Fate of the chemical warfare agent VX in asphalt: a novel approach for the quantitation of VX in organic surfaces.

VX is one of the most toxic chemical warfare agents. Its low volatility and its persistence in the environment raise the issue of long-term exposure risks, either by inhalation or by transdermal penetration. Therefore, a topic of acute interest is the fate of VX in preservative environmental surfaces. In this work, the fate of VX in asphalt pavement, a suspected preservative matrix, was explored, by applying a novel quantitative method for the extraction of trapped VX from "digested" asphalt. It is based on dissolution of asphalt in toluene, precipitation of the heavy components by basic methanol followed by GC-NPD analysis. This method is complementary to methanol extraction of VX from the outer surface of asphalt, and enabled us to explore the total amount of viable VX both on and inside the matrix. Using this method, bis-diisopropylaminoethyl-disulfide [(DES)2], a degradation product of VX, was also assayed. Small chunks of Asphalt were spiked with VX, sealed and analyzed after various aging periods up to 425 days. The level of VX on the outer surface of the asphalt was found to be diminishing with time following a single-exponential decay. The level inside the asphalt increases during the first day, decays steeply to a level of about 5% during the following two weeks, and declines moderately during all the period up to 425 days following a bi-exponential decay. The total recovery of VX from the asphalt declined from almost 100% after 30 minutes to about 2% after 425 days, with a half-life of about 14 days.

Detection of specific Bacillus anthracis spore biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied for the characterization of Bacillus anthracis spore biomarkers. B. anthracis spores were extracted under a simple procedure, followed by linear mode analysis, using sinapinic acid as the matrix. Several markers with a mass range of 4-7 kDa were detected in three B. anthracis strains: Vollum, Sterne and V770-NP1-R. Similar spectra were also obtained for spore extracts of two members of the B. cereus group: B. thuringiensis and B. cereus, but not for B. mycoides, B. subtilis or B. licheniformis, suggesting that these markers are specific to closely related members of the B. cereus group. When alpha-cyano-4-hydroxycinnamic acid was used as the matrix, at least four additional new markers within a mass range of 2-4 kDa could be detected in all B. anthracis spore extracts. These markers, corresponding to a molecular weight of 2528.3, 2792.4, 3077.4, and 3590.7 Da, have not been observed in extracts of the three closely related Bacillus species - B. cereus, B. thuringiensis and B. mycoides. These unique B. anthracis biomarkers, which were isotopically resolved and reproducibly detected in the highly accurate MALDI-TOFMS reflectron mode, may be useful as a basis for rapid and specific identification of B. anthracis strains.

Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases: MALDI-TOF/MS analysis of various nerve agent phosphyl adducts.

Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE with toxic organophosphate compounds is both a biochemical and a pharmacological challenge. Here we describe experiments which allowed to resolve some of the less well understood reaction pathways of phosphylation and "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N agents) such as tabun or the widely used pesticide methamidophos. Tryptic digests of phosphylated AChEs (from human and Torpedo californica), ZipTip peptide fractionation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) enabled reproducible signal enrichment of the isotopically resolved peaks of organophosphoroamidate conjugates of the AChE active site Ser peptides. For tabun and its hexadeuterio analogue, we find, as expected, that the two phosphoramidate adducts of the active site peptide differ by 6.05 mass units but following aging we find that the two corresponding phospho-peptides have identical molecular weights. We further show that the aging product of paraoxon-AChE adduct is identical to the aging product of the tabun-AChE conjugate. These results unequivocally demonstrate that the pathway of aging of tabun adducts of the human or the Torpedo californica AChEs proceeds through P-N bond scission. For methamidophos, we show that phosphylation of AChE involves elimination of the thiomethyl moiety and that the spontaneous reactivation of the resulting organophosphate adduct generates the phosphorus free AChE active site Ser-peptide.

Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis.

