Improved paclitaxel production by in situ extraction and elicitation in cell suspension cultures of Taxus chinensis

Dibutyl phthalate, oleic acid and terpineol were used to extract paclitaxel in situ fromTaxus chinensis suspension cultures. Oleic acid/terpineol (1:1, v/v) added to the cultures gave a higher paclitaxel concentration, compared with either of them alone. Oleic acid/terpineol (1:1, v/v) incorporated into the cultures at 3:50 (v/v) 4 days after elicitation, which was carried out by adding 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ to 10-day-old cultures, resulted in the greatest paclitaxel production of 48 mg l^–1 at day 10 after elicitation. This was double that of the culture by elicitation, and 7-fold higher than that of the culture by in situ extraction.     

Improved cell growth and Taxol production of suspension-cultured Taxus chinensis var. mairei in alternating and direct current magnetic fields

The growth of suspension cultures of Taxus chinensis var. mairei and Taxol production were promoted both by a sinusoidal alternating current magnetic field (50 Hz, 3.5 mT) and by a direct current magnetic field (3.5 mT). Taxol production increased rapidly from the 4th d with the direct current magnetic field but most slowly with the alternating current magnetic field. The maximal yield of Taxol was 490 microg l(-1) with the direct current magnetic field and 425 microg l(-1) with the alternating current magnetic field after 8 d of culture, which were, respectively, 1.4-fold and 1.2-fold of that without exposure to a magnetic field.     

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder： Cloning, Characterization and Functional Complementation

Farnesyl dlphosphate synthase （FPS; EC 2.5.1.10） catalyzes the production of 15-carbon farnesyl dlphosphate which Is a branch-point Intermediate for many terpenoids. This reaction Is considered to be a ratelimiting step In terpenold biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl dlphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 （GenBank accession number： AY461811） was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptlde with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Biolnformatlc analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetlc analysis showed that farnesyl dlphosphate synthases can be divided Into two groups： one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature Is the arrangement of 13 core helices around a large central cavity In which the catalytic reaction takes place. Our blolnformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPSgene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, Including the needles, stems and roots of T. media. Subsequently, functional complementatlon with TmFPS1 in a FPS-deflclent mutant yeast demonstrated that TmFPS1 did encode farnesyl dlphosphate synthase, which rescued the yeast mutant. This study will be helpful In future Investigations aiming at understanding the detailed role of FPS In terpenold biosynthesis flux control at the molecular genetic level.     

Molecular cloning and expression analyses of a new gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Taxus × media

A new full-length cDNA encoding 3-hydroxy-3-methylglutaryl-CoA synthase (designated as TmHMGS, GenBank Accession No. AY644708), which catalyses the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the taxol biosynthetic pathway, was isolated from young leaves of Taxus × media by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of TmHMGS contained a 1431 bp open reading frame (ORF) encoding a deduced protein of 476 amino acid residues. The deduced protein had an isoelectric point of 5.23 and a calculated molecular mass of about 53 kDa. Amino acid sequence comparison analysis showed that TmHMGS had high similarity with a number of HMGSs ranging from Schizosaccharomyces pombe to humans, with much higher identity with other HMGSs from plants than those from yeast and humans. Phylogenic analysis showed that TmHMGS had closest relationship with HMGS from Pinus sylvestris. Tissue expression pattern analysis showed that TmHMGS expressed in needles and stems at similar level, but no expression could be detected in roots. Expression of TmHMGS was all induced by under different elicitors such as silver nitrate, ammonium ceric sulphate and methyl jasmonate, revealed that TmHMGS was an elicitor-responsive gene.     

云南红豆杉(Taxus Yunnanensis)皮下真菌类群初步研究

分离纯化与云南红豆杉（Taxus Yunnanensis)内共生的皮下真菌,获得了52株内生真菌,对52株菌进行了分类研究。结果是：无孢类各属是优势菌群（共29株）,其次是木霉属（5株）和茎点霉属（4株）,首次报道了红豆杉属植物皮下真菌的类群和区系特点。     

Novel Taxa-4(20),12-diene and 2(3→20)Abeotaxane from Needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Novel taxa-4(20),12-diene and 2(3→20)abeotaxane from needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Cloning of taxane 2α-O-benzoyltransferase (TBT) genomic DNA from Taxus

Based on the cDNA sequence encoding taxane 2α-O-benzoyltransferase (TBT) from Taxus yunnanensis (Gen-Bank Accession No.: AY970522), genomic sequences of TBTs from T. yunnanensis (TyTBT) and T. cuspidata (TcuTBT) were cloned for the first time. They both contain only one intron. The finding that the introns of TyTBT and TcuTBT are more diverse than their exons implies that the gene diversity is more within introns than within exons, which may be important for keeping the functions of the genes.     

