Massetolide A biosynthesis in Pseudomonas fluorescens.

Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

Evaluation of antioxidative properties of Geranium macrorrhizum and Potentilla fruticosa extracts in Dutch style fermented sausages.

Antioxidative properties of Geranium macrorrhizum, Potentilla fruticosa and Rosmarinus officinalis (as a reference) extracts were evaluated in Dutch style fermented sausages. Extracts were incorporated into sausages during preparation. The sausages were subsequently fermented, tested and compared to a standard spices mix, traditionally used for the production of such sausages. Formation of the primary oxidation products - peroxides, and secondary - TBARS and hexanal was monitored. The polar extracts from Potentilla showed some antioxidant activity, especially in combination with ascorbate, however the activity was low compared to the standard spices mix. Polar extracts from Geranium showed only negligible antioxidant activity.

Elimination and detoxification of softwood extractives by white-rot fungi.

The ability of several white-rot fungal strains to remove and detoxify acetone extractives (pitch or resin) in Scots pine sapwood was investigated in stationary laboratory batch assays. Fungal pretreatment provided up to 62% total pitch reduction and significant decreases in pitch toxicity. The best strains were Bjerkandera sp. strain Stereum hirsutum and Trametes versicolor that eliminated over 93% of the problematic triglyceride fraction and 58-87% of other lipophilic extractive classes in only 2 weeks. Fungal removal of the wood extractives was accompanied by a 7.4-16.9-fold decrease in their inhibitory effect, as determined in the Microtox bioassay. Wood pretreatment by Bjerkandera sp. and T. versicolor caused limited losses of woody mass (less than 4% in 4 weeks); whereas S. hirsutum led to somewhat higher mass losses (7% in 4 weeks). These results indicate the potential of white rot fungi to control pitch deposition problems in pulping and to reduce the aquatic toxicity caused by naturally-occurring lipophilic extractives in forest industry effluents.

Screening of the needles of different yew species and cultivars for paclitaxel and related taxoids.

The needles of several yew species and cultivars were analysed by high-pressure liquid chromatography for paclitaxel, 10-deacetylpaclitaxel, cephalomannine, baccatin III, 10-deacetylbaccatin III and brevifoliol. About 750 samples were collected from five different locations in the Netherlands and the UK. The results of this screening show a large variation in taxane content between the different species and cultivars. The content of paclitaxel and 10-deacetylbaccatin III varied from 0 to 500 micrograms/g and 0 to 4800 micrograms/g dried needles, respectively. Brevifoliol was found in a very high concentration in Taxus brevifolia. 10-Deacetylpaclitaxel, cephalomannine and baccatin III were found in concentrations ranging from 0 to 500 micrograms/g dried needles.

Preparative isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba.

A chromatographic procedure for the preparative isolation of six different 6-alkylsalicylic acids (syn. ginkgolic acids) with as alkyl substituents C13:0, C15:0, C15:1, C17:1, C17:2 and, tentatively C17:3 from Ginkgo biloba leaves was developed. The procedure consisted of a combination of normal-phase, reversed-phase and argentation chromatography. The compounds were characterised by means of UV, 1H-NMR and 13C-NMR spectroscopy, and mass spectrometry after silylation. A 15 cm C18 RP-HPLC column connected in series with a 20 cm silver(I) loaded cation exchanger HPLC column in combination with the solvent methanol-water (93:7) acidified with 0.1% formic acid was capable of separating the ginkgolic acids C13:0, C15:1, C17:2, C15:0 and C17:1 within 21 min on an analytical scale. The separation is based on a combination of reversed-phase mechanisms and double bond complexation. Detection took place by UV at 311 nm. The separation is a good starting point for the development of a quantitative procedure for the five major ginkgolic acids in Ginkgo leaves and standardised extracts.

Evaluation and comparison of two improved techniques for the on-line detection of antioxidants in liquid chromatography eluates.

