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Minimization of water vapor interference in the analysis of non-methane volatile organic compounds by solid adsorbent sampling.

Water vapor can be a significant interference in the analysis of air for non-methane volatile organic compounds (NMVOCs) using solid-adsorbent sampling techniques. The adsorbent materials used in sampling cartridges have different hydrophobic characteristics, and it is therefore necessary to characterize solid-adsorbent cartridges over a wide range of humidity. Controlled humidity experiments were performed to assess the extent of water vapor interference when samples are collected onto AirToxics solid-adsorbent cartridges. It was found that elevating the temperature of the cartridge to 10 degrees C above the temperature of the air sample greatly reduced water vapor adsorption and interferences and resulted in > or = 90% recovery of NMVOCs, biogenic VOCs and chlorofluorocarbons. Similar collection efficiencies were obtained at ambient temperature by reducing the relative humidity to > or = 60% in the sample by dilution with dry, scrubbed ambient air. A procedure also was developed and optimized for dry-purging cartridges prior to analysis. However, under optimized conditions, significant losses of C3-C5 compounds still occurred under highly humid conditions. It was determined that these losses were due to reduced retention during sampling rather than loss during the dry purge procedure. The dry purge method was shown to be adequate at high humidities for sampling NMVOCs with retention indices greater than 500.

Adsorption↗

Determination of destruxins, cyclic peptide toxins, produced by different strains of Metarhizium anisopliae and their mutants induced by ethyl methane sulfonate and ultraviolet using HPLC method.

Metarhizium anisopliae produces a family of cyclic peptide toxins, destruxins (DTXs), which exhibit various insecticidal activity. Four major DTXs have been separated by HPLC and identified by the liquid chromatography electrospray mass spectrometry (LC-ESI-MS) methods. Strain F061 of M. anisopliae produced large amounts of (DTXs), especially DTX-A (12.84+/-0.04 microg/ml), DTX-B (66.89+/-2.57 microg/ml) and DMDB (1.41+/-0.13 microg/ml). High levels of DTX-E (4.19+/-0.13 microg/ml) were produced by strain F007 of M. anisopliae. The results of our studies also showed that either ethyl methane sulfonate (EMS) or ultraviolet (UV) can significantly increase the production of DTXs. Mutant 61E-9 produced high levels of DTX-A (30.05+/-1.97 microg/ml), DTX-B (110.37+/-10.02 microg/ml) and DMDB (8.30+/-0.45 microg/ml). High levels of DTX-E (20.59+/-2.65 microg/ml) were produced by mutant 7E-3. Both mutant strains are suitable for industrial fermentation processes and possess a wide range of potential applications in the area of metabolic toxin production.

Chromatography, High Pressure Liquid↗

Efficacy of vitamin-C (L-ascorbic acid) in reducing genotoxicity in fish (Oreochromis mossambicus) induced by ethyl methane sulphonate.

The genotoxic effects of ethyl methane sulphonate (EMS) have been assessed in a fish, Oreochromis mossambicus with endpoints including chromosome aberrations, abnormal red blood cell nuclei, abnormal sperm morphology, and protein content (both qualitative and quantitative) of selected tissues, namely, muscle, heart, eye, brain, gill, liver, spleen and kidney. EMS caused chromosomal aberrations, nuclear anomalies in red blood cells, abnormal sperm morphology, and alteration of protein synthesis in various tissues. Some of the EMS toxicity appeared to be modulated and ameliorated in this fish by vitamin-C treatment.

Animals↗

Methyl-coenzyme M reductase genes: unique functional markers for methanogenic and anaerobic methane-oxidizing Archaea.

In many anoxic environments, methanogenesis is the predominant terminal electron accepting process involved in the mineralization of organic matter, which is catalyzed by methanogenic Archaea. These organisms represent a unique but phylogenetically diverse guild of prokaryotes, which can be conveniently tracked in the environment by targeting the mcrA gene as a functional marker. This gene encodes the alpha subunit of the methyl-coenzyme M reductase (MCR), which catalyzes the last step in methanogenesis and is present in all methanogens. Cultivation-independent analysis of methanogenic communities involves the polymerase chain reaction (PCR) amplification of the mcrA gene from extracted community DNA, comparative analysis of mcrA clone libraries, or PCR-based fingerprinting analysis by terminal restriction fragment polymorphism analysis (T-RFLP). It has also been suggested that anaerobic methane-oxidizing Archaea possess MCR, which facilitates detection of this novel group of "reverse methanogens" as well using the mcrA gene as a functional marker.

