Physiological relevance of successive hydroxylations of toluene by toluene para-monooxygenase of Ralstonia pickettii PKO1

We have recently found that toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1 (encoded by tbuA1UBVA2C) performs successive hydroxylations of benzene (Appl. Environ. Microbiol. 70: 3814, 2004) as well as hydroxylates toluene to a mixture of 90% p-cresol and 10% m-cresol which are then further oxidized to 100% 4-methylcatechol (J. Bacteriol. 186: 3117, 2004) whereas it was thought previously that TpMO forms 100% m-cresol and is not capable of successive hydroxylations. Here we propose a modification of the degradation pathway originally described by Olsen et al. (J. Bacteriol. 176: 3749, 1994) that now relies primarily on TpMO for conversion of toluene to 4-methylcatechol (instead of m-cresol) since both m-cresol and p-cresol are shown here to be good substrates for Escherichia coli expressing TpMO (V_max/K_m=0.046, 0.036, and 0.055 mL min^-1 mg^-1 protein for the oxidation of toluene, m-cresol, and p-cresol, respectively). In light of the broader activity of TpMO, phenol hydroxylase (encoded by tbuD) appears to facilitate conversion of any m-cresol or p-cresol formed from toluene oxidation by TpMO to 4-methylcatechol; hence, the cell has a redundant method for making this important intermediate 4-methylcatechol. Further, it is suggested that the physiological relevance of the 10% m-cresol formed from toluene oxidation by TpMO is needed for induction of the meta cleavage operon tbuWEFGKIHJ to enable full metabolism of toluene since p-cresol (and o-cresol) do not induce the meta-cleavage pathway. Therefore both the successive hydroxylation of toluene by TpMO and the product distribution are of physiological relevance to the cell.     

Polyphenolases in the 1000g fraction of Papaver somniferum latex

A polyphenolase complex was isolated from the 1000-g organelle fraction of the latex of the opium poppy. This enzyme oxidized a variety of phenolic substrates, including p-cresol, catechol, p-coumaric acid, hydroquinone and tyrosine, but would not oxidize the opium alkaloid intermediates reticuline and salutaridinol. The activity of the enzyme complex was enhanced by techniques which involved the mechanical damage of the organelles and by solubilization of the organelle membrane with the detergent Triton X-100. The enzyme activity was inhibited by KCN and DIECA.     

Incorporation of p-cresol into lignins via peroxidase-catalysed copolymerization in nonaqueous media

Lignin, the second most abundant biopolymer after cellulose, is a low value by-product of agricultural and wood conversion processes, including wood pulp manufacture. Copolymerization with phenols has the potential to convert by-product lignins to higher-value phenolic resins. In this initial investigation, we have studied the use of horseradish peroxidase (HRP) in aqueous dioxane to catalyse the grafting of p-cresol (p-methylphenol) onto milled wood lignin, kraft lignin, and a lignin selectively o-demethylated by a brown-rot fungus. Advantages of this system are (1) the mild reaction conditions employed and (2) the ability of HRP to function in the dioxane: water solutions which solubilize lignin. The reaction is monitored by gel permeation chromatography using a reaction system of [¹⁴C]-p-cresol with unlabeled lignins. We have found that optimal incorporation of cresol into high-molecular-weight polymer occurs at 50–70% dioxane in water under the conditions used; a maximum incorporation of ca. 4 mol% (e.g., p-cresol incorporated per C₉ lignin unit) was obtained. Blocking the phenolic hydroxyl groups of the lignin inhibits copolymerization, consistent with the proposed mechanism of phenoxy radical copolymerization for this reaction.     

Plasmalemma-mitochondrial complexes involved in water transport in the hindgut of a milliped,Scaphiostreptus sp.

Summary Plasmalemma-mitochondrial complexes (PMC) are known to be very active in water transport in different tissues. The PMC in the hindgut cells of the milliped, Scaphiostreptus sp., differ from those found in other transporting epithelia of hindguts; they are closely connected with cisternae of the endoplasmic reticulum. This arrangement is thought to be very efficient in ion and water transport.     

