Fate of the chemical warfare agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX) on soil following accelerant-based fire and liquid decontamination.

In the event of alleged use of organophosphorus nerve agents, all kinds of environmental samples can be received for analysis. These might include decontaminated and charred matter collected from the site of a suspected chemical attack. In other scenarios, such matter might be sampled to confirm the site of a chemical weapon test or clandestine laboratory decontaminated and burned to prevent discovery. To provide an analytical capability for these contingencies, we present a preliminary investigation of the effect of accelerant-based fire and liquid decontamination on soil contaminated with the nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX). The objectives were (a) to determine if VX or its degradation products were detectable in soil after an accelerant-based fire promoted by aviation fuel, including following decontamination with Decontamination Solution 2 (DS2) or aqueous sodium hypochlorite, (b) to develop analytical methods to support forensic analysis of accelerant-soaked, decontaminated and charred soil and (c) to inform the design of future experiments of this type to improve analytical fidelity. Our results show for the first time that modern analytical techniques can be used to identify residual VX and its degradation products in contaminated soil after an accelerant-based fire and after chemical decontamination and then fire. Comparison of the gas chromatography-mass spectrometry (GC-MS) profiles of VX and its impurities/degradation products from contaminated burnt soil, and burnt soil spiked with VX, indicated that the fire resulted in the production of diethyl methylphosphonate and O,S-diethyl methylphosphonothiolate (by an unknown mechanism). Other products identified were indicative of chemical decontamination, and some of these provided evidence of the decontaminant used, for example, ethyl 2-methoxyethyl methylphosphonate and bis(2-methoxyethyl) methylphosphonate following decontamination with DS2. Sample preparation procedures and analytical methods suitable for investigating accelerant and decontaminant-soaked soil samples are presented. VX and its degradation products and/or impurities were detected under all the conditions studied, demonstrating that accelerant-based fire and liquid-based decontamination and then fire are unlikely to prevent the retrieval of evidence of chemical warfare agent (CWA) testing. This is the first published study of the effects of an accelerant-based fire on a CWA in environmental samples. The results will inform defence and security-based organisations worldwide and support the verification activities of the Organisation for the Prohibition of Chemical Weapons (OPCW), winner of the 2013 Nobel Peace Prize for its extensive efforts to eliminate chemical weapons.

Chemical analysis of bleach and hydroxide-based solutions after decontamination of the chemical warfare agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX).

Detailed chemical analysis of solutions used to decontaminate chemical warfare agents can be used to support verification and forensic attribution. Decontamination solutions are amongst the most difficult matrices for chemical analysis because of their corrosive and potentially emulsion-based nature. Consequently, there are relatively few publications that report their detailed chemical analysis. This paper describes the application of modern analytical techniques to the analysis of decontamination solutions following decontamination of the chemical warfare agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX). We confirm the formation of N,N-diisopropylformamide and N,N-diisopropylamine following decontamination of VX with hypochlorite-based solution, whereas they were not detected in extracts of hydroxide-based decontamination solutions by nuclear magnetic resonance (NMR) spectroscopy or gas chromatography-mass spectrometry. We report the electron ionisation and chemical ionisation mass spectroscopic details, retention indices, and NMR spectra of N,N-diisopropylformamide and N,N-diisopropylamine, as well as analytical methods suitable for their analysis and identification in solvent extracts and decontamination residues.

Analysis of R-Ras signalling pathways.

R-Ras has a high degree of sequence homology to Ras and to other members of the Ras subfamily including Rap, TC21 and M-Ras. Activated versions of Ras and TC21 are highly transforming in a variety of cell lines and mutated forms of both proteins have been found in human tumours. R-Ras interacts with many of the same proteins as Ras and TC21, including c-Raf1, and can induce transformed foci, although this activity is weak compared to Ras and appears to be cell-type specific. Here, we have investigated R-Ras signalling pathways in a variety of cell types. We find that microinjection of activated R-Ras into quiescent fibroblasts stimulates cell cycle progression through G(1) phase and subsequent DNA synthesis. However, unlike Ras, R-Ras does not activate the ERK MAP kinase pathway nor does it activate the JNK or p38/Mpk2 MAP kinase pathways. Microinjection of R-Ras into PC12 cells does not induce terminal differentiation, but instead causes extensive cell spreading, consistent with R-Ras having a role in integrin activation. Finally, in a macrophage cell line, R-Ras activates the alpha(M)beta(2) integrin via the small GTPase Rap1, leading to phagocytosis of opsonized red blood cells, whereas Ras does not. These results indicate that R-Ras has an important role in the regulation of cell growth and adhesion, but that this is mediated through downstream signals distinct from those used by Ras.

