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PubMed2004

Comparative analysis of the conventional and novel pmo (particulate methane monooxygenase) operons from methylocystis strain SC2.

In addition to the conventional pmoA gene (pmoA1) encoding the active site polypeptide of particulate methane monooxygenase, a novel pmoA gene copy (pmoA2) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene cluster (pmoCAB1, pmoCAB2). A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either pmoCAB1 or pmoCAB2 were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. A total of 190 of the 203 amino acid residues representing a highly conserved consensus sequence of the currently known PmoCAB1 and AmoCAB sequence types could be identified in PmoCAB2. The amoCAB gene cluster encodes ammonia monooxygenase and is evolutionarily related to pmoCAB. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that pmoCAB2 encodes a functional equivalent of PmoCAB1. In experiments in which we used 5' rapid amplification of cDNA ends we identified transcriptional start sites 320 and 177 bp upstream of pmoC1 and pmoC2, respectively. Immediately upstream of the transcriptional start sites of both pmoCAB1 and pmoCAB2, sequence motifs similar to Escherichia coli sigma(70) promoters were identified.

Amino Acid Sequence↗
PubMed1983

A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate.

Seven mutants of Escherichia coli were isolated that are sensitive to methyl methane sulfonate but not to UV light. They exhibited decreased host cell reactivation capacity for methyl methane sulfonate-treated phage lambda. Five of the mutations were mapped in the same region as alkA (previously called alk) and may indeed be identical to known mutations. Another mutation was found near nalA, and the gene responsible was named alkB. Its phenotype was different from that of ada, since the alkB mutant exhibited a normal adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine. A third type of mutation was mapped near polA, but this mutant contained an almost normal level of DNA polymerase I activity.

Drug Resistance, Microbial↗
PubMed2006

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M.

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.

Bacterial Proteins↗
PubMed1994

Biochemistry of the soluble methane monooxygenase.

The soluble form of methane monooxygenase (MMO) catalyzes the reaction NAD(P)H + O2 + CH4 + H+-->NAD(P)+ + H2O + CH3OH. Many other hydrocarbons serve as adventitious substrates. MMO consists of three protein components: component B, reductase, and hydroxylase (MMOH), the active site of which contains a hydroxo-bridged dinuclear iron cluster that is the site of catalysis. Such a cluster has not been previously associated with oxygenases, and spectroscopic studies have been conducted to ascertain its structural features and accessibility. The mechanism of MMO has been investigated through the use of diagnostic chemical reactions and transient kinetics. Both approaches are consistent with a mechanism in which the diiron cluster is first reduced to the diferrous state and then reacts with O2. The O-O bond is apparently cleaved heterolytically to yield water and an [Fe(IV).Fe(IV)]=O species, which purportedly abstracts a hydrogen atom from methane to yield a substrate radical and a diiron cluster-bound hydroxyl radical. Recombination of the radicals yields the product methanol. An intermediate with the properties of the novel [Fe(IV).Fe(IV)] = O species has been trapped and characterized. This is the first such species to be isolated in biology. Meanwhile, the reductase and component B have roles in catalysis beyond simple electron transfer from NAD(P)H. These roles appear to be related to regulation of catalysis, and are mediated by the formation of specific component complexes that alter the physical and catalytic properties of MMOH at different stages of the turnover cycle.

Models, Chemical↗
PubMed2000

Chlorinated methanes and liver injury: highlights of the past 50 years.

The chlorinated methanes, particularly carbon tetrachloride and chloroform, are classic models of liver injury and have developed into important experimental hepatoxicants over the past 50 years. Hepatocellular steatosis and necrosis are features of the acute lesion. Mitochondria and the endoplasmic reticulum as target sites are discussed. The sympathetic nervous system, hepatic hemodynamic alterations, and role of free radicals and biotransformation are considered. With carbon tetrachloride, lipid peroxidation and covalent binding to hepatic constituents have been dominant themes over the years. Potentiation of chlorinated methane-induced liver injury by alcohols, aliphatic ketones, ketogenic compounds, and the pesticide chlordecone is discussed. A search for explanations for the potentiation phenomenon has led to the discovery of the role of tissue repair in the overall outcome of liver injury. Some final thoughts about future research are also presented.

Animals↗
PubMed2003

Novel archaeal macrocyclic diether core membrane lipids in a methane-derived carbonate crust from a mud volcano in the Sorokin Trough, NE Black Sea.

