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During maternal recognition of pregnancy (MRP), a conceptus-derived signal leads to the persistence of the corpus luteum and the maintenance of gestation. In the horse, the nature of this signal remains to be elucidated. Several studies have focused on the changes in gene expression during MRP, but little information exists at the protein level. The aim of this study was to identify the proteins at the embryo-maternal interface around signalling of MRP in the horse (day 13) by means of mass spectrometry. A distinct influence of pregnancy was established, with 119 proteins differentially expressed in the uterine fluid of pregnant mares compared to cyclic mares and with upregulation of several inhibitors of the prostaglandin synthesis during pregnancy. By creating an overview of the proteins at the embryo-maternal interface in the horse, this study provides a solid foundation for further targeted studies of proteins potentially involved in embryo-maternal interactions, MRP and pregnancy loss in the horse.

The enzyme sphingomyelinase (SMase) is an important biomarker for several diseases such as Niemann Pick's, atherosclerosis, multiple sclerosis, and HIV. We present a two-component colorimetric SMase activity assay that is more sensitive and much faster than currently available commercial assays. Herein, SMase-triggered release of cysteine from a sphingomyelin (SM)-based liposome formulation with 60 mol % cholesterol causes gold nanoparticle (AuNP) aggregation, enabling colorimetric detection of SMase activities as low as 0.02 mU/mL, corresponding to 1.4 pM concentration. While the lipid composition offers a stable, nonleaky liposome platform with minimal background signal, high specificity toward SMase avoids cross-reactivity of other similar phospholipases. Notably, use of an SM-based liposome formulation accurately mimics the natural in vivo substrate: the cell membrane. We studied the physical rearrangement process of the lipid membrane during SMase-mediated hydrolysis of SM to ceramide using small- and wide-angle X-ray scattering. A change in lipid phase from a liquid to gel state bilayer with increasing concentration of ceramide accounts for the observed increase in membrane permeability and consequent release of encapsulated cysteine. We further demonstrated the effectiveness of the sensor in colorimetric screening of small-molecule drug candidates, paving the way for the identification of novel SMase inhibitors in minutes. Taken together, the simplicity, speed, sensitivity, and naked-eye readout of this assay offer huge potential in point-of-care diagnostics and high-throughput drug screening.

Bombolitin II is a member of a family of five structurally related heptadecapeptides, originally isolated from the venom of a bumblebee, acting at the membrane level and able to increase the activity of phospholipase A 2 . The biological activity of bombolitins seems to be related to their ability to form amphipathic helical structures. To complete our systematic studies on this class of peptides, in the present work we synthesized bombolitin II by solid phase methodology. The secondary structure of this peptide was fully characterized by circular dichroism, 1 H NMR, distance geometry and molecular dynamics simulation. The peptide tends to aggregate in aqueous solution at millimolar concentrations, with formation of bundles of amphiphilic helices. In dilute aqueous solution (10 -5 mol/L) bombolitin II adopts an amphipathic helical structure in the presence of sodium dodecyl sulfate. The helical segment spans the sequence from residue 2 to residue 16. The results are in line with the hypothesis that this class of peptides modulate the acitivity of phospholipase A 2 by altering the physical state of the aggregated substrate.

