Absolute configuration of the creatonotines and callimorphines, two classes of arctiid-specific pyrrolizidine alkaloids

Arctiids which as larvae sequester pyrrolizidine alkaloids (PAs) from their food plants are known to synthesize insect-specific PAs by esterifying necine bases derived from plant PAs with necic acids of insect origin. There are two classes of insect PAs, the creatonotines and the callimorphines. The creatonotines contain as necic acids either 2-hydroxy-3-methylbutyric acid (creatonotine A) or 2-hydroxy-3-methylpentanoic acid (creatonotine B). The three known callimorphines contain 2-hydroxy-2-methylbutanoic acid whose hydroxyl group can be either free (deacetylcallimorphine) or acetylated (callimorphine) or propionylated (homocallimorphine). Insect PAs are assumed to play an important role in the recycling of plant derived necine bases and the processing by trans-esterification of PA monoesters that cannot be directly transmitted to the insect's pupal and adult life-stages. The absolute configuration of the insect-specific necic acids was elucidated in the context of the suggested role of the insect PAs as insect-made mimics of plant monoester PAs of the lycopsamine type. For this purpose all needed stereoisomers were synthesized and a gas chromatography-mass spectrometry (GC-MS) method was established that allows the enantioselective separation and assignment of the stereochemistry of all insect specific necic acids as their methyl esters. The method could also be applied to the GC-MS analysis of the intact alkaloids which were hydrolyzed during injection and converted into their methyl esters. Analysis of the creatonotines and callimorphines isolated from the polyphagous arctiids Estigmene acrea and Grammia geneura that were fed with pure PAs and defined PA mixtures revealed the following absolute configuration: the callimorphines and creatonotine A were present in 2'R configuration, whereas creatonotine B was found as mixture of (2'R, 3'S)- and (2'S, 3'S)-stereoisomers. The ratio of 2'S to 2'R was extremely variable ranging from 98% S to 94% R. The cause of the lack of stereospecificity is discussed particularly in respect of a possible epimerization of the hydroxyl group at C-2' in analogy to the known epimerization at C-3' of plant acquired PAs of the lycopsamine type.     

Actin is not required for nanotubular protrusions of primary astrocytes grown on metal nano-lawn

We used sub-micron metal rod decorated surfaces, 'nano-lawn' structures, as a substrate to study cell-to-cell and cell-to-surface interactions of primary murine astrocytes. These cells form thin membranous tubes with diameters of less than 100 nm and a length of several microns, which make contact to neighboring cells and the substrate during differentiation. While membrane protrusions grow on top of the nano-lawn pillars, nuclei sink to the bottom of the substrate. We observed gondola-like structures along those tubes, suggestive of their function as transport vehicles. Elements of the cytoskeleton such as actin fibers are commonly believed to be essential for triggering the onset and growth of tubular membrane protrusions. A rope-pulling mechanism along actin fibers has recently been proposed to account for the transport or exchange of cellular material between cells. We present evidence for a complementary mechanism that promotes growth and stabilization of the observed tubular protrusions of cell membranes. This mechanism does not require active involvement of actin fibers as the formation of membrane protrusions could not be prevented by suppressing polymerization of actin by latrunculin B. Also theoretically, actin fibers are not essential for the growing and stability of nanotubes since curvature-driven self-assembly of interacting anisotropic raft elements is sufficient for the spontaneous formation of thin nano-tubular membrane protrusions.     

Crystal Structures of the p21-activated kinases PAK4, PAK5, and PAK6 reveal catalytic domain plasticity of active group II PAKs

p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4-6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix alphaC, a key regulatory element of kinase function, resulted in an additional helical turn at the alphaC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, alphaC, and the activation segment and firmly anchor alphaC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5.     

The structural coupling between ATPase activation and recovery stroke in the myosin II motor

Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates approximately 65 degrees . Simultaneous with this "recovery stroke," myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 A-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.     

Spontaneous regeneration of dry grasslands on set-aside fields

Dry grasslands are among the most threatened plant communities of Central Europe. We explore the time scale of spontaneous regeneration of dry grasslands on abandoned fields in an area of Central Europe, where also ancient grassland communities occur (Saxony-Anhalt, Germany). On three permanent plots with shallow soils we monitored during 10 years change of species composition and analysed whether spontaneous succession leads to assemblages similar to the ancient dry grassland communities in the direct surroundings. We found that dry grassland species are able to invade the permanent plots and during the 10 years of succession the number of dry grassland species increased. But even after 10 years there was a clear difference between ancient dry grassland communities and the assemblages on the permanent plots. Our findings suggest two important conclusions: First, spontaneous succession on abandoned fields is a cheap possibility for the conservation of some dry grassland species, at least on shallow soils. Second, the time scale of the regeneration process, however, is rather long. Hence, conservation of remnants of ancient grassland communities needs special attention.     

A plastid-localized glycogen synthase kinase 3 modulates stress tolerance and carbohydrate metabolism

Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress.     

Discovery of 5-HT6 receptor ligands based on virtual HTS

Based on a pharmacophore alignment on a 5-HT(6) ligand applying 4SCan technology, a new lead series was identified and further structurally investigated. K(i)s down to 8 nM were achieved.     

Centrosome-associated NDR kinase regulates centrosome duplication

Human NDR kinases are upregulated in some cancer types, yet their functions still remain undefined. Here, we report the first known function of a mammalian NDR kinase by demonstrating that human NDR directly contributes to centrosome duplication. A subpopulation of endogenous NDR localizes to centrosomes in a cell-cycle-dependent manner. Overexpression of NDR resulted in centrosome overduplication in a kinase-activity-dependent manner, while expression of kinase-dead NDR or depletion of NDR by small interfering RNA (siRNA) negatively affected centrosome duplication. By targeting NDR to the centrosome, we show that the centrosomal pool of NDR is sufficient to generate supernumerary centrosomes. Furthermore, our data indicate that NDR-driven centrosome duplication requires Cdk2 activity and that Cdk2-induced centrosome amplification is affected upon reduction of NDR activity. Overall, considering that centrosome overduplication is linked to cellular transformation, our observations may also provide a molecular link between mammalian NDR kinases and cancer.