A methane-derived carbonate crust was collected from the recently discovered NIOZ mud volcano in the Sorokin Trough, NE Black Sea during the 11th Training-through-Research cruise of the R/V Professor Logachev. Among several specific bacterial and archaeal membrane lipids present in this crust, two novel macrocyclic diphytanyl glycerol diethers, containing one or two cyclopentane rings, were detected. Their structures were tentatively identified based on the interpretation of mass spectra, comparison with previously reported mass spectral data, and a hydrogenation experiment. This macrocyclic type of archaeal core membrane diether lipid has so far been identified only in the deep-sea hydrothermal vent methanogen Methanococcus jannaschii. Here, we provide the first evidence that these macrocyclic diethers can also contain internal cyclopentane rings. The molecular structure of the novel diethers resembles that of dibiphytanyl tetraethers in which biphytane chains, containing one and two pentacyclic rings, also occur. Such tetraethers were abundant in the crust. Compound-specific isotope measurements revealed delta13C values of -104 to -111/1000 for these new archaeal lipids, indicating that they are derived from methanotrophic archaea acting within anaerobic methane-oxidizing consortia, which subsequently induce authigenic carbonate formation.

Archaea↗
PubMed2006

Multiline absorption spectroscopy for methane gas detection.

A multiline absorption spectroscopy technique was investigated based on the single-line absorption spectroscopy technique. An open-path methane-detecting system was designed. An LED was used as a broadband source, and a Fabry-Perot interferometer whose transmission peaks matched the methane R-branch absorption lines was used to enhance the detectable sensitivity. We demonstrate a minimum-detectable concentration of 7600 +/- 10% ppm (parts per million) with a multiline differential absorption spectroscopy technique and a concentration of 1000 +/- 10% ppm with a multiline wavelength modulation spectroscopy technique.

Journal Article↗
PubMed2006

In situ Raman spectroscopic study of the dissociation of methane hydrate at high pressure.

The dissociation of methane hydrate at high pressure is studied by in situ Raman spectroscopy in a diamond-anvil cell. As for the Raman spectrum of sI methane hydrate, the v(1) band of CH(4) is split into two peaks v'(1) and v''(1), indicating the partitioning of CH(4) between the large (5(12)6(2)) and small (5(12)) cavities, respectively. With increasing temperature, the intensity ratio of Iv'(1)/Iv''(1) decreases obviously, and the d(Iv'(1)/Iv''(1))/dT is -0.079 K(-1). Additionally, the v(1) band of the dissolved CH(4) is close to v''(1) of the CH(4) molecule engaged in the small 5(12) cavity. This implies that, in the initial stage of hydrate formation, the abundance of small 5(12) cavities is greater than that of large 5(12)6(2) cavities.

Journal Article↗
PubMedDate not supplied

Evaluation of genotoxic activity of maleic hydrazide, ethyl methane sulfonate, and N-nitroso diethylamine in Tradescantia.

OBJECTIVE: To assess the genotoxic activity of N-nitroso diethylamine (NDEA), maleic hydrazide (MH), and ethyl methane sulfonate (EMS) using two systems: the comet assay on nuclei from Tradescantia, and the pink mutation test on Tradescantia staminal hairs (clone 4430). MATERIAL AND METHODS: Tradescantia cups was obtained from Laboratorio de Citogenética y Mutagénesis del Centro de Ciencias de la Atmósfera de la Universidad Nacional Autónoma de México and treated with: N-nitroso diethylamine (NDEA) at 1, 5, 10 mM, maleic hydrazide (MH) at 1, 5, 10 mM and ethyl methane sulfonate (EMS) at 15, 30 and 45 mM; and used in both pink mutation assay and comet assay using cellular nuclei from Tradescantia staminal hairs. The observation of staminal hair was realized along eight days (6-14) after treatment), flowers produced day 14 after treatment were utilized done according to Underbrink. In previous reports on plants, were comet assay was used, breaking cellular wall and separating by centrifugation gradient are necessary. Here, nuclei from staminal hairs were obtained by squashing the cells (is not necessary to utilize to break special procedure cellular wall), collected using a nylon mesh of 80 Mm and next the comet assay was applied. Student's T test was the statistical test used for analyzing the comet assay data. RESULTS: Both assays showed a great sensitivity to the studied mutagens. A relationship between the dose-pink event and the dose-tail length was evident. Even though the Tradescantia mutation assay is a sensitive test with MH and EMS, low doses of NDEA were not able to induce a significant increase in the pink event frequencies; however, the comet assay was able to detect the mutagenic effect of NDEA at the same dose. Thus, it is clear that the comet assay is highly sensitive to the lowest dose of chemical mutagens. CONCLUSIONS: The comet assay on nuclei from Tradescantia staminal hairs is a useful tool to monitor genotoxic agents; it is simple, highly sensitive, and faster than the pink mutation test.

Comet Assay↗
PubMedDate not supplied

[Formation of the taxa of methane-oxidizing bacteria by numerical analysis methods].

