Detoxification of naturally occurring chromenes in larvae of the generalist herbivore Spodoptera littoralis (Noctuidae)

Five naturally occurring chromenes from the Asteraceae including the insecticidal compounds precocene II (1) and encecalin (2) were administered to last instar larvae of Spodoptera littoralis via the food or by topical contact. Metabolites formed and excreted via the frass were analysed by GC-MS and by direct comparison with reference compounds obtained by partial synthesis. In total 28 different metabolites were identified, many of them reported here for the first time. All metabolites detected originated from phase I reactions (most probably catalysed by Cytochrom P-450-dependent monooxygenases) by hydroxylation, demethylation or reduction of the parent chromenes. The resulting metabolites can be regarded as detoxification products based on previous structure-activity studies. The increased polarity of the metabolites will furthermore facilitate their excretion by the larvae compared to the more apolar parent chromenes. The largest number of metabolites (eight for each compound) was detected following oral treatment with precocene II and encecalin respectively. 3-Monool as well as 3,4-trans-diol derivatives predominated in the frass of larvae treated with the latter compounds whereas the 6-hydroxyethyl derivatives were the major metabolites of the other chromenes investigated. The patterns of metabolites originating from precocene II or encecalin were the same following oral application or topical treatment.     

Dating branches on the Tree of Life using DNA

The use of DNA sequences to estimate the timing of evolutionary events is increasingly popular, although it is fraught with practical difficulties. But the exponential growth of relevant information and improved methods of analysis are providing increasingly reliable sequence-derived dates, and it may become possible to reconcile fossil-derived and molecular estimates of divergence times within the next few years.     

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg⁶², from an adjacent subunit. Notably, Arg⁶² must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.     

Accumulation of the chalcone isosalipurposide in primary leaves of barley flavonoid mutants indicates a defective chalcone isomerase

Mutants defective in flavonoid biosynthesis have become increasingly useful in elucidating the potential functions of these compounds in plants. To define the role of flavonoids as UV-B protectants in barley, we have screened part of the collection of proanthocyanidin-free barley mutants at the Carlsberg Research Laboratory, Copenhagen, Denmark. The four mutants ant 30–245, ant 30–272, ant 30–287 and ant 30–310 showed drastically reduced flavonoid levels in the primary leaf as compared to their corresponding parent varieties, and in addition accumulated a new mutant-specific phenolic compound which was identified as the chalcone glucoside isosalipurposide. Results from diallelic crosses indicate that all four mutants belong to the same new complementation group, which is designated as the Ant 30 locus. This gene has not earlier been described in barley. The data presented suggest a defective chalcone isomerase gene for the observed flavonoid pattern in leaves of ant 30 mutants.     

Assay of molybdenum cofactor of barley

Conditions for assay of molybdenum cofactor in barley shoot extracts in the presence of molybdate (25 mM N₂MoO₄) and the sulphydryl-group protector, reduced glutathione (5 mM) were optimized. Both total Mo-cofactor (assayed after heat-treatment of cell-free extracts) and ‘free’ Mo-cofactor (assayed in untreated cell-free extracts) were assayed. Compared to control plants grown in the absence of an exogenous nitrogen source total Mo-cofactor levels increased around 70 % when plants were grown for 4 days in the presence of either 15 mM KNO₃ or 15 mM NH₄NO₃. Growth in the presence of 15 mM (NH₄)₂SO₄ did not affect the Mo-cofactor level. Very similar results were seen when plants were transferred to these nitrogen sources for 24 hr after previous growth in the absence of an exogenous nitrogen source. In contrast ‘free’ Mo-cofactor levels of both KNO₃ and NH₄NO₃-treated plants were increased 2-3-fold over untreated controls. Growth in the presence of (NH₄)₂SO₄ did not affect the ‘free’ Mo-cofactor level.     

Determination and quantification of monoterpenoids secreted into the medium of cell cultures of thuja occidentalis

Cell cultures of Thuja occidentalis excrete monoterpenoids into the culture medium. Highly volatile monoterpene hydrocarbons were only detectable in the presence of Miglyol, a water-insoluble non-toxic trap for lipophilic compounds. In the presence of Miglyol the levels of oxygenated monoterpenoids were two-fold higher than in dichloromethane-extracts of culture media. The cells excrete up to 3 mg regular monoterpenoids/g dry wt/day. Continuous extraction of lipophilic compounds by Miglyol also allowed quantification of a Tris—thujaplicinato—(Fe III)-complex.