The GTPase Rap1 controls functional activation of macrophage integrin alphaMbeta2 by LPS and other inflammatory mediators.

BACKGROUND: beta2 integrins mediate many aspects of the inflammatory and immune responses, including adhesion of leukocytes to the endothelium, complement-mediated phagocytosis in macrophages and neutrophils, and antigen-specific conjugate formation between cytotoxic T cells and their targets. A variety of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF), and lipopolysaccharide (LPS) and other bacterial products induce the functional activation of beta2 integrins, but the signaling events that link membrane receptors to integrin activation are poorly understood. RESULTS: We report here that expression of the constitutively active small GTPases Rap1 or R-ras, but not Ras or RalA, is sufficient for functional activation of alphaMbeta2, the complement receptor 3 (CR3), in macrophages, allowing phagocytosis of C3bi-opsonized targets. Inhibition of Rap1, but not other Ras-like or Rho-like small GTPases, abolishes activation of alphaMbeta2 induced by phorbol esters, LPS, TNF-alpha or PAF. Finally, Rap1 activation specifically controls the binding properties of alphaMbeta2 towards its physiological ligand, namely the complement-opsonized phagocytic targets. CONCLUSIONS: In macrophages, the Rap1 GTPase regulates activation of the alphaMbeta2 integrin in response to a wide variety of inflammatory mediators.

Characterization of rhoGAP. A GTPase-activating protein for rho-related small GTPases.

GTPase-activating proteins or GAPs play an important role in signal transduction pathways regulated by GTP-binding proteins. In addition to acting as down-regulators of GTPases, there is growing evidence that they also act as effector molecules required for downstream signaling. PLC-beta 1, the target protein regulated by the heterotrimeric GTPase Gq, has been shown to be a GAP, whereas rasGAP, a down-regulator of the small GTPase ras, may be required for the ras-mediated signals. We have purified a GAP specific for the rho subfamily of small GTPases. Partial sequence analysis of rhoGAP has led to the identification of a family of related proteins which now includes bcr, chimaerin, p190, p85, and 3BP-1. We report here the isolation of a cDNA clone encoding human rhoGAP and the expression of recombinant protein. The full-length protein is 50 kDa and is ubiquitously expressed in mammalian cells. At least three members of the rho family are substrates for rhoGAP, rho, rac, and G25K/CDC42, and they each bind equally well to the protein. In vitro GTPase assays, however, reveal that G25K/CDC42 is the preferred substrate. RhoGAP contains a proline-rich sequence, suggesting that it is an SH3-binding protein.

rho family GTPase activating proteins p190, bcr and rhoGAP show distinct specificities in vitro and in vivo.

rho family GTPases link extracellular signals to changes in the organization of cytoskeletal actin. Serum stimulation of quiescent Swiss 3T3 fibroblasts leads to rho-dependent actin stress fibre formation and focal adhesions, whilst several growth factors initiate signalling pathways leading to rac-dependent actin polymerization at the plasma membrane, and membrane ruffling. The product of the breakpoint cluster region gene bcr, rho GTPase accelerating protein (rhoGAP) and rasGAP-associated p190 share structurally related rho GAP domains, and possess GAP activity for rho family members in vitro. We have directly compared the activities of the isolated GAP domains of these three proteins in regulating different rho family GTPases, both by in vitro assays and by microinjection, to address their possible physiologic functions. We show that bcr accelerates the GTPase activity of rac, but not rho in vitro, and inhibits rac-mediated membrane ruffling, but not rho-mediated stress fibre formation, after microinjection into Swiss 3T3 fibroblasts. In vitro, rhoGAP has a striking preference for G25K as a substrate, whilst p190GAP has marked preferential activity for rho. Furthermore, p190 preferentially inhibits rho-mediated stress fibre formation in vivo. Our data suggest that p190, rhoGAP and bcr play distinct roles in signalling pathways mediated through different rho family GTPases.

