ntn genes determining the early steps in the divergent catabolism of 4-nitrotoluene and toluene in Pseudomonas sp. strain TW3

Pseudomonas sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes, ntnWCMAB*, which are very similar to and in the same order as the xyl operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (ntnW), an NAD+-linked benzaldehyde dehydrogenase (ntnC), a two-gene toluene monooxygenase (ntnMA), and part of a benzyl alcohol dehydrogenase (ntnB*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding xylWCMAB genes. The xylB homolog on the TW3 genome (ntnB*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the beta-ketoadipate pathway.     

钯掺杂α-MnO2无溶剂下催化氧化苯甲醇的性能

通过共沉淀和原位煅烧转化方法, 将Pd掺杂δ-MnO₂前驱体煅烧后制备得到Pd掺杂α-MnO₂纳米棒催化材料.通过氮气物理吸附、X射线衍射、透射电子显微镜、扫描电子显微镜、热重分析、X射线光电子能谱等技术对催化材料进行了表征.扫描电镜和透射电镜结果显示, α-MnO₂纳米棒表面没有明显的Pd纳米颗粒, 表明Pd可能掺杂到α-MnO₂晶格中.纯α-MnO₂的还原温度在390℃左右, 但Pd掺杂可以极大地促进α-MnO₂还原, 还原温度可低至约200℃左右.研究了所制备催化剂在无溶剂条件下对于以分子氧为氧化剂选择性催化氧化苯甲醇为苯甲醛的催化性能.结果表明: 在无溶剂及用纯氧气为氧化剂条件下, Pd掺杂α-MnO₂纳米棒对苯甲醇氧化显示出增强的催化活性; 所掺杂的氧化态Pd物质可增强催化材料中的氧迁移率; 在这些Pd掺杂α-MnO₂催化材料中, 当以Pd (3%, 质量分数) -MnO₂为催化剂时, 在110℃反应4 h后, 苯甲醇的转化率为39%, 远高于同条件下以纯α-MnO₂为催化剂时18. 3%的苯甲醇转化率.      

Novel nuclear magnetic resonance spectroscopy methods demonstrate preferential carbon source utilization by Acinetobacter calcoaceticus

Novel nuclear magnetic resonance spectroscopy techniques, designated metabolic observation, were used to study aromatic compound degradation by the soil bacterium Acinetobacter calcoaceticus. Bacteria which had been rendered spectroscopically invisible by growth with deuterated (2H) medium were used to inoculate cultures in which natural-abundance 1H hydrogen isotopes were provided solely by aromatic carbon sources in an otherwise 2H medium. Samples taken during the incubation of these cultures were analyzed by proton nuclear magnetic resonance spectroscopy, and proton signals were correlated with the corresponding aromatic compounds or their metabolic descendants. This approach allowed the identification and quantitation of metabolites which accumulated during growth. This in vivo metabolic monitoring facilitated studies of catabolism in the presence of multiple carbon sources, a topic about which relatively little is known. A. calcoaceticus initiates aromatic compound dissimilation by forming catechol or protocatechuate from a variety of substrates. Degradation proceeds via the beta-ketoadipate pathway, comprising two discrete branches that convert catechol or protocatechuate to tricarboxylic acid cycle intermediates. As shown below, when provided with several carbon sources simultaneously, all degraded via the beta-ketoadipate pathway, A. calcoaceticus preferentially degraded specific compounds. For example, benzoate, degraded via the catechol branch, was consumed in preference to p-hydroxybenzoate, degraded via the protocatechuate branch, when both compounds were present. To determine if this preference were governed by metabolites unique to catechol degradation, pathway mutants were constructed. Studies of these mutants indicated that the product of catechol ring cleavage, cis,cis-muconate, inhibited the utilization of p-hydroxybenzoate in the presence of benzoate. The accumulation of high levels of cis,cis-muconate also appeared to be toxic to the cells.     

