ATP inhibits hydroxyl radical formation and the inflammatory response of stimulated whole blood even under circumstances of severe oxidative stress

We recently reported that Adenosine-5′-triphosphate (ATP) is able to inhibit the inflammatory reaction in stimulated whole blood. Many diseases, in which inflammatory reactions are involved, are associated with oxidative stress. In the present study, we therefore, investigated the effect of ATP on cytokine release in stimulated whole blood under conditions of oxidative stress, as simulated by pre-incubation of blood with hydrogen peroxide (H₂O₂). In the presence of H₂O₂, ATP at concentrations of 100 and 300 μM inhibited Tumour Necrosis factor-alpha (TNF-α) release and stimulated IL-10 release in LPS-PHA stimulated whole blood. Moreover, electron spin resonance (ESR) measurements showed that ATP and its breakdown product Adenosine-5′-diphosphate (ADP) attenuated spin trap-hydroxyl radical adduct formation in the Fenton reaction. Our results demonstrate that even in circumstances of severe oxidative stress, ATP has marked anti-inflammatory properties in stimulated whole blood. Moreover, the inhibition of the hydroxyl radical ESR signal indicates a direct attenuation of oxidative stress by ATP.     

A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies

Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.     

Alpha-helix formation is required for high affinity binding of human apolipoprotein A-I to lipids

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.     

Metabolism and Plant Hormone Action During Clubroot Disease

Infection of Brassicaceae with the obligate biotrophic pathogen Plasmodiophora brassicae results in the development of root galls (clubroots). During the transformation of a healthy root to a root gall a plethora of changes in primary and secondary metabolism occur. The upper part of an infected plant is retarded in growth due to redirection of assimilates from the shoot to the root. In addition, changes in the levels of plant growth regulators, especially auxins and cytokinins, contribute to the hypertrophy of infected roots. Also, defense reactions are manipulated after inoculation of suitable host plants with P. brassicae. This review summarizes our current knowledge on the changes in these parameters. A model is presented for how primary metabolism and secondary metabolism, including plant hormones, interact to induce clubroot formation.     

Multi‐scale habitat modification by coexisting ecosystem engineers drives spatial separation of macrobenthic functional groups

By changing habitat conditions, ecosystem engineers increase niche diversity and have profound effects on the distribution and abundances of other organisms. Although many ecosystems contain several engineering species, it is still unclear how the coexistence of multiple engineers affects the physical habitat and the structure of the community on a landscape scale. Here, we investigated through a large‐scale field manipulation how three coexisting engineers on intertidal flats (cockles Cerastoderma edule; lugworms Arenicola marina; blue mussels Mytilus edulis) influence the functional composition of the local macrobenthic community and what the consequences are at the landscape level. By using biological trait analysis (BTA), we show that on the local scale biogenic changes in sediment accumulation and organic matter content translated into specific shifts in the distribution of functional traits within the community. At a landscape scale, the co‐occurrence of multiple ecosystem engineers resulted in the spatial separation of different functional groups, i.e. different functional groups dominated unique complementary habitats. Our results emphasize the role of co‐occurring multiple engineers in shaping natural communities, thus contributing to a better knowledge of community assembly rules. This understanding can profitably be used to improve ecosystem‐based management and conservation actions.     

A Combination of Fertility Signals and Aggression Regulates Reproduction in the Ant <Emphasis Type=Italic>Gnamptogenys striatula</Emphasis>

Approximately 150 ant species are facultatively or obligately queenless whereby mated workers assume the role of the queen. In many of these species a reproductive dominance hierarchy is established by way of aggressive interactions. Top-ranking workers, which are typically the most fecund, acquire a characteristic cuticular hydrocarbon profile. We studied the temporal dynamics of this chemical change and associated interplay with observed aggressive interactions in an experimentally orphaned colony of the facultatively queenless ant Gnamptogenys striatula. Our observations and chemical analyses demonstrate that chemical fertility signals played a major role in the establishment of a dominance hierarchy and aggression settled dominance relationships only when ants had identical hydrocarbon profiles. Moreover, individuals with a higher potential fertility, in this experiment reflected in a higher ovariole number, are shown to have a better chance of becoming a reproductive.     

Cadmium stress: an oxidative challenge

At the cellular level, cadmium (Cd) induces both damaging and repair processes in which the cellular redox status plays a crucial role. Being not redox-active, Cd is unable to generate reactive oxygen species (ROS) directly, but Cd-induced oxidative stress is a common phenomenon observed in multiple studies. The current review gives an overview on Cd-induced ROS production and anti-oxidative defense in organisms under different Cd regimes. Moreover, the Cd-induced oxidative challenge is discussed with a focus on damage and signaling as downstream responses. Gathering these data, it was clear that oxidative stress related responses are affected during Cd stress, but the apparent discrepancies observed in between the different studies points towards the necessity to increase our knowledge on the spatial and temporal ROS signature under Cd stress. This information is essential in order to reveal the exact role of Cd-induced oxidative stress in the modulation of downstream responses under a diverse array of conditions.     

The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier

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