Archaea↗

The Cu(II) acetate complex of the heteroscorpionate ligand (2-hydroxy-3-t-butyl-methylphenyl)bis(3,5-dimethylpyrazolyl)methane: a structural model for copper-substituted serralysin and astacin.

Reaction of Cu(II) acetate with the deprotonated ligand (2-hydroxy-3-t-butyl-methylphenyl)bis(3,5-dimethylpyrazolyl)methane (L1O(-)) in methanol produced the complexes [(L1O)Cu(OAc)], a=9.275(2), b=11.641(5), c=13.532(3) A, alpha=69.62(1) degrees, beta=89.49(2) degrees, gamma=87.12(1) degrees, P1. The Cu adopts a distorted five-coordinate geometry where the two pyrazole nitrogen ligands and a bidentate acetate occupy the pseudoequatorial plane with the phenoxy oxygen in an apical position. This complex has a number of features in common with galactose oxidase and the copper-substituted endopeptidases serralysin and astacin.

Crystallography, X-Ray↗

Mechanistic insights into C-H activation from radical clock chemistry: oxidation of substituted methylcyclopropanes catalyzed by soluble methane monooxygenase from Methylosinus trichosporium OB3b.

The soluble methane monooxygenase (MMO) system isolated from Methylosinus trichosporium OB3b catalyzes the adventitious oxidation of alkyl substituted methylcyclopropanes. If the chemical mechanism of C-H activation by MMO involves formation of a radical or carbocation intermediate at the methyl C-H of these 'radical clock' substrates, then cyclopropyl ring opened alcohols may appear in the product mixture due to rearrangement of the intermediate. The lifetime of radical intermediates can be determined from known rearrangement rate constants, k(r). Rearrangement was observed during the oxidation of 1,1,2,2-tetramethylcyclopropane (k(r)=1.7-17. 5x10(8) s(-1), 30 degrees C) but not for cis- or trans-1, 2-dimethylcyclopropane (k(r)=1.2-6.4x10(8) s(-1), 30 degrees C) or the very fast radical clock, trans-2-phenylmethylcyclopropane (k(r)=3.4x10(11) s(-1), 30 degrees C). The results show that the occurrence of rearranged products fails to correlate with either the chemical nature of the C-H bond being broken, which is very similar for all of the methylcyclopropanes studied here, or the magnitude of the radical k(r) value. This study suggests that the steric properties of the substrate play an important role in determining the outcome of the reaction. Substrates with bulky substituents near the C-H bond that is attacked appear to yield intermediates with sufficient lifetimes to rearrange. In contrast, substrates with less steric bulk are postulated to be able to approach the reactive oxygen species in the MMO active site more closely so that intermediates are either rapidly quenched or undergo subsequent interaction with the dinuclear iron cluster of MMO that prevents rearrangement.

Cyclopropanes↗

Tris (4-chlorophenyl) methane and tris (4-chlorophenyl) methanol in marine mammals from the Estuary and Gulf of St. Lawrence.

Levels of tris (4-chlorophenyl) methanol (TCPM) and its presumed precursor tris (4-chlorophenyl) methane (TCPMe) are reported in marine mammals from the Estuary and Gulf of St. Lawrence, Canada. These compounds were measured in blubber samples of seals and whales using ion trap mass spectrometry (MS/MS) detection. Detectable concentrations of both TCPM and TCPMe were observed in all of the samples analysed. Concentrations of these compounds varied with species ranging from 1.7 to 153 and from 1.3 to 50.6 ng/g lipid wt. for TCPM and TCPMe, respectively. TCPM was from 1.3 to 10 times more concentrated than TCPMe. The highest levels of both TCPM and TCPMe were observed in adult male beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, while adult female beluga whales from the same area showed levels similar to those in the seals examined. Among the four seal species investigated, TCPM and TCPMe levels were the highest in grey (Halichoerus grypus) and hooded (Cystophora cristata) seals, and lowest in harp seals (Phoca groenlandica). Intermediate levels were found in harbour seals (Phoca vitulina); however, their concentrations might be underestimated considering the younger mean age of these animals. Ratios of both 4,4'-DDE/sigma DDT and TCPM/sigma TCP were very similar between animals from the same species. Strong correlations between sigma TCP and sigma DDT were also observed for each species of mammals, most likely indicating that both sigma TCP and sigma DDT are bioaccumulated in marine mammals. The relationships between sigma DDT and sigma TCP also demonstrate that sigma TCP are less bioaccumulated than sigma DDT by the marine mammal species examined.

Adipose Tissue↗

Codisposal of municipal refuse, sewage sludge and marine dredgings for methane production.

