manoalide and 4-bromophenacyl-bromide

Topics: Acetophenones; Animals; Cell Line; Eicosanoids; Fatty Acids; Macrophages; Mice; Phospholipases A; Phospholipases A2; Quinacrine; Substrate Specificity; Terpenes

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A2 (PLA2) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA2 enzymatic activity and how these critical residues contribute to its PLA2 activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca2+-dependent secreted PLA2-like (sPLA2) activity, which was inhibited by sPLA2-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA2 activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA2-like enzymatic activity, and these residues are crucial for its sPLA2-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.

Topics: Acetophenones; Amino Acid Motifs; Amino Acid Sequence; Bocavirus; Calcium; Cloning, Molecular; Humans; Molecular Sequence Data; Mutation; Parvoviridae Infections; Parvovirus B19, Human; Phospholipase A2 Inhibitors; Phospholipases A2; Terpenes; Viral Proteins

4-Bromophenacyl bromide (BPB) is generally used as a phospholipase A(2) (PLA2) inhibitor. In the present study, we demonstrate that BPB induces Ca2+ influx in human gingival fibroblasts. In fura-2-loaded human gingival fibroblasts, BPB evoked a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. The BPB-induced Ca2+ mobilization was also shown in a single fluo-3-loaded-fibroblast. The BPB-induced increase in [Ca2+]i was completely abolished by the elimination of the external Ca2+. Ca2+ influx induced by the Ca2+-mobilizing agonist histamine was markedly enhanced in the presence of BPB. These suggest that the BPB-induced Ca2+ mobilization is due to the influx of extracellular Ca2+. However, it is unlikely that the effect of BPB is dependent on the inhibition of PLA2 activity, because other PLA2 inhibitors, such as AACOCF3, quinacrine dihydrochloride and manoalide, failed to induce Ca2+ mobilization. Chemical compounds similar to BPB, but which have no -CH2-Br at position 1 in the benzene ring failed to evoke Ca2+ mobilization, indicating that the position of -CH2--Br in BPB is important for causing the Ca2+ influx.

Topics: Acetophenones; Aniline Compounds; Arachidonic Acids; Bradykinin; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Fluorescent Dyes; Gingiva; Histamine; Humans; Kinetics; Phospholipases A; Phospholipases A2; Quinacrine; Spectrometry, Fluorescence; Structure-Activity Relationship; Terpenes; Xanthenes

The molecular mechanism of cytotoxic effect exerted by the lethal toxin (LeTx) of Bacillus anthracis is not well understood. In the present study, using primary culture of mouse peritoneal macrophages, we have investigated possible cytotoxic mechanisms. LeTx was not found to induce high levels of nitric oxide (NO) production for NO-mediated toxicity. Fragmentation of DNA, a biochemical marker of apoptosis, was not observed in LeTx-treated cells. Pretreatment of cells with antioxidants such as melatonin and dehydroepiandrosterone (DHEA) did not protect the LeTx-induced cytotoxicity. However, addition of phospholipase A2 (PLA2) inhibitors (quinacrine, p-bromophenacyl bromide, manoalide, butacaine) to the culture medium resulted in the inhibition of cytotoxicity of LeTx in a dose-dependent manner. LeTx-induced cytotoxicity was also inhibited by the tyrosine-specific protein kinase inhibitor genistein, but not by the protein kinase C inhibitors staurosporine or H-7. The results of these studies indicate a role for PLA2 and protein kinase in the cytotoxic mechanism of macrophages by anthrax lethal toxin.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 4-Aminobenzoic Acid; Acetophenones; Aminobenzoates; Animals; Anthrax; Antigens, Bacterial; Antioxidants; Apoptosis; Bacterial Toxins; Cells, Cultured; Dehydroepiandrosterone; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Macrophages, Peritoneal; Melatonin; Mice; Mice, Inbred ICR; Nitric Oxide; para-Aminobenzoates; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Protein Kinase Inhibitors; Quinacrine; Reactive Oxygen Species; Staurosporine; Terpenes

Adhesion to solid substrata has been shown to increase intracellular pH (pH(i)) of fibroblasts and of other cells (FEBS Lett. (1988) 234, 449-450; Proc. Natl. Acad. Sci. USA (1989) 86, 4525-4529; J. Biol. Chem. (1990) 265, 1327-1332; Exp. Cell Res. (1992) 200, 211-214; FEBS Lett. (1995) 374, 17-20). We have found that the inhibitors of PLA2, 4-bromophenacyl bromide and manoalide, completely blocked the increase of pH(i) and spreading of neutrophils upon adhesion to solid substrata. Inhibition of phospholipase C with neomycin or removal of extracellular Ca2+ affects neither neutrophil spreading nor their pH(i). Inhibition of PKC with H-7 or staurosporin increased pH(i). PMA, an activator of PKC, dramatically decreased pH(i) but did not impair the spreading of neutrophils. The effect of arachidonic acid, a product of PLA2 activity, on neutrophil pH(i) and spreading was similar to that of PMA. H-7, an inhibitor of PKC, partially blocked the effect of arachidonic acid (AA) on pH(i). BW755C, an inhibitor of AA metabolism by cyclooxygenase or lipoxygenase, affected neither the pH(i) nor cell spreading. We propose that the increase of pH(i) upon neutrophil adhesion is mediated by PLA2 activity, while PKC decreased pH(i). AA produced by PLA2 activates PKC, thus forming a feedback regulation of pH(i).

