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.     

Why does the bud-galling sawfly,Euura mucronata,attack long shoots?

Summary The bud-galling sawfly, Euura mucronata, attacked longer shoot length classes on its host, Salix cinerea, more frequently than shorter shoots. Shoot length accounted for 76 to 93 percent of the variance in number of galls per 100 shoots in three habitats: forest, watermeadow, and lakeside. The reasons for this pattern were addressed with studies on shoot length in relation to: 1. Number of resources (buds) per shoot; 2. Success in establishment of larvae in galls; 3. Gall size and resources per gall; and 4. Survival of larvae after establishment as influenced by plant resistance and natural enemy attack. The most important factors proved to be success in establishment of larvae, with percent of variance accounted for ranging from 57 to 77 percent in three of four sites where relationships were significant, and survival after establishment of larvae, with variance accounted for ranging from 40 to 54 percent in the same three sites. The pattern of survival was dictated by plant resistance and not by natural enemies. These two additive factors resulted in a general relationship across all sites of increasing emergence of fully developed larvae per cohort as shoot length increased, accounting for 78 percent of the variance. These adaptive advantages to attacking longer shoots are sufficient to account for the pattern of increased probability of shoots being attacked as they increase in length.     

Ethmoidal endocranium in primitive triassic amphibians

Three-dimensionally preserved and chemically prepared skulls and natural casts of representatives of the families Benthosuchidae, Melosauridae, and Capitosauridae yield data on the structure of the ethmoidal endocranium, i. e. of those nasal cranial structures that consisted originally of cartilage. This study demonstrates that the ethmoidal endocranium was principally a dorsoventrally compressed plate, pierced by a broad and oblique canal which communicated anteriorly with the outer dorsal surface by the fenestra endonarina and posteriorly with the mouth cavity by the fenestra endochoanalis(seu foris). The canal was very short, and housed the olfactory organ. The ethmoidal endocranium was connected with the palatoquadrate by the commissura quadratocranialis anterior; there was no lateral ethmoidal commissure, however, in older individuals the anterior section of the palatoquadrate might also contact the postchoanal part of the nasal endocranial skeleton.     

The C/EBP-binding region and adjacent sites regulate expression of the adipose P2 gene in human preadipocytes.

Human preadipocytes contain nuclear factors that specifically bind to the AE-1 sequence, previously demonstrated as an enhancer element in the regulation of adipose P2 gene expression during 3T3 adipose differentiation. By transient transfection and in vivo competition experiments, the trans-acting factors were found to bind either to the C/EBP recognition site in the AE-1 sequence and act as a negative regulator or to the adjacent site (termed 3' AE-1) and act as a positive regulator of adipose P2 gene activity in human preadipocytes.     

Magnetic interaction of manganese with pheophytin anion-radical and chlorophyll cation-radical in reaction centers of the photosystem II

The influence of Mn on saturation curves of ESR spectra of Ph(-) and P(+)(680) at 1-200K in samples with different content of Mn has been studied. An analysis of these data and those on photoinduced changes of fluorescence yield of chlorophyll leads to the conclusion that the Mn-containing centre in Photosystem 2 is a cluster of 4 Mn atoms, two of which can be replaced by Mg(2+) or any other divalent metal. The distances between Mn Na Ph as well as between Mn and P(680) have been estimated.     

The computer program package "SAMSON" for analysis of primary structure of biopolymers

A program package "SAMSON" for the computer analysis of biopolymer primary structures is described. All possible modes of sequence investigation are considered. The programs for sequence comparison are described in some details. The general principles of a program package organisation and of its user interface are also mentioned. For more complete information see Vernoslov S.E. et al. "Program package "SAMSON" for the analysis of the polymer primary structures", parts 1 and 2, Poustchino, ONTI NCBI, 1989.     

Big flies, small repeats: the "Thr-Gly" region of the period gene in Diptera

The region of the clock gene period (per) that encodes a repetitive tract of threonine-glycine (Thr-Gly) pairs has been compared between Dipteran species both within and outside the Drosophilidae. All the non- Drosophilidae sequences in this region are short and present a remarkably stable picture compared to the Drosophilidae, in which the region is much larger and extremely variable, both in size and composition. The accelerated evolution in the repetitive region of the Drosophilidae appears to be mainly due to an expansion of two ancestral repeats, one encoding a Thr-Gly dipeptide and the other a pentapeptide rich in serine, glycine, and asparagine or threonine. In some drosophilids the expansion involves a duplication of the pentapeptide sequence, but in Drosophila pseudoobscura both the dipeptide and the pentapeptide repeats are present in larger numbers. In the nondrosophilids, however, the pentapeptide sequence is represented by one copy and the dipeptide by two copies. These observations fulfill some of the predictions of recent theoretical models that have simulated the evolution of repetitive sequences.      

An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.