Two methods for the on-line detection in HPLC eluates of analytes possessing radical scavenging activity were improved and compared. The instrumental set-up of the method that is based on on-line inhibition of luminol chemiluminescence (CL) by antioxidants was improved using better quality syringe pumps, employing a diode array detector, and introducing a mixing/neutralisation coil and a pulse damper. Sensitivity of the HPLC-CL detection increased by a factor of 4. Post-column neutralisation of eluates improved compatibility of this detection method with acidified HPLC eluents. The second method, which is based on the post-column quenching of 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH*), was improved by readjusting composition and flow-rate of the reagent, mounting an additional pulse damper and detecting unreacted DPPH* with a detector equipped with a tungsten lamp. Purging of the DPPH* solution with He gas prior to analysis was introduced. This led to 30-fold better detection limits. The improved methods were compared with respect to limits of detection, the radical scavenging mechanism involved, compatibility with common HPLC solvents and pH range, and some technical aspects. The techniques described have high potential for the rapid identification of radical scavengers in complex samples like plant extracts.

Phytochemical investigation of Tabernaemontana crassa.

From the stembark of Tabernaemontana crassa the alkaloid ibogaine was isolated as the major component. Ibogaine showed activity against the gram-positive Bacillus subtilis. Conopharyngine was identified as one of the minor compounds.

Screening of antimicrobial activity of some plants belonging to the Apocynaceae and Loganiaceae.

A review is given of the literature on the antimicrobial activity of various indole alkaloids. The data are briefly discussed. A screening was performed of a series of plants belonging to the indole alkaloid rich plant families Apocynaceae and Loganiaceae, 25 materials representing 14 Apocynaceae species and 54 materials representing 41 Strychnos species were investigated. No activity against the yeast or the fungus, used as test organisms was found. A number of extracts showed activity against the gram-positive test organisms, 4 species showed activity against gram-negative bacteria.

Tabernaemontana L. (Apocynaceae): a review of its taxonomy, phytochemistry, ethnobotany and pharmacology.

The taxonomy, phytochemistry, ethnobotany, and pharmacology of the genus Tabernaemontana L. (Apocynaceae) is reviewed. The genus is currently being revised taxonomically; most of the segregate genera are being reunited with it and the number of species that will ultimately be recognized will probably be about 100. All the names encountered in the chemical and ethnobotanical literature have been evaluated as far as possible, and a list is presented of the recognized species and their synonyms. The biogenesis and classification of the indole alkaloids found in Tabernaemontana species is set out and some problems in the determination of their stereochemistry are discussed. To facilitate access to the information, three lists have been compiled: the alkaloids in alphabetical order; the alkaloids in order of increasing molecular weight; and the alkaloids grouped according to their biogenetic classification, together with the species and plant part(s) in which they are known to occur. Biogenetic and chemotaxonomic aspects are briefly considered. A table of the non-alkaloidal constituents is also included. The ethnobotany of individual Tabernaemontana species is outlined and an overall assessment made. Likewise, information on the pharmacology of crude extracts and individual alkaloids from Tabernaemontana species has been assembled and appraised.

Analysis of pharmaceutically relevant taxoids in wild yew trees from Sardinia.

Needles from a series of wild yews (Taxus baccata L.) from Sardinia were investigated for their contents of 10-deacetyl baccatin III (DAB-III, 1), paclitaxel (Taxol) (2) and taxine (3). Despite a common geographical origin, ample variation of the taxoid profile was discovered, and several samples were surprisingly devoid of all terpenoid markers above. This finding is unprecedented within the European yew, while the general lack of taxine might rationalize the observation that most plants investigated are actively and impunently browsed by goats.

A Monte Carlo tool for evaluating VMAT and DIMRT treatment deliveries including planar detectors.

