Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L.

Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated, exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.     

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.     

Imprinting defects in mouse embryos: stochastic errors or polymorphic phenotype?

Summary: Defects in expression of imprinted genes are believed to cause developmental abnormalities and play a role in carcinogenesis. To determine whether spontaneous imprinting defects may occur in mouse embryos, we studied the expression of two imprinted genes H19 and Igf2 in individual postimplantation 7.5 d.p.c. and 8.5 d.p.c. embryos. Biallelic expression of H19 was found in 1.6% of the embryos, whereas biallelic expression of Igf2 was found in 0.5% of the embryos. The loss of H19 imprinting (LOI) observed in a small fraction of early postimplantation embryos may be purely stochastic. Alternatively, since we never observed it in an inbred background, it may depend on genetic factors acting in trans. Either mechanism could explain the occurrence of polymorphic imprinting as well as the genesis of sporadic imprinting defects, including cancer. The frequency of LOI of H19 was higher than the incidence of sporadic imprinting disorders in humans (about 1 in 20,000). This contradiction may be explained by different incidence of imprinting errors in different imprinted regions of the genome, in different species, or by loss of the majority of nonmosaic embryos with imprinting defects before birth. genesis 31:11–16, 2001. © 2001 Wiley‐Liss, Inc.     

Age-associated change in mitochondrial DNA damage

There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previous reports from this lab have shown a decrease in mitochondrial cytochrome c oxidase (COX) activity associated with a reduction in COX gene and protein expression and a similar decrease in the rate of mitochondrial protein synthesis. Damage to mitochondrial DNA may contribute to this decline.

Using the HPLC-Coularray system (ESA, USA), we measured levels of nuclear and mitochondrial 8-oxo-2'-deoxyguanosine (8-oxodG) from 6-month (young) and 23-month-old (senescent) rat liver DNA. We measured the sensitivity of the technique by damaging calf thymus DNA with photoactivated methylene blue for 30s up to 2h. The levels of damage were linear over the entire time course including the shorter times which showed levels comparable to those expected in liver. For the liver data, 8-oxodG was reported as a fraction of 2-deoxyguanosine (2-dG). There was no change in the levels of 8-oxodG levels in the nuclear DNA from 6 to 23-months of age. However, the levels of 8-oxodG increased 2.5-fold in the mitochondrial DNA with age. At 6 months, the level of 8-oxodG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. These findings agree with other reports showing an age-associated increase in levels of mtDNA damage; however, the degree to which it increases is smaller. Such damage to the mitochondrial DNA may contribute to the age-associated decline in mitochondrial function.     

Clomazone does not inhibit the conversion of isopentenyl pyrophosphate to geranyl, farnesyl, or geranylgeranyl pyrophosphate in vitro

Clomazone, an herbicide that reduces the levels of leaf carotenoids and chlorophylls, is thought to act by inhibiting isopentenyl pyrophosphate isomerase or the prenyltransferases responsible for the synthesis of geranylgeranyl pyrophosphate. Cell-free extracts prepared from the oil glands of common sage (Salvia officinalis) are capable of converting isopentenyl pyrophosphate to geranylgeranyl pyrophosphate. Clomazone at 250 micromolar (a level that produced leaf bleaching) had no detectable effect on the activity of the relevant enzymes (isopentenyl pyrophosphate isomerase and the three prenyltransferases, geranyl, farnesyl, and geranylgeranyl pyrophosphate synthases). Thus, inhibition of geranylgeranyl pyrophosphate biosynthesis does not appear to be the mode of action of this herbicide.     

Antigenic cross-reactivity among monoterpene cyclases from grand fir and induction of these enzymes upon stem wounding.

A major wound response in grand fir (Abies grandis) sapling stems is the rapid increase in monoterpene production at the site of injury. Monoterpene cyclases (synthases) catalyze the formation of monoterpenes from geranyl pyrophosphate, and total cyclase activity increases markedly on wounding. At least six distinct cyclases, producing different monoterpene products, have been isolated from wounded grand fir saplings and characterized. The predominant wound-inducible cyclase produces both alpha- and beta-pinene. This pinene cyclase was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. The antibody preparation was found to cross-react by Western blotting with other grand fir monoterpene cyclases that produce different olefinic products, but not with monoterpene cyclases from related conifer species (Pinus contorta and P. ponderosa) or from angiosperms (Mentha piperita and M. spicata). The increase in monoterpene cyclase activity after wounding was closely correlated with the appearance of new cyclase protein as determined by immunoblotting. These results indicate that the wound-dependent increase in monoterpene cyclase activity is a consequence of de novo synthesis of cyclase protein.     

Biosynthesis of monoterpenes: inhibition of (+)-pinene and (-)-pinene cyclases by thia and aza analogs of the 4R- and 4S-alpha-terpinyl carbocation.

(+)-Pinene cyclase (synthase) from Salvia officinalis leaf catalyzes the cyclization of geranyl pyrophosphate, via (3R)-linalyl pyrophosphate and the (4R)-alpha-terpinyl cation, to (+)-alpha-pinene and to lesser quantities of stereochemically related monoterpene olefins, whereas (-)-pinene cyclase converts the same achiral precursor, via (3S)-linalyl pyrophosphate and the (4S)-alpha-terpinyl cation, to (-)-alpha-pinene and (-)-beta-pinene and to lesser amounts of related olefins. Racemic thia analogs of the linalyl and alpha-terpinyl carbocation intermediates of the reaction sequence were previously shown to be good uncompetitive inhibitors of monoterpene cyclases, and inhibition was synergized by the presence of inorganic pyrophosphate. These results suggested that the normal reaction proceeds through a series of carbocation:pyrophosphate anion paired intermediates. Both the (4R)- and the (4S)-thia and -aza analogs of the alpha-terpinyl cation were prepared and tested as inhibitors with the antipodal pinene cyclases, both in the absence and in the presence of inorganic pyrophosphate. Although the inhibition kinetics were complex, cooperative binding of the analogs and inorganic pyrophosphate was demonstrated, consistent with ion pairing of intermediates in the course of the normal reaction. Based on the antipodal reactions catalyzed by the pinene cyclases, stereochemical differentiation between the (4R)- and the (4S)-analogs was anticipated; however, neither enzyme effectively distinguished between enantiomers of the thia and aza analogs of the alpha-terpinyl carbocation. Enantioselectivity in the enzymatic conversion of (RS)-alpha-terpinyl pyrophosphate to limonene by the pinene cyclases was also examined. Consistent with the results obtained with the thia and aza analogs, the pinene cyclases were unable to discriminate between enantiomers of alpha-terpinyl pyrophosphate in this unusual reaction. Either the alpha-terpinyl antipodes are too similar to allow differentiation by the pinene cyclases, or these enzymes lack an inherent requirement to distinguish the (4R)- and (4S)-forms because they encounter only one enantiomer in the course of the normal reaction from geranyl pyrophosphate.     

The chemical composition of the cuticular wax of cranberry

Cuticular wax of cranberry fruit (Vaccinium macrocarpon var. Howes) was analysed by thin layer and gas-liquid chromatography in conjunction with mass spectrometry and infrared spectroscopy. The wax was shown to contain n-paraffins (10·7%), n-aldehydes (14·3%), n-alcohols (6·5%), n-fatty acids (9·7%), sterols (5·0%) and pentacyclic triterpene alcohols (5·8%), acetates (6·1%) and acids (30·7%).