It is well established that the response regulator of the chemotaxis system of Escherichia coli, CheY, can undergo acetylation at lysine residues 92 and 109 via a reaction mediated by acetyl-CoA synthetase (Acs). The outcome is activation of CheY, which results in increased clockwise rotation. Nevertheless, it has not been known whether CheY acetylation is involved in chemotaxis. To address this question, we examined the chemotactic behaviour of two mutants, one lacking the acetylating enzyme Acs, and the other having an arginine-for-lysine substitution at residue 92 of CheY - one of the acetylation sites. The Deltaacs mutant exhibited much reduced sensitivity to chemotactic stimuli (both attractants and repellents) in tethering assays and greatly reduced responses in ring-forming, plug and capillary assays. Likewise, the cheY(92KR) mutant had reduced sensitivity to repellents in tethering assays and a reduced response in capillary assays. However, its response to the addition or removal of attractants was normal. These observations suggest that Acs-mediated acetylation of CheY is involved in chemotaxis and that the acetylation site Lys-92 is only involved in the response to repellents. The observation that, in the cheY(92KR) mutant, the addition of a repellent was not chemotactically equivalent to the removal of an attractant also suggests that there are different signalling pathways for attractants and repellents in E. coli.

Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax.

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase.

Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P-N agents can undergo aging through scission of the P-N bond.

Chemotactic-like response of Escherichia coli cells lacking the known chemotaxis machinery but containing overexpressed CheY.

We describe a chemotactic-like response of Escherichia coli strains lacking most of the known chemotaxis machinery but containing high levels of the response regulator CheY. The bacteria accumulated in aspartate-containing capillaries, they formed rings on tryptone-containing semisolid agar, and the probability of counterclockwise flagellar rotation transiently increased in response to stimulation with aspartate (10(-10)-10(-5) M; the response was inverted at > 10(-4) M). The temporal response was partial and delayed, as was the response of a control wild-type strain having a high CheY level. alpha-Methyl-DL-aspartate, a non-metabolizable analogue of aspartate as well as other known attractants of E. Coli, glucose and, to a lesser extent, galactose, maltose and serine caused a similar response. So did low concentrations of acetate and benzoate (which, at higher concentrations, act as repellents for wild-type E. coli). Other tested repellents such as indole, Ni2+ and CO2+ increased the clockwise bias. These observations raise the possibility that, at least when the conventional signal transduction components are missing, a non-conventional chemotactic signal transduction pathway might be functional in E. coli. Potential molecular mechanisms are discussed.

Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers.

The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the PS-soman diastereomers was 4.0-7.5 x 10(4)-fold higher than that toward the PR-diastereomers. Aging of the PSCS-somanyl-HuAChE conjugate was also >1.6 x 10(4)-fold faster than that of the corresponding PRCS-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group Calpha of either PS- or PR-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the PR-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the PRCR-soman over the PRCS-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the PRCS-soman resembles that of the wild type HuAChE. Residue substitutions in the H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS- over the PSCR-stereomer. Aging of the PS-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the Calpha of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate-determining step of the dealkylation process, possibly because both the PSCS- and the PSCR-somanyl moieties yield the same carbocationic intermediate.

Normal melatonin levels in patients with fibromyalgia syndrome.

OBJECTIVE: To assess urine levels of melatonin measured by 6-sulphatoxymelatonin (aMT6s) in patients with fibromyalgia (FM). METHODS: Nocturnal aMT6s urine levels were measured by ELISA, in a sample of urine collected from 10 PM to 7 AM from 39 female patients with FM and 39 age matched healthy female controls. All subjects were interviewed and assessed for nonarticular tenderness, FM symptoms, quality of life, and physical functioning. RESULTS: Nocturnal aMT6s levels of patients with FM were not statistically different from those of controls: 16.7+/-9.2 vs 16.0+/-11.3 microg, respectively. No association was observed between aMT6s levels of patients with FM and disease duration, reproductive status, sleep and mood disturbances. CONCLUSION: Nocturnal urine aMT6s levels were similar in patients with FM and controls. Studies are needed to elucidate the possible role of melatonin in FM and should include larger samples of newly diagnosed untreated patients with FM.

Both acetate kinase and acetyl coenzyme A synthetase are involved in acetate-stimulated change in the direction of flagellar rotation in Escherichia coli.

