Sesquiterpene synthases in Artemisia annua: Cloning, Expression, and Characterisation of the Recombinant Enzymes

Plants produce a multitude of different compounds that may have biotic effects. Artemisia annua, produces a compound used as a drug against multi-drug-resistant strains of Plasmodium falciparum, the parasite causing malaria. The compound, artemisinin, is a sesquiterpene and is an essential component for the production of artemisinin drugs against malaria. In a first critical step of the biosynthesis of artemisinin, a sesquiterpene carbon structure is formed from the substrate farnesyl diphosphate. Enzymes catalysing sesquiterpene formation, are called sesquiterpene synthases. The common substrate farnesyl diphosphate may, enzymatically, be converted to over 300 different sesquiterpene hydrocarbons. Sesquiterpene synthases are thought to be regulatory enzymes for the intracellular carbon metabolic flux in plants. Epi-cedrol synthase and amorpha-4,11-diene synthase have been cloned from Artemisia annua. The cDNA for these clones have been expressed in E. coli and the recombinant enzymes have been identified and characterised. Recombinant epi-cedrol synthase catalyses the formation of both olefinic (3%) and oxygenated (97%) products from farnesyl diphosphate. The major product formed by this enzyme is 8-epi-cedrol. The other recombinant enzyme catalyses the synthesis of amorpha-4,11-diene, as a major product. Amorpha-4,11-diene has the correct structure to be the precursor for artemisinin. A reaction mechanism starting with a C1,C6 closure is proposed for both recombinant terpene synthases. Both recombinant enzymes demonstrate kinetic characteristics comparable to other cloned sesquiterpene synthases. Fusing the open reading frames of farnesyl diphospahte synthase and epi-aristolochene synthase (a sesquiterpene synthase from tobacco) in different orders resulted in two bifunctional enzymes. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (G-S-G) to introduce a linker between the two open reading frames. A channeling of the intermediate farnesyl diphosphate by the bifunctional enzymes was established

Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes

Effect of different radiation fractionation schedules on metastases from an oesophageal carcinoma

Subcutaneous metastases from an oesophageal carcinoma were irradiated using different schedules. The results have to be evaluated with greatest caution but indicate that with the same CRE value, few fractions caused less skin reactions than several, and the size of the shoulder of the cell survival curve was of the order of 0.7 Gy

Combined Transcript and Metabolite Analysis Reveals Genes Involved in Spider Mite Induced Volatile Formation in Cucumber Plants

Many plants have an indirect defense against herbivores by emitting volatiles that attract carnivorous enemies of the herbivores. In cucumber (Cucumis sativus) the production of carnivore attractants can be induced by herbivory or jasmonic acid spraying. From the leaves of cucumber plants with and without spider mite infestation, two subtractive cDNA libraries were made that were enriched in cDNA fragments up- or down-regulated by spider mite infestation. A total of 713 randomly selected clones from these libraries were used to make a cDNA microarray. Subsequently, cucumber plants were sprayed with jasmonic acid, mechanically damaged, infested with spider mites, or left untreated (control). Leaf samples were taken at a range of different time points, and induced volatile compounds and mRNA (from the same leaves) were collected. cDNAs prepared from the mRNA were hybridized to the clones on the microarray. The resulting gene expression profiles were analyzed in combination with volatile production data in order to gain insight in the possible involvement of the studied genes in the synthesis of those volatiles. The clones on the microarray and the induced cucumber volatiles could be grouped into a number of clusters in which specific biosynthetic genes clustered with the product of that pathway. For example, lipoxygenase cDNA clones clustered with the volatile (Z)-3-hexenyl acetate and the volatile sesquiterpene (E,E)- α-farnesene clustered with an up-regulated sesquiterpene synthase fragment. This fragment was used to screen a cDNA library which resulted in the cloning of the cucumber (E,E)-α-farnesene and (E)-β-caryophyllene synthases. The use of combined global gene expression analysis and metabolite analysis for the discovery of genes involved in specific biosynthetic processes is discussed

The quality assurance process for the ARTSCAN head and neck study - A practical interactive approach for QA in 3DCRT and IMRT

Aim: This paper describes the quality assurance (QA) work performed in the Swedish multicenter ARTSCAN (Accelerated RadioTherapy of Squamous cell CArcinomas in the head and Neck) trial to guarantee high quality in a multicenter study which involved modern radiotherapy such as 3DCRT or IMRT. Materials and methods: The study was closed in June 2006 with 750 randomised patients. Radiation therapy-related data for every patient were sent by each participating centre to the QA office where all trial data were reviewed, analysed and stored. In case of any deviation from the protocol, an interactive process was started between the QA office and the local responsible clinician and/or physicist to increase the compliance to the protocol for future randomised patients. Meetings and workshops were held on a regular basis for discussions on various trial-related issues and for the QA office to report on updated results. Results and discussion: This review covers the 734 patients out of a total of 750 who had entered the study. Deviations early in the study were corrected so that the overall compliance to the protocol was very high. There were only negligible variations in doses and dose distributions to target volumes for each specific site and stage. The quality of the treatments was high. Furthermore, an extensive database of treatment parameters was accumulated for future dose-volume vs. endpoint evaluations. Conclusions: This comprehensive QA programme increased the probability to draw firm conclusions from our study and may serve as a concept for QA work in future radiotherapy trials where comparatively small effects are searched for in a heterogeneous tumour population

Two-year results from a Swedish study on conventional versus accelerated radiotherapy in head and neck squamous cell carcinoma The ARTSCAN study

Background and purpose: Studies on accelerated fractionation (AF) in head and neck cancer have shown increased local control and survival compared with conventional fractionation (CF), while others have been non-conclusive. In 1998 a national Swedish group decided to perform a randomised controlled clinical study of AF. Materials and methods: Patients with verified squamous cell carcinoma of the oral cavity, oropharynx, larynx (except glottic T1 -T2, N0) and hypopharynx were included. Patients with prior chemotherapy or surgery were excluded. Patients were randomised to either CF (2 Gy/day, 5 days/week for 7 weeks, total dose 68 Gy) or to AF (1.1 Gy + 2.0 Gy/day, 5 days/week for 4.5 weeks, total dose 68 Gy). An extensive quality assurance protocol was followed throughout the study. The primary end point was loco-regional tumour control (LRC) at two years after treatment. Results: The study was closed in 2006 when 750 patients had been randomised. Eighty-three percent of the patients had stages III-IV disease. Forty eight percent had oropharyngeal, 21% laryngeal, 17% hypopharyngeal and 14% oral cancers. There were no significant differences regarding overall survival (OS) or LRC between the two regimens. The OS at two years was 68% for AF and 67% for CF. The corresponding figures for LRC were 71% and 67%, respectively. There was a trend towards improved LRC for oral cancers treated (p = 0.07) and for large tumours (T3-T4) (p = 0.07) treated with AF. The AF group had significantly worse acute reactions, while there was no significant increase in late effects. Conclusion: Overall the AF regimen did not prove to be more efficacious than CF. However, the trend towards improved results in AF for oral cancers needs to be further investigated. (c) 2011 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 100 (2011) 41-4