Cytochrome P-450-catalyzed desaturation of valproic acid in vitro. Species differences, induction effects, and mechanistic studies

The cytochrome P-450-mediated desaturation of valproic acid (VPA) to its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid (4-ene-VPA), was examined in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnover was found with microsomes from rabbits (44.2 +/- 2.7 pmol of product/nmol P-450/15 min), while lower activities were observed in preparations from human, mouse, and rat liver, in that order. Pretreatment of animals with phenobarbital led to enhanced rates of formation of 4-ene-VPA in vitro and yielded induction ratios for desaturation ranging from 2.5 to 8.4, depending upon the species. Comparative studies in the rat showed that phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The mechanism of the desaturation reaction was studied by inter- and intramolecular deuterium isotope effect experiments, which demonstrated that removal of a hydrogen atom from the subterminal C-4 position of VPA is rate limiting in the formation of both 4-ene- and 4-hydroxy-VPA. Hydroxylation at the neighboring C-5 position, on the other hand, was highly sensitive to deuterium substitution at that site, but not to deuteration at C-4. Based on these findings, it is proposed that 4-ene- and 4-hydroxy-VPA are products of a common P-450-dependent metabolic pathway, in which a carbon-centered free radical at C-4 serves as the key intermediate. 5-Hydroxy-VPA, in contrast, derives from an independent hydroxylation reaction.     

The metabolic sulfonation and side-chain oxidation of 3'-hydroxyisosafrole in the mouse and its inactivity as a hepatocarcinogen relative to 1'-hydroxysafrole

The chemically synthesized sulfuric acid esters of 1'-hydroxysafrole and 3'-hydroxyisosafrole, 1'-sulfooxysafrole and 3'-sulfooxyisosafrole, respectively, are both strong electrophiles. Each ester reacted with deoxyguanosine (dGuo) in aqueous solution to form both safrol-1'-yl- and isosafrol-3'-yl-deoxyguanosine adducts. Both 1'-hydroxysafrole and 3'-hydroxyisosafrole were also formed from each ester in the presence of water. When either 1'-[3H]hydroxysafrole or 3'-[3H]hydroxyisosafrole was incubated with mouse liver cytosols fortified with 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and RNA, similar levels of RNA- and protein-bound adducts were formed; thus, the hepatic sulfotransferase activities for these two substrates appear to be similar. In contrast, the levels of hepatic nucleic acid and protein adducts formed after administration of 3'-[3H]hydroxyisosafrole to mice were only 2-4% and 8-14%, respectively, of those obtained after an equimolar dose of 1'-[3H]hydroxysafrole. Likewise, when 3'-hydroxyisosafrole was injected into 12-day-old male B6C3F1 mice at a level of 0.1 or 2.5 mumol/g body wt., the average numbers of hepatomas per mouse (0.2 and 0.4, respectively) were not significantly increased over the average number for mice treated only with the solvent (0.2). By contrast, mice that received 0.1 mumol of 1'-hydroxysafrole/g body wt. developed about 2 hepatomas per mouse. The metabolism of 3'-hydroxyisosafrole in the rat and mouse differed markedly from that of 1'-hydroxysafrole. 3'-Hydroxyisosafrole rapidly underwent side-chain oxidation to yield 3,4-methylenedioxycinnamic acid and 3,4-methylenedioxybenzoic acid. In the first 4 h, 3,4-methylenedioxybenzoyl glycine and 3,4-methylenedioxycinnamoyl glycine, the major urinary metabolites, together accounted for 39% and 63% of the dose administered to rats and mice, respectively. The glucuronide of 3'-hydroxyisosafrole was not detected in the urine, whereas urinary excretion of the glucuronide of 1'-hydroxysafrole at 2 h accounted for approx. 40% of a dose of 1'-hydroxysafrole.     

Formation of C21 bile acids from plant sterols in the rat

Formation of bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of 4-14C-labeled sitosterol and sitosterol labeled with 3H in specific positions. The major part (about 75%) of the 14C radioactivity recovered as bile acids in bile after intravenous administration of [4-14C]sitosterol was found to be considerably more polar than cholic acid, and only trace amounts of radioactivity had chromatographic properties similar to those of cholic acid and chenodeoxycholic acid. It was shown that polar metabolites were formed by intermediate oxidation of the 3 beta-hydroxyl group (loss of 3H from 3 alpha-3H-labeled sitosterol) and that the most polar fraction did not contain a hydroxyl group at C7 (retention of 3H in 7 alpha,7 beta-3H2-labeled sitosterol). Furthermore, the polar metabolites had lost at least the terminal 6 or 7 carbon atoms of the side chain (loss of 3H from 22,23-3H2- and 24,28-3H2-labeled sitosterol). Experiments with 3H-labeled 7 alpha-hydroxysitosterol and 4-14C-labeled 26-hydroxysitosterol showed that none of these compounds was an efficient precursor to the polar metabolites. By analysis of purified most polar products of [4-14C] sitosterol by radio-gas chromatography and the same products of 7 alpha,7 beta-[2H2]sitosterol by combined gas chromatography-mass spectrometry, two major metabolites could be identified as C21 bile acids. One metabolite had three hydroxyl groups (3 alpha, 15, and unknown), and one had two hydroxyl groups (3 alpha, 15) and one keto group. Considerably less C21 bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. The C21 bile acids formed in male rats did not contain a 15-hydroxyl group. Conversion of a [4-14C]sitosterol into C21 bile acids did also occur in adrenalectomized and ovariectomized rats, indicating that endocrine tissues are not involved. Experiments with isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids occurs in this organ. Intravenously injected 7 alpha,7 beta-3H-labeled campesterol gave a product pattern identical to that of 4-14C-labeled sitosterol. Possible mechanisms for hepatic conversion of sitosterol and campesterol into C21 bile acids are discussed.     