As marine disposal of sewage sludge and dredged sediments may impose serious adverse effects to marine ecosystems, landfilling seems to be the most feasible method for the final disposal of these wastes. A batch experiment was conducted to study waste degradation and gas production after sewage sludge and marine dredgings were mixed with municipal refuse at 13 different ratios for 36 days. The addition of sludge and dredgings to municipal refuse enhanced gas production, compared with the degradation of refuse or sludge alone. A proper mixing ratio of wastes can also shorten the time to reach the final phase of anaerobiosis. The highest gas production was obtained from the ratio of 75-20-5 (refuse-sludge-dredgings) (wet weight basis). Its average daily gas production rate was 1.42 l kg(-1) waste mixture; methane content was 68.3%. The results indicated that codisposal of the three wastes would be beneficial for energy recovery from landfill gas.

Journal Article↗

Widespread contamination by tris(4-chlorophenyl)methane and tris(4-chlorophenyl)methanol in cetaceans from the North Pacific and Asian coastal waters.

For understanding global distribution, transport and behavior of tris(4-chlorophenyl)methane (TCPMe) and tris(4-chlorophenyl)methanol (TCPMOH), the two newly identified microcontaminants, the present study determined their concentrations and other persistent organochlorines (OCs) in the blubber of nine species of adult male cetaceans collected from various locations in the North Pacific Ocean and coastal waters of some Asian countries, during 1985-97. Concentrations of TCPMe and TCPMOH were found to be highest in northern right whale dolphins, which may be attributable to wide distribution of this species, including some heavily polluted areas such as coastal California. Elevated residue levels of TCPMe and TCPMOH were observed in both off-shore and coastal species, suggesting widespread contamination of these compounds in the marine environment. Higher contamination of TCPMe and TCPMOH was found in cetaceans from temperate and cold waters than those from tropical regions. The latitudinal distribution of TCPMe and TCPMOH in cetaceans from the North Pacific and Asian coastal waters was similar to that of DDTs, suggesting less transportable nature of TCPMe and TCPMOH in the marine environment. Data on the occurrence of TCPMe and TCPMOH further indicated high bioaccumulation potential of these compounds, which was comparable to DDTs. Relative concentrations of TCPMe/TCPMOH in cetaceans were apparently higher than those observed in seals, suggestive of lower metabolic capacity for these compounds in cetaceans than in pinnipeds, which is similar to classic contaminants like polychlorinated biphenyls and DDTs.

Journal Article↗

Simultaneous determination of phencyclidine and monohydroxylated metabolites in urine of man by gas chromatography-mass fragmentography with methane chemical ionization.

Phencyclidine and monohydroxy metabolites were measured in human urine using gas chromatography-mass fragmentography with methane chemical ionization. Samples were extracted either untreated or following acid hydrolysis, derivatized with heptafluorobutyric anhydride, separated on a 3% SE-30 column and analyzed by mass fragmentography. The assay was sensitive to ca. 0.01 microgram/ml for phencyclidine and ca. 0.05 microgram/ml for the metabolites. Urine samples from five human subjects enrolled in a methadone maintenance program who had ingested phencyclidine were analyzed. The phencyclidine concentration ranged from 0.3 to 23.7 microgram/ml. The concentrations of metabolites ranged from 0 to 1.8 microgram/ml. A new monohydroxy metabolite was detected in the samples, but its structure was not fully elucidated. The specificity of the assay was examined.

Gas Chromatography-Mass Spectrometry↗

Quantum chemical studies of methane monooxygenase: comparision with P450.

The catalytic pathways of soluble methane monooxygenase (sMMO) and cytochrome P450CAM, iron-containing enzymes, are described and compared. Recent extensive density functional ab initio electronic structure calculations have revealed many similarities in a number of the key catalytic steps, as well as some important differences. A particularly interesting and significant contrast is the role played by the protein in each system. For sMMO, the protein stabilizes various species in the catalytic cycle through a series of carboxylate shifts. This process is adequately described by a relatively compact model of the active site ( approximately 100 atoms), providing a reasonable description of the energetics of hydrogen atom abstraction. For P450CAM, in contrast, the inclusion of the full protein is necessary for an accurate description of the hydrogen atom abstraction.

Binding Sites↗

Methane monooxygenase and its related biomimetic models.

The past year has seen significant advances in the understanding of the dioxygen-activating chemistry of non-heme diiron enzymes, such as methane monooxygenase. Recent spectroscopic and structural studies on various biomimetic model compounds have provided new and valuable insights into this enzyme's mechanism of action and the important dioxygen-activation process.

Catalysis↗

Aluminium triggers genotoxic adaptation to methyl mercuric chloride and ethyl methane sulfonate, but not to maleic hydrazide in plant cells in vivo.

