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A novel human homologue of Escherichia coli, yeast and plant 1-acylglycerol-3-phosphate acyltransferase has been isolated from U937 cell cDNA. Expression of the cloned sequence in 1-acylglycerol-3-phosphate acyltransferase-deficient E. coli resulted in increased incorporation of oleic acid into cellular phospholipids. Membranes made from COS7 cells transfected with the cDNA exhibited higher acyltransferase activity towards a range of donor fatty acyl-CoAs and lysophosphatidic acid. Northern-blot analysis of the cDNA sequence indicated high levels of expression in immune cells and epithelium. Rapid amplification of cDNA ends revealed differentially expressed splice variants, which suggests regulation of the enzyme by alternative splicing. This cDNA therefore represents the first described sequence of a mammalian gene homologous to non-mammalian lysophosphatidic acid acyltransferases.

When human peripheral blood neutrophils were stimulated with various agonists which activate and/or prime neutrophils, we found that Cl − efflux was enhanced with a dramatic (50%) loss of intracellular Cl − . Interestingly, the Cl − efflux was enhanced by both agonists which induce a rapid transient increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) [class I, e.g. N -formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL8), platelet-activating factor, leukotriene B 4 and C5a] and those which do not induce such an [Ca 2+ ] i elevation (class II, e.g. tumor necrosis factor α-(TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. The time course of agonist-stimulated Cl − efflux differed depending on the agonist. Class I agonists such as IL8 and fMLP exhibited a 1 min lag phase before the onset of Cl − efflux; class II agonists such as GM-CSF and TNF displayed a 2 and 5 min lag phase, respectively. Both IL8 (class I)- and TNF (class II)-stimulated CF efflux exhibited similar sensitivity to inhibition by different types of ion transport inhibitors [ethacrynic acid (EA), amiloride, 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid, anthracene-9-carboxylic acid, and 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid). On the other hand, natural Cl − efflux, which is thought to be mainly mediated by Cl − /Cl − self exchange, was not inhibited by EA (0.5 mM) or amiloride (0.3 mM). These results imply that both class I and class II agonist-stimulated Cl − efflux occurs via a common Cl − transporter which is different from that reported previously in resting human neutrophils. Although all agonists which induced a Cl − efflux also induced shape change of neutrophils, there did not appear to be a causal relationship between shape change and agonist-stimulated Cl − efflux. However, a temporal correlation was found to exist between agonist-stimulated Cl − efflux and intracellular alkalinization following agonist stimulation. Agonist-stimulated Cl − efflux therefore seems to be a common phenomenon activated by several agonists which act through different signal transduction pathways.

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157–165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10–50 NY-ESO-1/LAGE-1157–165 epitopes per cell.

The elevation of intracellular levels of cyclic AMP by forskolin stimulation of adenylate cyclase regulates early and late phase neutrophil responses differentially. Early phase neutrophil responses as measured by shape change in response to chemotactic factors, transmigration across a polycarbonate membrane and priming were unaffected by forskolin-induced elevation of intracellular cAMP. Late phase neutrophil responses such as release of Superoxide anions, activation of phospholipase A2 and platelet activating factor (PAF) synthesis were inhibited by increasing intracellular cAMP through the addition of 10 μM forskolin for 10 min prior to stimulation. N-Formyl-methionyl-leucyl-phenylalanine-stimulated arachidonic acid release fell from 9.3% (untreated cells) to 4.6% in forskolin-treated cells. PAF generation was also inhibited from 430 pg 10 6 cells in untreated cells to background levels in forskolin-treated cells (110 pg 10 6 cells). Also, the reduction of cytochrome c by Superoxide anions fell from 4.2 solnmol 10 6 cells in the absence of forskolin to 2.0 nmol 10 6 cells following forskolin treatment. These results indicate that in neutrophils the elevation of cAMP acts differentially on cellular responses, not affecting early activation events, but markedly inhibiting late events such as the release of inflammatory mediators.