This study was carried out to identify the cellular component activating the histamine-stimulated Ca2+ entry in vas-deferens-derived DDT1 MF-2 cells. H-1-histaminoceptor stimulation resulted in a rise in intracellular Ca2+ concentration, caused by Ca2+ release from inositol phosphate-sensitive Ca2+ stores and Ca2+ entry from the extracellular space, accompanied by a transient Ca2+-activated outward K+ current. The histamine-evoked K+ current was still observed after preventing inositol phosphate-induced Ca2+ mobilization by intracellularly applied heparin. This current was activated by Ca2+ entry from the extracellular space, because it was abolished in the presence of the Ca2+-channel blocker La3+ or under Ca2+-free conditions. H-1 histaminoceptor-activated Ca2+ entry was also observed in the presence of intracellularly applied Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4, depleting their respective Ca2+ stores and pre-activating the inositol phosphate-regulated Ca2+ entry. Thus the ability of histamine to activate Ca2+ entry independently of Ca2+ mobilization and the formation of inositol phosphates suggests that another component is involved to initiate the Ca2+-entry process. It was observed that H-1-histaminoceptor stimulation resulted in a pronounced release of arachidonic acid (AA) in DDT1 MF-2 cells. Exogenously applied AA induced a concentration-dependent increase in internal Ca2+ due to activation of Ca2+ entry from the extracellular space. Slow inactivation of the AA-sensitive Ca2+ channels is suggested by the slow decline in Ca2+ entry. In accord, the histamine-induced Ca2+ entry was not observed with AA-pre-activated Ca2+ channels. Inhibition of the lipoxygenase and cyclo-oxygenase pathway did not affect the AA-induced Ca2+ entry. The histamine-induced change in internal Ca2+ and the concomitant Kf current were decreased in the presence of AA and caused by Ca2+ mobilization from internal stores. Blocking this internal Ca2+ release by heparin, in the presence of AA, resulted in abolition of the histamine-induced Ca2+-regulated K+ current. These observations show that AA, released on H-1-histaminoceptor stimulation in DDT1 MF-2 cells, is functioning as a second messenger to activate plasmamembrane Ca2+ channels promoting Ca2+ entry from the extracellular space.

The highly homologous bovine and porcine pancreatic phospholipases A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an α-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 310-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine•HCl. The F63V mutant was crystallized in space group P212121 with cell dimensions a = 79.88 Å, b = 65.23 Å, c = 52.62 Å, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 Å resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.

Tyr52 and Tyr73 are conserved amino acid residues throughout all vertebrate phospholipases A2. They are part of an extended hydrogen bonding system that links the N-terminal alpha-NH+-group to the catalytic residues His48 and Asp99. These tyrosines were replaced by phenylalanines in a porcine pancreatic phospholipase A2 mutant, in which residues 62 - 66 had been deleted (DELTA62 - 66PLA2). The mutations did not affect the catalytic properties of the enzyme, nor the folding kinetics. The stability against denaturation by guanidine hydrochloride was decreased, however. To analyse how the enzyme compensates for the loss of the tyrosine hydroxyl group, the X-ray structures of the DELTAY52F and DELTAY73F mutants were determined. After crystallographic refinement the final crystallographic R-factors were 18.1% for the DELTAY52F mutant (data between 7 and 2.3 angstrom resolution) and 19.1% for the DELTAY73F mutant (data between 7 and 2.4 angstrom resolution). No conformational changes occurred in the mutants compared with the DELTA62 - 66PLA2, but an empty cavity formed at the site of the hydroxyl group of the former tyrosine. In both mutants the Asp99 side chain loses one of its hydrogen bonds and this might explain the observed destabilization.

Snakebites are a relevant public health problem in Central and South America. Snake bite envenomations cause intense pain, not relieved by anti-venom. The fangs of many species are short, causing subcutaneous injection. Fangs of larger species inflict subcutaneous or intramuscular envenomation. To understand pain induced by subcutaneous venom, this study examined spinal mechanisms involved in pain-enhancing effects of subcutaneous Lys49 and Asp49 secretory phospholipase-A2 (sPLA2), two components of Bothrops asper snake venom showing highly different enzymatic activities. Unilateral intraplantar sPLA2-Lys49 (catalytically inactive) or sPLA2-Asp49 (catalytically active) into rat hindpaws each induced mechanical hyperalgesia (Randall–Selitto test), whereas only catalytically active sPLA2-Asp49 caused mechanical allodynia (von Frey test). Effects induced by both sPLA2s were inhibited by intrathecal fluorocitrate, a reversible glial metabolic inhibitor. In support, immunohistochemical analysis revealed activation of dorsal horn astrocytes and microglia after intraplantar injection of either sPLA2. Spinal proinflammatory cytokines, nitric oxide, and prostanoids each appear to be involved in the pain-enhancing effects of these sPLA2s. Blockade of interleukin-1 (IL1) inhibited hyperalgesia induced by both sPLA2s, while leaving allodynia unaffected. Blockade of tumor necrosis factor reduced responses to sPLA2-Asp49. An inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7-NI), inhibited hyperalgesia induced by both sPLA2s, without interfering with allodynia induced by sPLA2-Asp49. On the other hand, l-N6-(1-iminoethyl)lysine (l-NI), an inhibitor of the inducible nitric oxide synthase, did not alter any sPLA2-induced effect. Lastly, celecoxib, an inhibitor of cyclooxygenase-2, attenuated sPLA2 actions. These data provide the first evidence of spinal mediators involved in pain facilitation induced by subcutaneous venoms. International Association for the Study of Pain//IASP/Estados Unidos Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/[0596/02-7]/CAPES/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[99/08432-8]/FAPESP/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[00/12303-8]/FAPESP/Brasil UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAc beta 1-->4(Fuc alpha 1 -->3)GlcNAc beta 1-Asn (core alpha 1-->3-Fuc) and Xyl beta 1-2Man beta 1 --> 4GlcNAc beta 1-R (core beta 1 --> 2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha 1-->3-Fuc epitope, Evidence is presented that core alpha 1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H, contortus infected sheep. These results suggest that the core alpha 1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE, In addition we show here that IV-glycans carrying core alpha 1-->3-Fuc and beta 1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha 1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope, (C) 1999 Federation of European Biochemical Societies.