Non-toxic, conditioning doses of aluminium chloride were tested for induction of adaptive response to the genotoxic challenge doses of methyl mercuric chloride (MMCl), maleic hydrazide (MH) and ethyl methane sulfonate (EMS). Embryonic shoot cells of Hordeum vulgare and root meristem cells of Allium cepa were employed as the assay systems. Plant tissues fixed at different recovery hours following the challenge treatments with or without prior Al-conditioning were analyzed for cells with genotoxicity markers that include spindle and/or chromosome aberrations and micronuclei (MNC). The results provided evidence that Al(3+) triggered adaptive response that protected the plant cells from the genotoxicity of MMCl and EMS. Al(3+), however, failed to induce adaptive response against the genotoxicity of MH. A comparison of Al-induced adaptive response with that induced by heavy metals: Cd(2+), Cu(2+), Hg(2+), Ni(2+), Pb(2+), Zn(2+) and oxidative agents: hydrogen peroxide (H(2)O(2)) and paraquat (PQ) pointed to the similarity of Al-adaptive response to that of PQ rather than to other heavy metals or H(2)O(2). Al-induced adaptive response demonstrated in the present study to MMCl and EMS possibly involved antioxidant defense and DNA repair systems, respectively.

Adaptation, Physiological↗

A laboratory-scale test of anaerobic digestion and methane production after phosphorus recovery from waste activated sludge.

In enhanced biological phosphorus removal (EBPR) processes, activated sludge microorganisms accumulate large quantities of polyphosphate (polyP) intracellularly. We previously discovered that nearly all of polyP could be released from waste activated sludge simply by heating it at 70 degrees C for about 1 h. We also demonstrated that this simple method was applicable to phosphorus (P) recovery from waste activated sludge in a pilot plant-scale EBPR process. In the present study, we evaluated the effect of this sludge processing (heat treatment followed by calcium phosphate precipitation) on anaerobic digestion in laboratory-scale experiments. The results suggested that the sludge processing for P recovery could improve digestive efficiency and methane productivity at both mesophilic (37 degrees C) and thermophilic (53 degrees C) temperatures. In addition, heat-treated waste sludge released far less P into the digested sludge liquor than did untreated waste sludge. It is likely that the P recovery step prior to anaerobic digestion has a potential advantage for controlling struvite (magnesium ammonium phosphate) deposit problems in sludge handling processes.

Journal Article↗

Morphological changes in Schistosoma mansoni caused by the mutagen ethyl methane sulphonate.

Three week old Schistosoma mansoni, cultured for up to sixty days after exposure to ethyl methane sulphonate, showed extensive ballooning which often involved most of the worm's body. Other changes included tegumental breakdown which was expressed in varying degrees from mild hernia to total disruption of body shape. Increase in gut size involved stretching of the epithelium and inclusions ranging from masses of disrupted tissue to large, dark opaque masses. Masses were also occasionally seen in the fluid filled spaces of worms. Evidence of hyperplasia was not observed in any of the histological examinations performed.

Animals↗

Oxidation-reduction potentials of the methane monooxygenase hydroxylase component from Methylosinus trichosporium OB3b.

Methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of hydroxylase (MMOH), reductase (MMOR), and "B" (MMOB) protein components. MMOH contains two oxygen-bridged dinuclear iron clusters that are the sites of O2 activation and hydrocarbon oxidation. Each cluster can be stabilized in diferric [Fe(III).Fc(III)], mixed-valence [Fe(II).Fe(III)], and diferrous [Fe(II).Fe(II)] redox states. We have correlated the EPR spin quantitation of the S = 1/2 mixed-valence state with the system electrode potential to determine both formal redox potential values for MMOH at 4 degrees C: E1 degrees' = +76 +/- 15 mV and E2 degrees' = +21 +/- 15 mV (Em = +48 mV, 61% maximum mixed-valence state). Complementary Mössbauer studies of 57Fe-enriched MMOH allowed all three redox states to be quantitated simultaneously in individual samples and revealed that the distribution of redox states was in accord with the measured potential values. EPR spectra of partially reduced MMOH showed that the apparent midpoint potential values of MMOH-MMOR, MMOH-MMOR-MMOB, and MMOH-MMOR-MMOB-substrate complexes were slightly more positive than that of MMOH alone. In contrast, the MMOH-MMOB complex appeared to have a substantially more negative redox potential. The formal redox potential values of the latter complex were determined to be E1 degrees' = -52 +/- 15 mV and E2 degrees' = -115 +/- 15 mV, respectively, at 4 degrees C (Em = -84 mV, 65% maximum mixed-valence state). This negative 132-mV shift in the midpoint potential of MMOH coupled to MMOB binding suggests that MMOB binds approximately 10(4) more strongly to the diferric state of MMOH than to the diferrous state. Since the potential shift is strongly negative, and since a nearly constant separation between the two formal potential values of MMOH is maintained when MMOB binds, the role of the MMOB-MMOH complex must not be to thermodynamically stabilize the formation of the diferrous cluster which is the form that reacts with O2 during catalysis. However, MMOB binding may provide kinetic stabilization of the diferrous state and/or modulation of the interaction of MMOH with O2 and hydrocarbon substrates. Such roles may be effected through cyclic association and dissociation of the MMOB-MMOH complex as MMOH oscillates between redox states during catalysis, thereby dynamically altering the affinity of this complex.

