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Examines changes in the Swedish public's preferences and behavior with regard to forest recreation, by comparing studies conducted in 1977 and 1997. Frequency of forest visits; Decrease in the amount of wild berries picked for home consumption; Change in public use from harvesting to purely recreational; Decrease in the conflict between recreational and biodiversity goals in forestry.

Manganese lipoxygenase is secreted by the fungus Gaeumannomyces graminis. We expressed the enzyme in Pichia pastoris, which secreted approximately 30 mg Mn-lipoxygenase/L culture medium in fermentor. The recombinant lipoxygenase was N- and O-glycosylated (80-100 kDa), contained approximately 1 mol Mn/mol protein, and had similar kinetic properties (K(m) approximately 7.1 microM alpha-linolenic acid and V(max) 18 nmol/min/microg) as the native Mn-lipoxygenase. Mn-lipoxygenase could be quantitatively converted, presumably by secreted Pichia proteases, to a smaller protein (approximately 67 kDa) with retention of lipoxygenase activity (K(m) approximately 6.4 microM alpha-linolenic acid and V(max) approximately 12 nmol/min/microg). Putative manganese ligands were investigated by site-directed mutagenesis. The iron ligands of soybean lipoxygenase-1 are two His residues in the sequence HWLNTH, one His residue and a distant Asn residue in the sequence HAAVNFGQ, and the C-terminal Ile residue. The homologous sequences of Mn-lipoxygenase are H274VLFH278 and H462HVMN466QGS, respectively, and the C-terminal amino acid is Val-602. The His274Gln, His278Glu, His462Glu, and the Val-602 deletion mutants of Mn-lipoxygenase were inactive, and had lost >95% of the manganese content. His-463, Asn-466, and Gln-467 did not appear to be critical for Mn-lipoxygenase activity, as His463Gln, Asn466Gln, Asn466Leu, and Gln467Asn mutants metabolized alpha-linolenic acid to 11- and 13-hydroperoxylinolenic acids. We conclude that His-274, His-278, His-462, and Val-602 likely coordinate manganese.

A proteomics approach was used to identify liver proteins that displayed altered levels in mice following treatment with a candidate drug. Samples from livers of mice treated with candidate drug or untreated were prepared, quantified, labeled with CyDye DIGE Fluors, and subjected to two-dimensional electrophoresis. Following scanning and imaging of gels from three different isoelectric focusing intervals (3-10, 7-11, 6.2-7.5), automated spot handling was performed on a large number of gel spots including those found to differ more than 20% between the treated and untreated condition. Subsequently, differentially regulated proteins were subjected to a three-step approach of mass spectrometry using (a) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprinting, (b) post-source decay utilizing chemically assisted fragmentation, and (c) liquid chromatography-tandem mass spectrometry. Using this approach we have so far resolved 121 differentially regulated proteins following treatment of mice with the candidate drug and identified 110 of these using mass spectrometry. Such data can potentially give improved molecular insight into the metabolism of drugs as well as the proteins involved in potential toxicity following the treatment. The differentially regulated proteins could be used as targets for metabolic studies or as markers for toxicity.

Manganese lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full-length clone of the Mn-Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino-acid sequence of the Mn-Lipoxygenase precursor (618 amino acids, 67.7 kDa) could be aligned with mammalian and plant lipoxygenases with 23-28% identity over 350-400 amino-acid residues of the catalytic domains. Lipoxygenases have one water molecule and five amino acids as Fe ligands. These are two histidine residues in the highly conserved 30 amino-acid sequence WLLAK-X15-H-X4-H-X3-E of alpha helix 9, one histidine and usually an asparaine residue in the sequence H-X3-N-X-G of alpha helix 18, and the carboxyl oxygen of the C-terminal isoleucine (or valine) residue. The homologous sequence of alpha helix 9 of Mn-Lipoxygenase [WLLAK-X14-H(294)-X3-H(297)-X3-E] contained two single-amino-acid gaps, but otherwise His294 and His297 aligned with the two His residues, which coordinate iron. Mn-Lipoxygenase [H(478)-X3-N(482)-X-G] could be aligned with the two metal ligands of alpha helix 18, and the C-terminal residue was Val618. We conclude that Mn-Lipoxygenase belongs to the lipoxygenase gene family and that its unique biochemical properties might be related to structural differences in the metal centre and alpha helix 9 of lipoxygenases rather than to the metal ligands.

