Biosynthesis of pentahydroxystearic acid of cutin from linoleic acid in Rosmarinus officinalis

The hydroxyfatty acid polymer, cutin, is the structural component of plant cuticle. Combined gas chromatography-mass spectrometry of the hydrogenolysis and deuterolysis products of rosemary cutin (Rosmarinus officinalis) revealed a series of components suggesting the conversion of linoleic acid to 9,10,12,13,18-pentahydroxy-stearic acid. [U-¹⁴C]Linoleic acid was incorporated into the insoluble residue of rapidly expanding rosemary leaves. Depolymerization of the insoluble material followed by isolation of individual components and chemical degradation studies showed that linoleic acid was directly converted into 18-hydroxylinoleic acid, 18-hydroxy-9, 10-epoxyoctadec-12-enoic acid, 9,10,18-trihydroxyoctadec-12-enoic acid, 9,10,18-trihydroxy-12,13-epoxystearic acid, and 9,10,12,13,18-pentahydroxystearic acid. These results strongly suggest that, in the biosynthesis of the phytopolymer, linoleic acid is first converted into 18-hydroxylinoleic acid and that this precursor then undergoes sequential epoxidation-hydration at the Δ⁹ and Δ¹² double bonds to yield 9,10,12,13,18-pentahydroxystearic acid.     

Simulation modifies prehension: evidence for a conjoined representation of the graspable features of an object and the action of grasping it

Movement formulas, engrams, kinesthetic images and internal models of the body in action are notions derived mostly from clinical observations of brain-damaged subjects. They also suggest that the prehensile geometry of an object is integrated in the neural circuits and includes the object's graspable characteristics as well as its semantic properties. In order to determine whether there is a conjoined representation of the graspable characteristics of an object in relation to the actual grasping, it is necessary to separate the graspable (low-level) from the semantic (high-level) properties of the object. Right-handed subjects were asked to grasp and lift a smooth 300-g cylinder with one hand, before and after judging the level of difficulty of a "grasping for pouring" action, involving a smaller cylinder and using the opposite hand. The results showed that simulated grasps with the right hand exert a direct influence on actual motor acts with the left hand. These observations add to the evidence that there is a conjoined representation of the graspable characteristics of the object and the biomechanical constraints of the arm.     

Global and comparative proteomic profiling of overwintering and developing mountain pine beetle,Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae

BackgroundMountain pine beetles, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), are native to western North America, but have recently begun to expand their range across the Canadian Rocky Mountains. The requirement for larvae to withstand extremely cold winter temperatures and potentially toxic host secondary metabolites in the midst of their ongoing development makes this a critical period of their lives.ResultsWe have uncovered global protein profiles for overwintering mountain pine beetle larvae. We have also quantitatively compared the proteomes for overwintering larvae sampled during autumn cooling and spring warming using iTRAQ methods. We identified 1507 unique proteins across all samples. In total, 33 proteins exhibited differential expression (FDR < 0.05) when compared between larvae before and after a cold snap in the autumn; and 473 proteins exhibited differential expression in the spring when measured before and after a steady incline in mean daily temperature. Eighteen proteins showed significant changes in both autumn and spring samples.ConclusionsThese first proteomic data for mountain pine beetle larvae show evidence of the involvement of trehalose, 2-deoxyglucose, and antioxidant enzymes in overwintering physiology; confirm and expand upon previous work implicating glycerol in cold tolerance in this insect; and provide new, detailed information on developmental processes in beetles. These results and associated data will be an invaluable resource for future targeted research on cold tolerance mechanisms in the mountain pine beetle and developmental biology in coleopterans.     

Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.     

Human RECQL5β stimulates flap endonuclease 1

Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5β, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5β dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5β and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4’s stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.     

Purification and characterization of a mitochondrial thymine glycol endonuclease from rat liver

Mitochondrial DNA is exposed to oxygen radicals produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions in mitochondrial DNA may lead to structural genomic alterations, mitochondrial dysfunction, and associated degenerative diseases. The pyrimidine hydrate thymine glycol, one of many oxidative lesions, can block DNA and RNA polymerases and thereby exert negative biological effects. Mitochondrial DNA repair of this lesion is important to ensure normal mitochondrial DNA metabolism. Here, we report the purification of a novel rat liver mitochondrial thymine glycol endonuclease (mtTGendo). By using a radiolabeled oligonucleotide duplex containing a single thymine glycol lesion, damage-specific incision at the modified thymine was observed upon incubation with mitochondrial protein extracts. After purification using cation exchange, hydrophobic interaction, and size exclusion chromatography, the most pure active fractions contained a single band of approximately 37 kDa on a silver-stained gel. MtTGendo is active within a broad KCl concentration range and is EDTA-resistant. Furthermore, mtTGendo has an associated apurinic/apyrimidinic-lyase activity. MtTGendo does not incise 8-oxodeoxyguanosine or uracil-containing duplexes or thymine glycol in single-stranded DNA. Based upon functional similarity, we conclude that mtTGendo may be a rat mitochondrial homolog of the Escherichia coli endonuclease III protein.     

Regioselectivity of taxoid-O-acetyltransferases: heterologous expression and characterization of a new taxadien-5alpha-ol-O-acetyltransferase

In addition to the anticancer drug Taxol, yew (Taxus) species produce a large variety of other taxane diterpenoids which differ mainly in the type of acyl and aroyl groups appended to the many hydroxyl functions on the taxane core; acetate esters are particularly common. Taxol bears an acetate at C10 and another at C4 thought to originate by intramolecular migration of a C5 acetate function in the process of oxetane ring formation, but many other naturally occurring taxoids bear acetate groups at C1, C2, C7, C9, and C13, in addition to C5 and C10. cDNAs encoding a taxoid 5alpha-O-acetyltransferase (taxadien-5alpha-ol as substrate) and a taxoid 10beta-O-acetyltransferase (10-deacetylbaccatin III as substrate) have been acquired from a recently isolated family of Taxus acyl/aroyltransferase clones. To explore the origins of other acetylated taxoids, the group of recombinant Taxus acyltransferases was investigated with a range of polyhydroxylated taxoids as substrates. From this survey, a new acetyltransferase clone (denoted TAX19) was identified that was capable of acetylating taxadien-5alpha-ol with activity comparable to that of the previously identified 5alpha-O-acetyltransferase (clone TAX1). However, when these two recombinant enzymes were presented with taxadien-triol and tetraol substrates, they exhibited different regiospecificities. The TAX1 enzyme preferentially acetylates the "northern" hemisphere hydroxyls at C9 and C10, whereas the TAX19 enzyme preferentially acetylates the "east-west" pole positions at C5 and C13. The TAX1 enzyme possesses the lowest KM value with taxadien-5alpha-ol (an early pathway metabolite) as substrate, with much higher KM values for the polyhydroxylated taxoid substrates, whereas the TAX19 enzyme possesses lower KM values (than the TAX1 transferase) for all taxoid substrates tested. These results suggest that both TAX1 and TAX19 acyltransferases may function at the early C5 acetylation step of taxoid metabolism, and that the TAX19 acyltransferase, because of its broader specificity for polyhydroxylated taxoids, may also function later in metabolism and be responsible for the production of many other acetylated taxoids.     

Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis

Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.     

Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase

We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.