Different structural organization of Ras and Rho effector domains.

Ras regulates proliferation and differentiation signals in cells, and activation of the protein can lead to malignant transformation. Activation of the related protein, Rho, affects cell morphology, and it has been suggested that it may also have some oncogenic potential. We show here that Rho does not induce a malignant phenotype in NIH3T3 cells but instead is a potent activator of actin stress fibre formation. The limited homology between Ras and Rho has enabled us to determine the amino acids specifying their different biological activities and GTPase-activating protein (GAP) protein sensitivities using chimeras. Rho substituted with amino acids 23-46 of Ras induces transformed foci in NIH3T3 monolayers, and we conclude that Ras has a single effector domain required for downstream signalling. Although mutational analysis of Rho has revealed that residues 32-42 are also essential for its biological activity, Ras substituted with amino acids 25-48 of Rho does not induce actin stress fibre formation. On the basis of these experiments, we propose that Rho may have two effector domains: one at amino acids 32-42 and corresponding to the effector region of Ras and the second located elsewhere in the carboxy-terminal two-thirds of the molecule.

Microinjection of recombinant p21rho induces rapid changes in cell morphology.

The rho proteins, p21rho, are ubiquitously expressed guanine nucleotide binding proteins with approximately 30% amino acid homology to p21ras, but their biochemical function is unknown. We show here that microinjection of constitutively activated recombinant rho protein (Val14rho) into subconfluent cells induces dramatic changes in cell morphology: 15-30 min after injection cells adopt a distinct and novel phenotype with a contracted cell body and finger-like processes still adherent to the substratum. Ribosylation of Val14rho with the ADP-ribosyltransferase C3 from clostridium botulinum, before microinjection, renders the protein biologically inactive, but it has no effect on either its intrinsic biochemical properties or on its interaction with the GTPase activating protein, rho GAP. Micro-injection of ribosylated normal rho, on the other hand, has a similar effect of injection of C3 transferase and induces complete rounding up of cells. We also report striking biochemical changes in actin filament organization when contact-inhibited quiescent 3T3 cells are injected with Val14rho protein. The effects induced by activation or inactivation of p21rho described here, suggest that the biological function of this protein is to control some aspect of cytoskeletal organization.

Scrape-loading of Swiss 3T3 cells with ras protein rapidly activates protein kinase C in the absence of phosphoinositide hydrolysis.

Scrape-loading has been used to analyse the biochemical function of purified p21ras protein. We have shown that scrape-loading oncogenic p21ras into quiescent Swiss 3T3 cells causes morphological transformation of 90% of the cell population within 15 h. Since large numbers of cells can be loaded with p21ras, early induced biochemical changes can be analysed. In this way we have shown that oncogenic p21ras causes rapid activation of protein kinase C five minutes after introduction of protein, but that ras protein fails to stimulate measurable inositol phosphate formation. It appears, therefore, that the stimulation of protein kinase C activity is due to a ras induced increase in diacylglycerol from a source other than inositol phospholipids. Efficient stimulation of DNA synthesis by oncogenic p21ras only occurs in the presence of insulin. This stimulation of DNA synthesis by ras is absolutely dependent on functional protein kinase C activity.

Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins.

The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP.

The effect of Mg2+ on the guanine nucleotide exchange rate of p21N-ras.

There is growing evidence that the protein products of the ras gene family, p21ras, can couple growth factor receptors to intracellular second messenger production and in particular to phosphoinositol lipid turnover. So far, however, there has been no direct proof that the ras proteins function as typical regulatory G proteins. We show here that the human p21N-ras protein, isolated from an Escherichia coli expression system, can exist as a stable GDP complex which exchanges very slowly with exogenous GTP, the half-life of the p21N-ras X GDP complex being around 20 min. However, in low Mg2+ (0.5 microM) the exchange rate is dramatically increased and the half-life of the p21N-ras X GDP complex is less than 30 s. Furthermore, in low Mg2+, the relative binding affinity of the protein for GTP as compared to GDP is increased 10-fold. The effect of low Mg2+ on the exchange rate of both normal and oncogenic mutant p21ras molecules is identical. We propose that removal of Mg2+ in vitro induces a similar conformational change to stimulation in vivo. The properties described here are consistent with a G protein-like activity for p21N-ras.