Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.     

Biosynthesis of 1-alkenes in the defensive secretions ofTribolium confusum (Tenebrionidae); stereochemical implications

Summary The terminally unsaturated hydrocarbons of the defensive secretion ofTribolium confusum are biosynthesized from fatty acids by oxidative decarboxylation. The process involves an enantiospecific cleavage of the C–H bond of thepro-(S) C(3)–H atom and simultaneous decarboxylation of the acid into an 1-alkene and carbon dioxide via ananti-periplanar transition state geometry (anti-elimination). The stereochemistry of this biotranformation is identical in all respects with the same reaction in higher plants. The mechanism seems to be of general importance for the biosynthesis of many vinylic substructures of natural products from oxygen-containing precursors.     

Airborne methyl jasmonate stimulates the biosynthesis of furanocoumarins in the leaves of celery plants (Apium graveolens)

When leaves ofApium graveolens are exposed to vapours of methyl jasmonate (MeJa) or fed through the petiole with aqueous solutions of jasmonic acid (Ja), the levels of natural furanocoumarins, in particular xanthotoxin and bergapten, are greatly increased. The induction becomes manifest after application of 20 nmol of either Meja or Ja. Levels of xanthotoxin and bergapten begin to increase approximately 24 h after application of the inducer. Maximum concentration of the two furanocoumarins (40–70 fold increase) is reached after 4–6 days. This pronounced effect of volatile MeJa on the physiological status of the celery leaf may be considered as representative of a build-up of chemical defenses, in an undamaged plant, after receipt of airborne signals from damaged or undamaged, but fragrant plants.     

Identification and biological significance of 4-methyl-3Z,5-hexadienoic acid produced by males of the gall-forming tephritidsUrophora cardui (L.) andUrophora stylata (Fab.) (Diptera: Tephritidae)

Summary A new natural substance has been identified in the rectal ampullae of gall-forming fruit flies. The substance was found to be the only volatile compound in the rectal ampulla of maleUrophora cardui andUrophora stylata. GC-MS methods were used to characterize its structure as 4-methyl-3Z,5-hexadienoic acid. Physiological parameters such as the amount of the acid at different ages and under different conditions were investigated. The biological significance of the new volatile as an arresting pheromone was tested in several bioassays. The arrestant function could not be established, but the results gave hints of a territorial function between conspecific males. The results are discussed with respect to gland morphology and predictions of communication models among fruit flies.     

Giffordene, 2Z, 4Z, 6E, 8Z-undecatetraene,is the odoriferous principle of the marine brown algaGiffordia mitchellae

Summary Giffordene, 2Z, 4Z, 6E, 8Z-undecatetraene, and five stereoisomers are new odoriferous C₁₁H₁₆-hydrocarbons from the marine brown algaGiffordia mitchellae. Their isolation and identification is described.Acknowledgments. We thank Prof. G. Ohloff for kindly providing us with synthetic 1,3,5,7-undecatetraenes and Prof. P. Schreier for GC/FTIR-analyses.     

Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis

Tumour growth requires accompanying expansion of the host vasculature, with tumour progression often correlated with vascular density. Vascular endothelial growth factor (VEGF) is the best-characterized inducer of tumour angiogenesis. We report that VEGF dynamically regulates tumour endothelial expression of Delta-like ligand 4 (Dll4), which was previously shown to be absolutely required for normal embryonic vascular development. To define Dll4 function in tumour angiogenesis, we manipulated this pathway in murine tumour models using several approaches. Here we show that blockade resulted in markedly increased tumour vascularity, associated with enhanced angiogenic sprouting and branching. Paradoxically, this increased vascularity was non-productive-as shown by poor perfusion and increased hypoxia, and most importantly, by decreased tumour growth-even for tumours resistant to anti-VEGF therapy. Thus, VEGF-induced Dll4 acts as a negative regulator of tumour angiogenesis; its blockade results in a striking uncoupling of tumour growth from vessel density, presenting a novel therapeutic approach even for tumours resistant to anti-VEGF therapies.     

Herbivory-induced volatiles elicit defence genes in lima bean leaves

In response to herbivore damage, several plant species emit volatiles that attract natural predators of the attacking herbivores. Using spider mites (Tetranychus urticae) and predatory mites (Phytoseiulus persimilis), it has been shown that not only the attacked plant but also neighbouring plants are affected, becoming more attractive to predatory mites and less susceptible to spider mites. The mechanism involved in such interactions, however, remains elusive. Here we show that uninfested lima bean leaves activate five separate defence genes when exposed to volatiles from conspecific leaves infested with T. urticae, but not when exposed to volatiles from artificially wounded leaves. The expression pattern of these genes is similar to that produced by exposure to jasmonic acid. At least three terpenoids in the volatiles are responsible for this gene activation; they are released in response to herbivory but not artificial wounding. Expression of these genes requires calcium influx and protein phosphorylation/dephosphorylation.     

A QTL influencing F cell production maps to a gene encoding a zinc-finger protein on chromosome 2p15

F cells measure the presence of fetal hemoglobin, a heritable quantitative trait in adults that accounts for substantial phenotypic diversity of sickle cell disease and beta thalassemia. We applied a genome-wide association mapping strategy to individuals with contrasting extreme trait values and mapped a new F cell quantitative trait locus to BCL11A, which encodes a zinc-finger protein, on chromosome 2p15. The 2p15 BCL11A quantitative trait locus accounts for 15.1% of the trait variance.