Microglia Remodelling and Neuroinflammation Parallel Neuronal Hyperactivation Following Acute Organophosphate Poisoning.

Organophosphate (OP) compounds include highly toxic chemicals widely used both as pesticides and as warfare nerve agents. Existing countermeasures are lifesaving, but do not alleviate all long-term neurological sequelae, making OP poisoning a public health concern worldwide and the search for fully efficient antidotes an urgent need. OPs cause irreversible acetylcholinesterase (AChE) inhibition, inducing the so-called cholinergic syndrome characterized by peripheral manifestations and seizures associated with permanent psychomotor deficits. Besides immediate neurotoxicity, recent data have also identified neuroinflammation and microglia activation as two processes that likely play an important, albeit poorly understood, role in the physiopathology of OP intoxication and its long-term consequences. To gain insight into the response of microglia to OP poisoning, we used a previously described model of diisopropylfluorophosphate (DFP) intoxication of zebrafish larvae. This model reproduces almost all the defects seen in poisoned humans and preclinical models, including AChE inhibition, neuronal epileptiform hyperexcitation, and increased neuronal death. Here, we investigated in vivo the consequences of acute DFP exposure on microglia morphology and behaviour, and on the expression of a set of pro- and anti-inflammatory cytokines. We also used a genetic method of microglial ablation to evaluate the role in the OP-induced neuropathology. We first showed that DFP intoxication rapidly induced deep microglial phenotypic remodelling resembling that seen in M1-type activated macrophages and characterized by an amoeboid morphology, reduced branching, and increased mobility. DFP intoxication also caused massive expression of genes encoding pro-inflammatory cytokines Il1ß, Tnfα, Il8, and to a lesser extent, immuno-modulatory cytokine Il4, suggesting complex microglial reprogramming that included neuroinflammatory activities. Finally, microglia-depleted larvae were instrumental in showing that microglia were major actors in DFP-induced neuroinflammation and, more importantly, that OP-induced neuronal hyperactivation was markedly reduced in larvae fully devoid of microglia. DFP poisoning rapidly triggered massive microglia-mediated neuroinflammation, probably as a result of DFP-induced neuronal hyperexcitation, which in turn further exacerbated neuronal activation. Microglia are thus a relevant therapeutic target, and identifying substances reducing microglial activation could add efficacy to existing OP antidote cocktails.

Engineering Dynamic Surface Peptide Networks on ButyrylcholinesteraseG117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis.

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase.

Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed ß-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Identification of the catalytic residues of carboxylesterase from Arthrobacter globiformis by diisopropyl fluorophosphate-labeling and site-directed mutagenesis.

The role of amino acid residues in the enzymatic activity of carboxylesterase from Arthrobacter globiformis was analyzed by diisopropyl fluorophosphate (DFP) labeling and site-directed mutagenesis. The electrospray ionization mass spectrometric (ESI-MS) analysis of the esterase, covalently labeled by DFP, showed stoichiometric incorporation of the inhibitor into the enzyme. The further comparison of endopeptidase-digested fragments between native and DFP-labeled esterase by fast atom bombardment mass spectrometric (FAB-MS) analysis as well as site-directed mutagenesis indicated that Ser59 in the consensus sequence Ser-X-X-Lys, which is conserved exclusively in penicillin-binding proteins and some esterases, served as a catalytic nucleophile. In addition, the results obtained from analysis of the mutants at position 62 suggested the importance of the basic amino acid side chain at this position, and suggested the significance of this residue acting directly as a general base rather than its involvement in the maintenance of the optimum hydrogen-bonding network at the active site.

Detection, quantification, and microlocalisation of targets of pesticides using microchannel plate autoradiographic imagers.