Mechanism of aldehyde oxidation catalyzed by horse liver alcohol dehydrogenase

The mechanism of oxidation of benzaldehyde to benzoic acid catalyzed by horse liver alcohol dehydrogenase (HLADH) has been investigated using the HLADH structure at 2.1 A resolution with NAD+ and pentafluorobenzyl alcohol in the active site [Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations with HLADH were obtained by a best-fit superimposition of benzaldehyde or its hydrate on the pentafluorobenzyl alcohol bound to the active site Zn(II)ion. Equilibrium bond lengths, angles, and dihedral parameters for Zn(II) bonding residues His67, Cys46, and Cys174 were obtained from small-molecule X-ray crystal structures and an ab initio-derived parameterization of zinc in HLADH [Ryde, U. (1995) Proteins: Struct., Funct., Genet. 21,40-56]. Dynamic simulations in CHARMM were carried out on the following three constructs to 100 ps: (MD1) enzyme with NAD+, benzaldehyde, and zinc-ligated HO-in the active site; (MD2) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-R oxygen, with a proton residing on the pro-S oxygen; and (MD3) enzyme with NAD+ and hydrated benzaldehyde monoanion bound to zinc via the pro-S oxygen, with a proton residing on the pro-R oxygen. Analyses were done of 800 sample conformations taken in the last 40 ps of dynamics. Structures from MD1 and MD3 were used to define the initial spatial arrangements of reactive functionalities for semiempirical PM3 calculations. Using PM3, model systems were calculated of ground states and some transition states for aldehyde hydration, hydride transfer, and subsequent proton shuttling. With benzaldehyde and zinc-bound hydroxide ion in the active site, the oxygen of Zn(II)-OH resided at a distance of 2.8-5.5 A from the aldehyde carbonyl carbon during the dynamics simulation. This may be compared to the PM3 transition state for attack of the Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, which has an O...C distance of 1.877 A. HLADH catalysis of the aldehyde hydration would require very little motion aside from that in the ground state. Two simulations of benzaldehyde hydrate ligated to zinc (MD2 and MD3) both showed close approach of the aldehyde hydrate hydrogen to NAD+C4, varying from 2.3 to 3.3 A, seemingly favorable for the hydride transfer reaction. The MD2 configuration does not allow proton shuttling. On the other hand, when the pro-S oxygen is ligated to zinc (MD3), the proton on the pro-R oxygen averages 2.09 A from the hydroxyl oxygen of Ser48 such that initiation of shuttling of protons via Ser48 to the ribose 2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen is sterically favorable. PM3 calculations suggest that this proton shuttle represents a stepwise reaction which occurs subsequent to hydride transfer. The PM3 transition state for hydride transfer based on the MD3 configuration has the transferring hydride 1.476 A from C4 of NAD+ and 1.433 A from the aldehyde alpha-carbon.     

Quantitative determination of benzyl benzoate in benzyl benzoate lotion NF

The development of a simple, shorter and more accurate method than the NF method for the determination of benzyl benzoate in Benzyl Benzoate Lotion NF is discussed. Hydrolyzed benzyl benzoate was measured spectrophotometrically. Interference from other ingredients of the lotion, oleic acid and triethanolamine was almost negligible. The proposed method was completed in approximately 15 minutes, as opposed to the two hours required by the NF procedure.     

Thermodynamic and fluorescence studies of the underlying factors in benzyl alcohol-induced lipid interdigitated phase

To further investigate factors contributing to the action of alcohol in the solute-induced lipid interdigitation phase, thermodynamic and fluorescence polarization measurements were carried out to study the interaction of benzyl alcohol with dipalmitoyl phosphatidylcholine bilayer vesicles. The obtained results were compared with those previously reported for ethanol and cyclohexanol (L. G. Roth and C-H. Chen, Arch. Biochem. Biophys. 296, 207, 1992). Similar to ethanol, benzyl alcohol was found to exhibit a biphasic effect on the enthalpy (delta Hm) and the temperature (tm) of the lipid-phase transition and the steady-state fluorescence polarization (P) monitored by 1,6-diphenyl-1,3,5-hexatriene. At a total concentration of benzyl alcohol < 30 mg/ml (the alcohol concentration in lipid phase < 21 mg/ml), benzyl alcohol was found to exhibit large increases in delta Hm and P, which were correlated with the formation of a lipid interdigitated phase, as evidenced by reported X-ray diffraction data. Combining the results with benzyl alcohol and ethanol suggested that simultaneously large changes in delta Hm and P can be used as an indication of the occurrence of a solute-induced lipid interdigitated phase. The overall interacting force in the formation of this lipid phase, as derived from the interactions of the hydroxyl portion of an alcohol with the lipid phosphate head group and the hydrophobic portion of an alcohol with the lipid hydrocarbon chains, may or may not be dominated by hydrophobic interaction. Although lipid/water partition coefficients and the contribution of hydrophobic interaction to the overall interacting force were comparable between benzyl alcohol and cyclohexanol, benzyl alcohol induced lipid interdigitated phase, but not for cyclohexanol. This was due to the ability of benzyl alcohol to be more effective than cyclohexanol in simultaneously interacting with the phosphate head group and the hydrocarbon chains of lipid.     