Obligate methane oxidizing bacteria were classified within groups using numerical analysis and computer techniques. Employment of properties coinciding for all the species of the bacterial group under study was found to result in an insignificant (5--10%) change in the degree of similarity rather than in the redistribution of interrelationship; it had no effect on the formation of phenons. The Smirnov taxonomical analysis according to which the weight of a property is inversely proportional to its frequency has a number of advantages over other numerical methods where all the properties are assumed to be of equal value. Comparative evaluation of the data obtained by different methods which have been used by us to calculate the coefficients for coupled similarity of species (the coefficient of association, the coefficient of correlation, the index of distance, the coefficient of taxonomic ratio) shows that similar results are found in all the cases with respect to group division of the species belonging to the family Methylomonadaceae Leadbetter 1974. The species of methane oxidizing bacteria should be divided into the following genera: Methylosinus Whittenbury, Phillips a. Wilkinson 1970, Methylocystis Whittenbury, Phillips a. Wilkinson 1970, Methylococcus Foster a. Davis 1966, Methylomonas Leadbetter 1974.

Computers↗
PubMed2001

Field measurements of internal pressurization in Phragmites australis (Poaceae) and implications for regulation of methane emissions in a midlatitude prairie wetland.

Emergent aquatic macrophytes in vegetated wetlands provide routes for methane (CH(4)) transport from sites of production in oxygen-poor sediments, where CH(4) concentrations are relatively high, to the atmosphere, which typically has much lower CH(4) concentrations. Transport can occur through aerenchymatous tissue via simple diffusion. Recently, the importance of convective throughflow (i.e., mass transport of gases through plants driven by pressure gradients) in enhancing gas transport has been demonstrated in several genera (e.g., Nuphar, Nymphaea, Nelumbo, Typha, and Phragmites). This study was conducted to elucidate the governing plant-mediated gas transport mechanisms in a midlatitude prairie wetland and to determine both their diel and seasonal variations and the importance of environmental controlling factors. Pressures inside culms of the two dominant emergent aquatic macrophytes (Scirpus acutus and Phragmites australis) were measured directly throughout the growing season and on selected days in midseason. Supporting measurements included solar radiation, air temperature, relative humidity, and windspeed. Results indicated pressures inside green healthy culms of Phragmites were above atmospheric pressure by up to 1650 Pa during the day. At night culm pressures were at or slightly above atmospheric. No pressurization was detected in Scirpus. Highest pressures in Phragmites occurred during midseason when biomass and foliage area index were at their maxima (920 g/m(2) and 2.8, respectively). High internal pressures also coincided with periods of high solar radiation (>500 W/m(2)), high temperature (>20°C), and low relative humidity (<60%). Periods of high internal pressures also coincided with periods of high CH(4) efflux from the wetland as measured in concomitant studies. Convective throughflow driven by internal pressure gradients in Phragmites thus explains much of the diel variation in methane efflux previously reported from this wetland.

Journal Article↗
PubMed1975

Bis-(p-hydroxyphenyl)-pyridyl-2-methane: The common laxative principle of Bisacodyl and sodium picosulfate.

After both oral and rectal administration in humans (4,4'-diacetoxy-diphenyl)-(pyridyl-2)-methane (bisacodyl, Dulcolax) and 4,4'-(2-pyridyl-methylene)-diphenol-disulfuric acid semiester disodium (sodium picosulfate, Laxoberal) are hydrolyzed to bis-(p-hydroxyphenyl)-pyridyl-2-methane (BHPM). In both cases BHPM is responsible for the laxative action. Experiments in rats and guinea pigs have shown that the hydrolysis of picosulfate, in contrast to that of bisacodyl, is attributable to the microorganisms of the intestinal flora.

Administration, Oral↗
PubMed2004

[A study on the effect of methanal during determination of carbon monoxide in blood].

OBJECTIVE: To confirm whether formaldehyde disturb detecting carbon monoxide in blood. To give an evidence that can be used for detecting carboxyhemoglobin more accurately in carbon monoxide posioning appraises. METHODS: Blood samples came from carbon monoxide poisoning and the health were collected. Regular methods for detecting carboxyhemoglobin were used. Observing and comparing the detection results between which were spiked with methanal and no spiked one were performed. RESULTS: Methanal will affect the result of following experiments such as heating, adding NaOH, absorbed by PdCl2 and spectrophotometry. CONCLUSION: The samples which contaminated by formaldehyde couldn't be used for detecting carboxyhemoglobin.

Carbon Monoxide↗
PubMed1998

[Raman spectrum characteristics of methane].

Two characteristic Raman peaks of methane at 2917 and 3020cm(-1) were measured. The results show that the four C-H bonds of methane molecular are unequal. The assignment of Raman bands at 2917 and 3020cm(-1) was discussed in this paper.

English Abstract↗
PubMed1990

Preparative slab electrofocusing of methane monooxygenase from a type I methanotroph Methylomonas GYJ3.

