Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus

Bacteriophages have evolved specific mechanisms that redirect bacterial metabolic pathways to the bacteriophage reproduction cycle. In this study, we characterized the bactericidal mechanism of two polypeptides from bacteriophages Twort and G1 that target the DNA sliding clamp of Staphylococcus aureus. The DNA sliding clamp, which tethers DNA polymerase to its template and thereby confers processivity upon the enzyme, was found to be essential for the viability of S. aureus. Expression of polypeptides TwortORF168 and G1ORF240 in S. aureus selectively inhibited DNA replication which in turn resulted in cell death. Both polypeptides specifically inhibited the S. aureus DNA replicase that was reconstituted in vitro but not the corresponding replicase of Streptococcus pyogenes. We demonstrated that inhibition of DNA synthesis is multifaceted and occurs via binding the DNA sliding clamp: TwortORF168 and G1ORF240 bound tightly to the DNA sliding clamp and prevented both its loading onto DNA and its interaction with DNA polymerase C. These results elucidate the impact of bacteriophage polypeptide expression upon DNA replication in the growing cell.     

Use of cryopreserved cells for enabling greater flexibility in compound profiling

Measurement of intracellular calcium release following agonist challenge within cells expressing the relevant membrane protein is a commonly used format to derive structure-activity relationship (SAR) data within a compound profiling assay. The Fluorometric Imaging Plate Reader (FLIPR) has become the gold standard for this purpose. FLIPR traditionally uses cells that are maintained in continuous culture for compound profiling of iterative chemistry campaigns. This supply dictates that assays can only be run on 4 of 5 weekdays, or alternative cell culture machinery is required such that plating can occur remotely at the weekend. The data reported here demonstrate that high-quality compound profiling data can be generated from the use of cryopreserved cells and that these cells can also be plated at various densities to generate equivalent data between 24 and 72 h post-plating. Hence, the authors report a method that allows data generation throughout the week and without the requirement of highly automated cell culture or continuous culture.     

Identification of small molecule agonists of the motilin receptor

High-throughput screening resulted in the identification of a series of novel motilin receptor agonists with relatively low molecular weights. The series originated from an array of biphenyl derivatives designed to target 7-transmembrane (7-TM) receptors. Further investigation of the structure-activity relationship within the series resulted in the identification of compound (22) as a potent and selective agonist at the motilin receptor.     

Stress-responsive alpha-dioxygenase expression in tomato roots

Alpha-dioxygenase (alpha-DOX) enzymes catalyse the oxygenation of fatty acids to yield a newly identified group of oxylipins that play a role in protecting tissues from oxidative damage and cell death. In tomato (Lycopersicon esculentum Mill.) alpha-DOX was identified as salt-regulated by differential display of mRNA, and is represented by a small gene family comprising at least three members: LEalpha-DOX1, -2, and -3 of which only LEalpha-DOX1 was salt-responsive. The enhancement of LEalpha-DOX1 expression in roots by salt, wounding and challenge with Pythium aphanidermatum (Edson) Fitzp. suggests that alpha-DOX-generated oxylipins may mediate the response of roots to these environmental stresses. In roots, LEalpha-DOX1 was abscisic acid (ABA)-responsive. However, in the ABA-deficient mutant flacca salt-responsive expression was equivalent to that in the wild type. Similarly, in roots exposed to fluridone (FLU) salt up-regulated expression; however, in this case salt-responsive LEalpha-DOX1 expression was greater than that in roots that were not exposed to FLU. A possible explanation for this is provided by the role of ABA in suppressing ethylene accumulation in osmotically stressed roots. The ethylene-generating agent ethephon and precursor 1-aminocyclopropane-1-carboxylic acid markedly elevated LEalpha-DOX1 expression, and this enhanced expression was suppressed by ABA. LEalpha-DOX1 expression in salt-stressed roots was not markedly affected by AVG indicating that ABA may be responsible for enhanced alpha-DOX expression in salt-treated roots.     

Comparison of human TRPC3 channels in receptor-activated and store-operated modes. Differential sensitivity to channel blockers suggests fundamental differences in channel composition

Capacitative calcium entry or store-operated calcium entry in nonexcitable cells is a process whereby the activation of calcium influx across the plasma membrane is signaled by depletion of intracellular calcium stores. Transient receptor potential (TRP) proteins have been proposed as candidates for store-operated calcium channels. Human TRPC3 (hTRPC3), an extensively studied member of the TRP family, is activated through a phospholipase C-dependent mechanism, not by store depletion, when expressed in HEK293 cells. However, store depletion by thapsigargin is sufficient to activate hTRPC3 channels when expressed in DT40 avian B-lymphocytes. To gain further insights into the differences between hTRPC3 channels generated in these two expression systems and further understand the role of hTRPC3 in capacitative calcium entry, we examined the effect of two well characterized inhibitors of capacitative calcium entry, Gd3+ and 2-aminoethoxydiphenyl borane (2APB). We confirmed that in both DT40 cells and HEK293 cells, 1 microm Gd3+ or 30 microm 2APB completely blocked calcium entry due to receptor activation or store depletion. In HEK293 cells, 1 microm Gd3+ did not block receptor-activated hTRPC3-mediated cation entry, whereas 2APB had a partial (approximately 60%) inhibitory effect. Interestingly, store-operated hTRPC3-mediated cation entry in DT40 cells was also partially inhibited by 2APB, whereas 1 microm Gd3+ completely blocked store-operated hTRPC3 activity in these cells. Furthermore, the sensitivity of store-operated hTRPC3 channels to Gd3+ in DT40 cells was similar to the endogenous store-operated channels, with essentially 100% block of activity at concentrations as low as 0.1 microm. Finally, Gd3+ has a rapid inhibitory effect when added to fully developed hTRPC3-mediated calcium entry, suggesting a direct action of Gd3+ on hTRPC3 channels. The distinct action of these inhibitors on hTRPC3-mediated cation entry in these two cell types may result from their different modes of activation and may also reflect differences in basic channel structure.     

Antibody binding in altered gravity: implications for immunosorbent assay during space flight

A single antibody-incubation step of an indirect, enzyme-linked immunosorbent assay (ELISA) was performed during microgravity, Martian gravity (0.38 G) and hypergravity (1.8 G) phases of parabolic flight, onboard the NASA KC-135 aircraft. Antibody-antigen binding occurred within 15 seconds; the level of binding did not differ between microgravity, Martian gravity and 1 G (Earth's gravity) conditions. During hypergravity and 1 G, antibody binding was directly proportional to the fluid volume (per microtiter well) used for incubation; this pattern was not observed during microgravity. These effects in microgravity may be due to "fluid spread" within the chamber (observed during microgravity with digital photography), leading to greater fluid-surface contact and subsequently antibody-antigen contact. In summary, these results demonstrate that: i) ELISA antibody-incubation and washing steps can be successfully performed by human operators during microgravity, Martian gravity and hypergravity; ii) there is no significant difference in antibody binding between microgravity, Martian gravity and 1 G conditions; and iii) a smaller fluid volume/well (and therefore less antibody) was required for a given level of binding during microgravity. These conclusions indicate that reduced gravity would not present a barrier to successful operation of immunosorbent assays during spaceflight.