Alternative pathways foro-xylene orm-xylene andp-xylene degradation in aPseudomonas stutzeri strain

Pseudomonas stutzeri OX1 is able to grow ono-xylene but is unable to grow onm-xylene andp-xylene, which are partially metabolized through theo-xylene degradative pathway leading to the formation of dimethylphenols toxic to OX1.P. stutzeri spontaneous mutants able to grow onm-xylene andp-xylene have been isolated. These mutants soon lose the ability to grow ono-xylene. Data from HPLC analyses and from induction studies suggest that in these mutantsm-xylene andp-xylene could be metabolized through the oxidation of a methyl substituent.P. stutzeri chromosomal DNA is shown to share homology with pWW0 catabolic genes. In the mutant strains the region homologous to pWW0 upper pathway genes has undergone a genomic rearrangement.Abbreviations BADH benzylalcohol dehydrogenase - cat catechol - C23O catechol 2,3-dioxygenase - 2,3-,3,4-,2,4-,2,6-,3,5-2,5-DMP 2,3-,3,4-,2,4-,2,6-,3,5-,2,5-dimethylphenol - 2-MBOH 2-methylbenzyl alcohol - 3-MBOH 3-methylbenzyl alcohol - 4-MBOH 4-methylbenzyl alcohol - m-,p-tol m-,p-toluate - o-,m-,p-xyl o-,m-,p-xylene     

Time courses of changes of para-, meta-, and ortho-tyrosine in septic patients: A pilot study

Objectives: Sepsis is associated with oxidative stress. Due to oxidative stress, three tyrosine isoforms, para-, meta-, and ortho-tyrosine (p-, m-, and o-Tyr), can be formed non-enzymatically in smaller amounts. p-Tyr is mainly formed physiologically in the kidneys through the activity of the phenylalanine hydroxylase enzyme. The three tyrosine isoforms may undergo different renal handling.

Methods: Twenty septic patients were involved in the study and 25 healthy individuals served as controls. Blood and urine levels of p-, m-, and o-Tyr were measured on admission and four consecutive days.

Results: Serum m-Tyr levels were higher in septic patients than in controls on days 2 (P = 0.031) and 3 (P = 0.035). Serum p-Tyr levels were lower in the cases than in controls on days 1 (P = 0.005) and 2 (P = 0.040), and subsequently normalized due to a day-by-day elevation (P = 0.002). The tendency of urinary m-Tyr concentration was decreasing (P = 0.041), while that of urinary p-Tyr concentration was increasing (P = 0.001). Fractional excretion of m-Tyr (FE_m-Tyr) showed a decreasing tendency (P = 0.009), and was, on all days, higher than FE_p-Tyr, which remained near-normal, less than 4%. Procalcitonin showed significant correlation with FE_m-Tyr (r = 0.454; P < 0.001).

Discussion: Our data suggest that the oxidative stress marker m-Tyr and physiologic p-Tyr may be handled differently in septic patients. The excretion of m-Tyr correlates with inflammation. m-Tyr may be actively secreted or produced in the kidney in some patients, whereas the decreased serum level of p-Tyr is a consequence of diminished renal production and not of renal loss.     

Antioxidation and Tyrosinase Inhibition of Polyphenolic Curcumin Analogs

A series of polyphenolic curcumin analogs were synthesized and their inhibitory effects on mushroom tyrosinase and the inhibition of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical formation were evaluated. The results indictated that the analogs possessing m-diphenols and o-diphenols exhibited more potent inhibitory activity on tyrosinase than reference compound rojic acid, and that the analogs with o-diphenols exhibited more potent inhibitory activity of DPPH free-radical formation than reference compound vitamin C. The inhibition kinetics, analyzed by Lineweaver–Burk plots, revealed that compounds B₂ and C₂ bearing o-diphenols were non-competitive inhibitors, while compounds B₁₁ and C₁₁ bearing m-diphenols were competitive inhibitors. In particular, representative compounds C₂ and B₁₁ showed no side effects at a dose of 2,000 mg/kg in a preliminary evaluation of acute toxicity in mice. These results suggest that such polyphenolic curcumin analogs might serve as lead compounds for further design of new potential tyrosinase inhibitors.     

Modified method for determination of hippuric acid and methylhippuric acid in urine by gas chromatography

A modified method for the simultaneous determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (o-, m- and p-MHAs) in urine is described. These metabolites were extracted, derivatized into their methyl ester derivatives and analyzed using a gas chromatograph equipped with flame ionization detector and a DB-1 capillary column. The derivatives of HA, o-, m- and p-MHAs were well separated within 11 min. The accuracy and precision in the present method were sufficient for quantitative analysis, and the results obtained by the GC method were highly correlated with those by the HPLC method (NIOSH 8301).     