Hairy root cultures of Taxus × media var. Hicksii Rehd. as a new source of paclitaxel and 10-deacetylbaccatin III

Hairy root cultures of Taxus × media var. Hicksii were established by infection of the plantlets with the Agrobacterium rhizogenes strain LBA 9402. The paclitaxel accumulation in hairy root cultures increased after 100 M methyl jasmonate treatment from 69 to 210 g g^–1 dry wt while the 10-deacetybaccatin III content was not affected by the elicitor.     

天然东北红豆杉(Taxus cuspidata)种内和种间竞争

竞争是植物种内和种间关系的主要形式之一。通过对黑龙江穆棱东北红豆杉自然保护区的95株东北红豆杉对象木及980株竞争木的调查,运用Hegyi的单木竞争指数计算分析了东北红豆杉的种内和种间竞争强度。东北红豆杉的种内竞争强度不大,占总竞争的4%。竞争压力更多的来自于种间竞争,占总竞争的96%。与东北红豆杉竞争激烈的树种主要是冷杉、紫椴、色木槭和红松等地带性植被的优势种。随着东北红豆杉胸径的增大,所受到的竞争压力逐渐减小,胸径在20cm以前所受到的竞争压力最大,竞争强度与对象木胸径符合幂函数（CI=AD^-B）关系。     

云南红豆杉(Taxus Yunnanensis)内生真菌中产紫杉醇真菌的筛选

报道了从云南红豆杉（Taxus Yunnanensis)树皮和枝条中分离得到的52株内生真菌中,有19株菌的发酵产物经TLC分析和HPLC检测验证后产紫杉醇或紫杉烷类物质。其中紫杉醇含量在1‰以上的菌株有8株。TAX-47（茎点霉属）和TAX-49（组丝核菌属）的紫杉醇得率分别为47.302μg/L和31.06μg/L,TAX-25（茎叶核菌属）巴可亭Ⅲ得率为744.2μg/L,这些菌株值得进一步研究。     

红豆杉(Taxus chinensis (Pilger) Rehd.)化学成分研究(简报)

自七十年代发现了紫杉醇（taxol）独特的抗癌机制一促进微管蛋白聚合、抑制微管蛋白解聚后,紫杉醇及其类似物紫杉烷类二该化合物的资源调查和化学研究倍受重视。紫杉醇主要存在于红豆杉科的大多数植物中,红豆杉科（Taxaceae）红豆杉属（TaxuS）植物全球大约有11种,我国有4种和1个变种。我们从采自湖北神农架的红豆杉（Tchinens周树皮中分离得到8个结晶性物质,本文报道其中已鉴定的3个成分,即taxinineJ（l）,ldiydroxybaccatinl（2）和taxol（3）。据文献报道,已经从红豆杉分离得到二十多种紫杉烷类化合物l'l,而我们分离得到的1和2…     

红豆杉(Taxus chinensis (Pilger) Rehd.)中的非紫杉烷类化合物

从红豆杉（Ｔａｘｕｓ ｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇｅｒ）Ｒｅｈｄ．）树皮中分离到三个非萜类化合物,波同它们提吴茱次碱（ｒｕｔａｅｃａｒｐｉｎｅ）（１）。山奈黄＝－４＇－甲醚（ｋａｅｍｐｆｅｒｏｌ－４＇－ｍｅｔｈｙｌ ｅｔｈｅｒ）（２）和谷甾醇（β－Ｄ－吡喃葡萄糖甙（ｓｉｔｏｓｔｅｒｒｙＩ－３－Ｏ－βｇｌｕｃｏｓｉｄｅ）（３）。吴茱次碱是首次在红豆杉属植物中发现。     

Untersuchungen üben die Rückreaktionen im Xanthophyll-Cyclus bei Chlorella,Spinacia und Taxus

