Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.     

Disruption of the bacteriophage T4 Mre11 dimer interface reveals a two-state mechanism for exonuclease activity

The Mre11-Rad50 (MR) complex is a central player in DNA repair and is implicated in the processing of DNA ends caused by double strand breaks. Recent crystal structures of the MR complex suggest that several conformational rearrangements occur during its ATP hydrolysis cycle. A comparison of the Mre11 dimer interface from these structures suggests that the interface is dynamic in nature and may adopt several different arrangements. To probe the functional significance of the Mre11 dimer interface, we have generated and characterized a dimer disruption Mre11 mutant (L101D-Mre11). Although L101D-Mre11 binds to Rad50 and dsDNA with affinity comparable with the wild-type enzyme, it does not activate the ATP hydrolysis activity of Rad50, suggesting that the allosteric communication between Mre11 and Rad50 has been interrupted. Additionally, the dsDNA exonuclease activity of the L101D-MR complex has been reduced by 10-fold under conditions where processive exonuclease activity is required. However, we unexpectedly found that under steady state conditions, the nuclease activity of the L101D-MR complex is significantly greater than that of the wild-type complex. Based on steady state and single-turnover nuclease assays, we have assigned the rate-determining step of the steady state nuclease reaction to be the productive assembly of the complex at the dsDNA end. Together, our data suggest that the Mre11 dimer interface adopts at least two different states during the exonuclease reaction.     

Multiple sclerosis and pregnancy: what does the patient think? a questionnaire study

Background

The first objective of a DNA microarray experiment is typically to generate a list of genes or probes that are found to be differentially expressed or represented (in the case of comparative genomic hybridizations and/or copy number variation) between two conditions or strains. Rank Products analysis comprises a robust algorithm for deriving such lists from microarray experiments that comprise small numbers of replicates, for example, less than the number required for the commonly used t-test. Currently, users wishing to apply Rank Products analysis to their own microarray data sets have been restricted to the use of command line-based software which can limit its usage within the biological community.

Findings

Here we have developed a web interface to existing Rank Products analysis tools allowing users to quickly process their data in an intuitive and step-wise manner to obtain the respective Rank Product or Rank Sum, probability of false prediction and p-values in a downloadable file.

Conclusions

The online interactive Rank Products analysis tool RankProdIt, for analysis of any data set containing measurements for multiple replicated conditions, is available at: http://strep-microarray.sbs.surrey.ac.uk/RankProducts     

The redox‐sensing regulator YodB senses quinones and diamide via a thiol‐disulfide switch in Bacillus subtilis

The MarR/DUF24‐type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox‐sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6‐Ala mutant that the conserved Cys6 apparently contributes to the DNA‐binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox‐sensing in response to diamide and 2‐methylhydroquinone (MHQ) in vivo. DNA‐binding experiments indicate that YodB is regulated by a reversible thiol‐modification in response to diamide and MHQ in vitro. Redox‐regulation of YodB involves Cys6‐Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C‐terminal Cys101' or Cys108' residues. This study reveals a thiol‐disulfide switch model of redox‐regulation for the YodB repressor to sense electrophilic compounds in vivo.     

Optimum allocation of resources for QTL detection using a nested association mapping strategy in maize

In quantitative trait locus (QTL) mapping studies, it is mandatory that the available financial resources are spent in such a way that the power for detection of QTL is maximized. The objective of this study was to optimize for three different fixed budgets the power of QTL detection 1 − β* in recombinant inbred line (RIL) populations derived from a nested design by varying (1) the genetic complexity of the trait, (2) the costs for developing, genotyping, and phenotyping RILs, (3) the total number of RILs, and (4) the number of environments and replications per environment used for phenotyping. Our computer simulations were based on empirical data of 653 single nucleotide polymorphism markers of 26 diverse maize inbred lines which were selected on the basis of 100 simple sequence repeat markers out of a worldwide sample of 260 maize inbreds to capture the maximum genetic diversity. For the standard scenario of costs, the optimum number of test environments (E _opt) ranged across the examined total budgets from 7 to 19 in the scenarios with 25 QTL. In comparison, the E _opt values observed for the scenarios with 50 and 100 QTL were slightly higher. Our finding of differences in 1 − β* estimates between experiments with optimally and sub-optimally allocated resources illustrated the potential to improve the power for QTL detection without increasing the total resources necessary for a QTL mapping experiment. Furthermore, the results of our study indicated that also in studies using the latest genomics tools to dissect quantitative traits, it is required to evaluate the individuals of the mapping population in a high number of environments with a high number of replications per environment.     

Field evaluation of entomopathogenic nematodes for the biological control of the annual bluegrass weevil,Listronotus maculicollis (Coleoptera: Curculionidae), in golf course turfgrass

The ability of entomopathogenic nematodes to suppress larval populations of the annual bluegrass weevil, Listronotus maculicollis, was investigated under field conditions over a 3-year period (2006–2008). Combination of nematode species, application rate and timing produced strong numerical yet few statistically significant reductions. Steinernema carpocapsae Weiser, S. feltiae Filipjev, and Heterorhabditis bacteriophora Poinar applied at 2.5×10⁹ IJs/ha reduced first generation late instars between 69 and 94% in at least one field trial. Steinernema feltiae provided a high level of control (94%) to low densities (~20 larvae per 0.09 m²), but gave inadequate control for higher densities (24 and 50% suppression). No significant differences were found among treatment timings. However, applications timed to coincide with the peak of larvae entering the soil (fourth instars) generally performed better than applications made prior to (preemptive) or after the majority of the population advanced from the fourth instar. Nematode populations declined sharply between 0 and 14 days after treatment (DAT). Although nematode populations later increased (at 28 DAT), indicating an ability to recycle within hosts in the environment, they were nearly undetectable 56 DAT when the second generation host larvae were present in the soil. Applying commercially available nematode species at standard field rates cannot reliably reduce L. maculicollis immature densities on golf courses, nor will single applications suppress multiple generations. Future research will need to identify application strategies to improve biocontrol consistency.     

Dimerization of the hepatitis C virus nonstructural protein 4B depends on the integrity of an aminoterminal basic leucine zipper

The hepatitis C virus (HCV) nonstructural (NS) protein 4B is known for protein–protein interactions with virus and host cell factors. Only little is known about the corresponding protein binding sites and underlying molecular mechanisms. Recently, we have predicted a putative basic leucine zipper (bZIP) motif within the aminoterminal part of NS4B. The aim of this study was to investigate the importance of this NS4B bZIP motif for specific protein–protein interactions. We applied in silico approaches for 3D‐structure modeling of NS4B‐homodimerization via the bZIP motif and identified crucial amino acid positions by multiple sequence analysis. The selected sites were used for site‐directed mutagenesis within the NS4B bZIP motif and subsequent co‐immunoprecipitation of wild‐type and mutant NS4B molecules. Respective interaction energies were calculated for wild‐type and mutant structural models. NS4B‐homodimerization with a gradual alleviation of dimer interaction from wild‐type towards the mutant‐dimers was observed. The putative bZIP motif was confirmed by a co‐immunoprecipitation assay and western blot analysis. NS4B‐NS4B interaction depends on the integrity of the bZIP hydrophobic core and can be abolished due to changes of crucial residues within NS4B. In conclusion, our data indicate NS4B‐homodimerization and that this interaction is facilitated by the aminoterminal part containing a bZIP motif.