N-Oxidation of arylamines to nitrosobenzenes using chloroperoxidase purified from Musa paradisiaca stem juice

Abstract

N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase purified from Musa paradisiaca stem juice has been examined. The enzymatic characteristics of the stem chloroperoxidase using 4-chloroaniline as substrate were determined. The K_m values for 4-chloroaniline and H₂O₂ were 770 μM and 154 μM respectively, while the pH and temperature optima were 4.4 and 30°C respectively. The substrate specificities of the enzyme for the arylamines 3,4-dichloroamine, p-aminobenzoic acid, p-toluidine, p-anisidine, m-anisidine, p-aminophenol, o-aminophenol and m-aminophenol have been characterized. The feasibility of using concentrated M. paradisiaca stem juice for the specific conversion of 4-chloroaniline to 4-chloronitrosobenzene has been demonstrated. This enzyme can be used for the N-oxidation of other arylamines.     

Kinetics of azide attack on organonitriles coordinated to pentaamminecobalt(III)

Four new 5-substituted tetrazole complexes of pentaamminecobalt(III) have been synthesized. The N-1 bonded complexes of 5-(p-methylphenyl)tetrazole, 5-(p-chlorophenyl)tetrazole, 5-(p-nitrophenyl)tetrazole and the N-2 bonded complex of 5-(m-formylphenyl)tetrazole. Kinetic studies of the complex formation reaction, wherein azide ion attacks coordinated organonitrile ligands, have been shown to follow a second order rate law, first order in nitrile complex and azide. Rate constants and activation parameters have been determined for five organonitrile complexes and a Hammett type correlation has been established for the formation reaction described by the equation log k = 2.42σ – 2.44 at 25 °C and ionic strength 1.0 M. Two mechanisms are proposed for complex formation which are consistent with our observations.     

New metabolic pathway for o-cresol degradation by Pseudomonas sp. CP4 as evidenced by 1H NMR spectroscopic studies

Degradation intermediates of o-, m- and p-cresols extracted from resting cells of Pseudomonas sp. CP4, a potent cresol- and phenol-degrading laboratory isolate, were analysed by using ¹H NMR spectroscopy at 270 MHz. Ortho-, meta- and para-cresols were found to be degraded to 2-methyl-4-oxalocrotonate. 3-Methylcatechol from o-cresol was degraded further to 2-ketohex-cis-4-enoate, 4-methylcatechol from m- and p-cresol was degraded to 2-ketohex-cis-4-enoate. Also 2-ketopent-4-enoate was found to be formed from p-cresol. Formation of 2-methyl-4-oxalocrotonate was envisaged as taking place from 5-hydroxy-2-methylmuconic semialdehyde, the ring-cleavage product of 4-methylresorcinol, a possible product by hydroxylation of o-cresol along with 3-methylcatechol. This is a deviation from the hitherto known pathways of o-cresol degradation. Based on these observations, pathways for the degradation of all three isomers of cresol are proposed.     

Novel Base-Labile Protecting Groups for 5′-Hydroxy Function in Solid-Phase Oligonucleotide Synthesis

Abstract

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile protection for the 5′-hydroxy function in solid-phase oligonucleotide synthesis. A comparative study of the LMMoNBz, LMMpNBz and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting groups for oligonucleotide synthesis proved strong feasibility for the LMMoNBz group.     

Substituent effects in the enantioselective reduction of acetophenones with nabh4 in the presence of β-cyclodextrin

The effect of substituent groups on asymmetric induction by β-cyclodextrin (β-CD) was investigated in the reduction of a series of o-, m-, and p-substituted acetophenones (X = H, Br, Cl, CH₃, NO₂, OCH₃) with aqueous NaBH₄. The inclusion of the ketones studied in β-CD led to water-insoluble compounds so that the reaction proceeded in the solid state. The substitutions resulted generally in higher enantioselectivities than that obtained for acetophenone indicating stronger host—guest interactions. Acetophenone and its m- and p-derivatives gave preponderantly the (?)-alcohol while the prevailing enantiomer was the (+)-alcohol in the case of the o-derivatives. The enantioface selectivity was found to be mainly governed by steric demands imposed by the size and the shape of the β-CD cavity in the case of the o-substituted acetophenones and by hydrophobic interactions in the case of the m-derivatives. A more complicated situation arose from the asymmetic reduction of p-derivatives where a combination of these factors with hydrogen bonding of the carbonyl group to the hydroxyls of β-CD are responsible for the enantioselectivity. © 1994 Wiley-Liss, Inc.     