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ratio. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-14C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.

Human, monkey, and rat liver microsomes catalyze bisallylic hydroxylations of arachidonic and linoleic acids. The cytochrome P450 gene family of these hydroxylases has not been determined. We examined whether inducers of cytochrome P450 could augment the bisallylic hydroxylation activity of male rat liver microsomes. The microsomes were incubated with [14C]linoleic acid and NADPH and the monohydroxy metabolites were characterized. Microsomes prepared from control rats yielded mainly 18-hydroxyoctadecadienoic acid (18-HODE) and 17-HODE and microsomes from clofibrate-treated rats 18-HODE. Microsomes from beta-naphthoflavone-treated rats hydroxylated linoleic acid without position specificity, i.e., at carbons 8, 11, 14, 16, 17, and 18. 11-HODE, 17-HODE, and 18-HODE were major metabolites. Microsomes from rats treated with phenobarbital, isopropanol, imidazole, or acetone also formed these three products along with many other hydroxy metabolites. The synthetic glucocorticoid dexamethasone increased the biosynthesis of 11-HODE selectively. Microsomes from male Sprague-Dawley and Fischer rats treated with dexamethasone mainly formed 11-HODE and 18-HODE. The biosynthesis of 11-HODE was increased 10-fold and troleandomycin (50 microM) inhibited the biosynthesis of 11-HODE by 90%. The bisallylic hydroxylases were also investigated with 14C-labeled arachidonic and eicosapentaenoic acids as substrates. Microsomes from rats treated with dexamethasone converted 20:4n-6 to 13-hydroxyeicosatetraenoic acid (13-HETE), 10-HETE, 7-HETE, 19-HETE, and 20-HETE. Induction by acetone yielded the same products. Microsomes from dexamethasone-treated rats metabolized 20:5n-3 to 16-hydroxyeicosapentaenoic acid (16-HEPE), 13-HEPE, 10-HEPE, 19-HEPE, and 20-HEPE as major products, while microsomes from control and acetone-treated rats mainly formed 19-HEPE and 20-HEPE. We conclude that microsomes from dexamethasone-treated rats catalyze bisallylic hydroxylations of 18:2n-6, 20:4n-6, and 20:5n-3, possibly by induction of bisallylic hydroxylases of the CYP3A subfamily.

Lipoxygenases of bovine and human corneal epithelia were investigated. The bovine epithelium contained an arachidonate 12-lipoxygenase and a 15-lipoxygenase. The 12-lipoxygenase was found in the microsomal fraction, while the 15-lipoxygenase was mainly present in the cytosol (100,000 x g supernatant). 12S-Hydroxyeicosatetraenoic acid (12S-HETE) and 15S-hydroxyeicosatetraenoic acid (15S-HETE) were identified by GC-MS and chiral HPLC. BW A4C, an acetohydroxamic acid lipoxygenase inhibitor, reduced the biosynthesis of 12S-HETE and 15S-HETE by over 90% at 10 microM. IC50 for the 12-lipoxygenase was 0.3 microM. The bovine corneal 12-lipoxygenase was compared with the 12-lipoxygenases of bovine platelets and leukocytes. All three enzymes metabolized 14C-labelled linoleic acid and alpha-linolenic acid poorly (5-16%) in comparison with [14C]arachidonic acid. [14C]Docosahexaenoic acid and [14C]4,7,10,13,16-docosapentaenoic acid appeared to be less efficiently converted by the corneal enzyme than by the platelet and leukocyte enzymes. Immunohistochemical analysis of the bovine corneal epithelium using a polyclonal antibody against porcine leukocyte 12-lipoxygenase gave positive staining. The cytosol of human corneal epithelium converted [14C]arachidonic acid to one prominent metabolite. The product co-chromatographed with 15S-HETE on reverse phase HPLC, straight phase HPLC and chiral HPLC. Our results suggest that human corneal epithelium contains a 15-lipoxygenase and that bovine corneal epithelium contains both a 15-lipoxygenase and a 12-lipoxygenase. The corneal 12-lipoxygenase appears to differ catalytically from earlier described bovine 12-lipoxygenases.

