Effects of Gamma Stress and Carbon Dioxide on Eight Bioactive Flavonoids and Photosynthetic Efficiency in <Emphasis Type="Italic">Centella asiatica</Emphasis>

Increased atmospheric CO₂ and gamma irradiation have a significant impact on the plant photosynthetic apparatus and organic compound production. In this study, we evaluated the effect of elevated CO₂ on the photosynthetic efficiency and production of defensive secondary metabolites (flavonoids) induced by gamma irradiation as a physical elicitor in Centella asiatica. Irradiated and non-irradiated 10-week-old plants of C. asiatica were exposed to 400 and 800 μmol mol^?1 of atmospheric CO₂ in growth chambers for 2 h every day until six weeks. A CO₂-enriched atmosphere initially improved the photosynthetic efficiency and ameliorated the detrimental impact of gamma irradiation on the photosynthetic apparatus, increasing carbon allocation into the flavonoid pathway. Elevated CO₂ combined with gamma irradiation resulted in the highest concentration of flavonoids in C. asiatica tissues compared with the other treatments. There was an enhancement in rutin (2.49 fold), naringin (2.15 fold), fisetin (4.07 fold), and morin (4.62 fold) with rising CO₂ concentrations from 400 to 800 μmol mol^?1 in the irradiated plants. With increasing CO₂ concentration, the compensation point and the respiration declined, whereas the apparent quantum yield and the maximum net photosynthesis (A _max) rate increased. The efficiency of photosystem II (PSII) was improved in the irradiated plants grown under high concentrations of CO₂. The total carbohydrate concentration reached the maximum value at the highest level of CO₂, followed by gamma irradiation combined with the highest level of CO₂. Irradiated plants of C. asiatica grown under elevated CO₂ could be superior to non-irradiated plants due to increased carbon availability both for the flavonoid biosynthesis and for the photosynthetic pathway.     

A rare polyglycine type II‐like helix motif in naturally occurring proteins

Common structural elements in proteins such as α‐helices or β‐sheets are characterized by uniformly repeating, energetically favorable main chain conformations which additionally exhibit a completely saturated hydrogen‐bonding network of the main chain NH and CO groups. Although polyproline or polyglycine type II helices (PP_II or PG_II) are frequently found in proteins, they are not considered as equivalent secondary structure elements because they do not form a similar self‐contained hydrogen‐bonding network of the main chain atoms. In this context our finding of an unusual motif of glycine‐rich PG_II‐like helices in the structure of the acetophenone carboxylase core complex is of relevance. These PG_II‐like helices form hexagonal bundles which appear to fulfill the criterion of a (largely) saturated hydrogen‐bonding network of the main‐chain groups and therefore may be regarded in this sense as a new secondary structure element. It consists of a central PG_II‐like helix surrounded by six nearly parallel PG_II‐like helices in a hexagonal array, plus an additional PG_II‐like helix extending the array outwards. Very related structural elements have previously been found in synthetic polyglycine fibers. In both cases, all main chain NH and CO groups of the central PG_II‐helix are saturated by either intra‐ or intermolecular hydrogen‐bonds, resulting in a self‐contained hydrogen‐bonding network. Similar, but incomplete PG_II‐helix patterns were also previously identified in a GTP‐binding protein and an antifreeze protein.     

Preventive application of an entomopathogenic fungus in cover crops for wireworm control

Efficacy of the Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae) strain ART2825 for control of wireworms (Agriotes obscurus (L.), Coleoptera: Elateridae) was examined in a semi-field pot experiment. Pots were treated in late summer during sowing of spring oat as a cover crop. Survival of wireworms was assessed four weeks after their release in October 2013, and 30 weeks after release in April 2014. Viability and persistence of the fungus was determined by counting colony forming units from substrate samples and microsatellite analyses of recovered Metarhizium isolates. The number of colonies detected in the substrate in October 2013 increased with increasing concentrations of applied conidia, and no significant reduction was observed at the second evaluation date in April 2014. Increasing conidia application rates significantly increased mycosis and reduced wireworm survival, to a level comparable to that of treatment using insecticide-coated oat seeds. The preventive application of M. brunneum conidia to reduce wireworm populations in cover crops, preceding a damage-sensitive crop like potatoes, may be a promising biocontrol strategy.     

Narrowing the Gap between Theoretical and Practical Capacities in Li‐Ion Layered Oxide Cathode Materials

Although layered lithium oxides have become the cathode of choice for state‐of‐the‐art Li‐ion batteries, substantial gaps remain between the practical and theoretical energy densities. With the aim of supporting efforts to close this gap, this work reviews the fundamental operating mechanisms and challenges of Li intercalation in layered oxides, contrasts how these challenges play out differently for different materials (with emphasis on Ni–Co–Al (NCA) and Ni–Mn–Co (NMC) alloys), and summarizes the extensive corpus of modifications and extensions to the layered lithium oxides. Particular emphasis is given to the fundamental mechanisms behind the operation and degradation of layered intercalation electrode materials as well as novel modifications and extensions, including Na‐ion and cation‐disordered materials.     

Construction, verification and experimental use of two epitope-tagged collections of budding yeast strains

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein-protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available.     

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a beta-barrel structure

Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.     

Conformational sampling of the botulinum neurotoxin serotype A light chain: implications for inhibitor binding

Botulinum neurotoxins (BoNTs) are the most potent of the known biological toxins, and consequently are listed as category A biowarfare agents. Currently, the only treatments against BoNTs include preventative antitoxins and long-term supportive care. Consequently, there is an urgent need for therapeutics to counter these enzymes--post exposure. In a previous study, we identified a number of small, nonpeptidic lead inhibitors of BoNT serotype A light chain (BoNT/A LC) metalloprotease activity, and we identified a common pharmacophore for these molecules. In this study, we have focused on how the dynamic movement of amino acid residues in and surrounding the substrate binding cleft of the BoNT/A LC might affect inhibitor binding modes. The X-ray crystal structures of two BoNT/A LCs (PDB refcodes=3BTA and 1E1H) were examined. Results from these analyses indicate that the core structural features of the examined BoNT/A LCs, including alpha-helices and beta-sheets, remained relatively unchanged during 1 ns dynamics trajectories. However, conformational flexibility was observed in surface loops bordering the substrate binding clefts in both examined structures. Our analyses indicate that these loops may possess the ability to decrease the solvent accessibility of the substrate binding cleft, while at the same time creating new residue contacts for the inhibitors. Loop movements and conformational/positional analyses of residues within the substrate binding cleft are discussed with respect to BoNT/A LC inhibitor binding and our common pharmacophore for inhibition. The results from these studies may aid in the future identification/development of more potent small molecule inhibitors that take advantage of new binding contacts in the BoNT/A LC.     

Synthesis and characterization of a sulfated pentasaccharide containing the Lewisx motif

We report the synthesis of a sulfated pentasaccharide containing the Lewis(x) motif used for an NMR study described in Carbohydr. Res. 2003, 338, this issue, see following communication: doi:10.1016/S0008-6215(03)00243-X, using the dibutylstannylene acetal methodology.