Zusammenfassung Die als Rückreaktion bezeichnete Epoxidierung des Zea. ²über das Monoepoxid Anth. zum Diepoxid Viol. wird an verschiedenen pflanzlichen Objekten und unter verschiedenen Bedingungen untersucht.In Chlorella und den Nadeln von Taxus baccata ist sofort nach Beendigung der starken Belichtung die Rückumwandlung zu beobachten. Diese ist durch Begasung des Pflanzenmaterials mit reinem O₂ oder durch schwaches Licht noch etwas zu beschleunigen.In Blattscheibchen von Spinacia oleracea ist eine solche Epoxidierung unter Normalbedingungen (Dunkelheit, Luft) nicht festzustellen. Sie setzt erst unter dem Einfluß von schwachem Licht oder bei Zufuhr von O₂ (100%) ein. Das Fehlen der Rückreaktion hat seine Ursache in einem unter den gegebenen Versuchsbedingungen stattfindenden Spaltöffnungsverschluß, der während der Starklichtgabe beginnt und während der nachfolgenden Dunkelheit noch anhält, und einer daraus resultierenden Anaerobiose in den plastidenhaltigen Zellen. Durch eine Erhöhung der O₂-Spannung in diesen Zellen, entweder durch von außen zugeführten reinen O₂ oder durch intracelluläre Entwicklung von Photosynthese-O₂ (verbunden mit einer teilweisen Öffnung der Stomata), kommt es auch in den Spinatblättern zu einer Rückreaktion. Schwaches Licht kann also auf indirektem Weg die Rückreaktion beschleunigen.Die Konzentration des Zwischenproduktes Anth. nimmt bei der O₂-oder schwachlichtgeförderten Rückreaktion anfänglich stärker zu als das Endprodukt Viol. Daraus folgt, daß bei der Epoxidierung die beiden O-Atome nicht gleichzeitig, sondern nacheinander in die 5,6- und 5,6-Stellung des Zea. eingebaut werden.In isolierten Chloroplasten oder Zellfragmenten konnte bisher unter verschiedensten Bedingungen keine Rückreaktion nachgewiesen werden. Eine in gefriergetrockneten Zellen oder Chloroplasten durch Licht und O₂-Atmosphäre ausgelöste scheinbare Rückreaktion ist auf eine verschieden schnelle photooxidative Zerstörung der Carotinoide zurückzuführen.Die Epoxidierung des Zea., welche durch ein wahrscheinlich Cuhaltiges Enzym katalysiert wird, ist in vivo an das Vorhandensein von molekularem O₂ gebunden.

---

Studies on the backward-reactions in the xanthophyll-cycle of Chlorella, Spinacia and Taxus

Summary The epoxidation of zeaxanthin to the di-epoxide violaxanthin via the mono-epoxide antheraxanthin (called the backward-reaction), is examined with several plant objects and under various conditions.In Chlorella and in the needles of Taxus baccata a backward-conversion can be observed immediately after the termination of strong illumination. The reaction can be accelerated somewhat by exposure of the plant material to pure O₂ or dim light.One cannot observe such an epoxidation in leaf disks of Spinacia oleracea under normal conditions (dark, air). It begins only under the influence of dim light or when pure oxygen is supplied. The absence of the backward-reaction under the given experimental conditions is a consequence of a closure of the stomata, which begins during the strong illumination and continues in the succeeding dark period; it is therefore a consequence of anaerobiosis in the plastid-containing cells. Yet a backward-reaction starts if the O₂-tension in the cells is increased either by pure O₂ given from outside or by the intracellular evolution of photosynthetic O₂ (associated with a partial opening of the stomata).The concentration of the intermediate antheraxanthin increases strongly more at the beginning of the O₂- or dim-light-promoted backward-reaction than the concentration of the endproduct violaxanthin. Hence it follows that during epoxidation the two O-atoms are added to the 5,6- and the 5,6-position of the zeaxanthin not simultaneously but one after the other.In isolated chloroplasts or cell fragments no backward-reaction could be observed under various conditions tested. An apparent backward-reaction in lyophilized cells or chloroplasts, which is triggered by light or O₂-atmosphere, is a result of the different velocity of the photooxidative destruction of carotenoids.The in vivo epoxidation of zeaxanthin, which probably is catalysed by a Cu-containing enzyme, only proceeds in the presence of molecular O₂.

