Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well known are GA, GB, GD, GF, VX and VR. All of these compounds contain a chiral phosphorus center with the S P -enantiomers being significantly more toxic than the R P -enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R P -enantiomers. A series of enantiomerically pure chiral nerve agent analogues has been developed containing the relevant phosphoryl centers found in GB, GD, GF, VX and VR. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed stereoselectivity, have been identified. A number of variants showed dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S P -enantiomers. Improvements of up to three orders of magnitude relative to the wild type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study.Organophosphorus compounds have been utilized for more than 50 years as insecticides for the protection of agricultural crops (1) and similar compounds have been developed as chemical warfare agents (2). The structures of these latter compounds are presented in Scheme 1 and include tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX and VR. GA has a cyanide leaving group, the three remaining G-agents (GB, GD, and GF) have a fluoride leaving group, and the two versions of VX have a thiolate leaving group. The toxicity of these organophosphonates is due to the inactivation of acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of acetylcholine at neural synapses, through the phosphonylation of an active site serine residue (3). GA, GB, GF, VX, and VR contain a chiral phosphorus center and thus each of these nerve agents has two stereoisomers, while soman has four stereoisomers because of an additional chiral center within the pinacolyl substituent. The enantiomers are differentially toxic; the S Pstereoisomer of sarin reacts with AChE approximately ~10 4 times faster than the R Pstereoisomer and the two S P -stereoisomers of soman react ~10 5 times faster than the two † This work was supported by the NIH (GM 68550).

The base excision repair (BER) that repairs oxidative damage is upregulated as an adaptive response in maintaining tumorigenesis of RAS-transformed cancer cells.

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.countermeasures | nonconventional warfare agents | organophosphorous pesticides | protein engineering | transgenic plants B utyrylcholinesterase (BChE) is the major cholinesterase (ChE) in the serum of humans (1, 2). Although the closely related enzyme acetylcholinesterase (AChE) is well described as the primary synaptic regulator of cholinergic transmission, a definitive physiological role for BChE has not yet been demonstrated (3). BChE is catalytically promiscuous and hydrolyzes not only acetylcholine (ACh), but also longer-chain choline esters (e.g., butyrylcholine, its preferred substrate, and succinylcholine) and a variety of non-choline esters, such as acetylsalicylic acid (aspirin) and cocaine (4, 5). Moreover, BChE binds most environmentally occurring ChE inhibitors as well as man-made organophosphorous (OP) pesticides and nerve agents (NAs) (6, 7-10).The systemic biodistribution and affinity for ChE inhibitors allow endogenous BChE to provide broad-spectrum protection against various toxicants by their sequestration before they reach cholinergic synapses. However, under realistic high-dose exposure scenarios, BChE serum levels are too low to afford adequate protection, resulting in persistent cholinergic excitation due to irreversible inhibition of AChE and subsequent accumulation of ACh. Sublethal manifestations of this state include unregulated exocrine secretion and gastrointestinal hypermotility. Death usually results from unregulated stimulation at neuromuscular junction leading to hemodynamic instability and tetanic contraction of the respiratory muscles (11,12).Current OP poisoning therapy consists of atropine for muscarinic ACh receptor blockade and diazepam for symptomatic management of convulsions (12). Additionally, oxime therapy with 2-pralidoxime (2-PAM) can effectively reactivate some but not all OP-AChE adducts (13)(14)(15). This standard therapeutic approach can reduce mortality, but insufficiently prevents the incapacitation associated with OP toxicity (12, 16).Prophylaxis by administration of exogenous ChEs has proven successful in reducing OP-associated morbidity and mortality, but requires the availability of rel...

In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.The molecular mechanisms by which transformed cells become migratory and invasive during tumor progression are beginning to be unraveled (reviewed in Ref. 1). Some events are reminiscent of an important developmental process termed epithelial-to-mesenchymal transition (EMT) 3 (reviewed in Refs. 2, 3). During EMT, tumor cells redistribute or down-regulate their epithelium-specific proteins such as adherent and tight-junction proteins, including E-cadherin and occludin, and start to express mesenchymal proteins, such as vimentin and N-cadherin. As a result, cell-cell contacts are disrupted causing a loss of apico-basal polarity, and cells acquire mesenchymal and migratory properties necessary for invasion. Transcriptional repression has emerged as a fundamental mechanism for silencing of E-cadherin and occludin, and several transcriptional repressors have been identified (reviewed in Ref. 4). Snail and Slug, which belong to the Snail superfamily of zinc finger transcriptional repressors, are the most characterized E-cadherin repressors (5-9). The zinc fingers present at the carboxyl terminus of the proteins function as the sequencespecific DNA-binding domains that recognize consensus E2 box-type elements. Their repressor capacity is mediated by the SNAG domain present at the amino-terminal part of the proteins (reviewed in Ref. 10).A requirement for the CUX1 homeodomain ...