This study was carried out to identify the cellular component activating the histamine-stimulated Ca2+ entry in vas-deferens-derived DDT1 MF-2 cells. H-1-histaminoceptor stimulation resulted in a rise in intracellular Ca2+ concentration, caused by Ca2+ release from inositol phosphate-sensitive Ca2+ stores and Ca2+ entry from the extracellular space, accompanied by a transient Ca2+-activated outward K+ current. The histamine-evoked K+ current was still observed after preventing inositol phosphate-induced Ca2+ mobilization by intracellularly applied heparin. This current was activated by Ca2+ entry from the extracellular space, because it was abolished in the presence of the Ca2+-channel blocker La3+ or under Ca2+-free conditions. H-1 histaminoceptor-activated Ca2+ entry was also observed in the presence of intracellularly applied Ins(1,4,5)P-3 and Ins(1,3,4,5)P-4, depleting their respective Ca2+ stores and pre-activating the inositol phosphate-regulated Ca2+ entry. Thus the ability of histamine to activate Ca2+ entry independently of Ca2+ mobilization and the formation of inositol phosphates suggests that another component is involved to initiate the Ca2+-entry process. It was observed that H-1-histaminoceptor stimulation resulted in a pronounced release of arachidonic acid (AA) in DDT1 MF-2 cells. Exogenously applied AA induced a concentration-dependent increase in internal Ca2+ due to activation of Ca2+ entry from the extracellular space. Slow inactivation of the AA-sensitive Ca2+ channels is suggested by the slow decline in Ca2+ entry. In accord, the histamine-induced Ca2+ entry was not observed with AA-pre-activated Ca2+ channels. Inhibition of the lipoxygenase and cyclo-oxygenase pathway did not affect the AA-induced Ca2+ entry. The histamine-induced change in internal Ca2+ and the concomitant Kf current were decreased in the presence of AA and caused by Ca2+ mobilization from internal stores. Blocking this internal Ca2+ release by heparin, in the presence of AA, resulted in abolition of the histamine-induced Ca2+-regulated K+ current. These observations show that AA, released on H-1-histaminoceptor stimulation in DDT1 MF-2 cells, is functioning as a second messenger to activate plasmamembrane Ca2+ channels promoting Ca2+ entry from the extracellular space.

Bombolitins are five naturally occurring heptadecapeptides acting at the membrane level and able to increase the activity of phospholipase A2. As for other peptides with similar function, the biological activity of bombolitins seems to be mainly due to their ability to form amphipathic helical structures. We synthesized and tested the retro sequence of bom-bolitin I (retro-bombolitin I). This peptide showed an activity similar to that of the natural sequence and was able to adopt a helical structure in the presence of an amphipathic environment consisting of SDS micelles. The secondary structure of this peptide was fully characterized by CD and nmr spectroscopy. © 1994 John Wiley & Sons, Inc.

Upon induction of metamorphosis, larvae of the marine hydroid Hydractinia echinata release [14C]-arachidonic acid from previously labeled endogenous sources. The lipoxygenase inhibitors nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid inhibited metamorphosis induced by Cs+ and 1,2-sn-dioctanoylglycerol, whereas the inhibitors of cyclooxygenase, indomethacin, and acetylsalicylic acid were ineffective, suggesting a role for lipoxygenase metabolites of arachidonic acid in induction of metamorphosis. Lipoxygenase products in Hydractinia echinata were isolated and identified by gas chromatography/mass spectrometry. 8- and 12-HETE were the most abundant metabolites. In cytosolic fractions from larvae activity of an arachidonic acid metabolizing enzyme, presumably a lipoxygenase, was found. The metabolic product was identified by 1H-NMR and chiral phase HPLC as 8(R)-HETE. Its production was strongly inhibited by NDGA, but not by indomethacin. © 1994 Wiley-Liss, Inc.

The highly homologous bovine and porcine pancreatic phospholipases A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an α-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 310-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine•HCl. The F63V mutant was crystallized in space group P212121 with cell dimensions a = 79.88 Å, b = 65.23 Å, c = 52.62 Å, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 Å resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.

Tyr52 and Tyr73 are conserved amino acid residues throughout all vertebrate phospholipases A2. They are part of an extended hydrogen bonding system that links the N-terminal alpha-NH+-group to the catalytic residues His48 and Asp99. These tyrosines were replaced by phenylalanines in a porcine pancreatic phospholipase A2 mutant, in which residues 62 - 66 had been deleted (DELTA62 - 66PLA2). The mutations did not affect the catalytic properties of the enzyme, nor the folding kinetics. The stability against denaturation by guanidine hydrochloride was decreased, however. To analyse how the enzyme compensates for the loss of the tyrosine hydroxyl group, the X-ray structures of the DELTAY52F and DELTAY73F mutants were determined. After crystallographic refinement the final crystallographic R-factors were 18.1% for the DELTAY52F mutant (data between 7 and 2.3 angstrom resolution) and 19.1% for the DELTAY73F mutant (data between 7 and 2.4 angstrom resolution). No conformational changes occurred in the mutants compared with the DELTA62 - 66PLA2, but an empty cavity formed at the site of the hydroxyl group of the former tyrosine. In both mutants the Asp99 side chain loses one of its hydrogen bonds and this might explain the observed destabilization.

Our study provides evidence for the existence of an acylhydrolase activity in Tetrahymena pyriformis cells, capable of hydrolizing the sn-2 ester bond of the PAF molecule. This activity is mainly distributed in the microsomal fraction (76.5% of total) and has properties similar to the mammalian PAF-acetylhydrolase since it is Ca2+-independent, acid-labile, is inhibited by DFP and PMSF but it is not affected by egg yolk phosphatidylcholine. This microsomal acylhydrolase has apparent K(m) and V(max) values of 1.56-mu-M and 373 pmols . mg . min respectively. This is the first report of the existence of a PAF-acetylhydrolase activity in a non-mammalian cell. FEBS Lett

The effects of H2O2 on platelet function were investigated in vitro and ex vivo. H2O2 (0.5 to 5 mumol/L) alone did not influence platelet function, but when it was combined with subthreshold concentrations of arachidonic acid or collagen, it induced platelet aggregation and serotonin release in a dose-dependent fashion. The increase in platelet aggregation was associated with thromboxane A2 production and was prevented by 100 mumol/L aspirin. The amplification of platelet response by H2O2 was also inhibited 2 hours after 300 mg aspirin was given to healthy subjects. H2O2 alone did not affect intraplatelet Ca++ influx or mobilization but, combined with subthreshold concentrations of arachidonic acid, it increased Ca++ mobilization. In platelets prelabeled with tritiated arachidonic acid, H2O2 induced tritium release in a dose-dependent fashion; this effect was prevented by mepacrine, an inhibitor of the phospholipase A2 enzyme. Platelet function was not affected by using H2O2 in combination with other agonists such as thrombin, calcium ionophore, or adenosine diphosphate. This study suggests that H2O2 triggers activation of platelets preexposed to agonists at subthreshold levels by stimulating arachidonic acid metabolism, likely by stimulating the phospholipase A2 enzyme. The stimulation of platelets by concentrations of H2O2 similar to those released by activated leukocytes may give new insights into the functional cooperation between leukocytes and platelets.