The aim of this work is to describe and validate a new general research tool that performs Monte Carlo (MC) simulations for volumetric modulated arc therapy (VMAT) and dynamic intensity modulated radiation therapy (DIMRT), simultaneously tracking dose deposition in both the patient CT geometry and an arbitrary planar detector system. The tool is generalized to handle either entrance or exit detectors and provides the simulated dose for the individual control-points of the time-dependent VMAT and DIMRT deliveries. The MC simulation tool was developed with the EGSnrc radiation transport. For the individual control point simulation, we rotate the patient/phantom volume only (i.e. independent of the gantry and planar detector geometries) using the gantry angle in the treatment planning system (TPS) DICOM RP file such that each control point has its own unique phantom file. After MC simulation, we obtained the total dose to the phantom by summing dose contributions for all control points. Scored dose to the sensitive layer of the planar detector is available for each control point. To validate the tool, three clinical treatment plans were used including VMAT plans for a prostate case and a head-and-neck case, and a DIMRT plan for a head-and-neck case. An electronic portal imaging device operated in 'movie' mode was used with the VMAT plans delivered to cylindrical and anthropomorphic phantoms to validate the code using an exit detector. The DIMRT plan was delivered to a novel transmission detector, to validate the code using an entrance detector. The total MC 3D absolute doses in patient/phantom were compared with the TPS doses, while 2D MC doses were compared with planar detector doses for all individual control points, using the gamma evaluation test with 3%/3 mm criteria. The MC 3D absolute doses demonstrated excellent agreement with the TPS doses for all the tested plans, with about 95% of voxels having γ <1 for the plans. For planar dosimetry image comparisons, we defined an acceptable pass rate of >90% of percentage pixels with γ <1. We found that over 90% of control points in the plans passed this criterion. In general, our results indicate that the simulation tool is suitable for accurately calculating both patient/phantom doses and planar doses for VMAT dose delivery. The tool will be valuable to check performance and advance the development of in vivo planar detectors for use in measurement-based VMAT dose verification. In addition, the tool can be useful as an independent research tool for VMAT commissioning of the TPS and delivery system.

Desorption electrospray ionisation mass spectrometry: A rapid screening tool for veterinary drug preparations and forensic samples from hormone crime investigations.

Hormone and veterinary drug screening and forensics can benefit from the recent developments in desorption electrospray ionisation (DESI) mass spectrometry (MS). In this work the feasibility of DESI application has been studied. Using a linear ion trap or quadrupole time-of-flight (TOF) MS instrument both full-scan and data-dependent collision-induced dissociation MS(n) spectra were acquired in seconds without sample preparation. Preliminary data are presented for the rapid screening of (pro)hormone supplement samples, an illegal steroid cocktail and forensic samples from veterinary drug investigations. The potential of this DESI approach is clearly demonstrated since compounds observed could be independently confirmed by liquid chromatography/TOFMS with accurate mass measurement, and/or proton nuclear magnetic resonance spectroscopy. Specific concerns related to false-positive and false-negative findings due to limitations in quantification and memory-effects are briefly discussed. It is envisaged that DESI will achieve a prominent role in hormone and veterinary drug analysis in the near future.

Determination of limonin in grapefruit juice and other citrus juices by high-performance liquid chromatography.

A method has been developed for the quantitation of the bitter component limonin in grapefruit juice and other citrus juices. The sample clean-up consisted of centrifugation, filtration and a selective, rapid and reproducible purification with a C2 solid-phase extraction column. The limonin concentration was determined by high-performance liquid chromatography on a C18 column with UV detection at 210 nm. A linear response was obtained from 0.0 to 45 ppm limonin. The minimum detectable amount was 2 ng. The minimum concentration which was detected without concentration with good precision was 0.1 ppm. The method was also used for the determination of limonin in different types of oranges, including navel oranges, mandarins, lemons, limes, pomelos and uglis.

Concentration of Ginkgolides and Bilobalide in Ginkgo biloba Leaves in Relation to the Time of Year.

The concentrations of ginkgolides A, B, and C, and bilobalide were determined in the leaves of 3 different Dutch GINKGO BILOBA trees from late spring until late autumn 1990. The concentration versus harvest time plots were roughly the same for all the compounds in each of the 3 trees. Concentration was lowest in spring and then gradually increased until a maximum in late summer or early autumn was reached. Thereafter the concentration declined until leaf fall. The difference in ginkgolide and bilobalide content between the 3 trees was very high. For ginkgolide B, the pharmacologically most potent compound, the maximum concentration was less than 3, 68 and 204 ppm respectively. Possible reasons for these differences and consequences for phytotherapy are discussed briefly.

