Chromatographic separation and NMR characterization of the isomers of MMB-4, a bis-(pyridiniumaldoxime).

1,1'-Methylenebis{4-[(hydroxyimino)methyl]pyridinium) dichloride (MMB-4), a promising antidote for organophosphate poisoning, has been shown by chromatography and NMR to be a mixture of geometric isomers, predominantly the E/E form. The chromatographically separated isomers have been isolated, directly characterized by NMR to be E/E and E/Z isomers of high purity, and shown by HPLC and NMR to re-equilibrate in solution to the isomeric mixture found in bulk MMB-4. These findings clearly show that a minor component in MMB-4 is not an impurity, but a geometric isomer of the principal component and demonstrate the need to understand equilibrium processes for drug characterizations and isomer distributions of chemicals proposed for animal and human clinical trials.

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting.

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo.

Enhancement of an analytical method for the determination of squalene in anthrax vaccine adsorbed formulations.

Specific lots of anthrax vaccine adsorbed administered to members of the U.S. Armed Forces have been alleged to contain squalene, a chemical purported to be associated with illnesses of Gulf War veterans.

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle.

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface.

Biodegradation of the hexahydro-1,3,5-trinitro-1,3,5-triazine ring cleavage product 4-nitro-2,4-diazabutanal by Phanerochaete chrysosporium.

Initial denitration of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 produces CO2 and the dead-end product 4-nitro-2,4-diazabutanal (NDAB), OHCNHCH2NHNO2, in high yield. Here we describe experiments to determine the biodegradability of NDAB in liquid culture and soils containing Phanerochaete chrysosporium.

Selection of small-molecule mediators of the RNA regulation of PKR, the RNA-dependent protein kinase.

The RNA-dependent protein kinase (PKR) is a component of the interferon antiviral response and a member of the class of RNA-binding proteins with a double-stranded RNA binding motif. PKR is activated when it binds to double-stranded RNA (dsRNA) or viral replicative intermediates that comprise dsRNA and this activation results in the inhibition of protein synthesis. Some viruses circumvent this activity through the synthesis of highly structured decoy RNAs that bind PKR and block activation.

The binding site of the RNA-dependent protein kinase (PKR) on EBER1 RNA from Epstein-Barr virus.

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the eukaryotic initiation factor 2alpha, rendering the translation machinery inactive. Viruses have developed strategies for preventing the action of PKR, one of which is the production of small RNAs that inhibit the enzyme. Epstein-Barr virus (EBV) encodes EBER1, a 167 nucleotide non-coding RNA that is constitutively expressed by the EBV-infected cells.

Development and application of an analytical method for the determination of squalene in formulations of anthrax vaccine adsorbed.

Specific lots of Anthrax Vaccine Adsorbed, administered to members of the US Armed Forces, have been described on various Internet sites and in news articles as a source of squalene, a chemical purported by these media to be associated with the Gulf War Syndrome. We have developed and validated a method using high-performance liquid chromatography with ultraviolet detection for the determination of squalene in anthrax vaccine preparations. The method has a limit of detection of 140 parts per billion and has been successfully applied to a commercial vaccine known to contain squalene.

Identification of binding sites for both dsRBMs of PKR on kinase-activating and kinase-inhibiting RNA ligands.

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR has a double-stranded RNA-binding domain (dsRBD) composed of two copies of the dsRNA binding motif (dsRBM). PKR's dsRBD is involved in the regulation of the enzyme as dsRNAs of cellular and viral origins bind to the dsRBD, leading to either activation or inhibition of PKR's kinase activity.

Selective binding by the RNA binding domain of PKR revealed by affinity cleavage.

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood.

Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR.

RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD).

Mutagenicity of tetranitroazoxytoluenes: a preliminary screening in Salmonella typhimurium strains TA100 and TA100NR.

Tetranitroazoxytoluenes are polynitroaromatic compounds that can be produced during the microbial reduction of the explosive, 2,4,6-trinitrotoluene (TNT). The three major tetranitroazoxytoluenes were synthesized and tested in Salmonella typhimurium strains TA100 and TA100NR. All compounds were mutagenic in TA100 but not in TA100NR, indicating the need for nitroreductase activity to induce mutagenicity.

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT.

Structure-activity relationship for the intrinsic hepatotoxicity of dinitrotoluenes.

The relation of various structural parameters to hepatotoxic potential was investigated by using six dinitrotoluene (DNT) isomers and isolated rat hepatocyte suspensions as the biological test system. DNT-induced hepatotoxicity was found to correlate with an inhibition of protein synthesis and an increase in lactate dehydrogenase (LDH) release but not with lipid peroxidation. With each isomer, protein synthesis inhibition was the most sensitive indicator of cytotoxicity.

Comparative mutagenicity of halogenated pyridines in the Salmonella typhimurium/mammalian microsome test.