Organophosphorus (OP) compounds are a diverse chemical group that includes nerve agents and pesticides. They share a common chemical signature that facilitates their binding and adduction of acetylcholinesterase (AChE) within nerve synapses to induce cholinergic toxicity. However, this group diversity results in non-uniform binding and inactivation of other secondary protein targets, some of which may be adducted and protein activity influenced, even when only a relatively minor portion of tissue AChE is inhibited. The determination of individual OP protein binding targets has been hampered by the sensitivity of methods of detection and quantification of protein-pesticide adducts. We have overcome this limitation by the employment of a microchannel plate (MCP) autoradiographic detector to monitor a radiolabelled OP tracer compound. We preincubated rat thymus tissue in vitro with the OP pesticides, azamethiphos-oxon, chlorfenvinphos-oxon, chlorpyrifos-oxon, diazinon-oxon, and malaoxon, and then subsequently radiolabelled the free OP binding sites remaining with 3H-diisopropylfluorophosphate (3H-DFP). Proteins adducted by OP pesticides were detected as a reduction in 3H-DFP radiolabelling after protein separation by one dimensional polyacrylamide gel electrophoresis and quantitative digital autoradiography using the MCP imager. Thymus tissue proteins of molecular weights -28 kDa, 59 kDa, 66 kDa, and 82 kDa displayed responsiveness to adduction by this panel of pesticides. The 59 kDa protein target (previously putatively identified as carboxylesterase I) was only significantly adducted by chlorfenvinphos-oxon (p < 0.001), chlorpyrifos-oxon (p < 0.0001), and diazinon-oxon (p < 0.01), the 66 kDa protein target (previously identified as serum albumin) similarly only adducted by the same three pesticides (p < 0.0001), (p < 0.001), and (p < 0.01), and the 82 kDa protein target (previously identified as acyl peptide hydrolase) only adducted by chlorpyrifos-oxon (p < 0.0001) and diazinon-oxon (p < 0.001), when the average values of tissue AChE inhibition were 30%, 35%, and 32% respectively. The -28 kDa protein target was shown to be heterogeneous in nature and was resolved to reveal nineteen 3H-DFP radiolabelled protein spots by two dimensional polyacrylamide gel electrophoresis and MCP autoradiography. Some of these 3H-DFP proteins spots were responsive to adduction by preincubation with chlorfenvinphos-oxon. In addition, we exploited the useful spatial resolution of the MCP imager (-70 mm) to determine pesticide micolocalisation in vivo, after animal dosing and autoradiography of brain tissue sections. Collectively, MCP autoradiographic imaging provided a means to detect targets of OP pesticides, quantify their sensitivity of adduction relative to tissue AChE inhibition, and highlighted that these common pesticides exhibit specific binding character to protein targets, and therefore their toxicity will need to be evaluated on an individual compound basis. In addition, MCP autoradiography afforded a useful method of visualisation of the localisation of a small radiolabelled tracer within brain tissue.

Serine enzymes released by cultured neoplastic cells.

Serine proteases or esterases released from cell cultures into the growth medium were converted to radioactive derivatives by active site labeling with tritiated DFP, both in the presence and absence of other competing active site reagents. The individual labeled enzymes were then identified by SDS-polyacrylamide gel electrophoresis and scintillation autoradiography. Conditioned medium from embryonal mouse fibroblasts transformed by mouse sarcoma virus contained five serine enzymes that were not present in medium from normal cells; two serine enzymes were released by both cell types, and one serine enzyme was found only in medium from normal cells. Two of the enzymes released by transformed cells were identified as plasminogen activators; these accounted for most of the serine enzyme labeling in transformed culture media and for most of the serine enzyme difference between normal and transformed cultures. The culture fluids from two cell strains of human neoplastic origin were examined by the same method. A rhabdomyosarcoma strain released eight serine enzymes (mol wt ranging from 22,500 to 102,000), four of which were plasminogen activators; seven serine enzymes (mol wt 26,000-102,000), including two plasminogen activators, were detected in medium from human melanoma cultures. In terms of electrophoretic mobility two of the plasminogen activators from rhabdomyosarcoma were identical with those from melanoma cultures, while the remaining two rhabdomyosarcoma activators coincided with activators found in commerical urokinase.

The autoactivation of rabbit Hageman factor.

