Ralph W. Jack

NK Cells Are Required for Dendritic Cell–Based Immunotherapy at the Time of Tumor Challenge

Increasing evidence suggests that NK cells act to promote effective T cell–based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria–dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.

Mode of action of dysgalacticin: a large heat-labile bacteriocin

OBJECTIVES The mode of action of dysgalacticin, a large (21.5 kDa), heat-labile bacteriocin that is active against the human pathogen Streptococcus pyogenes, was investigated. METHODS We used recombinant dysgalacticin to determine its mode of action against S. pyogenes. Antimicrobial activity of dysgalacticin was determined by MIC assays and viability counts. The extracellular pH of glucose-energized S. pyogenes cell suspensions was measured to determine the influence of dysgalacticin on glucose fermentation. To examine the effect of dysgalacticin on glucose transport, uptake of [14C]glucose and the non-metabolizable analogue [3H]2-deoxyglucose (2DG) was measured. Furthermore, the effect of dysgalacticin on membrane integrity, intracellular potassium concentration, membrane potential and [14C]serine uptake was determined. RESULTS Dysgalacticin was bactericidal towards S. pyogenes and inhibited glucose fermentation by non-growing cell suspensions. Dysgalacticin blocked transport of both glucose and 2DG, indicating that dysgalacticin targets the phosphoenolpyruvate-dependent glucose- and mannose-phosphotransferase system (PTS) of S. pyogenes. This inhibitory activity was voltage-independent, and in addition to the inhibition of glucose transport, dysgalacticin increased the permeability of the cytoplasmic membrane mediating leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [14C]serine uptake, a membrane potential-dependent process in S. pyogenes. CONCLUSIONS Taken together, these data indicate that dysgalacticin targets the glucose- and/or mannose-PTS as a receptor leading to inhibition of sugar uptake. As a result of this interaction, dysgalacticin perturbs membrane integrity leading to loss of intracellular K+ ions and dissipation of the membrane potential, ultimately leading to cell death.

Structure, evolution and expression of a second subfamily of protein phosphatase 2A catalytic subunit genes in the rice plant (Oryza sativa L.).

Protein phosphatase 2A (PP2A) is one of the major serine/threonine protein phosphatases in the cell and plays a variety of regulatory roles in metabolism and signal transduction. Previously, we described the structure and expression of two genes encoding PP2A catalytic subunits (PP2Ac)—OsPP2A-1 and OsPP2A-3—in the rice plant (Yu et al. 2003). Here, we report the isolation and characterisation of a second structurally distinguishable PP2Ac subfamily comprised of three additional isogenes, OsPP2A-2, OsPP2A-4 (each containing ten introns) and OsPP2A-5 (which contains nine introns). Northern blot analysis demonstrated that the three isogenes are ubiquitously expressed in all rice tissues during plant development, and differentially expressed in response to high salinity and the combined stresses of drought and heat. Phylogenetic analyses indicated that the two PP2Ac subfamilies are descended from two ancient lineages, which derived from gene duplications that occurred after the monocotyledon–dicotyledon split. In the second subfamily, it is proposed that two duplication events were involved; in which, the initial duplication of a ten-intron primordial gene yielded OsPP2A-2 and the progenitor of OsPP2A-4 and OsPP2A-5. The OsPP2A-4/OsPP2A-5 progenitor, in turn, underwent a second duplication event, resulting in the present day OsPP2A-4 and OsPP2A-5. It is proposed that loss of the 5′-most intron from OsPP2A-5 occurred after these two duplication events.

Identification of a haemolysin-like peptide with antibacterial activity using the draft genome sequence of Staphylococcus epidermidis strain A487

Our interest in Staphylococcus epidermidis strain A487 was prompted by the unusual nature of its inhibitory activity in screening tests against methicillin-resistant Staphylococcus aureus isolates. The inhibitory activity was detected in deferred antagonism tests only if the agar plate was preheated for at least 35 min at ≥ 55 °C before inoculation of the indicator bacteria, this phenomenon indicating possible involvement of a heat-labile immunity agent or protease. The inhibitor was purified to homogeneity by ammonium sulphate precipitation, followed by cation-exchange and reversed-phase chromatography. Tandem MS revealed a novel peptide of molecular weight 2588.4 Da. The draft genome sequence of strain A487 was determined using 454 GS FLX technology, allowing the identification of the structural gene (hlp) encoding the mature peptide MQFITDLIKKAVDFFKGLFGNK. The deduced amino acid sequence of peptide 487 exhibited 70.8% similarity to that of a putative haemolysin from Staphylococcus cohnii. Analysis of the genome of strain A487 showed several additional inhibitor-encoding genes, including hld, the determinant for staphylococcal δ-lysin. This work indicates that potentially useful inhibitors could be overlooked in agar-based inhibitor screening programmes lacking a heat pretreatment step and also highlights the utility of draft genome sequence examination in antibacterial agent discovery.