Escherichia coli strains overproducing the response regulator CheY respond to acetate by increasing their clockwise bias of flagellar rotation, even when they lack other chemotaxis proteins. With acetate metabolism mutants, we demonstrate that both acetate kinase and acetyl coenzyme A synthetase are involved in this response. Thus, a response was observed when one of these enzymes was missing but not when both were absent.

Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray-ionization mass spectrometry.

While non-reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post-inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methylphosphonofluoridates, were measured by electrospray-ionization mass spectrometry (ESI-MS). The measured mass of the free enzyme was 64,700 Da (calculated 64,695 Da) and those of the methylphosphono-HuAChE adducts, bearing isopropyl, isobutyl, 1,2-dimethylpropyl and 1,2,2-trimethylpropyl substituents, were 64,820, 64,840, 64,852 and 64,860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64,780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2-dimethylpropyl methylphosphono-HuAChE, the change in the molecular mass and the kinetics of non-reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to 'aging' of phosphonyl-AChE adducts.

The specificity of fumarate as a switching factor of the bacterial flagellar motor.

Fumarate restores to flagella of cytoplasm-free, Che Y-containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemotaxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.

Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.

The value of precise preoperative localization of colonic arteriovenous malformation in childhood.

Massive hemorrhage from the gastrointestinal tract in a 12-yr-old boy caused by a congenital atypically located colonic arteriovenous malformation is described. Guided and "clean" resection of the involved colon was possible due to preoperative selective angiography, which proved to be the most efficient diagnostic tool. Histologic documentation of this rare pathology in childhood is presented, and the classification and features of the disease are briefly reviewed.

Mass spectrometric investigation of the presence of 7-methyl ring-opened guanine derivatives in urine.

The involvement of chemical alkylating agents in tumorigenesis and chemotherapy is well established and it was shown that one of the main sites of alkylation is the N-7 of guanine in DNA. Though excision of damaged bases is regarded as one of the repair mechanisms in damaged DNA there is a scarcity of information concerning the excised final metabolites in body fluids. This study attempts to demonstrate the usefulness of CAD MS/MS for the detection of the final metabolite-deformylated ring-opened 7 alkylguanine in urine. Such mass spectrometric methods can be used in biomedical studies.

Penetrating stab wound of the gluteus--a potentially life-threatening injury: case reports.

Presented are two cases of stab wounds through the gluteus in which serious damage was incurred by internal organs: in one case there was penetration of the rectum with the appearance of signs of acute abdomen; in the other there was tearing of retroperitoneal muscles and the internal iliac vessels with resultant hypovolemic shock. These observations suggest that there should be greater awareness of the life-threatening nature of stab wounds of the gluteus under similar circumstances.

Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.

Structure-activity studies of the thrombin receptor activating peptide.

Cleavage of the human platelet thrombin receptor by thrombin exposes a new N-terminal which acts as a putative tethered ligand. A synthetic peptide--"SFLL" (SFLLRNPNDKYEPF), corresponding to the new N-terminal region, activates and induces platelet aggregation and serotonin secretion. We have found that the pentapeptide--SFLLR is the minimal peptide length which retains full activity in inducing [14C]serotonin secretion. Structure-activity relationship studies were performed on this pentameric peptide. Systematic replacement of all amino acids with L-Ala indicated the importance of F-2, L-3 and R-5 for activity. Further studies demonstrated that the positive charge at the N-terminus, but not at the C-terminus of the pentapeptide, is crucial for activity.

Familial chronic recurrent pancreatitis in identical twins. Case report and review of the literature.

Familial presentation of chronic recurrent pancreatitis in childhood is rare. The etiology of this illness is obscure, and its hereditary properties are not well defined. Simultaneous occurrence of chronic recurrent pancreatitis in identical twins with the same clinical presentation and similar typical pancreatographic abnormalities is exceptional. Twin sisters, aged 9 years, were admitted to the hospital because of recurrent attacks of pancreatitis. Ultrasound examination revealed an enlarged irregular pancreatic duct in both girls, and endoscopic retrograde cholangiopancreatography showed a distorted duct with multiple strictures and dilatations similar to a "chain of lakes" pattern. Both patients underwent longitudinal pancreatojejunostomy within a month. The therapeutic regimen and preoperative and surgical treatment of such patients are discussed, as is the optimal timing of intervention.