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Nan C. Vance - One of the best experts on this subject based on the ideXlab platform.
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factors limiting seed production of Taxus brevifolia taxaceae in western oregon
American Journal of Botany, 1998Co-Authors: Stephen P. Difazio, M. V. Wilson, Nan C. VanceAbstract:Seed production of Pacific yew (Taxus brevifolia), an understory conifer, was studied at four sites in western Oregon over 2 yr. The effects of pollen supplementation, overstory canopy, and predator exclusion on ovule attrition were examined. Supplemental hand-pollination of ten trees at two sites resulted in significantly increased rates of ovule development and a doubling of seed efficiency (ratio of seeds to ovules). However, seed efficiency still averaged ,15% on branches receiving supplemental pollen, so pollination was not a primary factor limiting seed production. The number of developing ovules was positively associated with overstory openness, but seed production was not. Seed efficiency was negatively associated with overstory openness. Branches bagged to exclude vertebrate seed predators had higher seed production than unbagged branches at three of four sites for 2 yr. In contrast to unbagged branches, seed production on bagged branches was positively associated with overstory openness, as was the effectiveness of bagging. Therefore, both vertebrate predation and overstory were important in limiting seed production, and these factors interacted. Factors limiting seed production varied in importance among the four sites and between years, illustrating the importance of examining multiple limiting factors over several sites and years.
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Variation in sex expression of Taxus brevifolia in western Oregon
Canadian Journal of Botany, 1996Co-Authors: Stephen P. Difazio, M. V. Wilson, Nan C. VanceAbstract:Sex expression of Taxus brevifolia Nutt. was studied at four sites in western Oregon. Of 115 trees examined, 17 were cosexual. All cosexual trees had predominantly male sex expression, suggesting that male sterility is much more constant than female sterility for T. brevifolia. Some implications of the observed patterns are discussed, along with suggestions for further research. Keywords: Taxus brevifolia, yew, sex expression, dioecy.
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Physiological aspects of Taxus brevifolia seeds in relation to seed storage characteristics
Physiologia Plantarum, 1996Co-Authors: Christina Walters-vertucci, Jennifer Crane, Nan C. VanceAbstract:Water relations, desiccation tolerance and longevity of Taxus brevifolia (Nutt.) seeds were studied to determine the optimal stage of development and storage conditions for seeds of this species. Seeds equilibrated to a range of relative humidities (RHs) had unusually low water contents which can be accounted for by the high lipid content of gametophyte tissues (71% of the dry mass). Water relations of embryonic tissue were more typical of those reported for other seed species. The water content below which freezing transitions were not observable in the embryo was ca 0.24 g H 2 O (g dry weight) -1 (g g -1 ) for all maturity classes studied. Embryos did not achieve significant levels of desiccation tolerance (survival to water contents less than 0.5 g g -1 ) until the latter stages of development when dry matter was maximal. Mature embryos could be dried to 0.025 g g -1 (seed water content of 0.010 g g -1 ) with no loss of viability. Thus, at the latter stages of development, embryo water content could be optimized to avoid both desiccation and freezing damage. Survival of mature seeds declined over a 2-year period when seeds were stored at temperatures between 5 and 35°C and RHs between 14 and 75%, corresponding to seed water contents between 0.015 and 0.07 g g -1 . The deterioration rate was slowest for seeds stored at the lowest RH and temperature. Our data indicate that seeds of Taxus brevifolia show orthodox rather than recalcitrant storage characteristics, but that the optimum water content for storage was extremely low. The results suggest that even if stored at optimal water contents and low temperatures, T brevifolia seeds will be relatively short lived. The high quantity of lipids or reducing sugars may be contributing factors in the poor storage characteristics.
