Archaeobotanic evidence of the preincaic Chiribaya culture. Determination of capsaicinoids in archaeological samples of Capsicum frutescens and votive foods

The lack of Capsicum species or paprika (a basic ingredient of Peruvian foodstuff) in preincaic archaeological samples and votive foods, as evidenced by archaeobotanic studies, has stimulated the chemical analyses of these samples by HPLC methods. The results confirmed the absence of capsaicinoids in these samples whereas they were detected in more ancient fossil fruit.     

Weathering-Associated Bacteria from the Damma Glacier Forefield: Physiological Capabilities and Impact on Granite Dissolution

Several bacterial strains isolated from granitic rock material in front of the Damma glacier (Central Swiss Alps) were shown (i) to grow in the presence of granite powder and a glucose-NH₄Cl minimal medium without additional macro- or micronutrients and (ii) to produce weathering-associated agents. In particular, four bacterial isolates (one isolate each of Arthrobacter sp., Janthinobacterium sp., Leifsonia sp., and Polaromonas sp.) were weathering associated. In comparison to what was observed in abiotic experiments, the presence of these strains caused a significant increase of granite dissolution (as measured by the release of Fe, Ca, K, Mg, and Mn). These most promising weathering-associated bacterial species exhibited four main features rendering them more efficient in mineral dissolution than the other investigated isolates: (i) a major part of their bacterial cells was attached to the granite surfaces and not suspended in solution, (ii) they secreted the largest amounts of oxalic acid, (iii) they lowered the pH of the solution, and (iv) they formed significant amounts of HCN. As far as we know, this is the first report showing that the combined action of oxalic acid and HCN appears to be associated with enhanced elemental release from granite, in particular of Fe. This suggests that extensive microbial colonization of the granite surfaces could play a crucial role in the initial soil formation in previously glaciated mountain areas.Glaciers in alpine regions are highly sensitive to changes in climatic conditions (29). Increasing global atmospheric temperatures over the last decades have resulted in the recession of alpine glaciers (18). Forefields of temperate alpine glaciers provide unique opportunities to study initial soil formation as well as microbial and plant succession along the chronosequences (12, 26, 34, 36). The forefields close to the glacier terminus are initially vegetation free and consist mainly of rock material with high fractions of silt-sized grains with low C and N content and small amounts of available nutrients (14). Mineral weathering is a key process in the formation of soils (1, 26), and the crucial importance of microbially promoted mineral weathering for nutrient acquisition is increasingly recognized (2, 4, 39, 46). Recently exposed rock surfaces can be considered primary ecosystems where only a few microbes that are adapted due to their mineral-weathering abilities can grow (17). Some cations of rock-forming minerals are essential for proper cell functions. However, our understanding of geochemically significant microbes in forefields of temperate alpine glacier is still very limited but is crucial for increasing our knowledge of nutrient mobilization and the buildup of organic matter that is essential for the development of macroorganisms.The area of the Damma glacier in Central Switzerland is characterized by a relatively homogenous granitic rock basement and is used as field site of the interdisciplinary research project “Biosphere-Geosphere interactions: Linking climate change, weathering, soil formation and ecosystem evolution (BigLink)” (5). In the frame of this research project, we studied the functional roles of granite-colonizing microbes as biotic weathering agents in previously glaciated areas. So far, relatively little is known about microbe-granite interactions, especially regarding the release of trace elements. Several studies have examined the dissolution of specific granite-forming minerals in the presence of actively metabolizing bacteria or compounds that simulate metabolic activity (24, 30, 31, 37, 38, 44). There is a general agreement that microbially produced organic acids, siderophores, and extracellular polysaccharides can all promote dissolution of minerals. Previous dissolution experiments have mainly been performed with (i) commercially obtained minerals (23, 45), (ii) model microorganisms that were commercially obtained from culture collections (3, 35, 45), or (iii) laboratory strains, such as those of Bacillus subtilis (23) and Burkholderia fungorum (47). Most studies have focused on individual mineral specimens rather than on the mixture of minerals that are present in granite rock (47). Few studies observed mineral weathering of collected rock and bacteria isolated from volcanic areas covered with vegetation (30, 31). Moreover, there are no studies on microbial weathering for such immediately deglaciated environments combining functional and taxonomic investigations, probably due to the difficulties in obtaining heterotrophic bacterial isolates from granitic glacier forefields. In spite of this, a comprehensive culture collection containing approximately 500 bacterial strains, which were isolated from the glacier tongue of the Damma glacier, was established. Full-length 16S rRNA gene sequences of 120 isolates revealed that many isolates obtained from oligotrophic media were closely related to readily cultivable heterotrophic bacteria (e.g., Arthrobacter sp., Collimonas sp., Paenibacillus sp., and Pseudomonas sp.). These bacteria have been found to enhance mineral dissolution (39).Our aim was to characterize the impact of microorganisms on granite weathering. We performed laboratory dissolution experiments with sterile crushed granite rock material, 12 bacterial strains, and 1 algal strain. To investigate the potential weathering abilities of these isolates, granite dissolution experiments were performed abiotically with model agents, such as HCl for proton-promoted weathering or oxalate and citrate and KCN for ligand-promoted weathering.     