Alkenes↗

Formation of a polymethylene bis(disulfide) intersubunit cross-link between cysteine-281 residues in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase using octamethylene bis(methane[35S]thiosulfonate).

The synthesis of a radioactive cross-linking agent, S,S'-octamethylene bis(methane[35S]thiosulfonate) (OBMTS), is described. The route of synthesis can be generally used in the synthesis of 35S-labeled thiosulfonates for the selective modification of thiols in proteins. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) reacts asymmetrically with the bifunctional inhibitor. Initially two molecules of OBMTS react rapidly with the active-site thiol, Cys-149, on two of the four subunits to inhibit the enzyme completely without cross-linking. This is followed by the modification of four Cys-281 residues to incorporate two cross-links into the tetramer. Reduction of modified G3PD with 5 mM dithioerythritol under nondenaturing conditions released the inhibitor blocking the active-site thiol and completely restored enzyme activity while leaving the cross-link intact. Sodium dodecyl sulfate (Na-DodSO4) gel electrophoresis of the cross-linked enzyme under nonreducing conditions showed a dimer (Mr 72000) as the major species which was only cleaved by reduction in Na-DodSO4 containing beta-mercaptoethanol. The monomer formed was still radioactive, showing that the first disulfide in the cross-link was reduced at a much faster rate than the second disulfide. The latter was only reduced by using vigorous conditions. The location of the intersubunit cross-linked residues was established by isolation of the cyanogen bromide and tryptic subdigest peptides containing modified Cys-281. There were identified by molecular weight, amino terminal sequence, and amino acid composition.

Alkanesulfonates↗

Residues in Methylosinus trichosporium OB3b methane monooxygenase component B involved in molecular interactions with reduced- and oxidized-hydroxylase component: a role for the N-terminus.

Methane monooxygenase (MMO) is a non-heme-iron-containing enzyme which consists of 3 protein components: a hydroxylase (MMOH), an NAD(P)H-linked reductase (MMOR), and a 138-residue regulatory protein, component B (MMOB). Here, NMR spectroscopy has been used to derive interactions between MMOB and reduced and oxidized states of MMOH (245 kDa). Differential broadening of MMOB resonances in 1H-15N HSQC spectra acquired at different molar ratios of MMOH indicates interaction of both proteins, with MMOB binding more tightly to oxidized MMOH as observed previously. The most broadened backbone NH resonances suggest which residues in MMOB are part of the MMOH-binding interface, particularly when those residues are spatially close or clustered in the structure of MMOB. Although a number of different residues in MMOB appear to be involved in interacting with oxidized- and reduced-MMOH, some are identical. The two most common segments, proximal in the structure of MMOB, are beta-strand 1 with turn 1 (residues 36-46) and alpha-helix 3 going into loop 2 (residues 101-112). In addition, the N-terminus of MMOB is observed to be involved in binding to MMOH in either redox state. This is most strongly evidenced by use of a synthetic N-terminal peptide from MMOB (residues 1-29) in differential broadening 1H TOCSY studies with MMOH. Binding specificity is demonstrated by displacement of the peptide from MMOH by parent MMOB, indicating that the peptide binds in or near the normal site of N-terminal binding. The N-terminus is also observed to be functionally important. Steady-state kinetic studies show that neither a delta2-29 MMOB deletion mutant (which in fact does bind to MMOH), the N-terminal peptide, nor a combination of the two elicit the effector functions of MMOB. Furthermore, transient kinetic studies indicate that none of the intermediates of the MMOH catalytic cycle are observed if either the delta2-29 MMOB mutant or the N-terminal peptide is used in place of MMOB, suggesting that deletion of the N-terminus prevents reaction of reduced MMOH with O2 that initiates catalysis.

Amino Acid Sequence↗