     

Efficacies of the new Paclitaxel-eluting Coroflex Please™ Stent in percutaneous coronary intervention; comparison of efficacy between Coroflex Please™ and Taxus™ (ECO-PLEASANT) trial: study rationale and design

Background

Almost 50% of Americans have elevated low-density lipoprotein cholesterol (LDL-C). The behaviors required to lower LDL-C levels may be difficult to adhere to if they are inconsistent with spouses' health practices, and, alternatively, may be enhanced by enlisting support from the spouse. This trial extends previous trials by requiring spouse enrollment, teaching spouses how to provide emotional and instrumental support, allowing patients to decide which component of the intervention they would like to receive, and having patients determine their own goals and action plans.

Methods

Veteran outpatients with above-goal LDL-C (N = 250) and their spouses are randomized, as a couple, to receive printed education materials only or the materials plus an 11-month, nurse-delivered, telephone-based intervention. The intervention contains four modules: medication adherence, diet, exercise, and patient-physician communication. Patients decide which modules they complete and in which order; modules may be repeated or omitted. Telephone calls are to patients and spouses separately and occur monthly. During each patient telephone call, patients' progress is reviewed, and patients create goals and action plans for the upcoming month. During spouse telephone calls, which occur within one week of patient calls, spouses are informed of patients' goals and action plans and devise strategies to increase emotional and instrumental support. The primary outcome is patients' LDL-C, measured at baseline, 6 months, and 11 months. Linear mixed models will be used to test the primary hypothesis that an 11-month, telephone-based patient-spouse intervention will result in a greater reduction in LDL-C as compared to printed education materials. Various process measures, including social support, self-efficacy, medication adherence, dietary behavior, and exercise, are also assessed to explain any change, or lack thereof, in LDL-C.

Discussion

Given the social context in which self-management occurs, interventions that teach spouses to provide instrumental and emotional support may help patients initiate and adhere to behaviors that lower their LDL-C levels. Moreover, allowing patients to retain autonomy by deciding which behaviors they would like to change and how may improve adherence and clinical outcomes.

Trial Registration

The ClinicalTrials.gov registration number is NCT00321789.     

Seasonal changes of excitation energy transfer and thylakoid stacking in the evergreen tree Taxus cuspidata: how does it divert excess energy from photosynthetic reaction center?

Photosystems must efficiently dissipate absorbed light energy under freezing conditions. To clarify the energy dissipation mechanisms, we examined energy transfer and dissipation dynamics in needles of the evergreen plant Taxus cuspidata by time-resolved fluorescence spectroscopy. In summer and autumn, the energy transfer processes were similar to those reported in other higher plants. However, in winter needles, fluorescence lifetimes became shorter not only in PSII but also in PSI, indicating energy dissipation in winter needles. In addition, almost the same fluorescence spectra were obtained with different excitation wavelengths. In contrast, the fluorescence spectrum showed a large difference due to excitation wavelength in spring needles. The fluorescence spectrum of spring needles in 550-nm excitation showed similar spectra to that of winter needles, however, red-chlorophyll fluorescence was not observed in chlorophyll excitation. These observations suggest that some complexes with some kind of red-shifted carotenoid and red-chlorophyll unlink from the core complex in spring. Seasonal changes of excitation energy dynamics are also discussed in relation to changes in thylakoid stacking.     

Establishment of cell suspension cultures of yew (<Emphasis Type="Italic">Taxus × media</Emphasis> Rehd. and assessment of their genomic stability

Summary Yew cell suspension culture is used as an alternative source of paclitaxel, an anti-cancer drug. To optimize the initiation protocol, highly dormant yew seeds were germinated in vitro and the seedlings used to establish callus culture. The best sources of explant for callus initiation and growth were seedling stems and roots, and the most successful medium was modified B5 medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Calluses were friable and suitable for establishing cell suspension cultures, which were maintained for over 3 yr. Flow cytometric analysis of nuclear DNA content revealed that 2-yr-old cell suspension cultures consisted predominantly of putative euploid and aneuploid cells coexisting as sub-populations. Additional measurements performed 3 and 7 mo. later revealed further genomic instability, with a tendency towards a higher proportion of cells with elevated nuclear DNA content. In a selected cell line, which showed significant taxane production, the addition of 100 μM jasmonic acid strongly enhanced total taxane production and slightly inibited growth while no effect on nuclear DNA content was noted.