The chemistry of the abdominal gland secretion of six species of the rove beetle genus Bledius

The chemistry of the abdominal defensive secretions of six species of the rove beetle genus Bledius was examined. In all species the secretion contains the solid toxin p-toluquinone and its precursor p-toluhydroquinone dissolved in different solvents. In B. furcatus, B. tricornis and B. dissimilis these solvents are mainly alkenes and lactones, especially 1-undecene and γ-dodecalactone. In addition to alkenes and lactones the secretion contains decanoic acid and 3-methyl-2-butenoic acid in B. opacus and B. subterraneus. In B. arenarius the quinones are dissolved in octanoic acid and octyloctanoate, exclusively. In B. arenarius and B. opacus the secretion's content of p-toluquinone is reduced in comparison with the other species. Application experiments with the natural predators revealed that in B. arenarius this quinone reduction does not involve a reduction of irritation efficiency. This is probably due to the irriation properties of the B. arenarius solvent octanoic acid. The results are discussed with respect to the evolution of the different secretion types within the genus Bledius and within the subfamily Oxytelinae.     

Micellar catalysis of polyphenol oxidase in AOT/cyclohexane

The catalytic behaviour of mushroom polyphenol oxidase has been studied in dioctylsulphosuccinate (AOT)/cyclohexane reverse micelles. The steady-state conditions were accomplished up to 20 min and 17 μg protein in the assay towards 4-methylcatechol and no loss of specific activity was observed relative to aqueous medium. The pH activity profile of the enzyme was kept in reverse micelles as in water, showing a plateau between 5 and 6.5. The stability of polyphenol oxidase to pH was also studied and about 20% inactivation was found in reverse micelles relative to aqueous medium at neutral pHs. Moreover there was a decrease of stability at acidic pHs. The optimum W_o obtained was 20 and the enzyme was nearly independent of the surfactant concentration at constant W_o.

Kinetic studies of polyphenol oxidase towards several substrates showed that the substrate inhibition by p-cresol and 4-methylcatechol observed in buffer was not kept in AOT/cyclohexane reverse micelles. Moreover, the K_m increased and the catalytic efficiency (V/K_m) of the enzyme decreased as the hydrophobicity of substrates was increased.     

Biohydroxylation of N,N-dialkylarylamines by the isolated topsoil bacterium Bacillus megaterium

Topsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and was identified as Bacillus megaterium by 16S rDNA analysis and DNA/DNA-hybridization. In contrast to the well-known C-hydroxylation by liver microsomes, leading to p-hydroxylation, B. megaterium formed o- and p-monohydroxylated products, i.e. N,N-dimethyl-2-aminophenol and N,N-dimethyl-4-aminophenol, both identified by gas chromatography–mass spectrometry (GC–MS) using synthesized reference compounds. The observed hydroxylation showed slight regioselectivity in favour of the o-hydroxylated product. Two further substrates, N,N-diethylaniline and N-ethyl-N-methylaniline, were also successfully biohydroxylated by B. megaterium with corresponding regioselectivity. Interestingly, aniline, known to be transformed easily by cytochrome P-450meg into p-aminophenol, was not accepted as substrate.     

Oxidation of both termini of p- and m-xylene by Escherichia coli transformed with xylene monooxygenase gene

Xylene monooxygenase (XMO) from Pseudomonas putida mt-2 catalyzes oxidation of methyl group of toluene and xylenes. While it has been postulated that this enzyme oxidizes one methyl group of xylene, we observed that both methyl groups in p- and m-xylene were oxidized to alcohol and aldehyde when the relevant genes (xylM and xylA) were co-expressed in Escherichia coli C600 and MC4100. When p-xylene was used as a substrate, p-hydroxymethylbenzaldehyde and p-xylyleneglycol were identified, in addition to p-methylbenzylalcohol and p-tolualdehyde. When m-xylene was used as a substrate, m-hydroxymethylbenzaldehyde and m-xylyleneglycol were identified, in addition to m-methylbenzylalcohol and m-tolualdehyde. Ratio of the products varied significantly according to the reaction condition and host strain, presumably reflecting the relative activity of XMO and host-derived dehydrogenase(s). Using various oxidized compounds as substrates, it was indicated that dialcohol (p- or m-xylyleneglycol) was formed via p- or m-hydroxymethylbenzaldehyde, respectively, rather than directly from corresponding monoalcohol (p- or m-methybenzylalcohol).     