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Seasonal and tissue variation in taxane concentrations of Taxus brevifolia
Phytochemistry, 1994Co-Authors: Nan C. Vance, Rick G. Kelsey, Thomas E. SabinAbstract:Analysis of seven taxanes: taxol, baccatin III, 10-deacetyltaxol, 10-deacetylbaccatin III, 7-xylosyl-10-deacetyltaxol, cephalomannine and brevifoliol in extracts from bark and foliage of pacific yew (Taxus brevifolia) showed a gradient of decreasing concentration from stem base to branch tip. This decrease is attributed to the generally higher concentration of taxanes in the phloem tissue and the decrease in inner bark thickness from base to branch tip. Analysis of taxanes extracted from stem bark and needles sampled over a growing season showed that most taxane concentrations were significantly lower in the needles than in the bark. Typically, taxane concentrations in bark increased from May through August; whereas, in needles, concentrations changed little during that period. Two exceptions were baccatin III, which in the summer reached levels equivalent to bark, and brevifoliol which increased from March to August, reaching levels in needles nine times greater than bark.
Gary A Strobel - One of the best experts on this subject based on the ideXlab platform.
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The search for a taxol-producing microorganism among the endophytic fungi of the Pacific yew, Taxus brevifolia.
Journal of natural products, 1995Co-Authors: Andrea A Stierle, Gary A Strobel, Donald B Stierle, Paul G. Grothaus, Gary S. BignamiAbstract:Endophytic microbes associated with the Pacific yew tree, Taxus brevifolia, were examined as potential sources of the anticancer drug taxol [1], a secondary metabolite of the host organism. The first promising organism found was the novel fungus, Taxomyces andreanae, which was isolated from the inner bark of a yew tree growing in northwestern Montana. It appears to produce taxol and other taxanes in de novo fashion when grown in semi-synthetic liquid media. The presence of 1 in the fungal extract was confirmed by mass spectrometry, comparative chromatographic behavior with "yew" taxol, reactivity with taxol-specific monoclonal antibodies, and 9KB cytotoxicity studies. Both acetate-1-14C and phenylalanine UL-14C served as precursors of taxol-14C in fungal culture labeling studies, confirming the de novo synthesis of 1 by the fungus. Immunoassay techniques are currently being used to screen extracts of Taxomyces andreanae for new taxanes, and to determine if other endophytic fungi are taxol producers.
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The stimulation of taxol production in Taxus brevifolia by various growth retardants
Plant Science, 1994Co-Authors: Gary A Strobel, Andrea A Stierle, Wilford M. HessAbstract:Abstract We have shown that chlorocholine chloride (CCC), succinic acid, 2,2-dimethylhydrazide (Alar®) and tetramethylammonium bromide (TMAB) stimulate [14C]acetate incorporation into taxol in intact pieces of the inner bark of Taxus brevifolia (Pacific yew). Both CCC and Alar also stimulated taxol production in yew logs as measured by the incorporation of [1-14C]acetate into [14C]taxol. An increase in taxol accumulation also occured in silica gel placed under flaps of yew trees in the forest treated with these two compounds. CCC also caused an increase, in an 8-week treatment period, of recoverable taxol from Pacific yew bark in a forest setting. CCC, alar and TMAB are all known as growth retardants, but may cause an effect on taxol biosynthesis via an inhibition of sterol biosynthesis.
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taxol and taxane production by taxomyces andreanae an endophytic fungus of pacific yew
Science, 1993Co-Authors: Andrea A Stierle, Gary A Strobel, Donald B StierleAbstract:Taxomyces andreanae, a fungal endophyte, was isolated from the phloem (inner bark) of the Pacific yew, Taxus brevifolia. The fungus is hyphomyceteous and, when grown in a semi-synthetic liquid medium, produced taxol and related compounds. Taxol was identified by mass spectrometry, chromatography, and reactivity with monoclonal antibodies specific for taxol. Both [1-14C]acetic acid and L-[U-14C]phenylalanine served as precursors of [14C]taxol in fungal cultures. No taxol was detected in zero-time cultures or in the small agar plugs used to inoculate the culture flasks.
