Unbinding forces of single pertussis toxin-antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance

An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant k(a) and the dissociation constant k(d) . A significant reduction in k(a) was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the k(d) value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate k(d) values. The use of DFS as a technique to study ultratight binding is discussed.     

Identification of a chemoreceptor that specifically mediates chemotaxis toward metabolizable purine derivatives

Chemotaxis is an essential mechanism that enables bacteria to move toward favorable ecological niches. Escherichia coli, the historical model organism for studying chemotaxis, has five well‐studied chemoreceptors. However, many bacteria with different lifestyle have more chemoreceptors, most of unknown function. Using a high throughput screening approach, we identified a chemoreceptor from Pseudomonas putida KT2440, named McpH, which specifically recognizes purine and its derivatives, adenine, guanine, xanthine, hypoxanthine and uric acid. The latter five compounds form part of the purine degradation pathway, permitting their use as sole nitrogen sources. Isothermal titration calorimetry studies show that these six compounds bind McpH‐Ligand Binding Domain (LBD) with very similar affinity. In contrast, non‐metabolizable purine derivatives (caffeine, theophylline, theobromine), nucleotides, nucleosides or pyrimidines are unable to bind McpH‐LBD. Mutation of mcpH abolished chemotaxis toward the McpH ligands identified – a phenotype that is restored by complementation. This is the first report on bacterial chemotaxis to purine derivatives and McpH the first chemoreceptor described that responds exclusively to intermediates of a catabolic pathway, illustrating a clear link between metabolism and chemotaxis. The evolution of McpH may reflect a saprophytic lifestyle, which would have exposed the studied bacterium to high concentrations of purines produced by nucleic acid degradation.     

The carbon count of 2000 years of rice cultivation

More than 50% of the world's population feeds on rice. Soils used for rice production are mostly managed under submerged conditions (paddy soils). This management, which favors carbon sequestration, potentially decouples surface from subsurface carbon cycling. The objective of this study was to elucidate the long‐term rates of carbon accrual in surface and subsurface soil horizons relative to those of soils under nonpaddy management. We assessed changes in total soil organic as well as of inorganic carbon stocks along a 2000‐year chronosequence of soils under paddy and adjacent nonpaddy management in the Yangtze delta, China. The initial organic carbon accumulation phase lasts much longer and is more intensive than previously assumed, e.g., by the Intergovernmental Panel on Climate Change (IPCC). Paddy topsoils accumulated 170–178 kg organic carbon ha^?1 a^?1 in the first 300 years; subsoils lost 29–84 kg organic carbon ha^?1 a^?1 during this period of time. Subsoil carbon losses were largest during the first 50 years after land embankment and again large beyond 700 years of cultivation, due to inorganic carbonate weathering and the lack of organic carbon replenishment. Carbon losses in subsoils may therefore offset soil carbon gains or losses in the surface soils. We strongly recommend including subsoils into global carbon accounting schemes, particularly for paddy fields.     

NAMPT pathway is involved in the FOXO3a-mediated regulation of GADD45A expression

Nicotinamide-phosphoribosyltransferase (NAMPT), induced under stress, converts nicotinamide (NA) to nicotinamide mononucleotide (NMN), which then reacts with ATP to regenerate NAD(+). Despite the pivotal role of NAD(+) in metabolic reactions, the molecular pathways triggered by the intracellular changes in NAD(+) level in cancer cells are largely unknown. Growth Arrest and DNA Damage-inducible Gene (GADD45A) is regulated by multiple cellular factors which play an important role in the control of cell-cycle checkpoint, DNA repair process and signal transduction. The present study was designed to assess the significance of intracellular NAD(+) levels on the regulation of GADD45A expression. The results of this study demonstrate an inverse relationship between NAMPT expression and the regulation of GADD45A gene. Thus, an overexpression of NAMPT led to a decreased expression of GADD45A, whereas, the inhibition of NAMPT by the known chemical inhibitor FK866 increased the expression of GADD45A in cells. Inhibition of SIRT1, an NAD(+)-dependent deacetylase, using shRNA also led to an increased expression of GADD45A gene. In further experiments we could show that the increased expression of GADD45A under the above experimental conditions, NAMPT inhibition by FK866, involves acetylation of FOXO3a, a member of the important family of forkhead (FOXO) proteins. This knowledge should contribute to our understanding of the role played by NAMPT and SIRT1 in the regulation of GADD45A expression by FOXO3a.     

On the mechanism of apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> production during lignin formation and elicitation in cultured spruce cells—peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H₂O₂ generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H₂O₂ were present in the culture medium during lignin formation. Elicitation induced a burst of H₂O₂, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H₂O₂ from O₂, NADH stimulated H₂O₂ production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H₂O₂, but usually increased or prolonged elicitor-induced H₂O₂ formation. Culture medium peroxidases were not able to generate H₂O₂ in vitro with Cys or GSH as reductants. These thiols, however, generated H₂O₂ non-enzymically at pH 4.5. [³⁵S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H₂O₂ formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H₂O₂ generation but strongly delayed the elicitor-induced H₂O₂ accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H₂O₂ production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H₂O₂ generation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12

The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ insignificantly in genome structure. The growth patterns of these two sub-strains were very similar in a well-equipped bioreactor, and their genome structures were shown to be almost identical by DNA microarray. However, detailed profiling of protein and gene expression by 2-dimensional gel electrophoresis and microarray analysis showed many differentially expressed genes and proteins, combinations of which were highly correlated. The differentially regulated genes and proteins belonged to the following functional categories: genes regulated by sigma subunit of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in central metabolism. Genes involved in central cell metabolism - the glycolysis pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were differentially regulated at both the mRNA and proteome levels. The strains differ significantly in central metabolism and thus in the generation of precursor metabolites and energy. This high plasticity probably represents a universal feature of metabolic activities in closely related species, and has the potential to reveal differences in regulatory networks. We suggest that unless care is taken in the choice of strains for any validating experiment, the results might be misleading.     

Conversion of carbazole carboxamide based reversible inhibitors of Bruton’s tyrosine kinase (BTK) into potent,selective irreversible inhibitors in the carbazole,tetrahydrocarbazole, and a new 2,3-dimethylindole series

Incorporation of a suitably-placed electrophilic group transformed a series of reversible BTK inhibitors based on carbazole-1-carboxamide and tetrahydrocarbazole-1-carboxamide into potent, irreversible inhibitors. Removal of one ring from the core of these compounds provided a potent irreversible series of 2,3-dimethylindole-7-carboxamides having excellent potency and improved selectivity, with the additional advantages of reduced lipophilicity and molecular weight.     

Receptor assay guided structure‐activity studies of helicokinin insect neuropeptides and peptidomimetic analogues

Neuropeptides control numerous physiological processes in insects. The regulation of water balance is a crucial aspect of homeostasis in terrestrial insects and has been shown to be under endocrine control, primarily by corticotrophin releasing factor (CRF)‐related peptides and kinins. For helicokinin I, a diuretic neuropeptide from the economically important insect pest Heliothis virescens, detailed structure‐activity relationships have been established based on truncated structures, diverse amino acid scans and peptidomimetic analogues. The activities of selected compounds on functional expressed helicokinin receptors are compared with the results of a Malphigian tubule assay. Implications for further peptidomimetic variations are provided. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.