Purification and properties of lipase from a Bacillus strain for catalytic resolution of (R)-Naproxen

A Bacillus strain was screened for asymmetric resolution of (R)-Naproxen. The optical purity (ee (%)) of (R)-Naproxen was found to be 86.47% and conversion rate was 40–50% in bacterial cells PBS reaction system. The dissolved lipase was clarified from the Bacillus bacterial cells by centrifugation and loaded on a phenyl-Sepharose CL-4B column. After purification by a single hydrophobic chromatography, the activity of lipase was approximately 43 times higher than the crude one. The hydrolytic activity of lipase using Naproxen ethyl ester and p-nitrophenyl acetate (p-NPA) as substrate remained essentially constant during the purification procedure. A Bacillus strain with stereochemical selectivity was obtained.     

Chlorogenic acid modifies plasma and liver concentrations of: cholesterol,triacylglycerol, and minerals in (fa/fa) Zucker rats

Chlorogenic acid, a phenolic compound found ubiquitously in plants, is an in vitro antioxidant and metal chelator. Some derivatives of chlorogenic acid are hypoglycemic agents and may affect lipid metabolism. Concentrations of cholesterol and triacylglycerols are of interest due to their association with diseases such as non-insulin-dependent-diabetes- mellitus and obese insulin resistance. As little is known about the effects of chlorogenic acid in vivo, studies using obese, hyperlipidemic, and insulin resistant (fa/fa) Zucker rats were conducted to test the effect of chlorogenic acid on fasting plasma glucose, plasma and liver triacylglycerols and cholesterol concentrations. Aditionally, the effects of chlorogenic acid on selected mineral concentrations in plasma, spleen, and liver were determined. Rats were implanted with jugular vein catheters. Chlorogenic acid was infused (5 mg/Kg body weight/day) for 3 weeks via intravenous infusion. Chlorogenic acid did not promote sustained hypoglycemia and significantly lowered the postprandial peak response to a glucose challenge when compared to the same group of rats before Chlorogenic acid treatment. In Chlorogenic acid-treated rats, fasting plasma cholesterol and triacylglycerols concentrations significantly decreased by 44% and 58% respectively, as did in liver triacylglycerols concentrations (24%). We did not find differences (p > 0.05) in adipose triacylglycerols concentration. Significant differences (p < 0.05) in the plasma, liver, and spleen concentration of selected minerals were found in chlorogenic acid-treated rats. In vivo, chlorogenic acid was found to improve glucose tolerance, decreased some plasma and liver lipids, and improve mineral pool distribution under the conditions of this study.     

Proteases released by Lucilia cuprina during egg hatch

Products released by Luclia cuprina (sheep blowfly) during egg hatch and early larval development were found to contain a variable number of proteases, probably reflecting their different functions during these developmental phases. Moreover, a number of the developmentally regulated proteases produced only during egg hatch appeared to be strongly egg shell associated, indicating the egg shell proteins to be their specific substrates. Several proteases were tested for their ability to enhance the rate of egg hatch. The commercial proteases chymotrypsin and trypsin were able to significantly enhance (p≤0.01) egg hatch by 36% and 44%, respectively, when compared to an untreated control. Interestingly, fluids containing the egg shell associated proteases were able to significantly (p≤0.001) enhance L. cuprina egg hatch by up to 70%. This hatch enhancement activity could be significantly (p≤0.001) reversed by the addition of Phenylmethylsulphonyl fluoride (PMSF). Fractionation of the egg-associated proteases by gelatin affinity chromatography suggested that gelatin-binding molecules were responsible for the majority of the egg hatch enhancement activity in this preparation.     

A novel esterase from a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, that belongs to the amidase signature family

A novel esterase that belongs to the amidase signature family was found in a psychrotrophic bacterium, Acinetobacter sp. strain no. 6, isolated from Siberian soil. The gene coding for the esterase, named EstA8, was cloned, and an open reading frame of 1488 bp corresponding to 496 amino acid residues was identified. EstA8 showed 30% sequence identity with 6-aminohexanoate-cyclic-dimer hydrolases from Pseudomonas sp. strain NK87 and Flavobacterium sp. strain K172, which degrade a by-product of the nylon-6 industry. EstA8 was overproduced in Escherichia coli JM109 under the control of the lac promoter of pUC118 and purified. Consistent with the fact that the source microorganism is cold-adapted, the enzyme was unstable at moderate temperatures. It lost 75% of its original activity by incubation at 40 °C for 30 min. Despite its structural similarity to 6-aminohexanoate-cyclic-dimer hydrolase, 6-aminohexanoate cyclic dimer did not serve as the substrate. EstA8 is a member of the amidase signature family, but its esterase activity toward p-nitrophenyl esters, such as p-nitrophenyl acetate, was much higher than its amidase activity toward p-nitroanilides, such as p-nitroacetanilide.     