Proteolytic cleavage and activation of isolated, single chain, zymogen Hageman factor was observed in the presence of kaolin alone. The rate of cleavage of kaolin-bound Hageman factor was enhanced 50-fold by the presence of prekallikrein and high molecular weight kininogen. The two-chain 82,000 dalton form of activated Hageman factor (alpha-HF(a)) also cleaved kaolin- bound single-chain Hageman factor in a dose-dependent manner, yielding fragments of 28,000 and, 50,000 dahons under reducing conditions. Cleavage of kaolin-bound single-chain Hageman factor was not inhibited by preincubation with diisopropylfluorophosphate (12 mM) for 10 min, but long-term incubation of Hageman factor with diisopropylfluorophosphate (up to 48 h) resulted in inhibition of cleavage of kaolin-bound Hageman factor to an extent proportional to the inhibition of procoagulant Hageman factor activity. Hageman factor cleavage was maximal when the kaolin concentration was {approximately} 10-fold greater than the Hageman factor concentration (wt:wt), and was partially inhibited by high molecular weight kininogen. Kaolin-bound Hageman factor cleaved clotting factor XI in an amount which correlated with the extent of cleavage of the Hageman factor. These findings are compatible with the concept that single-chain Hageman factor and alpha- HF(a), are both capable of cleaving and activating kaolin-bound Hageman factor and that a close molecular association of kaolin-bound Hageman factor molecules is required for this reaction.

Factors influencing hematopoietic spleen colony formation in irradiated mice. I. The normal pattern of endogenous colony formation.

Data pertaining to the endogenous mouse spleen colony system, 10 days postirradiation, are presented. The D(o) for visible colonies is 78 R, while that for 6 hr iron uptake over a range of 600-800 R is 50 R. The D(o) for spleen weight is 196 R and that for IUdR uptake is 193 R. These measurements increase with the age of the mouse. Hypertransfusion decreases spleen iron uptake and colony number. DF-(32)P and sodium sulfate-(35)S are not useful indicators of splenic hematopoiesis in this system. Visible hematopoietic colonies in the spleen are not produced by vinblastine, nitrogen mustard, methotrexate, or cyclophosphamide.

Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes.

Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular mass; dot blot hybridization with synthetic oligonucleotides corresponding to the active sites of two known murine CTL esterases suggests homology to the murine enzyme HF. However, serine esterase was present at only approximately 10% of the level found in murine CTLs, and was not secreted during CTL-target cell interaction; moreover, hemolytic activity could not be detected in any of the seven cell lines tested. The results suggest that the human CTLs examined here kill their target cells by a mechanism different from that used by most cloned murine CTLs.

Secretion of plasminogen activator by stimulated macrophages.

Cultured thioglycollate-stimulated peritoneal macrophages synthesize, accumulate, and continuously release high levels of plasminogen activators for at least 4 days whereas cultures of unstimulated macrophages do not; the higher specific catalytic activity of released vs. cell-associated enzyme suggests that the plasminogen activators are actively secreted. The major macrophage plasminogen activator is a serine protease of mol wt 48,000, and thus resembles the comparable enzyme released by virally transformed fibroblasts. Macrophages release a second plasminogen activator of mol wt 28,000 that is also a serine enzyme. The secretion products released by stimulated and unstimulated macrophages have been compared by SDS-polyacrylamide gel electrophoresis after chemical labeling with (3)H-DFP or biosynthetic labeling with (14)C-amino acids. These procedures show that some proteins are formed in both cultures, whereas others are uniquely secreted by each type of macrophage. The serine enzymes released by the two kinds of macrophages differ in specificity and electrophoretic mobility.

Mass spectral characterization of organophosphate-labeled lysine in peptides.

Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.

Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates, measured by radioautography.

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-(32)P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10(7) sites per endplate, in the diaphragm, about 3 x 10(7). Reaction with DFP-(32)P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10(7) and 1.1 x 10(7)per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.

Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. II. Confirmation of radioautographic measurement by liquid-scintillation counting.

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-(3)H in conditions known to saturate all the available DFP-sensitive reaction sites. After being extensively washed, the enzyme acetylcholinesterase (AChase) was specifically reactivated by treatment with pyridine-2-aldoxime methiodide (2-PAM). The radioactive DP-groups released into solution by 2-PAM were measured by liquid scintillation counting, and related to the known number of motor endplates present. Considerable difficulty was encountered in reducing the excess, adsorbed radioactivity to acceptable levels: long washing routines, extraction with organic solvents, and removing excess muscle fiber by microdissection were necessary. Six experiments gave a mean value of 2.4 x 10(7)molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.