The anaerobic biodegradation ofo-, m- andp-cresol by sulfate-reducing bacterial enrichment cultures obtained from a shallow anoxic aquifer

Summary Sulfate-reducing bacterial enrichments were obtained from a shallow anoxic aquifer for their ability to metabolize eithero-, m-, orp-cresol. GC/MS and simultaneous adaptation experiments suggested that the anaerobic decomposition ofp-cresol proceeds by the initial oxidation of the aryl methyl group to formp-hydroxybenzoic acid. This intermediate was then converted to benzoic acid. Benzoic acid and a hydroxybenzaldehyde were also found in spent culture fluids from ano-cresol-degrading enrichment culture. This result, in addition to others, suggested thato-cresol may also be anaerobically degraded by the oxidation of the methyl substituent. An alternate pathway for anaerobicm-cresol decomposition might exist. Enrichment cultures obtained with eitherp- oro-cresol degraded both of these substrates but notm-cresol. In contrast, am-cresol enrichment culture did not metabolize theortho orpara isomers. Anaerobic biodegradation in all enrichment cultures was inhibited by molybdate and oxygen, and was dependent on the presence of sulfate as a terminal electron acceptor. The stoichiometry of sulfate-reduction and substrate depletion by the various enrichment cultures indicated that the parent cresol isomers were completely mineralized. This result was confirmed by the conversion of¹⁴C-labeledp-cresol to¹⁴CO₂. These results help clarify the fate of alkylated aromatic chemicals in anoxic aquifers.     

Physicochemical Parameters for Structure-Activity-Studies of Substituted Phenyl N-Methylcarbamates

The electronic and hydrophobic substituent parameters of a number of o-, m- and p- substituted phenyl N-methylcarbamates were estimated by measuring the alkaline catalyzed hydrolysis rate constant and 1-octanol/water partition coefficient. Since some o-substituted derivatives are very potent insecticides, emphasis was placed on determining the ortho substituent effects in terms of the existing free-energy related parameters. The information obtained should be useful in structure-activity studies of carbamate insecticides.     

Microbial Models of Mammalian Metabolism: Aromatic Hydroxylation of Isomeric Xylenes

The ability of 11 microorganisms to convert o-, m-, and p-xylene to monophenolic metabolites was evaluated. Four organisms were found to produce qualitatively identical products to those observed with mammalian systems.     

Synthesis and biological evaluation of novel series of aminopyrimidine derivatives as urease inhibitors and antimicrobial agents

A novel series of carbazole substituted aminopyrimidines (5a-p) were synthesized and screened for their in vitro urease inhibition and antimicrobial activity. Among the compounds, 4-(2,4-dichlorophenyl)-6-(9-methyl-9H-carbazol-3-yl)-pyrimidin-2-amine (5i) was found to be the most potent showing urease inhibitory activity with an IC₅₀ value 19.4?±?0.43 µM. Compounds 5c, 5g, 5j and 5o showed good activity against all selected bacterial strains and compounds 5b, 5c, 5m and 5o showed good activity against selected fungal strains. All the compounds were subjected for ADME predictions by computational method.     

Phenol and cresol mixture degradation by the yeast <Emphasis Type="Italic">Trichosporon cutaneum</Emphasis>

Most industrial wastes contain different organic mixtures, making important the investigation on the microbial destruction of composite substrates. The capability of microbes to remove harmful chemicals from polluted environments strongly depends on the presence of other carbon and energy substrates. The effect of mixtures of phenol- and methyl-substituted phenols (o-, m-, p-cresol) on the growth behaviour and degradation capacity of Trichosporon cutaneum strain was investigated. The cell-free supernatants were analysed by HPLC. It was established that the presence of o-, m- and p- cresol has not prevented complete phenol assimilation but had significant delaying effect on the phenol degradation dynamics. The mutual influence of phenol and p-cresol was investigated. We developed the kinetic model on the basis of Haldane kinetics, which used model parameters from single-substrate experiments to predict the outcome of the two-substrate mixture experiment. The interaction coefficients indicating the degree to which phenol affects the biodegradation of p-cresol and vice versa were estimated. Quantitative estimation of interaction parameters is essential to facilitate the application of single or mixed cultures to the bio-treatment of hazardous compounds.     

Sulfonamide derivatives of thiazolidin-4-ones with anticonvulsant activity against two seizure models: synthesis and pharmacological evaluation

A series of 4-thiazolidinones bearing a sulfonamide group (4a–w) were prepared by cyclizing various 5-bromo-2-methoxy-N’-[(1E)-arylmethylene/arylethylidene]benzenesulfonohydrazides. All the compounds were characterized by IR, ¹H NMR, and elemental analysis. The compounds were tested for their anticonvulsant activity utilizing MES and scPTZ animal models. The majority of the compounds exhibited significant activity against both animal models; however, compounds 4c, 4m, and 4o displayed promising activity and could be considered as leads for further investigations.     