Dichomine, a novel type of iboga alkaloid*,**.

From the leaves of TABERNAEMONTANA DICHOTOMA and the leaves and twigs of T. EGLANDULOSA a new alkaloid was isolated which was assigned structure 1 on the basis of spectroscopic and chemical evidence.

Alkaloids from Tabernaemontana psorocarpa.

The isolation of the alkaloids from two different samples of stem bark of Tabernaemontana psorocarpa is described. Both samples contained 16-epi-isositsirikine as the major alkaloid but differed from each other in the number and quantity of the other alkaloids. The following minor alkaloids were identified: 12-methoxy-14,15-dehydro-vincamine, vallesiachotamine, isovallesiachotamine, tetrahydroalstonine, coronaridine and voacangine.

On-line detection of antioxidative activity in high-performance liquid chromatography eluates by chemiluminescence.

Luminol chemiluminescence (CL) was employed for the on-line detection of radical scavengers in HPLC eluates. Optimization of CL reagents and instrumental setup resulted in a steady postcolumn luminol photochemical reaction in the presence of microperoxidase and hydrogen peroxide at pH 10. Quenching of the CL signal was utilized to detect radical scavenging activity of both natural and synthetic antioxidants at the nanogram level. The detection system can be used with isocratic or gradient elution. Several antioxidative compounds were detected in thyme and sage acetone extracts. Quantitative results can be obtained when antioxidants are analyzed at certain concentrations. The method is suitable for rapid screening of antioxidants in crude extracts.

Attraction of Phytoseiulus persimilis (Acari: Phytoseiidae) towards volatiles from various Tetranychus urticae-infested plant species.

Plants infested with the spider mite Tetranychus urticae Koch, may indirectly defend themselves by releasing volatiles that attract the predatory mite Phytoseiulus persimilis Athias-Henriot. Several plants from different plant families that varied in the level of spider mite acceptance were tested in an olfactometer. The predatory mites were significantly attracted to the spider mite-infested leaves of all test plant species. No differences in attractiveness of the infested plant leaves were found for predatory mites reared on spider mites on the different test plants or on lima bean. Thus, experience with the spider mite-induced plant volatiles did not affect the predatory mites. Jasmonic acid was applied to ginkgo leaves to induce a mimic of a spider mite-induced volatile blend, because the spider mites did not survive when incubated on ginkgo. The volatile blend induced in ginkgo by jasmonic acid was slightly attractive to predatory mites. Plants with a high degree of direct defence were thought to invest less in indirect defence than plants with a low degree of direct defence. However, plants that had a strong direct defence such as ginkgo and sweet pepper, did emit induced volatiles that attracted the predatory mite. This indicates that a combination of direct and indirect defence is to some extent compatible in plant species.

Antimicrobially active alkaloids from Tabernaemontana chippii.

From Tabernaemontana chippii root bark, forty-five alkaloids were isolated; thirty-four were fully characterized by means of their spectral data and/or co-tlc; eight alkaloids were new, four of them being 3-hydroxy derivatives of known dimeric voacamine type alkaloids. Most of the twenty-six known alkaloids belonged to the corynanthean, ibogan, or bisindole classes. The structures of eleven other alkaloids--all minor--were only partially elucidated, most of them being new alkaloids. All the dimeric alkaloids were shown to possess strong antimicrobial activity against gram-positive bacteria and moderate to weak activity against gram-negative bacteria.

Phloem sap collection from lettuce (Lactuca sativa L.): Methodology and yield.

Three methods to collect phloem sap on different lettuce lines were optimized and are described in detail. The success ratio for stylectomy of aphids was over 80% through the combination of a specially designed setup and electrical penetration graphs to monitor phloem sap ingestion. For unknown reasons on some lettuce lines stylets never showed sustained exudation. There were clear differences in stylet exudation between two aphid species on the same lettuce line. Honeydew collection in hexadecane made accurate quantitative analysis possible; samples were large and clean, but biotransformed. The EDTA chelation method produced large samples, but dilution, oxidation, and impurities from the wound surface reduced the reliability.