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acetophenones; Albumins; Arachidonic Acid; Cell Adhesion; Fibronectins; Humans; Hydrogen-Ion Concentration; Lysophosphatidylcholines; Neutrophils; Phospholipases A; Phospholipases A2; Protein Kinase C; Terpenes; Tetradecanoylphorbol Acetate

The effect on human herniated intervertebral disc phospholipase A2 (HD-PLA2) of a number of retinoids, antirheumatic drugs and reported PLA2 inhibitors was evaluated using autoclaved [1-14C]-oleate-labeled Escherichia coli membranes as the substrate. Dexamethasone, non-steroidal antiinflammatory drugs, aristolochic acid and retinol were inactive, whereas a marked inhibition was found for manoalide, retinal, nordihydroguaiaretic acid and p-bromophenacyl bromide after preincubation with the enzyme (IC50 values 0.25, 4, 5 and 5 microM, respectively). The results are parallel to those obtained with the PLA2 purified from human synovial fluid.

Topics: Acetophenones; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Humans; Intervertebral Disc; Intervertebral Disc Displacement; Laminectomy; Masoprocol; Phospholipases A; Phospholipases A2; Retinaldehyde; Synovial Fluid; Terpenes

The molecular mechanisms of depolarization-induced calcium-dependent acetylcholine (ACh) release and its inhibition by botulinum neurotoxin type A (BoTx) are not clear. We studied these mechanisms in an in vitro cholinergic neuronal pheochromocytoma PC12 cell line model. Cultured monolayer PC12 cells were differentiated by treatment with 50 ng/ml nerve growth factor (NGF) for 4 days to enhance cellular ACh synthesis and release. Stimulation of these cells with high K+ (80 mM) in the perfusion medium caused a marked increase (three to four times) in [3H]ACh release in a Ca(2+)-dependent manner. K(+)-stimulated [3H]ACh release was totally inhibited by pretreatment of cells with BoTx (2 nM) for 2 h. High K+ also stimulated the release of arachidonic acid ([3H]AA) from the cell membrane, which was inhibited by BoTx (2 nM). Addition of phospholipase A2 (PLA2) inhibitors (quinacrine, 4-bromophenacyl bromide, manoalide) to the perfusion medium inhibited K(+)-stimulated [3H]ACh and [3H]AA release in a dose-dependent manner. Inclusion of exogenous AA, the PLA2 activator melittin, or PLA2 itself prevented the effect of BoTx. These results demonstrate that in NGF-differentiated PC12 cells, AA release is associated with ACh release, BoTx inhibits both processes, and increased AA can protect against BoTx.

Topics: Acetophenones; Acetylcholine; Animals; Arachidonic Acid; Botulinum Toxins; Cell Differentiation; Dose-Response Relationship, Drug; Ethacrynic Acid; Indomethacin; Kinetics; Masoprocol; Melitten; Models, Biological; Nerve Growth Factors; PC12 Cells; Phospholipases A; Phospholipases A2; Potassium; Protein Kinase C; Quinacrine; Terpenes

Phagocytosis is a specialized function of neutrophils and macrophages that requires coordination of multiple biochemical and biophysical events. Considerable progress has been made in identifying the membrane receptors involved in phagocytosis, but the intracellular signaling pathways that are necessary for particle ingestion are poorly understood. In an effort to address this complex question, we investigated the role of arachidonic acid (AA) in the uptake of yeast and IgG-coated E (EIgG) or C-coated E. Human monocytes, labeled with 3H AA, released this label during phagocytosis of yeast and EIgG, but not in response to EC3b. The PL inhibitors bromophenacyl bromide and manoalide abolished the release of 3H and inhibited phagocytosis of EIgG in parallel. Both drugs caused a similar inhibition of yeast-mediated 3H release but had little effect on yeast ingestion. Similar results were obtained with the inhibitor quinacrine (mepacrine). Exogenously added AA and dihomo-gamma-linolenic acid restored bromophenacyl bromide-inhibited EIgG ingestion; arachidonate analogs eicosatrienoic acid and eicosapentanoic acid did not. Inhibition of the cyclooxygenase and lipoxygenase pathways for AA metabolism by indomethacin or BW755C did not affect EIgG phagocytosis, demonstrating that these major AA metabolic pathways are not involved in phagocytic signaling. These experiments suggest that release of AA is essential for EIgG ingestion and that phagocytosis in monocytes proceeds by at least two mechanisms, one dependent on AA (EIgG) and one independent of it (yeast).

Topics: Acetophenones; Antigen-Antibody Complex; Antigens, Differentiation; Arachidonic Acid; Arachidonic Acids; Complement C3b; Eicosanoids; Humans; Immunoglobulin G; In Vitro Techniques; Monocytes; Phagocytosis; Phospholipases; Quinacrine; Receptors, Fc; Receptors, IgG; Signal Transduction; Structure-Activity Relationship; Terpenes; Yeasts

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostagl

Topics: Acetophenones; Calcimycin; Cell Line; Dinoprostone; Fatty Acids, Unsaturated; Kinetics; Macrophages; Melitten; Phospholipases; Phospholipases A; Phospholipases A2; Platelet Activating Factor; Substrate Specificity; Terpenes