Zinc proteomes, phylogenetics and evolution

Evolution has not been studied in detail with reference to the changing environment. This requires a study of the inorganic chemistry of organisms, especially metalloproteins. The evolution of organisms has been analysed many times previously using comparative studies, fossils, and molecular sequences of proteins, DNA and 16s rRNA (Zhang and Gladyshev, Chem. Rev., 2009, 109, 4828). These methods have led to the confirmation of Darwin's original proposal that evolution followed from natural selection in a changing environment often pictured as a tree. In all cases, the main tree in its upper later reaches has been well studied but its lower earlier parts are not so well defined. To approach this topic we have treated evolution as due to the intimate combination of the effect of chemical changes in the environment and in the organisms (Williams and da Silva, The Chemistry of Evolution, 2006, Elsevier). The best chemicals to examine are inorganic ions as they are common to both. As a more detailed example of the chemical study of organisms we report in this paper a bioinformatic approach to the characterization of the zinc proteomes. We deduce them from the 821 totally sequenced DNA of organisms available on NCBI, exploiting a published method developed by one of us (Andreini, Bertini and Rosato, Acc. Chem. Res., 2009, 42, 1471). Comparing the derived zinc-finger-containing proteins and zinc hydrolytic enzymes in organisms of different complexity there is a correlation in their changes during evolution related to environmental change.     

Interaction of the two soluble metal-binding domains of yeast Ccc2 with copper(I)-Atx1

Yeast Ccc2 is a P-type ATPase responsible for transport of copper(I) from the cytosol to the trans-Golgi network. It possesses a soluble cytosolic N-terminal region containing two copper(I)-binding domains. Homologous eukaryotic copper-transporting ATPases have from one to six domains. We have expressed a fragment encompassing residues 1-150 of Ccc2, which corresponds to the two domains, and found that the second domain was substantially less structured than the first. The first domain could bind copper(I) and interact with the partner protein Atx1 at variance with the second. Similar results are found in ATPases from other organisms and may represent a general feature, whose biochemical implications are not yet fully appreciated.     

Non-heme iron through the three domains of life

Metalloproteins are proteins capable of binding one or more metal ions, which are often required for their biological function or for regulation of their activities or for structural purposes. In high-throughput genome-level protein investigation efforts, such as Structural Genomics, the systematic experimental characterization of metal-binding properties (i.e. the investigation of the metalloproteome) is not always pursued, and remains far from trivial. In the present work we have applied a bioinformatic approach to investigate the occurrence of (putative) non-heme iron-binding proteins in 57 different organisms spanning the entire tree of life. It is found that the non-heme iron-proteome constitutes between 1% and 10% of the entire proteome of an organism. However, the iron-proteome constitutes a higher fraction of the proteome in archaea (on average 7.1% +/- 2.1%) than in bacteria (3.9% +/- 1.6%) and in eukaryota (1.1% +/- 0.4%). The analysis of the function of each putative iron-protein identified suggests that extant organisms have inherited the large majority of their iron-proteome from the last common ancestor.     

Shp2 in PC12 cells: NGF versus EGF signalling

The balance between specific signals from different growth factors dictates the biological response of mammalian cells including cell proliferation, differentiation and survival. PC12 cells represent a model of choice to compare the signalling of differentiative growth factors, as NGF, and of mitogenic growth factors, as EGF. In these cells the prolonged activity of the ERK kinase dictates the decision of cells to differentiate. Here we focused on the cytosolic tyrosine phosphatase Shp2 as an established regulator of the Ras-ERK cascade, to elucidate its involvement in determining the stimulation-dependent PC12 cell fate. To this end, we generated PC12 derived cell lines that express the interfering mutant of Shp2 under a tetracycline-inducible promoter. Our findings show that Shp2 participates to the opposite effects induced in PC12 cells by EGF and NGF and that the interactions with the multidocking Gab2 protein mediate such effects.     

Copper exposure effects on yeast mitochondrial proteome

Mitochondria play an important role on the entire cellular copper homeostatic mechanisms. Alteration of cellular copper levels may thus influence mitochondrial proteome and its investigation represents an important contribution to the general understanding of copper-related cellular effects. In these study we have performed an organelle targeted proteomic investigation focusing our attention on the effect of non-lethal 1mM copper concentration on Saccharomyces cerevisiae mitochondrial proteome. Functional copper effects on yeast mitochondrial proteome were evaluated by using both 2D electrophoresis (2-DE) and liquid chromatography coupled with tandem mass spectrometry. Proteomic data have been then analyzed by different unsupervised meta-analysis approaches that highlight the impairment of mitochondrial functions and the activation of oxidative stress response. Interestingly, our data have shown that stress response generated by 1mM copper treatment determines the activation of S. cerevisiae survival pathway. To investigate these findings we have treated yeast cells responsiveness to copper with hydrogen peroxide and observed a protective role of this metal. These results are suggestive of a copper role in the protection from oxidative stress possibly due to the activation of mechanisms involved in cellular survival and growth.     

Principles and patterns in the interaction between mono-heme cytochrome c and its partners in electron transfer processes

Cytochromes c are very widespread proteins that play key roles in the electron transfer events associated to a wide variety of physiological redox processes. The function of cytochromes c is, at the broad level, to interact with different partners in order to allow electrons to flow from one protein to another. Here, we focused our attention on the protein-protein interactions that involve mono-heme cytochrome c domains in order to identify possible general vs. specific patterns of intermolecular interactions at the structural level. We observed that a number of physico-chemical properties are statistically different in transient vs. permanent and fused complexes. These include the extent of the protein interface area, the amino acid composition and the packing density at the interface. The understanding of the features of transient redox complexes is of particular importance because of the difficulty of obtaining co-crystals that preserve the physiologically relevant configuration. In addition, we identified three different structural modes of interaction that cover all the structurally characterized cytochrome c interactions except one. The mode of interaction does not correlate with the nature of the complex (transient, permanent, fused). Regardless of the mode of interaction, the distance between the heme iron and the partner metal center or organic cofactor center of mass is typically around 19-20 ? for complexes permitting direct electron transfer between the two sites.