Chemical heterogeneity of soldier defensive secretions in the desert subterranean termite, Amitermes wheeleri

Soldier defensive secretions were analyzed by GC/MS in eight spatially separated groups of Amitermes wheeleri collected in Arizona and California. Eleven sesquiterpenoids, four of known structure, were isolated. Quantitative and qualitative differences among the groups of termites were extensive; composition of the defensive secretions among colonies varied from one to six components. Intraspecific differences between pairs of sympatric colonies at six sites were much less pronounced. The variability found in A. wheeleri suggests that soldier defensive secretions in the genus Amiterines are not reliable markers for interspecific systematic comparisons.     

Deracemization of aryl ethanols and reduction of acetophenones by whole fungal cells of Aspergillus terreus CCT 4083, A. terreus CCT 3320 and Rhizopus oryzae CCT 4964

The enantioselective deracemization of a number of p-substituted aryl ethanols and the reduction of p-substituted acetophenones were carried out with whole fungal cells of Aspergillus terreus CCT 4083, A. terreus CCT 3320 and Rhizopus oryzae CCT 4964 giving the corresponding alcohols in enantiomeric excesses up to >99%.     

Carbon,nitrogen and phosphorus stoichiometry of Myrica nana in Yunnan,China

Aims Carbon (C), nitrogen (N) and phosphorus (P) play important roles in plant growth and physiological functions. We aimed at exploring the intrinsic relationships of C, N and P in Myrica nana—a common shrub in Yunnan Province—as well as their relationships with pant biomass and soil nutrients.
Methods We measured the concentration of C, N and P of M. nana from 29 sites for their magnitudes and correlations with soil nutrients.
Important findings 1) The arithmetic mean value of C, N and P concentration in the roots, stems and leaves of M. nana was 45.94%, 0.54%, 0.03%, and 46.32%, 0.58%, 0.03%, and 49.05%, 1.70%, 0.06%, respectively. C, N and P concentrations in the leaves were significantly higher than those in the roots and the stems. The C:N:P in roots, stems and leaves was 1531:18:1, 1544:19:1, and 818:10:1, respectively. 2) The C concentration and N:P in leaves of M. nana decreased with the increase of biomass of M. nana; the leaf C concentration was significantly correlated with biomass (p < 0.01), while the correlation between N:P and biomass was not significant (p > 0.05). The leaf N increased with the increase of plant biomass, the P was significantly correlated with biomass (p < 0.05), but the correlation between N concentration and biomass was not significant (p > 0.05). N:P in leaves was 34.2, suggesting that plant growth was limited by P. 3) C, N and P concentration in the roots were significantly correlated with soil P (p < 0.05), with N, P concentrations correlated with soil P positively (p < 0.01) and C negatively (p < 0.05). C concentration in the stems was significantly and negatively correlated with soil C, N, with significant correlation with C, N, and P concentration (p < 0.01). P concentration in the stems was significantly and positively correlated with soil P concentration (p < 0.01), while leaf P significantly and positively correlated with soil C, N and P (p < 0.01); leaf C concentration was significantly and negatively correlated with soil P (p < 0.01).     

The Influence on Survival of Season of Onset of Childhood Acute Lymphoblastic Leukemia (All)

Rhythmicity has been found to be an important characteristic of leukocytes. We tested, therefore, whether rhythmicity could be found to be reflected in one or more of the developmental stages of childhood acute lymphoblastic leukemia. The patient material comprised 205 children 96 girls and 109 boys) whose disease was diagnosed in Israel during the years 1976-1981. Their mean age was 5.9 years ± 3.94. No seasonal onset of disease was found, either in the whole group or in subgroups divided by sex or lymphocyte surface markers. Survival analyses however, revealed that the season of diagnosis influenced survival. Excluded from the study were 9 cases (4.4%) due to lack of some items. The follow-up period was between 1 and 72 months (median 26 months). It appeared that the life table curves were running in their temporal order, each following curve representing a consecutive 3-month period. The difference between the curves was statistically significant (p = 0.025/0.009). The survival percentages at the end of the follow-up increased steadily, from a trough during November-January (56.5%) to a peak in August-October (80.0%). Analysis by sex and white blood cell (WBC) count showed that the difference between the life table curves was primarily due to girls presenting less than 50,000 cells per cumm (n = 73, p = 0.005/0.0028). The survival percentages in the same order as mentioned earlier were: 47.1%, 68.4%, 89.5% and 94.4%. The steady rise in the different female time series suggests a circannual pattern. In the male series no temporal order could be detected nor did the difference between seasonal survival reach a level of significance. The significance of the participation of natural environmental factors in causing the seasonal variations in malignancy is discussed.     