β-Galactosidase from Lactobacillus pentosus: Purification,characterization and formation of galacto-oligosaccharides

A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U_oNPG/mg protein. The K_m, k_cat and k_cat/K_m values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 38 mM, 20 s^-1, 530 M^-1·s^-1 and 1.67 mM, 540 s^-1, 325 000 M^-1·s^-1, respectively. The temperature optimum of β-galactosidase activity was 60–65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg²⁺ ions enhanced both activity and stability significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D -Glc, β-D-Galp-(1→3)-D -Glc, β-D -Galp-(1→6)-D -Gal, β-D -Galp-(1→3)-D -Gal, and the trisaccharides β-D -Galp-(1→3)-D -Lac, β-D -Galp-(1→6)-D -Lac.     

Degradation of monohydroxylated benzoates by strain KUFI-6N of the yeast-like fungus Exophiala jeanselmei

Strain KUFI-6N of Exophiala jeanselmei, a cyclohexanol-utilizing yeast-like fungus, was found to grow on 3 isomers of hydroxybenzoate that functioned as the sole carbon sources. Distinct and highly specific hydroxylases converted p- and m-hydroxybenzoate to protocatechuate and o-hydroxybenzoate to catechol.     

Leucine aminopeptidase (bovine lens): Synthesis and kinetic properties of ortho-, meta-, and para-substituted leucyl-anilides

The synthesis of a number of leucyl derivatives of substituted anilides and their properties as substrates and inhibitors of Zn²⁺-Mg²⁺ leucine aminopeptidase (EC 3.4.11.1) at pH 8.5 and 30 °C are described. The compounds include leucyl-X where X is o-, m-, or p-aminobenzenesulfonic acid, o-, m-, or p-anisidine, and m- or p-aminobenzenesulfonyl fluoride. The latter two sulfonyl fluorides, designed to be active site-directed irreversible inhibitors, turned out to be good substrates for leucine aminopeptidase. The K_m and V values of the above compounds as substrates for leucine aminopeptidase are reported. N-Leucyl-m-aminobenzenesulfonate exhibits desirable properties (solubility much greater than K_m, Δ? at 295 nm of 2000 m^?1 cm^?1, and V of 300 μmol min^?1 mg^?1) as a substrate for a spectrophotometric assay of leucine aminopeptidase. With the exception of N-leucyl-p-aminobenzenesulfonate, all of the above compounds are inhibitors of the hydrolysis of leucyl-p-nitroanilide by leucine aminopeptidase with K_i values approximately their K_m values when they are used as substrates. Despite wide variability in steric bulk, chemical composition, and electrical charge of the substituted anilides, the K_m values of the above compounds vary over a narrow range (0.5 to 4.8 mm), which indicates that the leucyl moiety plays the predominant role in the determination of K_m values. Although the K_m values of m- substituents are similar to those of o- substituents, the V values for m-substituents are much greater than those for o- substituents, which suggests that o-substituents interfere with the catalytic process. N-Leucyl-p-aminobenzenesulfonate and N-alanyl-p-aminobenzenesulfonate as well as the nonsubstrate p-aminobenzenesulfonate stimulate rather than inhibit the proteolysis of leucyl-p-nitroanilide. The stimulation has no effect on V but lowers the K_m for the hydrolysis of leucyl-p-nitroanilide, which is compatible with these compounds' serving as nonessential activators.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.     

Metabolism of nitrophenols by bacteria isolated from parathion-amended flooded soil

Two bacterial isolates from parathion-amended flooded soil, Pseudomonas sp. and Bacillus sp., were examined for their ability to decompose nitrophenols. Uniformly labelled ¹⁴C-p-nitrophenol was metabolized by both bacteria, ¹⁴CO₂ and nitrite being end products. A substantial portion (23% for Pseudomonas sp. and 80% for Bacillus sp.) of radioactivity applied as p-nitrophenol was accounted for as ¹⁴CO₂ at the end of a 72-h period; 8 to 16% remained in the water phase after solvent extraction. Pseudomonas sp. produced nitrite also from 2,4-dinitrophenol, but only after a lag, and not from o- and m-nitrophenols. Interestingly, m-nitrophenol, known for its resistance to biodegradation because of meta substitution, was decomposed by Bacillus sp., resulting in the formation of nitrite and phenol; o-nitrophenol and 2,4-dinitrophenol resisted degradation by this bacterium.