The Gam protein of bacteriophage Mu is an orthologue of eukaryotic Ku

Mu bacteriophage inserts its DNA into the genome of host bacteria and is used as a model for DNA transposition events in other systems. The eukaryotic Ku protein has key roles in DNA repair and in certain transposition events. Here we show that the Gam protein of phage Mu is conserved in bacteria, has sequence homology with both subunits of Ku, and has the potential to adopt a similar architecture to the core DNA-binding region of Ku. Through biochemical studies, we demonstrate that Gam and the related protein of Haemophilus influenzae display DNA binding characteristics remarkably similar to those of human Ku. In addition, we show that Gam can interfere with Ty1 retrotransposition in Saccharomyces cerevisiae. These data reveal structural and functional parallels between bacteriophage Gam and eukaryotic Ku and suggest that their functions have been evolutionarily conserved.     

Resolving neutral and deterministic contributions to genomic structure in <Emphasis Type="Italic">Syntrichia ruralis</Emphasis> (Bryophyta,Pottiaceae) informs propagule sourcing for dryland restoration

Syntrichia ruralis is a cosmopolitan moss that occupies steep environmental gradients. In arid to semi-arid regions of the world it is a key component of biological soil crusts, which are fundamental to healthy dryland ecosystem processes. As such, S. ruralis has attracted the attention of conservationists seeking to restore degraded biological soil crust communities and their associated vascular flora. Here, we generate genomic data for S. ruralis populations that span climatic gradients across the Colorado Plateau of the southwestern USA to investigate the contributions of neutral and deterministic processes to the partitioning of genomic structure. Although S. ruralis appears to be highly dispersible, geographic proximity significantly predicts genomic similarity. In addition, even when taking into account apparently high migration rates among populations and spatial autocorrelation of allele frequencies, some genomic variation is explained by environmental gradients correlated with elevation and latitude. Consequently, efforts to restore dryland ecosystems by establishing S. ruralis as a foundation should include strategies to ensure that propagule sources of this moss are environmentally stratified and targeted to the current/future climates of restoration sites.     

An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture

Oligosaccharides attached to Asn297 in each of the C_H2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.     

The discovery of 3-(N-alkyl)aminopropylphosphonic acids as potent S1P receptor agonists

3-(N-Alkyl)aminopropylphosphonic acids are potent agonists of four of the five known sphingosine-1-phosphate (S1P) receptor subtypes.     

The terminology problem for T cells: a discussion paper

The school of thought that owes allegiance to Ludwig Wittgenstein teaches that language conditions perceptions. When we use the term ''cytotoxic T lymphocyte'' or ''helper T cell'' we tend to orientate our own thinking processes, and those of listeners or readers, down particular paths. Part of the problem is that we are often describing cell populations by functions that may either be a property of only a proportion of those that are being assayed, or are simply inferred from the expression of various cell-surface markers. The consequence can be a measure of confusion that might be avoided if we could communicate with greater clarity. Is it possible to achieve a better terminology that will be accepted generally? The following are some examples of why there may be some value in thinking about this.     

Nick recognition by DNA ligases

Phage T7 DNA ligase seals nicked DNA substrates and is a representative member of the ATP-dependent class of DNA ligases. Although the catalytic mechanism of DNA ligases has been delineated, little is known about the nature of nick recognition by these enzymes. Here, we show that T7 ligase discriminates, at the nick-binding step, between nicks containing either a 5'-phosphate or a 5'-OH. T7 ligase binds preferentially to phosphorylated nicks and catalyses the sealing reaction. We also show using DNA footprinting studies, that T7 ligase binds asymmetrically to nicks as a monomer, with the protein interface covering between 12 and 14 bp of DNA. Based on molecular modelling studies we propose a structural model of the ligase-DNA complex consistent with these and other data. Using photo-crosslinking and site-directed mutagenesis we have identified two residues, K238 and K240, critical for the transadenylation and nick-sealing reactions. Sequence conservation and structural analysis supports the premise that these two lysine residues are critical for both nucleotide binding and DNA nick recognition. The implications of these results on the ligation mechanism are discussed.     

(E)-4-Hydroxy-3-methyl-but-2 enyl pyrophosphate-stimulated Vγ9Vδ2 T cells possess T helper type 1-promoting adjuvant activity for human monocyte-derived dendritic cells

Vγ9Vδ2 T cells respond to pyrophosphate antigens and display potent antitumour activity in vitro. We have investigated the potential of the most potent phosphoantigen known to activate Vγ9Vδ2 T cells, (E)-4-hydroxy-3-methyl-but-2 enyl pyrophosphate (HMB-PP), as an adjuvant for dendritic cell (DC)-based vaccines. A single stimulation of peripheral blood mononuclear cells with HMB-PP and IL-2 was sufficient to generate lines of effector memory Vγ9Vδ2 T cells that retained their cytolytic and cytokine secretion activities. These cells induced differentiation of DC into semi-mature antigen-presenting cells expressing CD86, CD11c, CD54, HLA-DR, CD83 and CD40, which secreted low levels of bioactive IL-12 but no IL-10. Vγ9Vδ2 T cells also strongly costimulated IL-12 release but inhibited IL-10 production by lipopolysaccharide (LPS)-stimulated DC. When substituted for Vγ9Vδ2 T cells, IFN-γ did not induce full DC maturation but it augmented IL-12 and inhibited IL-10 release by LPS-stimulated DC, in a manner similar to HMB-PP-activated Vγ9Vδ2 T cells. Our findings indicate that Vγ9Vδ2 T cells, stimulated with nanomolar concentrations of HMB-PP, strongly promote T helper type 1 (Th1) responses through their ability to induce DC maturation and IL-12 secretion. This adjuvant activity may prove useful in DC-based cancer therapies.     

Anti-Thy-1 plus complement-treated, cultured bone marrow cells resemble fetal thymocytes in killer cell function and marker expression

Anti-Thy-1.2 plus complement treated bone marrow cells were tested after short-term culture for their ability to lyse allogeneic target cells. Significant lytic activity was generated after 9 days, and required both CAS and splenic or PEC feeders as culture supplements. Allogeneic as well as syngeneic-specific cytotoxic cells were generated polyclonally under such conditions, and could be separated by using limiting dilution protocols. When 65 clones were tested for lytic activity toward three targets bearing H-2k, H-2d, and H-2b haplotypes, respectively, only two clones lysed all three targets; 53 clones showed specificity toward one target only. Targets low in class I H-2 expression were lysed only minimally compared with high H-2 expressors. Allogeneic-kill by C57BL/6 bone marrow cells grown on AKR feeder cells was destroyed by treating effectors with anti-Thy-1.2, but not anti-Thy-1.1, antibody plus complement, suggesting 1) a de novo generation of surface Thy-1 during culture and 2) that effectors were derived from bone marrow, but not feeder, populations. Partial inhibition of kill occurred by treatment of effectors with anti-asialo-GM1 (approximately 80%), anti-Lyt-2 (approximately 60%), or anti-Ly-5.1 (approximately 30%) antibodies plus complement; treatment of effectors with anti-L3T4 or anti-NK-1.1 antibodies plus complement had no effect. When precursor populations were treated with either anti-Thy-1.2 alone or a combination of anti-Thy-1.2 and anti-Lyt-2 antibodies plus complement, killers were easily demonstrated. However, the addition of an anti-asialo-GM1 antibody plus complement treatment before culture abolished function. The characteristics of these effectors showed a resemblance to those described previously for day 14 to 17 fetal thymocytes, designated pCTL.