Search

High-throughput screening of 3.87 million compounds delivered a novel series of non-steroidal GR antagonists. Subsequent rounds of optimisation allowed progression from a non-selective ligand with a poor ADMET profile to an orally bioavailable, selective, stable, glucocorticoid receptor antagonist.

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.

In vitro phosphorylation of platelet subcellular fractions revealed that most of the alkali-resistant phosphoproteins and the majority of pp60c-src were in the surface membrane fraction. An alkali-resistant phosphoprotein of about 100 kDa was also immune precipitated by an anti-phosphotyrosine antibody and comigrated with gpIIIa. The phosphorylation of gpIIIa, but not gpIIb, was confirmed by the comparison of reduced and non-reduced gels, and this protein was phosphorylated exclusively on tyrosine. In contrast, both gpIIb and gpIIIa were phosphorylated when the purified complex was added to immunopurified, immobilised pp60c-src. A synthetic peptide with partial homology to a putative tyrosine phosphorylation site in the cytoplasmic domain of gpIIIa was phosphorylated by antibody-purified pp60c-src. Our results indicate that tyrosine-specific phosphorylation of gpIIIa by pp60c-src may play a role in the regulation of platelet function.

In this study the substrate specificity of a pyroglutamate aminopeptidase from synaptosomal membranes of guinea-pig brain was investigated. The enzyme was found to be specific for tripeptides, tripeptide-amides and tetrapeptides which possess the N-terminal sequence Glp-His and as such is specific for Thyrotropin Releasing Hormone or only very closely related peptides. The enzyme was found not to hydrolyse a number of analogues of Thyrotropin Releasing Hormone which have been shown to have therapeutical value in certain neuronal disorders.

In this article, the enzymes of brain and associated tissues that can degrade thyrotropin-releasing hormone (TRH) and luteinising hormone-releasing hormone (LH-RH) are reviewed. As both TRH and LH-RH are considered to act as neurotransmitters or neuromodulators in the CNS, attention is paid to the subcellular location of the enzymes described and how their topographies and substrate specificities fit them to playing roles as inactivating agents for TRH and LH-RH or as regulators of intracellular concentrations of TRH and LH-RH. Consideration is also given to enzymes involved in biotransformation of TRH to secondary metabolites that exhibit biological activity and to enzymes involved in the metabolism of secondary metabolites.