Hydrolysis of nerve gas by squid-type diisopropyl phosphorofluoridate hydrolyzing enzyme on agarose resin.

An enzyme purified from squid nerve that hydrolyzes the cholinesterase inhibitor diisopropyl phosphorofluoridate (DFP) has now been coupled to agarose beads. A column of this agarose-DFPase hydrolyzes the nerve gas 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman). Although the more inhibitory of the four diastereoisomers of Soman are hydrolyzed least rapidly, a column of sufficient length will accomplish 95 percent hydrolysis whether measured by fluoride release or loss of cholinesterase-inhibiting power. The results suggest a means for detoxifying unwanted chemical warfare agents.

Selective inactivation of influenza C esterase: a probe for detecting 9-O-acetylated sialic acids.

The influenza C virus (INF-C) hemagglutinin recognizes 9-O-acetyl-N-acetylneuraminic acid. The same protein contains the receptor-destroying enzyme (RDE), which is a 9-O-acetyl-esterase. The RDE was inactivated by the serine esterase inhibitor di-isopropyl fluorophosphate (DFP). [3H]DFP-labeling localized the active site to the heavy chain of the glycoprotein. DFP did not alter the hemagglutination or fusion properties of the protein, but markedly decreased infectivity of the virus, demonstrating that the RDE is important for primary infection. Finally, DFP-treated INF-C bound specifically and irreversibly to cells expressing 9-O-acetylated sialic acids. This provides a probe for a molecule that was hitherto very difficult to study.

Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin: a potential mechanism of long term toxicity by organophosphorus agents.

Chronic low dose exposure to organophosphorus poisons (OP) results in cognitive impairment. Studies in rats have shown that OP interfere with microtubule polymerization. Since microtubules are required for transport of nutrients from the nerve cell body to the nerve synapse, it has been suggested that disruption of microtubule function could explain the learning and memory deficits associated with OP exposure. Tubulin is a major constituent of microtubules. We tested the hypothesis that OP bind to tubulin by treating purified bovine tubulin with sarin, soman, chlorpyrifos oxon, diisopropylfluorophosphate, and 10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide (FP-biotin). Tryptic peptides were isolated and analyzed by mass spectrometry. It was found that OP bound to tyrosine 83 of alpha tubulin in peptide TGTYR, tyrosine 59 in beta tubulin peptide YVPR, tyrosine 281 in beta tubulin peptide GSQQYR, and tyrosine 159 in beta tubulin peptide EEYPDR. The OP reactive tyrosines are located either near the GTP binding site or within loops that interact laterally with protofilaments. It is concluded that OP bind covalently to tubulin, and that this binding could explain cognitive impairment associated with OP exposure.

Hydrolyzing activities of phenyl valerate sensitive to organophosphorus compounds paraoxon and mipafox in human neuroblastoma SH-SY5Y cells.

The molecular targets of best known neurotoxic effects associated to acute exposure to organophosphorus compounds (OPs) are serine esterases located in the nervous system, although there are other less known neurotoxic adverse effects associated with chronic exposure to OPs whose toxicity targets are still not identified. In this work we studied sensitivity to the non-neuropathic OP paraoxon and to the neuropathic OP mipafox of phenyl valerate esterases (PVases) in intact and lysed human neuroblastoma SH-SY5Y cells. The main objective was to discriminate different unknown pools of esterases that might be potential targets of chronic effects from those esterases already known and recognized as targets to these acute neurotoxicity effects. Two components of PVases of different sensitivities were discriminated for paraoxon in both intact and lysed cells; while the two components inhibitable by mipafox were found only for intact cells. A completely resistant component to paraoxon of around 30% was found in both intact and lysed cells; while a component of slightly lower amplitude (around 20%) completely resistant to mipafox was also found for both preparations (intact and lysed cells). The comparison of the results between the intact cells and the lysed cells suggests that the plasma membrane could act as a barrier that reduced the bioavailability of mipafox to PVases. This would imply that the discrimination of the different esterases should be made in lysed cells. However, those studies which aim to determine the physiological role of these esterases should be necessarily conducted in intact cultured cells.

Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor.

N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF's divergent effects on FPR1 phosphorylation as well as PMN function.

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters.

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.