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.     

Phylogeny of the millipede genus Sphaeriodesmus Peters, 1864 (Polydesmida: Sphaeriodesmidae) based on morphological characters

In order to understand the evolutionary relationships among the species encompassed within the genus Sphaeriodesmus Peters, 1864, a cladistic analysis including 63 species was conducted. Ninety-five morphological characters were used for the phylogenetic reconstruction. The results suggested that the current composition of the genus Sphaeriodesmus does not circumscribe a monophyletic group; instead, the genera Eusphaeriodesmus, Colobodesmus, and Proeilodesmus are here synonymized under Sphaeriodesmus. Although raw morphological data had suggested the genus Lophocyclus as the sister taxon of Sphaeriodesmus, the phylogenetic analysis under implied weight identified the genus Cyphodesmus as the taxon most closely related to Sphaeriodesmus. Sphaeriodesmus isolatus Chamberlin, 1940 is a subjective synonym of Sphaeriodesmus conformans Chamberlin, 1925. The putative subdivisions previously proposed within Sphaeriodesmus do not hold as monophyletic either. Low stability was observed concerning the higher-level phylogenetic relationships of Sphaeriodesmus. Sphaeriodesmus crucis (Loomis, 1974), S. mecistonyx (Hoffman, 1990), and S. triramus (Kraus, 1954) are new combinations.     

Elevated breath pentane in heart failure reduced by free radical scavenger

Pentane, a product of lipid peroxidation, has been detected in situations involving ischemic injury. Such injury may be limited if lipid peroxidation can be controlled by antioxidants. The role of lipid peroxidation in chronic heart failure (CHF) was assessed by measuring breath pentane in patients with CHF vs. age matched controls. The effect of a free radical scavenger on pentane released during CHF was also measured. Pentane levels were correlated with the daily dose of captopril, a sulfhydril-containing drug used to treat CHF, which is an angiotensin converting enzyme inhibitor. To separate the scavening effects of captopril from the pharmacologic effects of converting enzyme inhibitors, a crossover study using a nonsulfhydril inhibitor was used. Patients with CHF excreted (p < 0.005) hich concentrations of pentane (5.7 ± 2.1 vs. control 3.6 ± 1.2 nmol/l). Patients treated with captopril also had significantly higher (p < 0.05) excretion of pentane than the control patients (4.7 ± 1.3 vs. 3.6 ± 1.2 nmol/l). The dose of captopril was inversely proportional to the concentration of pentane excreted (r = 0.55, p < 0.05). Pentane excretion during captopril therapy was significantly lower before (p < 0.01) and after (p < 0.02) nonsulfhydril inhibitor therapy. Conclusion: breath pentane is elevated in CHF and it can be reduced by a free radical scavenger. This reduction of pentane excretion is not a converting enzyme inhibitor class effect.     

Tree size and flowering intensity as affected by nitrogen fertilization in non-bearing orange trees grown under Mediterranean conditions

A field experiment was conducted in the South of Portugal with young (0-3 years old) non-bearing ‘Lane Late’ orange trees. Five nitrogen (N) rates were used in a randomised block design with three replicates. The 180 g N tree⁻¹ over three years led to the greatest canopy width (176 cm) and volume (2,697 dm³). The greatest rate applied (720 g N tree⁻¹ in the three years) led to the largest flower yield. Nitrogen concentration in the flowers significantly increased with fertilizer N, and also with the flowering period up to the 23^rd day, declining thereafter. Flower yield was strongly correlated (r = 0.99, p < 0.001) with flower N concentration. Nutrient composition of flowers and of mature leaves from the spring flush was compared. Significant correlations were found for N (r = 0.47, p <0.01), P (r = -0.49, p <0.01), K (r = 0.44, p <0.05) and Ca (r = 0.87, p <0.001), suggesting that flowers can be used as a tool to diagnose the nutritional status of trees. Canonical analysis (with N treatment as dummy-variables) showed strong relationships between canopy width and N, which were greater at the larger rates of fertilizer application, and strong and inverse relationships between K and Mg, also with the greatest N rates.