Incubation of 5,8,11-[1-14C]eicosatrienoic acid with prostaglandin endoperoxide synthase of ram vesicular gland microsomes led to formation of a number of polar metabolites. Four major compounds were characterized by chemical and physical methods and found to be: (11R)-hydroxy-5,8,12-eicosatrienoic acid, 8,9,11-trihydroxy-5,12-eicosadienoic acid (two diastereoisomers), and 8,9-epoxy-11-hydroxy-5,12-eicosadienoic acid. On the basis of previous studies on the mechanism of prostaglandin biosynthesis it seemed likely that the initial step of conversion of 5,8,11-eicosatrienoic acid consisted of removal of the pro-S hydrogen from C-13. The resulting carbon-centered radical was apparently attacked by dioxygen at C-13 to provide a (13R)-(hydro)peroxy derivative, which served as the precursor of (13R)-hydroxyeicosatrienoic acid. Alternatively, attack by dioxygen occurred at C-11 to produce an (11R)-peroxy radical. This intermediate was further converted to (11R)-hydroxyeicosatrienoic acid by reduction, into two 8,9,11-trihydroxy-5,12-eicosadienoic acids by successive cyclization, oxygenation, and reduction, and into the epoxy-hydroxy acid by cyclization and intramolecular epoxidation. The relative abundance of (13R)-hydroxy-5,8,11-eicosatrienoic acid, (11R)-hydroxy-5,8,12-eicosatrienoic acid, and the epoxy alcohol plus the two 8,9,11-triols was 51, 9, and 40%, respectively. The oxygenation at C-13 and C-11 of 5,8,11-eicosatrienoic acid was inhibited by 90% in the presence of diclofenac, an inhibitor of prostaglandin endoperoxide synthase. The two diastereomeric 8,9,11-trihydroxy acids and the epoxy-hydroxy acid are novel oxylipins and their formation provides independent chemical evidence for the existence of an 11-peroxy radical intermediate in prostaglandin endoperoxide synthase catalysis. Oxygenation of 5,8,11-eicosatrienoic acid by cytochrome P450 from liver microsomes of cynomolgus monkeys and phenobarbital-treated rats was also investigated. The metabolites formed included 19- and 20-hydroxyeicosatrienoic acid, 8,9- and 11,12-dihydroxyeicosadienoic acids (formed by enzymatic hydrolysis of the corresponding epoxides), and (12R)-hydroxy-5,8,10-hydroxyeicosatrienoic acid.

[14C]Linoleic acid was incubated with phenobarbital-induced rat liver microsomes and formation of cis-trans-conjugated hydroxy fatty acids was investigated. 13-Hydroxy-9Z,11E-octadecadienoic acid (13-HODE), 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE), and three novel metabolites were identified, viz. 11-hydroxy-9Z,12Z-octadecadienoic acid (11-HODE), 8-HODE, and 14-HODE. 11-HODE (59% R), the main product, was unstable and converted to 9(R, S)-HODE and 13(R, S)-HODE in acidic media. All metabolites contained oxygen from O2. Experiments under oxygen-18 gas showed that 13-HODE and 9-HODE contained equal or less amounts of oxygen-18 than the other metabolites. In the former case, 9-HODE and 13-HODE were formed with stereo-selectivity (80-82% R). [11S-2H]Linoleic acid was metabolized to 13R-HODE with loss of deuterium (24% 2H) and to 9R-HODE with deuterium retention (95% 2H), while [11R-2H]linoleic acid was metabolized to 13R-HODE that largely retained the label (71% 2H) and to 9R-HODE that lost most of the label (22% 2H). These data indicated that P450 catalyzed abstraction of the pro-R hydrogen at C11, double bond migration and suprafacial oxygen insertion at C9 to give 9R-HODE, while abstraction of the pro-S hydrogen at C11, followed by double bond migration and oxygen insertion, yielded 13R-HODE. Hepatic microsomes of the cynomolgus monkey metabolized 18:2n-6 as above and 20:4n-6 to 13-hydroxyeicosatetraenoic acid, likely formed in analogy with 11-HODE. In summary, one mechanism in the biosynthesis of cis-trans-conjugated hydroxy fatty acids by P450 involves suprafacial hydrogen abstraction and oxygen insertion. In addition, hydrolysis of the unstable bis-allylic hydroxy metabolites may contribute to the formation of conjugated hydroxy fatty acids.