The Salmonella/microsome assay with strains TA97, TA98, TA100 and TA102 was used to examine the potential mutagenicity and structure-activity of 16 mono- and di-halogenated pyridines. The chemical reactivity of the halopyridines suggests that nucleophilic displacement of halogens can occur with halogens at positions 2, 4 and 6 being displaced in addition-elimination reactions. 2-Chloropyridine gave a positive result with rat-liver metabolic activation, and 2-fluoropyridine gave equivocal results under these conditions.

Short-term oral toxicity of a 2,4,6-trinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine mixture in mice, rats, and dogs.

The oral toxicity of a mixture of 2,4,6-trinitrotoluene and hexahydro-1,3,5-trinitro-1,3,5-triazine (1:0.62, w/w) compounds typically found in munitions plant effluents, was evaluated in mammalian species. Single-dose oral LD50s of the mixture were 574 and 594 mg/kg in male and female rats and 947 and 1130 mg/kg in male and female mice, respectively.

Short-term oral toxicity of 2,4,6-trinitrotoluene in mice, rats, and dogs.

The short-term oral toxicity of 2,4,6-trinitrotoluene (alpha-TNT) was determined in dogs, rats, and mice. Single-dose oral LD50s for alpha-TNT in corn oil were 1320 and 794 mg/kg in male and female rats, respectively, and 660 mg/kg in both male and female mice. For multiple-dose studies, dogs were dosed daily for up to 13 wk with alpha-TNT at 0, 0.

Single-dose and repeated-exposure toxicity of a complex wastewater from munitions manufacturing plants.

The single-dose and repeated-exposure toxicity of a synthetic mixture of 30 nitrotoluene analogs, representative of a complex industrial wastewater termed condensate water, was evaluated in dogs, rats, and mice. The single-dose oral LD50s for the synthetic condensate water (CW) were 447 and 295 mg/kg in male and female rats, respectively. In the repeated-exposure studies, dogs were given 0, 0.

Mutagenicity in Salmonella typhimurium and structure-activity relationships of wastewater components emanating from the manufacture of trinitrotoluene.

The mutagenicity of 36 polynitroaromatic compounds was investigated with five strains of Salmonella typhimurium. Isomeric trinitrotoluenes (TNT), with the exception of 2,4,6-TNT and 2,3,4-TNT, exhibit mutagenicity independently of nitroreductase enzymes, but isomeric aminodinitrotoluenes (ADNT) and isomeric dinitrotoluenes (DNT) need nitroreductase to induce mutation. Within groups of isomeric TNTs, DNTs, and ADNTs, mutagenic response was enhanced by a para orientation of nitro groups.

Application of high-pressure liquid chromatography and thermal energy analyzer to analysis of trinitroglycerin and its metabolites in blood.

A highly selective and sensitive analytical procedure for the determination of trinitroglycerin and four metabolites in whole blood was developed. Trinitroglycerin and its metabolites were extracted from whole blood with ethyl acetate and analyzed by high-pressure liquid chromatography using the thermal energy analyzer detector. Linearity of response was observed over the 1-1000-ng range.

N-formyliminodiacetic acid, a new compound from the reaction of nitrilotriacetic acid and chlorine.

It has been proposed that nitrilotriacetic acid be substituted for trisodium polyphosphates in detergents as a way to reduce the rate of eutrophication in the Great Lake Basin. The reaction of nitrilotriacetic acid with chlorine-containing solutions produces a hitherto unknown degradation production, N-formyliminodiacetic acid, in high yield. The toxicological and environmental implications of this reaction are unclear.

Cometabolism of DDT analogs by a Pseudomonas sp.

A Pseudomonas sp. capable of growth on several nonchlorinated and mono-p-chloro-substituted analogs of DDT as a sole carbon source degraded bis(p-chlorophenyl)methane and 1,1-bis(p-chlorophenyl)ethane only in the presence of diphenylethane. The products p-chlorophenylacetic acid and 2-(p-chlorophenyl)-propionic acid were not further metabolized by the bacterium.

Some observations on biodegradation of pollutants in aquatic systems.

The biodegradability of pollutants introduced into aquatic environments is subject to many variabilities characteristic of microbial processes. Some observations have been reported on studies with p-cresol, methyl parathion, benzo[b]thiophene, dibenzothiophene, 9H-carbazole, quinoline, benzo[f]quinoline, benz[a]anthracene, benzo[a]pyrene, 7H-dibenzo[c,g]carbazole, Mirex, 2,4-dichlorophenoxyacetic acid, Lindane and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane analogs. Water reservoirs included a eutrophic stream, a eutrophic pond, an oligotrophic lake and effluents from waste-water treatment plants.

Metabolism of DDT analogues by a Pseudomonas sp.

A Pseudomonas sp. rapidly metabolized several nonchlorinated analogues of DDT, with the exception of 2,2-diphenylethanol, as the sole carbon source. Several of the mono-p-chloro-substituted diphenyl analogues were also metabolized as the sole carbon source by the bacterium.