Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris and the 3D structure was solved by NMR. The cysteine stabilized ?/?-fold with a unique disulfide connectivity and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.
A five-dimensional (5D) APSY (automated projection spectroscopy) HCNCH experiment is presented, which allows unambiguous correlation of sugar to base nuclei in nucleic acids. The pulse sequence uses multiple quantum (MQ) evolution which enables long constant-time evolution periods in all dimensions, an improvement that can also benefit non-APSY applications. Applied with an RNA with 23 nucleotides the 5D APSY-HCNCH experiment produced a complete and highly precise 5D chemical shift list within 1.5 h. Alternatively, and for molecules where the out-and-stay 5D experiment sensitivity is not sufficient, a set of out-and-back 3D APSY-HCN experiments is proposed: an intra-base (3D APSY-b-HCN) experiment in an MQ or in a TROSY version, and an MQ sugar-to-base (3D APSY-s-HCN) experiment. The two 3D peak lists require subsequent matching via the N1/9 chemical shift values to one 5D peak list. Optimization of the 3D APSY experiments for maximal precision in the N1/9 dimension allowed matching of all (15)N chemical shift values contained in both 3D peak lists. The precise 5D chemical shift correlation lists resulting from the 5D experiment or a pair of 3D experiments also provide a valuable basis for subsequent connection to chemical shifts derived with other experiments.
Summary Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca(2+)-dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca(2+) and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N-terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins.
Community differentiation is a fundamental topic of the social sciences, and its prehistoric origins in Europe are typically assumed to lie among the complex, densely populated societies that developed millennia after their Neolithic predecessors. Here we present the earliest, statistically significant evidence for such differentiation among the first farmers of Neolithic Europe. By using strontium isotopic data from more than 300 early Neolithic human skeletons, we find significantly less variance in geographic signatures among males than we find among females, and less variance among burials with ground stone adzes than burials without such adzes. From this, in context with other available evidence, we infer differential land use in early Neolithic central Europe within a patrilocal kinship system.
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