Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.

Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

The presence of mitochondria increased the incorporation of [2-14C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (α-p-chlorophenoxyisobutyrate) and Clofenapate [α,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Amine biosynthesis in Lathyrus sativus seedlings

The biosynthesis of certain amines in Lathyrus sativus seedlings was studied in isolated shoots and cotyledons. In shoots, arginine was about 14 times more efficient than ornithine for the synthesis of agmatine, putrescine, spermidine and spermine. Isotope dilution experiments, and the changes in specific activities of the 4 amines with time when 14C-arginine served as the precursor, indicated that putrescine and the polyamines were formed mainly from arginine, via agmatine. Similar experiments showed that cadaverine was formed at least in part from homoarginine, though lysine was ca 4 times more effective as a precursor. The pattern of changes in specific activity of homoagmatine and cadaverine with time when 14C-homoarginine served as the precursor support the conclusion that homoarginine and arginine follow analogous metabolic routes in the biosynthesis of putrescine and cadaverine respectively.

Development of a Rapid Multiplex PCR Assay To Genotype Pasteurella multocida Strains by Use of the Lipopolysaccharide Outer Core Biosynthesis Locus

Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains.

Inhibition of the biosynthesis of isoprenoid compounds by phenolic acids in the rat brain

The incorporation of [2-14C]mevalonate into nonsaponifiable lipids by rat brain homogenates is inhibited by phenolic acids derived from tyrosine. The phenyl acids derived from phenylalanine are inhibitory only at very high concentrations compared with phenolic acids. The brain is more sensitive to inhibition by the phenolic acids than the liver. These studies indicate a possible role for phenolic acids in the impairment of cerebral sterol metabolism in phenylketonuria.

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.

The regulation of the biosynthesis of ubiquinone in the rat

The urinary excretion of p-hydroxybenzoate was not altered by ubiquinone feeding, but, although decreased considerably, was not eliminated in protein deficiency. The incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone in vivo increased in cold-exposed and p-chlorophenoxyisobutyrate (clofibrate)-fed rats, and these changes were parallel with the changes in the incorporation of [2-14C]mevalonate under these conditions. Starvation, cholesterol feeding and cholic acid feeding resulted in the decreased incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, confirming the decreased ubiquinone synthesis. Feeding exogenous ubiquinone increased the hepatic ubiquinone concentration, but did not cause any decrease in the incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, indicating the absence of a feedback control.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.