL-6, malondialdehyde and myeloperoxidase, increased the Nrf2 downstream effector glutathione in both plasma and lungs, and reversed the decrease of superoxide dismutase in both plasma and lungs induced by blast limb trauma. Our data indicated that NaHS protects against lung injury following blast limb trauma which is likely associated with suppression of the inflammatory and oxidative response and activation of Nrf2 cellular signal.     

麦冬皂苷药理作用研究进展

麦冬作为一种滋阴药,在传统中药中占有重要地位。随着对麦冬研究的不断深入,其主要有效成分皂苷类的药理活性也不断被发现。综述近年来麦冬皂苷在抗心脑血管疾病、抗衰老、改善学习记忆障碍、抗肿瘤、抗辐射、抗炎、免疫调节、镇咳、改善肝肺病理性损伤等方面的药理作用研究进展,以期为今后对麦冬及其皂苷类活性成分的深入研究和开发利用提供参考。     

Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM

d-Lactic acid and pyruvic acid are two important building block intermediates. Production of d-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l^?1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l^?1 dl-lactic acid into 25.23 g l^?1 d-lactic acid and 19.70 g l^?1 pyruvic acid in 10 h. Using a simple ion exchange process, d-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.