Inhibition of rat lung mixed-function oxidase activity following repeated low-level toluene inhalation: possible role of toluene metabolites

Toluene is a commonly used solvent that has been shown to alter mixed-function oxidase (MFO) activity, in an organ- and isozyme-specific pattern, following intraperitoneal administration. The purpose of this study was to determine whether similar changes occurred following repeated, low-level inhalation exposure, and to investigate the role of toluene metabolites in these alterations. Exposure to 375 ppm toluene, 6 h/d for up to 5 d, resulted in significant inhibition of the activity of pulmonary arylhydrocarbon hydroxylase (AHH), cytochrome P-4502B1 (CYP2B1), and CYP4B1, but not CYP1A1. After exposure to lower toluene levels (125 ppm, 6 h/d, 3 d), the activities of lung AHH, CYP2B1, and CYP4B1 were also significantly decreased, but in a dose-related manner. MFO activity was not consistently altered in liver. Control pulmonary or liver microsomes were incubated with various concentrations (0.01-10 mM) of toluene or its metabolites and CYP2B1, CYP1A1, and/or CYP4B1 activities were subsequently determined. Benzaldehyde produced a significant dose-related inhibition in the activity of all three lung P-450s examined (IC50 10(-3) M). Toluene was found to be a more potent inhibitor of lung CYP2B1 and CYP1A1 (IC50, 10(-4) M) than benzaldehyde, but neither toluene nor benzyl alcohol was an effective inhibitor of lung CYP4B1. Toluene and its metabolites were weaker inhibitors of CYP1A1 than of CYP2B1. For CYP2B1 and CYP1A1, the order of inhibitory potency was toluene > benzaldehyde > benzyl alcohol and suggests that both the parent molecule and its metabolites may act in concert to inhibit catalytic activity of these cytochromes. The MFO inhibition seen after repeated low-level toluene inhalation exposure could result in altered metabolic profiles of other xenobiotics in an organ-specific fashion.     

Antiarrhythmic effect of solvents: propylene glycol, benzyl alcohol

Propylene glycol and benzyl alcohol, the main constituents of most solvent vehicles, display a pronounced antiarrhythmic-antifibrillatory effects, when injected intravenously into animals (dogs, rats) with spontaneous or drug-induced arrhythmias. The antiarrhythmic dose for propylene glycol amounts to 0.2-0.3 ml/kg of a 70 per cent solution and, for benzyl alcohol to 0.2-0.4 ml/kg of a 4 per cent solution in physiologic saline, respectively. Similar effects were also obtained by the combined injection of propylene glycol + benzyl alcohol, in proportions which correspond to the formulae of numerous commercial "solvents" (vehicles): 2 to 20 per cent solutions of benzyl alcohol in 70 per cent propylene glycol (0.05-0.2 ml/kg). The mechanisms which might be responsible for the antiarrhythmic activity of solvents are discussed: lengthening of the effective refractory period, local and general anaesthetic effects, changes of osmolarity. The intravenous injection of propylene glycol and/or benzyl alcohol, in high doses, produces intravascular haemolysis. Clinical investigations are recommended as to the potential, beneficial or toxic effects of drug solvents, especially upon the cardiocirculatory system.     

Blunt impact causes changes in bone and cartilage in a regularly exercised animal model

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mouse embryos were harvested on gestation day 8 or 9 and stained with the vital lysosomal dye, LysoTracker Red. Following incubation in the stain, embryos were fixed in 2% paraformaldehyde overnight, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. The entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. To test this approach, the chemotherapeutic drug hydroxyurea was added to day 8 embryos in vitro to induce apoptosis. Our results demonstrated specific regions undergoing programmed cell death in normal development and increased apoptosis in embryos exposed to hydroxyurea. The observed patterns of LysoTracker Red staining correlate well with previous studies of cell death using other lysosomotropic dyes such as Nile blue sulfate, acridine orange, or neutral red. LysoTracker Red has the advantages of being aldehyde-fixable and highly fluorescent (bleaching was not observed even after multiple scans). This procedure allows for the optical imaging of whole day 9 (approximately 22 somites) embryos that were greater than 500 microns thick in the Z-axis.     

The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation

The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty. The first enzymes of the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a plasmid-located transposon Tn5280. Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB. Homology comparisons indicated that these genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and benzoate degradation. Similar to the tod-encoded enzymes, chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene, naphthalene, and biphenyl, but not benzoate, to the corresponding dihydrodiol and dihydroxy intermediates. These data strongly suggest that the chlorobenzene dioxygenase and dehydrogenase originated from a toluene or benzene degradation pathway, probably by horizontal gene transfer. This evolutionary event left its traces as short gene fragments directly outside the tcbAB coding regions.     

A Genetic Dissociation of Learning and Recall in Caenorhabditis elegans.

