Christine Suquet

Aggregation of equine platelets by PAF (platelet-activating factor)

Platelet-activating factor (PAF), a lipid released as a result of immediate allergic reactions from basophils and mast cells as well as by a variety of other cell types and stimuli, is one of the most potent platelet agonists and hypotensive agents known. Equine platelets stimulated over a wide range of PAF concentrations aggregated in a time- and dose-dependent manner. Maximum aggregation was observed at concentrations of PAF as low as 3.58×10−14 M with platelet-rich plasma (PRP) and 3.58×10−16 M with washed platelets. Furthermore, the aggregation observed did not appear to be breed-dependent. Finally, the platelet arachidonate pathway appeared to play no role in PAF-induced aggregation as exogenous arachidonate did not enhance the reaction, nor were equine platelets pretreated with 38 μM aspirin inhibited in their response to PAF. This level of aspirin totally inhibited the equine platelet aggregation response to arachidonate.

A superoxide dismutase of metacestodes of Taenia taeniaeformis

Superoxide dismutase was purified from Taenia taeniaeformis metacestodes by sequential ion exchange chromatography on quaternary-amino-ethyl-cellulose, gel filtration chromatography on ACA 44 and ion exchange chromatography on DEAE-cellulose, followed by chromatofocusing on polybuffer exchanger 94. This isolation procedure resulted in the detection of a single protein-staining band on alkaline gels, coincident with enzyme activity. We have, however, detected what appear to be two peaks of enzyme activity within this single protein-staining band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using gradient slab gels and analysis under reducing conditions, resulted in the detection of only one protein at an apparent Mr of 16,600, while analysis under non-reducing conditions, gave a single protein of an apparent Mr of 64,000. The isoelectric point of the purified protein is 6.6. Boiling for 3 min completely destroyed the enzyme, whereas incubation for 2 h at 37 degrees C resulted in the loss of 56% of the enzymic activity. Incubation with 10 mM KCN resulted in 83% inhibition of the enzyme. We have detected up to 168 U ml-1 of enzyme activity in the cyst fluid surrounding the parasite in situ. This is the first instance in which any parasite superoxide dismutase has been purified to apparent homogeneity. Parasite-mediated enzymic destruction of superoxide anion can not only protect against oxygen toxicity as a result of normal parasite respiratory processes but also may serve as yet another mechanism used by tissue-dwelling parasites to evade host immunologic attack.

Parasite defense mechanisms for evasion of host attack; A review

This review covers some of the basic mechanisms whereby parasites evade host responses. These mechanisms include; antigenic variation, repeated antigenic determinants, induction of suppressor cells, acquisition of host proteins or molecular mimicry, proteinase destruction of host effector molecules, proteinase inhibitor-mediated inhibition of humoral and cellular immune effector arms and immunosuppressive products of parasite arachidonic acid metabolism. Vet. Parasitol.

Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor

Abstract Suquet C. , Green-Edwards C. and Wes Leid R. 1984. Isolation and partial characterization of a Taenia taeniaeformis metacestode proteinase inhibitor. International Journal for Parasitology 14 : 165–172. A proteinase inhibitor from the metacestode of Taenia taeniaeformis was purified 136-fold to apparent homogeneity as evidenced by one Coomassie Blue protein staining band on 10% SDS slab gels under both reducing and non-reducing conditions. The apparent molecular weight under dissociating conditions was 19,500. This parasite protein inhibited esterolysis of TAME and BTEE by bovine pancreatic trypsin and chymotrypsin respectively in a time and dose-dependent manner. Proteolysis of casein by both enzymes was also inhibited in a time and dose-dependent manner. The parasite inhibitor was stable from pH 2.2 to 10.5 and was fully active after heating at 56 °C for 3 h. The proteases pronase and thermolysin, at concentrations of 1 mg ml −1 , completely inactivated the metacestode inhibitor. Two sulfhydryl proteases, papain and chymopapain, used at concentrations of 1 mg ml −1 were without effect. The irreversible proteinase inhibitors TLCK, TPCK and PMSF at concentrations up to 10 mM had no effect on the parasite inhibitor.

Inhibition of equine neutrophil chemotaxis and chemokinesis by a Taenia taeniaeformis proteinase inhibitor, taeniaestatin

Summary Taeniaestatin, a recently isolated Taenia taeniaeformis proteinase inhibitor, was used to inhibit equine neutrophil migration. Taeniaestatin itself was not chemotactic when used as a chemotactic factor but taeniaestatin did inhibit neutrophil chemokinesis when tested in a Zigmond‐Hirsch checkerboard assay. A dose‐dependent inhibition of both chemokinesis and chemotaxis was observed when zymosan activated bovine sera (ZABS) was used as the chemotactic factor. This inhibition was > 95% when 5 u of taeniaestatin was present on both the cell and chemotactic factor side of the chambers. Equine neutrophils gave dose‐ and time‐dependent migration responses to purified bovine C5a with an ED50 of 1±04 ± 10–7 M. Taeniaestatin inhibited the C5a‐mediated chemotactic and chemokinetic neutrophil responses (51% using 1 u and > 95% with 5 u of taeniaestatin). The inhibition of leucocyte motility by taeniaestatin was reversible and without cytotoxicity at the highest doses of taeniaestatin tested.

Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells.

The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH.-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking.

Inhibition of neutrophil aggregation by taeniaestatin, a cestode proteinase inhibitor

Abstract A metacestode proteinase inhibitor, taeniaestatin, was tested for its ability to inhibit neutrophil aggregation. A dose-dependent inhibition of Zymosan activated bovine serum (ZABS) and C5a-mediated aggregation was observed with an ID 50 of 1 and 1.5 units of taeniaestatin, respectively. Complete inhibition of C5ainduced aggregation was observed using 3 units of taeniaestatin, while 5 units were required for total inhibition of ZABS-induced aggregation.

Cloning, purification, crystallization and preliminary X-ray analysis of DOS heme domain, a new heme oxygen sensor in Escherichia coli

The heme-containing PAS domain of the direct oxygen-sensor protein (DOS(H)), a bona fide oxygen-sensor protein, has been cloned from Escherichia coli strain K12 and successfully purified. The oxidized form of this protein was crystallized by the hanging-drop method with a PEG 8000-based precipitant. Preliminary X-ray diffraction studies of the PAS-domain crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.1, b = 68.1, c = 82.6 A. A complete diffraction data set was collected to 1.9 A for MAD phasing. The electron-density map shows two molecules in an asymmetric unit and a unique six-coordination of the heme iron.