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Taxol formation in yew — Taxus
Plant Science, 1993Co-Authors: Gary A Strobel, Andrea Stierle, W M HessAbstract:Abstract In Pacific yew, Taxus brevifolia , taxol is found most abundantly in the vascular cambial region followed by the phloem, sapwood and heartwood. It is in this order that ‘in vitro’ taxol biosynthetic activity was also demonstrated using [1- 14 C]acetate as a precursor. However, only traces of taxol biosynthesis could be demonstrated in the xylem (sapwood-heartwood) suggesting that taxol may be mobilized from its place of greatest biosynthetic activity (vascular cambial region) to the xylem; perhaps via ray parenchyma. Taxol could also be effectively trapped under ‘bark flaps’, over a period of several weeks, using silica gel powder as an adsorbant. In vitro taxol biosynthesis using [1- 14 C]acetate in outer bark samples (cambium-phloem) could only be demonstrated in Taxus brevifolia, Taxus floridana and Taxus canadensis , among 10 of the 11 native Taxus species tested.
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Factors influencing the in vitro production of radiolabeled taxol by Pacific yew, Taxus brevifolia
Plant Science, 1992Co-Authors: Gary A Strobel, Andrea A Stierle, Frederik J.g.m. Van KuijkAbstract:Abstract A system for the production of [14C]taxol in vitro has been devised. The aseptic system utilized an appropriate 14C-labeled precursor, a reducing environment (dithiothreitol) and aseptically prepared pieces of the inner bark of Taxus brevifolia (Pacific yew). [14C]Phenylalanine and [14C]leucine are the best precursors, of the compounds tested, for [14C]taxol production. However, in the standard assay sodium [1-14C]acetate is used because of its relatively low price and its ability to label taxol uniformly. Chlorocholine chloride, an inhibitor of plant steroid metabolism, was an effective stimulator of [14C]taxol production, as were certain fungal elicitors. Taxol biosynthetic activity is greatest in the bark from lower portions of the main stem (trunk: 1 m from ground level).
N.g. Lewis - One of the best experts on this subject based on the ideXlab platform.
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Purification and Characterization of Taxa-4(5),11(12)-diene Synthase from Pacific Yew (Taxus brevifolia) that Catalyzes the First Committed Step of Taxol Biosynthesis
Archives of Biochemistry and Biophysics, 1995Co-Authors: Mehri Hezari, N.g. LewisAbstract:Abstract The first step in the biosynthesis of taxol in Pacific yew (Taxus brevifolia) is the cyclization of the universal diterpene precursor geranylgeranyl pyrophosphate to taxa-4(5),11(12)-diene. This parent olefin of the taxane diterpenoids is then elaborated to taxol and related compounds by a complex series of reactions involving oxidations and side-chain acylations. Cyclization activity is located principally in yew stem bark and adhering cambium. The operationally soluble cyclization enzyme was partially purified (∼600-fold) by combination of anion exchange, hydrophobic interaction, and dye-ligand chromatography. Nondenaturing, followed by denaturing, polyacrylamide gel electrophoresis, in combination with gel permeation chromatography, allowed the identification of taxadiene synthase as a monomeric protein of molecular weight 79,000. In general properties (divalent metal ion requirement, kinetic constants, molecular weight), the taxadiene synthase of Pacific yew is similar to the diterpene cyclase abietadiene synthase involved in resin acid biosynthesis in other gymnosperms. However, in pH optimum and response to inhibitors, these two diterpene cyclases are distinctly different. The activity (and enzyme protein) levels of Pacific yew taxadiene synthase are much lower than those for abietadiene synthase of lodgepole pine stem (constitutive) or of grand fir stem (wound-inducible) and the enzyme is not inducible to higher levels by stem wounding or elicitor treatment.
Ralph Quatrano - One of the best experts on this subject based on the ideXlab platform.
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Production of taxa-4(5),11(12)-diene by transgenic Physcomitrella patens
Transgenic Research, 2009Co-Authors: Aldwin Anterola, Erin Shanle, Pierre-françois Perroud, Ralph QuatranoAbstract:Taxadiene synthase gene from Taxus brevifolia was constitutively expressed in the moss Physcomitrella patens using a ubiquitin promoter to produce taxa-4(5),11(12)-diene, the precursor of the anticancer drug paclitaxel. In stable moss transformants, taxa-4(5),11(12)-diene was produced up to 0.05% fresh weight of tissue, without significantly affecting the amounts of the endogenous diterpenoids ( ent -kaurene and 16-hydroxykaurane). Unlike higher plants that had been genetically modified to produce taxa-4(5),11(12)-diene, transgenic P. patens did not exhibit growth inhibition due to alteration of diterpenoid metabolic pools. Thus we propose that P. patens is a promising alternative host for the biotechnological production of paclitaxel and its precursors.