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil,shale oil or diesel fuel

The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.     

Fungal disease incidence along tree diversity gradients depends on latitude in European forests

European forests host a diversity of tree species that are increasingly threatened by fungal pathogens, which may have cascading consequences for forest ecosystems and their functioning. Previous experimental studies suggest that foliar and root pathogen abundance and disease severity decrease with increasing tree species diversity, but evidences from natural forests are rare. Here, we tested whether foliar fungal disease incidence was negatively affected by tree species diversity in different forest types across Europe. We measured the foliar fungal disease incidence on 16 different tree species in 209 plots in six European countries, representing a forest‐type gradient from the Mediterranean to boreal forests. Forest plots of single species (monoculture plots) and those with different combinations of two to five tree species (mixed species plots) were compared. Specifically, we analyzed the influence of tree species richness, functional type (conifer vs. broadleaved) and phylogenetic diversity on overall fungal disease incidence. The effect of tree species richness on disease incidence varied with latitude and functional type. Disease incidence tended to increase with tree diversity, in particular in northern latitudes. Disease incidence decreased with tree species richness in conifers, but not in broadleaved trees. However, for specific damage symptoms, no tree species richness effects were observed. Although the patterns were weak, susceptibility of forests to disease appears to depend on the forest site and tree type.     

Different Methods Yielded Two-Fold Difference in Compliance with Physical Activity Guidelines on School Days

Introduction

The aim was to compare the average and the days method in exploring the compliance of children with physical activity guidelines and describe their physical activity patterns in different school day segments.

Methods

Physical activity was objectively measured in 472 children aged 6–13 for one school week. Children were compliant when fulfilling PA recommendations 1) on average over all measured days (average method) or 2) on at least four measured days (days method). To explore the difference in moderate to vigorous physical activity (MVPA) minutes between compliant and non-complaint children (using both the average and days method) in various day segments, linear mixed models was used.

Results

Compliance with physical activity guidelines was significantly higher with the average compared to the days method (51.7% and 23.7%, respectively). In segmented-day analysis, compliant children accrued more MVPA minutes in all day segments, especially during after-school. Gender differences appeared only during the in-school segments, where girls spent less time in MVPA (average method: -4.39 min, 95% CI = -5.36,-3.42, days method: -4.45 min, 95%CI = -5.46,-3.44). Older children accrued more MVPA minutes during physical education classes, but less during breaks, compared to younger children.

Conclusions

The used methods yielded remarkably different prevalence estimates for compliance to physical activity recommendations. To ensure comparability between studies, interventions and reports, there is a need for internationally agreed operationalization and assessment methods of physical activity guidelines. As non-compliant children had lower MVPA during all day segments, greater efforts should be made to provide physical activity opportunities both during and after school.     

Dieback of riparian alder caused by the Phytophthora alni complex: projected consequences for stream ecosystems

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Expression and characterization of recombinant 2',5'-oligoadenylate synthetase from the marine sponge Geodia cydonium

2',5'-oligoadenylate (2-5A) synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A synthetase/bacterial RNA complex activity.     

2′-phosphodiesterase and 2′,5′-oligoadenylate synthetase activities in the lowest metazoans,sponge [porifera]

Sponges [porifera], the most ancient metazoans, contain modules related to the vertebrate immune system, including the 2′,5′-oligoadenylate synthetase (OAS). The components of the antiviral 2′,5′-oligoadenylate (2–5A) system (OAS, 2′-Phosphodiesterase (2′-PDE) and RNAse L) of vertebrates have not all been identified in sponges. Here, we demonstrate for the first time that in addition to the OAS activity, sponges possess a 2′-PDE activity, which highlights the probable existence of a premature 2–5A system. Indeed, Suberites domuncula and Crella elegans exhibited this 2–5A degrading activity. Upon this finding, two out of three elements forming the 2–5A system have been found in sponges, only a endoribonuclease, RNAse L or similar, has to be found. We suspect the existence of a complex immune system in sponges, besides the self/non-self recognition system and the use of phagocytosis and secondary metabolites against pathogens.