Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

A rapid method for the purification of supercoiled PM2 DNA by affinity chromatography on H1 histone covalently coupled to agarose.

A simple and rapid method is described for the purification of supercoiled PM2 DNA by affinity chromatography on columns of H1 histone covalently coupled to agarose. The method does not require the use of intercalating agents or ultracentrifugation procedures. Under the conditions most appropriate for purification, elution is carried out in a single step with buffered 0.7 M NaCl after the sample has been loaded onto the column in buffered 0.2 M NaCl. The DNA eluted at the higher salt concentration consists of supercoiled closed circular DNA at greater than 90% purity independently of the ratio of supercoiled to nicked circular DNA in the input mixture.     

Metazoan parasites of Engraulis ringens as tools for stock discrimination along the Chilean coast

The value of the metazoan parasites of the anchoveta Engraulis ringens as biological tags for stock discrimination is assessed. Three hundred and fifty-nine specimens, obtained from six landing ports in northern and central Chile (Arica, Antofagasta, Caldera, Coquimbo, Valparaiso and Talcahuano) covering a latitudinal gradient of c . 18°, were analysed. Six metazoan parasite species (three ectoparasites and three endoparasites) were obtained. A correspondence analysis suggested that anchoveta from northern and central Chile are comprised of two stocks. Identification of stocks was largely based on qualitative differences (absence in southern localities of the monogenean Pseudanthocotyloides heterocotyle , a parasite proper of engraulids, and the copepod Caligus sp.) and quantitative differences in the prevalence of infection for the remaining species. Inertia and mass value for the correspondence analysis supported the hypothesis of two stocks.     

Characteristics of Pustula tragopogonis (syn. Albugo tragopogonis) Newly Occurring on Cultivated Sunflower in Germany

In temperate climates, Pustula tragopogonis is rarely found on cultivated sunflower. In Europe, it was so far of little economic impact on other Asteraceae, except for some regions in the Mediterranean. In 2003, P. tragopogonis was found for the first time in sunflower fields in southern Germany. The pathogen has a widespread occurrence there, especially in the region around Stuttgart, BW. Fatty acid profiling, ultrastructural investigation and ITS sequencing revealed a high similarity to an 2002 isolate from southern Africa and an 2005 isolate from Australia, but revealed significant differences to P. tragopogonis s.l. on Cirsium arvense, a common weed, growing on or in the vicinity of sunflower fields in Germany. P. tragopogonis from this host can therefore be excluded from being the source of the reported infection.     

Inhibition of dihydropyridine-sensitive calcium channels by the plant alkaloid ryanodine

At micromolar concentrations, ryanodine interacts with the dihydropyridine receptor of rabbit skeletal muscle transverse tubules. Ryanodine displaces specifically bound [3H]PN200-110 with an apparent inhibition constant of approx. 95 microM and inhibits dihydropyridine-sensitive calcium channels in the same preparation with an IC50 of approx. 45 microM. These concentrations of ryanodine are approximately three orders of magnitude higher than those required to saturate binding of the alkaloid to the ryanodine receptor of sarcoplasmic reticulum and to open the calcium release channel of sarcoplasmic reticulum (i.e. 20 nM (1988) J. Gen. Physiol. 92, 1-26). Thus at sufficiently high dose, ryanodine may affect SR as well as plasma membrane Ca permeabilities.     

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.     

Determinants of epithelial cell volume

Epithelial cell volume is determined by the concentration of intracellular, osmotically active solutes. The high water permeability of the cell membrane of most epithelia prevents the establishment of large osmotic gradients between the cell and the bathing solutions. Steady-state cell volume is determined by the relative rates of solute entry and exit across the cell membranes. Inhibition of solute exit leads to cell swelling because solute entry continues; inhibition of solute entry leads to cell shrinkage because solute exit continues. Cell volume is then a measure of the rate and direction of net solute movements. Epithelial cells are also capable of regulation of the rate of solute entry and exit to maintain intracellular composition. Feedback control of NaCl entry into Necturus gallbladder epithelial cells is demonstrable after inhibition of the Na,K-ATPase or reduction in the NaCl concentration of the serosal bath. Necturus gallbladder cells respond to a change in the osmolality of the perfusion solution by rapidly regulating their volume to control values. This regulatory behavior depends on the transient activation of quiescent transport systems. These transport systems are responsible for the rapid readjustments of cell volume that follow osmotic perturbation. These powerful transporters may also play a role in steady-state volume regulation as well as in the control of cell pH.