Worldwide, increasing numbers of insects have evolved resistance to a wide range of pesticides, which hampers their control in the field and, therefore, threatens agriculture. Members of the carboxylesterase and cytochrome P450 monooxygenase superfamilies are prominent candidates to confer metabolic resistance to pyrethroid insecticides. Both carboxylesterases and P450 enzymes have been shown to be involved in pyrethroid resistance in Australian Helicoverpa armigera, the noctuid species possessing by far the most reported resistance cases worldwide. However, specific enzymes responsible for pyrethroid resistance in field populations of this species have not yet been identified. Here, we show that the resistance toward fenvalerate in an Australian strain of H. armigera is due to a unique P450 enzyme, CYP337B3, which arose from unequal crossing-over between two parental P450 genes, resulting in a chimeric enzyme. CYP337B3 is capable of metabolizing fenvalerate into 4′-hydroxyfenvalerate, which exhibits no toxic effect on susceptible larvae; enzymes from the parental P450 genes showed no detectable fenvalerate metabolism. Furthermore, a polymorphic H. armigera strain could be bred into a susceptible line possessing the parental genes CYP337B1 and CYP337B2 and a resistant line possessing only CYP337B3. The exclusive presence of CYP337B3 in resistant insects of this strain confers a 42-fold resistance to fenvalerate. Thus, in addition to previously documented genetic mechanisms of resistance, recombination can also generate selectively advantageous variants, such as this chimeric P450 enzyme with an altered substrate specificity leading to a potent resistance mechanism.pest management | cotton bollworm O ne of the main threats of agriculture nowadays is the rapid development of resistance of pest insect species to control agents worldwide. The cotton bollworm, Helicoverpa armigera (Hübner), is the noctuid species possessing by far the most reported cases of insecticide resistance worldwide with evolved resistance against pyrethroids, organophosphates, carbamates, organochlorines (www.pesticideresistance.org), and recently against the macrocyclic lactone spinosad (1) and Bacillus thuringiensis-derived toxins (2). This capacity is partly due to its distribution, which is one of the widest for any agricultural insect pest species, covering Africa, the Middle East, southern Europe, India, central and southeastern Asia, eastern and northern Australia, New Zealand, and many eastern Pacific Islands (3). In addition, H. armigera is a significant pest of cotton, the single crop most intensively sprayed with insecticides. Almost 30% of all pesticides used worldwide are directed against this insect pest (4). In Australia, the economic losses due to direct yield reduction and pest management of H. armigera and endemic H. punctigera were estimated to be approximately A$150 million in 1990-1991 (5). In addition, H. armigera is highly polyphagous, feeding on 72 known host plant species distributed in 29 families in Australia (6).Af...

No abstract

Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts by a series of extraction and prepurification steps. The pharmacological activity is attributed to a number of flavonoid glycosides and unique terpene trilactones (TTLs), with largely uncharacterized pharmacological profiles on targets involved in neurological disorders. It is therefore important to complement existing targeted analytical methods for analysis of Ginkgo biloba preparations with alternative technology platforms for their comprehensive and global characterization. In this work, 1H NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that 1H NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone-d6. Unexpected or unwanted extract constituents were also easily identified in the 1H NMR spectra, which contrasts traditional methods that depend on UV absorption or MS ionizability and usually require availability of reference standards. Automated integration of 1H NMR spectral segments (buckets or bins of 0.02 ppm width) provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that 1H NMR-based metabolomics is an attractive method for non-selective and comprehensive analysis of Ginkgo extracts.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-009-0195-x) contains supplementary material, which is available to authorized users.

Here we provide evidence that aATA(8,24) (3a-acetyloxy-tir-8,24-dien-21-oic acid) inhibits Akt/mammalian target of rapamycin (mTOR) signaling. aATA(8,24) and other tirucallic acids were isolated from the acetylated extract of the oleo gum resin of Boswellia serrata to chemical homogeneity. Compared with related tirucallic acids, aATA(8,24) was the most potent inhibitor of the proliferation of androgen-insensitive prostate cancer cells in vitro and in vivo, in prostate cancer xenografted onto chick chorioallantoic membranes. aATA(8,24) induced loss of cell membrane asymmetry, caspase-3 activation, and DNA fragmentation in vitro and in vivo. These effects were selective for cancer cells, because aATA(8,24) exerted no overt toxic effects on peripheral blood mononuclear cells or the chick embryo. At the molecular level, aATA(8,24) inhibited the Akt1 kinase activity. Prior to all biochemical signs of cellular dysfunction, aATA(8,24) induced inhibition of the Akt downstream target mTOR as indicated by dephosphorylation of S6K1. This event was followed by decreased expression of cell cycle regulators, such as cyclin D1, cyclin E, and cyclin B1, as well as cyclindependent kinases CDK4 and CDK2 and phosphoretinoblastoma protein, which led to inhibition of the cell-cycle progression. In agreement with the mTOR inhibition, aATA(8,24) and rapamycin increased the volume of acidic vesicular organelles. In contrast to rapamycin, aATA(8,24) destabilized lysosomal and mitochondrial membranes and induced reactive oxygen species production in cancer cells. The ability of aATA(8,24) to inhibit Akt/ mTOR signaling and to induce simultaneously oxidative stress could be exploited for the development of novel antitumor therapeutics with a lower profile of toxic side effects.

Hairy root (HR) cultures are quickly evolving as fundamental research tool and as bio-based production system for secondary metabolites. In this study, an efficient protocol for establishment and elicitation of anthocyanin-producing HR cultures from black carrot was established. Taproot and hypocotyl explants of four carrot cultivars were transformed using wild-type Rhizobium rhizogenes. HR growth performance on plates was monitored to identify three fast-growing HR lines, two originating from root explants (lines NB-R and 43-R) and one from a hypocotyl explant (line 43-H). The HR biomass accumulated 25- to 30-fold in liquid media over a 4-week period. Nine anthocyanins and 19 hydroxycinnamic acid derivatives were identified and monitored using UPLC-PDA-TOF during HR growth. Adding ethephon, an ethylene-releasing compound, to the HR culture substantially increased the anthocyanin content by up to 82% in line 43-R and hydroxycinnamic acid concentrations by over 20% in line NB-R. Moreover, the activities of superoxide dismutase and glutathione-S-transferase increased in the HRs in response to ethephon, which could be related to the functionality and compartmentalisation of anthocyanins. These findings present black carrot HR cultures as a platform for the in vitro production of anthocyanins and antioxidants and add insight into the regulation of secondary metabolism in black carrot.