Koppaka V. Rao - One of the best experts on this subject based on the ideXlab platform.
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Taxanes from the bark of Taxus brevifolia
Phytochemistry, 1998Co-Authors: Koppaka V. Rao, John JuchumAbstract:Abstract Large-scale processing of the chloroform extract of the bark of Taxus brevifolia by reversed phase column chromatography on C-18 bonded silica using acetonitrile/water mixtures, gave a taxane-rich fraction which emerged just after the elution of paclitaxel. Re-chromatography of this fraction yielded 15 taxane constituents, many of which were obtained in crystalline from, and variously belonging to the 11(15 → 1)-abeotaxane group, taxinine J group, brevifoliol group and those with an oxygenation pattern at the 14 position. Five of these were new and several others were isolated for the first time from this source.
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Semi-synthesis of paclitaxel from naturally occurring glycosidic precursors
Journal of Heterocyclic Chemistry, 1997Co-Authors: Koppaka V. RaoAbstract:Paclitaxel, an antitumor drug effective on ovarian and breast carcinomas, is currently being produced both by direct isolation from the bark of Taxus brevifolia and by semi-synthesis from a natural precursor, 10-deacetyl baccatin III. Although other potential precursors such as 10-deacetyl paclitaxel-7-xyloside were known since 1984, their conversion to paclitaxel could not be achieved because of the lack of suitable methodology for hydrolyzing the xylose residue, compatible with the stability of the compound. A method is described here using periodate, followed by phenylhydrazine, to effect deglycosidation of 10-deacetyl paclitaxel-7-xyloside to form 10-deacetyl paclitaxel. In addition, by including an intermediate acetylation step before the reaction with phenylhydrazine, “direct” conversion of this xyloside to paclitaxel itself, is described. Because 10-deacetyl paclitaxel-7-xyloside occurs at >0.1% in the bark of Taxus brevifolia, its successful hydrolytic conversion to paclitaxel represents an extremely important reaction for the enhanced availability of this drug.
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A New Large-Scale Process for Taxol and Related Taxanes from Taxus brevifolia
Pharmaceutical research, 1995Co-Authors: Koppaka V. Rao, Jampani B. Hanuman, Claudio Alvarez, Mark Stoy, John Juchum, Richard M. Davies, Ronald BaxleyAbstract:Purpose. In view of the demonstrated antitumor activity of taxol, ready availability of the drug is important. The current isolation methods starting from the bark of Taxus brevifolia involve multiple manipulations, leading to only taxol and in a yield of 0.01%. A new process consisting of a single reverse phase column is introduced here, and the present purpose is to determine its large scale applicability.
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Taxol and related taxanes. I. Taxanes of Taxus brevifolia bark.
Pharmaceutical research, 1993Co-Authors: Koppaka V. RaoAbstract:The published procedures for the isolation of taxol from the Pacific yew (Taxus brevifolia) and other species of Taxus are cumbersome, and the yields of taxol are in the range of 0.0075–0.01%. This paper describes a simple and efficient procedure for the isolation of taxol and its major natural analogues from the bark of T. brevifolia consisting of a single chromatographic column (using silica gel, Florisil, or a reverse-phase C18-silica), followed by crystallization. Isolated yields of taxol from five “pooled” bark samples (blended from many different batches by the supplier) were in the range of 0.02–0.04%, and from bark collected from a more restricted locale, yields reached 0.06%. The procedure also yielded taxol analogues, such as 10-deacetylbaccatin III (0.02–0.04%), 10-deacetyltaxol-7-xyloside (0.06-0.1%), taxol-7-xyloside (0.005–0.01%), 10-deacetyltaxol (0.01–0.02%), 10-deacetylcephalomannine-7-xyloside (0.006–0.01%), and cephalomannine (0.005–0.007%). Of these, 10-deacetyltaxol-7-xyloside is the most abundant taxane in the Pacific yew bark.