The isolation and purification of methane monooxygenase from a type I methanotroph Methylomonas GYJ3 to near homogeneity is reported. The isoelectric focusing in flat-bed granulated gels has resolved the methane monooxygenase into three protein components. The specific activity of the enzyme is 198.4 n mol per min per mg protein, degree of purification 4.13-fold. Recovery of the focused proteins is high and elution simple. Several purification steps may be omitted from the previously published scheme by other techniques.

Isoelectric Focusing↗
PubMed1990

Sequential events in the pathogenesis of streptococcal cell wall-induced arthritis and their modulation by bis(5-amidino-2-benzimidazolyl)methane (BABIM).

This report builds on the authors' earlier discovery of bis(5-amidino-2-benzimidazolyl)methane (BABIM) as a strong suppressive agent for streptococcal cell wall fragment-induced arthritis in the Lewis rat. As a synthetic inhibitor of trypsinlike proteases, BABIM opens up a new route to the control of inflammatory joint disease. To gain a deeper insight into the function of the compound, the authors have now studied its influence on the sequential development of the joint changes and the associated lesions in spleen and liver. Bis(5-amidino-2-benzimidazolyl)methane is shown to block acute synovitis, to retard and reduce granuloma formation in spleen and liver, to decrease neutrophilic leukocytosis, and to diminish hemopoietic hyperplasia in the bone, and thus also to mitigate the distinctive osteoclastic and chondroclastic events. The compound does not interfere with the splenic immune response, the temporary rise in hepatocytic mitotic activity, or the organ deposition of streptococcal cell walls.

Animals↗
PubMed1989

Methane monooxygenase from Methylosinus trichosporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph.

Methane monooxygenase has been purified from the Type II methanotroph Methylosinus trichosporium OB3b. As observed for methane monooxygenase isolated from Type I methanotrophs, three protein components are required: a 39.7-kDa NADH reductase containing 1 mol each of FAD and a [2Fe-2S] cluster, a 15.8-kDa protein factor termed component B that contains no metals or cofactors, and a 245-kDa hydroxylase which appears to contain an oxo- or hydroxo-bridged binuclear iron cluster. Through the use of stabilizing reagents, the hydroxylase is obtained in high yield and exhibits a specific activity 8-25-fold greater than reported for previous preparations. The component B and reductase exhibit 1.5- and 4-fold greater specific activity, respectively. Quantitation of the hydroxylase oxo-bridged cluster using EPR and Mössbauer spectroscopies reveals that the highest specific activity preparations (approximately 1700 nmol/min/mg) contain approximately 2 clusters/mol. In contrast, hydroxylase preparations exhibiting a wide range of specific activities below 500 nmol/min/mg contain approximately 1 cluster/mol on average. Efficient turnover coupled to NADH oxidation requires all three protein components. However, both alkanes and alkenes are hydroxylated by the chemically reduced hydroxylase under single turnover conditions in the absence of component B and the reductase. Neither of these components catalyzes hydroxylation individually nor do they significantly affect the yield of hydroxylated product from the chemically reduced hydroxylase. Hydroxylase reduced only to the mixed valent [Fe(II).Fe(III)] state is unreactive toward O2 and yields little hydroxylated product on single turnover. This suggests that the catalytically active species is the fully reduced form. The data presented here provide the first evidence based on catalysis that the site of the monooxygenation reaction is located on the hydroxylase. It thus appears likely that the oxo-bridged iron cluster is capable of catalyzing oxygenase reactions without the intervention of other cofactors. This is a novel function for this type of cluster and implies a new mechanism for the generation of highly reactive oxygen capable of insertion into unactivated carbon-hydrogen bonds.

Cell-Free System↗
PubMedDate not supplied

Contact allergy to 2,4'-dihydroxy-(hydroxymethyl)-diphenyl methanes. Sensitizers in a phenol-formaldehyde resin.

A particular phenol-formaldehyde resin of the resol type (P-F-R-2) was shown to be a good marker for patients with contact allergy. Eleven patients with sensitivity to P-F-R-2 participated in the study in which chromatographically separated fractions of P-F-R-2 were patch tested. This procedure was repeated until a positive reaction to a single substance was noted. In this study 2 sensitizers are reported. Investigations by mass-spectrometry and nuclear magnetic resonance spectrometry identified the compounds to be 2,4'-dihydroxy-3,3'-di-(hydroxymethyl)-diphenyl methane (2,4'-H-3,3'-HPM) and 2,4'-dihydroxy-3',5'-di-(hydroxymethyl)-diphenyl methane (2,4'-H-3',5'-HPM). Six of 11 patients reacted to at least one of the compounds and simultaneous reactions were noted in 4 patients. By patch testing with serial dilutions of the two sensitizers positive reactions were noted down to 0.14 X 10(-3)% and 0.41 X 10(-3)% w/v for 2,4'-H3,3'-HPM and 2,4'-H-3',5'-HPM respectively.

Benzhydryl Compounds↗