A learning event can be dissociated into 3 components: acquisition, storage, and recall. When the laboratory wild-type strain of Caenorhabditis elegans (N2 strain) is exposed to benzaldehyde in the absence of food, the worms display a reduction of their attractive response to this volatile odorant. This results from the association between benzaldehyde and a nutrient-deficient environment. Another wild-type isolate, the CB4856 strain, fails to display this decreased response to benzaldehyde after exposure to benzaldehyde in the absence of food. However, like the N2 strain, when tested to isoamyl alcohol after benzaldehyde conditioning, the CB4856 strain displays a decreased isoamyl alcohol response. Therefore, the CB4856 strain does not have an acquisition deficit, but it suffers from a recall deficit specific to benzaldehyde. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of lipid solubility on the development of chronic cross-tolerance between ethanol and different alcohols and barbiturates

Tolerance to ethanol and cross-tolerance to other alcohols (n-propanol, n-butanol, t-butanol, isobutanol, t-amyl alcohol, n-amyl alcohol, and benzyl alcohol) and barbiturates (pentobarbital, secobarbital, amobarbital, thiopental, barbital and phenobarbital) that differ in lipid:water partition coefficient was examined in rats after chronic pretreatment with ethanol. Tolerance and cross-tolerance were studied with three different measures (hypothermia, tilt-plane, and rotarod). Tolerance to ethanol resulted in significant cross-tolerance to alcohols with low lipid solubility (n-propanol and t-butanol), whereas no cross-tolerance was seen with alcohols of high lipid solubility (isobutanol, n-amyl alcohol, t-amyl alcohol and benzyl alcohol). Cross-tolerance to n-butanol (which has intermediate lipid solubility) appeared to be metabolic rather than functional. Tolerance to ethanol also resulted in significant cross-tolerance to barbital and phenobarbital, but not to pentobarbital, secobarbital, amobarbital or thiopental. These studies suggest that lipid solubility is an important factor in relation to specificity of cross-tolerance to alcohols and barbiturates.     

Benzoyl cyanide in the defensive secretion of polydesmoid millipeds

A novel cyanogenetic compound benzoyl cyanide, was isolated from the defensive secretion of 3 polydesmoid millipeds (Pseudopolydesmus seratus, Apheloria corrugata and A. trimaculata). The secretion of the 3 species also contains mandelonitrile and benzaldehyde, and that of P. serratus contains mandelonitrile benzoate, benzoic acid, isovaleric acid, myristic acid and stearic acid.     

Protein fluorescence and electronic energy transfer in the determination of molecular dimensions and rotational relaxation times of native and coenzyme-bound horse liver alcohol dehydrogenase

The intrinsic fluorescence lifetimes of horse liver alcohol dehydrogenase (EC 1.1.1.1) and pig heart isocitrate dehydrogenase (EC 1.1.1.42) have been determined to be 5.36 ns and 4.84 ns, respectively. When reduced coenzyme is bound, the fluorescence lifetime of alcohol dehydrogenase is reduced to 4.98 ns while that of isocitrate dehydrogenase remains unchanged. Oxidized coenzymes have no effect on fluorescence lifetimes of alcohol and isocitrate dehydrogenases. This virtual constancy of protein fluorescence lifetimes has allowed the conclusion to be reached that in protein-ligand complexes with equilibrium constants in the range of 10(4)-10(6) M(-1), the static mode of quenching is substantial. The observation of resonance energy transfer in alcohol dehydrogenase-NADH complex facilitates the determination of the distance between tryptophan and the reduced nicotinamide ring involved in the transfer as 30.6 A, compared to the effective molecular radius of 36.2 A for alcohol dehydrogenase. The increased rotational relaxation times of coenzyme-bound alcohol dehydrogenase relative to the unliganded form (sigmah = 72 ns) indicate in this protein structural fluctuations occurring in the time range of nanoseconds.     

Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts

Methyl formate synthase, which catalyzes methyl formate formation during the growth of methylotrophic yeasts, was purified to homogeneity from methanol-grown Candida boidinii and Pichia methanolica cells. Both purified enzymes were tetrameric, with identical subunits with molecular masses of 42 to 45 kDa, containing two atoms of zinc per subunit. The enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl group of the hemiacetal adduct [CH2(OH)OCH3] of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate. Although neither methanol nor formaldehyde alone acted as a substrate for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogenase activity toward aliphatic long-chain alcohols such as octanol, showing that they belong to the class III alcohol dehydrogenase family. The methyl formate synthase activity of C. boidinii was found in the mitochondrial fraction in subcellular fractionation experiments, suggesting that methyl formate synthase is a homolog of Saccharomyces cerevisiae Adh3p. These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in methylotrophic yeasts. The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed.